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The RNA world, and the origins of life

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1The RNA world, and the origins of life Empty The RNA world, and the origins of life on Wed May 20, 2015 3:28 pm

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The RNA world, and the origins of life

https://reasonandscience.catsboard.com/t2024-the-rna-world-and-the-origins-of-life

Leslie Orgel at the University of Oxford, UK, was among the first to propose  RNA as a catalyst of the chemical reactions to make itself. A new theory was born, later dubbed the ‘RNA world hypothesis’.

The RNA world hypothesis, to be true, however, has to overcome  major hurdles:

1. Life uses only right-handed RNA and DNA. The homochirality problem is unsolved. This is an “intractable problem” for chemical evolution
2. RNA has been called a “prebiotic chemist's nightmare” because of its combination of large size, carbohydrate building blocks, bonds that are thermodynamically unstable in water, and overall intrinsic instability. Many bonds in RNA are thermodynamically unstable with respect to hydrolysis in water, creating a “water problem”. Finally, some bonds in RNA appear to be “impossible” to form under any conditions considered plausible for early Earth.   In chemistry, when free energy is applied to organic matter without Darwinian evolution, the matter devolves to become more and more “asphaltic”, as the atoms in the mixture are rearranged to give ever more molecular species. In the resulting “asphaltization”, what was life comes to display fewer and fewer characteristics of life.
3. Systems of interconnected software and hardware like in the cell are irreducibly complex and interdependent. There is no reason for information processing machinery to exist without the software and vice versa.
4. A certain minimum level of complexity is required to make self-replication possible at all; high-fidelity replication requires additional functionalities that need even more information to be encoded
5. RNA catalysts would have had to copy multiple sets of RNA blueprints nearly as accurately as do modern-day enzymes
6. In order a molecule to be a self-replicator, it has to be a homopolymer, of which the backbone must have the same repetitive units; they must be identical. On the prebiotic world, the generation of a homopolymer was however impossible.
7. Not one self-replicating RNA has emerged to date from quadrillions (10^24) of artificially synthesized, random RNA sequences.  
8. Over time, organic molecules break apart as fast as they form
9. How could and would random events attach a phosphate group to the right position of a ribose molecule to provide the necessary chemical activity? And how would non-guided random events be able to attach the nucleic bases to the ribose?  The coupling of a ribose with a nucleotide is the first step to form RNA, and even those engrossed in prebiotic research have difficulty envisioning that process, especially for purines and pyrimidines.”
10. L. E. Orgel:  The myth of a self-replicating RNA molecule that arose de novo from a soup of random polynucleotides. Not only is such a notion unrealistic in light of our current understanding of prebiotic chemistry, but it should strain the credulity of even an optimist's view of RNA's catalytic potential.
11.Macromolecules do not spontaneously combine to form macromolecules
125. The transition from RNA to DNA is an unsolved problem. 
13. To go from a self-replicating RNA molecule to a self-replicating cell is like to go from a house building block to a fully build house. 
14. Arguably one of the most outstanding problems in understanding the progress of early life is the transition from the RNA world to the modern protein based world. 31 
15. It is thought that the boron minerals needed to form RNA from pre-biotic soups were not available on early Earth in sufficient quantity, and the molybdenum minerals were not available in the correct chemical form. 33
16. Given the apparent limitation of double-stranded RNA (dsRNA) genomes to about 30 kb, together with the complexity of DNA synthesis, it appears dif¢cult for a dsRNA genome to encode all the information required before the transition from an RNA to a DNA genome. Ribonucleotide reductase itself, which synthesises deoxyribonucleotides from ribonucleotides, requires complex protein radical chemistry, and RNA world genomes may have reached their limits of coding capacity well before such complex enzymes had evolved. 33 

Proponents of the RNA world hypothesis commonly argue that it has been proven that RNA's could self-replicate. Let's suppose that were true, that is as if self-replication could produce a hard drive. To go from a hard drive ( which by itself requires complex information to be assembled, in case of biology, DNA, not RNA since it's too unstable, ) that does not explain the origin of the information to make all life essential parts in the cell.
It is as to go just from a hard drive storage device to a self replicating factory with the ability of self replication of the entire factory once ready, to respond to changing environmental demands and regulate its metabolic pathways, regulate and coordinate all cellular processes, such as molecule and building block biosynthesis according to the cells demands, depending on growth, and other factors.
The ability of uptake of nutrients, to be structured, internally compartmentalized and organized, being able to check replication errors and minimize them, and react to stimuli, and changing environments. That's is, the ability to adapt to the environment is a must right from the beginning. If just ONE single protein or enzyme - of many - is missing, no life. If topoisomerase II or helicase are missing - no replication - no perpetuation of life.
Why would a prebiotic soup or hydrothermal vents produce these proteins - if by their own there is no use for them?

Shapiro, 1986, p.186.
"`In other words,' I said, `if you want to create life, on top of the challenge of somehow generating the cellular  components out of non-living chemicals, you would have an even bigger problem in trying to it the ingredients
together in the right way.' `Exactly! ... So even if you could accomplish the thousands of steps between the amino acids in the Miller tar-which probably didn't exist in the real world anyway-and the components you need for a
living cell-all the enzymes, the DNA, and so forth-you's still immeasurably far from life. ... the problem of  assembling the right parts in the right way at the right time and at the right place, while keeping out the wrong
material, is simply insurmountable.'"

Paul Davies The Algorithmic Origins of Life
Despite the conceptual elegance of the RNA world, the hypothesis faces problems, primarily due to the immense challenge of synthesizing RNA nucleotides under plausible prebiotic conditions and the susceptibility of RNA oligomers to degradation via hydrolysis 21 Due to the organizational structure of systems capable of processing algorithmic (instructional) information, it is not at all clear that a monomolecular system – where a single polymer plays the role of catalyst and informational carrier – is even logically consistent with the organization of information flow in living systems, because there is no possibility of separating information storage from information processing (that being such a distinctive feature of modern life). As such, digital-first systems (as currently posed) represent a rather trivial form of information processing that fails to capture the logical structure of life as we know it. 

Replicator first, and metabolism first scenarios
https://reasonandscience.catsboard.com/t1428-replicator-first-and-metabolism-first-scenarios

No evidence that RNA molecules ever had the broad range of catalytic activities
https://reasonandscience.catsboard.com/t2243-no-evidence-that-rna-molecules-ever-had-the-broad-range-of-catalytic-activities

The hardware and software of the cell, evidence of design
https://reasonandscience.catsboard.com/t2221-the-hardware-and-software-of-the-cell-evidence-of-design

The origin of replication and translation and the RNA World
https://reasonandscience.catsboard.com/t2234-the-origin-of-replication-and-translation-and-the-rna-world

Tom Robbins: The time argument is worthless. As over time, organic molecules break apart as fast as they form - thus the monkey's on a typewriter argument does not work, as the INFORMATION represented on the paper when they strike a key, disappears off the paper as they type. Given enough time, CERTAIN things will probably happen, but only things that are not impossible (and of course there was a finite amount of time from the creation of the earth). Nature can't create specified, dedicated, self-replicating, self-repairing, self-editing information - THE ONLY source that we know of that can do this, is MIND..

Given the complexity of the simplest ribozymes mediating transcription and translation and the ongoing failure to obtain activated ribonucleotides from ribose and nucleobases, the RNA-world hypothesis faces substantial challenge 23

Asphalt, Water, and the Prebiotic Synthesis of Ribose, Ribonucleosides, and RNA 25
RNA has been called a “prebiotic chemist's nightmare” because of its combination of large size, carbohydrate building blocks, bonds that are thermodynamically unstable in water, and overall intrinsic instability. Many bonds in RNA are thermodynamically unstable with respect to hydrolysis in water, creating a “water problem”. Finally, some bonds in RNA appear to be “impossible” to form under any conditions considered plausible for early Earth.   In chemistry, when free energy is applied to organic matter without Darwinian evolution, the matter devolves to become more and more “asphaltic”, as the atoms in the mixture are rearranged to give ever more molecular species. In the resulting “asphaltization”, what was life comes to display fewer and fewer characteristics of life.

Biologists routinely observe the opposite. In the biosphere, when free energy is provided to organic matter that does have access to Darwinian evolution, that matter does not become asphaltic. Instead, “life finds a way” to exploit available raw materials, including atoms and energy, to create more of itself and, over time, better of itself. This observation is made across the Earth, from its poles to the equator, from high in the atmosphere to the deepest oceans, and in humidities that cover all but the very driest. The contrast between these commonplace observations in chemistry versus commonplace observations in biology embodies the paradox that lies at the center of the bio-origins puzzle. Regardless of the organic materials or the kinds of energy present early on Earth, chemists expect that a natural devolution took them away from biology toward asphalt. To escape this asphaltic fate, this devolution must have transited a chemical system that was, somehow, able to sustain Darwinian evolution. Otherwise, the carbon on Earth would have ended up looking like the carbon in the Murchison meteorite (or the La Brea tar pits without the fossils).

We have not addressed the “chirality problem”. Here, the challenge arises as we attempt to select from among a large number of possible approaches for chiral enrichment, ranging from the interaction of organic species with chiral minerals (e.g., quartz) to the resolution at the time of oligomerization. Current experiments suggest that RNA molecules that catalyze the degradation of RNA are more likely to emerge from a library of random RNA molecules than RNA molecules that catalyze the template-directed synthesis of RNA, especially given cofactors (e.g., Mg2þ). This could, of course, be a serious (and possibly fatal) flaw to the RNA-first hypothesis for bioorigins.

We need to explain the origin of both the hardware and software aspects of life, or the job is only half finished. Explaining the chemical substrate of life and claiming it as a solution to life’s origin is like pointing to silicon and copper as an explanation for the goings-on inside a computer. It is this transition where one should expect to see a chemical system literally take on “a life of its own”, characterized by informational dynamics which become decoupled from the dictates of local chemistry alone (while of course remaining fully consistent with those dictates). Thus the famed chicken-or-egg problem (a sole hardware issue) is not the true sticking point. Rather, the puzzle lies with something fundamentally different, a problem of a causal organization having to do with the separation of informational and mechanical aspects into parallel causal narratives. The real challenge of life’s origin is thus to explain how instructional information control systems emerge naturally and spontaneously from mere molecular dynamics.

But now we hit a snag. The second step on the road to life, or at least the road to proteins, is for amino acids to link together to form molecules known as peptides. A protein is a long peptide chain, or a polypeptide. Whereas the spontaneous formation of amino acids from an inorganic chemical mixture is an allowed downhill process, coupling amino acids together to form peptides is an uphill process. It therefore heads in the wrong direction, thermodynamically speaking. Each peptide bond that is forged requires a water molecule to be plucked from the chain. In a watery medium like a primordial soup, this is thermodynamically unfavorable. Consequently, it will not happen spontaneously: work has to be done to force the newly extracted water molecule into the water saturated medium. Obviously, peptide formation is not impossible because it happens inside living organisms. But there the uphill reaction is driven along by the use of customized molecules that are pre-energized to supply the necessary work. In a simple chemical soup, no such specialized molecules would be on hand to give the reactions the boost they need. So a watery soup is a recipe for molecular disassembly, not self-assembly.

Systems of interconnected software and hardware like in the cell are irreducibly complex and  interdependent. There is no reason for information processing machinery to exist without the software and vice versa.


Proof by self-replicating RNA
1. Till now, after more than 50 years of biochemical experiments, there were no self-replicating RNA molecules generated in any different laboratory conditions that resemble the prebiotic period of creation.
2. RNA has no self-replicating power.
3. Without self-replicating RNA there is neither natural selection nor evolution.
4. Therefore, there must have been another original cause of existence and that cause is God.

The classic evolutionary problem of 'which came first, protein or DNA' has not been solved by the 'self-reproducing' RNA theory as many textbooks imply. The theory is not credible as it was based on laboratory simulations which were highly artificial, and was carried out with a 'great deal of help from the scientists'.
19

For 40 years, efforts to understand the prebiotic synthesis of the ribonucleotide building blocks of RNA have been based on the assumption that they must have assembled from their three molecular components: a nucleobase (which can be adenine, guanine, cytosine or uracil), a ribose sugar and phosphate. Of the many difficulties encountered by those in the field, the most frustrating has been the failure to find any way of properly joining the pyrimidine nucleobases — cytosine and uracil — to ribose3 . The idea that a molecule as complex as RNA could have assembled spontaneously has therefore been viewed with increasing scepticism. 20

The issue of the complete synthesis of RNA nucleotides has been a major stumbling block for the RNA World Hypothesis.  The sugar found in the backbone of both DNA and RNA, ribose, has been particularly problematic, as the most prebiotically plausible chemical reaction schemes have typically yielded only a small amount of ribose mixed with a diverse assortment of other sugar molecules. 16

The most widely accepted hypothesis among biologists, the RNA world hypothesis, still has strong supporters [1] but difficulties with the hypothesis are recognized, especially the problem of synthesizing RNA in the absence of enzymes 15

Let's have a closer look at RNA world  - origin of life proposals: Here from the book of Bruce Alberts:  The Evolution of the Cell

One of the principal problems concerning the hypothesis of the RNA world is that it appears quite unlikely that a prebiotic environment could have existed containing the mixture of activated nucleotides favoring the formation and replication of ribozymes, as well as their evolution through natural selection. Even if there were several candidate reactions for an efficient prebiotic synthesis of nucleic bases, access to monomeric nucleotides by chemical pathways, in fact, comes up against several obstacles. If one goes no further than mimicking the biochemical pathway, the first difficulty that occurs is that of synthesizing ribose, which is formed in just negligible quantities within the complex mixture obtained by polymerization of formaldehyde, and, what is more, has a limited lifetime. The bond between a nucleic base and ribose that produces a nucleoside is then a very difficult reaction. There still remains the matter of obtaining a nucleotide by phosphorylation, which leads to mixtures because three positions remain available on the ribose, and then there is its activation 24

The RNA world, and the origins of life Rna_bi11
The RNA world, and the origins of life U8MyFKf
The daunting problem  how to make the pentose ring of RNA and DNA
How could and would random events attach a phosphate group to the right position of a ribose molecule to provide the necessary chemical activity? And how would non-guided random events be able to attach the nucleic bases to the ribose? 
 
In the case of RNA, not only must phosphodiester links be repeatedly forged (if we assume joining reactions involving P–O bond formation), but they must ultimately connect the 5ʹ‑oxygen of one nucleotide to the 3ʹ‑oxygen, and not the 2ʹ‑oxygen, of the next nucleotide. 2ʹ,5ʹ‑Linkages can be tolerated functionally at low levels in certain RNAs31, but they are not inheritable in a sequence-specific manner, and for most intents and purposes, extant biology
uses 3ʹ,5ʹ‑linkages. Although we have demonstrated that 3ʹ,5ʹ‑linkages can be preferentially formed by prebiotically selective 2ʹ‑O‑acetylation and ligation of those oligonucleotides with 3ʹ‑phosphate termini in mixtures of oligonucleotides with 2ʹ‑ and 3′-phosphate termini, the synthetic selectivities and preferences are not enough to explain how RNA with all 3ʹ,5ʹ‑linkages might first have been produced. 29

James Tour wrote : 
“The coupling of a ribose with a nucleotide is the first step [in abiogenesis], and even those engrossed in prebiotic research have difficulty envisioning that process, especially for purines and pyrimidines.” 27
A further problem lies in the synthesis and preservation of ribose, with the right chirality. ribose is not particularly preferred over other sugars nor is it stable. Hence, an autocatalytic cycle designed to produce large amounts of carbohydrates from formaldehyde will not preferentially make ribose nor preserve it. One then faces the question of how ribose molecules were maintained against chemical processes that tend to decompose them quickly into a nondescript assemblage of polymeric mixtures. The ribose produced must have the correct handedness or chirality; on Earth, d-sugars are exclusively involved in living processes. Production of a mixture of d- and l-sugars produces nucelotides that do not fit together properly, producing a very open, weak structure that cannot survive to replicate, catalyze, or synthesize other biological molecules. In fact, the synthesis of the RNA molecule itself is interrupted by mixing nucleotides of different chirality; only in a controlled laboratory experiment or theoretical model can such an assemblage be realized 26
To create a properly functioning RNA molecule out of a batch of heterochiral l- and d-sugars is a daunting challenge. The genetic template that sustains a particular kind of chemistry and set of structures is quickly lost after just one generation.
The RNA world, and the origins of life Dna_fo11


Problematic Chemical Postulates of the RNA World Scenario

Postulate 1: 
There was a prebiotic pool of beta-D-ribonucleotides.   8

Beta-D-ribonucleotides  are compounds made up of a purine (adenine or guanine) or a pyrimidine (uracil or cytosine) linked to the 1'-position of ribose in the beta-configuration.

There is, in addition, a phosphate group attached to the 5'-position of the ribose. For the four different ribonucleotides in this prebiotic scenario, there would be hundreds of other possible isomers.

But each of these four ribonucleotides is built up of three components: a purine or pyrimidine, a sugar (ribose), and phosphate. It is highly unlikely that any of the necessary subunits would have accumulated in any more than trace amounts on the primitive Earth. Consider ribose. The proposed prebiotic pathway leading to this sugar, the formose reaction, is especially problematic.

prebiotically plausible sequences of steps to the precursors of this ribose derivative and from it to the standard nucleotides are not obvious. 17
Sugars
While sugars have a variety of important roles in biochemistry, they are important components of nucleic acid backbones in the form of ribose and deoxyribose. 28 All canonical sugars share the empirical formula (CH2O)n, formally making them oligomers of formaldehyde (HCHO). Ribose, the sugar used in RNA, is but one of the isomeric pentamers where n = 5, as each sugar may contain a number of stereo centers. Early in the development of organic chemistry as an empirical science, it was found that basic solutions of formaldehyde could give rise to a complex mixture of compounds, which included various sugars (Butlerow 1861). The mechanism of this synthesis has since been explored extensively (Breslow 1959) (Figure 5.13).

The RNA world, and the origins of life FeHRglA


Early in its consideration as a prebiotic process for the production of ribose and other sugars, it was pointed out that the extreme diversity of products the reaction gives rise to, as well as the ultimate instability of sugars under the conditions of synthesis, may render this an implausible source of prebiotic carbohydrates . Recently, there has been a resurgence of interest in this pathway as several new mechanisms have been discovered that produce a less diverse mixture, give rise to a higher yield of ribose, and importantly make the ultimate sugar derivatives considerably more stable than they are in the free form. For example, conducting the reaction in the presence of borate selectively gives rise to a good yield of ribose–borate derivatives

Formose reaction
The formose reaction is of great importance to the question of the origin of life as it explains part of the path from simple formaldehyde to complex sugars like ribose and from there to RNA. In one experiment simulating early Earth conditions, pentoses formed from mixtures of formaldehyde, glyceraldehyde, and borate minerals such as colemanite. Both formaldehyde and glycolaldehyde have been observed spectroscopically in outer space, making the formose reaction of particular interest to the field of astrobiology.
The Butlerov synthesis of sugars, also known as the formose reaction, is very complex. It depends on the presence of a suitable inorganic catalyst, most commonly calcium hydroxide (Ca[OH]2) or calcium carbonate (CaCO3). In the absence of such inorganic catalysts, little or no sugar is produced. The Butlerov synthesis is autocatalytic—that is, catalyzed by its own products. It proceeds in a series of steps from formaldehyde through glycoaldehyde, glyceraldehyde, and dihydroxyacetone (four-carbon sugars), to pentoses (five-carbon sugars), to hexoses (six-carbon sugars) such as glucose and fructose. These six-carbon, simple sugars are important constituents of biological carbohydrates. The detailed reaction sequence is not yet understood, but may proceed as shown in scheme 3.7.
The RNA world, and the origins of life Formos10
The ribose formed is a racemic mixture, consisting of D-ribose (the configuration found in the biological nucleic acids RNA and DNA) and its mirror image L-ribose (a form not found in biological systems). All sugars have fairly similar chemical properties; thus, it is difficult to envision simple physicochemical mechanisms that could (1) preferentially concentrate ribose from a complex mixture or (2) enhance the yield of the d-ribose relative to that of its biologically inactive mirror image. The inherent instability of ribose poses yet another problem with respect to its prebiotic availability. Under neutral conditions (pH 7), the half-life for the decomposition of ribose is 73 minutes at 100C and only 44 years at 0C (Larralde, Robertson, and Miller 1995). As summarized in table 3.6, other pentose and hexose sugars are similarly unstable, as is ribose-2,4-diphosphate. Although many ways have been suggested to stabilize sugars, attaching the sugar to a purine or pyrimidine—that is, linking the sugar to a nucleoside—is the most biologically interesting. But the synthesis of such sugar–base nucleosides is notoriously difficult to achieve under truly prebiotic conditions. Thus, ribose-containing nucleosides are unlikely to have been components of the earliest prebiotic informational macromolecules (Shapiro 1988). 32

Aldol reaction
The aldol reaction is a means of forming carbon–carbon bonds in organic chemistry.Discovered independently by Charles-Adolphe Wurtz and Alexander Borodin in 1872, the reaction combines two carbonyl compounds (the original experiments used aldehydes) to form a new β-hydroxy carbonyl compound. These products are known as aldols, from the aldehyde + alcohol, a structural motif seen in many of the products. Aldol structural units are found in many important molecules, whether naturally occurring or synthetic. For example, the aldol reaction has been used in the large-scale production of the commodity chemical pentaerythritol[11] and the synthesis of the heart disease drug Lipitor (atorvastatin, calcium salt).
https://en.wikipedia.org/wiki/Aldol_reaction

and aldose-ketose isomerizations.
In carbohydrate chemistry, the Lobry de Bruyn–van Ekenstein transformation also known as the Lobry de Bruyn–Alberda–van Ekenstein transformation is the base or acid catalyzed transformation of an aldose into the ketose isomer or vice versa, with a tautomeric enediol as reaction intermediate. Ketoses may be transformed into 3-ketoses, etcetera. The enediol is also an intermediate for the epimerization of an aldose or ketose.
https://en.wikipedia.org/wiki/Lobry_de_Bruyn%E2%80%93van_Ekenstein_transformation

The improbability of prebiotic nucleic acid synthesis.
Many accounts of the origin of life assume that the spontaneous synthesis of a self-replicating nucleic acid could take place readily. Serious chemical obstacles exist, however, which make such an event extremely improbable. Prebiotic syntheses of adenine from HCN, of D,L -ribose from adenosine, and of adenosine from adenine and D-ribose have in fact been demonstrated. However these procedures use pure starting materials, afford poor yields, and are run under conditions which are not compatible with one another. Any nucleic acid components which were formed on the primitive earth would tend to hydrolyze by a number of pathways. Their polymerization would be inhibited by the presence of vast numbers of related substances which would react preferentially with them. It appears likely that nucleic acids were not formed by prebiotic routes

The nitrogenous substances react with formaldehyde, the intermediates in the pathways to sugars, and with sugars themselves to form non-biological materials10. Furthermore, as Stanley Miller and his colleagues recently reported, "ribose and other sugars have surprisingly short half-lives for decomposition at neutral pH, making it very unlikely that sugars were available as prebiotic reagents."

Or consider adenine. Reaction pathways proposed for the prebiotic synthesis of this building block start with HCN in alkaline (pH 9.2) solutions of NH4OH.12 These reactions give small yields of adenine (e.g., 0.04%) and other nitrogenous bases provided the HCN concentration is greater than 0.01 M. However, the reaction mixtures contain a great variety of nitrogenous substances that would interfere with the formose reaction. Therefore, the conditions proposed for the prebiotic synthesis of purines and pyrimidines are clearly incompatible with those proposed for the synthesis of ribose. Moreover, adenine is susceptible to deamination and ring-opening reactions (with half-lives of about 80 years and 200 years respectively at 37º C and neutral pH), making its prebiotic accumulation highly improbable. This makes it difficult to see how any appreciable quantities of nucleosides and nucleotides could have accumulated on the primitive Earth. If the key components of nucleotides (the correct purines and pyrimidines, ribose, and phosphate) were not present, the possibility of obtaining a pool of the four beta-D-ribonucleotides with correct linkages would be remote indeed.

If this postulate, the first and most crucial assumption, is not valid, however, then the entire hypothesis of an RNA World formed by natural processes becomes meaningless.
Though attractive, there are several serious objections to the notion that life began with RNA: 18

Pentose sugars, constituents of RNA and DNA, can be synthesised in the formose reaction, given the presence of formaldehyde (HCHO). The products are a melange of sugars of various carbon lengths which are optically left- and right-handed (d and l). With few exceptions sugars found in biological systems are of the d type; for instance, β-d-ribose of RNA, which is always produced in small quantities abiotically.Hydrocyanic acid (HCN) undergoes polymerisation to form diaminomaleonitrile which is on the pathway to producing adenine, hypoxanthine, guanine, xanthine and diaminopurine. These are purines: there is difficulty in producing pyrimidines (cytosine, thymine and uracil) in comparable quantities Neither preformed purines nor pyrimidines have been successfully linked to ribose by organic chemists. An attempt to make purine nucleosides resulted in a “dizzying array of related compounds”.39 This is expected if sugars and bases were randomly coupled. The prebiotic production of numerous isomers and closely related molecules hinders the likelihood of forming desirable mononucleosides. Furthermore, unless ribose and the purine bases form nucleosides rapidly they would be degraded quite quickly.
The chemical structure and constituents of RNA.
Signature in the Cell, Stephen C. Meyer, page 241:
Producing ribose under realistic conditions has proven even more problematic. Prebiotic chemists have proposed that ribose could have arisen on the early earth as the by-product of a chemical reaction called the formose reaction. The formose reaction is a multistep chemical reaction that begins as molecules of formaldehyde in water react with one another. Along the way, the formose reaction produces a host of different sugars, including ribose, as intermediate by-products in the sequence of reactions. But, as Shapiro has pointed out, the formose reaction will not produce sugars in the presence of nitrogenous substances.11 These include peptides, amino acids, and amines, a category of molecules that includes the nucleotide bases. This obviously poses a couple of difficulties. First, it creates a dilemma for scenarios that envision proteins and nucleic acids arising out of a prebiotic soup rich in amino acids. Either the prebiotic environment contained amino acids, which would have prevented sugars (and thus DNA and RNA) from forming, or the prebiotic soup contained no amino acids, making protein synthesis impossible. Of course, RNA-first advocates might try to circumvent this difficulty by proposing that proteins arose well after RNA. Yet since the RNA-world hypothesis envisions RNA molecules coming into contact with amino acids early on within the first protocellular membranes (see above), choreographing the origin of RNA and amino acids to ensure that the two events occur separately becomes a considerable problem.

Nucleosides and Nucleotides
The construction of nucleosides depends on the union of a nitrogenous base, via the correct linkage, with a sugar derivative. 28 Some success in prebiotic synthesis has been achieved in this area. For example, it has been found that heating pure ribose with purines gives rise to small yields of purine ribosides, though a variety of isomers are produced. The equivalent reaction using pyrimidines does not work well, however. Recently, though, it has been found that a nonnatural pyrimidine can be linked under the same condition to give a pyrimidine nucleoside . The phosphorylation of nucleosides to give nucleotides has been accomplished in a variety of manners that are conceivably prebiotic. Dry heating various mixtures of nucleosides in the presence of ammonium salts and orthophosphate or apatite and cyanate gives decent yields of pyrimidine nucleotides. The limitations of the synthesis of pyrimidine nucleosides led Orgel and coworkers to examine other disconnects to find less obvious, unorthodox syntheses, which have been elucidated and explored further more recently. This approach is novel in that the sugar and nitrogenous base are constructed simultaneously and phosphate is incorporated prior to the completion of the nucleoside’s synthesis. This has proven effective for the pyrimidines, but to date, a complete analogous synthesis for the purine nucleotides has proven elusive.

Postulate 2: 
Beta-D ribonucleotides spontaneously form polymers linked together by 3', 5'-phosphodiester linkages (i.e., they link to form molecules of RNA

nucleotides do not link unless there is some type of activation of the phosphate group. The only effective activating groups for the nucleotide phosphate group (imidazolines, etc.), however, are those that are totally implausible in any prebiotic scenario. In living organisms today, adenosine-5'-triphosphate (ATP) is used for activation of nucleoside phosphate groups, but ATP would not be available for prebiotic syntheses. Joyce and Orgel note the possible use of minerals for polymerization reactions, but then express their doubts about this possibility

Joyce and Orgel then note that if there were activation of the phosphate group, the primary polymer product would have 5', 5'-pyrophosphate linkages; secondarily 2', 5'-phosphodiester linkages -- while the desired 3',5'-phosphodiester linkages would be much less abundant. However, all RNA known today has only 3',5'-phosphodiester linkages, and any other linkages would alter the three-dimensional structure and possibilities for function as a template or a catalyst.

Even waiving these obstacles, and allowing for minute amounts of oligoribonucleotides, these molecules would have been rendered ineffective at various stages in their growth by adding incorrect nucleotides, or by reacting with the myriads of other substances likely to have been present. Moreover, the RNA molecules would have been continuously degraded by spontaneous hydrolysis and other destructive processes operating on the primitive Earth.

In brief, any movement in the direction of an RNA World on a realistically-modeled early Earth would have been continuously suppressed by destructive cross-reactions.

One of the more enigmatic and difficult problems confronting the prebiotic chemistry community is identifying how the monomers of RNA, or pre-RNA, or even non-related polymeric components selectively formed and self-assembled out of the presumed random prebiotic mixtures. It is in this assembly into informational polymers (Figure 4) where significant selection processes must have occurred not only for the base composition but also for the other components of nucleic acids (or nucleic acid alternatives and precursors). Focusing on just a narrow view of RNA precursors, the linking of a nucleo-base to a ribose sugar is one such pressure. There are multiple ways in which a nucleobase can be attached to ribose via an N-glycosidic bond, but only one is found in contemporary nucleic acids (via the N9 of purines and N1 of pyrimidines). 14

Achieving regio- and stereochemical selectivity of glycosylation reactions under simulated prebiotic conditions has plagued the community ever since Orgel and others began working on this problem

Complex Chemical Systems Can Develop in an Environment That Is Far from Chemical Equilibrium 7
Simple organic molecules such as amino acids and nucleotides can associate to form polymers. One amino acid can join with another by forming a peptide bond, and two nucleotides can join together by a phosphodiester bond.

Protein synthesis (condensation of amino acids through sequential peptide bond formation) is a fundamental and ubiquitous reaction in biology. Aqueous media are the required environments in which this chemistry takes place; however, protein synthesis is unfavorable in aqueous solution. In modern biology, the condensation reactions necessary in the formation of peptide bonds are facilitated catalytically by the large subunit of the ribosome.

Peptide bond synthesis occurs in the 50S subunit ( of the ribosome ) at the peptidyl transferase center, (PTC) 4

The crucial peptide bond formation of protein synthesis is catalyzed by the ribosome in all organisms. 5

The activation of amino acids and the formation of peptides under primordial conditions is one of the great riddles of the origin of life.  

The famous pioneer of evolutionary origin-of-life experiments, Stanley Miller, points out that polymers are ‘too unstable to exist in a hot prebiotic environment’ 10

Question: How could simple organic molecules such as amino acids and nucleotides  associate to form polymers,  one amino acid  joining with another by forming a  peptide bond, if peptide bonds are synthesized in the probably most complex protein complex known, the ribosome, but the ribosome was not there at this stage ?


The RNA world, and the origins of life Journal_of_Cosmology222




The RNA world, and the origins of life Journal_of_Cosmology



Prebiotic Peptide synthesis was likely initiated in a simple way, yet must have evolved into the contemporary complexity of the ribosome. Of course. There is no other explanation, since an Intelligent designer is excluded a priori. In order to know how the current ribosome-catalyzed reaction evolved from a primitive system, model systems based on the RNA world hypothesis with the molecules like the minihelix and tRNA were postulated. Elucidation of the evolutionary route from the simple system to the present complex ribosome is a big challenge in modern science; this gap may be filled by the concept of the proto-ribosome, which is composed of a symmetrical tRNA-like dimer. 5

The peptide synthesis hypotheses must jump over a large gap to attain ribosome-based peptide synthesis. 5

We have right at the beginning of naturalistic proposals of the origin of life the typical guess work which extends all over the key issues of the origin of life, the transition to the 3 domains of life, from unicellular to multicellular, and biodiversity on earth. That seems to be a RED LiNE extending through all scientific papers, which deal with key issues in biology. Naturalism is simply not tenable, the explanations all end at a dead end, where guesswork is common, and evolution of the gaps arguments.  

A restricted set of 20 amino acids constitute the universal building blocks of the proteins, while RNA and DNA molecules are constructed from just four types of nucleotides each.   It is uncertain why these particular sets of monomers were selected for biosynthesis in preference to others that are chemically similar

The earliest polymers may have formed in any of several ways - for example, by the heating of dry organic compounds or by the catalytic activity of high concentrations of inorganic polyphosphates or other crude mineral catalysts. Under laboratory conditions, the products of similar reactions are polymers of variable length and random sequence in which the particular amino acid or nucleotide added at any point depends mainly on chance

The origin of life requires that in an assortment of such molecules there must have been some possessing, if only to a small extent, a crucial property: the ability to catalyze reactions that lead, directly or indirectly, to production of more molecules of the catalyst itself. Production of catalysts with this special self-promoting property would be favored, and the molecules most efficient in aiding their own production would divert raw materials from the production of other substances. In this way one can envisage the gradual development of an increasingly complex chemical system of organic monomers and polymers that function together to generate more molecules of the same types, fueled by a supply of simple raw materials in the environment. Such an autocatalytic system would have some of the properties we think of as characteristic of living matter: it would comprise a far from random selection of interacting molecules; it would tend to reproduce itself; it would compete with other systems dependent on the same feedstocks; and if deprived of its feedstocks or maintained at a wrong temperature that upsets the balance of reaction rates, it would decay toward chemical equilibrium and "die."

What we see here, is a typical fairy tale story, based on no evidence, just fantasy without a shred data to back up the story. Why should someone be credule towards such a scenario? Words like must have been, would be, one can envisage, would, would have, we think of, it would do not invoke much credibility.....


The RNA world, and the origins of life Www_bioon_com_book_biology_mboc_mboc_cgi_action
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Formation of polynucleotides and polypeptides.   Nucleotides of four kinds (here represented by the single letters A, U, G, and C) can undergo spontaneous polymerization with the loss of water. The product is a mixture of polynucleotides that are random in length and sequence. Similarly, amino acids of different types, symbolized here by three-letter abbreviated names, can polymerize with one another to form polypeptides. Present-day proteins are built from a standard set of 20 types of amino acids.

Polynucleotides Can Both Store Information and Catalyze Chemical Reactions 2
Polynucleotides have properties that contrast with those of polypeptides. They have more limited capabilities as catalysts, but they can directly guide the formation of exact copies of their own sequence. This capacity depends on the complementary pairing of nucleotide subunits, which enables one polynucleotide to act as a template for the formation of another. In the simplest case, a polymer composed of one nucleotide (for example, polycytidylic acid, or poly C) can line up the subunits required to make another polynucleotide (in this example, polyguanylic acid, or poly G) along its surface, thereby promoting their polymerization into poly G

The RNA world, and the origins of life Www_bioon_com_book_biology_mboc_mboc_cgi_action


Polynucleotides as templates.    Preferential binding occurs between pairs of nucleotides (G with C and U with A) by relatively weak chemical bonds (above). This pairing enables one polynucleotide to act as a template for the synthesis of another (left).

This capacity depends on the complementary base pairing of nucleotide subunits, which enables one polynucleotide to act as a template for the formation of another. As we have seen in this and the preceding chapter, such complementary templating mechanisms lie at the heart of DNA replication and transcription in modern-day cells. But the efficient synthesis of polynucleotides by such complementary templating mechanisms requires catalysts to promote the polymerization reaction: without catalysts, polymer formation is slow, error-prone, and inefficient.


Consider now a polynucleotide with a more complex sequence of subunitsspecifically, a molecule of RNA strung together from four types of nucleotides, containing the bases uracil (U), adenine (A), cytosine (C), and guanine (G), arranged in some particular sequence. Because of complementary pairing between the bases A and U and between the bases G and C, this molecule, when added to a mixture of activated nucleotides under suitable conditions, will line them up for polymerization in a sequence complementary to its own. The resulting new RNA molecule will be rather like a mold of the original, with each A in the original corresponding to a U in the copy and so on. The sequence of nucleotides in the original RNA strand contains information that is, in essence, preserved in the newly formed complementary strands: the second round of copying, with the complementary strand as a template, restores the original sequence 2

The RNA world, and the origins of life Www_bioon_com_book_biology_mboc_mboc_cgi_action
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Replication of a polynucleotide sequence (here an RNA molecule).    In step 1 the original RNA molecule acts as a template to form an RNA molecule of complementary sequence. In step 2 this complementary RNA molecule itself acts as a template, forming RNA molecules of the original sequence. Since each templating molecule can produce many copies of the complementary strand, these reactions can result in the "multiplication" of the original sequence.

Such complementary templating mechanisms are elegantly simple, and they lie at the heart of information transfer processes in biological systems. The genetic information contained in every cell is encoded in the sequences of nucleotides in its polynucleotide molecules, and this information is passed on (inherited) from generation to generation by means of complementary base-pairing interactions.

Templating mechanisms, however, require additional catalysts to promote polymerization; without these the process is slow and inefficient and other, competing reactions prevent the formation of accurate replicas. Today, the catalytic functions that polymerize nucleotides are provided by highly specialized catalytic proteins that is, by enzymes. In the "prebiotic soup" primitive polypeptides might perhaps have provided some catalytic help. But molecules with the appropriate catalytic specificity would have remained rare unless the RNA itself were able somehow to reciprocate and favor their production. We shall come back to the reciprocal relationship between RNA synthesis and protein synthesis, which is crucially important in all living cells. But let us first consider what could be done with RNA itself, for RNA molecules can have a variety of catalytic properties, besides serving as templates for their own replication. In particular, an RNA molecule with an appropriate nucleotide sequence can act as a catalyst for the accurate replication of another RNA molecule - the template - whose sequence can be arbitrary.

The special versatility of RNA molecules is thought to have enabled them to play a central role in the origin of life. We have however to ignore what has stated above, namely that without catalysts the process is slow and inefficient which makes this hypothesis remotely possible. That makes the scenario very unlikely. And so far, the sequence would be completely random, no coded information.




Linking the RNA sugar-phosphate backbones together

Phosphodiester bonds


The sugar-phosphate backbone forms the structural framework of nucleic acids, including DNA and RNA. This backbone is composed of alternating sugar and phosphate groups, and defines directionality of the molecule. 3

The RNA world, and the origins of life Asdasas
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The RNA world, and the origins of life Asasddas

1) http://www.bioon.com/book/biology/mboc/mboc.cgi@action=figure&fig=1-4.htm
2) http://www.bioon.com/book/biology/mboc/mboc.cgi@code=010103174953651.htm
3) http://www.nature.com/scitable/definition/phosphate-backbone-273
3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465415/
4) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2926754/
5) http://journalofcosmology.com/Abiogenesis130.html
6) Alberts, molecular biology of the cell, pg.401
7) http://gowiki.tamu.edu/wiki/index.php/Category:GO:0090501_!_RNA_phosphodiester_bond_hydrolysis
7) http://www.bioon.com/book/biology/mboc/mboc.cgi@code=010102442543328.htm
8 ) http://www.arn.org/docs/odesign/od171/rnaworld171.htm
9) R. Shapiro, "The improbability of prebiotic nucleic acid synthesis," Origins of Life 14 (1984): 565-570; R. Shapiro, "Prebiotic ribose synthesis: a critical analysis," Origins of Life 18 (1988): 71-85.   http://www.ncbi.nlm.nih.gov/pubmed/6462692
10) http://creation.com/origin-of-life-the-polymerization-problem
11) https://www.promega.com/~/media/files/resources/product%20guides/cloning%20enzymes/ligases.pdf?la=en
12) http://en.wikipedia.org/wiki/Ligase_ribozyme
13) http://en.wikipedia.org/wiki/Hairpin_ribozyme
14) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181368/
15) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390864/
16) http://exploringorigins.org/nucleicacids.html
17) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC18793/
18) http://creation.com/origin-of-life-critique
19) Scientific American, February, 1991 p:100-109
20) Systems chemistry on early Earth, Jack W. Szostak, nature, Vol 459|14 May 2009,
21) http://arxiv.org/pdf/1207.4803v2.pdf
22) http://arxiv.org/pdf/1207.4803v2.pdf
23) https://www.mpg.de/9333399/Origin_of_Life_basetext.pdf
24) M. Gargaud · H. Martin · P. López-García T. Montmerle · R. Pascal Young Sun, Early Earth and the Origins of Life , page 116
25) http://pubs.acs.org.sci-hub.tw/doi/abs/10.1021/ar200332w
26) Earth Evolution of a Habitable World, Second edition, page 156
27) http://inference-review.com/article/two-experiments-in-abiogenesis#endnote-1
28 ) ASTROBIOLOGY An Evolutionary Approach, page 106
29) http://sci-hub.tw/https://www.nature.com/articles/s41570-016-0012
30) https://cos.gatech.edu/hg/item/575811
31) http://www.weizmann.ac.il/sb/Pages/Yonath/Belousoff-2010SpringerBOOK.pdf
32) https://en.wikipedia.org/wiki/Formose_reaction
33. http://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S1074552100000429


further readings: http://creation.com/native-folds-in-polypeptide-chains-1
http://www.evolutionnews.org/2015/06/on_the_origin_o_7097191.html
The Origin of RNA and “My Grandfather’s Axe”
Insuperable Problems Of The Genetic Code Initially Emerging In An RNA World
http://biorxiv.org/content/early/2017/05/22/140657
The RNA World: molecular cooperation at the origins of life



Last edited by Admin on Mon Sep 02, 2019 12:10 pm; edited 90 times in total

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We have seen that the expression of hereditary information requires extraordinarily complex machinery and proceeds from DNA to the protein through an RNA intermediate. This machinery presents a central paradox: if nucleic acids are required to synthesize proteins and proteins are required, in turn, to synthesize nucleic acids, how did such a system of interdependent components ever arise? One view is that an RNA world existed on Earth before modern cells arose. According to this hypothesis, RNA both stored genetic information and catalyzed the chemical reactions in primitive cells. Only later in evolutionary time did DNA take over as the genetic material and proteins become the major catalyst and structural component of cells. Heredity is perhaps the central feature of life. Not only must a cell use raw materials to create a network of catalyzed reactions, it must do so according to an elaborate set of instructions encoded in the hereditary information. The replication of this information ensures that the complex metabolism of cells can accurately reproduce itself.  6

The emergence of life requires a way to store information, a way to duplicate it, a way to change it, and a way to convert the information through catalysis into favorable chemical reactions. But how could such a system begin to be formed?

In present-day cells, the most versatile catalysts are polypeptides, composed of many different amino acids with chemically diverse side chains and, consequently, able to adopt diverse three-dimensional forms that bristle with reactive chemical groups. Polypeptides also carry information, in the order of their amino acid subunits. But there is no known way in which a polypeptide can reproduce itself by directly specifying the formation of another of precisely the same sequence.

DNA and RNA are composed of nucleotides that are linked to one another in a chain by chemical bonds, called ester bonds, between the sugar base of one nucleotide and the phosphate group of the adjacent nucleotide. The sugar is the 3' end, and the phosphate is the 5' end of each nucleiotide. The phosphate group attached to the 5' carbon of the sugar on one nucleotide forms an ester bond with the free hydroxyl on the 3' carbon of the next nucleotide. These bonds are called phosphodiester bonds, and the sugar-phosphate backbone is described as extending, or growing, in the 5' to 3' direction when the molecule is synthesized.

Both DNA and RNA ligases catalyze the formation of a phosphodiester bond between adjacent nucleotides with the concomitant hydrolysis of ATP to AMP and inorganic pyrophosphate. 

The ligation mechanism is essentially identical for both DNA and RNA ligases, and occurs in three stages:

First is the formation of an enzyme-nucleotide intermediate through the transfer of an adenylyl group (AMP) from either ATP or NAD to the epsilon-amine group of a lysine residue in the enzyme. This results in the release of pyrophosphate when ATP is the cofactor and NMN when NAD is used.

Second, the adenylyl group is transferred from the enzyme to the 5′-phosphate of the DNA (DNA ligases) or donor polynucleotide (RNA ligases), thereby activating it.

Third, a phosphodiester bond is formed by nucleophilic attack of the 3′- hydroxyl group of the DNA (DNA ligases) or acceptor polynucleotide (RNA ligases) on the
activated 5′-phosphate, with concomitant release of AMP.

The RNA world, and the origins of life Https_www_promega_com_media_files_resou_Page_1



The RNA Ligase Ribozyme was the first of several types of synthetic ribozymes produced by in vitro evolution and selection techniques. They are an important class of ribozymes because they catalyze the assembly of RNA fragments into phosphodiester RNA polymers, a reaction required of all extant nucleic acid polymerases and thought to be required for any self-replicating molecule. Ideas that the origin of life may have involved the first self-replicating molecules being ribozymes are called RNA World hypotheses. Ligase Ribozymes may have been part of such a pre-biotic RNA world. 12

In order to copy RNA, fragments or monomers (individual building blocks) that have 5'-triphosphates must be ligated together. This is true for modern (protein-based) polymerases and is also the most likely mechanism by which a ribozyme self-replicase in an RNA world might function. Yet no one has found a natural ribozyme that can perform this reaction.
surprise, surprise......

No evidence that RNA molecules ever had the broad range of catalytic activities

http://reasonandscience.heavenforum.org/t2024-the-rna-world-and-the-origins-of-life#3415


  1. A protein must be able to fold into a specific 3-dimensional shape in order to have biological activity. But the forces holding the folded protein in shape are so weak that many amino acids need to be involved - imposing a minimum length on their sequence of about 70 (Kyte), and maybe 50 for nucleic acids. So trying to improve the odds of finding a biologically active macromolecule by starting with short ones, just will not work.

  2. A similar misperception is that the first replicator need only have had poor replicating ability, which could gradually have improved (by mutation and selection of improved versions). But it is important to note that a poor replicator is more likely to degrade through miscopying than to improve its performance, and this poses a dilemma for the production of a primitive replicator. Although the common presumption is that a crude replicator can gradually improve its performance through a natural selection sort of process, in fact there is a threshold before that could take place. That is, a replicator must already have a reasonably good performance in order to be able to improve on that performance.

    In other words, natural selection cannot take place until there is a reasonably reliable replicating system. So the first replicating system would need to have arisen exclusively by chance.



The RNA world hypothesis: the worst theory of the early evolution of life 
http://www.ncbi.nlm.nih.gov/pubmed/22793875

(i) RNA is too complex a molecule to have arisen prebiotically;
(ii) RNA is inherently unstable;
(iii) catalysis is a relatively rare property of long RNA sequences only; and
(iv) the catalytic repertoire of RNA is too limited.


The "RNA World" is essentially a hypothetical stage of life between the first replicating molecule and the highly complicated DNA-protein-based life. The chief problem facing an RNA world is that RNA cannot perform all of the functions of DNA adequately to allow for replication and transcription of proteins.

New findings challenge assumptions about origins of life
http://reasonandscience.heavenforum.org/t1428-replicator-first-and-metabolism-first-scenarios
There is currently no known chemical pathway for an "RNA world" to transform into a "DNA/protein world."

http://phys.org/news/2013-09-assumptions-life.html#jCp
But for the hypothesis to be correct, ancient RNA catalysts would have had to copy multiple sets of RNA blueprints nearly as accurately as do modern-day enzymes. That's a hard sell; scientists calculate that it would take much longer than the age of the universe for randomly generated RNA molecules to evolve sufficiently to achieve the modern level of sophistication. Given Earth's age of 4.5 billion years, living systems run entirely by RNA could not have reproduced and evolved either fast or accurately enough to give rise to the vast biological complexity on Earth today.

OOL theorist Leslie Orgel notes that an "RNA World" could only form the basis for life, "if prebiotic RNA had two properties not evident today: a capacity to replicate without the help of proteins and an ability to catalyze every step of protein synthesis." The RNA world is thus a hypothetical system behind which there is little positive evidence, and much materialist philosophy: "The precise events giving rise to the RNA world remain unclear … investigators have proposed many hypotheses, but evidence in favor of each of them is fragmentary at best. The full details of how the RNA world, and life, emerged may not be revealed in the near future. 

The best claimed evidence of an "RNA World" includes the fact that there are RNA enzymes and genomes, and that cells use RNA to convert the DNA code into proteins. However, RNA plays only a supporting role in the cell, and there is no known biochemical system completely composed of RNA.

RNA experts have created a variety of RNA molecules which can perform biochemical functions through what is commonly termed "test tube evolution." However, "test tube evolution" is just a description for what is in reality nothing more than chemical engineering in the laboratory employing Darwinian principles; that does not imply that there is some known pathway through which these molecules could arise naturally.

In order a molecule to be a self replicator , it has to be a homopolymer, of which the backbone must have the same repetitive units; they must be identical. On the prebiotic world, the generation of a homopolymer was however impossible.

Steven A. Benner, Ph.D. Chemistry, Harvard, prominent origin-of-life researcher and creator of the Foundation for Applied Molecular Evolution, was posted on Huffington Post on December 6, 2013.  In it he said,

"We have failed in any continuous way to provide a recipe that gets from the simple molecules that we know were present on early Earth to RNA." 

That lead Leslie Orgel to say :
It would take a miracle if a strand of RNA ever appeared on the primitive Earth.

(Dover, 1999, p. 218).
I would have thought it relevant to point out for biologists in general that not one self-replicating RNA has emerged to date from quadrillions (1024) of artificially synthesized, random RNA sequences 

How  could the first living cells with DNA-based molecular biology have originated by spontaneous chemical processes on the prebiotic Earth? Primordial DNA synthesis would have required the presence of specific enzymes, but how could these enzymes be synthesized without the genetic information in DNA and without RNA for translating that information into the amino acid sequence of the protein enzymes? In other words, proteins are required for DNA synthesis and DNA is required for protein synthesis.

This classic "chicken-and-egg" problem made it immensely difficult to conceive of any plausible prebiotic chemical pathway to the molecular biological system. Certainly no such chemical pathway had been demonstrated
  2

Joyce and Orgel note, it seems unlikely that a structure with fewer than 40 nucleotides would be sufficient. Suppose, then, that "there is some 50-mer [RNA molecule of 50 nucleotides length]," Joyce and Orgel speculate, that "replicates with 90% fidelity. ... Would such a molecule be expected to occur within a population of random RNAs?"

Perhaps: but one such self-replicating molecule will not suffice.

"Unless the molecule can literally copy itself," Joyce and Orgel note, "that is, act simultaneously as both template and catalyst, it must encounter another copy of itself that it can use as a template." Copying any given RNA in its vicinity will lead to an error catastrophe, as the population of RNAs will decay into a collection of random sequences. But to find another copy of itself, the self-replicating RNA would need (Joyce and Orgel calculate) a library of RNA that "far exceeds the mass of the earth."18

In the face of these difficulties, they advise, one must reject the myth of a self-replicating RNA molecule that arose de novo from a soup of random polynucleotides. Not only is such a notion unrealistic in light of our current understanding of prebiotic chemistry, but it should strain the credulity of even an optimist's view of RNA's catalytic potential. If you doubt this, ask yourself whether you believe that a replicase ribozyme would arise in a solution containing nucleoside 5'-diphosphates and polynucleotide phosphorylase!

G. F. Joyce, L. E. Orgel, "Prospects for Understanding the Origin of the RNA World," In the RNA World, Cold Spring Harbor Laboratory Press, New York, 1993, p. 13.

This discussion… has, in a sense, focused on a straw man: the myth of a self-replicating RNA molecule that arose de novo from a soup of random polynucleotides. Not only is such a notion unrealistic in light of our current understanding of prebiotic chemistry, but it would strain the credulity of even an optimist's view of RNA's catalytic potential
Even if we suppose that there was self-replicating RNA in the primordial world, that numerous amino acids of every type ready to be used by RNA were available, and that all of these impossibilities somehow took place, the situation still does not lead to the formation of even one single protein. For RNA only includes information concerning the structure of proteins. Amino acids, on the other hand, are raw materials. Nevertheless, there is no mechanism for the production of proteins. To consider the existence of RNA sufficient for protein production is as nonsensical as expecting a car to assemble itself by simply throwing the blueprint onto a heap of parts piled up on top of each other. A blueprint cannot produce a car all by itself without a factory and workers to assemble the parts according to the instructions contained in the blueprint; in the same way, the blueprint contained in RNA cannot produce proteins by itself without the cooperation of other cellular components which follow the instructions contained in the RNA.

The problem of the origin of the RNA World is far from being solved. One can sketch out a logical order of events, beginning with prebiotic chemistry and ending with DNA/protein-based life. However, it must be said that the details of this process remain obscure and are not likely to be known in the near future.   3

in the early 1980s, the discovery of self-splicing, catalytic RNA molecules (in the ciliated protozoan Tetrahymena thermophila), put molecular flesh on the speculative bones of the idea of an early evolutionary stage dominated by RNA. These catalytic RNA molecules have subsequently been termed "ribozymes." "One can contemplate an RNA World," wrote Walter Gilbert in 1986, "containing only RNA molecules that serve to catalyze the synthesis of themselves." 
Micromolecules do not spontaneously combine to form macromolecules. 

It is said that DNA is the secret of life. DNA is not the secret of life. Life is the secret of DNA. Evolutionists persistently claim that the initial stage in the origin of life was the origin of a self-replicating DNA or RNA molecule. There is no such thing as a self-replicating molecule, and no such molecule could ever exist.The formation of a molecule requires the input of a highly selected type of energy and the steady input of the building blocks required to form it. To produce a protein, the building blocks are amino acids. For DNA and RNA these building blocks are nucleotides, which are composed of purines, pyrimidines, sugars, and phosphoric acid. If amino acids are dissolved in water they do not spontaneously join together to make a protein. That would require an input of energy. If proteins are dissolved in water the chemical bonds between the amino acids slowly break apart, releasing energy (the protein is said to hydrolyze). The same is true of DNA and RNA. To form a protein in a laboratory the chemist, after dissolving the required amino acids in a solvent, adds a chemical that contains high energy bonds (referred to as a peptide reagent). The energy from this chemical is transferred to the amino acids. This provides the necessary energy to form the chemical bonds between the amino acids and releases H and OH to form H2O (water). This only happens in a chemistry laboratory or in the cells of living organisms. It could never have taken place in a primitive ocean or anywhere on a primitive Earth. Who or what would be there to provide a steady input of the appropriate energy? Destructive raw energy would not work. Who or what would be there to provide a steady supply of the appropriate building blocks rather than just junk? In speaking of a self-replicating DNA molecule, evolutionists are reaching for a pie in the sky.


The primordial...conundrum -- which came first, informational polynucleotides or functional polypeptides -- was obviated by the simple but elegant compaction of both genetic information and catalytic function into the same molecule

RNA molecules are not just strings of symbols that carry information in an abstract way. They also have chemical personalities that affect their behavior. In particular, the specific sequence of nucleotides governs how the molecule folds up in solution. Just as the nucleotides in a polynucleotide can pair with free complementary nucleotides in their environment to form a new polymer, so they can pair with complementary nucleotide residues within the polymer itself. A sequence GGGG in one part of a polynucleotide chain can form a relatively strong association with a CCCC sequence in another region of the same molecule. Such associations produce complex three-dimensional patterns of folding, and the molecule as a whole takes on a specific shape that depends entirely on the sequence of its nucleotides

The RNA world, and the origins of life Sem_t_tulo
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Conformation of an RNA molecule.Nucleotide pairing between different regions of the same polynucleotide (RNA) chain causes the molecule to adopt a distinctive shape.

The three-dimensional folded structure of a polynucleotide affects its stability, its actions on other molecules, and its ability to replicate, so that not all polynucleotide shapes will be equally successful in a replicating mixture.

An RNA molecule therefore has two special characteristics: it carries information encoded in its nucleotide sequence that it can pass on by the process of replication, and it has a specific folded structure that enables it to interact selectively with other molecules and determines how it will respond to the ambient conditions.

The only problem is that random sequences are not information.....

The odds of suddenly having a self-replicating RNA pop out of a prebiotic soup are vanishingly low," says evolutionary biochemist Niles Lehman of Portland State University in Oregon.


Moreover, errors inevitably occur in any copying process, and imperfect copies of the originals will be propagated. With repeated replication, therefore, new variant sequences of nucleotides will be continually generated. Thus, in laboratory studies, replicating systems of RNA molecules have been shown to undergo a form of natural selection in which different favorable sequences eventually predominate, depending on the exact conditions.

"Polymerization" thus requires "dehydration synthesis." Many have proposed alternatives to get around this stumbling block. Since polymerization reactions also require an input of energy, heating and drying has been theorized to input energy, and remove the water. However, this heating and drying has to take place in such a way as to not wipeout the created polymers. Some theorized locations for this reaction have been intertidal pools or volcanic ridges where repeated cycles of heating and drying can take place. The problem is that all the water must be removed, but you don’t want to over-cook the polymers you are creating. Organic molecules tend to break down rapidly (i.e. cook) in the presence of heat. This would have to be a very fine balancing act that would also requires rapid input of organic material to overcome the rate at which the heat would destroy the molecules. A successful scenario is very difficult to imagine. Even under ideal laboratory conditions using pure monomers and carefully measured heating and drying cycles, only small amounts of polymers have been created. 1




The most interesting RNA molecule synthesized is perhaps an RNA "polymerase" which can replicate 14 base pairs of RNA.42 Yet, the polymerase itself is 200 pairs long.42 As Gerald Joyce noted, OOL theorists are thus 14 / 200 towards achieving a possible model molecule for the RNA World. $2 However, Joyce also noted that the replication accuracy of this molecule is too poor to allow for it to persist as a functional form of life.42

These purely speculative scenarios aren't bad on their own merits, but they are just another reminder of the philosophical presupposition driving this research in the first place: naturalism. Only when scientists assume there must be a natural explanation do they turn to completely unfalsifiable unverifiable and incomplete speculatory hypotheses.

The theory then says that some unknown precursor of RNA turned into RNA through an unknown process. This "RNA-world hypothesis" states that life then arose from a population of self-replicating RNA molecules. RNA is a sister molecule to DNA, used when DNA breaks up to create proteins or replicate. Like a copy from the library, RNA has a complementary code to DNA and goes out to do the dirty work. A few types of RNA have been known to have auto-catalytic self-replicating abilities, however this scenario inevitably encounters a chicken and egg problem18.

But these molecules must be encapsulated within a "cell wall structure" or a small protective enclosure from the outside world. But, the protective cell requires replicating genetic machinery to be created. Thus, we now have a "chicken and egg scenario"--which came first? the self-replicating machinery (which needs a cell to operate), or the cell itself, which protects (and is created by) the cellular machinery? The answer is neither came first for both are required for self-replication. How could self-replicating RNA arise naturally when it essentially is an irreducibly complex system that cannot functionally replicate without other distinct components.


The RNA world, and the origins of life Www_bioon_com_book_biology_mboc_mboc_cgi_action
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What do Ribozyme Engineering Experiments Really Tell Us About the Origin of Life?

In vitro RNA selection does not demonstrate that complex ribozymes could have arisen naturally in a prebiotic soup, because the in vitro experimental conditions are wholly unrealistic, revealing at every turn the fingerprints of intervening intelligence. RNA World researchers have taken their own engineering of ribozymes as analogous to plausible prebiotic processes, when in fact the two situations are profoundly different. Indeed, aspects of ribozyme engineering, together with other lines of evidence, support a very different view of biological origins from that advocated by RNA World researchers.

Ribozyme engineering involves two broad experimental strategies. The "rational design" approach modifies existing types of ribozymes to produce better or even novel RNA catalysts. The "irrational design" approach, on the other hand, uses pools of partially randomized RNA molecules, which are screened -- "selected" -- for functional activity of a desired sort. Those molecules catalyzing the desired reaction are then used as the basis for the next round of "evolution." This randomization-selection process may be repeated several times, to yield increasingly faster RNA catalysts.

These experiments certainly add to our knowledge of RNA chemistry. A simple question directly illuminates the doubtful relevance of these experiments to prebiotic chemistry, however. How did pools of 1015 RNA molecules (to cite a value from a recent ribozyme engineering experiment4) accumulate on the early earth? How, for that matter, did any RNA accumulate?

Here an analogy may be helpful. Suppose you learn about a blackjack player who routinely beats the casinos in Las Vegas. You would not be impressed to find that the casinos had inexplicably made an exception for this person. They allowed him to fill parking lots, stadiums, and indeed the open desert around Las Vegas with millions of dealers who each dealt thousands of hands. The player monitored these millions of dealers electronically. Whenever a good hand turned up, he would play that hand, and ignore all the others.

Is that winning at blackjack? Not at all. The player contrives to "win" only by violating the actual rules of the game. In the case of prebiotic chemistry, the actual rules of the game govern the formation of RNA molecules without the help of biochemists. And, according to those rules (see discussion of postulates 1-4, main text, and below), RNA does not arise from its chemical constituents except (a) in organisms, and (b) in laboratories where intelligent organisms synthesize it. 4

Physicist Paul Davies points out that there are immense thermodynamic problems in producing the peptide chains of amino acids. The Second Law of Thermodynamics describes the natural tendency of closed systems to degenerate, to lose information, order and complexity; that is, to increase their entropy. Heat flows from hot to cold, water flows downhill, cars rust, etc. Now the second law has a statistical character – it does not absolutely forbid physical systems going against the flow ‘uphill’, but it stacks the odds
very much against it. Davies says, ‘It has been estimated that, left to its own devices, a concentrated solution of amino acids would need a volume of fluid the size of the observable universe, to go against the thermodynamic tide, and create a single small polypeptide spontaneously.


Stephen Meyer :

The (historical) question that must be asked about biological origins is not "Which materialistic scenario will prove adequate?" but "How did life as we know it actually arise on earth?" Since one of the logically appropriate answers to this latter question is that "Life was designed by an intelligent agent that existed before the advent of humans," I believe it is anti-intellectual to exclude the "design hypothesis" without consideration of all the evidence, including the most current evidence, that would support it
Let us presume that a soup enriched in the building blocks of all of these proposed replicators has somehow been assembled, under conditions that favor their connection into chains. They would be accompanied by hordes of defective building blocks, the inclusion of which would ruin the ability of the chain to act as a replicator. The simplest flawed unit would be a terminator, a component that had only one "arm" available for connection, rather than the two needed to support further growth of the chain.

There is no reason to presume than an indifferent nature would not combine units at random, producing an immense variety of hybrid short, terminated chains, rather than the much longer one of uniform backbone geometry needed to support replicator and catalytic functions. Probability calculations could be made, but I prefer a variation on a much-used analogy. Picture a gorilla (very long arms are needed) at an immense keyboard connected to a word processor. The keyboard contains not only the symbols used in English and European languages but also a huge excess drawn from every other known language and all of the symbol sets stored in a typical computer. The chances for the spontaneous assembly of a replicator in the pool I described above can be compared to those of the gorilla composing, in English, a coherent recipe for the preparation of chili con carne. With similar considerations in mind Gerald F. Joyce of the Scripps Research Institute and Leslie Orgel of the Salk Institute concluded that the spontaneous appearance of RNA chains on the lifeless Earth "would have been a near miracle." I would extend this conclusion to all of the proposed RNA substitutes that I mentioned above.6

Origins of Life, Hugh Ross, pg.81

High-energy phosphate compounds. Phosphate groups assume an integral role in the linkages that form the backbone of DNA and RNA. They also comprise the head-group region of key cell membrane components (phospholipids). In addition to their structural importance, phosphates also serve a critical role in the cell’s metabolic processes. Phosphate chains, called polyphosphates, form a relatively unstable high-energy chemical structure in which the cell’s metabolic systems store energy. The breakage of these highenergy phosphate bonds releases energy used by the cell to power its operation. All organisms continuously produce and consume massive amounts of ATP (adenosine triphosphate) and similar compounds in which polyphosphate groups are constituents. Many researchers speculate that more primitive prebiotic polyphosphate compounds played a similar role to ATP during the origin-of-life process and later evolved into ATP. Because high-energy compounds that could transfer phosphate groups to the RNA and DNA backbones were essential to the RNA- and DNA-protein-world scenarios, a phosphate source must have been present on early Earth. Researchers propose several possible prebiotic chemical routes to polyphosphates. The most common include  the heating of apatite (a phosphate-containing mineral);  the high-temperature heating (from 392 to 1,112 °F, 200 to 600 °C) of dihydrogen phosphates; and  the phosphates’ reaction with high-energy organic compounds.

Although several plausible routes to polyphosphates exist, researchers wonder if these chemical pathways have any relevance to early Earth. For example, to produce polyphosphates from apatite and dihydrogen phosphate, water must be completely driven from the system—an impossibility for phosphate minerals confined to rocks. Furthermore, the high temperatures needed to form polyphosphates would in turn destroy any organic material. The suggested production of polyphosphates from high-energy chemicals (allegedly formed in spark-discharge reactions on early Earth) lacks chemical robustness. These reactions require unrealistically high levels of starting materials and produce low yields. Laboratory spark-discharge experiments performed under a wide range of chemical conditions failed to yield polyphosphates when phosphates were included in the reaction vessel. Even if a means existed on primordial Earth to form polyphosphates, their availability for prebiotic reactions is unlikely because calcium ions drive polyphosphates to precipitate out of solutions. These ions would have been everywhere on early Earth. Given the extreme rarity (or nonexistence) of polyphosphate minerals on Earth today, the conclusion that prebiotic polyphosphate synthesis could not have taken place on early Earth seems justifiable. Studies on possible prebiotic production of cytosine, ribose, and polyphosphates demonstrate that even though researchers have identified chemical pathways to them, the lack of available starting materials, plus chemical interference by other environmental materials and rapid decomposition, would have precluded formation. In other words, viable chemical routes to these key life molecules have not been found.
The origin-of-life community widely acknowledges the prebiotic production of ribose, cytosine, and polyphosphates as painfully problematic. In fact, at the opening plenary lecture of ISSOL 2002, after summarizing these and other problems, distinguished origin-of-life researcher Leslie Orgel stated, “It would be a miracle if a strand of RNA ever appeared on the primitive Earth.”20 As a preface to this conclusion, Orgel remarked that he “hoped no creationists [were] in the audience.” Laughter erupted throughout the room. Orgel did not advocate a supernatural explanation for life’s origin. Rather, he acknowledged the intractable problem of accounting for its emergence through natural processes. However, the problems are not limited to the prebiotic production of chemical compounds. Critical analysis of any proposed prebiotic route exposes similar problems. For the sake of argument, however, one might ask, “What if these molecules were freely available? Can unattended chemical events account for the emergence of the cell’s metabolic systems and the origin of self-replicating molecules?”

the assumption that life began with RNA molecules brewed in the prebiotic environment from random abiotic reactions faces major problems, including 

(a) the instability of ribose (with a half-life of 73 minutes at 100°C, and of 44 years at 0 0c) and other sugars; 
(b) the difficulties of robust prebiotic synthesis of the glycosidic bonds of activated nucleotides 
(c) the lack of efficient two-way non-enzymatic template-directed polymerizations. Although there are alternative views, the difficulties associated with the prebiotic origin of RNA have prompted the search for alternative genetic macromolecules, i.e., for a pre-RNA world where the informational polymers had a backbone different from ribose-phosphate and perhaps even with other nucleobases.

Offering more criticisms of the RNA world hypothesis,  Nita Sahai admits that RNA self-replication has never been achieved, nor has monomer polymerization to yield RNA enzymes 5
1) http://www.ideacenter.org/contentmgr/showdetails.php/id/838
2) http://www.arn.org/docs/odesign/od171/rnaworld171.htm
3) http://cshperspectives.cshlp.org/content/4/5/a003608.full
4) http://www.arn.org/docs/odesign/od171/ribo171.htm
5) http://www.evolutionnews.org/2015/07/researcher_almo097301.html
6) http://www.scientificamerican.com/article/a-simpler-origin-for-life/
7) http://www.icr.org/article/few-reasons-evolutionary-origin-life-impossible/
8  https://www.c4id.org.uk/index.php?option=com_content&view=article&id=211:the-problem-of-the-origin-of-life&catid=50:genetics&Itemid=43



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Information Flows from Polynucleotides to Polypeptides

How could the information encoded in a polynucleotide specify the sequence of a polymer of a different type? Clearly, the polynucleotides must act as catalysts to join selected amino acids together. In present-day organisms a collaborative system of RNA molecules plays a central part in directing the synthesis of polypeptides - that is, protein synthesis - but the process is aided by other proteins synthesized previously. The biochemical machinery for protein synthesis is remarkably elaborate. One RNA molecule carries the genetic information for a particular polypeptide in the form of a code, while other RNA molecules act as adaptors, each binding a specific amino acid. These two types of RNA molecules form complementary base pairs with one another to enable sequences of nucleotides in the coding RNA molecule to direct the incorporation of specific amino acids held on the adaptor RNAs into a growing polypeptide chain. Precursors to these two types of RNA molecules presumably directed the first protein synthesis without the aid of proteins  1

Today, these events in the assembly of new proteins take place on the surface of ribosomes - complex particles composed of several large RNA molecules of yet another class, together with more than 50 different types of protein. The ribosomal RNA in these particles plays a central catalytic role in the process of protein synthesis and forms more than 60% of the ribosome's mass.

How could these 50 proteins have come into existence  ??

It seems likely, then, that RNA guided the primordial synthesis of proteins, perhaps in a clumsy and primitive fashion.

Haha. Isnt that a joke ?? Likely based on what exactly ?  

In this way RNA was able to create toolsin the form of proteinsfor more efficient biosynthesis, and some of these could have been put to use in the replication of RNA and in the process of tool production itself.

These seem really helpless just so stories......

The synthesis of specific proteins under the guidance of RNA required the evolution of a code by which the polynucleotide sequence specifies the amino acid sequence that makes up the protein. This code - the genetic code - is spelled out in a "dictionary" of three-letter words: different triplets of nucleotides encode specific amino acids. The code seems to have been selected arbitrarily (subject to some constraints, perhaps); yet it is virtually the same in all living organisms.

That sets the cream on the ice...... Anyone to believe this ??



1) http://www.bioon.com/book/biology/mboc/mboc.cgi@code=01010699553128.htm

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4The RNA world, and the origins of life Empty Four problems for RNA polymer formation on Thu Jun 04, 2015 7:02 pm

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From Stephen Meyers excellent book : Signature of the cell :

http://reasonandscience.heavenforum.org/t2024-the-rna-world-and-the-origins-of-life

Problem 1: RNA Building Blocks Are Hard to Synthesize and Easy to Destroy

Before the first RNA molecule could have come together, smaller constituent molecules needed to arise on the primitive earth. These include a sugar known as ribose, phosphate
molecules, and the four RNA nucleotide bases (adenine, cytosine, guanine, and uracil). It turns out, however, that both synthesizing and maintaining these essential RNA building
blocks, particularly ribose (the sugar incorporated into nucleotides) and the nucleotide bases, has proven either extremely difficult or impossible to do under realistic prebiotic
conditions.

Stanley Miller concluded in 1998 that

“a high temperature origin of life involving these compounds [the RNA bases] therefore is unlikely.”

Robert Shapiro:

the presumption that “the bases, adenine, cytosine, guanine and uracil were readily available on the early earth” is “not supported by existing knowledge of the basic chemistry of these substances.

The RNA-world hypothesis faces an even more acute, but related, obstacle—a kind of catch-22. The presence of the nitrogen-rich chemicals necessary for the production of
nucleotide bases prevents the production of ribose sugars. Yet both ribose and the nucleotide bases are needed to build RNA.

Dean Kenyon explains

“The chemical conditions proposed for the prebiotic synthesis of purines and pyrimidines [the bases] are sharply incompatible with those proposed for the synthesis of
ribose.”


Shapiro concludes:

“The evidence that is currently available does not support the availability of ribose on the prebiotic earth, except perhaps for brief periods of time, in low concentration as part of a complex mixture, and under conditions unsuitable for nucleoside synthesis.”


Problem 2: Ribozymes Are Poor Substitutes for Proteins

RNA can perform only a few minor functional roles and then usually as the result of scientists intentionally “engineering” or “directing” the RNA catalyst (or ribozyme) in question.

For this reason, claiming that catalytic RNA could replace proteins in the earliest stages of chemical evolution is extremely problematic. To say otherwise would be like asserting
that a carpenter wouldn’t need any tools besides a hammer to build a house, because the hammer performed two or three carpentry functions. True, a hammer does perform some
carpentry functions, but building a house requires many specialized tools that can perform a great variety of specific carpentry functions. In the same way, RNA molecules
can perform a few of the thousands of different functions proteins perform in “simple” single cells (e.g., in the E. coli bacterium), but that does not mean that RNA molecules
can perform all necessary cellular functions.

Problem 3: An RNA-based Translation and Coding System Is Implausible

To evolve beyond the RNA world, an RNA-based replication system eventually would have to begin to produce proteins, and not just any proteins, but proteins capable of template-directed protein manufacture. But for that to occur, the RNA replicator first would need to produce machinery for building proteins. In modern cells it takes many proteins to build proteins. So, as a first step toward building proteins, the primitive replicator would need to produce RNA molecules capable of performing the functions of the modern proteins involved in translation. Presumably, these RNA molecules would need to perform the functions of the twenty specific tRNA synthetases and the fifty ribosomal proteins, among the many others involved in translation. At the same time, the RNA replicator would need to produce tRNAs and the many mRNAs carrying the information for building the first proteins. These mRNAs would need to be able to direct protein synthesis using, at first, the transitional ribozyme-based protein-synthesis machinery and then, later, the permanent and predominantly protein-based protein-synthesis machinery. In short, the evolving RNA world would need to develop a coding and translation system based entirely on RNA and also generate the information necessary to build the proteins that later would be needed to replace it.

This is a tall order. The cell builds proteins from the information stored on the mRNA transcript (i.e., the copy) of the original DNA molecule. To do this, a bacterial cell depends upon a translation and coding system consisting of 106 distinct but functionally integrated proteins as well several distinct types of RNA molecules (tRNAs, mRNAs, and rRNAs).19 This system includes the ribosome (consisting of fifty distinct protein parts), the twenty distinct tRNA synthetases, twenty distinct tRNA molecules with their specific anticodons (all of which jointly embody the genetic code), various other proteins, free-floating amino acids, ATP molecules (for energy), and—last, but not least—information-rich mRNA transcripts for directing protein synthesis. Furthermore, many of the proteins in the translation system perform multiple functions and catalyze coordinated multistep chemical transformations.

Unlike RNA catalysts (ribozymes), the protein-based enzymes involved in translation perform multiple functions, often in closely integrated or choreographed ways. Ribozymes, however, are the one-trick ponies of the molecular world. Typically, they can perform one subfunction of the several coordinated functions that a corresponding enzyme can perform. But they cannot perform the entire range of necessary functions, nor can they do so with the specificity needed to execute the many sequentially coordinated reactions that occur during translation.

Producing the molecular complexes necessary for translation requires coupling multiple tricks—multiple crucial reactions—in a closely integrated (and virtually simultaneous) way. True enzyme catalysts do this. RNA and small-molecule catalysts do not.

Problem 4: The RNA World Doesn’t Explain the Origin of Genetic Information

Even if a system of ribozymes for building proteins had arisen from an RNA replicator, that system of molecules would still need information-rich templates for building specific proteins. RNA-world advocates give no account of the origin of that information beyond vague appeals to chance. chance is not a plausible explanation for the information necessary for building even one protein of modest length, let alone a set of RNA templates for building the proteins needed to establish a protein-based translation system and genetic code. Explaining how the building blocks of RNA might have arranged themselves into information-rich sequences has proven no easier than explaining how the parts of DNA
might have done so, given the requisite length and specificity of these molecules.



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The RNA World And Other Origin-of-Life Theories

http://www.panspermia.org/rnaworld.htm# 28ref

There is no evidence in life today of anything that produces huge quantities of new, random strings of nucleotides or amino acids, some of which are advantageous. But if precellular life did that, it would need lots of time to create any useful genes or proteins. How long would it need? After making some helpful assumptions we can get the ratio of actual, useful proteins to all possible random proteins up to something like one in 10^500 (ten to the 500th power). So it would take, barring incredible luck, something like 10^500 trials to probably find one. Imagine that every cubic quarter-inch of ocean in the world contains ten billion precellular ribosomes. Imagine that each ribosome produces proteins at ten trials per minute (about the speed that a working ribosome in a bacterial cell manufactures proteins). Even then, it would take about 10^450 years to probably make one useful protein. But Earth was formed only about 4.6 x 10^9 years ago. The amount of time available for this hypothetical protein creation process was maybe a few hundred million or ~10^8 years. And now, to make a cell, we need not just one protein, but a minimum of several hundred.

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6The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Thu Jul 09, 2015 12:18 pm

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The implausibility of prevital nucleic acid

https://answersingenesis.org/evidence-against-evolution/evolutionist-criticisms-rna-world-conjecture/

If it is hard to imagine polypeptides or polysaccharides in primordial waters it is harder still to imagine polynucleotides. But so powerful has been the effect of Miller’s experiment on the scientific imagination that to read some of the literature on the origin of life (including many elementary texts) you might think that it had been well demonstrated that nucleotides were probable constituents of a primordial soup and hence that prevital nucleic acid replication was a plausible speculation based on the results of experiments.

There have indeed been many interesting and detailed experiments in this area. But the importance of this work lies, to my mind, not in demonstrating how nucleotides could have formed on the primitive Earth, but in precisely the opposite: these experiments allow us to see, in much greater detail than would otherwise have been possible, just why prevital nucleic acids are highly implausible.

Let us consider some of the difficulties:

First, as we have seen, it is not even clear that the primitive Earth would have generated and maintained organic molecules. All that we can say is that there might have been prevital organic chemistry going on, at least in special locations.

Second, high-energy precursors of purines and pyrimidines had to be produced in a sufficiently concentrated form (for example at least 0.01 M HCN).

Third, the conditions must now have been right for reactions to give perceptible yields of at least two bases that could pair with each other.

Fourth, these bases must then have been separated from the confusing jumble of similar molecules that would also have been made, and the solutions must have been sufficiently concentrated.

Fifth, in some other location a formaldehyde concentration of above 0.01 M must have built up.

Sixth, this accumulated formaldehyde had to oligomerise to sugars.

Seventh, somehow the sugars must have been separated and resolved, so as to give a moderately good concentration of, for example, D-ribose.

Eighth, bases and sugars must now have come together.

Ninth, they must have been induced to react to make nucleosides. (There are no known ways of bringing about this thermodynamically uphill reaction in aqueous solution: purine nucleosides have been made by dry-phase synthesis, but not even this method has been successful for condensing pyrimidine bases and ribose to give nucleosides (Orgel & Lohrmann, 1974).)

Tenth, whatever the mode of joining base and sugar it had to be between the correct nitrogen atom of the base and the correct carbon atom of the sugar. This junction will fix the pentose sugar as either the alpha or beta-anomer of either the furanose or pyranose forms (see page 29). For nucleic acids it has to be the beta-furanose. (In the dry-phase purine nucleoside syntheses referred to above, all four of these isomers were present with never more than 8 % of the correct structure.)

Eleventh, phosphate must have been, or must now come to have been, present at reasonable concentrations. (The concentrations in the oceans would have been very low, so we must think about special situations—evaporating lagoons and such things (Ponnamperuma, 1978).)

Twelfth, the phosphate must be activated in some way—for example as a linear or cyclic polyphosphate—so that (energetically uphill) phosphorylation of the nucleoside is possible.

Thirteenth, to make standard nucleotides only the 5′hydroxyl of the ribose should be phosphorylated. (In solid-state reactions with urea and inorganic phosphates as a phosphorylating agent, this was the dominant species to begin with (Lohrmann & Orgel, 1971). Longer heating gave the nucleoside cyclic 2′,3′-phosphate as the major product although various dinucleotide derivatives and nucleoside polyphosphates are also formed (Osterberg, Orgel & Lohrmann. 1973).)

Fourteenth, if not already activated—for example as the cyclic 2′,3′-phosphate—the nucleotides must now be activated (for example with polyphosphate; Lohrmann, 1976) and a reasonably pure solution of these species created of reasonable concentration. Alternatively, a suitable coupling agent must now have been fed into the system.

Fifteenth, the activated nucleotides (or the nucleotides with coupling agent) must now have polymerised. Initially this must have happened without a pre-existing polynucleotide template (this has proved very difficult to simulate (Orgel & Lohrmann. 1974)); but more important, it must have come to take place on pre-existing polynucleotides if the key function of transmitting information to daughter molecules was to be achieved by abiotic means. This has proved difficult too. Orgel & Lohrmann give three main classes of problem:

While it has been shown that adenosine derivatives form stable helical structures with poly(U)—they are in fact triple helixes—and while this enhances the condensation of adenylic acid with either adenosine or another adenylic acid—mainly to di(A) stable helical structures were not formed when either poly (A) or poly(G) were used as templates.
It was difficult to find a suitable means of making the internucleotide bonds. Specially designed water-soluble carbodiimides were used in the experiments described above, but the obvious pre-activated nucleotides—ATP or cyclic 2′,3′-phosphates—were unsatisfactory. Nucleoside 5′-phosphorimidazolides, for example were more successful, but these now involve further steps and a supply of imidazole, for their synthesis (Lohrmann & Orgel, 1978).
Internucleotide bonds formed on a template are usually a mixture of 2′-5′ and the normal 3′-5′ types. Often the 2′-5′ bonds predominate although it has been found that Zn2+, as well as acting as an efficient catalyst for the template-directed oligomerisation of guanosine 5′-phosphorimidazolide also leads to a preference for the 3′-5′ bonds (Lohrmann, Bridson & Orgel, 1980).

Sixteenth, the physical and chemical environment must at all times have been suitable—for example the pH, the temperature, the M2+ concentrations.

Seventeenth, all reactions must have taken place well out of the ultraviolet sunlight; that is, not only away from its direct, highly destructive effects on nucleic acid-like molecules, but away too from the radicals produced by the sunlight, and from the various longer lived reactive species produced by these radicals.

Eighteenth, unlike polypeptides, where you can easily imagine functions for imprecisely made products (for capsules, ionexchange materials, etc.), a genetic material must work rather well to be any use at all—otherwise it will quickly let slip any information that it has managed to accumulate.

Nineteenth, what is required here is not some wild one-off freak of an event: it is not true to say ‘it only had to happen once’. A whole set-up had to be maintained for perhaps millions of years: a reliable means of production of activated nucleotides at the least.

Now you may say that there are alternative ways of building up nucleotides, and perhaps there was some geochemical way on the early Earth. But what we know of the experimental difficulties in nucleotide synthesis speaks strongly against any such supposition. However it is to be put together, a nucleotide is too complex and metastable a molecule for there to be any reason to expect an easy synthesis.

You might want to argue about the nineteen problems that I chose: and I agree that there is a certain arbitrariness in the sequence of operations chosen. But if in the compounding of improbabilities nineteen is wrong as a number that would be mainly because it is much too small a number. If you were to consider in more detail a process such as the purification of an intermediate you would find many subsidiary operations—washings, pH changes and so on. (Remember Merrifield's machine: for one overall reaction, making one peptide bond, there were about 90 distinct operations required.)

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7The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Fri Aug 07, 2015 5:53 pm

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Self-organizing biochemical cycles 1

How were ribonucleotides first formed on the primitive earth? This is a very difficult problem. Stanley Miller's synthesis of the amino acids by sparking a reducing atmosphere (2) was the paradigm for prebiotic synthesis for many years, so at first, it was natural to suppose that similar methods would meet with equal success in the nucleotide field. However, nucleotides are intrinsically more complicated than amino acids, and it is by no means obvious that they can be obtained in a few simple steps under prebiotic conditions. A remarkable synthesis of adenine (3) and more or less plausible syntheses of the pyrimidine nucleoside bases (4) have been reported, but the synthesis of ribose and the regiospecific combination of the bases, ribose, and phosphate to give β-nucleotides remain problematical.

Self-organizing biochemical cycles

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC18793/

The novel, potentially replicating polymers that have been described up to now, like the nucleic acids, are formed by joining together relatively complex monomeric units. It is hard to see how any could have accumulated on the early earth. A plausible scenario for the origin of life must, therefore, await the discovery of a genetic polymer simpler than RNA and an efficient, potentially prebiotic, synthetic route to the component monomers. The suggestion that relatively pure, complex organic molecules might be made available in large amounts via a self-organizing, autocatalytic cycle might, in principle, help to explain the origin of the component monomers. I have emphasized the implausibility of the suggestion that complicated cycles could self-organize, and the importance of learning more about the potential of surfaces to help organize simpler cycles.

1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC18793/



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The Origin of RNA and “My Grandfather’s Axe” 1

The origin of RNA is one of the most formidable problems facing prebiotic chemists.

The RNA world, and the origins of life 1-s2_010

Three Models for the Prebiotic Assembly of the First Informational Polymers

(A) The classic model. The recognition unit (RU), trifunctional connector (TC), and ionic linker (IL) assemble sequentially to produce nucleotides (or protonucleotides) before becoming polymerized to form RNA (or proto-RNA) polymers. Base pairing is not expected until polymers of a critical length are synthesized.

(B) The ribose-centric model. The cytosine base is built on a pre-existing sugar. Like the classic model, nucleosides are formed before being coupled into polymers and before base pairing. Unlike the classic model, the chemistry of the ribose-centric model is dependent on the exact structures used in the assembly pathway and, therefore, implies that RNA has not evolved from an earlier polymer.

(C) The polymer fusion model. Recognition units (RUs) form supramolecular assemblies that involve pairings, either as dyads or hexads, that are the same as those that will hold strands together in the informational polymers. Trifunctional connector (TCs) and ionized linkers (ILs) form covalent polymers, among the many other polymers that exist in the prebiotic chemical inventory. The match in the spacing of functional groups of the TCs in the TC-IL polymers with the RUs in their supramolecular assembly promotes the fusion of these polymers through the covalent linking of TCs and RUs. Note that only in this model is there a mechanism that guarantees that the RUs incorporated into polymers will be able to actually act as recognition units through their ability to form pairing structures prior to being linked by a backbone.

1) http://www.sciencedirect.com/science/article/pii/S1074552113001154

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There are problems in imagining an RNA-catalysed metabolic network 1

The question about the initial trigger is not trivial: to form an RNA nucleotide, not only one, but a series of reactions is necessary. An initial RNA-catalysed reaction system needed, in essence, to provide some sort of function, in order that genetics could select for it and improve catalysis and efficiency. In other words, evolution is selecting for the (functional) product and not for an intermediate step, so an initial RNA-based network could not have come into place one reaction at a time, but only as an already operational entity. This argument renders the origin of metabolism as an RNA-based metabolic reaction system not impossible, but substantially less probable. This problem is amplified by the notion that the least self-sustaining chemical networks lack evolvability, and evolutionary selection cannot change the chemistry in a reaction system, neither are the thermodynamic properties of metabolites subject to genetic selection. Thus RNA genetics has its limitations in optimizing chemical reaction networks.

1) http://www.biochemsoctrans.org/content/ppbiost/42/4/985.full.pdf

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The stability of the RNA bases: Implications for the origin of life 1

High-temperature origin-of-life theories require that the components of the first genetic material are stable. We therefore have measured the half-lives for the decomposition of the nucleobases. They have been found to be short on the geologic time scale. At 100°C, the growth temperatures of the hyperthermophiles, the half-lives are too short to allow for the adequate accumulation of these compounds (t1/2 for A and G ≈ 1 yr; U = 12 yr; C = 19 days). Therefore, unless the origin of life took place extremely rapidly (<100 yr), we conclude that a high-temperature origin of life may be possible, but it cannot involve adenine, uracil, guanine, or cytosine. The rates of hydrolysis at 100°C also suggest that an ocean-boiling asteroid impact would reset the prebiotic clock, requiring prebiotic synthetic processes to begin again. At 0°C, A, U, G, and T appear to be sufficiently stable (t1/2 ≥ 106 yr) to be involved in a low-temperature origin of life. However, the lack of stability of cytosine at 0°C (t1/2 = 17,000 yr) raises the possibility that the GC base pair may not have been used in the first genetic material unless life arose quickly (<106 yr) after a sterilization event. A two-letter code or an alternative base pair may have been used instead.

How the genetic code would have made the transition from two base pairs to four base pairs is another question......which is elucidated here:

Conclusions. Most atmospheric models generally predict a warm early Earth with high levels of CO2 or other greenhouse gases. In the absence of greenhouse warming, however, the Earth’s oceans would have been frozen because of a 30% less luminous sun (62). Our kinetic data on the stability of the nucleobases indicate that a cold or frozen early Earth would be more favorable for the accumulation of the nucleobases and therefore for the origin of life. An early frozen Earth may have been melted numerous times as a result of a large meteor or comet impacts (63). However, very large impactors could boil the Earth’s oceans. The rates of hydrolysis at 100°C, for all of the nucleobases measured, suggest that an ocean-boiling impact event would completely decompose the nucleobases in addition to a number of other biologically important compounds. This would require the whole prebiotic process to begin again. Ocean-boiling impacts therefore are more damaging to prebiotic chemistry than to an early biosphere (64–66), where the survival of a single organism (e.g., in a crustal environment) would be sufficient to reestablish the entire ecosystem.
Other stability problems also point to a low-temperature origin of life and early evolution in the pre-RNA and RNA world. These include the stability of ribose (67), the decomposition of nucleosides (28, 68), and the hydrolysis of the phosphodiester bonds of RNA (23). Similar stability considerations would apply to any alternative pre-RNA backbone, e.g., peptide nucleic acids. All of these factors point to a low-temperature accumulation of organic compounds on the primitive Earth and a low-temperature origin of life. Therefore, atmospheric models suggesting a cool early Earth (≈ 0°C) rather than a warm one (12, 13) need to be considered.

1) http://www.pnas.org/content/95/14/7933.full

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11The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Thu Nov 12, 2015 6:34 am

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A critique of some current evolutionary origin-of-life models


Evolutionary origin-of-life theories have many hurdles to overcome.1,2,3 To form a self-reproducing cell from non-living chemicals requires the generation of a large amount of information, or specified complexity. A cell must be able to perform many chemical reactions in the right order, place and degree, which requires a number of specific catalysts (enzymes). It must also be able to reproduce the information needed to produce these enzymes.
In all known cells, the specific catalysts are proteins, while the information storage/retrieval and reproduction tasks are carried out by the nucleic acids DNA and RNA. Proteins are polymers of amino acids, while nucleic acids are polymers of nucleotides. Nucleotides themselves are a combination of a sugar (deoxyribose for DNA, ribose for RNA), a nitrogenous base and a phosphate group.
But the DNA itself codes for the proteins, yet requires at least 50 proteins for the necessary decoding, and still others for replication. The noted philosopher of science, the late Sir Karl Popper, commented:

What makes the origin of life and of the genetic code a disturbing riddle is this: the genetic code is without any biological function unless it is translated; that is, unless it leads to the synthesis of the proteins whose structure is laid down by the code. But … the machinery by which the cell (at least the non-primitive cell, which is the only one we know) translates the code consists of at least fifty macromolecular components which are themselves coded in the DNA. Thus the code can not be translated except by using certain products of its translation. This constitutes a baffling circle; a really vicious circle, it seems, for any attempt to form a model or theory of the genesis of the genetic code.
Thus we may be faced with the possibility that the origin of life (like the origin of physics) becomes an impenetrable barrier to science, and a residue to all attempts to reduce biology to chemistry and physics.4

The obvious conclusion is that both the DNA and proteins must have been functional from the beginning, otherwise life could not exist.


RNA World?


To avoid this conclusion, some evolutionists have theorised that one type of molecule could perform both catalytic and reproductive roles. A recent discovery of some catalytic functions in RNA has led many evolutionists to postulate an ‘RNA world’. The idea is that the first life consisted mainly of RNA, which could not only reproduce but also carry out many of the functions now carried out by enzymes. But this model has several dubious postulates:
A pool of exclusively ‘right-handed’ ribose molecules could be produced, separated from a jumble of other sugars, and remain stable long enough; the bases could be produced in large quantities; and a high concentration of phosphate (PO43-) would be in solution rather than precipitated out.[1]Ribose could combine with the bases and phosphate to produce β-D-ribonucleotides.[2]These β-D-ribonucleotides could spontaneously produce RNA polymers of the proper form.[3]That if such polymers form, they could replicate themselves.[4]That such self-replicating RNA molecules would have all the functions needed to sustain an organism.[5]That such an RNA organism could give rise to a modern organism with protein catalysts, coded on the reproducing material, and the means to decode them.[/list]

These postulates are all contrary to experimental evidence.5 It is no wonder that one of the leading researchers into ‘RNA World’ models, Gerald Joyce, wrote:


The most reasonable assumption is that life did not start with RNA …. The transition to an RNA world, like the origins of life in general, is fraught with uncertainty and is plagued by a lack of experimental data.6

A Self-replicating Molecule


A group led by Julius Rebek synthesized a molecule called amino adenosine triacid ester (AATE), which itself consists of two components, pentafluorophenyl ester and amino adenosine. When AATE molecules are dissolved in chloroform with the two components, the AATE molecules act as templates for the two components to join up and form new AATE molecules.7 There are a number of reasons why this is irrelevant to an evolutionary origin of life


This system carries very little information, in contrast to even the simplest cell. Mycoplasma genitalium has the smallest known genome of any living organism, which contains 482 genes comprising 580,000 bases.8 This organism is an obligate parasite. A free-living organism would need many more genes.The new AATE molecule binds too strongly to the parent, so no new reactants can come in and join, as Rebek himself admits.9Replication only occurred in highly artificial, unnatural conditions.10 A reaction in chloroform is irrelevant to living organisms. In particular, chloroform would not hinder condensation reactions as water does. Most polymerisation reactions in life are condensation reactions, that is, they eject a small molecule like water. If there is much water around as there is with all living things, the reverse reaction is favoured, that is the hydrolysis (break-up) of polymers. [For more information, see my later paper, Origin of Life: The Polymerization Problem].The molecule reproduced too accurately—there is no possibility of neo-Darwinian evolution by mutation and natural selection.11[/list]

Self-replicating Peptides?


Amino acids can be formed (with difficulty12) in Miller-type experiments where reducing gases are sparked, unlike ribose and the nitrogenous bases. Thus some evolutionists are investigating protein-first rather than nucleic-acid-first theories of the origin of life. But proteins do not have anything analogous to the base-pairing in nucleic acids. So there was a surprise in August 1996, when some newspapers and science journals reported a peptide that can reproduce itself. David Lee et al. reported that a short peptide derived from part of a yeast enzyme can catalyse its own formation.13
Lee et al. made a 32-unit-long a-helical peptide based on the leucine-zipper domain of the yeast transcription factor GCN4. They found that it catalysed its own synthesis in a neutral, dilute water solution of 15 and 17-unit fragments. This was an ingenious experiment, but it does not help the evolutionary cause because:

Where would the first 32-unit long chain of 100 % left-handed amino acid residues come from? Amino acids are not formed as easily as Lee et al. claim. If they form at all, they are extremely dilute and impure, as well as racemic (50–50 mix of left and right-handed forms). Such amino acids do not spontaneously polymerise in water.Where would a supply of the matching 15 and 17-unit chains come from? Not only does the objection above apply, but what mechanism is supposed to produce the right sequences? Even if we had a mixture of the right homochiral (all the same handedness) amino acids, the chance of getting one 15-unit peptide right is one in 2015 (= one in 3 x 1019). If it is not necessary to get the sequences exactly right, then it would mean that the ‘replication’ is not specific, and would thus allow many errors.The 15 and 17-unit peptides must be activated, because condensation of ordinary amino acids is not spontaneous in water. Lee et al. used a thiobenzyl ester derivative of one peptide. As they say, this also circumvents potential side reactions. The hypothetical primordial soup would not have had intelligent chemists adding the right chemicals to prevent wrong reactions!The particular 32-unit chain was an a-helix, where hydrogen bonds between different amino acid residues cause the chain to helicize. This common structure is more likely to be able to act as a template under artificial conditions. Lee et al. claim that b-sheets, which also depend on hydrogen bonding, might also be able to act as templates. This seems plausible. a-helices and b-sheets are known as the secondary structure of the protein.14The exact way in which the protein folds is called the tertiary structure, and this determines its specific properties. Although Lee et al. say:[/list]

we suggest the possibility of protein self-replication in which the catalytic activity of the protein could be conserved,

they present no experimental proof.

Complexity Theory


This has been promoted by Stuart Kauffman.15 It claims that large numbers of interacting components spontaneously organise themselves into ordered patterns. Sometimes a small perturbation of a system could cause it to switch from one pattern to another. Kauffman proposes that his idea could account for the origin of life, body shapes and even cultural patterns and economics. Complexity theorists point to computer simulations of the patterns of clam shells and other shapes found in nature.[/size]
But this has little relevance to the real world of chemicals. Chemicals obey the Second Law of Thermodynamics, and do not arrange themselves into self-sustaining metabolic pathways. Living cells have molecular machinery to channel the chemistry in the right direction and amounts. If the clam shell pattern on the computer screen was enlarged, there would be no traces of cells with cilia, mitochondria, DNA, etc.16
[size=13]It is small wonder that even most sections of the evolutionary establishment are sceptical of complexity theory. The cover of the June 1995 issue of Scientific American asked ‘Is Complexity Theory a Sham?’. This issue contained an article called ‘From Complexity to Perplexity’, which said:

http://creation.com/self-replicating-enzymes

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12The RNA world, and the origins of life Empty Self-sustained Replication of an RNA Enzyme on Tue Nov 24, 2015 3:35 am

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Self-sustained Replication of an RNA Enzyme 1

Summary
RNA enzymes have been made to undergo self-sustained replication in the absence of proteins, providing the basis for an artificial genetic system.

An RNA enzyme that catalyzes the RNA-templated joining of RNA was converted to a format whereby two enzymes catalyze each other’s synthesis from a total of four component substrates. These cross-replicating RNA enzymes were optimized so that they can undergo self-sustained exponential amplification at a constant temperature and in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about one hour, and can be continued indefinitely. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates. During a serial transfer experiment in which the population underwent overall amplification of >10^25-fold, recombinant replicators arose and grew to dominate the population. RNA enzymes that undergo self-sustained replication can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.

The most fundamental process of biological systems is the replication of the genetic material, brought about by genetically-encoded enzymes. Genetic replication involves a plus-strand nucleic acid template that directs the synthesis of a complementary minus-strand, which in turn directs the synthesis of a new plus-strand. The number of both strands increases exponentially with repeated rounds of templated copying. A longstanding research goal has been to devise a non-biological system that undergoes replication in a self-sustained manner, that is, brought about by enzymatic machinery which is part of the system being replicated. One way to realize this goal, inspired by the notion of primitive RNA-based life, would be for an RNA enzyme to catalyze the replication of RNA molecules, including the RNA enzyme itself (1–4). This has now been achieved in a cross-catalytic system that involves two RNA enzymes that catalyze each other’s synthesis from a total of four component substrates. In this system, exponential growth continues indefinitely at constant temperature, with a doubling time of about 1 h. Furthermore, many such replicators can be constructed and allowed to compete for common resources, resulting in the emergence of new variants and survival of the fittest variants over time.

A well-studied class of RNA enzymes are the RNA ligases, which catalyze the RNA-templated joining of RNA molecules (5, 6). One such ligase is the “R3C” RNA enzyme, which was obtained using in vitro evolution (7). This enzyme binds two RNA substrates through Watson-Crick pairing and catalyzes nucleophilic attack of the 3′-hydroxyl of one substrate on the 5′-triphosphate of the other, forming a 3′,5′-phosphodiester and releasing inorganic pyrophosphate. The R3C ligase previously was configured so that it could self-replicate by joining two RNA molecules to produce another copy of itself (8 ). Self-replication was inefficient, however, because the substrates formed a non-productive complex that limited the extent of exponential growth. Even under the most favorable conditions, the doubling time was about 17 h and no more than two doublings could be achieved.

The R3C ligase then was converted to a cross-catalytic format (Fig. 1A), whereby a plus-strand RNA enzyme (E) catalyzed the joining of two substrates (A′ and B′) to form a minus-strand enzyme (E′), which in turn catalyzed the joining of two substrates (A and B) to form a new plus-strand enzyme (9). This too was inefficient because of the formation of non-productive complexes and the slow underlying rate of the two enzymes. Even with thermal cycling to disrupt the non-productive complexes and recycle the catalysts, it was not possible to achieve even a single doubling of the two enzymes (10). The enzyme E catalyzes the formation of E′ at a rate of 0.034 min−1 with a maximum extent of 20%, while E′ catalyzes the formation of E at a rate of 0.026 min−1 with a maximum extent of 11% (9). These reaction rates are about 10-fold slower than that of the parental R3C ligase (7), and when the two cross-catalytic reactions are carried out within a common mixture, the reaction rates are even slower (9).

Fig. 1
The RNA world, and the origins of life Nihms810
Scheme for cross-catalytic replication of RNA enzymes. 
(A) The enzyme E′ (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, while E catalyzes ligation of A′ and B′ to form E′. The two enzymes dissociate to provide copies of both E and E′ that each can catalyze another reaction. 
(B) Sequence and secondary structure of the complex formed between the cross-replicating RNA enzyme and its two substrates (E′, A, and B are shown; E, A′, and B′ are the reciprocal). Curved arrow indicates the site of ligation. Boxed residues indicate the sites of critical wobble pairs that provide enhanced catalytic activity compared to the parental R3C ligase.

Scheme for cross-catalytic replication of RNA enzymes. (A) The enzyme E′ (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, while E catalyzes ligation of A′ and B′ to form E′. The two enzymes ...
In order to achieve sustained exponential amplification, it thus became necessary to improve the catalytic properties of the cross-replicating RNA enzymes. This was done using in vitro evolution, optimizing the two component reactions in parallel and seeking solutions that would apply to both reactions when conducted in the cross-catalytic format (11). The 5′-triphosphate-bearing substrate was joined to the enzyme via a hairpin loop (B′ to E, and B to E′), and nucleotides within both the enzyme and the separate 3′-hydroxyl-bearing substrate (A′ and A) were randomized at a frequency of 12% per nucleotide position. The two resulting populations of molecules were subjected to six rounds of stringent in vitro selection, selecting for their ability to react in progressively shorter times, ranging from 2 h to 10 milliseconds. The shortest times were achieved using a quench-flow apparatus. Mutagenic PCR was performed after the third round to maintain diversity in the population. Following the sixth round, individuals were cloned from both populations and sequenced. There was substantial sequence variability among the clones, but all contained mutations just upstream from the ligation junction that resulted in a G•U wobble pair at this position.

The G•U wobble pair was installed in both enzymes and both 3′-hydroxyl-bearing substrates (Fig. 1B). In the trimolecular reaction (with two separate substrates), the optimized enzymes, E and E′, exhibited a rate of 1.3 and 0.3 min−1 with a maximum extent of 92% and 88%, respectively. This was deemed sufficient to initiate exponential amplification. A reaction mixture was prepared containing 0.1 µM each of unlabeled E and E′, 5.0 µM each of [5′-32P]-labeled A and A′, 5.0 µM each of unlabeled B and B′, 25 mM MgCl2, and 50 mM EPPS buffer (pH 8.5), which was incubated at 42 °C for 10 h. Samples were taken from the mixture at various times, and the yield of newly-synthesized E and E′ was determined by separating the radiolabeled materials in a denaturing polyacrylamide gel. Both enzymes exhibited robust exponential growth, with more than 25-fold amplification after 5 h, followed by a leveling off as the supply of substrates became depleted (Fig. 2A). The data fit well to the logistic growth equation:

[E]t = a / (1 + be−ct), where [E]t is the concentration of E (or E′) at time t, a is the maximum extent of growth, b is the degree of sigmoidicity, and c is the exponential growth rate.
This equation is commonly used in population ecology to model the exponential growth of organisms subject to the carrying capacity of the local environment. For the enzymes E and E′, the exponential growth rate was 0.92 and 1.05 h−1, respectively.

Fig. 2
The RNA world, and the origins of life Nihms811
Self-sustained amplification of cross-replicating RNA enzymes. 
(A) The yield of both E (black) and E′ (gray) increased exponentially before leveling off as the supply of substrates became exhausted. 
(B) Amplification was sustained by performing a serial transfer experiment, allowing ~25-fold amplification before transferring 1/25th of the mixture to a new reaction vessel that contained a fresh supply of substrates. The concentrations of E and E′ were measured at the end of each incubation.

Self-sustained amplification of cross-replicating RNA enzymes. (A) The yield of both E (black) and E′ (gray) increased exponentially before leveling off as the supply of substrates became exhausted. (B) Amplification was sustained by performing ...
Exponential growth can be continued indefinitely, so long as a supply of the four substrates is maintained. One way to achieve this is to carry out a serial transfer experiment in which a portion of a completed reaction mixture is transferred to a new reaction vessel that contains a fresh supply of substrates. Six successive reactions were carried out in this fashion, each 5 h in duration and transferring 1/25th of the material from one reaction mixture to the next. The first mixture contained 0.1 µM each of E and E′, but all subsequent mixtures contained only those enzymes that were carried over in the transfer. Exponential growth was maintained throughout 30 h total incubation, with an overall amplification of >108-fold for each of the two enzymes (Fig. 2B). This corresponds to 28 doublings in a process that was sustained by the enzymes themselves. No temperature cycling was required and the reaction mixtures did not contain any proteins or other biological materials.

A genetic system requires not only self-replication, but also the opportunity for many different genetic molecules to replicate, with their replication rate dependent on genetically-encoded functional properties. It is possible to construct many variants of the cross-replicating RNA enzymes that differ with respect to their “genotype” and associated “phenotype”. The genotype is defined as the regions of the enzyme that engage in Watson-Crick pairing with its cross-catalytic partner and that can vary in sequence without significantly affecting replication efficiency. These regions are located at the 5′ and 3′ ends of the enzyme (Fig. 1B). Other regions of Watson-Crick pairing between the two enzymes are tolerant of some sequence variation, albeit with some alteration of replication efficiency.

Four nucleotide positions at the 5′ end and four nucleotides at the 3′ end of the enzyme were chosen as the sites for genotypic variation (Fig. 3). A rule was adopted that each of these regions would contain one G•C and three A•U pairs so that there would be no substantial differences in base-pairing stability among the various genotypes. This allowed 32 possible pairs of complementary sequences for each region, of which 12 were chosen as a set of designated genotypes (Fig. 3). Each genotype was associated with a distinct phenotype, manifest as a particular sequence within the catalytic core of the enzyme. For simplicity, the same phenotype was associated with both members of a cross-replicating pair, although this need not be the case.

Fig. 3
The RNA world, and the origins of life Nihms812
Twelve pairs of cross-replicating RNA enzymes were constructed. 
Four nucleotides at the 5′ and 3′ ends of the enzyme were chosen as the sites for genotypic variation, and 11 nucleotides within the catalytic core were chosen as the corresponding sites for phenotypic variation (boxed regions). The sequence of these regions for each of the 12 E enzymes is shown at the right. The corresponding E′ enzymes have a complementary sequence in the genotype region and the same sequence in the catalytic core. Alterations of the catalytic core relative to the E1 enzyme are highlighted by black circles.


Twelve pairs of cross-replicating RNA enzymes were constructed. Four nucleotides at the 5′ and 3′ ends of the enzyme were chosen as the sites for genotypic variation, and 11 nucleotides within the catalytic core were chosen as the corresponding ...
Twelve pairs of cross-replicating enzymes were synthesized, as well as the 48 substrates (12 each of A, A′, B, and B′) necessary to support their exponential amplification. Each replicator was tested individually and demonstrated varying levels of catalytic activity and varying rates of exponential growth (fig. S1). Replication was somewhat faster in the presence of 25 compared to 15 mM MgCl2, but the lower concentration was chosen for subsequent studies because it is less likely to promote the use of mismatched substrates and renders the RNA less susceptible to hydrolysis. Of the 12 pairs of cross-replicating enzymes, the one shown in Fig. 1B (now designated E1 and E1′) had the fastest rate of exponential growth, achieving about 20-fold amplification after 5 h in the presence of 15 mM MgCl2. The various cross-replicating enzymes shown in Fig. 3 had the following rank order of replication efficiency: E1, E10, E5, E4, E6, E3, E12, E7, E9, E8, E2, E11. The top five replicators all achieved more than 10-fold amplification after 5 h, and all except E11 achieved at least 5-fold amplification after 5 h.

Two different serial transfer experiments were carried out involving mixtures of various cross-replicating enzymes and their corresponding substrates. The first was initiated with 0.1 µM each of E1–E4 and E1′–E4′, and 5.0 µM each of the 16 corresponding substrates. Sixteen successive reactions were carried out over a period of 70 h, transferring 1/20th of the material from one reaction mixture to the next (fig. S2A). Individuals were cloned from the population following the final reaction, and were sequenced to determine their genotype and to confirm the identity of their corresponding phenotype. Among 25 clones (sequencing E′ only), there was no dominant replicator (fig. S2B). E1′, E2′, E3′, and E4′ all were represented, as well as 17 clones that were the result of recombination between a particular A′ substrate and one of the three B′ substrates other than its original partner (or similarly for A and B).

Recombination occurs when an enzyme binds and ligates a mismatched substrate. In principle, any A could become joined to any B or B′, and any A′ could become joined to any B′ or B, resulting in 64 possible enzymes. The “genetic code” was designed so that cognate substrates have a binding advantage of several kcal/mol compared to non-cognate substrates (fig. S2C). However, once a mismatched substrate is bound and ligated, it forms a recombinant enzyme that can cross-replicate by drawing upon the corresponding set of four substrates. Recombinants can give rise to other recombinants, as well as revert back to non-recombinants. Mismatches are less likely to occur during the pairing of A and B′ regions compared to the pairing of A′ and B regions because the former enjoy the benefit of an additional base pair for matched substrates (Fig. 1B). Thus there are expected to be preferred pathways for mutation, primarily involving substitution among certain A′ and among certain B components (fig. S2D), although reflected in the identity of both members of a cross-replicating pair.

Another serial transfer experiment was initiated with 0.1 µM each of all 12 pairs of cross-replicating enzymes and 5.0 µM each of the 48 corresponding substrates. In this more complex mixture there was abundant opportunity for recombination, with 132 possible pairs of recombinant cross-replicating enzymes, as well as the 12 pairs of non-recombinant cross-replicators. Twenty successive reactions were carried out over a period of 100 h, transferring 1/20th of the material from one reaction mixture to the next, and achieving an overall amplification of >1025-fold (Fig. 4A). Again individuals were cloned from the final population and sequenced. Of 100 clones (sequencing 50 E and 50 E′), only 7 were non-recombinants (Fig. 4B). The distribution was highly non-uniform, with sparse representation of molecules containing components A6–A12 and B5–B12 (and reciprocal components B6′–B12′ and A5′–A12′). The most frequently represented components were A5 and B3 (and reciprocal components B5′ and A3′). The three most abundant recombinants were A5B2, A5B3, and A5B4 (and their cross-replication partners), which together accounted for one-third of all clones.

Fig. 4
The RNA world, and the origins of life Nihms813
Self-sustained amplification of a population of cross-replicating RNA enzymes, resulting in selection of the fittest replicators. 
(A) Beginning with 12 pairs of cross-replicating RNA enzymes (Fig. 3), amplification was sustained by serial transfer for 20 successive rounds of ~20-fold amplification and 20-fold dilution. The concentrations of all E (black) and E′ (gray) molecules were measured at the end of each incubation. 
(B) Graphical representation of the observed genotypes among 50 E and 50 E′ clones (dark and light columns, respectively) that were sequenced following the last incubation. The A and B (or B′ and A′) components of the various enzymes are shown on the horizontal axes, with non-recombinant enzymes indicated by shaded boxes along the diagonal. The number of clones containing each combination of components is shown on the vertical axis.


Self-sustained amplification of a population of cross-replicating RNA enzymes, resulting in selection of the fittest replicators. (A) Beginning with 12 pairs of cross-replicating RNA enzymes (Fig. 3), amplification was sustained by serial transfer for ...
The exponential growth rates of A5B2, A5B3, and A5B4 were compared to that of E1, the most efficient non-recombinant replicator. In the presence of their cognate substrates alone, E1 remained the most efficient replicator, but in the presence of all 48 substrates, the most efficient replicator was A5B3 (Fig. 5A). The exponential growth rate of E1 was 0.75 h−1 in the presence of its cognate substrates, but it exhibited only linear growth at a rate of 0.10 h−1 in the presence of all substrates. In contrast the exponential growth rate of A5B3 was 0.68 h−1 in the presence of its cognate substrates, and 0.33 h−1 in the presence of all substrates. When the A5B3 replicator was provided a mixture of substrates corresponding to the components of the three most abundant recombinants (A5, B2, B3, B4, B5′, A2′, A3′, and A4′), its exponential growth rate was 0.84 h−1, the highest measured for any replicator in the presence of 15 mM MgCl2 (Fig. 5B).

Fig. 5
The RNA world, and the origins of life Nihms814
Exponential amplification of the starting cross-replicating enzymes (E1 and E1′) and of the most efficient cross-replicator (A5B3 and B5′A3′) that emerged during the serial transfer experiment involving all 48 substrates (Fig. 4).
(A) Comparative growth of E1 (circles) and A5B3 (squares) in the presence of either their cognate substrates alone (filled symbols) or all substrates that were present during serial transfer (open symbols). 
(B) Growth of A5B3 (black) and B5′A3′ (gray) in the presence of the eight substrates (A5, B2, B3, B4, B5′, A2′, A3′, and A4′) that comprise the three most abundant cross-replicating enzymes.


Exponential amplification of the starting cross-replicating enzymes (E1 and E1′) and of the most efficient cross-replicator (A5B3 and B5′A3′) that emerged during the serial transfer experiment involving all 48 substrates ( ...
The fitness of a pair of cross-replicating enzymes depends on several factors, including their intrinsic catalytic activity, exponential growth rate with cognate substrates, ability to withstand inhibition by other substrates in the mixture, and net rate of production through mutation among the various cross-replicators. The A5B3 recombinant and its cross-replication partner B5′A3′ have different catalytic cores (Fig. 3), and both exhibit robust activity. The A5B3 enzyme has a rate of 0.58 min−1 and maximum extent of 90%, which is comparable to E1 with a rate of 0.63 min−1 and maximum extent of 90% (measured in the presence of 15 mM MgCl2). The B5′A3′ enzyme has a rate of 0.66 min−1 and maximum extent of 90%, which is considerably more active than E1′ with a rate of 0.11 min−1 and maximum extent of 92%. The nearly equal rates of the A5B3 and B5′A3′ enzymes may account for their well-balanced rate of production throughout the course of exponential amplification (Fig. 5B). Other factors, however, such as substrate binding and product release, can influence the rate of exponential growth, which may explain why amplification of E1 with its cognate substrates outpaces that of A5B3. The selective advantage that A5B3 enjoys appears to derive from its relative resistance to inhibition by other substrates in the mixture (Fig. 5A) and its ability to capitalize on facile mutation among substrates B2, B3, and B4 and among substrates A2′, A3′, and A4′ (fig. S2D).

A population of cross-replicating RNA enzymes can serve as an experimental model of a genetic system. This model is greatly simplified compared to biological genetics because it involves only two genetic loci with, at present, only 12 alleles per locus. It is likely, however, that the number of alleles could be increased by exploiting more than four nucleotide positions at the 5′ and 3′ ends of the enzyme, and by relaxing the rule that these nucleotides form one G•C and three A•U pairs. One could construct many different genetic systems with alternative rule sets, resulting in alternative behaviors during the course of selective amplification. Different mixtures of enzymes and substrates and different reaction conditions are expected to lead to different outcomes, and these could then be related to the underlying properties of the genetic system.

In order to support greater complexity in a system of cross-replicating RNAs it will be necessary to constrain the set of substrates so that each enzyme can secure its own substrates without being overwhelmed by other substrates in the mixture. One way to do this is to choose a set of substrates that are more distinguishable than the ones used here. Another approach is to adjust the concentrations of the various substrates in proportion to their utilization by the population of enzymes. It is not clear how this would be done within the system, but it could be achieved using a deconstructive PCR procedure in which the population of newly-formed enzymes is used to generate a corresponding population of substrates (11). In this way both the successful enzymes and their component substrates are inherited from one generation to the next.

Another important challenge for an artificial genetic system is to support a broad range of encoded functions, well beyond replication itself. It is possible to insert a functional domain within the central stem-loop of the cross-replicating enzymes so that replication is dependent on execution of that encoded function (Lam & Joyce, unpublished results). It would be much more powerful, however, to have a system in which novel function emerges during the course of selective amplification. The self-sustained evolution of RNA with open-ended opportunities for discovering novel function likely has not occurred on Earth since the time of the RNA world, and continues to present an intriguing research opportunity.

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652413/

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13The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Fri Dec 18, 2015 6:27 pm

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Problematic Chemical Postulates of the RNA World Scenario



Postulate 1: There was a prebiotic pool of beta-D-ribonucleotides.

Beta-D-ribonucleotides (see Figure 2) are compounds made up of a purine (adenine or guanine) or a pyrimidine (uracil or cytosine) linked to the 1'-position of ribose in the beta-configuration.
There is, in addition, a phosphate group attached to the 5'-position of the ribose. For the four different ribonucleotides in this prebiotic scenario, there would be hundreds of other possible isomers.
But each of these four ribonucleotides is built up of three components: a purine or pyrimidine, a sugar (ribose), and phosphate. It is highly unlikely that any of the necessary subunits would have accumulated in any more than trace amounts on the primitive Earth. Consider ribose. The proposed prebiotic pathway leading to this sugar, the formose reaction, is especially problematic9. If various nitrogenous substances thought to have been present in the primitive ocean are included in the reaction mixture, the reaction would not proceed. The nitrogenous substances react with formaldehyde, the intermediates in the pathways to sugars, and with sugars themselves to form non-biological materials10. Furthermore, as Stanley Miller and his colleagues recently reported, "ribose and other sugars have suprisingly short half-lives for decomposition at neutral pH, making it very unlikely that sugars were available as prebiotic reagents."11
Or consider adenine. Reaction pathways proposed for the prebiotic synthesis of this building block start with HCN in alkaline (pH 9.2) solutions of NH4OH.12These reactions give small yields of adenine (e.g., 0.04%) and other nitrogenous bases provided the HCN concentration is greater than 0.01 M. However, the reaction mixtures contain a great variety of nitrogenous substances that would interfere with the formose reaction. Therefore, the conditions proposed for the prebiotic synthesis of purines and pyrimidines are clearly incompatible with those proposed for the synthesis of ribose. Moreover, adenine is susceptible to deamination and ring-opening reactions (with half-lives of about 80 years and 200 years respectively at 37º C and neutral pH), making its prebiotic accumulation highly improbable13. This makes it difficult to see how any appreciable quantities of nucleosides and nucleotides could have accumulated on the primitive Earth. If the key components of nucleotides (the correct purines and pyrimidines, ribose, and phosphate) were not present, the possibility of obtaining a pool of the four beta-D-ribonucleotides with correct linkages would be remote indeed.
If this postulate, the first and most crucial assumption, is not valid, however, then the entire hypothesis of an RNA World formed by natural processes becomes meaningless.


Postulate 2: Beta-D ribonucleotides spontaneously form polymers linked together by 3', 5'-phosphodiester linkages (i.e., they link to form molecules of RNA;

 see figure 2).

Joyce and Orgel discuss candidly the problems with this postulate14. They note that nucleotides do not link unless there is some type of activation of the phosphate group. The only effective activating groups for the nucleotide phosphate group (imidazolides, etc.), however, are those that are totally implausible in any prebiotic scenario. In living organisms today, adenosine-5'-triphosphate (ATP) is used for activation of nucleoside phosphate groups, but ATP would not be available for prebiotic syntheses. Joyce and Orgel note the possible use of minerals for polymerization reactions, but then express their doubts about this possibility15:


Whenever a problem in prebiotic synthesis seems intractable, it is possible to postulate the existence of a mineral that catalyzes the reaction...such claims cannot easily be refuted.


In other words, if one postulates an unknown mineral catalyst that is not readily testable, it is difficult to refute the hypothesis.
Joyce and Orgel then note that if there were activation of the phosphate group, the primary polymer product would have 5', 5'-pyrophosphate linkages; secondarily 2', 5'-phosphodiester linkages -- while the desired 3',5'-phosphodiester linkages would be much less abundant. However, all RNA known today has only 3',5'-phosphodiester linkages, and any other linkages would alter the three-dimensional structure and possibilities for function as a template or a catalyst.
Even waiving these obstacles, and allowing for minute amounts of oligoribonucleotides, these molecules would have been rendered ineffective at various stages in their growth by adding incorrect nucleotides, or by reacting with the myriads of other substances likely to have been present. Moreover, the RNA molecules would have been continuously degraded by spontaneous hydrolysis and other destructive processes operating on the primitive Earth16.
In brief, any movement in the direction of an RNA World on a realistically-modeled early Earth would have been continuously suppressed by destructive cross-reactions.


Postulate 3: A polyribonucleotide (i.e. RNA molecule), once formed, would have the catalytic activity to replicate itself, and a population of such self-replicating molecules could arise.



The difficulty with this postulate is evident in the following quotation from Joyce and Orgel:
...it is assumed...that a magic catalyst existed to convert the activated nucleotides to a random ensemble of polynucleotide sequences, a subset of which had the ability to replicate. It seems to be implicit that such sequences replicate themselves but, for whatever reason, do not replicate unrelated neighbors.17
They refer to this as a component of "The Molecular Biologists Dream," and discuss the difficulties inherent in such a view. In order for a stable population of self-replicating RNA molecules to arise -- a prerequisite for further evolution -- the RNA molecules must be able to replicate themselves with high fidelity, or the sequence specificity which makes self-replication possible at all will be lost. While "it is difficult to state with certainty the minimum possible size of an RNA replicase ribozyme," Joyce and Orgel note, it seems unlikely that a structure with fewer than 40 nucleotides would be sufficient. Suppose, then, that "there is some 50-mer [RNA molecule of 50 nucleotides length]," Joyce and Orgel speculate, that "replicates with 90% fidelity. ... Would such a molecule be expected to occur within a population of random RNAs?"
Perhaps: but one such self-replicating molecule will not suffice.
"Unless the molecule can literally copy itself," Joyce and Orgel note, "that is, act simultaneously as both template and catalyst, it must encounter another copy of itself that it can use as a template." Copying any given RNA in its vicinity will lead to an error catastrophe, as the population of RNAs will decay into a collection of random sequences. But to find another copy of itself, the self-replicating RNA would need (Joyce and Orgel calculate) a library of RNA that "far exceeds the mass of the earth."18
In the face of these difficulties, they advise, one must reject


the myth of a self-replicating RNA molecule that arose de novo from a soup of random polynucleotides. Not only is such a notion unrealistic in light of our current understanding of prebiotic chemistry, but it should strain the credulity of even an optimist's view of RNA's catalytic potential. If you doubt this, ask yourself whether you believe that a replicase ribozyme would arise in a solution containing nucleoside 5'-diphosphates and polynucleotide phosphorylase!19


Postulate 4: Self-replicating RNA molecules wouild have all of the catalytic activities necessary to sustain a ribo-organism.

S.A. Benner et al. note20:
...one is forced to conclude that the last ribo-organism had a relatively complex metabolism that included oxidation and reduction reactions, aldol and Claison condensations, transmethylations, porphyrin biosynthesis, and an energy metabolism based on nucleoside phosphates, all catalyzed by riboenzymes...It should be noted that this reconstruction cannot be weakened without losing much of the logical and explanatory force of the RNA World model.
Although Benner et al. speak of the last "ribo-organism," surely the first ribo-organism would have required nearly all of the same metabolic capabilities in order to survive. It is also apparent that the scenario of Benner et al. would surely include enclosing the ribozymes within a membrane with the ability to transport ions and organic molecules across that membrane.
Anyone who is familiar with biochemistry would recognize that it would take hundreds of different ribozymes, each with a particular catalytic activity, to carry out the metabolic processes described above. It should also be apparent that most of these metabolic capabilities would have to be functional within a short period of time (certainly not hundreds of years), in the same microscopic region, or the ribo-organism would never survive.
When one recognizes that catalytic activities of RNA are just as dependent upon specific sequences of nucleotides in RNA21 as protein enzymes are of amino acid sequences, then the probability of postulate 4 being valid is seen to be vanishingly small.
Benner et al. note that the diverse catalytic properties of enzymes often require coenzymes or prosthetic groups. They mention particularly the iron-porphyrin, heme, and pyridoxal, but have no suggestion how these (and other co-enzymes) could have functioned in the catalytic activities of early RNA molecules.
The other unproven assumption of postulate 4 is that RNA molecules initially had all of these suggested catalytic activities, but nearly all of these activities have been subsequently lost. RNA molecules with catalytic activity that are known today predominantly have nuclease or nucleotidyl transferase activity with some minimal esterase actitivy22. There is no solid evidence that RNA molecules ever had the broad range of catalytic activities suggested by Benner et al., even though a number of the authors of The RNA World speak of present-day RNA molecules as being vestiges of that early RNA World.


Conclusion

We have more to learn about RNA, both in vivo (as used by organisms) and in vitro, in terms of its chemistry generally and functional properties in particular. RNA is a remarkable molecule.
The RNA World hypothesis is another matter. We see no grounds for considering it established, or even promising, except perhaps on the objectionable philosophical grounds of philosophical naturalism (and its operational offspring, methodological naturalism), according to which the best naturalistic hypothesis is perforce the hypothesis to be accepted. We consider that historical biology should be open to all empirical possibilities, including design -- and see the molecular biological system of organisms, of which RNA is so stunning a part, as exemplars of design.
We find ourselves, however, distinctly in the minority of biologists. If design exists at all, it is a matter of subjective intuition, the majority of our colleagues would claim, asserting with science writer George Johnson that "the point of science is...to explain the world through natural law."23
We would put the point rather differently. The point of science is to explain the world, through natural laws or whatever other causes best account for the phenomena at hand.
Philosopher of science Stephen Meyer captures the point well:


The (historical) question that must be asked about biological origins is not "Which materialistic scenario will prove adequate?" but "How did life as we know it actually arise on earth?" Since one of the logically appropriate answers to this latter question is that "Life was designed by an intelligent agent that existed before the advent of humans," I believe it is anti-intellectual to exclude the "design hypothesis" without consideration of all the evidence, including the most current evidence, that would support it.24


Detecting design is not a matter of subjective intuition.25 To see design as a real causal possibility, however, one must break free of the constraints of naturalism.


http://www.arn.org/docs/odesign/od171/rnaworld171.htm

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14The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Sat Jan 02, 2016 8:33 am

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The "RNA World"


https://pt-br.widbook.com/ebook/read/life-and-the-universe-by-intelligent-design


The RNA world, and the origins of life Readin20

The RNA world, and the origins of life 6cad633dc0794a699b69a1ee7365ad53_original


Figure 1. In the beginning RNA created both the proteins and the DNA, and the proteins were without form, and void; but when chaperones were available - who knows how - darkness was removed upon the face of Life, and Life began.
In the "RNA world", a "super molecule" of the chicken-egg type is assigned the task to starts Life on earth. In such "world" the elaborated mechanisms for protein synthesis as we observed today in Life did not yet exist; hence RNA would constitute both the enzymatic and genetic basis of the first organisms. Faced with one of the greatest chicken-egg dilemma of Life, the dillema of which came first, an RNA/DNA or a protein/enzyme, and with their inexhaustible imagination in finding hypothetical scenarios - a capacity in which evolutionists are unbeatable - such hypotheis1 was elaborated. In the "RNA world" it is therefore speculated that neither the chicken nor the egg came first, but the two are merged into one, and a single "super RNA" molecule emerges and makes the function of both the egg and the chicken. The RNA then acquires the status of a "chicken-egg" molecule: a "super primordial RNA".
The "RNA world" hypothesis is therefore fundamentally based on the ability of RNA to store, transmit, and duplicate genetic information - similarly to DNA - in addition to its catalytic ability, an ability also observed for some proteins. Called ribozymes, these super RNA break and catalyze phosphodiester bonds. It has also been observed that such "multifuction RNA" may also be able to catalyze peptide bonds between amino acids (AAs). Since such two properties were discovered for RNA, such as for the r-RNA found in ribosomes, plus its ability to self replication, and knowing that RNA, besides acting as enzymes, also works in Life as proteins molds transmitting via m-RNA and t-RNA the information stored in the DNA, it was imagined - what an imagination - the scenario of a "prebiotic world" ruled solely by RNA molecules. Figure 2 summarizes the evolutionary steps of such imaginary scenario.


The RNA world, and the origins of life B00c0b49ecf742b89d63e27cd6c85759_original


Figure 2. In the beginning was the super RNA, which was "magically" formed. It self replicates, and then starts doing a cascade of various "miracles" such as the simultaneous catalyze of the synthesis of both functional proteins and encoding DNA.
In the "RNA world" (1), and from nucleotides (Figure 3) in the primordial soup, takes place the polymerization of nucleotides - who knows how since chemistry prohibits such polymerization - forming "small" RNA polymers, which turn to be self-replicating and autocatalytic. Now catalyzed by ribozymes in step (2), the RNA replication process starts. Next, now functioning as templates, these "super RNAs" (3) capture prebiotic amino acids (AA) of the primordial soup and by doing so catalyze the synthesis of proteins. And amazingly, such proteins turn to be functional. Now in step (4), an even more spectacular and electrifying event occurs, in which the primordial RNA indeed demonstrates its super supreme powers since it starts "Life" on this planet by coding for the synthesis of both DNA and proteins. This is why we like to call such molecule as the "super-RNA", a so powerful molecule that it must have been also the ancestor of the "Superman" (Figure 4)!


The RNA world, and the origins of life 3fbb1d4cf47a43fdb67fb6e903facead_original


* Figure 3. Nucleotides, which are formed by three types of chemical species (anions phosphate, a sugar - ribose - and heterocyclic nitrogen bases). A molecule of extreme difficult synthesis which requires pure and specific reagents and controlled conditions plus enzymes to accelerate reactions. But such enzymes do not yet exist since they shall be synthesize latter by such RNA.


The RNA world, and the origins of life AAffA0nNPuCLAAAAAElFTkSuQmCCThe RNA world, and the origins of life Readin16


Figure 4. The "super primordial RNA". Was it also the molecular ancestor of Superman?
That is, at this point, the super chicken-egg molecule of RNA finally divides, in an egg (DNA) and a chicken (proteins or enzymes). Finally in step (E), a cell membrane shows up - of a bylayer phospholipid type because another type would not serve for the job - which encapsulates the prebiotic "Life". So LUCA is formed and the cycle we currently observed in Life is established: DNA → RNA → protein/enzyme → DNA. Such sequence must have been promoted by the "Tinker Bell of Darwin", I believe. Then to summarize the opera sonnet, the hypothesis about the origin of Life via "the super RNA world" is based in this double ability of RNA to both store genetic information and to catalyze chemical reactions. It is therefore believed - what an ardent faith - that such RNA would have preceded both DNA and proteins, which presumably evolved later than the RNA itself.
So would this "super RNA" - with all such super powers in catalysis, synthesis and coding -make chemical evolution at the molecular level viable? Did we find the solution for the chicken-egg dilemma of Life by merging the egg with the chicken? The answer is still no. And why not? Because there are very serious problems with such hypothesis, which are perceived when you know a little of Chemistry. Remember here the famous quote, which I once complemented: "Evolution hopes you do not know anything about Chemistry, and learn only Biology and Genetics, because there nothing makes sense except in the light of it". But what would be such problems?
Problem 1. Note that when we replace the "protein world" hypothesis by the "RNA world " hypothesis, in which Life begins not with proteins but with such "super RNA" molecule, we make our task much more complicated since the chemical synthesis now involved would be three times more demanding. Proteins are made of a single class of molecules - the amino acids (AAs). But RNA (Figure 2) is made of not only one but three classes of molecules: a sugar (ribose), a phosphate anion and four nitrogenated bases, those nitrogen-containing heterocyclic aromatic molecules of high chemical intelligence that are able to link themselves via two or three hydrogen bonds. So, as for the "protein world," we will face in the "RNA world" the challange of availability and high concentrations, but now not only for one, but for three different types of molecules. That is, step (1) of the RNA world is not so much a hypothesis, but it is better classified as a chemical delirium! For nucleotides and their polymers are "works of art" in synthesis. And even worse, nucleotides are unstable. And the worst of all: Chemits know very well that the conditions that favor the synthesis of sugars are incompatible with the conditions for the synthesis of nitrogenated bases. How can we imagine therefore a scenario in which two incompatible molecules are being synthesized and becoming both available in the primordial soup? At the same time and the same place? And the right sugar (ribose) and the right four nitrogenous bases (ATCC)? And with the "super RNA" being miraculously formed there and also being there miraculously preserved without degrading, and staying in the soup long enough to make its miracles? Would this scenario be considered viable?
Problem 2. For the synthesis of that super autocatalytic and self-replicating RNA, we must realize also that the first "triple miracle" of the availability of all three classes of molecules, incompatible in their synthesis, should be also followed by a second miracle. Which one? It is known that only a few specific sequences of nucleotides in RNA are autocatalytic. The "super RNA" would therefore have to be also a "super lucky RNA", which not only would be formed under these inhospitable conditions, but formed with the right sequence. And such a sequence could not be short, but one that would involve dozens of nitrogenated bases, at least 150 of them. At this point you could ask: what is the probability of this to happen? And the answer is: a likelihood of something close to that discussed in the dilemma of Émile Borel, of the monkeys typing Shakespeare! It would exhaust all probabilistic resources of this Universe, or a collection of them!
If you consider that statistics has been very cruel to the "RNA world", wait to see the cruelty of Chemistry. For to connect nucleotides, P-O-P bonds between phosphate anions need to be established. These reactions are extremely slow and occur in Life only with the help of enzyme catalysis, that is, they are greatly accelerated by enzymes, which are what? Such enzymes are proteins, exactly the type of molecules that the "RNA world" is "trying" to form but have not formed them yet. And for even greater trouble for the "RNA world", it is known that phosphate PO43- anions precipitated in the presence of Ca2+, forming the insoluble salt of Ca3(PO4)3. And Ca2+ should be abundant in the primordial soup.
Problem 3. Also note that in step (C) of Figure 2, the hyper mega "super lucky RNA" now needs to synthesize a protein. Well, but didn´t we mention that same problem before in the "protein world"? Yes! And it is precisely here that the whole rhetoric of the RNA world surrenders to facts, and seems to definetively fail. And such failure will be even more clear when the need to form the double helix of DNA appears. For now our "super RNA" will have to rely - again - in all the luck available in the universe! For it will need not to synthesize any protein but a functional protein! And such synthesis must be done without the help of ribosomes which, as we all know, is the only automated molecular factory can read the receipe delivered by DNA via RNA, and cybernetically perform this function with the help of their workers, the t-RNA, reacting amino esters and not AAs and avoiding the laterals killer reactions. What would therefore be the likelihood for it to form such a functional protein? For a protein with 150 AAs, the probability would be 1 in (20)150, that is, one in billions of billions! And there are more challanges to come, since at this point AAs must be available, and at proper concentrations, and all of them in just one place. And to put even more dark clouds to disturbe this "historic" day for Life on Earth, there should be only AAs there, and they had to be pure, and all AAs should be homochiral, and of the L type! And how could the "super powerful RNA" have learned to separate and select only homochiral type L AAs? Even worse, even with all the luck of this world, and from other worlds too, with such tremendous luck such RNA could guide for a functional protein, who would keep the recipe for repeating the synthesis with the same sequence if a storage system, and a system for reading and transmiting such storaged biochemical information was not yet in operatition? In other words, the world of RNA gives no relief, but triples the problems since it "drags" to it all chemical restrictions from the protein world, only making the problems triply as bad, making the RNA world even more chemically impossible.
Problem 4. More dark clouds should be put upon the "RNA world", indeed a world full of storms. Note that steps (1-4) occurr in an open environment, that is, in a extremely hostile environment, still unprotected from the bilayer membranes of Life. Such steps would therefore occurr outside a cellular environment where we know today there is no Life! But then, in step 5 of Figure 2, in an event of even greater luck, such a double layer membrane simply appears from nowhere to protect the RNA world. But did you know that the double layer membranes of Life are unstable unless a living organism controls its properties? And that such membrane is useful to cell only when input and output channels, such as Na+ channels and water filters, the aquaporins, are connected to it? But without giving any importance at all to chemical rationality, "super RNA" plays another magical trick a la Mandrake and simply forms next a double helix DNA, using now another type of sugar, desoxyribose, but still of the homochiral D type, and using a different set of bases: AGCT. For in DNA the ribose used in RNA is replaced by deoxyribose, by losing its hydroxyl group, and the RNA → DNA sequence is reversed to DNA → RNA - who knows how - and the LUCA finally appears in this planet (Figure 1). There is a saying declaring that paper accepts everything, and indeed when the evolution is explained rhetorically in paper as in Figure 2, it appears to make perfect sense. But when the chemistry that I know and respect and understand is used, it is cruel to the RNA wold, grading it by "zero".
Problem 5. In all known organisms - and not on those which are believed to have existed one day in this planet - is the DNA and not the RNA that carries the genetic inormation. DNA has many advantages over RNA, which make it a much more suitable molecule to store the genetic code. First, DNA is a molecule that forms a double helix whereas the RNA strand is formed by only a single molecular filament. The double-stranded helix gives DNA the ability to be replicated with greater simplicity and security. Most importantly, however, it is that the DNA and RNA differ in the sugar that makes up the backbone of the molecule. The deoxyribose - the sugar used in the DNA - is different from that used for RNA, that is, ribose (Figure 4). Ribose has an additional hydroxyl group (OH), which considerably decreases its stability as compared to DNA. This OH group is able to initiate a chemical reaction that promotes RNA degradation. For this reason, DNA is a much more stable and better genetic material. Obviously therefore, DNA -and not RNA- was "chosen" from the begining as the permanent storage of genetic information. To RNA it was given only the function of transmiting DNA information, and only temporarily (Figure 5). Evidence seems therefore to indicate that RNA was, is and will always be improper to start Life!


The RNA world, and the origins of life AAffA0nNPuCLAAAAAElFTkSuQmCCThe RNA world, and the origins of life Readin17


* Figure 5. The crucial question: what or who removed the hydroxyl group from ribose so as to allow DNA to be stable enough to store the information of Life?


The RNA world, and the origins of life AAffA0nNPuCLAAAAAElFTkSuQmCCThe RNA world, and the origins of life Readin18


Figure 5. The Central Dogma of Life. And as so it has always been, from the very beginning, as indicated by data.
But why there are so many scientific papers published about the RNA world? And so many announcements of scientific proofs, experiments, and more experiments showing that yes, the "primordial RNA" could indeeed self-replicate and present catalytic properties, and form long RNA sequences? So many papers in Nature in Science? You don't know how? Well, then let me explain to you how all of this takes place. In these experiements, the "smart" chemists of the "RNA world" do two things that in the primordial soup would correspond to two simultaneous true "chemical miracles"! They first a) begin their experiments with long RNAs - bearing a few dozen nucleotides- such complex molecules that are carefully synthesized in protected environments by intelligent chemists from pure, homochiral reagents. In addition, they b) select the sequences which are self-catalytic - since most RNA sequences are innocous. For these - and other tricks - that the RNA world is known, intra-waals, not to be a solution - but the nightmare of evolutionary biologists and chemists (Figure 6).


The RNA world, and the origins of life AAffA0nNPuCLAAAAAElFTkSuQmCCThe RNA world, and the origins of life Readin20


Figure 6. An evolutionary biologist dreaming about the chemical structures he has to explain in his classroon of chemical evolution, that lecture about the "super powerful and lucky RNA world".
_________________________
1. Gilbert, Walter (1986), "The RNA World," Nature, 319: 618.

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15The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Fri Jan 29, 2016 3:23 am

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A ribonucleic acid (RNA) world



RNA is a linear polymer of ribonucleotides, usually single stranded. Each ribonucleotide monomer contains the sugar ribose linked with a phosphate group and one of four bases: adenine, guanine, cytosine or uracil. RNA appears in both prokaryotic and eukaryotic cells as messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) which are involved in protein synthesis with DNA the source of information. Some viruses however contain genomes of RNA. The nuclei of eukaryotic cells carry two other types of RNA; heterogeneous nuclear RNA (hnRNA or pre-mRNA) and small nuclear RNA (snRNA).
In recent literature there is much excitement over the discovery that there are RNAs that can catalyse specific biochemical reactions. These are the ribozymes, that is, RNA with enzymatic functions.33 RNA can do this surprising feat by folding its linear chains to appropriate secondary and tertiary structures thereby conferring “domain” type catalytic structures as seen in protein enzymes.


The RNA world, and the origins of life 7807molecular-structures-DNA


Figure 3. The molecular structures of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are built using the nitrogenous bases adenine and guanine (purines), and thymine, cytosine and uracil (pyrimidines), which are the “letters” of the genetic code.

That RNA can act as a template and also now exhibits catalytic activity fuelled hypotheses for the evolution of an “RNA world”.34 In this scenario RNA is the primary polymer of life that replicates itself. DNA and proteins were later refinements. So the first genes were short strands of RNA that reproduced themselves, perhaps on clay surfaces. This conjecture is strengthened by the fact that in cells today there are segments of some eukaryotic pre-rRNAs which can cleave themselves off and join the two cut ends together to reform the mature rRNA. In 1982 Thomas Cech and his colleagues at the University of Colorado discovered this can take place in the absence of protein in the ciliated protozoan Tetrahymena thermophila.35 Just as remarkable are the small nuclear RNAs (snRNAs), which complex with protein to form small nuclear ribonucleoproteins (snRNPs; pronounced “snurps”). Particles called spliceosomes convert pre-mRNA to mRNA.36 Other ribozymes include the hammerhead variety and RNAse P, which generates the 5'ends of tRNAs. The former are found in certain plant viruses. Origin-of-life theories see prebiotic significance in these ‘vestigial’ post-translational mechanisms.
Though attractive, there are several serious objections to the notion that life began with RNA:


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[*]Pentose sugars, constituents of RNA and DNA, can be synthesised in the formose reaction, given the presence of formaldehyde (HCHO). The products are a melange of sugars of various carbon lengths which are optically left- and right-handed (d and l). With few exceptions sugars found in biological systems are of the d type; for instance, β-d-ribose of RNA, which is always produced in small quantities abiotically.


[*]Hydrocyanic acid (HCN) undergoes polymerisation to form diaminomaleonitrile which is on the pathway to producing adenine, hypoxanthine, guanine, xanthine and diaminopurine. These are purines: there is difficulty in producing pyrimidines (cytosine, thymine and uracil) in comparable quantities37,38 (see Figure 3).


[*]Neither preformed purines nor pyrimidines have been successfully linked to ribose by organic chemists. An attempt to make purine nucleosides resulted in a “dizzying array of related compounds”.39 This is expected if sugars and bases were randomly coupled. The prebiotic production of numerous isomers and closely related molecules hinders the likelihood of forming desirable mononucleosides. Furthermore, unless ribose and the purine bases form nucleosides rapidly they would be degraded quite quickly.


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Purine and Pyrimidine Nucleotide Biosynthesis

Purine ribonucleotides (for example, AMP, GMP) are synthesised from scratch by living systems in ways not remotely connected with the laboratory models. The purine ring system is built up stepwise from an intermediate 5'-phosphoribosyl-1-pyrophosphate (PRPP) to a larger molecule inosine monophosphate (IMP). This involves a pathway comprising 11 reactions.
The biosynthesis of pyrimidines is less complex, but again the process is elegantly dissimilar to the in vitro chemistry, with some of the enzymes on the pathway exercising regulatory functions.
The purine and pyrimidine biosynthetic pathways are finely tuned, and defects such as enzyme deficiencies, their mutant forms or loss of feedback inhibition, cause diseases in man.
Suppose that we already have mononucleosides—purines (or pyrimidines) linked to ribose. Heating these in a mixture of urea, ammonium chloride and hydrated calcium phosphate has been shown to produce mono-, di- and cyclic phosphates of the mononucleoside. The subsequent chemistry would yield a rich (or untidy, depending on how it is viewed) racemic mixture of d- and l-oligonucleotides in all sorts of combinations and permutations. Internal cyclisation reactions would destroy much of these oligonucleotides.40
Suppose further that we have a parent strand of RNA in a chirally-mixed pool of activated monoribonucleotides. By base-pairing, the strand correctly aligns on itself the incoming monomeric units in matching sequence. Phosphodiester bonds are spontaneously forged. The chief obstacles to efficient and faithful copying appear to be threefold.41
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[*]d-mononucleotides and l-mononucleotides hinder each other’s polymerisation on an RNA template.


[*]Short chains of nucleotides tend to fold back on themselves to form double helical Watson-Crick segments.


[*]Newly formed strands separate with difficulty from their parent RNA strands. The process grinds to a halt.


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Using activated monomers—both nucleotides and amino acids—Ferris and his co-workers could form oligomers up to 55 monomers long on mineral surfaces. Such surfaces bind monomers of one charge (negative in these experiments) and strength of binding increases with chain length. Desorption then becomes impossible.42
Joyce sums up the difficulties of conjuring up a hypothetical RNA world in these words.

“The most reasonable interpretation is that life did not start with RNA … The transition to an RNA world, like the origins of life in general, is fraught with uncertainty and is plagued by a lack of relevant experimental data. Researchers into the origins of life have grown accustomed to the level of frustration in these problems … It is time to go beyond talking about an RNA world and begin to put the evolution of RNA in the context of the chemistry that came before it and the biology that followed.”43

These sentiments are shared by Orgel, a long-time, well-known prebiotic chemist. In 1994 he wrote:

“The precise events giving rise to the RNA world remain unclear. As we have seen, investigators have proposed many hypotheses, but evidence in favour of each of them is fragmentary at best. The full details of how the RNA world, and life, emerged may not be revealed in the near future.”44

As we have seen, the intuition that an RNA world preceded DNA and protein is based on some features found in modern cells. But it appears to be contradicted by the available experimental evidence. In fact, the extra hydroxyl of ribose renders it more reactive than deoxyribose and, in principle, makes the more stable DNA a more likely progenitor.

Key points



  • The presumed rise of oxygen levels in a primitive reducing atmosphere formerly attributed to the evolution of photosynthesis can be explained by oxygen-independent biological iron oxidation.

  • Recent investigations indicate that the Earth’s atmosphere was never as reducing as previously thought.

  • Recent discovery of fossil stromatolites and algae from the Precambrian has reduced the time for evolution of the first cell ten-fold.

  • The atmosphere of 3.5 billion years ago could have contained significant quantities of oxygen.

  • Under oxidising conditions, the formation of organic compounds and their polymerisation do not occur.

  • Biological homochirality of sugars and amino acids remains an enigma.

  • Hypotheses of ribonucleic acids (RNAs) as the initial self-replicating molecule have serious unresolved difficulties.

  • Extrapolating results of in vitro synthesis of purines and pyrimidines should take into account that biosynthesis utilises different reaction pathways.Other Options

Attention switched to other molecules that can carry information and replicate themselves. In 1991 a team of Danish chemists led by Egholm strung the four familiar bases of nucleic acids along a peptide (polyamide) backbone forming a peptide nucleic acid (PNA).45,46 Unfortunately, PNAs bind natural DNA and RNA tightly (about 50 to 100 times stronger than the natural polymers bind among themselves) so that it is difficult to envisage their being a prebiotic replicating system. So strong is their affinity for DNA that they would disrupt nucleotide duplexes unless they were removed from an evolving RNA milieu. Their base-specificity for natural nucleic acids of oligomers of 10 units or more, and consequently their fidelity in copying RNA or DNA, is uncertain. This militates against the co-evolution of multiple genetic systems, a suggestion raised by Böhler and his coworkers.47 Using an unusual activated monomer, guanosine 5′-phosphoro (2-methyl) imidazolide, they formed 3'-5'-linked oligomers with PNA as template. In fact, because of problems of cyclisation the activated dimer rather than the monomer was used. No oligomers of more than 10 were formed, and there was present in the complex mixture short oligomers with unnatural 2'-5'-phosphodiester bonds, pyrophosphate linked oligomers and possibly cyclic oligomers.


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16The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Mon Jan 30, 2017 9:59 am

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The RNA world, and the origins of life Orgel_10

Earth life 'may have come from Mars

Scientists have long wondered how atoms first came together to make up the three crucial molecular components of living organisms: RNA, DNA and proteins. The molecules that combined to form genetic material are far more complex than the primordial "pre-biotic" soup of organic (carbon-based) chemicals thought to have existed on the Earth more than three billion years ago, and RNA (ribonucleic acid) is thought to have been the first of them to appear. Simply adding energy such as heat or light to the more basic organic molecules in the "soup" does not generate RNA. Instead, it generates tar. RNA needs to be coaxed into shape by "templating" atoms at the crystalline surfaces of minerals. The minerals most effective at templating RNA would have dissolved in the oceans of the early Earth, but would have been more abundant on Mars, according to Prof Benner. 1


1. http://www.bbc.com/news/science-environment-23872765

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17The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Sat Jul 01, 2017 4:00 pm

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Andrew Catherall

Some thoughts on the evidence for the RNA World hypothesis, following on from Frazer Blaxland's recent post. Much of this is taken, some paraphrased and some verbatim, from Higgs and Lehman's recent review which I have linked at the bottom. Text in square brackets are my additions.
Intro: "The RNA World is the conceptual idea that there was a
period in the early history of life on Earth when RNA,
or something chemically very similar, carried out most
of the information processing and metabolic [energy-producing biochemical reactions] transformations
needed for biology to emerge from chemistry.
This scenario, if it indeed existed, took place some 4 billion
years ago. By contrast, the realization that RNA is a
good candidate for the emergence of life is an idea that is
only ~50 years old. It was recognized early on by [Francis] Crick, [Leslie] Orgel and others that RNA has both a genotype and a phenotype, and that a system based on RNA would be a plausible precursor to the much more complex system of DNA–RNA–proteins on which current life is based. It was also realized that the ribonucleotide coenzymes now
used by many proteins may be molecular ‘fossils’ from an
RNA-based metabolism. Discoveries of naturally occurring
ribozyme [RNA-enzyme] catalysts, such as self-splicing introns [introns are non-coding sequences in genes that must be removed prior to translation] and the ribonuclease P catalyst, were made in the 1980s and, with the demonstration that ribosomal RNA catalyses peptide bond formation in the ribosome [the structure that captures mRNAs and uses the RNA code as a template to produce a peptide chain], the credentials of RNA as a catalyst became firmly established."
Evidence for RNA world hypothesis (this list is, as always,a simplification and by no means exhausative):
* RNA is capable of indepednent (auto)catalysis, self-replication and information storage, the three key features any early living system would require. Proteins can catalyze reactions but cannot self-replicate or transmit genetic information, DNA can transmit genetic information but cannot catalyze reactions.
* Catalysis (ribozyme activity): a stream of experimental support has arisen for the ability of RNA alone to act as a catalyst, ever since the first discovery that in the ciliate protozoan Tetrahymena: it was shown that splicing (removal of an intron sequence) of an RNA can occur autocatalytically.
* In what many consider the 'smoking gun', it was then shown that protein synthesis in the ribosome itself depends on RNA activity (ribozyme activity) - Robertson: "The active site for peptide-bond formation lies deep within a central core of RNA, whereas proteins decorate the outside of this RNA core and insert narrow fingers into it. No amino acid side chain comes within 18 Å of the active site . Clearly, the ribosome is a ribozyme , and it is hard to avoid the conclusion that, as suggested by Crick, “the primitive ribosome could have been made entirely of RNA” ". RNAs are also part of the spliceosome, there are many self-splicing introns that use RNA interemediates, the guide RNAs in Trypanosomes, RNAs provide the template for telomerases etc etc.
* Self-replication: (Howe lecture notes) : RNA molecules can also catalyse the replication of other RNA molecules without the requirement for protein. RNA replicase described by Johnston et al (RNA catalyzed RNA polymerization: accurate and general RNA-templated primer extension (2001) Science 292:1319-1325) 1088/1100 sequenced 11-nucleotide extension products were found to be accurate. The average accuracy was estimated to be around 96%. That RNA should have catalytic activity is not that surprising, really. It can fold to form quite elaborate structures, with a variety of potentially active groups
* Experimental evolution supports the idea that initially rudimentary RNA catalytic abilites can improve over time in response to selection - "The Holliger group had achieved notable success in this regard by engineering and selecting mutations in polymerase ribozymes that could catalyse the template directed polymerization of an RNA chain of roughly half of its own length....[they also showed under certain conditions that] they were able to achieve a substantial improvement in activity and showed, for the first time, that RNA could replicate strands of their own length (206 nucleotides in this case) or above".
*Mononucleotides, the building blocks of RNA, can self-polymerize up to 20 nucleotides long on clay! This has given rise to the idea of autocatalytic sets, which also has experimental support, but I won't go into it here.
* Lots of recent mathematical theory highlighted the importance of cooperation between early replicators, support for this emprically has been demonstrated by the fact that RNAs will 'cooperate' - e.g. Vaidya 2012 - "Here we show that mixtures of RNA fragments that self-assemble into self-replicating ribozymes spontaneously form cooperative catalytic cycles and networks. We find that a specific three-membered network has highly cooperative growth dynamics. When such cooperative networks are competed directly against selfish autocatalytic cycles, the former grow faster, indicating an intrinsic ability of RNA populations to evolve greater complexity through cooperation. We can observe the evolvability of networks through in vitro selection."
* The antiquity of the role of RNA in protein synthesis (i.e. RNA before protein is supported by paleogenetic experiments that resurrected components of 3-billion-year-old translation systems for study in the laboratory (see Gaucher 2003)! In modern organisms, DNA nucleotides are produced from RNA nucleotides, again supporting the idea that RNA preceeded DNA. Indeed the evolution of DNA from RNA simply required the reduction of a 2'OH.
* Other things that would make this post too long
How did prebiotic synthesis of RNA occur?
* There very well could have been prior precusors to RNA (see linked review). But, many origin of life chemists have shown that RNA nucleotides can form in plausibly prebiotic conditions I won't go into the chemistry in a facebook post, but those interested could look at this review,

http://link.springer.com.ololo.sci-hub.cc/article/10.1007%2Fs11084-017-9537-2

E.g. see Sutherland's recent demonstration that all 3 precursors of ribonucleotides, amino acids and lipids can all be derived by the reductive homologation of hydrogen cyanide in plausibly prebiotic conditions!

A good review - http://www.nature.com.ololo.sci-hub.cc/nrg/journal/v16/n1/abs/nrg3841.html

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Scientists prove plausibility of new pathway to life's chemical building blocks 1

For decades, chemists considered a chemical pathway known as the formose reaction the only route for producing sugars essential for life to begin, but more recent research has called into question the plausibility of such thinking. Now a group from The Scripps Research Institute has proven an alternative pathway to those sugars called the glyoxylate scenario, which may push the field of pre-life chemistry past the formose reaction hurdle.

1. https://www.sciencedaily.com/releases/2012/01/120131175629.htm

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19The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Sat Dec 30, 2017 4:55 am

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A fundamental problem

An RNA molecule, perhaps acting as a ribozyme, must be copied into its reverse complement and then recopied again to produce an identical (and functional) copy of the original molecule. Whatever the nature of the replicating mechanism, this requires the wasteful creation of an intermediate reverse complement (unless all the sequences are palindromes) and also a doubling of the error rate as there must be two copying steps. The former problem might be alleviated if the reverse complement is also a different functional ribozyme.

There is also a more fundamental problem associated with this replication mechanism, that a single replication step is unlikely to occur in isolation. This means that there will be copies of each molecule and its reverse complement in the same space at the same time. In the modern world, this does not cause any difficulty, as the nucleic acid is either double stranded or protected from hybridization by single-strand-binding proteins. Even if the single-stranded molecule quickly adopted a secondary and a tertiary structure, it must still remain sufficiently accessible to be unfolded and copied by the replicase. This degree of accessibility would also allow some hybridization with its complement and once completely base paired, the resulting duplex would have a lower energy than any internal bonding structure (Bartel 1999; Joyce & Orgel 1999). The duplex would therefore act as a low-energy sink, removing functional molecules from the population.

In a more primitive world, it is difficult to imagine a mechanism that would keep the template strand and its reverse complementary transcript strand apart. The physical screening of the replicase itself, whatever its nature, would provide some protection for the current template from its transcript, but this is only a local solution as an adjacent replicase could easily be making a complementary strand. Some physical separation such as a membrane might be imagined, but would require an unrealistic degree of synchronization to keep all complementary copies apart. As in the modern world, some single-stranded binding mechanism might be imagined, based on either random oligonucleotides or peptides. While the latter is not impossible, a solution is proposed later that also opens some further possibilities.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1664686/

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20The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Fri Oct 26, 2018 12:59 pm

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THE RNA WORLD,  AND THE ORIGINS OF LIFE

Leslie Orgel at the University of Oxford, UK, was among the first to propose RNA as a catalyst of the chemical reactions to make itself. A new theory was born, later dubbed the ‘RNA world hypothesis’. 

The RNA world hypothesis, to be true, however, has to overcome  major hurdles:

1. Life uses only right-handed RNA and DNA. The homochirality problem is unsolved. This is an “intractable problem” for chemical evolution
2. RNA has been called a “prebiotic chemist's nightmare” because of its combination of large size, carbohydrate building blocks, bonds that are thermodynamically unstable in water, and overall intrinsic instability. Many bonds in RNA are thermodynamically unstable with respect to hydrolysis in water, creating a “water problem”. Finally, some bonds in RNA appear to be “impossible” to form under any conditions considered plausible for early Earth.   In chemistry, when free energy is applied to organic matter without Darwinian evolution, the matter devolves to become more and more “asphaltic”, as the atoms in the mixture are rearranged to give ever more molecular species. In the resulting “asphaltization”, what was life comes to display fewer and fewer characteristics of life.
3. Systems of interconnected software and hardware like in the cell are irreducibly complex and interdependent. There is no reason for information processing machinery to exist without the software and vice versa.
4. A certain minimum level of complexity is required to make self-replication possible at all; high-fidelity replication requires additional functionalities that need even more information to be encoded
5.  RNA catalysts would have had to copy multiple sets of RNA blueprints nearly as accurately as do modern-day enzymes
6.  In order a molecule to be a self-replicator, it has to be a homopolymer, of which the backbone must have the same repetitive units; they must be identical. On the prebiotic world, the generation of a homopolymer was however impossible.
7. Not one self-replicating RNA has emerged to date from quadrillions (10^24) of artificially synthesized, random RNA sequences.  
8. Over time, organic molecules break apart as fast as they form
9. How could and would random events attach a phosphate group to the right position of a ribose molecule to provide the necessary chemical activity? And how would non-guided random events be able to attach the nucleic bases to the ribose?  The coupling of a ribose with a nucleotide is the first step to form RNA, and even those engrossed in prebiotic research have difficulty envisioning that process, especially for purines and pyrimidines.”
10.  L. E. Orgel:  The myth of a self-replicating RNA molecule that arose de novo from a soup of random polynucleotides. Not only is such a notion unrealistic in light of our current understanding of prebiotic chemistry, but it should strain the credulity of even an optimist's view of RNA's catalytic potential.
11. Macromolecules do not spontaneously combine to form macromolecules
125. The transition from RNA to DNA is an unsolved problem. 
13. To go from a self-replicating RNA molecule to a self-replicating cell is like to go from a house building block to a fully build house. 
14. If two amino acids are located within the peptidyl transferase center, they will easily form a peptide bond. But as soon as you do that in the absence of the ribosome, the ends of the amino acids come together, forming a cyclic structure. Polymers cannot form. But if the ends are kept apart, by a theoretical primitive ribosome, a chain of peptide bonds could grow into a polymer. 
15. Arguably one of the most outstanding problems in understanding the progress of early life is the transition from the RNA world to the modern protein based world. 
16. It is thought that the boron minerals needed to form RNA from pre-biotic soups were not available on early Earth in sufficient quantity, and the molybdenum minerals were not available in the correct chemical form.
17. Given the apparent limitation of double-stranded RNA (dsRNA) genomes to about 30 kb, together with the complexity of DNA synthesis, it appears dif¢cult for a dsRNA genome to encode all the information required before the transition from an RNA to a DNA genome. Ribonucleotide reductase itself, which synthesises deoxyribonucleotides from ribonucleotides, requires complex protein radical chemistry, and RNA world genomes may have reached their limits of coding capacity well before such complex enzymes had evolved. 

Proponents of the RNA world hypothesis commonly argue that it has been proven that RNA's could self-replicate. Let's suppose that were true, that is as if self-replication could produce a hard drive. To go from a hard drive ( which by itself requires complex information to be assembled, in case of biology, DNA, not RNA since it's too unstable, ) that does not explain the origin of the information to make all life essential parts in the cell.
It is as to go just from a hard drive storage device to a self replicating factory with the ability of self replication of the entire factory once ready, to respond to changing environmental demands and regulate its metabolic pathways, regulate and coordinate all cellular processes, such as molecule and building block biosynthesis according to the cells demands, depending on growth, and other factors.
The ability of uptake of nutrients, to be structured, internally compartmentalized and organized, being able to check replication errors and minimize them, and react to stimuli, and changing environments. That's is, the ability to adapt to the environment is a must right from the beginning. If just ONE single protein or enzyme - of many - is missing, no life. If topoisomerase II or helicase are missing - no replication - no perpetuation of life.
Why would a prebiotic soup or hydrothermal vents produce these proteins - if by their own there is no use for them?

Shapiro, 1986, p.186.
"`In other words,' I said, `if you want to create life, on top of the challenge of somehow generating the cellular  components out of non-living chemicals, you would have an even bigger problem in trying to it the ingredients
together in the right way.' `Exactly! ... So even if you could accomplish the thousands of steps between the amino acids in the Miller tar-which probably didn't exist in the real world anyway-and the components you need for a
living cell-all the enzymes, the DNA, and so forth-you's still immeasurably far from life. ... the problem of  assembling the right parts in the right way at the right time and at the right place, while keeping out the wrong
material, is simply insurmountable.'"


Paul Davies The Algorithmic Origins of Life
Despite the conceptual elegance of the RNA world, the hypothesis faces problems, primarily due to the immense challenge of synthesizing RNA nucleotides under plausible prebiotic conditions and the susceptibility of RNA oligomers to degradation via hydrolysis 21 Due to the organizational structure of systems capable of processing algorithmic (instructional) information, it is not at all clear that a monomolecular system – where a single polymer plays the role of catalyst and informational carrier – is even logically consistent with the organization of information flow in living systems, because there is no possibility of separating information storage from information processing (that being such a distinctive feature of modern life). As such, digital-first systems (as currently posed) represent a rather trivial form of information processing that fails to capture the logical structure of life as we know it. 

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21The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Fri Mar 08, 2019 8:31 am

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A final chapter has recently been opened with the discovery of autocatalytic RNA molecules. 

These were originally received with great excitement by the prebiological evolutionists because they gave hope of alleviating the need to make proteins in the first cell. These so-called "ribozymes" proved incapable of rising to the occasion, however, for not only are the molecules themselves very limited in what they have been shown capable of doing, but the production of the precursors of RNA by any prebiological mechanism considered thus far is a problem at least as difficult as the one ribozymes purport to solve:

1) While ribose can be produced under simulated prebiological conditions via the formose reaction, it is a rare sugar in formaldehyde polymers (the prebiological mechanism believed to have given rise to sugars). In addition the presence of nitrogenous substances such as amino acids in the reaction mixture would prevent sugar synthesis (Shapiro, 1988). Cairns-Smith (1993) has summarized the situation as follows:"Sugars are particularly trying. While it is true that they form from formaldehyde solutions, these solutions have to be far more concentrated than would have been likely in primordial oceans. And the reaction is quite spoilt in practice by just about every possible sugar being made at the same time - and much else besides. Furthermore the conditions that form sugars also go on to destroy them. Sugars quickly make their own special kind of tar - caramel - and they make still more complicated mixtures if amino acids are around."

2) When produced and condensed with a nucleotide base, a mixture of optical isomers results, only one of which is relevant to prebiological studies.

3) Polymerization of nucleotides is inhibited by the incorporation of such an enantiomorph.

4) While only 3'-5' polymers occur in biological systems, 5'-5' and 2'-5' polymers are favored in prebiological type synthetic reactions (Joyce and Orgel, 1993, but see Usher,et. al. for an interesting sidelight).

5) None of the 5 bases present in DNA/RNA are produced during HCN oligomerization in dilute solutions (the prebiological mechanism believed to give rise to nucleotide bases). And many other non-coding bases would compete during polymerization at higher concentrations of HCN.

In addition to the problems of synthesis of the precursors and the polymerization reactions, the whole scheme is dependent on the ability to synthesize an RNA molecule which is capable of making a copy of itself, a feat that so far has eluded strenuous efforts. The molecule must also perform some function vital to initiating life force. So far all of this talk of an "RNA World" remains wishful thinking best categorized as fiction. The most devastating indictment of the scheme however, is that it offers no clue as to how one gets from such a scheme to the DNA-RNA-Protein mechanism of all living cells. The fact that otherwise rational scientists would exhibit such rampant enthusiasm for this scheme so quickly reveals how little faith they have in all other scenarios for the origin of life, including the ones discussed above.

https://origins.swau.edu/papers/life/chadwick/default.html

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22The RNA world, and the origins of life Empty How Long Did It Take for Life to Appear? on Mon Feb 17, 2020 4:48 am

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How Long Did It Take for Life to Appear?

A 100-base long RNA molecule needs to be synthesized at least 100 times faster than the hydrolysis rate of a single phosphodiester bond. Even if highly stable precursors to the ribose phosphate backbone of RNA are proposed for the pre-RNA world, the bases themselves will decompose over long periods of time. For example, cytosine hydrolyses to uracil with a half-life of 300 years at pH 7 and 25°C in single-stranded DNA (Lindahl 1993). Adenine, which is usually thought to be very stable, deaminates to hypoxanthine with a half-life of 204 days at 100°C and pH 7 (Shapiro 1995). This is only about ten times slower than cytosine (t1/2 = 21 days at 100°C and pH 7). Given these stability constraints, there is no reason to assume that the self-organization of prebiotic compounds into a system capable of undergoing Darwinian evolution involved extended periods of time.

https://www.cell.com/cell/fulltext/S0092-8674(00)81263-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867400812635%3Fshowall%3Dtrue

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23The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Mon Mar 23, 2020 10:27 am

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https://www.youtube.com/watch?v=v5HQB3JFIZg

the first speaker is Steven Benner from
the University of Florida at the
foundation for molecular evolution did I
get that right Steve
I came close anyway I happy to have him
talk and go for it
people who introduced me yeah I'm gonna
try to first start by doing what's
necessary to do for an audience that is
not necessarily in the field that I'm
going to talk about which is to say what
I'm going to say first and what I want
to say is the following propositions
first sort of as the guiding theory
proposition that a poly electrolyte with
a uniform structure is a universal for
Darwinism and I haven't seen any of
those cards go up but maybe that's
because they're not out a poly
electrolyte means a molecule with many
of repeating backbone charges so DNA in
your case is a repeating minus minus
minus minus minus backbone charge in
principle on an alien planet you could
have a genetic molecule with a repeating
positive charge in the backbone but the
argument is the proposition is and
that's the guiding theory for this talk
is that
and a poly electrolyte with that uniform
structure will be universal in life and
water no matter where you find it the
second thing is a specific hypothesis to
provide context is that we assume in
this talk that the poly electrolyte has
supported the first Darwinism on earth
was actually ribonucleic acid RNA will
have a lot of talking about that the
next thing we're going to do is we're
going to structure problem selection and
this is a very important question right
this is a huge field and people just
wander through it saying that they're
working on the origin of life when
actually what they're doing is having
fun with something that they struck
their fancy and perhaps even if all the
work goes exactly as planned everything
turns out well they won't have actually
learned anything about the origins of
life so what we do here is try to come
up with things like the proposition that
settled science that says that RNA is
impossible to form free biotic li and if
you're guarding theory is this poly
electrolyte theory and your guiding
hypothesis
is that RNA was first if settled science
says that RNA is impossible to form
prebiotics you have a paradox you've got
to address that and that focus here's
your research on specific problems that
leaves all the other stuff aside and not
relevant so then we go back to
strategies for paradox resolution you'll
discover that in this field settled
science for a longest time has not
considered the mineral environment of
the processes that might allow the form
RNA to form and in fact we wave
paradoxes in the real world really our
result is they're not really paradoxes
they just you've just forgotten to say
something and your line of assumptions
or your line of premises and whatever
syllogism you think is true and mineral
guided processes actually are ways
around this paradox and we'll talk a
little bit about that the next thing I
want to tell you about is a natural
history contact seal turns out that the
needed chemistry mineral combination was
very short-lived was very transient on
earth and if you stick through this with
me your reward and this is always
important right this is without the
reward your reward will be a relatively
simple fact the form RNA probiotic Li a
relatively narrow date when RNA when
life on Earth originated prebiotic Li
and a relatively clear statement about
the next rounds of paradoxes that need
to be solved to bring this forward and
so these are the people who I will cite
frequently throughout the talk as well
as people who have been cited on
publications and we're especially
indebted to people like ELISA Biondi and
huge Indian Daniel cooter in my
laboratory who have been doing a lot of
the chemistry but a lot of input from
Steve Moises and Ramon grosser and
Dustin trail who's here and Kevin
Donnelly and David Catlin have been to
some extent our adversaries released
critics of the process and Rob Levinsky
is my mineral dealer and so I have also
bought a lot of rocks so I probably
won't pass them around because they are
it's just too large for that to be
working well all right now you're ready
this is gonna be a long morning
great let's talk about the guiding
theory that these polyelectrolytes with
a uniform structure are universal for
Darwinism and for those of you who
actually know the structures of DNA and
actually were forced to memorize it
there's a piece of DNA and the repeating
linking groups or phosphates and they
carry a natural negative charge on them
at neutral pH and that's what we mean by
a poly electrolyte it's a repeating
structure with minus minus minus minus
might not it's always surprising to
people when they realize that in your
DNA you form a double helix where a
backbone - - - - - - - another backbone
- - - - - and the students always say at
some point they realize oh wait a minute
Coulomb's law says the plusses attract
minuses so what are we doing and
trusting our genetic information to a
Pollyanna I'm bonding to another
polyatomic ion that seems to be a
repulsive interaction and indeed it is
but it actually turns out it is
responsible for Watson Crick pairing
rules because when these two strands get
together the repeating backbone negative
charge on one strand repels the backbone
from the other strand forces the
interaction between those two strands as
far away from the backbone as possible
and that's where we get these rules that
you learn in eighth grade where a pairs
with T and G pairs with C if you make an
analogue of this molecule and we've done
so without them repeating negative
charges the molecule doesn't have those
rules at all
I mean further the backbone negative
charges causes this molecule to not fold
right because by extending itself you
bring those backbone charges far away
let me put another picture up right so
it's this backbone backbone repulsion
that forces the base base interactions
as far away from the backbone as
possible and it also forces the Strand
to unfold and that's needed for
templating that's how you get another
molecule of DNA where R and I for that
matter it synthesized on a poly
electrolyte backbone and so this means
that for physical reasons just repeating
negative charge in the backbone here
represented by those Peas with a little
negative charge on them
quite as stupid as it might seem from
first glance but it's more important
than that experiments support this and I
know I'm just going to throw up on the
structure of this slide variants of this
molecule that have been made in our
laboratory and other people's
laboratories without repeating charges
and these things say oh now you could
say Steven if you just have been a
little bit more clever and made one of
the more interesting or correct
molecules that would have worked without
repeating backbone charge but at some
point you know you stopped making these
molecules in part because the funding
agency doesn't tolerate it anymore but
this is an empirical basis for what I've
told you a moment ago which is really
just for charge Coulomb theory and this
is how we know that that's this general
rule is likely to be universal now
there's another reason for this and this
is actually perhaps even the more
important thing that's because Darwinism
is equated to life and life is equated
to Darwinism itself a fundamental level
not the theory right
we believe that Darwinism that is this
process of reproduction with errors
where the errors themselves are
reproducible it's the only way in the
universe for allowing organic matter to
self-organize to give properties that we
value in life and that means that even
if you get on the enterprise and go to a
distant planet you know you're going to
find organisms that are organized in
their chemistry and they got there by
this process of random variation non
prospective random variation with errors
or the errors themselves are replicable
and that's actually a difficult thing to
get and so I just put up here some
well-known things in chemistry which may
not be well known to you but really you
have to be able to change the sequence
of your
of the basis of RNA AGC and you actually
it's constrained now to very short
period time not even a million years
it's a half a million years so this plus
minus is a statement about the physics
of the atmosphere returning to an
unproductive state from a very transient
time when the atmosphere was productive
for forming the basis in fact the
biggest source of uncertainty is the
availability of dry land which we
discussed yesterday from June cor Naga I
have by the way taking this out of corn
August paper with roses
I have reversed the timescale so Kevin
likes it from old-time to modern time
and of course there is this very nice
plot which gives us you know a
reasonable amount of the least crust as
1.0 not 0.1 because we flipped it
remember of course the amount of crust
is not the amount of dry land and but a
mere hand reducing the oceans with
impact your iron to hydrogen die
hydrogen will certainly dry some land
out and the question is only what the
temperature is at that dry land under
whatever atmosphere is doing whatever to
the greenhouse effect so there you go so
what I didn't done today is basically
told you the guiding theory
polyelectrolytes are important we've
told you that we are hypothesizing an
RNA was the poly electrolyte that formed
the first Darwinism on earth we focused
on settled science paradoxes that claim
that RNA could not have formed we've
given you mineral based solutions for
these things and by the way just for the
record I brought with me some prebiotic
soup so if you would like to know what
was almost certainly on the surface of
any dry land that June can give us all
right there it is and you can do
whatever you want with it we even have a
little bit of the nickel and from the
meteorite impact which you can actually
take back to the lab and make RNA out of
um you need a natural history contacts I
giving you one scenario which is
involving impact two very similar to
what Kevin told you about and now it's
time to collect your reward a relatively
simple path to form RNA prebiotic Li a
relatively narrow date when life on
earth originated prebiotic Li and a
clear statement of the next round of
paradoxes and for that I mean I just
mentioned a few of them we don't know
enough about this borate controlled
carbohydrate process and we don't quite
we need to know more about that we
actually need to know more about the
availability of heavy and boring I'm
hoping that Dustin will tell me about
that we've not said a word about
chirality here and that's a big problem
and there's a range of paradoxes it also
arise from the poverty of RNA as a
catalyst it's actually not a very good
catalyst and when there is a catalyst it
tends to destroy RNA more than that
makes RNA but that's a detail so there's
what you said but let me just make a
comment about you know
multidisciplinarity I greatly love this
what we've done here is we've given you
one of my favorite slides I've been
using this now for years um oops I don't
want to do that let's go back we've used
information from natural history to talk
about why we are looking for RNA it has
to do with the fact that the RNA
ribosome machine is RNA we've done a lot
of synthetic biology to try to explain
how this works
and to confirm theories by doing
synthesis we focused on this and we've
used the synthesis to also help go out
and try an alien life form you know in
my career what's been striking to me is
how many times areas that were
considered to be separate disciplines
have merged into a single discipline and
it's not by getting conferences together
where someone who's an expert in geology
talks to somebody's an expert in
chemistry it's by having students
trained so that they move freely from
biology the chemistry to geology and to
various other things and that is of
course your challenge for the students
in the audience so let me stop there
thank you for your attention I'd be
happy to answer any questions
[Applause]
yeah excellent question and the answer
to that is yeah so the question is
whether we can now explain why we use
GAC and T and not some of these other
ones that we've made and you gotta
remember people are easy to fool and the
easiest person to fool is yourself so I
can come up with an explanation for that
and we about volumes we've written lots
and lots of papers based on the
experimental work with alternative
systems but you got to remember when you
ask an organic chemist to take an
organic chemistry course you woke up to
the professor afterwards and say hey why
is this this way and the professor will
give you an answer now you say oh no no
it's exactly the opposite and the
professor will give you just as
convincing an answer if it's the
opposite and that's how you know that
Organic Chemistry is a science so it
would be better so we have a great paper
from Earth's last mari who is actually
went out and said yep four is the
optimum number of pairs and he had an
argument for it would've been much more
comfortable for me if he did not know
before he made that argument that the
natural stuff had four bases so the
answer is yes we've got lots and lots of
explanations but this is a just so
problem you have to worry about whether
we can come up with something
independent to justify our favourite
theories yeah well to get something that
a chemistry flask a material that has to
get out of the flask go out into the you
know down to Lake Avenue and buy itself
some food right that's what you're
talking about here or are the
photosynthesize or to do any of these
other things that get you the food the
resources we in the laboratory are
feeding this nice stuff so you're all of
a sudden talking about
compartmentalization
we don't know how to do come
part mentalization you're having talked
about sensing I had to move in the
gradient of a food photosynthesis
where's camp oh there you are mean well
I mean I mean Blankenship's I mean
you're gonna hear all sorts of these are
hard problems and if I knew how to do
photosynthesis with RNA catalysis I
would be I don't know what I would be
doing I wouldn't be maybe standing yeah
so there are lots and lots of like going
from just a self of a system that can
create copies of itself with errors
where the errors of themselves are
replicable is a long way away from being
self-sustaining yeah I'm in the back
no yes sure why not
are there other people yeah I know Phil
Hager is the better person here than I
am so we cheat we take natural DNA
polymerases but guess what the natural
DNA polymerase is have evolved four four
letters they don't take eight letter
anything so so we had to the mutai
tee-ball we do a lot like what Francis
Arnold who is here at Caltech this you
we do directed evolution on these DNA
polymerases now Phil is a guy who really
is doing a good job of this he is
actually getting the RNA molecule to
itself be the enzyme so he does not have
to go to sigma-aldrich kromega or any of
these other companies and bringing a
polymerase it doesn't want to work
anyhow I have to mutate it so clearing
this cycle getting this replicates going
where the RNA molecule is itself
catalyzing the replication that is Phil
Hager and he is somebody who should have
invited to this conference instead of me

http://elshamah.heavenforum.com

24The RNA world, and the origins of life Empty Re: The RNA world, and the origins of life on Mon Mar 23, 2020 10:35 am

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https://www.youtube.com/watch?v=8RZsWVBQzOk

good morning everyone welcome to a nai
director seminar I just heard an open
mic which i'm pretty sure wasn't Steve
so you might just want to check your
mics I am very very pleased that Steve
Benner is here to give the NAI director
seminar this morning or this afternoon
depending on where you are Steve's group
at the foundation for applied molecular
evolution which is a foundation that he
began is addressing some of the most
profound questions in understanding our
nature of life and in particular the
question that Steve's group is
addressing is what's required of life in
that it couldn't have been any other way
and what's contingent and that it could
have been lots of other ways and Steve
as a chemist is approaching this through
synthetic biology which is a field which
steve has and his colleagues have been a
pioneer in developing in particular what
they have done is to use organic
chemical synthesis to prepare artificial
genetic systems and then actually
operate those systems to produce
proteins that have amino acids other
than the amino acids that are normally
used by proteins and they're applying
these in a wide variety of areas
including dressing haha y ok Steve
including addressing the big questions
and also in areas such as personalized
medicine where they are working on
developing ways of treating individuals
based on the individual's own genetic
makeup so we are really really
privileged to have Steve with us today
to give the talk steve has both a
bachelor's and a master's in molecular
biophysics and biochemistry from Yale
and a PhD in chemistry from Harvard and
he will be speaking with us today on for
general approaches I forget the exact
title here I know I have it for general
approaches to the nature of life is
probably close enough Steve I will just
turn it over to you ok thanks very much
Karl Hoffmann feel free I'm not sure how
these conferencing systems work but if
you have an opportunity to interrupt me
and ask the question as we go through
this talk will feel free to do so well I
guess you are looking at my first slide
what is like would that be correct is
that some place in front you yes it is
okay excellent and this is of course is
a question that is both provocative and
central is the part of the problem just
so that I now have advanced the slides
the next one correct all it's working
okay excellent so yeah keep in mind that
about five years ago John Burris and I
about John bearish persuaded me to join
this committee that was put together by
the National Research Council to talk
about the limits of organic life and
planetary systems was actually
commissioned by NASA and the National
Research Council put various people
together some of whom I guess are in
attendance here and you can see your
names listed one of the things that the
committee decided not to do at the
outset was to decide not to try to
define life I thought that was a bit
cowardly myself because if you're going
to argue about the limits of life you at
least have to have some criteria for
what a definition of life would be but
then Carol Cleland got involved in the
organization periphery and her point of
course was it in order to define life
you actually have to have a theory of
light and in fact if you talk to any of
these philosophers long enough you can
see the comment which i think is on the
bottom of the of the first slide which
is that please go make sure i have this
zoom to fit on my screen which is
actually when you talk to a philosopher
long enough they will persuade you that
you don't even know how to define
definition and you don't even know how
to define water and that's one of the
things that philosopher has i've done to
us so very much what you talk about is
very much connected to what you think is
possible and what you might think
actually exists and that of course is
himself as driven by theory and the
theory
may or may not be complete in fact we
know from the history of science that
many times with the theories that we
thought were quite reliable on which we
were making constructive actions turned
out to be not true at all so one of the
things that we would like to do is not
have a definition that is just a laundry
list of the attributes of known living
systems and I pulled the list here out
of a textbook you know the ability to
reproduce the ability to utilize energy
that you excrete waste that you move or
you have the ability to respond to
external stimuli as all a laundry list
of attributes it's not really much of a
definition um that's not because biology
doesn't have definitions and some of
them are now listed on this particular
slide so many of you for example
especially if you're coming from the
background of cell biology know about
the cell theory of life there's
obviously the evolutionary theory of
life their various information the
reason life and there's various
molecular theories of life and these
have operated over the last several
hundred years certainly the cell theory
of life has emerged after the microscope
came along and therefore was able to
identify a whole world of cellular life
that was not visible to the naked eye
well so so like theory of life is you
know certainly one that's quite popular
it's actually used for example when
people look for life in the cosmos
certainly NASA missions so Eve this is
actually a older slide this is actually
from book who is looking at cork cells
and in fact he is the individual who
came up with the word cell in English
language because he was looking at what
looked to him like small rooms or
chambers mostly implants schwann of
course and schleiden were the people who
actually looked at the unification of
life in 1847 exit was in 1840 this this
is actually an english translation of
their book which you can actually
download from the history of science
foundation in berlin and it's in just in
case you think that we put in the modern
world a gloss on what ancient people
were thinking 150 years ago it's
actually quite clear that these folks
were actually trying to unify life and a
theoretical sense in fact their whole
introduction to this
it's a discussion about how deplorable
it is that people who study plants and
people who said the animals don't talk
to each other and what he's going to use
is try to use the cell structure of both
of these as a way of trying to unify
life in some sort of theory now the fact
that this is driving and our idea of
what constitutes life at a very
fundamental level is the fact that a
couple months ago proto light announced
its plans this was last August to create
artificial life in three to five years
this caused quite a flurry in the press
not the least of which was because
people thought it was dangerous but what
did they consider will they put this
picture out there's a cell structure and
indeed when we go look for life in the
cosmos we also look for cell structures
very frequently and this is of course on
the right the famous picture from the
island hills meteorite which is looking
at structures it looks sort of cell like
him from that a large amount of
speculation was started as to whether
there might have been life that formed
those so the cell theory of life is fine
but really the as the debate over the
island Hills meteorite illustrates cell
like structures are really not
definitive biosignatures and they can be
generated by many non light living
processes as well well I'm not going to
dwell on the second theory it's one of
my favorites sort of the evolutionary
theory of life this is the animal room
at the natural history museum in Paris
where you can see people trying to
classify all forms of life that they
could find on earth upstairs from this
room you can actually find the original
fossils that Cuvier was actually looking
at when he came up with his first
concepts of evolution of course Lamarck
statue is out in front of this guy
what's quite clear is that the
evolutionary theory of life is
classically defined has really not been
as successful as it could be in
particular it really did not identify
the central fact that life on Earth is
monophyletic so it doesn't actually
transparently make obvious that
microbial life and macroscopic light
were actually related by common ancestor
even though they have cells because of
course at some point he decided life as
a natural state and that cells are
necessary for
I hear force of this conclusion that you
have plants based on cells and animals
based themselves it could just as easily
be the fact that those arose
independently by conversions rather than
by common ancestry and so it really
turned to molecular theories of life to
come up with the essential understanding
of monophyletic system you're looking
just at three structures the structure
in the middle upper part of the slide of
urea this is the compound that was made
in 1828 by bowler which is what
discarded anti-white to listen or
discarded vitalism as a theory of light
the metabolism in the lower left of
people are arguing that this is
essential feature of life and in fact if
you go through the laundry lists if you
learn in high school biology about what
like this the ability to take in energy
and just secrete waste is all part of
the metabolism definition tab metabolism
the concept of life then of course in
the right isn't one of my favorite
theories of life which is the so-called
gene theory of life which is the
argument that in order to have life you
have to have a molecule that contains
information that can be passed from
generation to generation perhaps with
some errors that allow the first steps
of a Darwinian evolution to proceed so
that's cool the question today and it's
a little ambitious but the question is
whether we can bring these together I
actually gave a talk on the top wedge of
this four-part diagram a couple days ago
at the nest apps icon so I won't go into
great detail of this but one of our
goals is to whether we can take the
bottom wedge which is really the Natural
History view may be supported by
molecular evolution to work backwards in
time so that we can go to a simpler form
of life now the theory here is really
very very very krups naive but that is
that if you go backwards in time to a
simpler form of life what will remain as
you examine more and more ancient life
is is life that is more essential that
is a simpler form of life is more
reflective of what is necessary for life
in general and that more derived forms
of life have all the baggage of history
super impose upon what is essential for
light and therefore make it
typical are more difficult to see what
is the Center for life so one of the
ideas that maybe is we can look at the
tree at the bottom of the four-part
diagram you can see how you can look at
the three kingdoms and maybe in for
ancestral life-forms and from those
resurrect them maybe even for laboratory
study combine them with the fossils on
the right to try to come up with
something more essential to life
something essential here meaning in the
essence is something more basic and
we've tried this I mean certainly
molecular evolution is what has brought
together sort of the classical
evolutionary with the molecular
evolution right theory for those of you
who are not familiar with this I really
three components of a molecular
evolution analysis especially when you
deal with proteins and I've drawn them
here in the upper left-hand corner
you're looking at amino acid sequences
or at least the first 20 amino acids of
proteins from box from sheep and from
camel um it's transparently obvious on
the similarities between those three
sequences that they are related by
common ancestry if you have any kind of
statistical model where you propose that
those sequences arose and their
similarities by random chance the
chances of that happening are quite
small you can notice of course that the
camel sequence is more different from
the ox and the sheep and the options of
sheep differ from each other and from
that comes the second part of the
phylogenetic analysis which you see on
the right which is the evolutionary tree
which shows the familial relationships
of these species that oxen sheep diverge
from each other after they both diverse
as an ancestral form from camel and of
course the whole set divers long after
Megan's split off from that lineage
leading the modern ruminants so that's
an important component the third thing
which is not as often discussed bits
than known from I guess ever since Linus
Pauling and meals
candolyn the 1960s proposed it was if
you have the sequences of the descendant
proteins you can infer the sequences of
the ancestral proteins and I'm just at
the bottom left of this slide pointing
to a an evolutionary reconstruction you
know if you want to know what is a
position one of this protein from oxen
we need the ancestor of oxen sheep will
the Ox has AK it's a lysine at position
1 the Sheep also has AK that's a lie
seen also in position one therefore it
is most parsimonious that it requires
the fewest number of amino acid
replacements to assume that the ancestor
also had a keg or a lysine in position
one and so the ancestral sequence which
has a lysine in position one a k is in
black at the bottom of that alignment e
is the second amino acid but you know
when you get to the position three in
the alignment you got a problem because
ox has a tee that's a three inning and
cheap have a nest that's a searing what
do you do because if you go to the tree
reconstruction of the lower left if you
infer that the ancestor had a tea or a
threonine in position three then there
was one change which produce the Syrian
the modern sheep in contrast that you
assume that the ancestor has a searing
at position 3 then there's one change in
the lineage leading the modern ox that
put a three inning there those two
inferences about the ancestral state
reconstructed at position 3 are equally
parsimonious that is they both require
one change in the tree and therefore in
classical analysis one would not know
whether to put a 39 or Siri at position
3 and the ancestral sequence and by the
way that's true even if you're a fan of
maximum likelihood the analyses you
still have about a forty-eight percent
chance that they're being a threonine at
the MSS or at that site may have
forty-seven percent chance that it's a
Syrian and the remaining five percent of
the probability is distributed over all
the other amino acids so never mind we
live in the age of the genome everybody
and his brother has have
his genome sequence and so if you
actually look at the database you're
likely to find an out-group in this case
camel which conveniently has a 39 in
position three and that resolves the
ambiguity and imprints of the ancestral
sequence and so these are the you know
the ways in which you can bring together
sort of a historical theory our sixth
oracle model for life based of
paleontology and the mullet molecular
structure of life in a productive way
well now um now we can try to go
backwards in time we can try to
understand the details of interactions
between chemistry and homology using
this and our real goal of course was to
try to get an experimental carlet here
for what we do in silico with the
sequences which you saw in the previous
slide one of your problems is that most
people think that because evolution
occurred in the past its hypotheses are
intrinsically untestable and therefore
essentially an unscientific and any of
you talk to creationists or intelligent
design people they will remind you of
this at the at the time but it's not
true there are clearly predictions that
can be made about future discoveries and
future analyses that can be made with
evolutionary theory but one of the major
questions is whether we could ever get
an experimental Carla in the laboratory
to test or these just so stories that
molecular evolutionists as well as their
their compatriots in classical evolution
are constantly created and that actually
is an idea that actually goes back to
the line is polynomials of can lure
about 4000 actually closer to 50 years
ago now which was it if you can infer
the sequences of ancient proteins using
the process I just described you to the
magic of recombinant DNA technology you
could resurrect them you can bring them
back to light in the laboratory where
you can study them and therefore bring
the power of experimental method to bear
on the questions that relate history
function in the
ancient world in particular to molecular
structure and of course one thing is
quite clear is that as a set of the
bottom of this slide what these
experiments are going to show is
something that perhaps a few people in
this conference need to have shown to
them and that is that evolution is
extremely powerful as a way at least in
modern Terran life of getting function
out of molecules and of course that is
one of the things that supports our view
that evolution if not the defining
feature of living system system you're
going to be a defining feature of living
systems well let me just take a look at
the the major problem in the planet over
the last 40 million years Al Gore
notwithstanding it is not global warming
it has been global cooling you're
looking on this plot to the left the
isotope ratio data that shows that
decomposition of the D the decline of
global temperature of tens of degrees
centigrade since the eocene let me see
if this pointer actually we're some of
this work there's the eocene here so
that's this so that's the eocene there's
the legacy and there's the myosin
there's the pliocene the line going up
indicates a ratio of isotopes and co
precipitated shirts which is showing the
cooling of the water in which they are
precipitating as well as the ice ages
which appears here but there's actually
a couple of global cooling from a time
when the planet was much much warmer
where they were tropical rainforest
pretty much everywhere in Nebraska
weather is now an open savanna or
Prairie cooling in the legacy and
romantically then cooling again the
myosin of course cooling in the modern
ages with the ice ages and we have on
the right and artist rendition of sort
of how this had an impact on you you as
a primate grew up evolutionarily in
tropical rainforests where you had a
bunch of vitamin C coming from fruits
and vegetables you lost your ability as
a result or at least without
evolutionary consequences to make
vitamin C when the planet cooled and
dried the rainforest 10
the primates who are making quite a good
living in the trees even as far north of
Scandinavia all of a sudden had their
source of vitamin C as well as other
things removed and so what happened of
course was a extinction of primates over
a large range part of their range and so
you can't belong as a toolmaker unable
to sort of take over the fun and despite
the global cooling the global climate
change should actually drive many things
and this is work from lynn margulis
referring to penetrate biology you're
talking about the interaction between
the planets and the life form and one of
the things it drove was what you could
sort of see in the back of this Savannah
which is the emergence of grasses
grasses really were not present more
than in any large abundance more than 40
million years ago in fact when they shot
Jurassic Park they had a hard time
getting an authentic background without
grasses which have taken over the place
but for those of you who have never
eaten grass you can should know that the
grass is about twenty-five percent
silica it's a low nutrition source and
one of the impacts of grasses taking
over large parts of what had previously
been tropical or semi-tropical forest
was it it drove the emergence of a new
kind of animal and I've already
mentioned them there the sheep's are the
Sheep the oxen and the camels these are
animals that pen actually eat grass and
make a living at well they don't
actually eat grass what the oxen do is
they collect the grass that's a low
nutrition food arising because of the
cooling and prairies about 40 million
years ago and then of course really in
the minus scene after the rooming
collects the grass they feed the grass
to bacteria that are growing in their
first stomach and then they of course
cough up the bacteria from time to time
and shoe it which gives the classical
ruminants physiology and then what the
rumen does is they eat fresh bacteria
they feed the bacteria to digestive
enzymes and the subsequent chambers of
their stomachs and in the small and
large intestines and the key point is
that this is a new lifestyle it's
something that I
the only emerged in the pan is a logical
record about 40 million years ago its
emerging at the time or shortly before
the time the grass has become important
and it requires a new kind of digested
entomology to support and that's because
as many of you know who are
microbiologist is a bacteria are
terribly rich in ribonucleic acid that
is RNA the ribonucleic acid is really
not present in the enemies and an
abundance and the food of other animals
and so the ox takes in about seventeen
percent of its nitrogen in one of our
and and therefore they need to digestive
ribonuclease than unions I'm in the
digestive tract that will break down the
RNA that's coming from their new
lifestyle that is eating freshly
fermenting bacteria which is coming from
a new lifestyle of eating grass which
requires bacteria and the cellulases
that are in the bacteria to convert the
low nutrition something a reasonable
refreshing so all of a sudden you have
ribonuclease which is shown in three
ways on this slide on the right hand
side you compute see a three-dimensional
structure of this protein below you see
the one amino acid sequence of these
proteins or whose sequence you've
already seen at least in the first 20
letters when I was describing the trees
then you can see the chemical mechanism
which shows how ribonuclease goes about
its business of breaking RNA into small
pieces yes ma'am cool well that's a just
so story okay that's a story that says
well planet cooled the grasslands
emerged animals that have room in a
digestion emerge they seem to have
enzymes that digest the grasses and they
have these ruins which contain bacteria
that that's just the RNA the coming it's
coming out of the which is which is
contained Vic Terry wit that have RNA
that need ribonuclease to tie Jess how's
that and the question is whether you can
now make that correlation very classical
physiology to actual intellectual or
chemistry and the answer as you can by
resurrecting ancient ribonucleases and I
just put down here some species
which look at the last four million
years of ribonuclease divergence so we
have a swamp Buffalo and the river
buffalo and the ox and then the Elon
does the out-group this is a non
domesticated remit the ancient species
called paki portex whose outline is
representative in that little black box
at the top is the ancestor now remember
no fossil corresponds exactly to a tree
determined by molecular data but it's
close enough and so you can resurrect an
ancient ribonuclease from this guy who
didn't live hasn't lived for four
million years and that's how the protein
behaves and the answer is well pretty
much like the modern proteins behave now
we have a couple of criteria to decide
whether or not a modern protein has a
digestive role in fact whenever you
resurrect an ancient protein since you
are not resurrecting the ancient
organism at the same time you have to do
something in vitro that sheds light on a
historical hypothesis here we're going
to be asking when this ribonucleic
became a knight chested ensign and to do
so we use much the same logic the
classical evolutionary people use which
is the statement that well if the
Tyrannosaurus Rex tooth looks like it is
optimum for tearing flesh then it's a
flesh eater here we're going to look at
kinetic properties of ribonucleases
modern and resurrected we're going to
look for example at their stability
against digestion but remember these are
proteins that are in the digestive tract
there are enzymes that hydrolyze
proteins also in the digestive tract so
one of our goals is going to be to make
sure that the ancient protein as well as
the modern protein is itself stable
advanced digestion which is of course a
requirement for approaching to be
digested we also look at its ability to
look at many different substrates and
digestive tract you have to take almost
all RNA sequences there are certain
things that a digestive enzyme does not
have to do and that's listed at the
bottom of this slide does not need to
digest double strand RNA the snow
to bind in double-stranded nucleic acids
of any types kind but we're going to
look at the ancient protein say if it
behaves like a digestive enzyme being
stable itself against a session and
having broad substrate specificity but
no interacting with double strand
nucleic acids then it was a digestive
enzyme and that's sort of where we go on
this well what's kind of amusing is that
digestive behavior in ribonuclease is in
fact found back to our humerus which is
this fellow over here on the left right
and overall this tree in fact if you
look at on the right hand side of this
tree you'll see all sorts of animals all
of which are ruminants all which chew
their cud all of them are descended from
ancestors ruminant which is represented
by the lower case number g in this tree
lived about 35 million years ago he was
probably a room in it as well but the
point was it his ribonuclease as
resurrected in the laboratory extinct
now for 35 million years behaves in the
laboratory like a digestive enzyme
should its table itself to digestion
attacks on digestive subjects and does
not act on non by chester substrate is
that clear so what we're doing here is
just you know making sort of the
groundwork it turns out that what I've
just said you is different the minute
you go farther back in time that is if
you go back to the acadec sis which is
this little fellow with a fossil here on
the left of the slide this guy is not a
room and as far as we can tell he's
actually coming up in the eocene he's a
ancestor of presumably not only the
classical ruminants but also the camels
and then maybe even the pig of the
hippopotamus the point is that the
resurrected ancestral protein is not a
digestive enzyme it does not act on non
digestive that's right it does not act
LM digestive subjects it's not itself
particularly stable the digestion but it
is actually curious enough about 10
times more active on non digestive
substrates that's a whole story about
what that enzyme was doing back in that
organism but it was not been Chesham so
okay so fair enough that's a way in
which we play
to show how effective evolution is to
manage in this case global cooling you
know it's not getting us all that far
back in time doing more essential or a
life one that is more representative of
the essence of life in fact that I
acadec sis doesn't look well he looks
like half sheep and half pig but he's
not in any sense permanent to get
something primitive by the strategy of
going backwards in time you got to go
back a lot farther and for this we have
had a marvelous collaboration with Aaron
Bouchet he's also here at the foundation
we looked at elongation factors and it's
a it's a great protein because as many
of you know there's a structure but it
happens to be present all over and all
sorts of life forms all three kingdoms
of life it was a present the last common
ancestor it is used to assist delivering
of charge transfer RNA to the ribosome
and we can therefore because it's
everywhere and because it's highly
concerned we can infer the sequences of
the ancient elongation factors from its
descendants we also have an in vitro
assay as i mentioned in order to when
you resurrect something ancient you're
not going to have the ancient organism
as a context but at least following the
idea the president is the key to the
past is true that the temperature that
the elongation factors work optimally in
modern bacteria are the temperatures at
which those bacteria live I mean just to
show you an example of that this isn't a
gtp-binding essay for the elongation
factor isolated from ecoli which is
living in your gut comfortably at 37
degrees centigrade the enzyme the
elongation factor works best at 37
degrees that's what the maximum of this
plot means that's the temperature where
it lives and of course if you go to
thermos which is living up 65 degrees
nice light the elongation factor from
that it has a max actually did the
maximum binding temperatures about 65
degrees as well so that means that if
you have a the ability to get into your
hands the elongation factor from any age
bacteria that you're interested in you
have the ability to infer the
temperature at which that ancient
bacteria lived and eric and mike and a
few other people working in the lab went
back and did that keep in mind is an
enormous amount of ambiguity in the
trees that you infer for in this
particular case we only were able to go
back as far as deep into the eubacterial
tree and then there's all sorts of
questions about where aquafx trees and
there's all sorts of issues but we've
looked at a couple of trees and we
sampled a couple of sequences to
represent the ambiguity the idea here is
to try to determine whether the
interpretation of the result that as the
temperature optimum is ambiguous with
respect to the ambiguity in the
evolutionary model or conversely to try
to determine whether or not the
inference in this particular case that
the ancestral bacterium lifted 65
degrees is robust with respect to
ambiguities in the trees inferred
ancestral sequences in the light and so
the green line was with one tree you can
read the paper which we call PSA the
blue line was a different tree MLS a and
the the result was real quite the same
the ancestral protein had a temperature
optimum also 65 degrees centigrade
indicating a bit at least with a part of
the tree that we have sampled sorry more
precise at least for the tree space that
we have sampled and the ancestral
sequences that we have simply the
conclusion that the ancestor it was a
thermo file but not a hyperthermic file
is reasonably robust well since that
time eric has gone back and looked at
resurrected elongation factors
throughout the tree this is just
extracted from a recent paper that just
came out a couple weeks ago in nature
you're looking at temperatures
everywhere and if you're interested for
example as I mentioned at the bottom the
temperature when the plants acquired
chloroplasts it's around here somewhere
it's over here someplace or when the
mitochondria became
in this severe you look at this tree get
an idea of the temperature history at
least in new bacterial images for which
we have descendants well alright there's
a big disappointment here I mean
obviously I think it don't get me wrong
I'm delighted to know something about
the temperature history of life on Earth
and to the extent to which this record
made by inferring the sequences of
ancient proteins and bringing them back
to life in the lab correlates the
geological records it's delightful and
Don low and Paul canal have come up with
geological statements as well about this
temperature history but of course we're
still not back to essence as far as we
can tell in metabolism the agent you
bacterium is I think it's probably
simpler than the modern diversity of
bacterial modern world but it's still
reasonably complicated it still is a
fellow who is able to make proteins by
translation it's still is able to you
don't do wide range of metabolisms is in
no sense primitive and it no sense
origins so so while we going back in
time we and gotten a lot of interesting
data and you can review a lot of this we
aren't yet to origins we're showing up
to essence but never in a mine we've
managed to make some progress we
certainly have dealt with this so-called
philosophical challenge that I mentioned
a few moments ago that most scientists
really don't view historical hypotheses
as being inherently not a scientific
effect you talk to most molecular
biologists you don't really get the
impression that they have a constructive
belief in evolution right they they
they'll tell you they believe in
evolution but it doesn't really
influence how they carry out their
professional lives and so certainly if
you can go back and in first structures
of ancient life-forms from the
structures of their descendants we can
come up with some of these stories one
of which I mentioned to you I haven't
mentioned me some of the others that you
see cool well alright not much simpler
not much in essence and so um we
certainly are prepared to go in the
direction that Jerry Joyce was one of
many authors of this so-called NASA
definition of life as a chemical system
capable of Darwinian evolution we've now
gotten kept it straight and evolution
together into our
area of life by the way we don't have
cells there and then it's interesting
question mr. whether they belong there
but of course you can ask the question
is if it's that simple I the essence of
life is a chemical system that can do
this Darwinian game how would you
establish this given intelligence really
cannot take us back to a truly essential
life form and that we're also by the way
having difficulty with this top wage
which I discussed a week ago at the apps
icon which is that we're still flailing
around trying to get the pieces of
origins together in a way that ties
origins with what NASA is telling us
about what's out here in the left wedge
and so this gets to this the right-hand
wedge this is sort of the remaining game
in town which which Carl mentioned
already which is this idea of synthetic
biology it's actually a very old
tradition and I mean right now people
are talking about this as being a new
field and pacsun think college is not
really a field it's a sweet search
strategy that complements other research
strategies that we understand the world
around us so people obviously use
observation and something biology has
used observation from the origin of the
species another approach is analysis
which is in some sense reductionist it's
your first thing you do with the living
species kill it and then you take it
apart and that's certainly been done
since the Enlightenment it's been done
the molecular sentence for the last 150
200 years very very very very productive
but one of the things is that without a
third will approach the third research
strategy is synthesis which is to create
life and it sort of goes by you know the
philosophy if you're so smart why don't
you rich right i mean synthesis says
that if you really understand life or
anything else for that matter you i'd be
from making if you understand why and
you know certain organic compounds our
red dyes you ought to be able to make a
red dye or if you under think that you
understand why particular compound has
pharmaceutical value out of they would
make it
compound which have the same
pharmaceutical value so synthesis
Android chemistry in particular as a way
of testing understanding by constructing
new forms of matter it is a special
value and it is a way and enforcing
discipline upon scientists in
contradiction to their human instincts
right the human instinct is that when
data are emerging that contradict your
theory you discard the data you don't
discard the theory it's a very common in
some sense it's necessary because most
of the data you collected doesn't agree
with your theory as an artifact is
arising because the instrument is broken
or because you haven't done right of the
experiment correctly but when synthesis
does is celexa put them on the moon goal
that forces scientists across uncharted
grounds where they're forced to
encounter and solve unscripted problems
in ways and do not allow self-deceptions
and my favorite example for this is
always the Mars climate orbiter that is
if the guidance software is metric in
the heart where it's English the rocket
crashes now all the way out if you look
at the mission reports you know they
were evidence there's reason to believe
if something was wrong it was put aside
what synthesis does by setting this
ambitious goal is to force you not to
follow your instinct you've got to
eventually have things work and for that
synthesis guys discovery innovation in
ways that analysis cannot and my
favorite quote from Paul wonder actually
from almost 30 years ago is it actually
just to show you that synthesis is not a
field it's the chemistry is almost a
subfield within synthesis now chemistry
has taken a tremendous advantage about
it because we are able to make new forms
of matter through synthesis as well and
imagine how much easier it would be to
do geology and to test a theory of plate
tectonics if you could you know like
Hitchhiker's Guide go to Magrathea and
have them make you a new planet with a
slightly different tweak that you could
then study to see whether your theory
help out how true here you're looking at
four structures there are four different
molecules to an organic chemist everyone
has meaning I've already mentioned the
one
I'm left-hand corner the synthesis of
urea was what led to the downfall of
vitalism this molecule here psycho
locket Etrian brand new synthesis by
Bill cetera that it it forms the
underpinnings of modern understanding of
a run aromaticity a feature of benzene
that you were forced to learn when you
took organic chemistry I mean this
structure all the way over here is
vitamin b12 it was to the attempted
synthesis of baton and you might imagine
that making that molecule was indeed
putting a man on the moon it was a
difficult molecule to make but the
principles of orbital symmetry emerged
from that synthesis as scientists were
dragged kicking and screaming across
uncharted territory they encountered
problems they tried to make that
molecule and their failure to solve them
with existing Theory force them to come
up with new theory well the same thing
is for life okay and certainly when we
started going back and trying to
understand the gene theory of life one
of the questions you have to ask
yourself is what is the chemical
structures necessary to support a gene
that will then support Darwinian
evolution which will then support life
and one of the things that we may was
the compound not the left-hand compound
but the next one in the left compound of
quarters natural DNA what we may was
natural DNA with a repeating negative
charge has an replace replaced by a
structural unit these s double bond o s
double bond o which is very similar in
structure to the phosphates that join
natural DNA but which lack the repeating
charge and if you go back and read this
article as well as articles of follow
that when you replace the repeating
charge in a backbone of DNA by a
non-repeating unit that is otherwise
hydrophilic the molecule ceases to
support molecular recognition rule-based
molecular recognition after a point
these molecules here actually will work
as small fragments but when you to
longer fragment be things start to fall
and you start to have non-genetic
behavior and so from this came what we
call the polyelectrolyte theory of a
gene the attempt to make a DNA molecule
that doesn't have a repeating charge in

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the backbone led to a failure
we can't we couldn't and of course that
forced us to rethink what was missing in
our existing theories and from that what
was missing in our existing theory was a
full understanding of why that DNA
molecule or RNA has these repeating
charges in the benefit they turn out to
be very important for the rule-based
molecular recognition properties that
are essential for inheritance that are
also essential for evolution and that's
captured under this idea of a
second-generation model for DNA
structure it also provides a way for
which you might look for the universal
gene not just the gene on earth by
looking for not the basis which we have
shown and we'll show you in a minute our
variable but by looking for the backbone
repeating charge that under at least a
polyelectrolyte theory of a gene is
required for all genes to work well cool
it's clear that other things don't
require it as as Carl mentioned in the
introduction I mean there you are
there's your for basis the base is under
the Watson and Crick theory of a gene
were central the backbone phosphates
were peripheral well we now know that
the back of phosphates are quite central
the bases are actually although not
peripheral are certainly less central in
the sense that they can certainly be
changed a lot easier actually the good
test for the students in the audience to
look at those four structures I have cgt
and a which you'll notice I only have a
in quotation marks the others are not in
quotation marks that's because the
structure of a is actually already been
changed from what is present in your DNA
and all the students should now mention
that the person sitting next to them in
authority why that structures wrong or
different it actually is better I'll
pause for two seconds that's enough
pause and then direct your attention for
those of you didn't know the answer to
this amino group down here at the bottom
that's actually not President add name
God made a mistake when she made this
come how she left at all and that's why
adenine and thymine forms only two
hydrogen bonds not the three hydrogen
bonds that hold the other GCC
spare one of the questions of course is
again you know if you're so smart why
don't you just design a new genetic
molecule and I've already told you that
we failed once we're not smart enough to
design a genetic molecule where the
repeating negative charge has been
removed but we turned out to be able to
design a new genetic system where we
shuffled around the hydrogen bond donors
and accepted groups let me just point
out the watson-crick rules for those of
you who are students right the
watson-crick base pairing rule is
following this two rules of
complementarity one is a size
complementarity principle that is at
large things pair with small things of
big purine pairs of a small perimeter
the other rule is in hydrogen bond
donors which I have here is these red
hydrogen's pair with hydrogen bond
acceptor which I have us the blue
nitrogen to oxygen so see as a small
thing presenting a hydrogen bond red
blue-blue that's a hydrogen bond donor
acceptor acceptor pairs with G a big
thing having a hydrogen bond acceptor
donor donor blue red red and that is how
you get to base pairs obviously a and T
even with you know deep down here at the
bottom have the same size
complementarity just as the hydrogen
bond patterns are different with T it's
blue red blue and with a in this
modified varmis red blue red and so that
means if this big thing a does not pair
with C and this big thing G does not
pair with see well by switching around
by shuffling by moving the red things
and the blue things a hydrogen bond
donors and acceptors back and forth you
can conjecture Lee create a new form of
genetic substance but of course
synthesis is how you test that
conjecture and so you make all these
compounds and you discover the yes
indeed you can make many of them and you
can do what we do in DNA synthesis is we
measure melting temperatures of DNA
strands that contain funny things I just
put up a whole bunch of stuff here which
includes all of the base pairs that we
made which contain three hydrogen bonds
between the big thing down here the
small thing the big
small thing this row contains base pairs
between big and small but there are two
hydrogen bonds because we leave
something off at the bottom or here we
leave something off at the top or there
will be something off at the bottom
another thing off at the bottom here's
something joined by exactly one hydrogen
bond and we have all sorts of other
structural parameters I won't go into
discussing but you can read about in
this paper that we wrote about five
years ago with Ron guard that creates
rules so if you want to go back and
design your own artificial genetic
system if these rules are correct again
this is a synthesis proposition or
synthesis testable proposition you ought
to be able to make anything following
rules but one of the rules is that yep
three hydrogen bonds is better than two
and two hydrogen bonds is better than
one that's what's shown in this
particular diagram where you're looking
on the left at melting temperatures of
some representative samples the red dots
are melting temperatures of species we
have a base in the middle which is
joined by three hydrogen bonds yellow
dots and that diagram or measuring
melling temperatures of species which
would contain base courage chillin by
two hydrogen bonds and the black are
either mismatches or one hydrogen bond
and you'll discover that three hydrogen
bonds is better than two which is better
than one that is red dots are better
than yellow dots there than black dots
even when when you have size
complementarity that is a purine pairs
of the primitive as big pairs with small
it's also true to some extent win big
pairs with big and small pairs are small
so we have some relatively stable
permanent criminal even when you have a
small thing paired with small thing
letters not sighs complimentary but if
you have three hydrogen bonds joining
them they still look pretty well well
that's kind of cool now I know I sure
how many cameras there on the audience
and I put these two slides up just in
case people are interested in how you go
back and do this right we don't get the
right answers right the first time we
identify trends we rationalize
exceptions we test hypotheses so as I
mentioned we have a small thing pair
with a small thing in
non sighs complementary fashion joined
by three hydrogen bonds but you know
this is actually more stable than we
expected to be based on trends and part
of the reason we think that's the case
is because this base has a positive
charge on the nitrogen as being
indicated here next but the positive
charge seems to be good in the stacking
of a base pair it turns out that if you
put a negative charge as we have here on
this red blue red thing it's less stable
and um come out you come up with this
rule that you're not allowed to have an
aniline or negative charge in the
nucleobase stack even though you can't
have a positive charge and there are all
sorts of other rules that we can go back
and test by making new forms of matter
this is the synthesis strategy right
complement analysis complements
observation we couldn't go back and make
these new forms of matter we would have
some problems actually trying to to get
this theory to be well grounded in our
heads there's a whole story which I
won't tell which you can read about in
this paper by Daniel hooter where it
turns out that some of these things this
is a donor donor acceptor hydrogen
bonding pattern on a small heterocycle
it turns out that some of those don't
work very well because of chemical
instability not not a hydrogen bonding
instability and so we fixed that by
doing chemical changes and we also had
problems which you find in modern bases
call tautomerization this is where
hydrogen's move around spontaneously you
don't want to have hydrogen moving
around in the molecule that you're using
to have kids especially if you're using
those hydrogen bonds tell what
information goes into the kids because
moving hydrogen's around changes the
information that's a mutation we had one
of these base pairs was extremely new
tunic ten percent of the time it was
causing a mutation and ninety percent of
the time only was it complementing the
correct compliment and so we had to fix
that so you can see all sorts of ways in
which Simmons synthesis is demonstrating
this predictive manipulative control
over base pairing using this sort of
metal language of organic chemistry this
is just a way of saying that our theory
is good
enough to create a DNA system with now
on additional eight letters that
watson-crick base brain really is pretty
much as simple as shuffling hydrogen
bond donors and acceptors back and forth
within the context of size
complementarity and that was not the
case as I mentioned a moment ago for the
backbone so our theory is good enough to
understand basis but Arthur is not good
tough to understand the backbone um and
this has been terribly useful I won't go
through all the studies if some of you
have had HIV hepatitis b your hepatitis
c you would be one of the four hundred
thousand patients last year that use
this non-standard genetic information to
help personalized healthcare no I'm the
time has gone to sleep on the frame
clock but so let's see how we're doing
with respect to time ok good well one of
the questions you can ask is okay great
that's the polyelectrolyte theory of the
gene but now the question is well can
you get this artificial genic system to
support Darwinian evolution of course at
some point you're going to say well you
can get it to support Darwinian
evolution can you get it to support life
um one of our problems of course was
that we did not have the stomach to
create a brand-new enzyme that would
accept a genetic alphabet with 12
nucleotides different before that we
already have plus the eight that we
invented and furthermore the natural
polymerizes I think God or evolution has
given us are well adapted for the four
bases that we already have and so we
spent a lot of time trying to get DNA
polymerases that would work with the
expenditure natick alphabet with 12
letters in addition to the four and it
all came down to a focus on this green
pair of electrons so this is done shared
pair of electrons is presented to the
minor groove by both C and G which are
shown here they're also presented by T
or just case uracil and a in this case
amino a and these unshared pair of
electrons in the minor
groove is a recognition spot for polymer
ases and we had really only two choices
one was to change the amino acids that
we're looking for those green electrons
in the minor groove or to go to one of
the synthetic pairs which is this donor
donor acceptor hydrogen bonding pattern
which has a green pair of electrons on
both the big component here and the
small component there now we've done
both of these at this point and and and
that that's the same slide so so and
there's the recognition element that's
where most polymerizes are looking for
that unshared pair of electrons and
those are the amino acid residues and
family a plume races on the left and
family be Clem races on the right that
are actually looking for that green pair
of electrons some of which some of the
extra letters in the genetic opera that
we have made don't have them here's one
that does not and some of them of the
extra letters in the genetic alphabet
that we may do have that green pair of
electrons so both strategies were
followed and I won't go to the detail
this is a case where we're doing a
Darwinian evolution actually an
artificial six-letter genetic system
those are the six letters four of them
are natural cytosine is natural gwanny
is natural timing is natural and Adnan
is natural this pair is a pair of
hydrogen bonding species that are a
fifth and six letter the genetic
alphabet if you look closely the red and
blue hydrogen bond donor and acceptor
things are not at the same spots as they
are here there's not a green pair of
electrons down where my laser pointer is
but we have engineered the reverse
transcriptase that is doing this PCR
type of amplification by replacing the
amino acid at position 1 88 in a
position amino acid at position 4 78 to
try to let the natural enzyme take these
unnatural substrates and it was from
this that Phillip ball gave us this
wonderful headline article enzyme stitch
non-natural DNA guided evolution
man-made stuff of life all right um
there were problems with this system as
well I've already mentioned about
tautomerism there's a work that we've
done to allow six-letter pcr to manage
that and there's now a six-letter pcr
that we have with a six letters being
Phi of T instead of comedy and I so see
Isis see and icg which are two
additional letters so there's this
second example of a six letter based
there and that's what got me called an
old school synthetic biologist meaning
somebody who tries to come up with new
genetic systems that work as a way of
testing these basic questions like how
life got started or whether forms in my
in my tank this sale for those of you
interested the just to close the circle
we do have now a case where we actually
have done a six letter P Sierra this is
just published last year where there's
an unshared pair of electrons the green
electrons in the minor groove which is
where the laser pointer is pointing
right now the other green pair of
electrons is over here now you can
decide for yourself whether or not the
system can undergo Darwinian evolution
we have in this paper and you can look
at this the fact that we have
polymerizes that will amplify a
six-letter genetic system where the
fifth and six letters are these two
bases we have mutations impact on
surveys this base over here and this
space over here what i've shown you here
is the possibility of this system doing
mutation about one percent of the time
this guy the funny small thing will not
find it's appropriate partner the funny
big thing but it will deprotonate and it
deprotonate it changes the hydrogen
bonding pattern so that the compliment /
here is not the funny big thing but
rather natural g and there's a mutation
process that we can look in this system
where g is replaced instead of that and
likewise this guy every now and then
we'll hair template opposite protonated
see these are mechanism
by which you've all this system and not
only replace the two additional bases by
the two standard basis but also convert
the two standard basis into the two
additional basis and you can see a
system which can undergo the basis of
point mutation and well keep in mind
that Jerry Joyce did not talk about
chemical system capable Darwinian
evolution which is certainly is when you
talk about with a self-sustaining
chemical system paper with Darwin
evolution what's absolutely clear right
now is in order for this is undergo
Darwinian evolution you have to have a
graduate student or postdoc sitting
there at every cycle and adding reagents
removing waste products some supporting
the metabolism and so the evolution of
this particular system is slow on two
surgeries so that's basically what we
have to say I mean there's are the four
paths I've obviously talked about the
bottom and the right triangle in large
part because I spent a lot of time on
the other two triangles just a week ago
for a conference that many of you were
at but with that let me stop them really
summarize just by saying that yes you
can go backwards in time to simpler life
but not to essentially it's really not
all that clear that it's all about
informative about what we think is
necessary for the essence of life
although it's quite clear the Darwinian
evolution is a very effective way of
doing things it may not be the only way
um but certainly this is what drives us
to construct artificial light in the
laboratory with a target on chemistry
and Darwin and not other things that we
may not what we thought of and certainly
reject like vitalism that certainly
other things that we haven't necessary
thought but by saying this ambitious
goal the thought is that we're being
dragged kicking and screaming across
uncharted territory where if we are
unable to get emergent properties out of
sight in vitro selection experiments
with a six letter or eight letter
genetic alphabet we're going to be
missing something in our theory of life
and that's of course what this emphasis
activity centers a strategy is supposed
to produce so let me stop oh thank you
for your attention I'll be happy to
answer any questions I can receive okay
thank you Steve I call the back right
speaker
okay if you have a question would you
please raise your hand on WebEx and I'll
also give folks an opportunity to just
jump in with questions but while I've
got the open mic here let me just put in
a plug first of all for the archives if
you would like to tell somebody about
Steve's talk and they weren't able to
hear it it's going to be archived within
a few days on the NAI website you'll be
able to see Steve's face and actually
see everything except the laser pointer
which he used excellently I must say and
unfortunately that's the one thing that
isn't present in the archive but I think
anybody who didn't see the talk and
would like to will enjoy being able to
do it on the archive and of course all
the other talks from this year are
archived there as well and let me also
while I have the open mic just put in a
plug for the next director seminar which
is going to be giovanna tinetti on jun
2nd and she's going to be talking about
her work on understanding the
characteristics of extrasolar planets
and with that marco do we have any hands
raised on WebEx we have a question from
Goddard God please go ahead when you
talk about the bases you are just say
you use a basis and then try to see if
they work with their four ways we took
up supernatural why do we don't you have
a system completely sign tip synthetic
and with the eight basis you know
interacting between them without their
for that we know well yeah that's a good
question the answer is because that's a
lot more work right adding two plus four
that are natural that can be purchased
from sigma-aldrich and where the
triphosphates are available from tri
link is a lot easier for anyone graduate
student or postdoc to do them to have
the poor graduate student wrote o'clock
have to make six triphosphates which is
actually the difficult synthesis of
making the nucleus size one thing making
the triphosphate is more difficult so
that's so we have not for example done
the pie EAD that is the permitting with
a donor acceptor donor and is
then the piña ad and I a dee da which is
the but that's only because what happens
is one of these base pairs gets assigned
to one individual who has to make both
components right and that's also enough
work the last thing that they want to do
before i let them graduate is to make
another pair of tripods and another pair
of tripod sites so that's the that's the
correct answer to your question the
question of course is whether or not it
would be easier to come up with an
artificial genexus and their reasons I
believe that it would if we hitched
aside the natural basis and part of that
reason is because way in which the
heterocycle is joined the sugar that
there are three base pairs where the
heterocycle is joined to the sugar by
carbon-nitrogen bond and those are the
forces standard the two standard base
pairs plus one of the unnatural ones and
there are three that are joined by
carbon-carbon bonds there's reason to
believe that uniformity would be easier
to achieve in a high fidelity genetic
system unfortunately all of the systems
that are enjoying by carbon carbon or
the non centered basis which would
require somebody to actually make them
all at the same time but the but that
might very well be an easier system to
implement so there are reason to think
that the chemical properties which are
distributed unevenly across these 12
letters in the genetic alphabet would we
be find it easier if we were to pick and
choose in a way that does not include
for standard into non center but just
from the point of view of beating a poor
graduate student into making them or a
postdoc the same and that's why we do it
okay thanks we have a question from
Colorado hi can you hear me safe I can
oh good just stealing I enjoyed your
talk like it though hi I have a question
about using microbial paler genetics to
infer things about the earliest forms of
life I noticed you didn't talk about
lateral gene transfer which makes it I
understand and perhaps I'm wrong about
this difficult to infer what the very
earliest forms of life were like
microbial life and even more importantly
up
Darwinian evolution presupposes a
complex cooperative arrangement among
proteins and nucleic acids and it's
achieved and legs nor through ribosomes
and so carlo's and others have argued
that you know whatever life came early
or couldn't have been able to do
Darwinian evolution there must be some
kind of approach unit which he
speculates might have done something
like Lamarckian evolution so I just
wondered whether you view these as
problems for the idea of microbial
phylogenetics I've microbial paleo
genetics yeah I do in their big problems
first I I don't think it I'm trying to
avoid saying I hope I did say that all
that we were really doing was going back
deep into the eubacterial tree which is
really all that we've done with the
elongation factors we actually are
nowhere well I know how close we are
we're we're not at the last common
ancestor of archaia a new bacteria for
example the the cat to that of course is
that as I mentioned we haven't gotten
into any sense a essential form of life
we still do rely on the autumn two
things actually we rely on the
definition of the tree in here that tree
is being really defined as the ribosomal
tree but it's like the elongation factor
tree right which happens to have very
nice congruence so the ribosomal trace
that we're not looking at least lateral
transfer elongation factors in there and
their immediate coach substrates or
coenzymes but what what is quite clear
is that we do not necessarily have a
speciation concepts we have defined
basically the species tree as the
elongation factor tree / ribosomal RNA
tree but look one of the big questions
about Darwinian evolution frankly and
what we have not addressed and i guess i
was trying to say this but I didn't say
it effectively in the talks let me try
to say it effectively now and that is
one of the big questions about whether
or not life can be simply a chemical
system capable of Darwinian evolution is
whether you do need to have both
proteins and nucleic acids to have to
our way in evolution so about seven
years ago we wrote a paper for a NASA
book white paper where
the argument was made that we didn't
have well that a genetic molecule is how
going to have a hard time being a
catalytic molecule catalytic molecule is
going to have a hard time being a
genetic molecule why well because of
genetic molecule templates where are
Catholic molecule fold genetic molecule
tries have the same physical properties
regardless of building block
substitution whereas a catalytic you'll
need to have different properties as a
function of building block substitution
and so all these things were because you
had to have a property a for a genetic
marker property not a for a catalytic
molecule and vice versa it was actually
very hard to conceive of molecular
systems that could do both catalysis and
genetics at RNA which has certainly the
ability demonstrated a Duke boat was
actually quite special in the world of
biopolymers it really is hard to find
any other biopolymer that does a good
job of both folding when it wants to and
not folding when it doesn't want to or
changing the physical property went once
and not change physical property when it
wants to and so the one essence of
Darwinian evolution that is really
missed right now and it is missed for
sure in this going backwards in time is
whether a single biopolymer can support
during evolution and so help me God the
answer that question which we found
entirely satisfactory 20 years ago was
that yes that molecule is RNA it did so
an early Earth problem solved okay it's
unfortunately not an acceptable answer
20 years later right now we have had the
most miserable time getting well let me
obviously gone Burke and various people
there's a large number of people Jack
szostak Jerry Joyce has gotten catalysis
out of nucleic acids but it's been
extremely difficult to get an RNA
molecule that catalyzes a great length
template directed synthesis of RNA p 10
round his student Hannah's a or have
come close to sort they've done the best
job today um and it's been extremely
difficult to find evidence out of the
paradox of the genetic molecule must do
x and a cowlick molecule must do not x
further when RNA wanders into a region
of highly G rich sequence pace it tends
to fold it doesn't tend the display rule
base
molecular evolution anymore so your
question is spot-on we wear the big
puzzle right now is between the
molecular description of evolution the
essence of light and then evolution as
we know in the modern world is whether
or not what we get in the modern world
can be gotten with a single biopolymer
I'd be a lot happier if a we could make
RNA from prebiotic precursors in
high-yield we can make it half-baked I
would be a lot happier if we can had an
RNA molecule that with facility
catalyzes the synthesis of RNA
especially it had a complete cycle I'd
be a lot better happier if we have a
reasonable Mathematica or theoretical
description of how catalytic power is
distributed within RNA sequence things I
many of you have this go ahead okay
there aren't any more hands raised so if
anybody would like to just jump in with
the question this is an opportunity to
isteve it's Lisa Pratt I'm particularly
interested in this this sulfur anchored
backbone and wondering if you can can
say anything about what happens if you
get away from from circum neutral pH
conditions if we were to think about
early evolution in a very acidic
environment can we uncouple ourselves
from phosphate backbones yeah yeah but
that's a very good question i'm there's
a paper that we wrote for again one of
these NASA things which discuss life at
very low pH in life at very high pH and
it's it's absolutely certain that you
must get away from certain features of
natural nucleic acid standard nucleic
acids standard Terran nucleic acids if
you want to go to higher acidity for
example certainly DNA doesn't do a very
good job at high at high acidity a low
pH the bases fall off of DNA and this
becomes a horrible mess of deep
urination and deeper imagination um it's
an interesting question in fact what was
the talk of that I gave I guess with
with Andrew poriyal session at the
site conference as to whether you could
get away with the repeating negative
charge by having a repeating dipole
where the positive ends of the dipole
we're all tucked inside the molecule on
the negative ends or outside these sort
of effective charge that without having
a net charge and we were actually
focused more on Titan as the place
because there you have to go to high pH
you have to go to a more hydrophobic
solvent and you have to go to lower
temperatures and so the idea is the
shortage of the question is but we don't
know but the speculation was that you
could get away from this pole
electrolyte model for the backbone at
low temperatures in hydrophobic solvents
where you just couldn't tolerate you
can't dissolve a repeating charge and
solvent like methane at any temperature
but the idea was yes you might be able
to get away from it that way the higher
acidity conditions that you find in the
solar system like Venus for example or
of course also very polar so you
wouldn't need to get away from the
higher acidity the me for repeating
sulfuric acid will dissolve a
polyelectrolyte just fine like it was
dissolved most things but then of course
you really have to worry about the
acid-base properties in the acid
stability of the of that of the
acid-base properties of the bases
paradoxically to find the nucleobases
and yeah we had proposed a whole number
of CGI ko Sai's carbon glycosides which
would be stable under Venusian
atmosphere type ph's what they would
still have the repeating negative charge
in the back row you will really need to
get away from that repeating charge if
you go to a hydrophobic solvent like
what you see on the surface notions of
Titan and then your problem is frankly
that nothing dissolves the temperatures
of the surface oceans of Titan that was
the point that William Baines made in
his talk at the absite conference last
week that anything cold is a bad solvent
not because it's a bad solvent just
because it's cold and and so managing
surface genetics genetics and the ocean
service oceans of Titan Estonia problem
Steve this discussion reminds me of a
talk at the lab
sturgeon of life Gordon research
conference in Ventura that was given by
felisa wolf Simon I think I real Anbar
and somebody else who may very well be
on this net we're co-authors in which
she was talking about the possibility of
arsenic substituting for phosphorus and
I'm just wondering if these approaches
of synthetic biology enable you to
investigate the theoretical possibility
of a system which is fundamentally
different in this other way or some
other ways for example with artesunate
substituting for phosphate well I mean I
is a good organic chemist I would say
not only is this strategy of synthesis a
way of investigating that question it's
a necessary way of investigating that
question that is there is no proposal
for that hypothesis in the chemical
community they will meet the chemists
standard of proof that will be
acceptable to the chemical community all
that is absent of an experiment where
you try to make the DNA that contains
arsenic in the background and and of
course when police your first mentioned
that to us and Paul Paul Davies also and
we went out and tried in trying to make
something and what we encountered
products this is the example is was
actually chemistry of arsenate that's
well known and that is at the arsenate
ester unlike the phosphate ester falls
apart with half-lives of minutes and
water at room temperature the phosphate
esters and DNA have half-lives on the
winter walks 10 to the 15 seconds is
what we are talking about that is 10 to
the 13th minutes not one to five minutes
so so the arsenate esters are orders of
magnitudes 10 to the 13th as Carl Sagan
was a billions and billions and billions
of times less stable than phosphate
esters meaning it is really unlikely she
would have arsenide based DNA in aqueous
mini at room temperature earth room
temperature but yeah I mean the point
here is that you can't you're not
allowed in the chemistry community I
mean you're allowed in the planetary
community right to propose a super-earth
a very large rocky earth without then
going out making one but no one will
complain to you and reject your paper
because you did not make an earth with a
the five times the current earth and
test your theory on it but in chemistry
if you propose a structure that you
control on a sheet of paper you have an
obligation or you have to give up your
union card to try to make it I in fact
it's worse than that you almost have an
obligation to make it you always have an
obligation to make it in the chemistry
community before you publish the privet
limit so in this sense the chemists have
a very different way of approaching
science than the planetary scientists or
the astrophysicists who don't have to
make a new star before they can publish
a paper about how a star might work
thanks Steve any further questions
please just jump in if you have one okay
if not then let's thank Steve again for
great talk

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