The RNA world, and the origins of life

The RNA world, and the origins of life

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

The Death Knell for Life from an RNA world
https://www.youtube.com/watch?v=xSewB3Tzpvs


Robert P. Bywater On dating stages in prebiotic chemical evolution 15 February 2012
Despite the wide repertoire of chemical and biological properties of RNA, which make it such an appealing contender for being the first type of molecular species to usher in life onto this planet, there is no explanation for how such a complex chemical species could have arisen in the absence of sophisticated chemical machinery. The generation of complex chemicals require many millions of cycles of synthesis, partial degradation, concentration, selection and reannealing in combinatorially new ways such that sufficiently diverse species could be produced and reproduced, from which particularly suitable entities survived 34

Claim: Donald R.Prothero, The evolving Earth, page 290
Many scientists have suggested that this is an overly complicated and implausible hypothesis: build a genetic code of proteins first, then replace it with another, more complex one. Instead, they argue, it makes more sense to evolve the genetic code in nucleic acids from the very beginning, even if nucleic acids are harder to produce in chemical reactions than are proteins. Thus, we have a classic “chicken or egg” problem. Which came first: the protein replication system or the nucleic acid replication system? Fortunately, there is a way to resolve this conundrum. In the early 1980s scientists discovered certain types of RNA, known as ribozymes, that perform multiple functions. The RNA in these molecules not only acts as a genetic code but also catalyzes reactions and binds together proteins. In fact, the functional part of the ribosome in the cell, which translates the cellular RNA into proteins, is a ribozyme. Thus, ribozymes perform not only their familiar role as replicators but also the role that proteins play. Further research led to the idea that the simplest scenario for the origin of living, self-replicating systems would be an “RNA world.” The very first self-replicating form of life would be a single-stranded RNA, perhaps enclosed in a lipid bilayer membrane and perhaps using simple carbohydrates for food storage. Using both its replication powers and enzymatic powers, it would make more copies of itself and perform the role of the proteins as well until later, more complex reactions involving many different proteins could evolve. Every year, more discoveries are made that add details to our understanding of the origin of life and the RNA world. For example, according to Lehmann and colleagues (2009), coding sequences of amino acids are easily built on small RNA templates in normal prebiotic conditions. Experiments by Kun and others (2005) show that the first ribozymes in the RNA world were much longer and more stable. According to Costanza and colleagues (2009), experiments have shown that nucleotides easily merge in water to form RNA over 100 nucleotides long. Pino and colleagues (2008) demonstrated that RNA molecules link up into long chains easily under normal earth conditions. And finally, a range of experiments by Long and colleagues (2003) and by Patthy (2003) showed that new genes have been produced repeatedly by evolution. The RNA world hypothesis is now accepted as the most likely scenario for the origin of the first selfreplicating system that can be truly called “life,” although there are still additional conundrums that are being worked on: How did the RNA world get replaced by the DNA world of today? And what preceded the RNA world? Could it have been (some suggest) a PNA world (peptide-nucleic acid) system that had amino acids in the nucleic acid chains instead of the sugar ribose? Or something else? Like any good scientific problem, the
solution of one mystery leads to new and more interesting problems to solve.

Response: 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. In 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 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 built 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 synthesizes 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 

Tan, Change; Stadler, Rob. The Stairway To Life:
The extra-hydroxyl group in the pentose ring of RNA makes it more susceptible to hydrolysis, especially in an alkaline environment. A weak solution of sodium hydroxide will destroy RNA but will not damage DNA. Finally, replacement of thymine in DNA with uracil in RNA makes RNA more sensitive to radioactive mutation. If DNA in living organisms requires active repair mechanisms, the more delicate prebiotic RNA, it stands to reason, must also require active repair mechanisms.

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?

Robert Shapiro: Origins, a Skeptic's Guide to the Creation of Life on Earth (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

Paul C. W. Davies The algorithmic origins of life 2013 Feb 6
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.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3565706/

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

[/b]

The daunting problem  how to make the pentose ring of RNA and DNA


 
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 : 


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.



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.ren/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
34. https://sci-hub.ren/10.1007/s00114-012-0892-6

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

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 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

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.

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.

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

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

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

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.

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.)

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/

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

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



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

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

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

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

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

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

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

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

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

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/

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

The "RNA World"


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






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.




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)!




* 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.




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!




* 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?




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).




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.

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.




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:


[list="margin: -1em 0px 1.5em 1.5em; padding-right: 0px; padding-left: 0px; border: 0px; font-stretch: inherit; font-size: 16px; line-height: 24px; font-family: 'PT Serif'; vertical-align: baseline; list-style: none; color: rgb(31, 9, 9);"]
[*]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.


[/list]

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
[list="margin: -1em 0px 1.5em 1.5em; padding-right: 0px; padding-left: 0px; border: 0px; font-stretch: inherit; font-size: 16px; line-height: 24px; font-family: 'PT Serif'; vertical-align: baseline; list-style: none; color: rgb(31, 9, 9);"]
[*]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.


[/list]
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.


http://creation.com/origin-of-life-critique

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

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

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

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/

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. 

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

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

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

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

1. Life uses only right-handed RNA and DNA. There is no selection process of only right-handed RNA and DNA on prebiotic earth.
2. RNA is thermodynamically unstable in water, and overall intrinsically unstable. It 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. How could and would information processing machinery come to exist without the software and vice versa?
4. A certain minimum level of complexity is required to make self-replication possible at all; how was that achieved prebiotically?
5. RNA catalysts would have had to copy multiple sets of RNA blueprints nearly as accurately as do modern-day enzymes. how did that supposedly happen prebiotically?
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. How was that possible In the prebiotic world?.
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. How was that overcome on early earth?
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 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. If you disagree, why?
11. Macromolecules do not spontaneously combine to form macromolecules. How do you think did it occur nonetheless prebiotically?
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 built 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.  
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.
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 synthesizes 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.