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Defending the Christian Worldview, Creationism, and Intelligent Design

Otangelo Grasso: This is my personal virtual library, where i collect information, which leads in my view to the Christian faith, creationism, and Intelligent Design as the best explanation of the origin of the physical Universe, life, and biodiversity


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Defending the Christian Worldview, Creationism, and Intelligent Design » Origin of life » The RNA & DNA World » The RNA world, and the origins of life

The RNA world, and the origins of life

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26The RNA world, and the origins of life - Page 2 Empty Re: The RNA world, and the origins of life Sun Jul 12, 2020 6:06 am

Otangelo


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"The RNA World is a widely-embraced hypothetical stage of molecular evolution, devoid of protein enzymes, in which all functional catalysts were ribozymes. Only one fact concerning the RNA World can be established by direct observation: if it ever existed, it ended without leaving any unambiguous trace of itself."

https://www.sciencedirect.com/science/article/abs/pii/S0303264717302332

Even this is a bit of an understatement. Because without the prior assumption of evolution, which can and has underwritten a wide range of speculation, there is precisely zero reason to believe this wild hypothesis. No organisms have ever been discovered that demonstrate the RNA World hypothesis in action. Nor have scientists ever constructed any such organisms in their laboratories. This is not too surprising because no one has even produced anything remotely close to a detailed design of how such organisms could function.

Wills and Carter also point out negative evidences such as catalysis (RNA enzymes lack the ability to function over a wide range of temperatures) and the “impossible obstacles” to the hypothetical yet necessary transition from the RNA World to something resembling today’s extant cells. As Carter explains:

"Such a rise from RNA to cell-based life would have required an out-of-the-blue appearance of an aaRS [aminoacyl-tRNA synthetase]-like protein that worked even better than its adapted RNA counterpart. That extremely unlikely event would have needed to happen not just once but multiple times — once for every amino acid in the existing gene-protein code. It just doesn’t make sense."

https://www.sciencedaily.com/releases/2017/11/171101160756.htm

Indeed, it just doesn’t make sense. And yet in spite of these obvious problems, the RNA World has been a textbook staple, presented as a plausible and likely example of how early life evolved.

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27The RNA world, and the origins of life - Page 2 Empty Re: The RNA world, and the origins of life Mon Nov 23, 2020 4:34 pm

Otangelo


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Nucleic acid instability challenges RNA world hypothesis 26 SEPTEMBER 2016
RNA and DNA have very different abilities to withstand chemical changes like depurination, deamination, and hydrolysis. The chemical stability of these two nucleic acids should also be considered when thinking about how they could become incorporated into the earliest forms of life.
https://www.chemistryworld.com/news/nucleic-acid-instability-challenges-rna-world-hypothesis/1017458.article

The stability of the RNA bases: Implications for the origin of life MATTHEW LEVY AND STANLEY L. MILLER , July 1998
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.
https://www.pnas.org/content/pnas/95/14/7933.full.pdf

PREBIOTIC RIBOSE SYNTHESIS: A CRITICAL ANALYSIS ROBERT SHAPIRO d 9 February, 1987
The evidence that is currently available does not support the availability of ribose on the prebiotic earth, except perhaps for brief periods of time, in low concentration as part of a complex mixture, and under conditions unsuitable for nucleoside synthesis.
https://sci-hub.st/https://link.springer.com/article/10.1007/BF01808782

Robert Shapiro:
The presumption that “the bases, adenine, cytosine, guanine and uracil were readily available on the early earth” is “not supported by existing knowledge of the basic chemistry of these substances. The RNA-world hypothesis faces an even more acute, but related, obstacle—a kind of catch-22. The presence of the nitrogen-rich chemicals necessary for the production of nucleotide bases prevents the production of ribose sugars. Yet both ribose and the nucleotide bases are needed to build RNA.

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

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

Naturally occurring RNA molecules possess very few of the specific enzymatic properties of proteins. Science has shown that ribozymes can perform a few of the inumerous functions performed by proteins. Some RNA molecules can
cleave other RNA molecules (at the phosphodiester bond) (hydrolysis). Ribosomal (rRNA) performs peptide-bond formation in the peptidyl transferase center, though only in association with an additional chemical catalyst. Beyond that, RNA can perform only a few minor functional roles and then usually as the result of engineering in the laboratory. Claiming that catalytic RNA could replace proteins in the earliest stages of chemical evolution is not evidence-based.

How do you go from an RNA based world, to produce proteins? That is a huge, unsolved gap. In order to do so, you need the molecular machines that do so. The primitive replicator would need to produce RNA molecules capable of performing the functions of proteins involved in translation. These RNA molecules would need to perform the functions of the twenty specific tRNA synthetases and the fifty ribosomal proteins, among the many others involved in translation. How do you go to substitute supposed ribozymes with proteins, that later would do the same job? That's a far fetched just so scenario, which bears no evidence that such a transition could/would occur. In other words, the evolving RNA world would need to develop a coding and translation system based entirely on RNA and also generate the information necessary to build the proteins that later would be needed to replace it.

Determination of the Core of a Minimal Bacterial Gene Set
A bacterial cell depends upon a translation and coding system consisting of 106 distinct but functionally integrated proteins as well several distinct types of RNA molecules (tRNAs, mRNAs, and rRNAs) The minimal number of ribosomal proteins required for proper functioning of the ribosome corresponds to the gene set present in M. genitalium, which includes 31 proteins for the large ribosomal subunit and 19 proteins for the small one.
https://mmbr.asm.org/content/68/3/518

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Otangelo


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The prebiotic origin of nucleotides, and the RNA World

https://reasonandscience.catsboard.com/t2024p25-the-rna-world-and-the-origins-of-life#9363

RNA & DNA: It's prebiotic synthesis: Impossible !! Part 1
https://www.youtube.com/watch?v=-ZFlmL_BsXE

RNA & DNA: It's prebiotic synthesis: Impossible !! Part 2
https://www.youtube.com/watch?v=dv4mUjmuRRU


DNA is one of the most intriguing and fascinating biomolecules found in nature. It forms the famous double helix which is elegant and beautiful. It is made of DNA (deoxyribonucleic acid) monomers, which are the molecules that make up the “alphabet” that specifies biological heredity. Life is information-driven. Specified complex information stored in genes dictates, instructs and directs the making of very complex molecular machines, autonomous robotic production lines, and chemical cell production plants, and it also directs and orders the cell to do its work, and how to operate and is as such of central importance in all life forms. Who wants to find answers about how life started, needs to find compelling explanations about how RNA and DNA first emerged on earth. The information stored in DNA is transcribed into RNA ( ribonucleic acid) and finally translated to make proteins. RNA has several other important roles in the cell. Interestingly, some viruses use RNA to store information. 

Nucleic acid research started in 1871, with a small sentence in the essay “Über die chemische Zusammensetzung der Eiterzellen” He characterized this substance as nitrogen-containing and being very rich in phosphorous. The following decades were marked by resolving the molecular structure of the “nuclein”. (“About the chemical composition of pus cells”) by Miescher 31 James Watson and Francis Crick discovered the structure of the DNA  molecule in 1953.  RNA is built of (almost) the same four-letter alphabet as DNA. It is more fragile, and as such, it could also be an information carrier, but less adequate long term. In all known living beings, genetic information flows from DNA to RNA to proteins. The work of  Watson and Crick on the structure of DNA was performed with some access to the X-ray crystallography of Maurice Wilkins and Rosalind Franklin at King's College London.  This information was critical for their further progress. They obtained this information as part of a report by Franklin to the Medical Research Council. Combining all of this work led to the deduction that DNA exists as a double helix. The report was by no means secret, but it put the critical data on the parameters of the helix (base spacing, helical repeat, number of units per turn of the helix, and diameter of the helix) in the hands of two who had contributed none of those data. With this information, they could begin to build realistic models. The big problem was where to put the purine and pyrimidine bases. Details of the diffraction pattern indicated two strands and indicated that the relatively massive phosphate ribose backbones must be on the outside, leaving the bases in the center of the double helix.

RNA and DNA  are chemically unlikely molecules that are composed of three parts: a nitrogenous base, a  five-carbon sugar (pentose), and phosphate.  DNA uses thymine as a base, and RNA uses uracil. These monomers are joined to form polymers by the phosphate group. In the genome, they form double strands with Watson-Crick base-pairing. 

How did RNA synthesize prebiotically?
A number of reasons have been given why a prebiotic synthesis of RNA, and even more, DNA, is too complex. In cells, the synthesis of RNA and DNA requires extremely complex energy-demanding, finely adjusted, monitored,  and controlled anabolic pathways. Since they were not extant prebiotically, RNA had to be synthesized spontaneously on early earth by abiotic alternative non-enzymatic pathways.  This is one of the major, among many other unsolved origin of life problems. Krishnamurthy points out that "there has been some common ground on what would be needed for organic synthesis of DNA/RNA (for example, the components of ribose and nucleobases to come from formaldehyde, cyanide and their derivatives) but none of the various approaches has found universal acceptance within the origins of life community at large. 26

Over the last decades, Extraterrestrial sources like meteorites, interplanetary dust particles, hydrothermal vents in the deep ocean, and warm little ponds, a prebiotic soup, have been a few of the proposals. High-energy precursors to produce purines and pyrimidines would have had to be produced in sufficient quantities, and concentrated at a potential building site of the first cells. As we will see, there has to be put an unrealistic demand for lucky accidents, and, de facto, there is no known prebiotic route to this plausibly happening by unguided means.  

An article published in 2014 summarizes the current status quo: The first, and in some ways the most important, problem facing the RNA World is the difficulty of prebiotic synthesis of RNA. This point has been made forcefully by Shapiro and has remained a focal point of the efforts of prebiotic chemists for decades. The ‘traditional’ thinking was that if one could assemble a ribose sugar, a nucleobase, and a phosphate, then a nucleotide could arise through the creation of a glycosidic bond and a phosphodiester bond. If nucleotides were then chemically activated in some form, then they could polymerize into an RNA chain. Each of these synthetic events poses tremendous hurdles for the prebiotic Earth, not to mention the often-invoked critique of the inherent instability of RNA in an aqueous solution. Thus, the issue arises of whether there could have been a single environment in which all these steps took place. Benner has eloquently noted that single-pot reactions of sufficient complexity lead to ‘asphaltization’ (basically, the production of intractable ‘goo’). 2 

Steve Benner (2013): The late Robert Shapiro found RNA so unacceptable as a prebiotic target as to exclude it entirely from any model for the origin of life. Likewise, Stanley Miller, surveying the instability of carbohydrates in water, concluded that ‘‘neither ribose nor any other carbohydrate could possibly have been a prebiotic genetic molecule’’ (Larralde et al., 1995). Many have attempted to awaken from the RNA nightmare by proposing alternative biomolecules to replace ribose, RNA nucleobases, and/or the RNA phosphate diester linkages, another source of prebiotic difficulty. These have encountered chemical challenges of their own. 47

Steve Benner (2012): Gerald Joyce called 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. No experiments have joined together those steps ( to make RNAs) without human intervention. Further, many steps in the model have problems. Some are successful only if reactive compounds are presented in a specific order in large amounts. Failing controlled addition, the result produces complex mixtures that are inauspicious precursors for biology, a situation described as the “asphalt problem”. 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. 48

De Duve confesses: "Unless we accept intelligent design, it is clear that the RNA precursors must have arisen spontaneously as a result of existing conditions" 21 - the problem is, - Science is clueless about how nucleotides could have been formed prebiotically.

Prof. Dr. Oliver Trapp (2019): Many questions arising from the RNA world hypothesis have not yet been answered. Among these are the transition from RNA to DNA and the pre-eminence of D-ribose in all coding polymers of life. 10

DNA and RNA: The only possible information storage molecules?
Steven A. Benner (2005): Starting in the 1980s, some synthetic biologists began to wonder whether DNA and RNA were the only molecular structures that could support genetics on Earth or elsewhere.   This knowledge, and the fact that the Watson–Crick model proposed no particular role for the phosphates in molecular recognition, encouraged the inference that the backbone could be changed without affecting pairing rules. The effort to synthesize non-ionic backbones changed the established view of nucleic acid structure. Nearly 100 linkers were synthesized to replace the 2′-deoxyribose sugar. Nearly all analogues that lacked the REPEATING CHARGE showed worse rule-based molecular recognition. Even with the most successful uncharged analogues (such as the polyamide-linked nucleic-acid analogues (PNA)) molecules longer than 15 or 20 building units generally failed to support rule-based duplex formation. In other uncharged systems, the breakdown occurs earlier. The repeating charge in the DNA backbone could no longer be viewed as a dispensable inconvenience. The same is true for the ribose backbone of RNA: The backbone is not simply scaffolding to hold the nucleobases in place; it has an important role in the molecular recognition that is central to genetics.  17

Lack of natural selection
The idea that nucleotides were readily laying around on the early earth, just waiting to be picked up, and concentrated on the building site of life, was mocked by Leslie Orgel as 'the Molecular Biologist's Dream. This is maybe the most stringent problem of prebiotic nucleotide synthesis: The materials on prebiotic earth were a mess of mixtures of lifeless chemicals, and nothing restricts the possibility of a great diversity of nucleotides with differing sugar moieties. There was no natural selection. Many science papers simply ignore this and resort nonetheless to little magic of selective pressure. It's like from Frankenstein to man. Some patchwork here and there, and chance does the rest and figures things out.  Szostak and colleagues were well aware of the problem. They wrote:

There are many nucleobase variations such as 8-oxo-purine, inosine, and the 2-thio-pyrimidines, as well as sugar variants including arabino-, 2′- deoxyribo-, and threonucleotides. The likely presence of byproducts leads to a significant problem with regard to the emergence of the RNA world, since the initially synthesized oligonucleotides would be expected to be quite heterogeneous in composition. How could such a heterogeneous mixture of oligonucleotides give rise to the relatively homogeneous RNAs that are thought to be required for the evolution of functional RNAs such as ribozymes? 30

So, in 2020, they presented a model, ignoring the fact made by Benner and others, that molecules simply disintegrate and randomize, they proposed that "  many versions of nucleotides merged to form patchwork molecules with bits of both modern RNA and DNA, as well as largely defunct genetic molecules, such as ANAThese chimeras, like the monstrous hybrid lion, eagle and serpent creatures of Greek mythology, may have been the first steps toward today's RNA and DNA." 29 Rather than focussing "on the consequences of coexisting activated arabino- and 2′-deoxy-nucleotides for nonenzymatic template-directed primer extension", the authors need to provide a plausible trajectory for how natural selection pressures provided the separation of non-canonical nucleotides to achieve a homogeneous state of affairs, where only RNA's and DNAs used in life polymerize. Often, the key questions in the mids of the often confusing technical jargon get lost.  

The nucleobases
The nucleobases are key components of RNA and DNA. The bases are divided into purines ( adenine (A) and guanine (G)) and pyrimidines [cytosine (C) and thymine (T) in DNA, and Cytosine (C) and uracil (U) in RNA]. While purines have a double ring structure and nine atoms, purines have a single ring structure with six atoms. The structural difference between these sugars is that ribonucleic acid contains a hydroxyl (-OH) group, whereas deoxyribonucleic acid contains only a hydrogen atom in place of this hydroxyl group.

Purines
Purines are one of the two compounds that are used to make the semantophoretic nucleotides RNA and DNA that store genetic information. Adenine and guanine are made of two nitrogen-containing rings. 

Adenine
One of the earliest experiments attempting to synthesize adenine in prebiotic conditions was made by Oró in 1961, where he presented evidence for the "synthesis of adenine from aqueous solutions of ammonium cyanide at temperatures below 100°." 18 In 1966, P. Ferris and L. E. Orgel pointed out, what the achilles heel was in Oró's experiment: "Adenine was formed in only 0.5% yield in Oro’s experiment; most of the cyanide formed an intractable polymer."19 Evidently, there was no prebiotic natural selection to sort out those bases that could later be used as nucleobases, from those with no function. 

Shapiro pointed out that: Useful yields of adenine cannot be obtained except in the presence of 1.0 M or stronger ammonia. The highest reasonable concentration of ammonia or ammonium ion that can be postulated in oceans and lakes on the primitive earth is about 0.01 M. Orgel  has put forward the following prerequisite for the very first information system: 'its monomeric components must have been abundant components of a prebiotic mixture of organic compounds.' Adenine does not seem to meet this requirement. The instability of adenine on a geological time scale makes its widespread prebiotic accumulation unlikely. Adenine synthesis requires unreasonable Hydrogen cyanide concentrations. Adenine plays an essential role in replication in all known living systems today and is prominent in many other aspects of biochemistry. Despite this, a consideration of its intrinsic chemical properties suggests that it did not play these roles at the very start of life. These properties include the low yields in known syntheses of adenine under authentic prebiotic conditions, its susceptibility to hydrolysis and to reaction with a variety of simple electrophiles, and its lack of specificity and strength in hydrogen bonding at the monomer and mixed oligomer level. 14

Elsewhere, Shapiro addressed an eventual extraterrestrial source: The isolation of adenine and guanine from meteorites has been cited as evidence that these substances might have been available as “raw material” on prebiotic Earth (18). However, acid hydrolyses have been needed to release these materials, and the amounts isolated have been low 5

In a recent paper from 2018, Annabelle Biscans mentions other routes investigated: Miyakama et al. suggest that purines have been formed in the atmosphere in the absence of hydrogen cyanide. They reported that guanine could have been generated from a gas mixture (nitrogen, carbon monoxide, and water) after cometary impacts. Also, it has been proposed that adenine was formed in the solar system (outside of Earth) and brought to Earth by meteorites, given the fact that adenine was found in significant quantity in carbonaceous chondrites - and concludes: Despite great efforts and impressive advancements in the study of nucleoside and nucleotide abiogenesis, further investigation is necessary to explain the gaps in our understanding of the origin of RNA. 20

Guanine
In 1984, Yuasa reported a 0.00017% yield of guanine after electrical discharge experiments. However, it is unknown if the presence of guanine was not simply resulted from a contaminant of the reaction. . S L Miller and colleagues made experiments in 1999, and yield trace amounts of guanine form by the polymerization of ammonium cyanide (0.0007% and 0.0035% depending on temperatures) indicating that guanine could arise in frozen regions of the primitive earth. 22

Abby Vogel Robinson reported in 2010: For scientists attempting to understand how the building blocks of RNA originated on Earth, guanine -- the G in the four-letter code of life -- has proven to be a particular challenge. While the other three bases of RNA -- adenine (A), cytosine (C) and uracil (U) -- could be created by heating a simple precursor compound in the presence of certain naturally occurring catalysts, guanine had not been observed as a product of the same reactions.

Pyrimidines
Pyrimidine bases are the second of the quartet that makes up DNA that stores genetic information. Uracil ( Thymine in DNA) and cytosine are made of one nitrogen-containing ring. In 2009, Sutherland, and Szostak published a paper on a high-yielding route to activated pyrimidine nucleotides under conditions thought to be prebiotic, claiming to be "an encouraging step toward the greater goal of a plausible prebiotic pathway to RNA and the potential for an RNA world." 27 Robert Shapiro disagrees:

Although as an exercise in chemistry this represents some very elegant work, this has nothing to do with the origin of life on Earth whatsoever.  The chances that blind, undirected, inanimate chemistry would go out of its way in multiple steps and use of reagents in just the right sequence to form RNA is highly unlikely. 28

Cytosine
Scientists have failed to produce cytosine in spark-discharge experiments.

Robert Shapiro (1999): The formation of a substance in an electric spark discharge conducted in a simulated early atmosphere has also been regarded as a positive indication of its prebiotic availability. Again, low yields of adenine and guanine have been reported in such reactions, but no cytosine. The failure to isolate even traces of cytosine in these procedures signals the presence of some problem with its synthesis and/or stability. The deamination of cytosine and its destruction by other processes such as photochemical reactions place severe constraints on prebiotic cytosine syntheses.  12

Rich Deem (2001):  
Cytosine has never been found in any meteorites.
Cytosine is not produced in electric spark discharge experiments using simulated "early earth atmosphere."
Synthesis based upon cyanoacetylene requires the presence of large amounts of methane and nitrogen, however, it is unlikely that significant amounts of methane were present at the time life originated.
Synthesis based upon cyanate is problematical, since it requires concentrations in excess of 1 M (molar). When concentrations of 0.1 M (still unrealistically high) are used, no cytosine is produced.
Synthesis based upon cyanoacetaldehyde and urea suffers from the problem of deamination of the cytosine in the presence of high concentrations of urea (low concentrations produce no cytosine). In addition, cyanoacetaldehyde is reactive with a number of prebiotic chemicals, so would never attain reasonable concentrations for the reaction to occur. Even without the presence of other chemicals, cyanoacetaldehyde has a half-life of only 31 years in water.
Cytosine deaminates with an estimated half-life of 340 years, so would not be expected to accumulate over time.
Ultraviolet light on the early earth would quickly convert cytosine to its photohydrate and cyclobutane photodimers (which rapidly deaminate). 49

Uracil
In 1961, Sidney Fox and colleagues synthesized Uracil under: "thermal conditions which yield other materials of theoretical prebiochemical significance. The conditions studied in the synthesis of uracil included temperatures in the range of 100° to 140°C, heating periods of from 15 minutes to 2 hours". 33  Other attempts to provide plausible prebiotic scenarios for the non-enzymatic synthesis of nucleotides and nucleobases continue to the present day. In 2019, Okamura and colleagues published a paper on pyrimidine nucleobase synthesis where their conclusion remarks is noteworthy:

We show that the cascade reaction proceeds under one-pot conditions in a continuous manner to provide SMePy 6. Importantly the key intermediate SMePy 6 gives rise not only to canonical but also to non-canonical bases arguing for the simultaneous prebiotic formation of a diverse set of pyrimidines under prebiotically plausible conditions.

This highlights a general problem mentioned before: chemical reactions are very common resulting in a mixture of molecules, of which most are not relevant for abiogenesis. There was no mechanism to sort out those detrimental in the process towards life. 32

Fast decomposition rate
Adenine deaminates at 37°C with a half-life of 80 years (half-life = time that a substance takes to decompose, and loses half of its physiologic activity). At 100°C its half-live is 1 year. For guanine, at 100°C its half-live is 10 months, uracil is 12 years, and thymine 56 years.  For the decomposition of a nucleobase, this is very short. For nucleobases to accumulate in prebiotic environments, they must be synthesized at rates that exceed their decomposition. Therefore, adenine and the other nucleobases would never accumulate in any kind of "prebiotic soup." 14

A paper published in 2015 points out that: 

Nucleotide formation and stability are sensitive to temperature. Phosphorylation of nucleosides in the laboratory is slower at low temperatures, taking a few weeks at 65 ◦C compared with a couple of hours at 100 ◦C. The stability of nucleotides, on the other hand, is favored in warm conditions over high temperatures. If a WLP is too hot (>80 ◦C), any newly formed nucleotides within it will hydrolyze in several days to a few years. At temperatures of 5 ◦C to 35 ◦C that either characterize more-temperate latitudes or a post snowball Earth, nucleotides can survive for thousand-to-million-year timescales. However, at such temperatures, nucleotide formation would be very slow.  25

That means, in hot environments, nucleotides might form, but they decompose fast. On the other hand, in cold environments, they might not degrade that fast, but take a long time to form. Nucleotides would have to be generated by prebiotic environmental synthesis processes at a far higher rate than they are decomposed and destroyed, and accumulated and concentrated at one specific construction site. Putting that into perspective, P.Ubique, the smallest known free-living cell, has a genome size of 1,3 million nucleotides. The best-studied mechanism relevant to the prebiotic synthesis of ribose is the formose reaction. Several problems have been recognized in ribose synthesis via the formose reaction, which reaction is very complex. It depends on the presence of a suitable inorganic catalyst. Ribose is merely an intermediate product among a broad suite of compounds including sugars with more or fewer carbons. There would have been no way to activate phosphate somehow, in order to promote the energy dispendious reaction.

Extraterrestrial nucleobase sources
In april 2022, nature magazine announced the identification of nucleobases in carbonaceous meteorites.  Guanine and adenine were detected in murchison meteorite extracts, and now various pyrimidine nucleobases such as cytosine, uracil, and thymine, and their structural isomers such as isocytosine, imidazole-4-carboxylic acid, and 6-methyluracil, respectively. They came to the conclusion that "a diversity of meteoritic nucleobases could serve as building blocks of DNA and RNA on the early Earth".23 An article of NASA echoed the authors conclusion: "This discovery demonstrates that these genetic parts are available for delivery and could have contributed to the development of the instructional molecules on early Earth."24 The fatal blow is the fact that the nucleobases relevant for life come always mixed together with isomers that are irrelevant. There was no prebiotic selection to sort out and concentrate exclusively those relevant for life. 

Selecting the right backbone sugar

The difficulty to get ribose prebiotically
One of the most debated questions concerns the availability and synthesis of prebiotic ribose. Pentose sugar is a 5-carbon monosaccharide. These form two groups: aldopentoses and ketopentoses. The pentose sugars found in nucleotides are aldopentoses. Deoxyribose and ribose are two of these sugars.  Ribose is a monosaccharide containing five carbon atoms. d-ribose is present in the six different forms. 

The formose reaction
Jim Cleaves II (2011): The formose reaction, discovered by Butlerow in 1861, is a complex autocatalytic set of condensation reactions of formaldehyde to yield sugars and other small sugar-like molecules. 46
Gaspar Banfalvi (2020): Among the best-known nonenzymatic pathways to ribose formation, we find the formose (from formaldehyde and aldose words) or Butlerow reaction 44 Gerald F Joyce (2012)The classical prebiotic synthesis of sugars is by the polymerization of formaldehyde (the “formose” reaction). It yields a very complex mixture of products including only a small proportion of ribose. This reaction does not provide a reasonable route to the ribonucleotides. A number of other studies have addressed the problems presented by the lack of specificity of the formose reaction and by the instability of ribose. 43

S.Islam (2017): Several problems have been recognized for ribose synthesis via the formose reaction. The formose reaction is very complex. It depends on the presence of a suitable inorganic catalyst. Ribose is merely an intermediate product among a broad suite of compounds including sugars with more or fewer carbons. The reality of the formose reaction is that it descends into an inextricable mixture. The vast array of sugars produced is overwhelming and the intrinsic lack of selectivity for ribose is its undoing. Ultimately, the formose reaction produces a disastrously complex mixture of linear and branched Aldo and keto-sugars in the racemic forms. The consequences of such uncontrolled reactivity are that ribose is formed in less than 1% yield among a plethora of isomers and homologs. The instability of ribose prevents its accumulation and requires it to undergo extremely rapid onward conversion to ribonucleosides before the free sugar is lost to rapid degradation. 36

Irina V Delidovich and colleagues (2014): The classical formose reaction (FR) is hardly applicable for any practical purposes outside of the history of chemical science. The typical “sugary substance” formed as a result of the catalytic oligomerization of formaldehyde nowadays known as “formose” comprises dozens of straight-chain and branched monosaccharides, polyols, and polyhydroxycarbonic acids.5 

There are no further alternatives: Either chance "choose" by fortuitous random events the five-membered ring ribofuranose backbone for DNA and RNA, or it was a choice by intelligence with specific purposes. What is more plausible and probable?  The formose reaction requires a high concentration of Formaldehyde, which, however, readily undergoes a variety of reactions in aqueous solutions. Another problem is that ribose is unstable and rapidly decomposes even at low temperature and neutral pH, and as well in water. Furthermore, as Stanley Miller and his colleagues reported:  Ribose and other sugars have surprisingly short half-lives for decomposition at neutral pH, making it very unlikely that sugars were available as prebiotic reagents. 4

Leslie Orgel (2004): We conclude that some progress has been made in the search for an efficient and specific prebiotic synthesis of ribose and its phosphates. However, in every scenario, there are still a number of obstacles to the completion of a synthesis that yields significant amounts of sufficiently pure ribose in a form that could readily be incorporated into nucleotides. 34

Cairns-Smith (1990) 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. 1990

There have been a wide variety of attempts and proposals to try to solve the riddle, but up to date, without success. Science magazine (2016): Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. 35 Annabelle Biscans (2018): Even if some progress has been made to understand ribose formation under prebiotic conditions, each suggested route presents obstacles, limiting ribose yield and purity necessary to form nucleotides. A selective pathway has yet to be elucidated. 6

RNA and DNA use a five-membered ribose ring structure as backbone. Rings containing six carbons instead of five carbons do not possess the capability of efficient informational Watson–Crick base-pairing. Therefore, these systems could not have acted as functional competitors of RNA in a genetic system, even though these six-carbon alternatives of RNA should have had a comparable chance of being formed under the conditions that formed RNA. The reason for their failure is the fact that six-carbon-six-membered-ring sugars are too bulky to adapt to the requirements of Watson–Crick base-pairing within oligonucleotide duplexes. In sharp contrast, an entire family of nucleic acid alternatives in which each member comprises repeating units of one of the four possible five-carbon sugars (ribose being one of them) turns out to be a highly efficient informational base-pairing system. But why and how would natural non-designed events on early earth select what works? Observe Albert Eschenmoser's end note in his science paper from 1986: Optimization, not maximization, of base-pairing strength, was a determinant of RNA's selection. [url=https://www.science.org/doi/10.1126/science.284.5423.2118#:~:text=Chemical etiology (2) of nucleic,the molecular basis of life's]8[/url] But how and why would unintended events select something, that on its own has no function? These ring structures would simply lay around and then soon disintegrate. The smuggling in of evolutionary jargon is widespread for sake of the lack of any alternative. The authors omit and do not ask these relevant questions. That permits keeping the naturalistic paradigm alive. But it should be evident how nonsensical evolutionary claims and such inferences are. 

Ribose - the best alternative 
Prof. Gaspar Banfalvi (2006): Ribose was not randomly selected but the only choice, since β-D-ribose fits best into the structure of physiological forms of nucleic acids. 16

Ribose sugar is the molecule of choice for nucleic acids, yet because it is difficult to imagine forming under plausible prebiotic conditions and has a short lifetime, origin-of-life researchers have searched diligently for alternatives, like glycerol, that might have served as scaffolding for prebiotic chemicals prior to the emergence of DNA.  Unfortunately, they don’t work.  Steven Benner: Over 280 alternative molecules have been tested, and they just do not work at all; those that might be better than ribose are implausible under prebiotic conditions.  “Ribose is actually quite good – uniquely good,” he said.  Deal with it: one’s chemical evolution model is going to have to include ribose.  That means figuring out how it can form, how it can avoid destruction in water, and how it can avoid clumping into useless globs of tar.  (RNA, the main player in the leading “RNA World” scenario for the origin of life, uses ribose; DNA uses a closely-related sugar, deoxyribose.) 38

Various possible ribose configurations: 

The RNA world, and the origins of life - Page 2 Ribose11
Ribose can exist in various forms: α-D-ribose, β-D-ribose  ( right-handed chiral form, dextrorotary) or α-L-ribose, β-L-ribose ( left-handed chiral form, levorotary).  it can form α-nucleosides, β-nucleoside, envelope or twisted conformations,

The RNA world, and the origins of life - Page 2 Ribose10
Ribose conformations and configurations. (a) Major conformers of cyclopentane. (b) Envelope and twisted conformers of tetrahydrofuran. (c) D-configuration as well as α and β anomeric configurations of D-ribose. (d) Twisted conformations in ribose, C3′-endo in A-DNA and C2′-endo conformations in B-DNA. 44

Selecting β-nucleosides
Life uses mostly β-nucleosides rather than α-nucleosides ( which are extremely rare in biological systems). In β-nucleosides, the ribose or deoxyribose is linked to nucleobases through β-glycosidic bonds, which means that the nucleobase at C1 is cis with respect to the hydroxymethyl group at C4, known as the β-configuration. In  α-nucleosides, the nucleobase and hydroxymethyl group in the ribose or deoxyribose are in a trans relationship

The RNA world, and the origins of life - Page 2 Alpha_10

Configuration of β-nucleosides and α-nucleosides

Life uses exclusively right-handed homochiral β-D-ribonucleotides. Roger D. Blandford (2020): The homochirality of the sugars has important consequences for the stability of the helix and, hence, on the fidelity or error control of the genetic code.  45

Prof. Gaspar Banfalvi (2006): Bases in α-anomeric position are unable to base-pair, eliminating the possibility of helix formation. 16

Tan, Change; Stadler, Rob. The Stairway To Life:
In all living systems, homochirality is produced and maintained by enzymes, which are themselves composed of homochiral amino acids that were specified through homochiral DNA and produced via homochiral messenger RNA, homochiral ribosomal RNA, and homochiral transfer RNA. No one has ever found a plausible abiotic explanation for how life could have become exclusively homochiral. 50

Emily Singer (2016): At a chemical level, a deep bias permeates all of biology. The molecules that make up DNA and other nucleic acids such as RNA have an inherent “handedness.” These molecules can exist in two mirror-image forms, but only the right-handed version is found in living organisms. Handedness serves an essential function in living beings; many of the chemical reactions that drive our cells only work with molecules of the correct handedness. DNA takes on this form for a variety of reasons, all of which have to do with intermolecular forces. 42

Abiogenesis researchers are in the dark when it comes to explaining how the molecules of life, amongst them, the life-essential RNAs and DNAs could have been selected without a mental agency. 

Phosphorus
Phosphorus is the third essential element making part of the structures of DNA and RNA. It is perfect to form a stable backbone for the DNA molecule. Phosphates can form two phosphodiester bonds with two sugars at the same time and connect two nucleotides. Phosphorus is difficult to dissolve, and that would be a problem both in an aquatic as-as well on a terrestrial environment. Phosphoesters form the backbone of DNA molecules. 

Libretexts explains: A phosphodiester bond occurs when exactly two of the hydroxyl groups in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. Phosphodiester bonds are central to all life on Earth as they make up the backbone of the strands of nucleic acid. In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA. Strong covalent bonds form between the phosphate group and two ribose 5-carbon rings over two ester bonds.  On prebiotic earth, however, there would have been no way to activate phosphate somehow, in order to promote the energy dispendious reaction.  

That adds up to the fact that concentrations on earth are very low. Kitadai (2017): So far, no geochemical process that led to abiotic production of polyphosphates in high yield on the Earth has been discovered. 39 The phosphate is connected to ribose which is connected to the nitrogenous base. Each of the 3 parts of nucleotides must be just right in size, form, and must fit together. The bonds must have the right forces in order to form the spiral form DNA molecule. And there would have to be enough units concentrated at the same place on the prebiotic earth of the four bases in order to be able to form a self-replicating RNA molecule if the RNA world is supposed to be true. The Albert team explains: A nucleotide is differentiated from a nucleoside by one phosphate group. Accordingly, a nucleotide can also be a nucleoside monophosphate. If more phosphates bond to the nucleotide (nucleoside monophosphate) it can become a nucleoside diphosphate (if two phosphates bond), or a nucleoside triphosphate (if three phosphates bond), such as adenosine triphosphate (ATP). 40 Adenosine triphosphate, or ATP, is the energy currency in the cell, a crucial component of respiration and photosynthesis, amongst other processes. The base, sugar, and phosphate need to be joined together correctly - involving two endothermic condensation reactions involved in joining the nucleotides, which means it has to absorb energy from its surroundings. In other words, compared with polymerization to make proteins, nucleotides are even harder to synthesize and easier to destroy; in fact, to date, there are no reports of nucleotides arising from inorganic compounds in primeval soup experiments.

Why phosphorus?
The selection of phosphorus as a backbone of RNA and DNA was a very smart choice. F H Westheimer (1987): The existence of a genetic material such as DNA requires a compound for a connecting link that is at least divalent. In order that the resulting material remain within a membrane, it should always be charged, and therefore the linking unit should have a third, ionizable group. The linkage is conveniently made by ester bonds, but, in order that the ester be hydrolytically stable, that charge should be negative and should be physically close to the ester groups. All of these conditions are met by phosphoric acid, and no alternative is obvious. Furthermore, phosphoric acid can form monoesters of organic compounds that can decompose by a mechanism other than normal nucleophilic attack, a mechanism that allows them sufficient reactivity to function in intermediary metabolism. 15

Bonding ribose to the nucleobase, to get nucleosides
Supposing that the parts were available, they would have had to be joined together at the same assembly site,  and sorted out from non-functional molecules.  Joining all three components together involves two difficult reactions: formation of a glycosidic bond, with the right stereochemistry linking the nucleobase and ribose, and phosphorylation of the resulting nucleoside. Nucleosides lack a phosphate at the C5′ position. There are no known ways of bringing about this thermodynamically uphill reaction in an 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.

John D. Sutherland (2010): It has normally been assumed that ribonucleotides arose on the early Earth through a process in which ribose, the nucleobases, and phosphate became conjoined. However, under plausible prebiotic conditions, condensation of nucleobases with ribose to give β-ribonucleosides is fraught with difficulties. The reaction with purine nucleobases is low-yielding and the reaction with the canonical pyrimidine nucleobases does not work at all. The route as operated thus far in the laboratory is associated with several steps, and the conditions for these steps are different. Furthermore, purification in between certain steps was carried out to make analysis of the chemistry easier. Clearly, these issues need to be addressed before the synthesis can be seen as geochemically plausible. 3

Brian J. Cafferty (2015): The coupling of ribose with a base is the first step to form RNA, and even those engrossed in prebiotic research have difficulty envisioning that process, especially for purines and pyrimidines. 40 
Terence N. Mitchell (2008):  Nucleosides are formed by linking an organic base ( guanine, adenine, uracil or cytosine) to a sugar (here D-ribose). This reaction looks simple, but how it could have occurred by an enzyme-free prebiotic synthesis, in particular involving pyrimidine bases, is an open question. 41 Fazale Rana (2011): In order for 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, for what reason would the generation of a homopolymer be useful? 37

Consider that only random non-designed events could account for the generation, which seems rationally extremely unlikely, if not impossible. The chance for that alone occurring by coincidence is extremely remote. Whatever the mode of joining base and sugar was, it had to be between the correct nitrogen atom of the base and the correct carbon atom of the sugar. The prebiotic synthesis of simple RNA molecules would, therefore, require an inventory of ribose and nucleobases. Assembly of these components into proto-RNA would further require a mechanism to link the ribose and nucleobase together in the proper configuration to form polymers, and then to activate the combined molecule (called a nucleoside) with a pyrophosphate or some other functional component that would promote the formation of a bond between the nucleoside.  There have been many imaginative ideas and attempts for its solution, all unsuccessful.   In most cases the nucleoside components generated in the experiments, attempting to join the bases to the ribose backbone represent only a minor fraction of a full suite of compounds produced, so the synthesis of a nucleoside would require either that the components be further purified or that some mechanism exist to selectively bring the components together out of a complex mixture. How would non-designed random events be able to attach the nucleic bases to the ribose and in a repetitive manner at the same, correct place?  

From nucleosides to nucleotides
Activated monomers are essential because polymerization reactions occur in an aqueous medium and are therefore energetically uphill in the absence of activation. A plausible energy source for polymerization remains an open question. Condensation reactions driven by cycles of anhydrous conditions and hydration would seem to be one obvious possibility but seem limited by the lack of specificity of the chemical bonds that are formed. 51

Libretexts: Phosphodiester bonds are central to most life on Earth, as they make up the backbone of the strands of DNA. In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA. Strong covalent bonds form between the phosphate group and two 5-carbon ring carbohydrates (pentoses) over two ester bonds. In order for the phosphodiester bond to be formed and the nucleotides to be joined, the tri-phosphate or di-phosphate forms of the nucleotide building blocks are broken apart to give off energy required to drive the enzyme-catalyzed reaction. When a single phosphate or two phosphates known as pyrophosphates break away and catalyze the reaction, the phosphodiester bond is formed. Hydrolysis of phosphodiester bonds can be catalyzed by the action of phosphodiesterases which play an important role in repairing DNA sequences. 52

Prebiotic phosphodiester bond formation
An often-cited claim is that RNA polymerization could be performed on clay. Robert Shapiro wrote a critique in regards to prebiotic proposals of clay-catalyzed oligonucleotide synthesis (2006): 
An extensive series of studies on the polymerization of activated RNA monomers has been carried out by Ferris and his collaborators. A recent publication from this group concluded with the statement: “The facile synthesis of relatively large amounts of RNA oligomers provides a convenient route to the proposed RNA world. The 35–40 oligomers formed are both sufficiently long to exhibit fidelity in replication as well as catalytic activity”. The first review cited above had stated this more succinctly: “The generation of RNAs with chain lengths greater than 40 oligomers would have been long enough to initiate the first life on Earth”. Do natural clays catalyze this reaction? The attractiveness of this oligonucleotide synthesis rests in part on the ready availability of the catalyst. Montmorillonite is a layered clay mineral-rich in silicate and aluminum oxide bonds. It is widely distributed in deposits on the contemporary Earth. If the polymerization of RNA subunits was a common property of this native mineral, the case for RNA at the start of life would be greatly enhanced. However, the “[c]atalytic activity of native montmorillonites before being converted to their homoionic forms is very poor”. The native clays interfere with phosphorylation reactions. This handicap was overcome in the synthetic experiments by titrating the clays to a monoionic form, generally sodium, before they were used. Even after this step, the activity of the montmorillonite depended strongly on its physical source, with samples from Wyoming yielding the best results. Eventually the experimenters settled on Volclay, a commercially processed Wyoming montmorillonite provided by the American Colloid Company 11

Selecting the binding locations
Once the three components would have been synthesized prebiotically, they would have had to be separated from the confusing jumble of similar molecules nearby, and they would have had to become sufficiently concentrated in order to move to the next steps, to join them to form nucleosides, and nucleotides. 

The phosphate/ribose backbone of DNA is hydrophilic (water-loving), so it orients itself outward toward the solvent, while the relatively hydrophobic bases bury themselves inside. 

Xaktly explains: Additionally, the geometry of the deoxyribose-phosphate linkage allows for just the right pitch, or distance between strands in the helix, a pitch that nicely accommodates base pairing. 46
Lots of things come together to create the beautiful right-handed double-helix structure. Production of a mixture of d- and l-sugars produces nucelotides that do not fit together properly, producing a very open, weak structure that cannot survive to replicate, catalyze, or synthesize other biological molecules. 46

Eduard Schreiner (2011): In DNA the atoms C1', C3', and C4' of the sugar moiety are chiral, while in RNA the presence of an additional OH group renders also C2' of the ribose chiral.47

A biological system exclusively uses d-ribose, whereas abiotic experiments synthesize both right- and lefthanded-ribose in equal amounts. But the pre-biological building blocks of life didn’t exhibit such an overwhelming bias. Some were left-handed and some right. So how did right-handed RNA emerge from a mix of molecules?  Some kind of symmetry-breaking process leading to enantioenriched bio monomers would have had to exist. But none is known. Gerald Joyce wrote a science paper that was published in Nature magazine, in 1984. His findings suggested that in order for life to emerge, something first had to crack the symmetry between left-handed and right-handed molecules, an event biochemists call “breaking the mirror.” Since then, scientists have largely focused their search for the origin of life’s handedness in the prebiotic worlds of physics and chemistry, not biology - but with no success. So what is the cop-out? Pure chance !! Luck did the job. That is the only thinkable explanation once God's guiding hand is excluded. How could that be a satisfying answer in face of the immense odds? It is conceivable that the molecules were short enough for all possible sequences, or almost, to be realized (by way of their genes) and submitted to natural selection. This is the way de Duve thought that Intelligent Design could be dismissed. This coming from a Nobel prize winner in medicine makes one wondering, to say the least.  De Duve dismissed intelligent design and replaced it with natural selection. Without providing a shred of evidence. But based on pure guesswork and speculation.

Prebiotic base-pairing
In order to create a stable genome which was necessary for life to start, bases need to be paired between pyrimidines and purines. In molecular biology, complementarity describes a relationship between two structures each following the lock-and-key principle. Complementarity is the base principle of DNA replication and transcription as it is a property shared between two DNA or RNA sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things. This complimentary base pairing is essential for cells to copy information from one generation to another. There is no reason why these structures could or would have emerged in this functional complex configuration by random trial and error. A paper from Nature magazine, in 2016, demonstrates the complete lack of explanations despite decades of attempts to solve the riddle. Brian J. Cafferty and colleagues write:

The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Despite decades of effort, the chemical origin of nucleosides and nucleotides (that is, nucleobases glycosylated with ribose and phosphorylated ribose) remains an unsolved problem.   They then proceed: Here we show that two plausible prebiotic heterocycles, melamine, and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. The data presented here demonstrate the efficient single-step syntheses of complementary nucleosides and nucleotides, starting with the plausible proto-nucleobases melamine and BA and ribose or R5P. 9



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1. R. Shapiro: Life: What]https://jsomers.net/life.pdf]What A Concept! 2008 , page 84
2. Paul G. Higgs: The RNA World: molecular cooperation at the origins of life 11 November 2014
3. John D Sutherland: Ribonucleotides 2010 Mar 10.
4. STANLEY L. MILLER: Rates of decomposition of ribose and other sugars: Implications for chemical evolution August 1995 
5. Irina V. Delidovich: Catalytic Formation of Monosaccharides: From the Formose Reaction towards Selective Synthesis 2014
6. Annabelle Biscans: Exploring the Emergence of RNA Nucleosides and Nucleotides on the Early Earth 2018 Dec; 8
7. Cornelia Meinert: Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs 2016 Apr 8
8. ALBERT ESCHENMOSER: [url=https://www.science.org/doi/10.1126/science.284.5423.2118#:~:text=Chemical etiology (2) of nucleic,the molecular basis of life's]Chemical Etiology of Nucleic Acid Structure[/url] 25 Jun 1999
9. Brian J. Cafferty: Spontaneous formation and base pairing of plausible prebiotic nucleotides in water 25 April 2016
10. Prof. Dr. Oliver Trapp: Direct Prebiotic Pathway to DNA Nucleosides 26 May 2019
11.  A. G. Cairns-Smith Seven Clues to the Origin of Life: A Scientific Detective Story  1990
12. Robert Shapiro: [url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC20907/#:~:text=At 100%C2%B0C the,and T is 56 yr.]Prebiotic cytosine synthesis: A critical analysis and implications for the origin of life[/url] April 13, 1999
13. SAbby Vogel Robinson: Study: Adding UV light helps form ?Missing G? of RNA building blocks June 14, 2010
14. R Shapiro The prebiotic role of adenine: a critical analysis 1995 Jun;25
15. F H Westheimer:Why nature chose phosphates[/size]  1987 Mar 6
16. Prof. Gaspar Banfalvi: Why Ribose Was Selected as the Sugar Component of Nucleic Acids 28 Mar 2006
17. Steven A. Benner: SYNTHETIC BIOLOGY 01 July 2005
18. J.Oró: Synthesis of adenine from ammonium cyanide  June 1960
19. James P. Ferris and L. E. Orgel: Studies in Prebiotic Synthesis. I. Aminomalononitrile and 4-Amino-5-cyanoimidazole”” 1966 Aug 20
20. Annabelle Biscans: Exploring the Emergence of RNA Nucleosides and Nucleotides on the Early Earth 6 November 2018
21. Christian de Duve: Singularities: Landmarks on the Pathways of Life 2005
22. Guanine
23. Yasuhiro Oba: Identifying the wide diversity of extraterrestrial purine and pyrimidine nucleobases in carbonaceous meteorites 26 April 2022
24. Anil Oza: Could the Blueprint for Life Have Been Generated in Asteroids? Apr 26, 2022
25. Ben K. D. Pearce: Origin of the RNA world: The fate of nucleobases in warm little ponds October 2, 2017
26. R. Krishnamurthy: Experimentally investigating the origin of DNA/RNA on early Earth 12 December 2018
27. J.D. Sutherland, and Jack W. Szostak: Chemoselective Multicomponent One-Pot Assembly of Purine Precursors in Water November 2, 2010
28. James Urquhart Insight into RNA origins May 13, 2009
29. Caitlin McDermott-Murphy: First building blocks of life on Earth may have been messier than previously thought January 22, 2020
30. Jack W. Szostak* A Model for the Emergence of RNA from a Prebiotically Plausible Mixture of Ribonucleotides, Arabinonucleotides, and 2′-Deoxynucleotides January 8, 2020
31. Florian M. Kruse: Prebiotic Nucleoside Synthesis: The Selectivity of Simplicity 19 May 2020
32. Hidenori Okamura: A one-pot, water compatible synthesis of pyrimidine nucleobases under plausible prebiotic conditions 07 Jan 2019
33. SIDNEY W. FOX Synthesis of Uracil under Conditions of a Thermal Model of Prebiological Chemistry 16 Jun 1961
34. Leslie E Orgel: Prebiotic chemistry and the origin of the RNA world Mar-Apr 2004
35. Cornelia Meinert: Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs 2016 Apr 8
36. Saidu lIslam: Prebiotic Systems Chemistry: Complexity Overcoming Clutter  13 April 2017
37. Fazale Rana: Creating Life in the Lab: How New Discoveries in Synthetic Biology Make a Case for the Creator February 1, 2011
38. Origin-of-Life Expert Jokes about Becoming a Creationist   11/05/2004
39. Norio Kitadai: Origins of building blocks of life: A review July 2018
40. Brian J. Cafferty: Was a Pyrimidine-Pyrimidine Base Pair the Ancestor of Watson-Crick Base Pairs? Insights from a Systematic Approach to the Origin of RNA 23 April 2015
41. Terence N. Mitchell: The “RNA World” 2008
42. Emily Singer New Twist Found in the Story of Life’s Start OCTOBER 11, 2016
43. Gerald F Joyce: The Origins of the RNA World 2012 May; 4
44. Gaspar Banfalvi: Ribose Selected as Precursor to Life 30 Jan 2020
45. Roger D. Blandford: The Chiral Puzzle of Life 2020
46. Jim Cleaves II: Encyclopedia of Astrobiology: Formose Reaction 2011
47. 
Steven A. Benner: The ‘‘Strong’’ RNA World Hypothesis: Fifty Years Old    2013 Apr;13
48. Steven A. Benner: Asphalt, Water, and the Prebiotic Synthesis of Ribose, Ribonucleosides, and RNA March 28, 2012
49. Rich Deem: Origin of life: latest theories/problems June 2001
50. Change Laura Tan, Rob

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The problem with such experiments is that they start with Ribose 5-phosphate (R5P) which is already a complex molecule that was not available on the prebiotic earth.  Once all the parts would have been available, they would have had to be joined together at the same assembly site,  and sorted out from non-functional molecules. 

Nucleotide biosynthesis regulation
Rani Gupta (2021):  Nucleotide biosynthesis is regulated by feedback inhibition, feed-forward activation as well as by cross-regulation. Nucleotide analogs, precursor/substrate analogs and inhibitors of folic acid pathway can inhibit nucleotide biosynthesis. 15

Since biosynthesis regulation had to be extant at LUCA, researchers have to explain the emergence of all these complex feedback systems before life started without invoking natural selection & evolution. Instantiating systems that can monitor, fine-tune and regulate complicated production systems is a major challenging task depending on the knowledge and pre-set and foresight of specific targets, and what is intended to be achieved.  

Srivatsan Raman (2014): Microbes can be made to produce industrially valuable chemicals in high quantities by engineering their central metabolic pathways. Through iterations of genetic diversification and selection, we increased the production of naringenin and glucaric acid 36- and 22-fold, respectively. Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers.  Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. 16

Engineering, selecting, optimizing, specifying an optimal design, evaluating, elaborating strategies, goal-oriented elimination and preservation and identifying, are all clear activities that require mental elaboration, and are best assigned to an intelligent setup. Daniel Charlier's scientific paper (2018) about the crossroad of arginine and pyrimidine biosynthesis in E.Coli bacteria gives us insight into how cells tackle this task: He writes:

In all organisms, carbamoylphosphate (CP) ( which is the second intermediate product in pyrimidine synthesis ) is a precursor common to the synthesis of arginine and pyrimidines. In Escherichia coli and most other Gram-negative bacteria, CP is produced by a single enzyme, carbamoylphosphate synthase (CPSase). This particular situation poses a question of basic physiological interest: what are the metabolic controls coordinating the synthesis and distribution of this high-energy substance in view of the needs of both pathways? The study of the mechanisms has revealed unexpected moonlighting gene regulatory activities of enzymes and functional links between mechanisms as diverse as gene regulation and site-specific DNA recombination. At the level of enzyme production, various regulatory mechanisms were found to cooperate in a particularly intricate transcriptional control of a pair of tandem promoters. Transcription initiation is modulated by an interplay of several allosteric DNA-binding transcription factors using effector molecules from three different pathways (arginine, pyrimidines, purines), nucleoid-associated factors (NAPs), trigger enzymes (enzymes with a second unlinked gene regulatory function), DNA remodeling (bending and wrapping), UTP-dependent reiterative transcription initiation, and stringent control by the alarmone ppGpp. At the enzyme level, CPSase activity is tightly controlled by allosteric effectors originating from different pathways: an inhibitor (UMP) and two activators (ornithine and IMP) that antagonize the inhibitory effect of UMP. Furthermore, it is worth noticing that all reaction intermediates in the production of CP are extremely reactive and unstable, and protected by tunneling through a 96 Å long internal channel. 17

The instantiation of complex network systems that autonomously coordinate, regulate, cooperate, modulate, remodel, control, and protect ( which are all processes to achieve specific results ), require careful planning and engineering skills in order to be instantiated.  In the list of ten things that can be safely attributed as signatures of intelligent setup & design are artifacts which use might be employed in different systems. In the above case, it is one metabolic network, that is used to manufacture different end-products, all needed in the overarching function of the system.

The difficult ( if not impossible) task of prebiotic RNA and DNA synthesis on early earth
Kepa Ruiz-Mirazo (2013): The chemistry of nucleotides seems to be, by far, the most complicated one among the different bio-monomers, involving many independent reactions that had to be optimized and coupled in order to give an overall efficient process. This is the reason why, up to now, modular approaches have failed to solve the question of whether it was possible that RNA monomers could be synthesized on the early Earth 13

ROBERT SHAPIRO clarifies some important points. He was interviewed by J.Craig Venter in 2008:

 I then spent decades running a laboratory in DNA chemistry, and so many people were working on DNA synthesis — which has been put to good use as you can see — that I decided to do the opposite, and studied the chemistry of how DNA could be kicked to Hell by environmental agents. Among the most lethal environmental agents I discovered for DNA — pardon me, I'm about to imbibe it — was water. Because water does nasty things to DNA. For example, there's a process called DNA animation, where it kicks off part of the coding part of DNA from the units — that was discovered in my laboratory. Another thing water does is help the information units fall off of DNA, which is called depurination and ought to apply only one of the subunits — but works under physiological conditions for the pyrimidines as well, and I helped elaborate the mechanism by which water helped destroy that part of DNA structure. 

Since then, so-called prebiotic chemistry, which is of course falsely named, because we have no reason to believe that what they're doing would ever lead to life — I just call it 'investigator influenced abiotic organic chemistry' — has fallen into the same trap. In the proceedings of the National Academy of Sciences about two months ago there was a paper — I think it was theoretical — they showed that in certain hydro-thermal events, convection forces and other attractive forces, about which I am unable to comment, would serve to concentrate organic molecules so that organic molecules would get much more concentrated in the bottom of this than they would in the ordinary ocean. Very nice, perhaps it's a good place for the origin of life, and interesting finding, but then there was another commentary paper in the Proceedings by another invited commentator, who said,
Great advance for RNA world because if you put nucleotides in, they'll be concentrated enough to form RNA; and if you put RNA in, the RNA will come together and form aggregates, giving you much more chance of forming a ribosome or whatever. I looked at the paper and thought, How did nucleotides come in? How did RNA come in? How did anything come in? The point is, you would take whatever mess prebiotic chemistry gives you and you would concentrate that mess so it's relevant to RNA or the origin of life — it's all in the eye of the beholder. And almost all of prebiotic chemistry is like this; they take chemicals of their own selection.

People were talking about Steve Benner and his borate paper where he selected, of his own free will, the chemical formaldehyde, the chemical acid-aldehyde, and the mineral borate, and he decided to mix them together and got a product that he himself said was significant in leading to the origin of RNA world, and I, looking at the same thing, see only the hands of Steve Benner reaching to the shelf of organic chemicals, picking formaldehyde, and from another shelf, picking acidaldehyde, etc. Excluding them carefully. Picking a mineral that occurs only in selective places on the Earth and putting it in heavy doses. And at the end getting a complex of ribose and borate, which by itself would be of no use for making RNA, because the borate loves to hold onto the ribose, and as long as it holds onto the ribose it can't be used to make RNA. If it lets go of the ribose, then the ribose becomes vulnerable to destruction by all the other environmental agents. The half-life of pure ribose in solution, a different experiment and a very good one, by Stanley Miller is of the order of one or two hours, and all of the other sugars prominent in Earth biology have similar instability.

I was publishing papers like this and I got the reputation, or the nickname in the laboratory of the prebiotic chemist, of 'Dr. No'. If someone wanted a paper murdered, send it to me as a referee. At some point, someone said, Shapiro, you've got to be positive somewhere. So how did life start? And do we have any examples of authentic abiotic chemistry, not subject to investigator interference? The only true samples we have are those meteorites, which are scooped up quickly and often fallen in an unspoiled place — there was a famous meteorite that fell in France in a sheep field in the 1840s and led to dreadful chemistry of people seeing all sorts of biomolecules in it, not surprisingly. But if you took pristine meteorites and look inside, what you see are a predominance of simple organic compounds. The smaller the organic compound, the more likely it is to be present. The larger it is, the less likely it is to be present. Amino acids, yes, but the simplest ones. Over a hundred of them. All the simplest ones, some of which, coincidentally, overlap the unique set of 20 that coincide with Earth life, but not containing the larger amino acids that overlap with Earth life. 

And no sample of a nucleotide, the building block of RNA or DNA, has ever been discovered in a natural source apart from Earth life. Or even take off the phosphate, one of the three parts, and no nucleoside has ever been put together. Nature has no inclination whatsoever to build nucleosides or nucleotides that we can detect, and the pharmaceutical industry has discovered this. Life had to start with the mess — a miscellaneous mixture of organic chemistry to begin with. How do you organize this? You have to have a preponderance of some chemicals or lacking others would be against the second law of thermo-dynamics — it violates a concept that as a non-physicist that I barely grasp called 'entropy'.

In the simplest case, and there may be many more elaborate cases, they found that the energy wouldn't be released unless some chemical transformations took place. If the chemical transformations took place then the energy was released, a lot of it is heat. If this just went on continuously, all you do is use up the energy. Release all of it and you've converted one chemical to another. Big deal. To get things interesting, you have to close the cycle where the chemicals can be recycled by processes of their own, and then go through it again, releasing more energy. And once you have that, you can then develop nodes — because organic chemistry is very robust, there are reaction pathways leading everywhere, which is why it's such a mess.

One doesn't need a freak set of perhaps a hundred consecutive reactions that will be needed to make an RNA, and life becomes a probable thing that can be generated through the action of the laws of chemistry and physics, provided certain conditions are met. You must have the energy. It's good to have some container or compartment because if your products just diffuse away from each other and get lost and cease to react with one another you'll eventually extinguish the cycle. You need a compartment, you need a source of energy, you need to couple the energy to the chemistry involved, and you need sufficiently rich chemistry to allow for this network of pathways to establish itself. Having been given this, you can then start to get evolution.

The RNA world, and the origins of life - Page 2 Robert11

Shapiro wrote in: 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. 5

A. Graham Cairns-Smith also lists several hurdles that would have to be overcome in his book: Genetic takeover, page 64:

What is missing from this story of the evolution of life on earth is the original means of producing such sophisticated materials as RNA. The main problem is that the replication of RNA depends on a clean supply of rather complicated monomers—activated nucleotides. What was required to set the scene for an RNA world was a highly competent, long-term means of production of at least two nucleotides. In practice the discrimination required to make nucleotide parts cleanly, or to assemble them correctly, still seems insufficient. 

The implausibility of prevital nucleic acid 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 to make RNA & DNA

1. 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.
2. high-energy precursors of purines and pyrimidines had to be produced in a sufficiently concentrated form (for example at least 0.01 M HCN).
3. the conditions must now have been right for reactions to give perceptible yields of at least two bases that could pair with each other.
4. 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.
5. in some other locations a formaldehyde concentration of above 0.01 M must have built up.
6. this accumulated formaldehyde had to oligomerize to sugars.
7. somehow the sugars must have been separated and resolved, so as to give a moderately good concentration of, for example, D-ribose.
8. bases and sugars must now have come together.
9. they must have been induced to react to make nucleosides. (There are no known ways of bringing about this thermo dynamically uphill reaction in an 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
10. 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 a- or fl-anomer of either the furanose or pyranose forms. For nucleic acids, it has to be the fl-furanose. (In the dry-phase purine nucleoside syntheses referred to above, all four of these isomers were present with never more than 8 ‘Z, of the correct structure.)
11. 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 etc.   
12. 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.
13. 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. Longer heating gave the nucleoside cyclic 2’,3’-phosphate as the major product although various dinucleotide derivatives and nucleoside polyphosphates are also formed
14. if not already activated — for example as the cyclic 2’,3’-phosphate — the nucleotides must now be activated (for example with polyphosphate) 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.
15. the activated nucleotides (or the nucleotides with coupling agent) must now have polymerized. Initially this must have happened without a pre-existing polynucleotide template (this has proved very difficult to simulate ; 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.
(i) 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.
(ii) 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 N/\ n K/N/P-r’o%OHN/\N were more successful, but these now involve further steps and a supply of imidazole, for their synthesis.
(iii) 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 Zn“, as well as acting as an eflicient catalyst for the template-directed oligomerization of guanosine 5’-phosphorimidazolide also leads to a preference for the 3’—5’ bonds.
16. the physical and chemical environment must at all times have been suitable — for example the pH, the temperature, the M2+ concentrations.
17. 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.
18. 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.
19. 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.


The RNA world, and the origins of life - Page 2 Cairns10

As the difficulties accumulate the stakes get higher: success would be all the more resounding, but it becomes less likely. Sooner or later it becomes wiser to put your money elsewhere.
 2

M. Gargaud and colleagues detail the size of the problem:

One of the principal problems concerning the hypothesis of the RNA world is that it appears quite unlikely that a prebiotic environment could have existed containing the mixture of activated nucleotides favoring the formation and replication of ribozymes, as well as their evolution through natural selection. Even if there were several candidate reactions for the efficient prebiotic synthesis of nucleic bases, access to monomeric nucleotides by chemical pathways in fact comes up against several obstacles. If one goes no further than mimicking the biochemical pathway, the first difficulty that occurs is that of synthesizing ribose, which is formed in just negligible quantities within the complex mixture obtained by polymerization of formaldehyde, and, what is more, has a limited lifetime. The bond between a nucleic base and ribose that produces a nucleoside is then a  very difficult reaction. There still remains the matter of obtaining a nucleotide by phosphorylation, which leads to mixtures because three positions remain available on the ribose, and then there is its activation. So there are two possibilities, either to envisage an easier pathway for the prebiotic synthesis of nucleotides or to squarely reject RNA as the initial bearer of information, in favor of an alternative bearer that has not left any evolutionary traces. 4

Unsolved issues regarding nucleic acid synthesis 
How would the early Earth have generated and maintained organic molecules? All that can be said is that there might have been prebiotic organic chemistry going on, at least in special locations.
How would prebiotic processes have purified the starting molecules to make RNA and DNA which were grossly impure? They would have been present in complex mixtures that contained a great variety of reactive molecules.
How did the synthesis of the nitrogenic nucleobases in prebiotic environments occur?
How did fortuitous accidents select the five just-right nucleobases to make DNA and RNA, Two purines, and three pyrimidines?
How did unguided non-designed events select purines with two rings, with nine atoms, forming the two rings: 5 carbon atoms and 4 nitrogen atoms, amongst almost unlimited possible configurations?
How did lucky coincidence pick pyrimidines with one ring, with six atoms, forming its ring: 4 carbon atoms and 2 nitrogen atoms, amongst an unfathomable number of possible configurations?
How did random trial and error foresee that this specific atomic arrangement of the nucleobases is required to get the right strength of the hydrogen bond to join the two DNA strands and form Watson–Crick base-pairing?
How did mechanisms without external direction foresee that this specific atomic arrangement would convey one of, if not the best possible genetic system to store information?
How would these functional bases have been separated from the confusing jumble of similar molecules that would also have been made?
How were high-energy precursors to produce purines and pyrimidines produced in a sufficiently concentrated form and joined to the assembly site?
How could the adenine-uracil interaction function in any specific recognition scheme under the chaotic conditions of a "prebiotic soup" considering that its interaction is weak and nonspecific?
How could the ribose 5 carbon sugar rings which form the RNA and DNA backbone have been selected, if 6 or 4 carbon rings, or even more or less, are equally possible but non-functional?
How would the functional ribose molecules have been separated from the non-functional sugars?
How could right-handed configurations of RNA and DNA have been selected in a racemic pool of right and left-handed molecules? Ribose must have been in its D form to adopt functional structures ( The homochirality problem )
How was exclusively β-D-ribofuranose chosen in nucleic acids over pyranose, given that the former species are substantially more stable at equilibrium?
How were the correct nitrogen atom of the base and the correct carbon atom of the sugar selected to be joined together?
How could random events have brought all the 3 parts together and bonded them in the right position ( probably over one million nucleotides would have been required ?)
How could prebiotic reactions have produced functional nucleosides? (There are no known ways of bringing about this thermodynamically uphill reaction in aqueous solution)
How could prebiotic glycosidic bond formation between nucleosides and the base have occurred if they are thermodynamically unstable in water, and overall intrinsically unstable?
How could  RNA nucleotides have accumulated, if they degrade at warm temperatures in time periods ranging from nineteen days to twelve years? These are extremely short survival rates for the four RNA nucleotide building blocks.
How was phosphate, the third element, concentrated at reasonable concentrations?. (The concentrations in the oceans or lakes would have been very low)
How would prebiotic mechanisms phosphorylate the nucleosides at the correct site (the 5' position) if, in laboratory experiments, the 2' and 3' positions were also phosphorylated?
How could phosphate have been activated somehow? In order to promote the energy dispendious nucleotide polymerization reaction, and (energetically uphill) phosphorylation of the nucleoside had to be possible.
How was the energy supply accomplished to make RNA? In modern cells, energy is consumed to make RNA.
How could a transition from prebiotic to biochemical synthesis have occurred? There are a huge gap and enormous transition that would be still ahead to arrive at a fully functional interlocked and interdependent metabolic network.
How could  RNA have formed, if it requires water to make them, but RNA cannot emerge in water and cannot replicate with sufficient fidelity in water without sophisticated repair mechanisms in place?
How would the prebiotic synthesis transition of RNA to the highly regulated cellular metabolic synthesis have occurred?  The pyrimidine synthesis pathway requires six regulated steps, seven enzymes, and energy in the form of ATP.
The starting material for purine biosynthesis is Ribose 5-phosphate, a product of the highly complex pentose phosphate pathway, which uses 12 enzymes. De novo purine synthesis pathway requires ten regulated steps, eleven enzymes, and energy in the form of ATP.
How did formaldehyde concentration of above 0.01 M build up?
How did accumulated formaldehyde oligomerise to sugars?
How were they induced to react to make nucleosides? (There are no known ways of bringing about this thermo dynamically 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


Linking the building blocks
During DNA replication, in order to make daughter cells, DNA monomers are linked together to form genomes, and chromosomes identical to the mother cell, the information-bearing molecule of life. Using the RNA polymerase machine complex, DNA is transcribed to messenger RNA (mRNA), which are long strands of joined RNA monomers, forming mRNA polymers,  that store the "message" which is sent to the ribosome where the message is translated. The ribosome based on the instructions from mRNA polymerizes amino acids, strands that fold to become proteins, the working horses of the cell. The sequence of mRNA dictates the sequence of amino acids, which is obtained through translation, using the genetic code. 3 nucleotides form a codon "word", that is assigned to one of the 20 amino acids used in life.  In modern cells, ultra-complex machinery does the polymerization work. But prebiotically, these machines were not extant. That raises the question: How did the first RNA, DNA, and amino-acid polymer strands emerge prebiotically? The synthesis of proteins and nucleic acids from small molecule precursors represents one of the most difficult challenges to the model of pre-biological ( chemical) evolution.

Tan, Change; Stadler, Rob. The Stairway To Life (2020): Consistent Linkage of Building Blocks in living organisms, RNA, DNA, and proteins are chains of monomers that are linked together with perfect consistency, like boxcars perfectly aligned on the tracks and interconnected to form a long train. This “homolinkage” of long biopolymers is very difficult to achieve abiotically, even in modern laboratories run by human intellect. Abiotic chemical reactions to link chains of monomers end up looking more like a train derailment unless complex and highly controlled chemical reactions are employed to connect each monomer correctly 1

Prebiotic RNA and DNA polymerization
Another major problem that origin of life research faces is how to explain the transition from monomer ribonucleotides to polynucleotides. The emergence and existence of catalytic polymers are fundamental. Postulates of how polymerization could have occurred on prebiotic earth are, therefore, another essential question that has not been elucidated.  Initially, this could not have happened with a pre-existing polynucleotide template. In the case of RNA, not only must phosphodiester links be repeatedly forged, but they must ultimately connect the 5 prime‑oxygen of one nucleotide to the 3 prime‑oxygen, and not the 2 prime‑oxygen, of the next nucleotide. How could and would random events attach a phosphate group to the right position of a ribose molecule to provide the necessary chemical activity?  Pierre-Alain Monnard (2012): A fundamental requirement of the RNA world hypothesis is a plausible nonenzymatic polymerization of ribonucleotides that could occur in the prebiotic environment, but the nature of this process is still an open issue. 6

In present-day cells, polymerization is carried out by enzymes with high efficiency and specificity. Enzymes are genetically encoded polymers requiring complex, protein-based synthetic machinery.
Observe what Dr. Pierre-Alain Monnard et al. (2012) write: Selection toward highly efficient catalytic peptides, which eventually resulted in present-day enzymes, could have started at a very early stage of chemical evolution. 31

This is an entirely unsupported claim.In living organisms today, adenosine-5'-triphosphate (ATP) is used for the activation of nucleoside phosphate groups, but ATP would not be available for prebiotic syntheses. Joyce and Orgel note the possible use of minerals for polymerization reactions, but then express their doubts about this possibility.

Robert P. Bywater (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 requires 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 32

Geoffrey Zubay, Origins of Life on the Earth and in the Cosmos (2000): Once the mononucleotide has been made, it must be converted to an activated derivative suitable for incorporation into a polynucleotide chain. In biochemical pathways, the nucleoside triphosphate derivative is usually used. The triphosphate derivative has more than enough chemical energy to power the formation of the phosphodiester linkages found in polynucleotides so there is no thermodynamic problem here. However, these compounds are not very reactive. In biosystems, sophisticated polymerases are essential to catalyze the polymerization of nucleoside triphosphates. Orgel and others have searched for other forms of activated nucleotides that would be reactive under mild conditions and would not require any more than a divalent cation catalyst. Their extensive search has led them to the use of imidazole-activated mononucleotides. Compounds of this type can be synthesized very efficiently by organo-chemical methods but a satisfactory prebiotic route for their synthesis has not been discovered. Because the reaction of imidazole and a mononucleotide involves the loss of a water molecule, a remote possibility is that the phosphorimidazolide is formed under dehydrating conditions. The formation of activated nucleotides by a prebiotically plausible route remains a most challenging problem. 25

Libretext: Phosphodiester bonds are central to all life on Earth as they make up the backbone of the strands of nucleic acid. In DNA and RNA, the phosphodiester bond is the linkage between the 3' prime carbon atom of one sugar molecule and the 5' prime carbon atom of another, deoxyribose in DNA and ribose in RNA. In modern cells, in order for the phosphodiester bond to be formed and the nucleotides to be joined, the tri-phosphate or di-phosphate forms of the nucleotide building blocks are broken apart to give off energy required to drive the enzyme-catalyzed reaction. Once a single phosphate or two phosphates (pyrophosphates) break apart and participate in a catalytic reaction, the phosphodiester bond is formed. 4 

Saidul Islam (2017): Laboratory-based chemical syntheses of ribonucleotides do most, if not all, require manipulation of sugars and nucleobases with protecting group strategies to overcome the thermodynamic and kinetic pitfalls that prevent their fusion. 2

Deamer (2010): The general problem regarding the condensation of small organic molecules to form macromolecules in an aqueous environment is the thermodynamically unfavorable process of water removal. In the current biosphere, these types of reactions are catalyzed by enzymes and energetically driven by pyrophosphate hydrolysis. 4

Deamer interviewed by Suzan Masur ( 2014): In a solution of monomers, such as monomers of RNA or DNA in solution, the laws of thermodynamics do not allow them to polymerize because there is a tremendous energy barrier to getting them to form bonds. 44

Weber, Arthur L.(1998): Obviously, biocatalysts and energy-rich inorganic phosphorus species were not extant on the Earth before life began. In all cases, the starting problem in a prebiotic synthesis would be the fact that materials would consist of an enormous amount of disparate molecules lying around unordered, and would have had to be separated and sorted out. 6

Allaboutscience: The intrinsic nature of the phosphodiester bonds is also finely-tuned. For instance, the phosphodiester linkage that bridges the ribose sugar of RNA could involve the 5’ OH of one ribose molecule with either the 2’ OH or 3’ OH of the adjacent ribose molecule. RNA exclusively makes use of 5’ to 3’ bonding. There are no explanations of how the right position could have been selected abiotically in a repeated manner in order to produce functional polynucleotide chains.  As it turns out, the 5’ to 3’ linkages impart far greater stability to the RNA molecule than do the 5’ to 2’ bonds. Nucleotides can polymerize via condensation reactions.  The activated nucleotides (or the nucleotides with coupling agent) now had to be polymerized. 7

Arthur V. Chadwick, Ph.D. (2005): When produced and condensed with a nucleotide base, a mixture of optical isomers results, only one of which is relevant to pre-biological studies. Polymerization of nucleotides is inhibited by the incorporation of such an enantiomorph. While only 3'-5' polymers occur in biological systems, 5'-5' and 2'-5' polymers are favored in pre-biological type synthetic reactions. 13

A recent paper from Steven Benner and co-workers (2022) claimed: This study shows that various mafic rock glasses almost certainly present on the surface of the Hadean Earth catalyze the formation of polyribonucleic acid in water starting from nucleoside triphosphates. 21

RNA spontaneously forms on basalt lava glass in the presence of nucleoside triphosphates. This is a simple reaction, which is expected to happen spontaneously in various conditions. Nucleoside triphosphates however are compounds that were not around prebiotically - only living cells synthesize them through very complex biosynthesis pathways. They require long chains of complex enzyme-catalyzed reactions, and also energy, which were not around on early earth.

The RNA world, and the origins of life - Page 2 Steve_10
Steven Benner (2008) The red bonds in RNA are each unstable in water. Each of these bonds represents a problem for the prebiotic synthesis of RNA in water, even after the building blocks are in hand since the synthesis of these bonds requires the loss of water. Further, even if the RNA could be made, the red bonds would break in water. In modern life, damage done by water to RNA  and DNA is repaired. Such repair systems were preusumably not present prebiotically. Another paradox. In water, adenine, guanine, and cytosine all eventually lose their NH2 units, the phosphate backbone of  RNA hydrolyzes, and the nucleobases will fall off of ribose. 22

Steven Benner (2012) 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., Mg2+). This could, of course, be a serious (and possibly fatal) flaw to the RNA-first hypothesis for bio-origins. 45

Steven Benner (2014)The Water Paradox: Water is commonly viewed as essential for life, and theories of water are well known to support this as a requirement. So are biopolymers, like RNA, DNA, and proteins. However, these biopolymers are corroded by water. For example, the hydrolytic deamination of DNA and RNA nucleobases is rapid and irreversible, as is the base-catalyzed cleavage of RNA in water. This allows us to construct a paradox: RNA requires water to function, but RNA cannot emerge in water, and does not persist in water without repair. Any solution to the “origins problem” must manage the paradox forced by pairing this theory and this observation; life seems to need a substance (water) that is inherently toxic to polymers (e.g. RNA) necessary for life 24

Westheimer (1987) Although RNA is a phosphodiester and carries a negative charge, it is relatively susceptible to hydrolysis; the rate of its spontaneous reaction with water, extrapolated to room temperature, is about 100 times greater than that of DNA 30

The instability problem
Pekka Teerikorpi (2009): A further problem in the accumulation of long RNA polymers is their inherent instability. RNA polymers are very easily broken into parts by hydrolysis, and their functional sequence could have been easily lost via multiple copying mistakes or mutations. Considering all these chemical obstacles, it seems that the whole reaction cascade for the formation of functional polynucleotides (including the synthesis of the nucleoside bases and ribose, assembly of nucleosides, their phosphorylation and activation, and finally, the polymerization and stabilization of the polymers) has been very difficult in the prebiotic conditions. These processes seem so unlikely that it has been proposed that some other information storing and transfer mechanisms preceded the RNA world and then “guided” the formation (or provided catalysts for) the RNA-based world. But it is not easy to explain how the transfer from a more primitive genetic system into RNA could have happened. 36

Phosphodiester bonds
Activated monomers are essential because polymerization reactions occur in an aqueous medium and are therefore energetically uphill in the absence of activation. A plausible energy source for polymerization remains an open question. Condensation reactions driven by cycles of anhydrous conditions and hydration would seem to be one obvious possibility but seem limited by the lack of specificity of the chemical bonds that are formed. 3

Libretexts: Phosphodiester bonds are central to most life on Earth, as they make up the backbone of the strands of DNA. In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA. Strong covalent bonds form between the phosphate group and two 5-carbon ring carbohydrates (pentoses) over two ester bonds. In order for the phosphodiester bond to be formed and the nucleotides to be joined, the tri-phosphate or di-phosphate forms of the nucleotide building blocks are broken apart to give off energy required to drive the enzyme-catalyzed reaction. When a single phosphate or two phosphates known as pyrophosphates break away and catalyze the reaction, the phosphodiester bond is formed. Hydrolysis of phosphodiester bonds can be catalyzed by the action of phosphodiesterases which play an important role in repairing DNA sequences. 8

Prebiotic phosphodiester bond formation
An often-cited claim is that RNA polymerization could be performed on clay. Robert Shapiro wrote a critique in regards to prebiotic proposals of clay-catalyzed oligonucleotide synthesis (2006): 
An extensive series of studies on the polymerization of activated RNA monomers has been carried out by Ferris and his collaborators. A recent publication from this group concluded with the statement: “The facile synthesis of relatively large amounts of RNA oligomers provides a convenient route to the proposed RNA world. The 35–40 oligomers formed are both sufficiently long to exhibit fidelity in replication as well as catalytic activity”. The first review cited above had stated this more succinctly: “The generation of RNAs with chain lengths greater than 40 oligomers would have been long enough to initiate the first life on Earth”. Do natural clays catalyze this reaction? The attractiveness of this oligonucleotide synthesis rests in part on the ready availability of the catalyst. Montmorillonite is a layered clay mineral-rich in silicate and aluminum oxide bonds. It is widely distributed in deposits on the contemporary Earth. If the polymerization of RNA subunits was a common property of this native mineral, the case for RNA at the start of life would be greatly enhanced. However, the “[c]atalytic activity of native montmorillonites before being converted to their homoionic forms is very poor”. The native clays interfere with phosphorylation reactions. This handicap was overcome in the synthetic experiments by titrating the clays to a monoionic form, generally sodium, before they were used. Even after this step, the activity of the montmorillonite depended strongly on its physical source, with samples from Wyoming yielding the best results. Eventually the experimenters settled on Volclay, a commercially processed Wyoming montmorillonite provided by the American Colloid Company 9

Selecting the binding locations
Once the three components would have been synthesized prebiotically, they would have had to be separated from the confusing jumble of similar molecules nearby, and they would have had to become sufficiently concentrated in order to move to the next steps, to join them to form nucleosides, and nucleotides. 

The phosphate/ribose backbone of DNA is hydrophilic (water-loving), so it orients itself outward toward the solvent, while the relatively hydrophobic bases bury themselves inside. 

Xaktly explains: Additionally, the geometry of the deoxyribose-phosphate linkage allows for just the right pitch, or distance between strands in the helix, a pitch that nicely accommodates base pairing. 10
Lots of things come together to create the beautiful right-handed double-helix structure. Production of a mixture of d- and l-sugars produces nucelotides that do not fit together properly, producing a very open, weak structure that cannot survive to replicate, catalyze, or synthesize other biological molecules. 

Eduard Schreiner (2011): In DNA the atoms C1', C3', and C4' of the sugar moiety are chiral, while in RNA the presence of an additional OH group renders also C2' of the ribose chiral.11

Rob Stadler (2021): Even in a very short DNA of just two nucleotides, there are dozens of incorrect possible arrangements of the components and only one correct arrangement. The probability of consistent arrangement decreases exponentially as the DNA lengthens. If natural processes could polymerize these monomers, the result would be chaotic “asphalt,” not highly organized, perfectly consistent biopolymers. Think about it — if monomers spontaneously polymerized within cells, the cell would die because all monomers would be combined into useless random arrangements. 18

Pier Luigi Luisi (2014): Attempts to obtain copolymers, for instance by a random polymerization of monomer mixtures, yield a difficult-to-characterize mixture of all different products. To the best of our knowledge, there is no clear approach to the question of the prebiotic synthesis of macromolecules with an ordered sequence of residues. 19

Stephen D. Fried (2022):The condensation reaction of nucleotides presents several challenges. First, the selective incorporation of 5′–3′ phosphodiester linkages represents a regioselectivity challenge, given the simultaneous presence of 2′ hydroxyls. Second, nucleobases possess numerous nucleophilic functional groups, which must compete with the 5′ and 3′ hydroxyl groups on the sugar as the donor to the phosphate group in condensation. Hence, the creation of linear polymers (as opposed to the combinatorially more facile highly branched structures) poses a statistical challenge. Third, the double negative charge present on terminal monophosphates render them quite unreactive without activation or catalysis. 48

Pekka Teerikorpi (2009): As described by Gerald Joyce (The Scripps Research Institute, La Jolla), a leading student of prebiotic RNA chemistry, the lack of specificity has indeed been a major problem of prebiotic reactions. The spontaneous reactions starting from hydrogen cyanide, or from cyanoacetylene, cyanate, and urea can lead to a number of different nucleobase analogs. But of all the analogs, only adenine and guanine purines, and cytosine and uracil pyrimidines were eventually used by nature for the formation of the functional nucleosides. In the composition of the nucleosides in prebiotic conditions, the existing bases could have been connected to the ribose components, just as well, both in α- and β-configuration, and the furanose (four-carbon) ring of ribose could have formed just as well in L and D isoforms (left- and right-handed). Ribose sugar could also have formed a five-carbon (pyranose) ring by binding the 5' and 1' carbons. Prebiotic polymerization reactions between all different nucleotide analogs and isoforms would have also led to a wide variety of different phosphate linkages between different carbon atoms of the ribose. Altogether, these reactions would have easily used different purine and pyrimidine variants, bound with different derivatives of different cyclic sugars, formed both in L- and D-configurations. These very random nucleoside analogs could then have been phosphorylated at different carbon positions, and then again, the randomly phosphorylated nucleotide analogs could have been connected to each other in a number of different ways as shown with light lettering in Fig. 30.5. None of these alternatives would have produced functional RNA polymers. Only the correctly formed and polymerized nucleotides would have been functional templates for replication via complementary base pairing. We do not understand how life, in the absence of any selective enzyme reactions, choose to use exactly these nucleotide components and their specific isoforms, or how it could control the formation of the phosphodiester bonds to occur only between the 5' and 3' carbons of the nucleotides. 36

The homochirality problem
A biological system exclusively uses d-ribose, whereas abiotic experiments synthesize both right- and lefthanded-ribose in equal amounts. But the pre-biological building blocks of life didn’t exhibit such an overwhelming bias. Some were left-handed and some right. So how did right-handed RNA emerge from a mix of molecules?  Some kind of symmetry-breaking process leading to enantioenriched bio monomers would have had to exist. But none is known. Gerald Joyce wrote a science paper that was published in Nature magazine, in 1984.  G. F. Joyce (1984):  This inhibition raises an important problem for many theories of the origin of life. 42 His findings suggested that in order for life to emerge, something first had to crack the symmetry between left-handed and right-handed molecules, an event biochemists call “breaking the mirror.” 

To polymerize proteins, it is essential that only left-handed amino acids are added to the chain. The same applies to ribozymes. First, a prebiotic ribozyme ( able to catalyze its self-replication as a template) would have had to emerge and form spontaneously from a pool of racemic mixture of left and right-handed RNA, selecting only enantiomeric pure monomers, incorporating them in the chain. Secondly, in huge sequence space of nonfunctional sequences, it would have to select one that bears function. Once it would start performing template-directed reactions it would have only a racemic mixture at its disposal, monomers of the opposite handedness to the template would be incorporated as chain terminators at the 2′(3′) end of the products. This would end the sequence, and no copy of itself would be the product.

Stu Borman (2014): No known modern-day RNA-based enzyme can assemble RNA from a racemic soup of left- and right-handed RNA building blocks, the form in which RNA likely would have existed prior to the origin of an RNA world. To develop such a ribozyme, chemical biologist Gerald F. Joyce and postdoc Jonathan T. Sczepanski of Scripps Research Institute California used directed evolution. Like modern RNAs, the new ribozyme has d chirality. But unlike them, it catalyzes the template-directed poly­merization of RNAs of opposite handedness, the joining together of l-RNA building blocks bound to an l-RNA template. It ignores d-RNA building blocks that may be around.

Gerald F. Joyce (2014): Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer. This finding poses a severe challenge for the spontaneous emergence of RNA-based life.  It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D- and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one-handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness.  41

The evident problem is outlined a bit later in Stu Borman's article: The study does not directly address how a cross-chiral ribozyme that itself has pure chirality “could have emerged de novo from an achiral mix of nucleotides. Early-world cross-chiral systems “would at some point have to transition to today’s homochiral systems” and that it is difficult to envisage how that could occur.” 

Since then, scientists have largely focused their search for the origin of life’s handedness in the prebiotic worlds of physics and chemistry, not biology - but with no success. So what is the cop-out? Pure chance !! Luck did the job. That is the only thinkable explanation once God's guiding hand is excluded. How could that be a satisfying answer in face of the immense odds? It is conceivable that the molecules were short enough for all possible sequences, or almost, to be realized (by way of their genes) and submitted to natural selection. This is the way de Duve thought that Intelligent Design could be dismissed. This coming from a Nobel prize winner in medicine makes one wondering, to say the least.  De Duve dismissed intelligent design and replaced it with natural selection. Without providing any of evidence. A claim, based on pure guesswork and speculation.



1. Robert Shapiro: Small Molecule Interactions were Central to the Origin of Life 2006
2. A. Graham Cairns-Smith: Genetic Takeover: And the Mineral Origins of Life 1988
3. 
4. M. Gargaud: Young Sun, Early Earth  and the Origins of Life 2012
5. Robert Shapiro: Origins : A Skeptic's Guide to the Creation of Life on Earth  January 1, 1986
6. 
7. Lena Vincent: The Prebiotic Kitchen: A Guide to Composing Prebiotic Soup Recipes to Test Origins of Life Hypotheses 11 November 2021
8. S.Maruyama: [url=https://researchoutreach.org/articles/origin-life-conditions-sparked-life-earth/#:~:text=energy%2Dmaterial circulation.-,The earliest Earth was a 'naked planet;' the Hadean,pelted by aqueous asteroid material.]The origin of life: The conditions that sparked life on Earth[/url] December 23, 2019
9. Bernard Marty: [url=https://www.sciencedirect.com/science/article/pii/S1631071318300221#:~:text=(1997) estimated that the Archaean,trapped in quartz%2Dgoethite deposits.]Salinity of the Archaean oceans from analysis of fluid inclusions in quartz [/url]12 December 2017
10. Dr. Stanley L. Miller: From Primordial Soup to the Prebiotic Beach: An interview with exobiology pioneer 1994
11. Kepa Ruiz-Mirazo: Prebiotic Systems Chemistry: New Perspectives for the Origins of Life December 30, 2011
12. Freeman Dyson:  Origins of Life 2nd Edition 2010
13. Kepa Ruiz-Mirazo: Prebiotic Systems Chemistry: New Perspectives for the Origins of Life October 31, 2013
14. Yasuji Sawada: A Thermodynamic Approach towards the Question “What is Cellular Life?” 20 Jul 2020
15. Rani Gupta: [url=https://link.springer.com/chapter/10.1007/978-981-16-0723-3_19#:~:text=other pyrimidine nucleotides.-,Nucleotide biosynthesis is regulated by feedback inhibition%2C feed%2Dforward activation,pathway can inhibit nucleotide biosynthesis.]Nucleotide Biosynthesis and Regulation[/url] 21 April 2021
16. Srivatsan Raman: Evolution-guided optimization of biosynthetic pathways December 1, 2014
17. Daniel Charlier: Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis 20 September 2018
18. David Deamer: [url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7133448/#:~:text=We present a testable hypothesis,and dehydration to form protocells.]The Hot Spring Hypothesis for an Origin of Life[/url] 2020 Mar 25
19. Norio Kitadai: Origins of building blocks of life: A review 2017
20. M. S. Dodd: Evidence for early life in Earth’s oldest hydrothermal vent precipitates 2017
21. Lena Vincent: The Prebiotic Kitchen: A Guide to Composing Prebiotic Soup Recipes to Test Origins of Life Hypotheses 11 November 2021



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31The RNA world, and the origins of life - Page 2 Empty The RNA world - a failed hypothesis Sun Jul 10, 2022 10:56 am

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The RNA world - a failed hypothesis

https://reasonandscience.catsboard.com/t2024p25-the-rna-world-and-the-origins-of-life#9366

The term “RNA world” was first coined in the science paper: Origin of life: The RNA world, published in 1986 by Walter Gilbert 29. It is probably not only the most extensively investigated hypothesis for the origin of life but is also the most popular, hailed by many as the most plausible of how cells emerged on the early earth, and kickstarted life. 

For example, Harold S Bernhardt (2012) wrote in a paper giving the title: The RNA world hypothesis: the worst theory of the early evolution of life (except for all the others) still wrote in the concluding remarks: 
I have argued that the RNA world hypothesis, while certainly imperfect, is the best model we currently have for the early evolution of life. [url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495036/#:~:text=However%2C the following objections have,of RNA is too limited.]33[/url] Others express the same opinion. For example:
Florian Kruse (2019): The RNA world hypothesis is the central consensus in the origins of life research, although many questions arising from this hypothesis have not yet been answered. 35
Jessica C. Bowman (2015): An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research.  

The RNA World Hypothesis is actually a group of related models, with a variety of assumptions and definitions. In all variations of the RNA World Hypothesis, RNA enzymes (ribozymes) predate protein enzymes. Ribozymes performed a variety of catalytic functions in the RNA World, from metabolite biosynthesis to energy conversion. The defining ribozyme of the RNA World, which unites all RNA World models, performed template-directed synthesis of RNA: in the RNA World, RNA self-replicated. 
36

Not all researchers are however that enthusiastic. Italian chemist and OoL researcher Pier Luigi Luisi, for example, interviewed by Suzan Mazur, responded (2012): The most popular view of Origin of Life, by way of the RNA world, to me and to many others is and always has been a fantasy. This is the theory by which self-replicating RNA arose by itself. Self-replicating also means Darwinian evolution. This, according to the story, produces ribozymes, nucleic acid also capable of catalysis. Ribozymes capable of catalyzing the synthesis of DNA and protein. How did self-replicating RNA arise? And, even granted that, how do we go from this to our DNA/protein cells? It is all in the air, still. 43

Life would coincide with the start of a first self-replicating entity, a jack-of-all-trades super-RNA molecule, which would as a world's first, rule and dominate, and somehow promote both, genetics and catalysis, operating initially both functions performed by DNA and proteins, starting to generate and process information, in parallel replicating, performing metabolic transformations and functions ( similar to proteins) and evolve through natural selection. 

Harris Bernstein:  (2020): In early protocellular organisms the genome is thought to have consisted of ssRNAs (genes) that formed folded structures with catalyic activity (ribozymes) 34

Such a transitional state of affairs, from non-life to life, has never been observed. The imagination led very far. According to the narrative, after the emergence of replicating molecules (replicases) that could self-replicate, short amino acid peptides from the prebiotic soup would have joined RNAs, and given rise to the RNA-peptide world, enhancing its catalytic efficiency.  Westheimer (1987) hypothesized that:  the greater structural variety of amino acids permitted better catalytic properties in protein enzymes than in those composed of RNA 30 and given rise to the much more complex DNA–RNA–proteins interdependence, genetic information directing the making and operation of proteins, and subsequent descendant generations would undergo further mutations, creating metabolic networks, promoting growth and division, the fittest survive, gaining new abilities and getting more complex, and evolve into a progenote, into a cenansestor, a first, and a last universal common ancestor, that then would give rise to the three domains of life. 

The RNA world, and the origins of life - Page 2 Rna_wo10
Creative Commons CC0 License; 
Hannes Mutschler (2019): A schematic representation of the classical RNA world hypothesis. 
Initially, synthesis and random polymerization of nucleotides result in pools of nucleic acid oligomers, in which template-directed non-enzymatic replication may occur. Recombination reactions result in the generation of longer oligomers. Both long and short oligomers can fold into structures of varying complexity, resulting in the emergence of functional ribozymes. As complexity increases, the first RNA replicase emerges, and encapsulation results in protocells with distinct genetic identities capable of evolution. In reality, it is likely that multiple processes occurred in parallel, rather than in a strictly stepwise manner, and encapsulation may have occurred at any stage. 34

Museum of science: Until relatively recently, it was thought that proteins were the only biological molecules capable of catalysis.  In the early 1980s, however, research groups led by Sidney Altman and Thomas Cech independently found that RNAs can also act as catalysts for chemical reactions. This class of catalytic RNAs is known as ribozymes, and the finding earned Altman and Cech the 1989 Nobel Prize in Chemistry. 39

Could RNA substitute proteins in an RNA world?
RNA can perform various catalytic functions. RNA riboswitches regulate gene expression and perform peptidyl-transfer reactions in the ribosome, self-splicing Group I intron ribozymes remove intron sequences in genes,  RNA ligases, and polymerase ribozymes ( they break and catalyze phosphodiester bonds), etc. The thing is, ribozymes are extremely good and specialized in what they are doing. They are all encoded in DNA in modern cells and preordained to do what they do with specificity.  How would they emerge spontaneously from a messy primordial soup by random chance?

Timothy J. Wilson (2020): What is arguably the most important reaction in the cell, the condensation of amino acids to form polypeptides by the peptidyl transferase activity of the ribosome is catalyzed by RNA in the large subunit . Another example is the splicing of mRNA, where the U2/U6 snRNA complex is a ribozyme. RNase P is a ribozyme that processes the 5' end of tRNA in all domains of life. Some of the small nucleolytic ribozymes are widespread, such as the hammerhead and twister ribozymes. RNA can accelerate phosphoryl transfer reactions by a millionfold or more. This is achieved by one or other of two main broad strategies. The group I self-splicing introns use divalent metal ions to organize the active center, activate the nucleophile, and stabilize the transition state and the group II introns and RNase P also appear to function as metalloenzymes. By contrast, the nucleolytic ribozymes use general acid-base catalysis most frequently utilizing nucleobases. Even though the natural pKa values of the nucleobases are either low (adenine and cytosine) or high (guanine and uracil), generally resulting in a low fraction of active catalyst at physiological pH, a ribozyme like twister has its active center to impose an in-line geometry for attack by the O20 nucleophile, stabilize the phosphorane transition state and perform nucleobase-mediated general acid-base catalysis to achieve a substantial rate acceleration. Peptidyl transferase activity in the large ribosomal subunit does not use nucleobase-mediated catalysis, but the reaction appears to involve proton transfer mediated by a 20 -hydroxyl of tRNA. 

This demonstrates how all extant ribozymes are highly complex and specified to perform their designated catalytic functions with high specificity, precision, and efficiency. All enzymes that use metal co-factors require enormously complex biosynthesis pathways that are similar to robotic production lines. They orchestrate the synthesis of the co-factors, and the precise insertion into the reaction centers, the pockets, where the catalytic reaction occurs. How could the origin of such a state of affairs be explained by chance events? 

Timothy J. Wilson continues: According to the simplest version of the RNA world hypothesis (W. Gilbert, 1986) ribozymes would have catalyzed all cellular chemical reactions in a primitive metabolism. This would have required RNA to catalyze a far wider range of chemistry than we currently are aware of in nature, and it would have required relatively difficult reactions such as carbon–carbon bond formation. Many of the reactions available to the organic chemist for this purpose would be highly improbable for RNA catalysts. 49

Limited catalytic possibilities of RNAs
Wan, C. (2022): An essential component of an RNA world scenario would be an RNA “replicase” – a ribozyme capable of self-replication as well as copying other RNA sequences. While such a replicase has not been found in nature 27

Ronald R. Breaker (2020): Only a few classes of ribozymes are known to contribute to the task of promoting biochemical transformations. The RNA World theory hypothesis encompasses the notion that earlier forms of life made use of a much greater diversity of ribozymes and other functional RNAs to guide complex metabolic states long before proteins had emerged in evolution. 47

Jessica C. Bowman (2015): Although RNA in extant biology is seen to catalyze only RNA cutting and ligation along with peptidyl transfer (within the ribosome), a wide variety of chemical transformations can be catalyzed by ribozymes selected in vitro. 36

Charles W Carter, Jr (2017): Catalytic RNA itself cannot fulfill the tasks now carried out by proteins. The term “catalytic RNA” overlooks three fundamental problems: 1) it vastly overestimates the potential catalytic proficiency of ribozymes (Wills 2016); and fails to address either 2) the computational essence of translation or 3) the requirement that catalysts not only accelerate, but more importantly, synchronize chemical reactions whose spontaneous rates at ambient temperatures differ by more than 10^20-fold 33

Selecting ribozymes in the laboratory
Timothy J. Wilson (2020):  To explore what might be possible by way of RNA-mediated catalysis of novel chemical reactions there have been many investigations in which in vitro evolution methods have been used to select RNA species that will accelerate a given reaction from a random pool of sequences. These experiments have generally been carried out in a similar manner in which one reactant is tethered to an RNA oligonucleotide whose sequence has been partially or totally randomized, while the other is linked to biotin. If an RNA within the pool can catalyze formation of a bond between the reactants this connects the RNA to the biotin, allowing it to be isolated by binding to streptavidin. This can then be amplified and a second round of selection performed. Something like 15–20 such cycles will be performed after which the reactant will be disconnected from the RNA to see if it will catalyze a reaction in trans. Clearly this strategy is limited to bond-forming reactions, and we can divide this into reactions leading to the formation of C-C, C-N, and C-S bonds. 

Carbon–carbon bond formation. Ribozymes have been selected that can catalyze C-C bonds by the non-natural Diels– Alder cycloaddition reaction, the aldol reaction and related Claisen condensation. 
Carbon–nitrogen bond formation. Selected ribozymes catalyzing C-N bond formation include one that alkylates itself at a specific guanine N7, amide and peptide bond formation and glycosidic bond formation. Very recently Höbartner and colleagues have selected an RNA that catalyzes methyl transfer from O6 -methylguanine to adenine N1. 
Carbon–sulfur bond formation. C-S bond formation has been demonstrated by selected RNA species catalyzing Michael addition and CoA acylation. The estimated rate enhancements vary, being strongly dependent on the estimation of the uncatalyzed rate, but are frequently around 1000-fold. They are probably relatively unsophisticated catalysts. It is likely to be much easier to find an RNA that can exploit metal ions in catalysis than one that uses nucleobases as chemical participants for example. 49

TM.Tarasow ( 1997): Carbon–carbon bond formation and the creation of asymmetric centres are both of great importance biochemically, but have not yet been accomplished by RNA catalysis. (DAase activity) was carried out with a library of 10^14 unique sequences. The RNA molecules were constructed of a contiguous 100-nucleotide randomized region 44

That means nature would have had to shuffle in sequence space of 10^14 possible combinations, to find one that would be able to catalyze a Carbon-carbon bond formation. Considering an estimate of the age of the universe which is 13,7 Billion years, that would be = 1 x 10^16 seconds. A prebiotic soup would have had to try potentially one reaction per second, for 13,7 billion years, to find a functional sequence. That far stretches plausibility. 

Requirement of cofactors and coenzymes for ribozyme function
The Achilles heel of all these experiments is that there was no prebiotic selection, and biotin is a Vitamin B7, an enzyme co-factor. The synthesis of biotin is very complex, depending on a series of complex enzymes ( BioC, BioH BioF, BioA, BioD, and BioB, and SAH, S-adenosylhomocysteine; SAM, S-adenosyl-L-methionine; AMTOD, S-adenosyl-2-oxo-4-thiomethylbutyrate; 5’-DOA, 5′-deoxyadenosine.) Evidently, these enzymes and cofactors were not swimming in the prebiotic soup, synthesizing biotin, ready to be linked to the random nucleotide chains.

Daniel N. Frank (1997): Despite the occurrence of a wide variety of structures and mechanisms among catalytic RNAs (ribozymes), most are metalloenzymes that require divalent metal cations for catalytic function. The ribozyme RNase P for example absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active site. Divalent metals are thought to play two critical roles in ribozyme function. First, they promote the proper folding of RNA tertiary structures. Second, metals can participate directly in catalysis by activating nucleophiles, stabilizing transition states, and stabilizing leaving groups  51

Gerald F. Joyce (2018): Divalent metal cations appear to be essential for efficient RNA copying, but the poor affinity of the catalytic metal for the reaction center means that very high concentrations of these ions are required, which causes problems for both the RNA (degradation, hydrolysis of activated monomers) and for the fatty acid–based membranes. RNA polymerase enzymes solve these problems by binding and precisely positioning the metal ion for catalysis . A prebiotically plausible means of achieving effective metal ion catalysis at low ambient concentration would greatly simplify the development of model protocells. 38

Coenzymes and cofactors are molecules that help an enzyme or protein function appropriately. Coenzymes are organic molecules and quite often bind loosely to the active site of an enzyme and aid in substrate recruitment, whereas cofactors do not bind the enzyme. Cofactors are "helper molecules" and can be inorganic or organic in nature. These include metal ions and are often required to increase the rate of catalysis of a given reaction catalyzed by the specific enzyme. These coenzymes and cofactors play an integral role in a number of cellular metabolism reactions playing both structural and functional roles to aid in the catalysis.  28




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1. Change Laura Tan, Rob Stadler: The Stairway To Life: An Origin-Of-Life Reality Check  March 13, 2020 
2. Saidu lIslam: Prebiotic Systems Chemistry: Complexity Overcoming Clutter  13 April 2017
3. David Deamer: Bioenergetics and Life's Origins 2010 Feb; 2
4. [url=https://chem.libretexts.org/Courses/Sacramento_City_College/SCC%3A_Chem_309_-_General_Organic_and_Biochemistry_(Bennett)/Text/13%3A_Functional_Group_Reactions/13.10%3A_Phosphoester_Formation#:~:text=an ester bonds.-,Phosphodiesters,the strands of nucleic acid.]Phosphoester Formation[/url]
5. David Deamer: Bioenergetics and Life's Origins  January 13, 2010
6. Weber, Arthur L.: Prebiotic Polymer Synthesis and the Origin of Glycolytic Metabolism 1998-01-01
7. All about science
8. Libretext: Phosphoester Formation
9. Robert Shapiro: Small Molecule Interactions Were Central to the Origin of Life Review 2006
10. Xaktly: DNA & RNA: The foundation of life on Earth
11. Eduard Schreiner: Stereochemical errors and their implications for molecular dynamics simulations 2011
12. David Deamer: The Role of Lipid Membranes in Life’s Origin 2017 Jan 17
13. Arthur V. Chadwick, Ph.D.: Abiogenic Origin of Life: A Theory in Crisis 2005
14. G. Waechtershaeuser: Peptides by Activation of Amino Acids with CO on (Ni,Fe)S Surfaces: Implications for the Origin of Life 31 JULY 1998
15. Elizabeth C. Griffith: In situ observation of peptide bond formation at the water–air interface August 6, 2012
16. Fabian Sauer: From amino acid mixtures to peptides in liquid sulphur dioxide on early Earth 2021 Dec 10
17. Hui Huang, Siwei Yang: Photocatalytic Polymerization from Amino Acid to Protein by Carbon Dots at Room Temperature October 22, 2019
18. Rob Stadler: Long Story Short — A Strikingly Unnatural Property of Biopolymers December 1, 2021
19. Pier Luigi Luisi : OPEN QUESTIONS IN ORIGIN OF LIFE: EXPERIMENTAL STUDIES ON THE ORIGIN OF NUCLEIC ACIDS AND PROTEINS WITH SPECIFIC AND FUNCTIONAL SEQUENCES BY A CHEMICAL SYNTHETIC BIOLOGY APPROACH February 2014
20. Michael Marshall: How the first life on Earth survived its biggest threat — water 09 December 2020
21. Steven A. Benner: Catalytic Synthesis of Polyribonucleic Acid on Prebiotic Rock Glasses 8 Jun 2022
22. Steven A. Benner: Life, the Universe and the Scientific Method 2008
23. Martina Preiner: The ambivalent role of water at the origins of life 16 May 2020
24. Steven A. Benner: Paradoxes in the Origin of Life 5 December 2014
25. Geoffrey Zubay:Origins of Life on the Earth and in the Cosmos  2000
26. S W FOX: A Theory of Macromolecular and Cellular Origins 1965
27. Wan, C.: Evolution and Engineering of RNA-based Macromolecular Machines 2022
28. 
29. Walter Gilbert: Origin of life: The RNA world 20 February 1986
30. F H Westheimer: Why nature chose phosphates  1987 Mar 6
31. Dr. Rafał Wieczorek: Formation of RNA Phosphodiester Bond by Histidine-Containing Dipeptides 18 December 2012
32. Robert P. Bywater writes in: On dating stages in prebiotic chemical evolution 15 February 2012
33. Charles W Carter, Jr: Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding  24 October 2017
34. Harris Bernstein: Origin of DNA Repair in the RNA World October 12th, 2020
35. Prof. Dr. Oliver Trapp: Direct Prebiotic Pathway to DNA Nucleosides 26 May 2019
36. Pekka Teerikorpi: The Evolving Universe and the Origin of Life: The Search for Our Cosmic Roots2009
37. Jonathan Wells: The Politically Incorrect Guide to Darwinism and Intelligent Design August 21, 2006
38. Gerald F. Joyce: Protocells and RNA Self-Replication 2018
39. [url=https://exploringorigins.org/ribozymes.html#:~:text=The discovery of ribozymes supported,and to catalyze chemical reactions.]Exploring life's origins[/url]
40. Stu Borman: Ribozyme May Hint At The Origin Of Life November 17, 2014 
41. Gerald F. Joyce A cross-chiral RNA polymerase ribozyme 29 October 2014
42. G. F. Joyce: Chiral selection in poly(C)-directed synthesis of oligo(G) 16 August 1984
43. Suzan Mazur: Pier Luigi Luisi: Origin of Life Mindstorms Needed 19 December 2012
44. T M Tarasow: RNA-catalysed carbon-carbon bond formation 1997 Sep 4
45. Steven A. Benner: Asphalt, Water, and the Prebiotic Synthesis of Ribose, Ribonucleosides, and RNA March 28, 2012
46. Steven A. Benner Paradoxes in the Origin of Life 5 Dec. 2014
47. Ronald R. Breaker: Imaginary Ribozymes 2020 Aug 21
48. Stephen D. Fried: Peptides before and during the nucleotide world: an origins story emphasizing cooperation between proteins and nucleic acids 09 February 2022
49. Timothy J Wilson: The potential versatility of RNA catalysis 2021 May 5

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HAROLD B. WHITE, III (1975): Coenzymes are complex organic molecules that are essential for many enzyme-catalyzed reactions. At least 52% of the nearly 1750 enzymes recently cataloged (IUPAC-IUB, 1972) require a coenzyme for activity. I propose that coenzymes are the surviving vestiges of nucleic acid enzymes which preceded the evolution of ribosomal protein synthesis. 34

If the RNA world were true, ribozymes would have had to catalyze a wide range of catalytic reactions which subsequently were substituted by proteins. About half would only obtain the necessary catalytic activity by recruiting and employing cofactors and coenzymes ( which, as we see, also depend on complex biosynthesis pathways, and alternative non-enzymatic emergence would be very unlikely).  It is as if a Software engineer had to learn to become a mechanical engineer and assemble complex machines. There is no evidence that RNAs, made of just four different building blocks ( the four nucleobases), were ever able to catalyze the widely different enzymatic and metabolic reactions required for life to thrive. There is no evidence, that that somehow, prebiotic shuffling created a pool of millions of repetitive complex nucleotides, all with the repetitive configuration of purine and pyrimidine bases. There is no chemical logic that makes it appear plausible, that a gradual chemical evolutionary process would have promoted the emergence of an RNA world, followed by an RNA-peptide world. There is a wide unexplained gap between the RNA world, and the modern DNA - RNA - protein state of affairs in modern cells. Can this gap be bridged with new hypotheses and the advance in abiogenesis investigations? That prediction looks rather unlikely. Only time will tell. 
 
Solving a chicken & egg problem?
Supposedly, the RNA world hypothesis solved a long-standing chicken & egg, or catch22 problem ( J.Wells [2006]: In Joseph Heller’s novel about World War II, Catch-22, an aviator could be excused from combat duty for being crazy. But a rule specified that he first had to request an excuse, and anyone who requested an excuse from combat duty was obviously not crazy, so such requests were invariably denied. The rule that made it impossible to be excused from combat duty was called “Catch-22.”  ) In 1965, Sidney Fox wrote a scientific article, asking: How, when no life existed, did substances come into being which today are absolutely essential to living systems, yet which can only be formed by those systems?  He was referring to a problem, outlined by Jordana Cepelewicz (2017): For scientists studying the origin of life, one of the greatest chicken-or-the-egg questions is: Which came first — proteins or nucleic acids like DNA and RNA? 33 This problem arises because DNA and RNA direct the synthesis of enzymes and proteins. But proteins synthesize the making of RNA and DNA. 

Jessica C. Bowman (2015) claimed: The RNA World Hypothesis resolves the putative chicken and egg dilemma: which came first, polynucleotide or polypeptide? The simultaneous emergence from whole cloth of two functional biopolymers, one encoding the other, seems improbable. A single type of ancestral biopolymer (polynucleotide), performing multiple roles, appears to be characterized by high parsimony. A ‘‘Polymer Transition’’, a progression of biology from one polymer type (polynucleotide) to two polymer types (polynucleotide and polypeptide), is consistent with an expectation that ancient biology transitioned from simple to complex. 32

Under naturalism, the only possible explanation for the complexity seen in biochemistry is gradualism: From the simple to the complex. Gradually moving from chemistry to biology. A sudden appearance of all the complex interdependent intricacies observed in the living cannot be explained by a biochemical Big bang. It is untenable. Therefore, under the framework of philosophical naturalism, answers have to be found that confer with the gradualistic scenario. Only the model of intelligent design permits the hypothesis of instant creation by an intelligent agency. Giving up gradualism means giving up naturalism. 

Eugene V Koonin (2007): The origin of the translation system is, arguably, the central and the hardest problem in the study of the origin of life, and one of the hardest in all evolutionary biology. The problem has a clear catch-22 aspect: high translation fidelity hardly can be achieved without a complex, highly evolved set of RNAs and proteins but an elaborate protein machinery could not evolve without an accurate translation system. 13

Paul C. W. Davies (2013): Because of 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.  The real challenge of life's origin is thus to explain how instructional information control systems emerge naturally and spontaneously from mere molecular dynamics. 15

Self-replication in the RNA world
Despite the current celebrity status, there are several reasons that raise doubts that RNAs would self-assemble into ribozyme polymerases with function-bearing sequences, obtaining the right chemical structures with autocatalytic properties, and being apt to start self-replication. 

Harold S Bernhardt (2012): The following objections have been raised to the RNA world hypothesis: (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. 12

Since the various problems of prebiotic RNA monomers have been outlined in the previous chapter, we will address now the issues with RNA self-replication.  Steven Benner (2013): Catalysis and genetics place contradicting demands on any single molecular system asked to do both. For example, catalytic molecules should fold, to surround a transition state. Genetic molecules should not fold, to allow them to template the synthesis of their complements. Catalytic molecules should have many building blocks, to create versatile catalytic potential. Genetic molecules should have few building blocks, to ensure that they are copied with high fidelity 11

Jack W Szostak (2012): The first RNA World models were based on the concept of an RNA replicase - a ribozyme that was a good enough RNA polymerase that it could catalyze its own replication. Although several RNA polymerase ribozymes have been evolved in vitro, the creation of a true replicase remains a great experimental challenge. 6

Hannes Mutschler (2019): It has not yet been possible to demonstrate robust and continuous RNA self-replication from a realistic feedstock (i.e. activated mono- or short mixed-sequence oligonucleotides). In the case of ribozymes, only ‘simple’ ligation or recombination-based RNA replication from defined oligonucleotides has been demonstrated. Such systems have only a limited ability to transmit heritable information and so are not capable of open-ended evolution — the ability to indefinitely increase in complexity like living systems. Open-ended evolution requires that a replicase must at least be able to efficiently copy generic sequences longer than that required to encode its own function. RNA in isolation (including ribozymes) is simply not sufficient to catalyze its own replication, and substantial help from either other molecules or the environment is essential. 7

The annealing problem
Jordana Cepelewicz (2019): As a first step toward making a copy of itself, a single strand of RNA can take up complementary nucleotide building blocks from its surroundings and stitch them together. But the paired RNA strands then tend to bind to each other so tightly that they don’t unwind without help, which prevents them from acting as either catalysts or templates for further RNA strands. “It’s a real challenge,” Sutherland said. “It’s held the field back for a long time.” 8

Gerald F. Joyce (2018): Because the product of template copying is a double-stranded RNA, there must be some means of either strand separation or strand displacement synthesis. Transient temperature fluctuations could lead to thermal strand separation, but long RNA duplexes (≥30 base pairs) are difficult to denature thermally. 10

In modern cells, ribonuclease H enzymes destroy annealed RNAs but evidently, they were not around on prebiotic earth.

The Eigen paradox
Jaroslaw Synak (2022): Another challenge is the maintenance of genetic information in RNA sequences over many rounds of imperfect replication. In order to survive, RNA polymerase must be copied faster than it is hydrolyzed and accurately enough to preserve its function. In the early stages of molecular evolution, due to the lack of reliable replication mechanisms, the mutation rate was likely very high and the critical amount of information could not have been stored in long RNA sequences; on the other hand, the short ones could not be efficient enzymes. Maynard Smith estimated that the maximum length of the RNA replicase is approximately 100 nucleotides, assuming nonenzymatic replication with a copying fidelity of one base of up to 0.99. In order to further increase this length, the copying fidelity would have to be increased, which requires the presence of specific enzymes. This is known as Eigen’s paradox and is often equivalently formulated as: no enzymes without a large genome and no large 5

Indeed. But the problem is not only to maintain genetic information but for the first replicator to obtain it in the first place!

Natalia Szostak (2017): Researchers have performed many attempts to create RNA polymerase ribozyme, recently resulting in a cross-chiral RNA polymerase ribozyme and a system of cooperative RNA replicators, as well as RNA polymerase ribozyme that is able to synthesize structured functional RNAs, including aptamers and ribozymes. However, these molecules are too large to be maintained in a quasispecies population, as they exceed the 100 nucleotide error threshold, which is the maximum length polynucleotide molecule that can be accurately replicated without high fidelity polymerases. Eigen suggested hypercycles as a solution to the error threshold problem mentioned above. However, even if the traditional hypercycle model formulation based on ordinary differential equations is ecologically stable, it is proved to be evolutionarily unstable. To evolve life as we know it, separation of the roles performed by replicases, information storage, and replication of the information, into two molecules appears to be one of the crucial events that had to occur early in the stages leading to life. 4

A remarkable admission !!

Sami EL Khatib (2021): Countless challenges are faced by an RNA self-replicating cycle; for it to be a fully chemically and  enzymatically free reaction, the cycle loses rate and fidelity, so much that it does not even reach the critical threshold for the sustenance of life, meaning the RNA nucleotides break apart faster than the incorporation of nucleotides takes place, thus is the case when experimenting with modern substrates, that do not leak out of cells and are very polar with the regularly known triphosphate ester, this is advantageous to the modern cell where it uses enzymes to catalyze the release of di-phosphate, but not for the primitive cell as the substrates are found in the environment and require continuous dynamic exchange. The RNA world hypothesis has been criticized mostly because of the belief that long RNA sequences are needed for the catalytic function of RNA. These long sequences are enormous and are needed to isolate the catalytic and biding functions of the overall ribozyme. For example the best ribozyme replicase created so far, which is able to replicate an impressive 95-nucleotide stretch of RNA, is ~190 nucleotides in length, which is by far too large a number to have risen in any random assembly, thus in vitro selection experiments had to be designed where 10,000,000,000,000 – 1,000,000,000,000,000 of randomized RNA molecules are required as the starting point for the isolation of ribozymic and/or binding activity. This experiment clearly contradicts the probable prebiotic situation. 14

Eugene Koonin (2012):  The primary incentive behind the theory of self-replicating systems that Manfred Eigen outlined was to develop a simple model explaining the origin of biological information and, hence, of life itself. Eigen’s theory revealed the existence of the fundamental limit on the fidelity of replication (the Eigen threshold): If the product of the error (mutation) rate and the information capacity (genome size) is below the Eigen threshold, there will be stable inheritance and hence evolution; however, if it is above the threshold, the mutational meltdown and extinction become inevitable (Eigen, 1971). The Eigen threshold lies somewhere between 1 and 10 mutations per round of replication; regardless of the exact value, staying above the threshold fidelity is required for sustainable replication and so is a prerequisite for the start of biological evolution. Indeed, the very origin of the first organisms presents at least an appearance of a paradox because 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. However, the replication fidelity at a given point in time limits the amount of information that can be encoded in the genome. What turns this seemingly vicious circle into the (seemingly) unending spiral of increasing complexity—the Darwin-Eigen cycle.  The crucial question in the study of the origin of life is how the Darwin-Eigen cycle started—how was the minimum complexity that is required to achieve the minimally acceptable replication fidelity attained? In even the simplest modern systems, such as RNA viruses with the replication fidelity of only about 10^3 and viroids that replicate with the lowest fidelity among the known replicons (about 10^2), replication is catalyzed by complex protein polymerases. The replicase itself is produced by translation of the respective mRNA(s), which is mediated by the immensely complex ribosomal apparatus. Hence, the dramatic paradox of the origin of life is that, to attain the minimum complexity required for a biological system to start on the Darwin-Eigen spiral, a system of a far greater complexity appears to be required. How such a system could evolve is a puzzle that defeats conventional evolutionary thinking, all of which is about biological systems moving along the spiral; the solution is bound to be unusual.

When considering the origin of the first life forms, one faces the proverbial chicken-and-egg problem: What came first, DNA or protein, the gene or the product? In that form, the problem might be outright unsolvable due to the Darwin-Eigen paradox: To replicate and transcribe DNA, functionally active proteins are required, but production of these proteins requires accurate replication, transcription, and translation of nucleic acids. If one sticks to the triad of the Central Dogma, it is impossible to envisage what could be the starting material for the Darwin-Eigen cycle. Even removing DNA from the triad and postulating that the original genetic material consisted of RNA (thus reducing the triad to a dyad), although an important idea, does not help much because the paradox remains. For the evolution toward greater complexity to take off, the system needs to somehow get started on the Darwin-Eigen cycle before establishing the feedback between the (RNA) templates (the information component of the replicator system) and proteins (the executive component). The brilliantly ingenious and perhaps only possible solution was independently proposed by Carl Woese, Francis Crick, and Leslie Orgel in 1967–68: neither the chicken nor the egg, but what is in the middle—RNA alone. The unique property of RNA that makes it a credible—indeed, apparently, the best—candidate for the central role in the primordial replicating system is its ability to combine informational and catalytic functions. Thus, it was extremely tempting to propose that the first replicator systems—the first life forms—consisted solely of RNA molecules that functioned both as information carriers (genomes and genes) and as catalysts of diverse reactions, including, in particular, their own replication and precursor synthesis. This bold speculation has been spectacularly boosted by the discovery and subsequent study of ribozymes (RNA enzymes), which was pioneered by the discovery by Thomas Cech and colleagues in 1982 of the autocatalytic cleavage of the Tetrahymena rRNA intron, and by the demonstration in 1983 by Sydney Altman and colleagues that RNAse P is a ribozyme. Following these seminal discoveries, the study of ribozymes has evolved into a vast, expanding research area. 

Despite all invested effort, the in vitro evolved ribozymes remain (relatively) poor catalysts for most reactions; the lack of efficient, processive ribozyme polymerases seems particularly troubling. An estimate based on the functional tolerance of well-characterized ribozymes to mutations suggest that, at fidelity of 10^3 errors per nucleotide per replicase cycle (roughly, the fidelity of the RNA-dependent RNA polymerases of modern viruses), an RNA “organism” with about 100 “genes” the size of a tRNA (80 nucleotides) would be sustainable. Such a level of fidelity would require only an order of magnitude improvement over the most accurate ribozyme polymerases obtained by in vitro selection 3

Lack of prebiotic RNA repair mechanisms
Harris Bernstein (2020): Persistence and replication of even the simplest forms of RNA life must have depended on preserving the information content of the RNA genome from damage (a form of informational noise). Damage to the RNA genome likely occurred in a variety of ways including spontaneous hydrolysis, exposure to UV light and exposure to reactive chemicals. 9

Where did the energy come from?
Jack Szostak, interviewed by Suzan Mazur (2014): The problem is RNA falls apart. The activated nucleotides we use to do the non-enzymatic replication -- they react with water, so they fall apart. There needs to be a way to bring energy back into the system to essentially keep the battery charged. To keep all the nucleotides activated and to keep things running. 1

PHILIP BALL (2020): It’s an alluring picture – catalytic RNAs appear by chance on the early Earth as molecular replicators that gradually evolve into complex molecules capable of encoding proteins, metabolic systems and ultimately DNA. But it’s almost certainly wrong. For even an RNA-based replication process needs energy: it can’t shelve metabolism until later. And although relatively simple self-copying ribozymes have been made, they typically work only if provided with just the right oligonucleotide components to work on. What’s more, sustained cycles of replication and proliferation require special conditions to ensure that RNA templates can be separated from copies made on them. Perhaps the biggest problem is that self-replicating ribozymes are highly complex molecules that seem very unlikely to have randomly polymerized in a prebiotic soup. And the argument that they might have been delivered by molecular evolution merely puts the cart before the horse. The problem is all the harder once you acknowledge what a complex mess of chemicals any plausible prebiotic soup would have been. It’s nigh impossible to see how anything lifelike could come from it without mechanisms for both concentrating and segregating prebiotic molecules – to give RNA-making ribozymes any hope of copying themselves rather than just churning out junk, for example. In short, once you look at it closely, the RNA world raises as many questions as it answers.  The best RNA polymerase the researchers obtained this way had a roughly 8% chance of inserting any nucleotide wrongly, and any such error increased the chance that the full chain encoded by the molecule would not be replicated. What’s more, making the original class I ligase was even more error-prone and inefficient – there was a 17% chance of an error on each nucleotide addition, plus a small chance of a spurious extra nucleotide being added at each position. These errors would be critical to the prospects of molecular evolution since there is a threshold error rate above which a replicating molecule loses any Darwinian advantage over the rest of the population – in other words, evolution depends on good enough replication. Fidelity of copying could thus be a problem, hitherto insufficiently recognized, for the appearance of a self-sustaining, evolving RNA-based system: that is, for an RNA world. Maybe this obstacle could have been overcome in time. But my hunch is that any prebiotic molecule will have been too inefficient, inaccurate, dilute and noise-ridden to have cleared the hurdle. 2

An RNA world could not explain the origin of the genetic code
Susan Lindquist (2010): An RNA-only world could not explain the emergence of the genetic code, which nearly all living organisms today use to translate genetic information into proteins. The code takes each of the 64 possible three-nucleotide RNA sequences and maps them to one of the 20 amino acids used to build proteins. 29

Without amino acids, there could not be an assignment. 64 trinucleotide codons are assigned to 20 amino acids. Both had to be present, in order for this assignment to occur. But having both would not be enough either. In reality, the entire system had to be created from the get-go, fully developed, and operational from the beginning. That led Fujio Egami (1981) to present a " working hypothesis on the interdependent genesis of nucleotide bases, protein amino acids, and the primitive genetic code: the primitive genetic code was dependent upon the concentration of different nucleotide bases and amino acids coexisting in the primeval environments and upon the selective affinity between bases and amino acids." 30
 
Charles W Carter, Jr (2017): Computational and structural modeling argue that some mutual, interdependent processes embedded information into proteins and nucleic acids.

While talking about evolutionary modification, Egami was probably not aware that an interdependent system cannot be the product of evolutionary change. An interdependent system must be right all at once, right from the start. Stepwise, gradual evolution of the system is not possible, since intermediate stages would confer no advantage, nor function. Mapping nucleotides to amino acids is only possible if all players are there. 31

The RNA-peptide world
The RNA-peptide world tries to build a bridge between the replication first, and metabolism first scenarios, advancing the RNA world and combining it with catalytic peptides and primitive metabolism.

Stephen D. Fried (2022): Diverse lines of research in molecular biology, bioinformatics, geochemistry, biophysics, and astrobiology provide clues about the progression and early evolution of proteins, and lend credence to the idea that early peptides served many central prebiotic roles before they were encodable by a polynucleotide template, in a putative ‘peptide-polynucleotide stage’. 23

The presupposition is that a result of chemical prebiotic conditions permitted the emergence of activated ribonucleotides and amino acids.  The proposal hypothesizes that RNAs started to interact and get into a relationship with small peptides ( small amino acid strands) right from the beginning, rather than everything starting exclusively with RNAs, that later would transition to mutually beneficial interaction with amino acids.  In modern cells, DNA that stores the genetic data using the genetic code is transcribed into messenger RNA (mRNA), subsequently translated in the ribosome apparatus into functional amino acid sequences, which form polypeptides, and in the end, proteins. The core problem is the origin of the codon-amino acid assignment through the genetic code. The RNA-peptide world attempts to address this current state of affairs, starting with an RNA-peptide world, which constitutes the first step to arrive at the end of the current solution, where the sophisticated translation is performed through the ribosome.    

Charles W. Carter, Jr. (2015): In the RNA-world scenario, the necessary catalysts were initially entirely RNA-based and did not include genetically encoded proteins. IN the RNA-peptide world, the idea that coded peptides functioned catalytically in the early stages of the origin of life directly contradicts the second central tenet of the “RNA World” scenario.  The important distinction between this scenario and the RNA World hypothesis is that the requisite specificity is low in the initial stages of the former but unacceptably high in the latter. Low specificity processes occur with greater frequency and hence are more likely to have occurred first. The unavailability of activated amino acids was the most critical barrier to the emergence of protein synthesis. 16

Dave Speijer (2015): Wery small RNAs (versatile and stable due to base-pairing) and amino acids, as well as dipeptides, coevolved. The “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a “pure RNA ribosome” evolution started out with. Though the oldest center of the ribosome seems “RNA only”, we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to base-pairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the “RNA world” view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. We argue that an RNA world completely independent of amino acids never existed.

Indeed, I agree, that an RNA world never existed. But did an RNA-peptide world?

Speijer: The idea of an independent RNA world without oligopeptides or amino acids stabilizing structures and helping in catalysis does not seem a viable concept. On the other hand, the idea of catalytic protein existing without RNA storing the polypeptide sequences, which have catalytic activity, and organizing the production of these sequences, also does not seem a viable concept. Here we argue for a “coevolutionary” theory in which amino acids and (very small) peptides, as well as small RNAs, existed together and where their separate abilities not only reinforced each other’s survival but allowed life to more quickly climbing the ladder of complexity.

Every naturalistic approach works only from the simple to the complex in a slow, gradual manner. Even if not linear but with ups and downs, the outcome is always that there is more functional complexity at the end. That is as well Speijers proposal: "Starting with small molecules (easily) derived from prebiotic chemistry, we will try to reconstruct a possible history in which every stage of increased complexity arises from the previous more simple stage because specific nucleotide/amino acid (RNA/peptide) interactions allowed it do so." Observe how Speijer introduces teleonomy into the explanation. As if RNAs and amino acids operated or behaved with the "aim" or purpose of keeping a state of affairs, that wasn't even there. RNAs and amino acids on their own are not alive. They are molecules used in biology. But molecules have no innate drive or "urge" to keep a specific state of affairs, that would favor a future outcome, the gradual complexification that would, in the end, result in the existence of self-replicating cells.

Speijer: We now come at a crucial and, we have to admit, somewhat theoretical juncture: coevolution is illustrated by the presumption that RNAs could not persist without peptide protection, that very short (very early) peptides were made more abundant by RNA-producing them, and that they co-evolve forming longer RNAs and peptides. This would constitute an RNA/peptide world of ribozymes and short oligopeptides. These oligopeptides had RNA protection functions (DADVDGD being the obvious ancestor sequence of the universal RNA polymerase active site sequence NADFDGD) This motif (Asn-Ala-Asp-Phe-Asp-Gly-Asp) is a specific stretch of amino acids that is central in all cellular life. RNA polymerases catalyze the transcription from DNA to mRNA. Dennis R. Salahub (2008): Most known RNA polymerases (RNAPs) share a universal heptapeptide, called the NADFDGD motif. The crystal structures of RNAPs indicate that in all cases this motif forms a loop with an embedded triad of aspartic acid residues. This conserved loop is the key part of the active site. 17

The odds to get this sequence randomly is one in 20^7 or one in 10^10, that is taking a pool of 20 selected amino acids used in life would have to be shuffled 10 billion times to get this specified functional sequence. Not forgetting, that it is incorporated in a much longer polymer sequence that also has to be functional, and embedded and working in a joint venture with other polymer subunit strands of RNA polymerase. A far fetch.

Kunnev (2018): The hypothesis assumes that ribonucleotides would polymerize leading to very short RNAs from 2 to about 40 bases. The polymerization would incorporate random sequences and random 3D structures. The process would preserve mostly stable ones. Wet-Dry cycles could facilitate the process of RNA polymerization. Compartmentalization is another important factor since most of the described events are unlikely to occur in very low concentrations. Some level of environmental separation would be expected, for example, micro-chambers out of porous surface of rocks or lipid vesicles or both. Surface adsorption might have facilitated RNA-RNA interactions, RNA-lipids interactions and some beneficial chemical reactions. Thus, clay surfaces have been shown to promote encapsulation of RNA into vesicles and grow by incorporating fatty acid supplied as micelles and can divide without dilution of their contents.  At temperatures between 1°C and to denaturation (about 55°C) temperature, short random RNA oligos would get stabilized via intra and intermolecular hybridization based on Watson-Crick base pairing, forming complexes of various 3D shape and size. Larger hybridized regions would confer greater stability and would be selected for. Highly self-complementary RNAs would be unlikely to exist, forcing intermolecular hybridization of short sequences and the emergence of complexes of several RNA oligos. The formation of RNA complexes also assumes a thermal cycle that would drive the process by sequential denaturation (~55–100°C) and re-annealing (<55°C) phases. Frequent repetition of the thermal cycle and stability selection would favor accumulation of complexes with higher degree of complementarity and higher GC content. Non-enzymatic aminoacylation between 2′ or 3′ positions of ribose and activated amino acids could occur. In addition, ribozymes capable of amino acid transfer from one RNA to another have been selected under laboratory conditions and similar molecules could have participated in aminoacylation of RNAs. Aminoacylated RNAs would be involved in complex formation, bringing some of the aminoacylated RNA 3′-ends in close proximity. This would promote peptide bond formation between two adjacent amino acids, most likely with the assistance of wet/dry natural cycles. All amino acids would have statistically equal probability to aminoacylate RNA. At that stage, any RNA molecule could be aminoacylated and could serve as a template. 18

That means, any available amino acid nearby could be involved in the reaction - inclusive amino acids not used in life, and they could be attached anywhere to the RNA molecule. There is also no restriction in regards of possible RNA configurations with any sort of nucleobases. There is no mechanism that would prevent other than the nucleobases used in life to be involved in the reactions.  It would result simply in a disordered random accumulation of RNA-peptides. 

Kunnev: We presume that following this initial stage all components of the translation system would co-evolve in a stepwise way. Specialization of ribosomal Large Subunit—LSU will start with evolution of peptidyl transferase center (PTC). The evolution of peptides to proteins would occur from small motif to domains and finally— folded proteins. 

Felix Müller (2022): The ability to grow peptides on RNA with the help of non-canonical vestige nucleosides offers the possibility of an early co-evolution of covalently connected RNAs and peptides, which then could have dissociated at a higher level of sophistication to create the dualistic nucleic acid–protein world that is the hallmark of all life on Earth. It is difficult to imagine how an RNA world with complex RNA molecules could have emerged without the help of proteins and it is hard to envision how such an RNA world transitions into the modern dualistic RNA and protein world, in which RNA predominantly encodes information whereas proteins are the key catalysts of life.22

This story, when it comes to elucidating the trajectory from these small RNA-peptides, to fully developed proteins is very "sketchy" and superficial. This is a common modus operandi to uphold a story, that by looking closer, does not withstand scrutiny.

Charles Carter, structural biologist (2017): For life to take hold, the mystery polymer would have had to coordinate the rates of chemical reactions that could differ in speed by as much as 20 orders of magnitude. 24

Marcel Filoche (2019): Enzymes speed up biochemical reactions at the core of life by as much as 15 orders of magnitude. Yet, despite considerable advances, the fine dynamical determinants at the microscopic level of their catalytic proficiency are still elusive. Rate-promoting vibrations in the picosecond range, specifically encoded in the 3D protein structure, are localized vibrations optimally coupled to the chemical reaction coordinates at the active site. Remarkably, our theory also exposes a hitherto unknown deep connection between the unique localization fingerprint and a distinct partition of the 3D fold into independent, foldspanning subdomains that govern long-range communication. The universality of these features is demonstrated on a pool of more than 900 enzyme structures, comprising a total of more than 10,000 experimentally annotated catalytic sites. Our theory provides a unified microscopic rationale for the subtle structure-dynamics-function link in proteins. The intricate networks of metabolic cascades that power living organisms ultimately rest on the exquisite ability of enzymes to increase the rate of chemical reactions by many orders of magnitude. Although many molecular machines contain intrinsically disordered domains, the 3D fold is central to enzyme functioning. In particular, increasing evidence is accumulating in the literature in favor of the existence of specific fold-encoded motions believed to govern the relevant collective coordinate(s) that are coupled to the chemical transformation. These motions typically correspond to localized vibrations of the protein scaffold that contribute to the catalytic reaction, i.e., modes that, if impeded, would lead to a deterioration of the catalytic efficiency.

The more the function of a machine depends on its precise setup and arrangement respecting very limited tolerances, the more efforts have to be undertaken to achieve the required precision, demanding engineering solutions where nothing can be left to chance. That is precisely the case with proteins. There is an extraordinarily limited tolerance upon which proteins have to be engineered and designed, a requirement to achieve the necessary catalytic functions. That sets the bar for the cause to instantiate this state of affairs very high, for which random events are entirely inadequate!! The situation becomes even worse when we consider what Mathieu E. Rebeaud described as (2021): the challenge of reaching and maintaining properly folded and functional proteomes. Most proteins must fold to their native structure in order to function, and their folding is largely imprinted in their primary amino acid sequence. However, many proteins, especially large multidomain polypeptides, or certain protein types such as all-beta or repeat proteins, tend to misfold and aggregate into inactive species that may also be toxic. Life met this challenge by evolving employing molecular chaperones that can minimize protein misfolding and aggregation, even under stressful out-of-equilibrium conditions favoring aggregation. 25

Hays S. Rye (2013): Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones assists in protein folding. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. 27

Chaperones do bear no function unless there are misfolded proteins, that need to be re-folded in order to function. But non-functional proteins accumulating in the cell would be toxic waste and eventually kill the cell. So this creates another chicken & egg problem. What came first: Protein synthesis, or chaperones helping proteins to fold correctly? Consider as well, that, as Jörg Martin puts it (2000): The intracellular assembly of GroEL-type chaperonins appears to be a chaperone-dependent process itself and requires functional preformed chaperonin complexes !! 26 There are machines in the cell, that help other machines to be folded correctly, and these machines are also employed to help other machines to fold in order to be able to operate properly! Amazing!

Thorsten Hugel (2020): In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We disentangle specific and non-specific effects that control Hsp90’s ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins. 28

Susan Lindquist (2010): Cells also require a ubiquitin-proteasome system, targeting terminally misfolded proteins for degradation, and with translocation machineries to get proteins to their proper locations. These protein folding agents constitute a large, diverse, and structurally unrelated group. Many are upregulated in response to heat and are therefore termed heat shock proteins (HSPs).  HSP90 is one of the most conserved HSPs, present from bacteria to mammals, and is an essential component of the protective heat shock response. The role of HSP90, however, extends well beyond stress tolerance. Even in nonstressed cells, HSP90 is highly abundant and associates with a wide array of proteins (known as clients) that depend on its chaperoning function to acquire their active conformations. 20% of yeast proteins are influenced by Hsp90 function, making it the most highly connected protein in the yeast genome, and GroES mediates the folding of ~10% of proteins in E. coli.29

Short RNA-peptides, or peptides on their own, are not functional and are useless in a supposed "proto-cell" unless they have the right size and sequence, able to fold into the functional 3D conformation.  In face of this evidence, supposing and theorizing intermediate states and transitions of growing size and complexity over long periods of time until a functional state of affairs is achieved, is untenable. It opposes the evidence just described. Sophisticated exquisite mechanisms have to be instantiated from the get-go, to guarantee the right setup and folding of proteins of the full length.  Such a hypothesized transition is never to work and going to happen. These RNA-peptides would simply lay around, and then sooner or later disintegrate.  These explanations not including an intelligent agent are entirely inadequate to account for the origin of this kind of these high-tech engineering marvel implementations on a molecular scale!

George Church, Professor of Genetics, described the ribosome as "the most complicated thing that is present in all organisms". The peptidyl transferase center (PTC) is the core of the ribosome, where peptide bond formation occurs, which is a central catalytic reaction in life, where proteins are synthesized, and is as such of particular importance.  The process is so intriguingly complex, that a science paper in 2015 had to admit that: "The detailed mechanism of peptidyl transfer, as well as the atoms and functional groups involved in this process are still in limbo." 19 The PTC is a ribozyme, which means it is composed of ribosomal RNAs ( rRNAs). Francisco Prosdocimi (2020): The PTC region has been considered crucial in the understanding about the origins of life. It has been described as the most significant trigger that engendered a mutualistic behavior between nucleic acids and peptides, allowing the emergence of biological systems. The emergence of this proto-PTC is a prerequisite to couple a chemical symbiosis between RNAs and peptides. Of 1434 complete sequences of 23S ribosomal RNAs analyzed, it was demonstrated that site A2451 from the 23S rRNA, which is the catalytic site of the PTC, is essential for the peptide bond to occur, and is absolutely preserved in each and every analyzed sequence. The PTC is known to be a flexible and efficient catalyst as it is capable of recognizing different, specific substrates (20 different amino acids bind to aminoacyl-tRNAs) and polymerizing proteins at a similar rate. 20  

Sávio T.Farias (2014): Studies reveal that the PTC has a symmetrical structure comprising approximately 180 nucleotides. Molecular structure models suggest that the catalytic portion of the 23S rRNA entities of the symmetrical region possesses the common stem-elbow-stem (SES) structural motif. 21

Let's suppose that this structure would have emerged in an RNA-peptide world. Let's also not consider, that finding a functional sequence of 180 RNAs would vastly exceed the resources in sequence space, exhausting the maximum number of possible events in a universe that is 18 Billion years old (10^16 seconds) where every atom (10^80) is changing its state at the maximum rate of 10^40 times per second is 10^139. If we had such a core PTC, it would have no function whatsoever, unless all other players would be in place to perform translation from RNA to amino acids, having as well the genetic code implemented, and the entire chain from DNA to mRNA, to then coming to the events in translation. All these proposals, the RNA world, and the RNA-peptide world are based on silly pipe dreams - that they call theories when they are not more than ideas, based on fertile minds, and not results based on scientific evidence, experimentation, and tests in the lab. These are just invented scenarios - out of the need to keep an explanatory framework based on philosophical naturalism to find answers that do not require invoking a supernatural entity. All these proposals have been shown to be inadequate and doomed to failure.  Biological cells are too complicated, sophisticated, integrated, and functional in order to warrant the belief that they could have originated by unguided means - the ribosome is a prime example to conclude this.

1. Suzan Mazur: The Origin of Life Circus: A How To Make Life Extravaganza  November 30, 2014
2. PHILIP BALL: Flaws in the RNA world  12 FEBRUARY 2020
3. Eugene Koonin: The Logic of Chance: The Nature and Origin of Biological Evolution  31 agosto 2011
4. Natalia Szostak: Simulating the origins of life: The dual role of RNA replicases as an obstacle to evolution July 10, 2017
5. Jaroslaw Synak: RNA World Modeling: A Comparison of Two Complementary Approaches 11 April 2022
6. Jack W Szostak: The eightfold path to non-enzymatic RNA replication 03 February 2012
7. Hannes Mutschler: The difficult case of an RNA-only origin of life AUGUST 28 2019
8. Jordana Cepelewicz: Origin-of-Life Study Points to Chemical Chimeras, Not RNA September 16, 2019
9. Harris Bernstein: Origin of DNA Repair in the RNA World October 12th, 2020
10. Gerald F. Joyce: Protocells and RNA Self-Replication 2018
11. Steven A. Benner: The ‘‘Strong’’ RNA World Hypothesis: Fifty Years Old 2013 Apr;13
12. Harold S Bernhardt:[url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495036/#:~:text=However%2C the following objections have,of RNA is too limited.] The RNA world hypothesis: the worst theory of the early evolution of life[/url] (except for all the others) 2012 Jul 13
13. Eugene V Koonin: On the origin of the translation system and the genetic code in the RNA world by means of natural selection, exaptation, and subfunctionalization 2007 May 31
14. Sami EL Khatib: Assumption and Criticism on RNA World Hypothesis from Ribozymes to Functional Cells March 12, 2021
15. Paul C. W. Davies: The algorithmic origins of life 2013 Feb 6
16. Charles W. Carter, Jr. What RNA World? Why a Peptide/RNA Partnership Merits Renewed Experimental Attention 23 January 2015
17. Dennis R. Salahub: Characterization of the active site of yeast RNA polymerase II by DFT and ReaxFF calculations 08 April 2008
18. Dimiter Kunnev: Possible Emergence of Sequence Specific RNA Aminoacylation via Peptide Intermediary to Initiate Darwinian Evolution and Code Through Origin of Life 2018 Oct 2;8
19. Hadieh Monajemi: The P-site A76 2′-OH acts as a peptidyl shuttle in a stepwise peptidyl transfer mechanism 2015
20. Francisco Prosdocimi: The Ancient History of Peptidyl Transferase Center Formation as Told by Conservation and Information Analyses 2020 Aug 5
21. Sávio T.Farias: Origin and evolution of the Peptidyl Transferase Center from proto-tRNAs 2014
22. Felix Müller: A prebiotically plausible scenario of an RNA–peptide world 11 May 2022
23. Stephen D. Fried: Peptides before and during the nucleotide world: an origins story emphasizing cooperation between proteins and nucleic acids 09 February 2022
24. Jordana Cepelewicz: The End of the RNA World Is Near, Biochemists Argue December 19, 2017
25. Mathieu E. Rebeaud:  On the evolution of chaperones and cochaperones and the expansion of proteomes across the Tree of Life May 17, 2021
26. Jörg Martin: Assembly and Disassembly of GroEL and GroES Complexes 2000
27. Hays S. Rye: GroEL-Mediated Protein Folding: Making the Impossible, Possible 2013 Sep 25
28. Thorsten Hugel: Controlling protein function by fine-tuning conformational flexibility 2020 Jul 22
29. Susan Lindquist: HSP90 at the hub of protein homeostasis: emerging mechanistic insights 2010 Jul;11
30. F Egami: A working hypothesis on the interdependent genesis of nucleotide bases, protein amino acids, and primitive genetic code 1981 Sep;11
31. Charles W Carter, Jr: Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding 24 October 2017
32. Jessica C. Bowman: The Ribosome Challenge to the RNA World  20 February 2015
33. Jordana Cepelewicz: Life’s First Molecule Was Protein, Not RNA, New Model Suggests November 2, 2017
34. Coenzymes & Cofactors

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34The RNA world, and the origins of life - Page 2 Empty Selecting the nucleobases used in life Wed Jul 13, 2022 6:19 am

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Selecting the nucleobases used in life

https://reasonandscience.catsboard.com/t2024p25-the-rna-world-and-the-origins-of-life#9371

1. On the early earth, in the existing "structure space", a limitless number of different molecules could have been generated by natural processes, like lightning, hydrothermal vents, volcanic gas eruptions, etc.  
2. Life uses exclusively a quartet of specified complex macromolecules, that are synthesized in modern cells by complex metabolic pathways, that were not extant, prebiotically. 
3. Selecting a specific set of complex macromolecules out of unlimited "structure space" by unguided means is theoretically remotely possible, but de facto, impossible. Therefore, these molecules were not selected naturally. They were designed.  

Maybe you are familiar with the concept of "sequence space". It relates to the fact that there is a huge combinatorial space (or possibilities) to put an amino acid strand together, but only a very limited number of sequences bear function, or eventually fold into 3D forms, and become functional proteins. That makes it very remotely possible, that random chance joined functional sequences together on the early earth. Analogously, the same goes for "Structure space" of the four macromolecular "bricks" or building blocks used in life. Adenine, for example, one of the five nucleobases used in RNA and DNA,  are purines, made of carbon, hydrogen, and nitrogen atoms. They have a six-membered nitrogen ring, fused to a five-membered nitrogen ring. The thymine nucleobase is a pyrimidine, and has just a one-ring structure, using carbon, hydrogen, and nitrogen atoms. There is no physical law, that restricts these molecules to have this isomeric ring structure and atomic composition. But in structure space, only a very small set or arrangement of nucleobases, with a specified chemical arrangement, bears function. How was the functional nucleobase quintet selected prebiotically? 

H. James Cleaves 2nd (2015): ‘‘Structure space’’ represents the number of molecular structures that could exist given specific defining parameters. For example, the total organic structure space, the druglike structure space, the amino acid structure space, and so on. Many of these chemical spaces are very large. For example, the total number of possible stable drug-like organic molecules may be on the order of 10^33 to 10^180. , The number of known naturally occurring or synthetic molecules is much smaller. As of July 2009, there were 49,037,297 unique organic and inorganic chemical substances registered with the Chemical Abstracts Service As a final comparison, a recent exploration of the organic contents of methanol extracts of the Murchison meteorite using high-resolution mass spectrometry revealed a complex though a relatively small set of compounds ranging from 100,000 to perhaps 10,000,000. Clearly, nature is constrained in its exploration of the vastness of chemical space by the reaction mechanisms available to it at any given point in time and the physicochemical stability of the resulting structures in their environmental context.

The number of molecules that could fulfill the minimal requirements of being ‘‘nucleic acid-like’’ is remarkably large and in principle limitless, though reasonable arguments could probably be made as to why monomers cannot
contain more than some given number of carbon atoms.

A variety of structural isomers of RNA could potentially function as genetic platforms. Ribonucleosides may have competed with a multitude of alternative structures whose potential proto-biochemical roles and abiotic syntheses remain to be explored. The rules of organic chemistry, though the set of possible molecules could be very large. If there were alternative molecules that could better fulfill these criteria, then extant genetic systems could be considered suboptimal. It is of interest to understand whether biology’s solution to these various problems is optimal, suboptimal, or arbitrary. To date, no one-pot reaction has yielded either the purine or pyrimidine ribonucleosides directly from likely prevalent prebiotic starting materials. Enumeration of the riboside BC5H9O4 space gives some appreciation of the size and dimensionality of nucleic acid-like molecule space and allows some consideration of the optimality or arbitrariness of biology’s choice of this particular isomer.

With respect to the atom choice explored here (using only carbon, hydrogen, and oxygen), we note first that C, H, and O are among the most cosmo- and geochemically abundant elements and that CHO isomers are in principle derivable from formose-type chemistry, which allows an obvious linkage to abiotic geochemistry. The evaluation of the BC5H9O4 isomer space must thus be viewed as a first practical example of an exploration of what is a much larger chemical space. Limiting the search to structural isomers with the molecular formula of the core sugar of RNA (BC5H9O4, where B= a nitrogenous base), the range and variety of possible structures is enumerated precisely with structure generation software. This gives a glimpse of what abiotic chemistry could produce.

The structural space explored here is restricted to the molecular formula of the core RNA riboside but nonetheless includes a large number of possible isomers. In the formula range from BC3H7O2 to BC5H9O4 (RNA’s) there are likely scores of valid formulas. These could collectively produce many thousands of structurally sound isomers. In turn, each of these isomers could yield many stereo- and macromolecular linkage-isomers, leading ultimately to perhaps billions of nucleic acid polymer types potentially capable of supporting base-pairing. It is likely that only a subset of these structural and stereoisomers would lead to stable base-pairing systems 50

Andro C. Rios (2014): The native bases of RNA and DNA are prominent examples of the narrow selection of organic molecules upon which life is based. How did nature “decide” upon these specific heterocycles? Evidence suggests that many types of heterocycles could have been present on the early Earth. The prebiotic formation of polymeric nucleic acids employing the native bases remains a challenging problem. Hypotheses have proposed that the emerging RNA world may have included many types of nucleobases. This is supported by the extensive utilization of non-canonical nucleobases in extant RNA and the resemblance of many of the modified bases to heterocycles generated in simulated prebiotic chemistry experiments. Nucleobase modification is a ubiquitous post-transcriptional activity found across all domains of life. These transformations are vital to cellular function since they modulate genetic expression 9

If we consider that basically any of the basic compounds and atoms extant on early earth or in meteorites could have been incorporated to make macromolecules, and a wide array of different ring structures and isomeric conformations to make nucleobases, for example, could be formed, then it becomes clear, that the structure space becomes basically limitless. 

The RNA world, and the origins of life - Page 2 No_ans12

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