A Peptide–Nucleic Acid Replicator Origin for Life
Authors: Bernard M.A.G. Piette, Jonathan G. Heddle
Journal: Trends in Ecology & Evolution (2020)

The first ancestor to all life was a self-replicating entity capable of evolving but must have been much simpler than a cell; that is, a molecular replicator.Recent work on RNA polymerase ribozymes supports the view that the molecular replicator was made of RNA, and no other molecule was required (the RNA World).The RNA World is challenged by the fact that a self-replicating RNA polymerase ribozyme has yet to be demonstrated and that little evidence for its existence is seen in life today as well as problems in how the transition from RNA-only to RNA–protein world could have occurred.A molecular replicator with two components –RNA and peptide – overcomes these problems and may be a better fit. Evolution requires self-replication. But, what was the very first self-replicator directly ancestral to all life? The currently favoured RNA World theory assigns this role to RNA alone but suffers from a number of seemingly intractable problems. Instead, we suggest that the self-replicator consisted of both peptides and nucleic acid strands. Such a nucleopeptide replicator is more feasible both in the light of the replication machinery currently found in cells and the complexity of the evolutionary path required to reach them. Recent theoretical and mathematical work supports this idea and provide a blueprint for future investigations. Evolution requires self-replication. But, what was the very first self-replicator directly ancestral to all life? The currently favoured RNA World theory assigns this role to RNA alone but suffers from a number of seemingly intractable problems. Instead, we suggest that the self-replicator consisted of both peptides and nucleic acid strands. Such a nucleopeptide replicator is more feasible both in the light of the replication machinery currently found in cells and the complexity of the evolutionary path required to reach them. Recent theoretical and mathematical work supports this idea and provide a blueprint for future investigations. Life as we understand it is cellular. The last universal common ancestor (LUCA) of all cells (not a single cell of course but a population) is understood in some detail; it possessed a cell membrane, DNA, the basic molecular machines for copying DNA (i.e., polymerase etc.), and a functional ribosome, among many more [1.Weiss M.C. et al.The physiology and habitat of the last universal common ancestor.Nat. Microbiol. 2016; 1: 16116Crossref PubMed Scopus (512) Google Scholar]. From this highly truncated list alone it is clear that LUCA was far too complex to spontaneously assemble. It must have evolved from simpler systems, themselves able to self-replicate with some tolerance for error (otherwise they would not be able to evolve). Indeed, it is difficult to imagine that anything recognisably a cell could have spontaneous origins. This means that they in turn must have evolved from even simpler self replicators; that is, molecular self-replicators. The first such replicator is referred to as the initial Darwinian ancestor (IDA) [2.Yarus M. Getting past the RNA world: the initial Darwinian ancestor.Cold Spring Harb. Perspect. Biol. 2011; 3a003590Crossref PubMed Scopus (51) Google Scholar]. The identity of the IDA has been cause for much speculation over the years. Nonbiological replicators such as clay crystals [3.Cairns-Smith A.G. Genetic Takeover: And the Mineral Origins of Life. Cambridge University Press, 1982Google Scholar] have been invoked but are unconvincing as they require a complete takeover of one substrate with another and lack a persuasive argument to show how this could have occurred. An IDA built from biological molecules is more convincing and necessitates physicochemical conditions compatible with their formation, and with the self-replicating reaction cycle of the IDA itself. The latter likely required relatively mild conditions approaching those of analogous biochemical reactions today. There is now ample evidence that biological building blocks such as amino acids, ribose, and deoxyribose, among others, were present on the early Earth [4.Zaucha J. Heddle J.G. Resurrecting the dead (molecules).Comput. Struct. Biotechnol. J. 2017; 15: 351-358Crossref PubMed Scopus (4) Google Scholar, 5.Nam I. et al.Abiotic synthesis of purine and pyrimidine ribonucleosides in aqueous microdroplets.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 36-40Crossref PubMed Scopus (69) Google Scholar, 6.Bada J.L. New insights into prebiotic chemistry from Stanley Miller's spark discharge experiments.Chem. Soc. Rev. 2013; 42: 2186-2196Crossref PubMed Scopus (128) Google Scholar, 7.Wachowius F. et al.Nucleic acids: function and potential for abiogenesis.Q. Rev. Biophys. 2017; 50e4Crossref PubMed Scopus (32) Google Scholar]. Potential chemistries for building block synthesis have been demonstrated, with cyanosulfidic chemistry [8.Sutherland J.D. The origin of life – out of the blue.Angew. Chem. Int. Ed. 2016; 55: 104-121Crossref PubMed Scopus (254) Google Scholar] showing great promise. Furthermore, recent work has shown convincing peptide ligation in prebiotic conditions [9.Canavelli P. et al.Peptide ligation by chemoselective aminonitrile coupling in water.Nature. 2019; 571: 546-549Crossref PubMed Scopus (81) Google Scholar]. Given these findings, it now seems reasonable to assume that the IDA was constructed of components highly similar or identical to those found in life today. Indeed, such a replicator must have occurred at some stage very early in evolution even if not at the very beginning. The scene being set for an IDA constructed of biological molecules to arise, our focus turns to the main issue of this work – deciding on its identity. Three approaches will be useful to us in this endeavour. (i) Consideration of current self-replicating biological systems. By looking at how cells currently achieve self-replication we may be able to extrapolate into the past and deduce the composition of the start point. (ii) Consideration of the simplest system. Which IDA is most likely able to achieve self-replication and has the simplest path to arrive at current self-replicating systems? (iii) Dynamical feasibility. It is not always obvious that a given self-replication scheme is actually dynamically feasible. For example, expected rates of substrate breakdown will determine if the mathematical model allows self-sustained self-replication. Where substrates break down more quickly than they can be synthesised, the mathematical model will rightly predict self-sustained self-replication to be unfeasible. A dynamically unfeasible scheme need not be considered further. A number of suggestions for the IDA have been made over the years. These include protein or peptide alone, such as thiol-rich peptides; amyloid (see Glossary) inspired in part by our understanding of prions [10.Wiltzius J.J. et al.Molecular mechanisms for protein-encoded inheritance.Nat. Struct. Mol. Biol. 2009; 16: 973Crossref PubMed Scopus (209) Google Scholar,11.Wickner R.B. et al.Amyloid diseases of yeast: prions are proteins acting as genes.Essays Biochem. 2014; 56: 193-205Crossref PubMed Google Scholar]; nucleic acid alone (mainly the RNA World [12.Gilbert W. The RNA world.Nature. 1986; 319: 618Crossref Scopus (1963) Google Scholar, 13.Higgs P.G. Lehman N. The RNA world: molecular cooperation at the origins of life.Nat. Rev. Genet. 2015; 16: 7-17Crossref PubMed Scopus (305) Google Scholar, 14.Orgel L.E. Prebiotic chemistry and the origin of the RNA world.Crit. Rev. Biochem. Mol. 2004; 39: 99-123Crossref PubMed Scopus (728) Google Scholar]); and a mixture of both [15.Fishkis M. Emergence of self-reproduction in cooperative chemical evolution of prebiological molecules.Orig. Life Evol. Biosph. 2011; 41: 261-275Crossref PubMed Scopus (12) Google Scholar, 16.Di Giulio M. On the RNA world: evidence in favor of an early ribonucleopeptide world.J. Mol. Evol. 1997; 45: 571-578Crossref PubMed Scopus (65) Google Scholar, 17.Carter C. What RNA world? Why a peptide/RNA partnership merits renewed experimental attention.Life. 2015; 5: 294-320Crossref PubMed Scopus (56) Google Scholar, 18.Carter Jr., C.W. Kraut J. A proposed model for interaction of polypeptides with RNA.Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 283-287Crossref PubMed Scopus (110) Google Scholar]. The most widely accepted is the RNA World which posits that the IDA was an RNA strand capable of folding into an active replicase. This is the main alternative to a nucleopeptide replicator considered in this work. The RNA World is immediately attractive when considering our three tests above. (i) RNA is intricately involved in current self-replicating systems (Figure 1). It carries the transcribed genetic information (and there is general agreement that the original substrate for the genetic code was RNA, not DNA) and it constitutes the majority of the ribosome, the heart of cellular self-replication, including the catalytic site itself [19.Nissen P. et al.The structural basis of ribosome activity in peptide bond synthesis.Science. 2000; 289: 920-930Crossref PubMed Scopus (1753) Google Scholar]. In addition, cells do contain RNA sequences (ribozymes) that are able to catalyse reactions; a function more typically associated with proteinaceous enzymes. (ii) Considerations of simplicity make the RNA World attractive because it is able to house both encoding of sequence information and catalytic activity in a single molecule in a way which proteins (which are poor at information storage) and DNA (poor at catalysis) are not. (iii) In principle, single-component self-replicating systems (RNA or otherwise) are not dynamically or chemically forbidden and indeed rudimentary systems have been demonstrated on the macroscopic scale [20.Griffith S. et al.Robotics: self-replication from random parts.Nature. 2005; 437: 636Crossref PubMed Scopus (112) Google Scholar]. However, a closer look at the RNA-only IDA in the context of these three considerations reveals serious problems, some of which appear insurmountable. Comparison with a nucleopeptide IDA suggest the latter is a more likely candidate. The concept of using current cells and molecules as a guide to predict the features of earlier (now extinct) ones and indeed, even resurrect extinct proteins is well established [4.Zaucha J. Heddle J.G. Resurrecting the dead (molecules).Comput. Struct. Biotechnol. J. 2017; 15: 351-358Crossref PubMed Scopus (4) Google Scholar]. Bioinformatics techniques [4.Zaucha J. Heddle J.G. Resurrecting the dead (molecules).Comput. Struct. Biotechnol. J. 2017; 15: 351-358Crossref PubMed Scopus (4) Google Scholar] allow phylogenetic trees to be reconstructed [21.Yang Z. Rannala B. Molecular phylogenetics: principles and practice.Nat. Rev. Genet. 2012; 13: 303-314Crossref PubMed Scopus (409) Google Scholar] and predictions to be made of the identity of ancient ancestor molecules. The extreme chronological distance between the IDA and the current day would make this challenging but perhaps possible if a simple self-replicator was present in any cells today. In fact, no such single molecule replicator exists. Instead, replication is split between nucleic acids and proteins with the general rule being that nucleic acids encode and transfer information (DNA and mRNA), and proteins (enzymes) carry out catalytic functions including synthesising new copies of the nucleic acids (Figure 1). A notable exception being the ribosome, the catalytic centre of which is a ribozyme [22.Moore P.B. Steitz T.A. After the ribosome structures: how does peptidyl transferase work?.RNA. 2003; 9: 155-159Crossref PubMed Scopus (54) Google Scholar]. This means that a basic cross-catalytic symmetry is observed: RNA makes protein, protein makes RNA. Thus, in the spirit of Spiegelman's Monster (Box 1), we can conceive that given the correct conditions, a supply of energy and suitable chemical building blocks, a self-replicating system using components from current cells could function and would include DNA, DNA polymerase, ribosome, RNA polymerase, tRNA and tRNA synthetases among others. Indeed, recent progress has been made towards this in experiments using liposome-based synthetic cells. These contained DNA replication machinery from Φ29 and were capable of self-sustained DNA amplification [23.van Nies P. et al.Self-replication of DNA by its encoded proteins in liposome-based synthetic cells.Nat. Commun. 2018; 9: 1583Crossref PubMed Scopus (119) Google Scholar]. However, such systems would not be indefinitely self-sustaining due to the eventual degradation of the components responsible for synthesising the protein machinery. Unsurprisingly, these preliminary functional self-replicating systems retain the nucleic acid–protein division of labour.Box 1Spiegelman's Monster and the Rise of In Vitro EvolutionQβ bacteriophage is an RNA virus whose genome is replicated by Qβ replicase, an RNA-dependent RNA polymerase. In 1965, Spiegelman carried out an interesting experiment – he mixed together Qβ replicase and the phage RNA together with RNA nucleotides. As a result he was able to observe in vitro replication of the genome [64.Haruna I. Spiegelman S. Autocatalytic synthesis of a viral RNA in vitro.Science. 1965; 150: 884-886Crossref PubMed Scopus (78) Google Scholar] – a breakthrough at the time. Furthermore, the system was able to evolve [65.Mills D.R. et al.An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule.Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 217Crossref PubMed Scopus (456) Google Scholar], freed from constraints and requirements of functioning in cells and encoding other virus proteins. Over the course of 75 serial transfers, the RNA evolved to become shorter, eventually reducing in length to 550 nt from an original of 3600 nt. Subsequent experiments were able to isolate a 218 nt RNA; essentially the minimum required to function. The system did not encode or synthesize the Qβ replicase, which was provided. Nevertheless, it showed the potential for simple, self-replicating systems to function and, importantly, to evolve outside of the cellular environment. Crucially, it showed that this could be achieved with a few constituents, comprising only building blocks, an RNA strand, and a protein. The spirit of Spiegelman's experiment lives on today as efforts continue to produce fully self-contained in vitro self-replicating systems [66.Li J. et al.Cogenerating synthetic parts toward a self-replicating system.ACS Synth. Biol. 2017; 6: 1327-1336Crossref PubMed Scopus (31) Google Scholar]. Qβ bacteriophage is an RNA virus whose genome is replicated by Qβ replicase, an RNA-dependent RNA polymerase. In 1965, Spiegelman carried out an interesting experiment – he mixed together Qβ replicase and the phage RNA together with RNA nucleotides. As a result he was able to observe in vitro replication of the genome [64.Haruna I. Spiegelman S. Autocatalytic synthesis of a viral RNA in vitro.Science. 1965; 150: 884-886Crossref PubMed Scopus (78) Google Scholar] – a breakthrough at the time. Furthermore, the system was able to evolve [65.Mills D.R. et al.An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule.Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 217Crossref PubMed Scopus (456) Google Scholar], freed from constraints and requirements of functioning in cells and encoding other virus proteins. Over the course of 75 serial transfers, the RNA evolved to become shorter, eventually reducing in length to 550 nt from an original of 3600 nt. Subsequent experiments were able to isolate a 218 nt RNA; essentially the minimum required to function. The system did not encode or synthesize the Qβ replicase, which was provided. Nevertheless, it showed the potential for simple, self-replicating systems to function and, importantly, to evolve outside of the cellular environment. Crucially, it showed that this could be achieved with a few constituents, comprising only building blocks, an RNA strand, and a protein. The spirit of Spiegelman's experiment lives on today as efforts continue to produce fully self-contained in vitro self-replicating systems [66.Li J. et al.Cogenerating synthetic parts toward a self-replicating system.ACS Synth. Biol. 2017; 6: 1327-1336Crossref PubMed Scopus (31) Google Scholar]. As a thought experiment, we can simplify further the extant nucleopeptide replicator system so that only indispensable components remain and assume that instead of multiple examples of each class there was one able to carry out all the functions currently undertaken by that class. That is, there existed (i) a single nucleic acid sequence that encoded a single amino acid sequence and acted as its own mRNA and its own ribosome; and (ii) a peptide sequence that acted to copy the mRNA that encoded the amino acid sequence of the same peptide (Figure 1). This is the most fundamental ancestral self-replicator that maintains the existing functional split between nucleic acids and peptides. What advantage does such a system have over the main alternative, the RNA World? Perhaps the most convincing answer comes when considering how they could have evolved into the current replication machinery. For the nucleopeptide IDA the path is straightforward. Each component simply maintains the same role, evolving greater specificity and efficiency over time, something that can be achieved by errors that would inevitably arise during replication. The part with mRNA functionality expands its code to encode for an increased number of amino acids. This leads to production of more efficient polymerases, thus ensuring better survival. The part with ribosome functionality separates from the mRNA portion and gains increasing catalytic efficiency, again increasing survival likelihood. For an RNA-only replicator, the story is less convincing. A self-replicating RNA molecule seems at first glance feasible, and if such an RNA-only IDA existed, it is likely that it would be a self-replicating ribozyme polymerase (Figure 1) [24.Horning D.P. Joyce G.F. Amplification of RNA by an RNA polymerase ribozyme.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 9786-9791Crossref PubMed Scopus (123) Google Scholar]. However, this is not what is seen in extant cells and it is difficult to understand the steps by which it gained mRNA functionality, and transferred polymerase functionality to peptides and information storage functionality to DNA. Of these, the first two present most difficulty as the ribozyme, being a self-sufficient self-replicating entity simply has no need for interaction with peptides or amino acids. This makes a gradual transfer of these functions to them difficult to envision. How could gain of mRNA functionality; that is, encoding of amino acid sequences in RNA occur in a ribozyme whose sequence is optimised for ribozyme self-copying functions? Any change to encode a helpful peptide would presumably decrease ribozyme effectiveness. It seems unlikely that by chance the sequence giving useful ribozyme functionality would also happen to encode a functional peptide that would increase the replication and survival of the ribozyme itself. If this did take place then it would consequently have required the ribozymal polymerase sequence to serve also as mRNA; encoding an amino acid sequence that could be translated into a functional peptide. We then come close to the nucleopeptide replicator outlined in Figure 1; the difference being that the system in Figure 1 has the advantage of not requiring the mRNA to also have polymerase functionality. Similar arguments pertain to transfer of polymerase functionality from RNA to peptides. Recent work shows that this is highly unlikely, suggesting such an RNA functionality never existed [25.Carter Jr., C.W. Wills P.R. Interdependence, reflexivity, fidelity, impedance matching, and the evolution of genetic coding.Mol. Biol. Evol. 2017; 35: 269-286Crossref Scopus (34) Google Scholar,26.Wills P.R. Carter Jr., C.W. Insuperable problems of the genetic code initially emerging in an RNA world.Biosystems. 2018; 164: 155-166Crossref PubMed Scopus (37) Google Scholar]. By asking which possible IDA is simplest we are considering which functional replicator is simple enough to be realistically feasible as the first, spontaneously occurring IDA. The simplest system does not have to be the real one but arguments for parsimony in biology are powerful [27.Crisci J.V. Parsimony in evolutionary theory: law or methodological prescription?.J. Theor. Biol. 1982; 97: 35-41Crossref Scopus (17) Google Scholar]. The simplest RNA World theory requires only a self-replicating ribozyme. This could be an RNA strand with ligase activity; that is, self-templating using pre-existing large fragments of complimentary sequences. Ligase (but not self-replicating ligase) ribozymes do exist in nature [28.Vicens Q. Cech T.R. A natural ribozyme with 3′,5′ RNA ligase activity.Nat. Chem. Biol. 2009; 5: 97-99Crossref PubMed Scopus (25) Google Scholar] and in vitro designed/evolved ribozyme ligases have been produced, beginning with the work of Bartel and Szostak [29.Bartel D.P. Szostak J.W. Isolation of new ribozymes from a large pool of random sequences [see comment].Science. 1993; 261: 1411-1418Crossref PubMed Scopus (703) Google Scholar]. Efforts have been made to produce minimal ligases, for example by Kurihara et al. [30.Kurihara E. et al.Development of a functionally minimized mutant of the R3C ligase ribozyme offers insight into the plausibility of the RNA World Hypothesis.Biology. 2014; 3: 452-465Crossref PubMed Scopus (10) Google Scholar] when they made an ~50 nt functional version of R3C ligase [31.Rogers J. Joyce G.F. A ribozyme that lacks cytidine.Nature. 1999; 402: 323-325Crossref PubMed Scopus (81) Google Scholar], similar in length to the small L1 ligase [32.Robertson M.P. et al.Optimization and optimality of a short ribozyme ligase that joins non-Watson-Crick base pairings.RNA. 2001; 7: 513-523Crossref PubMed Scopus (33) Google Scholar]. These ligases, however, do not self-replicate. Efforts to produce self-replicating ligases have borne fruit. Paul and Joyce, for example, modified R3C ligase ribozyme so that it could template two half copies of itself that it then ligated [33.Paul N. Joyce G.F. A self-replicating ligase ribozyme.Proc. Natl. Acad. Sci. U. S. 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This may seem unlikely, but in fact, recent work suggests that functional ribozyme ligases can be produced spontaneously (i.e., nonenzymatically) from short building blocks more likely to have been present on the early Earth [36.Zhou L. et al.Assembly of a functional ribozyme from short oligomers by enhanced non-enzymatic ligation.bioRxiv. 2019; : 700229Google Scholar,37.Wachowius F. Holliger P. Non-enzymatic assembly of a minimized RNA polymerase ribozyme.ChemSystemsChem. 2019; 1: 12-15Crossref Google Scholar]. However, again, these ligases do not replicate themselves. This may be an insurmountable problem, as Wachowius et al. stated: 'Fundamentally, emergence of new functions when assembling long sequences is confounded by the nature of such activities: ligases use less information to choose substrates than is required to define the ligase activity itself, so cannot copy themselves (or other components) from sequences lacking that information, i.e. random sequence' [7.Wachowius F. et al.Nucleic acids: function and potential for abiogenesis.Q. Rev. Biophys. 2017; 50e4Crossref PubMed Scopus (32) Google Scholar]. A ribozyme acting as a polymerase therefore seems more promising. This could copy any template strand from only short nucleotide building blocks (Figure 1). The first designed ribozyme able to convincingly do this was R18 [38.Johnston W.K. et al.RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.Science. 2001; 292: 1319-1325Crossref PubMed Scopus (491) Google Scholar,39.Wochner A. et al.Ribozyme-catalyzed transcription of an active ribozyme.Science. 2011; 332: 209-212Crossref PubMed Scopus (265) Google Scholar]. The tC9Y ribozyme made a breakthrough, being able to polymerise products slightly longer than itself [40.Attwater J. et al.In-ice evolution of RNA polymerase ribozyme activity.Nat. Chem. 2013; 5: 1011-1018Crossref PubMed Scopus (180) Google Scholar]. The most recent advance is the 24-3 ribozyme that can copy RNA sequences having secondary structure, although this is still possible only for short sequences; that is, they cannot copy themselves. These recent ribozymes, at close to 200 nt in length, are likely too big to spontaneously arise, although other recent work [41.Akoopie A. Müller U.F. Lower temperature optimum of a smaller, fragmented triphosphorylation ribozyme.Phys. Chem. Chem. Phys. 2016; 18: 20118-20125Crossref PubMed Google Scholar] suggests that, in some cases, ribozymes may have been able to function as fragments working together. In summary, if a self-replicating RNA-only ribozyme polymerase does prove possible, it may be that it is too long and complex to have arisen as the IDA. An alternative to an RNA-only IDA is that it consisted of both nucleic acid and peptide components, each able to catalyse polymerisation of the other. An early example of this idea was the Carter and Kraut model [18.Carter Jr., C.W. Kraut J. A proposed model for interaction of polypeptides with RNA.Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 283-287Crossref PubMed Scopus (110) Google Scholar], which proposed that a short double-stranded (ds)RNA sequence would be able to catalyse formation of a short β hairpin structure that in turn would catalyse dsRNA polymerisation. With a potential for partial coding [42.Carter C.W.J. Cradles for molecular evolution.New Sci. 1975; 27: 784-787Google Scholar], sustained self-replication could be possible. Our favoured nucleopeptide IDA model is a conceptual relative of the Carter and Kraut model (Figure 1). [43.Banwell E.F. et al.Reciprocal nucleopeptides as the ancestral Darwinian self-replicator.Mol. Biol. Evol. 2018; 35: 404-416Crossref PubMed Scopus (7) Google Scholar] It relies only on random production of short amino acids and RNAs (or possibly a mix of RNA, DNA, and other monomers since lost, known as XNA). Here, a short stretch of single-stranded (ss)RNA could act as both a primordial RNA (p-RNA) and a primordial ribosome (p-Rib), encoding a peptide sequence and catalysing its polymerisation. If the resulting peptide was able to act as a primordial polymerase (p-Pol) and copy the ssRNA then an IDA would result. The first and most fundamental problem with this concept is how specific amino acids (or classes of amino acids) can be encoded and located at specific mRNA sequences (the coding problem; Box 2). This is troublesome as tRNA and certainly tRNA synthetases did not then exist. The simplest answer to this problem is hard stereochemical selection, where there is a direct interaction between amino acids and their codons or anticodons [44.Gamow G. Possible relation between deoxyribonucleic acid and protein structures.Nature. 1954; 173: 318Crossref Scopus (192) Google Scholar,45.Woese C.R. et al.On the fundamental nature and evolution of the genetic code.Cold Spring Harb. Symp. Quant. Biol. 1966; 31: 723-736Crossref PubMed Scopus (207) Google Scholar]. Such an interaction could bring specific amino acids into close proximity on the p-mRNA, increasing the probability of polymerisation via peptide bond formation. This is a so called entropy trap and has been suggested in other models of early biological replicators wherein RNA-based carriers of amino acids align on mRNA sequences via codon–anticodon interactions [46.Carter Jr., C.W. Wills P.R. Hierarchical groove discrimination by Class I and II aminoacyl-tRNA synthetases reveals a palimpsest of the operational RNA code in the tRNA acceptor-stem bases.Nucleic Acids Res. 2018; 46: 9667-9683Crossref PubMed Scopus (23) Google Scholar]. The release of the produced peptide could occur stochastically or through periodic environmental changes (e.g., changes in temperature). There is some experimental support for entropy traps [47.Tamura K. Schimmel P. Peptide synthesis with a template-like RNA guide and aminoacyl phosphate adaptors.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8666-8669Crossref PubMed Scopus (35) Google Scholar,48.Jacobsen J.R. Schultz P.G. Antibody catalysis of peptide bond formation.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5888-5892Crossref PubMed Scopus (53) Google Scholar] and indeed they may even play a role in peptide bond formation in the extant ribosome [49.Sievers A. et al.The ribosome as an entropy trap.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7897-7901Crossref PubMed Scopus (252) Google Scholar].Box 2How Did the Link between Codons and Amino Acids Arise?In all life, triplet codons in mRNA code for specific amino acids that are brought to the ribosome as activated amino acids attached to a tRNA that contains the relevant, specific anticodon. That the correct amino acid is connected to the correct tRNA is ensured by enzymes called aminoacyl tRNA synthetases (aaRSs). These attach the amino acids to the 3′ end of the tRNA distal from the anticodon. This is a form of sym