At some stage in the origin of life, an informational polymer must have arisen by purely chemical means. According to one version of the 'RNA world' hypothesis this polymer was RNA, but attempts to provide experimental support for this have failed. In particular, although there has been some success demonstrating that 'activated' ribonucleotides can polymerize to form RNA, it is far from obvious how such ribonucleotides could have formed from their constituent parts (ribose and nucleobases). Ribose is difficult to form selectively, and the addition of nucleobases to ribose is inefficient in the case of purines and does not occur at all in the case of the canonical pyrimidines. Here we show that activated pyrimidine ribonucleotides can be formed in a short sequence that bypasses free ribose and the nucleobases, and instead proceeds through arabinose amino-oxazoline and anhydronucleoside intermediates. The starting materials for the synthesis-cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde and inorganic phosphate-are plausible prebiotic feedstock molecules, and the conditions of the synthesis are consistent with potential early-Earth geochemical models. Although inorganic phosphate is only incorporated into the nucleotides at a late stage of the sequence, its presence from the start is essential as it controls three reactions in the earlier stages by acting as a general acid/base catalyst, a nucleophilic catalyst, a pH buffer and a chemical buffer. For prebiotic reaction sequences, our results highlight the importance of working with mixed chemical systems in which reactants for a particular reaction step can also control other steps.

Amide bond formation is one of the most important reactions in both chemistry and biology 1-4 , but there is currently no chemical method to achieve α-peptide ligation in water that tolerates all twenty proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes the biological role of peptides predates Life's last universal common ancestor and that peptides played an essential role in the origins of Life 5-9 . The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis points towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of Life 5,9-13 . However, a robust mechanism for aminoacyl thioester formation has never been demonstrated 13 . Here, we report a chemoselective, high yielding a-aminonitrile ligation that exploits only prebiotically plausible molecules-hydrogen sulfide, thioacetate 12,14 and ferricyanide 12,14-17 or cyanoacetylene 8,14 -to yield apeptides in water. The ligation is extremely selective for a-aminonitrile coupling and tolerates all 20 proteinogenic amino acid residues. Two essential features enable the peptide ligation in water: 1) the reactivity and pKaH of a-aminonitriles makes them compatible with ligation at neutral pH, and 2) Nacylation stabilises the peptide product and activates the peptide precursor to (biomimetic) N®C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological a-peptides, suggesting short N-acyl peptide nitriles were plausible substrates during early evolution.To improve the efficiency and selectivity of peptide ligation in water we sought to develop a novel mechanism for non-enzymatic peptide synthesis, which would operate via biomimetic N®C ligation in near-neutral pH water, and we suspected that a combination of sulfur and nitrile chemistry would be required ( Fig. 1a) 8,9,14,[18][19][20][21] . Proteinogenic a-aminonitriles (AA-CN) are readily synthesised 8,18 , and their direct ligation would provide the simplest prebiotic pathway to peptides. Unfortunately, incubation of AA-CN in water results in extremely ineffective peptide synthesis 22 . a-Amino acids (AA) are widely assumed to be prebiotic precursors of peptides, but the harsh conditions (typically strongly acidic or alkaline solutions) required for AA formation from AA-CN are incompatible with the integrity of both peptides and electrophilic activating agents. Therefore, we sought a more congruent and direct pathway from a-aminonitriles to a-peptides, and although the conversion of AA-CN to AA-SH has never been reported 23 , harnessing the AA-CN nitrile moiety for thioacid synthesis seemed more prudent than dissipating its activation through exhaustive hydrolysis.Orgel has previously suggested that a-aminothioacids (AA-SH) 16 might offer an interesting alternative to biological thioesters 10,11 . AA-SH unite excellent aqueous stability with highly selective (electrophilic or oxidative) activation 12,14,16,24 , but their prebiotic synthesis...

A plausible process for non-enzymatic RNA replication would greatly simplify models of the transition from prebiotic chemistry to simple biology. However, all known conditions for the chemical copying of an RNA template result in the synthesis of a complementary strand containing a mixture of 2′-5′ and 3′-5′ linkages, rather than the selective synthesis of only 3′-5′ linkages as found in contemporary RNA. Here we show that such backbone heterogeneity is compatible with RNA folding into defined three-dimensional structures that retain molecular recognition and catalytic properties and, therefore, would not prevent the evolution of functional RNAs such as ribozymes. Moreover, the same backbone heterogeneity lowers the melting temperature of RNA duplexes that would otherwise be too stable for thermal strand separation. By allowing copied strands to dissociate, this heterogeneity may have been one of the essential features that allowed RNA to emerge as the first biopolymer.The ability of RNA molecules to fold into defined three-dimensional structures with exquisitely specific molecular recognition and catalytic properties is the conceptual basis of the RNA World hypothesis, an early stage in the evolution of life in which RNA served not only as the polymer of inheritance, but as the central functional polymer of biochemistry [1][2][3] . This model is most strikingly supported by the observation that all modern proteins are synthesized by the peptidyl transferase ribozyme at the heart of the ribosome 4,5 . With the RNA World hypothesis so strongly supported by this and other evidence 1 , the central question in the origin of life field concerns the pathway from the prebiotic chemistry of the early Earth to the emergence of simple forms of cellular life containing RNA genomes coding for RNA enzymes. While there has been considerable recent progress towards the elucidation of potentially prebiotic pathways for ribonucleotide synthesis 6-8 and the assembly of activated nucleotides into oligonucleotides 9,10 , the non-enzymatic replication of RNA oligonucleotides remains problematic. A series of seemingly intractable difficulties * Correspondence to: szostak@molbio.mgh.harvard.edu. † Current Address: Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London, WC1H 0AJ, UK Author ContributionsAll authors contributed to the design of the experiments and to writing the paper. Experiments were conducted by A.E.E. and M.W.P. Competing Financial Interests StatementThe authors declare no competing financial interests.Published as: Nat Chem. 2013 May ; 5(5): 390-394. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptcontinues to make a robust system for the chemical replication of RNA elusive [11][12][13][14] . These problems include the slow rate, poor fidelity and low regioselectivity of non-enzymatic RNA template copying; in addition, activated substrates typically hydrolyze on the same timescale as polymerization. The importance of the latter point ...