Non-enzymatic Replication Research Articles

Freeze-thaw cycles as drivers of complex ribozyme assembly.

The emergence of an RNA catalyst capable of self-replication is considered a key transition in the origin of life. However, how such replicase ribozymes emerged from the pools of short RNA oligomers arising from prebiotic chemistry and non-enzymatic replication is unclear. Here we show that RNA polymerase ribozymes can assemble from simple catalytic networks of RNA oligomers no longer than 30 nucleotides. The entropically disfavoured assembly reaction is driven by iterative freeze-thaw cycles, even in the absence of external activation chemistry. The steep temperature and concentration gradients of such cycles result in an RNA chaperone effect that enhances the otherwise only partially realized catalytic potential of the RNA oligomer pool by an order of magnitude. Our work outlines how cyclic physicochemical processes could have driven an expansion of RNA compositional and phenotypic complexity from simple oligomer pools.

Replacing uridine with 2-thiouridine enhances the rate and fidelity of nonenzymatic RNA primer extension.

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s2U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s2U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s2U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s2U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.

Generation of functional RNAs from inactive oligonucleotide complexes by non-enzymatic primer extension.

The earliest genomic RNAs had to be short enough for efficient replication, while simultaneously serving as unfolded templates and effective ribozymes. A partial solution to this paradox may lie in the fact that many functional RNAs can self-assemble from multiple fragments. Therefore, in early evolution, genomic RNA fragments could have been significantly shorter than unimolecular functional RNAs. Here, we show that unstable, nonfunctional complexes assembled from even shorter 3′-truncated oligonucleotides can be stabilized and gain function via non-enzymatic primer extension. Such short RNAs could act as good templates due to their minimal length and complex-forming capacity, while their minimal length would facilitate replication by relatively inefficient polymerization reactions. These RNAs could also assemble into nascent functional RNAs and undergo conversion to catalytically active forms, by the same polymerization chemistry used for replication that generated the original short RNAs. Such phenomena could have substantially relaxed requirements for copying efficiency in early nonenzymatic replication systems.

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

The mixture of 2'-5' and 3'-5' linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2'-5' linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2'-5' linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2'-linked sugars were in the expected 2'-endo conformation, some were partially or fully in the 3'-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2'-5' linkages. Our results contribute to the view that a low level of 2'-5' substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.

6 Crystal structure studies of RNA molecules containing 2′–5′-linkages

RNA can play dual roles as a carrier of genetic information and as a catalyst of specific reactions, and it may have been the first biopolymer to have emerged on the early earth. The non-enzymatic replication of RNA was likely a key step in the evolution of simple cellular life from prebiotic chemistry. In the current model of template-directed polymerization of activated monomers, the chemical copying of RNA always generates a mixture of 3′–5′ and 2′–5′ backbone linkages due to the similar nucleophilicity and orientation of the 2′ and 3′ hydroxyl groups on the ribose. This lack of regiospecificity has been regarded as a central problem for the evolution of functional RNAs, since the resulting backbone heterogeneity was expected to disrupt their folding, molecular recognition and catalytic properties of functional RNAs such as ribozymes. However, a recent study from our lab has demonstrated that RNAs with a certain percentage of 2′–5′ linkages can still retain RNA functions, for example, in a FMN-binding aptamer and a hammerhead ribozyme system. More interestingly, it has been known for a long time that 2′–5′ linkages can reduce the melting temperature of RNA duplexes, making it easier to separate the strands. Although the detailed mechanism is still not clear, considering that strand separation is another unsolved big problem for non-enzymatic RNA replication, this feature may actually afford a selective advantage to duplexes exhibiting backbone heterogeneity. In addition, previous studies have revealed that 2′–5′ linkages in a RNA duplex are more easily hydrolyzed compared to normal 3′–5′ linkages. Thus, there is a selective advantage for the evolution of homogeneous RNA systems with more accurate replication. Altogether, the coexistence of 2′–5′ and 3′–5′ linkages may be a central feature that allowed RNA to play a central role in the original stage of life. In this work, we will present several X-ray crystal structures of RNA duplexes and an aptamer that contain 2′–5′ linkages. These structures help us to understand how RNA can adjust its structure to accommodate the backbone heterogeneity.

A Polymer Reaction Engineering Approach to Polynucleotide Replication

AbstractIt is often interesting to look at specific scientific questions from the perspective of other, related technologies. The problem of nonenzymatic RNA replication can be viewed from the perspective of classical polymer science and with some interesting insights. This communication will show that current mechanism of stalling, which is used to account for the fact that the rate of insertion errors in nonenzymatic RNA replication are much lower than would be expected, can be modeled by using the Mayo–Lewis equation for calculating copolymer composition in copolymerizations of synthetic polymers. Using kinetic parameters from the literature, it is possible to independently predict the error threshold for RNA replication. Agreement with the accepted model for nonenzymatic replication is surprisingly good. magnified image

Whence Flavins? Redox-Active Ribonucleotides Link Metabolism and Genome Repair to the RNA World

Present-day organisms are under constant environmental stress that damages bases in DNA, leading to mutations. Without DNA repair processes to correct these errors, such damage would be catastrophic. Organisms in all kingdoms have repair processes ranging from direct reversal to base excision and nucleotide excision repair, and the recently characterized giant viruses also include these mechanisms. At what point in the evolution of genomes did active repair mechanisms become critical? In particular, how did early RNA genomes protect themselves from UV photodamage that would have hampered nonenzymatic replication and led to a mutation rate too high to pass on accurate sequence information from one generation to the next? Photolyase is a widespread and phylogenetically ancient enzyme that utilizes longer wavelength light to cleave thymine dimers in DNA produced via photodamage. The protein serves as a binding scaffold but does not contribute to the catalytic chemistry; the action of the dinucleotide cofactor FADH(2) breaks the chemical bonds. This small bit of RNA, hailed as a "fossil of the RNA World," contains the flavin heterocycle, whose redox activity has been harnessed for myriad functions of life from metabolism to DNA repair. In present-day biochemistry, flavin biosynthesis begins with guanosine and proceeds through seven steps catalyzed by protein-based enzymes. This leads to the question of how flavins originally evolved. Did the RNA world include ancestral RNA bases with greater redox activity than G, A, C, and U that were capable of photorepair of uracil dimers? Could those ancestral bases have chemically evolved to the current flavin structure? Or did flavins already exist from prebiotic chemical synthesis? And were they then co-opted as catalysts for repair sometime after metabolism was established? In this Account, we analyze simple derivatives of guanosine and other bases that show two prerequisites for flavin-like photolyase activity: a significantly lowered one-electron reduction potential and a red-shifted adsorption spectrum that facilitates excited-state electron transfer in a spectral window that does not produce cyclobutane pyrimidine dimers. Curiously, the best candidate for a primordial flavin is a base damage product, 8-oxo-7,8-dihydroguanine (8-oxoGua or "OG"). Other redox-active ribonucleotides include 5-hydroxycytidine and 5-hydroxyuridine, which display some of the characteristics of flavins, but might also behave like NADH.

Effect of stalling after mismatches on the error catastrophe in nonenzymatic nucleic acid replication.

The frequency of errors during genome replication limits the amount of functionally important information that can be passed on from generation to generation. During the origin of life, mutation rates are thought to have been quite high, raising a classic chicken-and-egg paradox: could nonenzymatic replication propagate sequences accurately enough to allow for the emergence of heritable function? Here we show that the theoretical limit on genomic information content may increase substantially as a consequence of dramatically slowed polymerization after mismatches. As a result of postmismatch stalling, accurate copies of a template tend to be completed more rapidly than mutant copies and the accurate copies can therefore begin a second round of replication more quickly. To quantify this effect, we characterized an experimental model of nonenzymatic, template-directed nucleic acid polymerization. We found that most mismatches decrease the rate of primer extension by more than 2 orders of magnitude relative to a matched (Watson−Crick) control. A chemical replication system with this property would be able to propagate sequences long enough to have function. Our study suggests that the emergence of functional sequences during the origin of life would be possible even in the face of the high intrinsic error rates of chemical replication.

Efficient and Rapid Template-Directed Nucleic Acid Copying Using 2′-Amino-2′,3′-dideoxyribonucleoside−5′-Phosphorimidazolide Monomers

The development of a sequence-general nucleic acid copying system is an essential step in the assembly of a synthetic protocell, an autonomously replicating spatially localized chemical system capable of spontaneous Darwinian evolution. Previously described nonenzymatic template-copying experiments have validated the concept of nonenzymatic replication, but have not yet achieved robust, sequence-general polynucleotide replication. The 5′-phosphorimidazolides of the 2′-amino-2′,3′-dideoxyribonucleotides are attractive as potential monomers for such a system because they polymerize by forming 2′→5′ linkages, which are favored in nonenzymatic polymerization reactions using similarly activated ribonucleotides on RNA templates. Furthermore, the 5′-activated 2′-amino nucleotides do not cyclize. We recently described the rapid and efficient nonenzymatic copying of a DNA homopolymer template (dC15) encapsulated within fatty acid vesicles using 2′-amino-2′,3′-dideoxyguanosine−5′-phosphorimidazolide as the activated monomer. However, to realize a true Darwinian system, the template-copying chemistry must be able to copy most sequences and their complements to allow for the transmission of information from generation to generation. Here, we describe the copying of a series of nucleic acid templates using 2′-amino-2′,3′-dideoxynucleotide−5′-phosphorimidazolides. Polymerization reactions proceed rapidly to completion on short homopolymer RNA and LNA templates, which favor an A-type duplex geometry. We show that more efficiently copied sequences are generated by replacing the adenine nucleobase with diaminopurine, and uracil with C5-(1-propynyl)uracil. Finally, we explore the copying of longer, mixed-sequence RNA templates to assess the sequence-general copying ability of 2′-amino-2′,3′-dideoxynucleoside−5′-phosphorimidazolides. Our results are a significant step forward in the realization of a self-replicating genetic polymer compatible with protocell template copying and suggest that N2′→P5′-phosphoramidate DNA may have the potential to function as a self-replicating system.

Self‐Replicating Amphiphilic β‐Sheet Peptides

Several different non-enzymatic molecular replication systems have been prepared and analyzed, including nucleic acids, fatty acids, peptides, and organic molecules. This research was evidently motivated by the chemists enthusiasm to shine light on plausible scenarios that may have lead to the origin of life on earth and early molecular evolution, and it also provided new opportunities for understanding fundamental principles, such as molecular recognition and autocatalysis. The study of non-enzymatic replication has been expanded recently beyond autocatalysis, to small molecular networks, in which the replication is also a product of template-assisted cross-catalysis. The design of replicating peptides has centered mainly on helical coiled-coil structures, in which monomeric or dimeric peptides, twenty-five to forty amino acids in length, serve as templates for substrate binding and thus for enhanced condensation and replication. However, it has been postulated that shorter peptides with simpler sequences may also serve as templates for self replication, provided that they are able to arrange themselves into unique and welldefined structures. We show herein that rather simple peptides, close analogues of the synthetic amphiphilic Glu(Phe-Glu)n peptides, [27] can form soluble, one-dimensional b-sheet aggregates in water, which serve to significantly accelerate peptide ligation and self replication. It has been postulated and demonstrated that native amyloid fibrils can replicate, albeit with moderate efficiency. Specific design of synthetic peptides can be used to make soluble aggregates with defined structures and higher replication efficiencies. Towards this aim, it has been shown that peptides comprising of repetitive dyads of hydrophilic and hydrophobic amino acid residues tend to adopt b-pleated sheet arrangements. Recently, it was further revealed that the terminal proline residue, which is rigid, does not have a backbone N H group available for hydrogen bonding, and often acts as a b-sheet breaker, can be used to enhance the formation of ordered b-sheet assemblies. Based on this evidence, we have synthesized the sequence Aba-Glu-(PheGlu)5-Pro of peptide 2 in which the N-terminus proline of the b-sheet forming peptide PFE-5 [34] has been replaced by the capping aromatic 4-acetamidobenzoate (ABA; Table 1). The

Chemical Primer Extension: Individual Steps of Spontaneous Replication

The replication of genetic information, as we know it from today's biology, relies on template-directed, polymerase-catalyzed extension of primers. It is known that short stretches of complementary RNA can form on templates in the absence of enzymes. This account summarizes recent work on efficient enzyme-free primer extension, both with 3'-amino-terminal deoxyribonucleotide primers and with primers made of unmodified RNA. Near-quantitative primer extension with half-life times on the order of hours has been demonstrated by using azaoxybenzotriazolides of nucleotides and downstream-binding oligomers. Further, small non-nucleosidic substituents placed on the terminus of the template or the downstream-binding oligomer have been shown to increase the rate and fidelity of primer-extension reactions. Since all four templating bases (A, C, G, T/U) direct sequence-selective primer-extension steps, we feel that there is renewed hope that full, nonenzymatic replication from monomers may eventually be achieved.

Errors and Alternatives in Prebiotic Replication and Catalysis

The work on nonenzymatic nucleic acid replication performed by Leslie Orgel and co-workers over the last four decades, now extended by work on artificial selection of RNA aptamers and ribozymes, is generating some pessimism concerning the 'naked gene' theories of the origin of life. It is suggested here that the low probability of finding RNA aptamers and ribozymes within pools of random sequences is not as disquieting as the poor gain in efficiency obtained with increases in information content. As acknowledged by Orgel and many other authors, primitive RNA replication and catalysis must have occurred within already complex and dynamic environments. I, thus, propose to pay attention to a number of possibilities that bridge the gap between 'naked gene' theories, on one side, and metabolic theories in which complex systems self-propagate by growth and fragmentation, on the other side. For instance, one can de-emphasize nucleotide-by-nucleotide replication leading to long informational polymers, and view instead long random polymers as storage devices, from which shorter oligomers are excised. Catalytic tasks would be mainly performed by complexes associating two or more oligomers belonging to the same or to different chemical families. It is proposed that the problems of stability, binding affinity, reactivity, and specificity could be easier to handle by heterogeneous complexes of short oligomers than by long, single-stranded polymers. Finally, I point out that replication errors in a primitive replication context should include incorporations of alternative nucleotides with interesting, chemically reactive groups. In this way, an RNA sequence could be at the same time an inert sequence when copied without error, and a ribozyme, when a chemically reactive nucleotide is inadvertently introduced during replication.

Solution structure of a HNA–RNA hybrid

Background: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. Results: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA–RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA–RNA duplex compared to canonical dsRNA or an RNA–DNA duplex. Conclusions: The P–P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA–RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA–RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

Peptide nucleic acids rather than RNA may have been the first genetic molecule.

Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

Nonenzymatic Oligomerization of Ribonucleotides on Guanosine‐Rich Templates: Suppression of the Self‐Pairing of Guanosine

Acridine-labeled primers have been shown to be a very valuable tool for the study of the template-directed oligomerization of RNA (shown schematically below). Conditions for the efficient incorporation of cytidine have been established by using such compounds. The aggregation of guanosine-rich templates, which has long been considered a major obstacle for nonenzymatic replication of RNA, can thus be reduced to a minimum.

The limits of template-directed synthesis with nucleoside-5′-phosphoro(2-methyl)imidazolides

In earlier work we have shown that C-rich templates containing isolated A, T or G residues and short oligo(G) sequences can be copied effectively using nucleoside-5'-phosphoro(2-methyl)imidazolides as substrates. We now show that isolated A or T residues within an oligo(G) sequence are a complete block to copying and that an isolated C residue is copied inefficiently. Replication is possible only if there are two complementary oligonucleotides each of which acts as a template to facilitate the synthesis of the other. We emphasize the severity of the problems that need to be overcome to make possible non-enzymatic replication in homogeneous aqueous solution. We conclude that an efficient catalyst was involved in the origin of polynucleotide replication.

Three stages in the evolution of the genetic code

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity those amino acids emerging later in a translation process are derived. Codon number and chemical complexity indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage 1 use purine-rich codons, while all the amino acids introduced in the second stage, in contrast, use pyrimidines in the third position of their codons. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non-enzymatic replication and interactions of hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids, which gradually decreased during their evolution. Amino acids independently available from prebiotic synthesis were thus correlated to purine-rich codons. Implications on the prebiotic replication are discussed also in the light of recent codon usage data.

Molecular replication.

Simple replicating molecules are likely to have played a critical role in the origin of life. Recent experiments show that non-enzymatic replication is conceivable in a wide range of synthetic chemical systems. The challenge now facing these studies is how to develop information-coding systems from simple prebiotic precursors.

Template-directed synthesis of high molecular weight polynucleotide analogues.

A GREAT deal is known about the structures of nucleic acids and the mechanisms involved in nucleic acid replication. This relatively new knowledge has led a number of authors to speculate that other linear heteropolymers might be able to serve as carriers of genetic information. A number of polynucleotide analogues have been synthesised by chemical or enzymatic methods1–7 and one of them has been used as a template for RNA polymerase4. Two studies, in particular, have some relationship to the present work. 3′–5′-Linked polynucleotides derived from 2′-amino-2′-deoxynucleoside 5′-phosphates have been synthesised enzymatically7. Letsinger et al.5,6 reported the chemical and enzymatic formation of polydeoxyribonucleotides containing N5′–O3′-phosphoramidate linkages. Here we discuss nucleic acid analogues derived from 2′-amino-2′-deoxynucleotides that we believe may be capable of non-enzymatic replication, and describe some experimental results which, although they are far from establishing the principle of self-replication, do show that it is possible to synthesise fairly long oligomers on templates in aqueous solution.

Prebiotic Polynucleotides and Polypeptides

AbstractWork on the non‐enzymatic replication of polynucleotides is discussed emphasizing sequence dependence and showing that alternating polynucleotides should replicate more easily than any others. Experimental work is described showing that polypeptides in which hydrophobic and hydrophilic residues alternate, adopt a β‐structure in aqueous solution. The possible relevance of these observations for the evolution of the genetic code is given.