Chemical Primer Extension in Seconds

Tam

Walton

et al. 2017

The nonenzymatic copying of RNA templates with imidazoleactivated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA primer-template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson-Crick base pairs, with the primer 3ʹ-hydroxyl oriented to attack the 5ʹ-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazoliumbridged dinucleotide intermediate would be preorganized to react with the primer by in-line S N 2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5ʹ-5ʹ linkage are important for optimal preorganization of the complex, whereas the position of the 5ʹ-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA primer extension.RNA self-replication | diguanosine dinucleotide | crystal structure | origin of life I n the RNA world hypothesis, the emergence of RNA-catalyzed RNA replication is thought to have been preceded by a stage in which RNA replication was driven purely through chemical processes (1, 2). The nonenzymatic RNA-templated polymerization of activated nucleotides or oligonucleotides has been extensively studied, with the intent of optimizing the rate, extent, and fidelity of nonenzymatic RNA/DNA polymerization (3, 4). Numerous phosphate-activating groups have been studied in the context of nonenzymatic RNA replication. For example, imidazoles such as 2-methylimidazole (5) and, more recently, 2-aminoimidazole (6), have been found to be useful phosphate activators. The potentially prebiotic synthesis of imidazoles under primitive Earth conditions has been investigated (7). On the other hand, Richert and coworkers reported the use of benzotriazole-activated monomers to improve the rate of primer extension (8). In an alternative approach, the in situ activation of monoribonucleotides, and subsequent template-guided polymerization, has been achieved by Richert and coworkers (9), using a carbodiimide reagent together with N-alkyl-imidazole catalysts.Many of the thermodynamic and kinetic parameters associated with nonenzymatic RNA replication have been quantitatively determined (10-14). Until recently, the general assumption has been that nonenzymatic primer extension with activated mononucleotides involves classical S N 2 nucleophilic substitution, in which the nucleophilic 3ʹ-hydroxyl group of the primer attack...

mentioning

confidence: 99%

Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex

Tam

Walton

et al. 2017

“…Work from our laboratory has extended this concept to the transcription of DNA into a complementary threose nucleic acid (TNA) polymer (10)(11)(12). More generally, the formation of Watson-Crick basepaired duplexes between different nucleic acids suggests that information transfer should be possible between many different nucleic acids, including variants such as 3′-amino RNA and DNA (13)(14)(15), 2′-amino RNA and DNA (16,17), 4′-3′ lyxopyranosyl-NA (18), glycerol nucleic acid (GNA) (19), TNA (20), and others. As long as two polymers can base-pair with each other, it seems reasonable that a mixed polymer containing monomers of each type could still engage in base-pair mediated replication.…”

mentioning

confidence: 99%

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity

Trevino

Elenko

et al. 2011

Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 1∶1 mixture of deoxy-and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy-and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.abiogenesis | genetic takeover | RNA progenitor | origin of life G iven that RNA is likely to have played a key role early in the evolution of life (1), understanding the origins of the RNAbased biosphere is a critical aspect of understanding the origin of life. The difficulties associated with the prebiotic synthesis of a macromolecule as complicated and fragile as RNA have stimulated interest in the hypothesis that RNA was preceded by simpler and/or more stable progenitor nucleic acids (2-4). The systematic exploration of nucleic acids that are structurally related to RNA has revealed that the formation of stable WatsonCrick base-paired, antiparallel duplex structures is compatible with a surprising degree of variation in the sugar-phosphate backbone (5). These studies imply that many distinct nucleic acids are in principle capable of mediating the inheritance of genetic information. On the other hand, recent advances in the prebiotic chemistry leading to the pyrimidine ribonucleotides (6) have revived the prospects of the RNA-first model, with the attendant advantage of avoiding a difficult genetic takeover. Both RNAfirst and RNA-late models assume that life began with a single, well-defined genetic polymer. Perhaps the greatest problem implicit in this assumption is the challenge of explaining the origin of pure nucleotide monomers on the early Earth. Indeed, the supp...

“…and coworkers of the extension of primers ending in a 3′-amino nucleotide show that the template-directed primer extension reaction can be very fast, and can occur with relatively high fidelity on both RNA and DNA templates (8,10). We have recently shown that short homopolymeric RNA and DNA templates can be very rapidly copied by nonenzymatic primer extension in the presence of 2-methylimidazole-activated 3′-amino nucleotides (7).…”

Section: Significancementioning

confidence: 99%

“…Obviating the requirement for a polymerase greatly simplifies the task of creating and assembling the components of an artificial cell, and thus of constructing simple living systems from inanimate materials. We and others have therefore explored the synthesis of a variety of phosphoramidatelinked nucleic acids, their corresponding amino-sugar monomers, and the characterization of nonenzymatic template-directed primer extension reactions in these systems (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Among these systems, we have examined 2′-amino versions of the acyclic glycerol nucleic acid (5), 2′-amino-2′,3′-dideoxyribonucleic acid (4,6), and 3′-amino-2′,3′-dideoxyribonucleic acid (7).…”

mentioning

confidence: 99%

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer

Blain

Zielińska

et al. 2013

Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N 3′ -P 5′ -linked phosphoramidate DNA (3′-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3′-NP-DNA templates can be copied into complementary 3′-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3′-NP-DNA template into a complementary strand of 3′-NP-DNA. Our results suggest that 3′-NP-DNA has the potential to serve as the genetic material of artificial biological systems.origin of life | nonenzymatic primer extension | artificial genetic systems | nucleotide modifications | mismatch T he phosphoramidate nucleic acids are of particular interest as potential genetic materials for artificial life-forms because of their potential for replication by the nonenzymatic polymerization of amino-sugar nucleotides. Because of the greater nucleophilicity of the amino group relative to the 3′-hydroxyl group of ribo-and deoxyribo-nucleotides, amino-sugar nucleotides exhibit more rapid spontaneous polymerization. Obviating the requirement for a polymerase greatly simplifies the task of creating and assembling the components of an artificial cell, and thus of constructing simple living systems from inanimate materials. We and others have therefore explored the synthesis of a variety of phosphoramidatelinked nucleic acids, their corresponding amino-sugar monomers, and the characterization of nonenzymatic template-directed primer extension reactions in these systems (1-11). Among these systems, we have examined 2′-amino versions of the acyclic glycerol nucleic acid (5), 2′-amino-2′,3′-dideoxyribonucleic acid (4, 6), and 3′-amino-2′,3′-dideoxyribonucleic acid (7).The structural simplicity of the acyclic sugar-phosphate nucleic acid backbones has made them attractive targets for study. Indeed, an acyclic nucleotide consisting of a glycerol-phosphate backbone linked to a formylated nucleobase (12) was among the first of such nucleic acids to be chemically synthesized, but incorporation of this nucleotide into oligomers caused a severe loss of duplex stability. Much later, the glycerol nucleic acids, in which...