Downstream Oligonucleotides Strongly Enhance the Affinity of GMP to RNA Primer–Template Complexes

Tam, Chun Pong; Fahrenbach, Albert C.; Björkbom, Anders; Prywes, Noam; Izgu, Enver Cagri; Szostak, Jack W.

doi:10.1021/jacs.6b09760

Cited by 23 publications

(35 citation statements)

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“…We therefore set out to measure the strength of GpppG binding to the RNA template relative to GMP. NMR methods have been used to measure the affinity of GMP for RNA primer-templates (10,(12)(13)(14). Here we used the same methods and RNA primer-template system (10) to determine the affinity of GpppG for an RNA template overhang consisting of two consecutive cytidines.…”

Section: Resultsmentioning

confidence: 99%

“…Many of the thermodynamic and kinetic parameters associated with nonenzymatic RNA replication have been quantitatively determined (10)(11)(12)(13)(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 attacks the activated phosphorus center of the adjacent template-bound monomer via an in-line mechanism (15).…”

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confidence: 99%

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

Zhang

Tam

Walton

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Zhang

Tam

Walton

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, the protecting effect of lipid was also shown on the aforementioned reactions. It has been reported that short oligomers (up to trimers) can enhance the rate of template-directed replication by three folds (Kaiser and Richert 2013;Li et al 2017;Tam et al 2017;O'Flaherty et al 2018). Thus, the intact oligomers reported in the present study, can, potentially assist the propagation of information in template-directed replication scenarios.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the formation and replication of RNA molecules on the early Earth would have been nonenzymatic, and driven by physico-chemical properties of molecules, resulting in short oligonucleotides. Such small oligoribonucleotides have been shown to not only enhance the rate of template-directed replication (Kaiser and Richert 2013;Li et al 2017;Tam et al 2017;O'Flaherty et al 2018), but are also thought to act as cofactor precursors that pervades extant biology (Puthenvedu et al 2015;Majerfeld et al 2016;Puthenvedu et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides

Dagar

Sarkar

Rajamani

2019

Preprint

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The spontaneous emergence of RNA on the early Earth continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic nucleotides under neutral to alkaline conditions, in fully dehydrated state. Herein, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, where starting reactants were subjected to repeated dehydration-rehydration (DH-RH) regimes, like they would have been on an early Earth. DH-RH conditions, a recurring geological theme, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions in terrestrial geothermal niches, which are hypothesized to be pertinent sites for the emergence of life. 2′-3′ and 3′-5′ cyclic nucleotides of one purine-based (adenosine) and one pyrimidine-based (cytidine) system were evaluated in this study. Additionally, the effect of amphiphiles was also investigated. Furthermore, to discern the effect of 'realistic' conditions on this process, the reactions were also performed using hot spring water samples from an early Earth analogue environment. Our results showed that the oligomerization of cyclic nucleotides under DH-RH conditions resulted in intact informational oligomers. Amphiphiles increased the stability of, both, the starting monomers and the resultant oligomers. In analogue condition reactions, oligomerization of nucleotides and back-hydrolysis of the resultant oligomers was pronounced.Altogether, this study demonstrates how nonenzymatic oligomerization of cyclic purine and pyrimidine nucleotides, under laboratory-simulated and early Earth analogous conditions, could have resulted in RNA oligomers of a putative RNA World.Dagar, S.

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“…[14] These structures revealed ap rogressive decrease in the distance from the 3'-OH to the adjacent phosphate,s uggesting that an activated downstream helper contributes additional structural preorganization that potentiates catalysis of the reaction between the primer and the intermediate ( Figure 4B). This effect, along with the higher observed affinity of template-binding by am onomer beside adownstream helper, [15] helps explain the improved rates and yields of nonenzymatic primer extension.…”

Section: Structural Studies Of the Reaction Mechanismmentioning

confidence: 91%

The Mechanism of Nonenzymatic Template Copying with Imidazole‐Activated Nucleotides

Walton

Zhang

et al. 2019

Angew Chem Int Ed

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The emergence of the replication of RNA oligonucleotides was a critical step in the origin of life. An important model for the study of nonenzymatic template copying, which would be a key part of any such pathway, involves the reaction of ribonucleoside‐5′‐phosphorimidazolides with an RNA primer/template complex. The mechanism by which the primer becomes extended by one nucleotide was assumed to be a classical in‐line nucleophilic‐substitution reaction in which the 3′‐hydroxyl of the primer attacks the phosphate of the incoming activated monomer with displacement of the imidazole leaving group. Surprisingly, this simple model has turned out to be incorrect, and the dominant pathway has now been shown to involve the reaction of two activated nucleotides with each other to form a 5′–5′‐imidazolium bridged dinucleotide intermediate. Here we review the discovery of this unexpected intermediate, and the chemical, kinetic, and structural evidence for its role in template copying chemistry.

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Downstream Oligonucleotides Strongly Enhance the Affinity of GMP to RNA Primer–Template Complexes

Cited by 23 publications

References 38 publications

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

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

Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides

The Mechanism of Nonenzymatic Template Copying with Imidazole‐Activated Nucleotides

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