Template-Free Assembly of Functional RNAs by Loop-Closing Ligation

Wu, Long-Fei; Liu, Ziwei; Roberts, Samuel J.; Su, Meng; Szostak, Jack W.; Sutherland, John D.

doi:10.1021/jacs.2c05601

Cited by 15 publications

(27 citation statements)

References 44 publications

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“…Following this observation, we predict that a system composed by a large collection of duplexes exposing only short toeholds will undergo an exceptionally long equilibration process. This finding is particularly relevant in the context of models for the nonenzymatic replication of genetic information, which is believed to be driven by chemical reactions occurring in highly heterogeneous and complex mixtures of oligoribonucleotides ( 12 , 69 ).…”

Section: Discussionmentioning

confidence: 97%

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

Todisco

Szostak

2022

Nucleic Acids Research

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Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life.

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Section: Discussionmentioning

confidence: 97%

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

Todisco

Szostak

2022

Nucleic Acids Research

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“…Oligonucleotides of this length can then be assembled into functional ribozymes, either by splinted ligation 15 or by iterated loop-closing ligation. 16 These ribozyme building blocks could be the end products of one or potentially multiple virtual circular genomes replicating together in a protocell in a prebiotically plausible environment. Here, we explore a model VCG system with a 12-nt long virtual genome represented by 247 different oligonucleotides, which range from 2 to 12 nucleotides in length.…”

Section: ■ Introductionmentioning

confidence: 99%

Experimental Tests of the Virtual Circular Genome Model for Nonenzymatic RNA Replication

et al. 2023

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The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model, the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extensions on a subset of kinetically trapped partially base-paired configurations, followed by the shuffling of these configurations to enable continued oligonucleotide elongation. Here, we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of lengths 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides and investigated the effect of factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require hightemperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is preactivated. We discuss the necessity of ongoing in situ activation chemistry for continuous and accurate VCG replication.

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“…Partially overlapping pairs of such oligonucleotides can anneal with each other, after which loop-closing ligation can lead to the formation of stem-loop structures. 12 The iteration of such processes could then lead to the assembly of complex structured RNAs, including ribozymes.…”

Section: Discussionmentioning

confidence: 99%

“…Our lab has recently demonstrated that splinted ligation and loop-closing ligation can form functional ribozymes from short oligonucleotides. 11,12 However, even the replication of shorter oligonucleotides faces problems that can lead to information loss at both ends of the sequence. First, the nonenzymatic copying of the last base of a sequence by primer extension is known to be very slow relative to the copying of internal nucleotides, 13 which could lead to progressive loss of 3ʹ-sequences over cycles of replication.…”

Section: Introductionmentioning

confidence: 99%

“…Oligonucleotides of this length can then be assembled into functional ribozymes, either by splinted ligation 11 or by iterated loop-closing ligation. 12 These ribozyme building blocks could be the end products of one or potentially multiple virtual circular genomes replicating together in a protocell in a prebiotically plausible environment.…”

Section: Introductionmentioning

confidence: 99%

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Experimental Tests of the Virtual Circular Genome Model for Non-enzymatic RNA Replication

Ding

Zhou

Mittal

et al. 2023

Preprint

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The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extension on a subset of kinetically trapped partially base-paired configurations, followed by shuffling of these con-figurations to enable continued oligonucleotide elongation. Here we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of length 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides, and investigated factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctua-tions would be essential for continuous templated extension of the entire VCG system, since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is pre-activated. We discuss the necessity of ongoing in-situ activation chemistry for continuous and accu-rate VCG replication.

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Template-Free Assembly of Functional RNAs by Loop-Closing Ligation

Cited by 15 publications

References 44 publications

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

Experimental Tests of the Virtual Circular Genome Model for Nonenzymatic RNA Replication

Experimental Tests of the Virtual Circular Genome Model for Non-enzymatic RNA Replication

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