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bioRxiv · 10.1101/2023.04.12.536642

Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides

Ding, D.·Zhang, S. J.·Szostak, J. W.
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Abstract

The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension, due to their higher intrinsic reactivity and their higher template affinity. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our findings pave the way for future research into the activation of complex mixtures of mono- and oligonucleotides for the enhanced copying and potentially the replication of arbitrary RNA sequences.

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Ding, D., Zhang, S. J., Szostak, J. W.. 2023-04-12. Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides. https://doi.org/10.1101/2023.04.12.536642

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