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

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; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. 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 demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.