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

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. Lay Summary The absence of a prebiotically plausible pathway for the efficient nonenzymatic copying of RNAs remains a major obstacle towards constructing self-replicating protocells that emulate early lifeforms. We demonstrate the activation of short oligonucleotides and the subsequent formation of monomer-bridged-oligonucleotide species, leading to efficient nonenzymatic template copying in the same reaction mixture. Our findings suggest that in-situ activated mixtures of mono- and oligo-nucleotides would significantly outperform mononucleotides in driving the copying of arbitrary RNA sequences. 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.

Automated summary

Research shows that the potential prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can efficiently synthesize activated mononucleotides and lead to template-directed primer extension. Additionally, short oligonucleotides can be activated through eutectic phase phospho-Passerini chemistry and promote the formation of monomer-bridged-oligonucleotide species, enabling efficient nonenzymatic template copying. These findings simplify the process of the origin of life by demonstrating that multiple steps from activation chemistry to RNA copying can occur together in a single complex environment.