Nonenzymatic Template-Directed Primer Extension Using 2'-3' Cyclic Nucleotides Under Wet-Dry Cycles

RNA World Hypothesis is centred around the idea of a period in the early history of life's origin, wherein nonenzymatic oligomerization and replication of RNA resulted in functional ribozymes. Previous studies in this endeavour have demonstrated template-directed primer extension using chemically modified nucleotides and primers. Nonetheless, similar studies that used non-activated nucleotides led to the formation of RNA only with abasic sites. In this study, we report template-directed primer extension with prebiotically relevant cyclic nucleotides, under dehydration-rehydration (DH-RH) cycles occurring at high temperature (90 °C) and alkaline conditions (pH 8). 2'-3' cyclic nucleoside monophosphates (cNMP) resulted in primer extension, while 3'-5' cNMP failed to do so. Intact extension of up to two nucleotide additions was observed with both canonical hydroxy-terminated (OH-primer) and activated amino-terminated (NH₂-primer) primers. We demonstrate primer extension reactions using both purine and pyrimidine 2'-3' cNMPs, with higher product yield observed during cAMP additions. Further, the presence of lipid was observed to significantly enhance the extended product in cCMP reactions. In all, our study provides a proof-of-concept for nonenzymatic primer extension of RNA, using intrinsically activated prebiotically relevant cyclic nucleotides as monomers.