Non-canonical 3′-5′ Extension of RNA with Prebiotically Plausible Ribonucleoside 2′,3′-Cyclic Phosphates

[Image: see text] Ribonucleoside 2′,3′-cyclic phosphates (N>p’s) are generated by multiple prebiotically plausible processes and are credible building blocks for the assembly of early RNA oligomers. While N>p’s can be polymerized into short RNAs by non-enzymatic processes with variable efficiency and regioselectivity, no enzymatic route for RNA synthesis had been described. Here we report such a non-canonical 3′-5′ nucleotidyl transferase activity. We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p’s (2′,3′-cyclic A-, G-, U-, and CMP) to the 5′-hydroxyl termini of RNA strands with 5′ nucleotide addition enhanced in all cases by eutectic ice phase formation at −7 °C. We also observed 5′ addition of 2′,3′-cyclic phosphate-activated β-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers. Our results establish a new mode of RNA 3′-5′ extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.