Template-Independent Enzymatic RNA Synthesis

A route to prepare ribonucleoside triphosphates featuring a 3’-aminoxy (3’-O-NH ₂ ) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3’-O-NH ₂ group within the extended primer regenerates the 3’-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Polθ. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.