Template-Independent Enzymatic RNA Synthesis

A route to prepare ribonucleoside triphosphates featuring a 3-aminoxy (3-O-NH2) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Pol{theta}) 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-NH2 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 Pol{theta}. 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.