A Platform for Controlled Template-Independent Enzymatic Synthesis of RNA Oligonucleotides and Therapeutics

Abstract Therapeutic RNA oligonucleotides have shown tremendous potential to manage and treat disease, yet current manufacturing methods cannot deliver on this promise. Here, we report the development and optimization of a novel, aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methodologies. Our platform is made possible by reversible terminator nucleoside triphosphates and an enzyme capable of their incorporation. We show that many common therapeutic RNA modifications are compatible with our process and demonstrate the enzymatic synthesis of natural and modified oligonucleotides in both liquid and solid phases. Our platform offers many unique advantages over chemical synthesis, including the realization of a more sustainable process to produce therapeutic RNA oligonucleotides. One-Sentence Summary An enzyme and novel nucleotide building blocks are used to synthesize RNA oligonucleotides template independently under aqueous conditions.