ENZNAT: Template-Independent Enzymatic Synthesis of Nucleic Acid Therapeutics

Nucleic acid-based therapeutics comprise a rapidly expanding category of drugs that have the potential to treat a broad range of

genetic and infectious diseases, cancer, cardiovascular disorders etc. Several antisense oligonucleotides (ASOs), short interfering RNAs

(siRNAs) and mRNA vaccines have recently been approved and many are under clinical trials. Therapeutic oligonucleotides contain

modified ribose moieties and phosphorothioate linkages for improved stability in the cellular environment. Many of the world's top

pharmaceutical and biotech companies are now engaged in developing ''safe and effective'' nucleic acid (NA) therapeutics. Currently,

modified NAs are produced by solid-phase oligonucleotide synthesis (SPOS) which uses toxic/deleterious reagents and solvents,

making the scale-up problematic and expensive. Herein, we propose to establish a novel sustainable bio-based route towards

modified nucleic acids, which will involve iterative oligo synthesis in water under mild conditions, utilising benign enzymes and

renewable precursors. Modified nucleotide monomers will be synthesised and added to an initiator oligo using template-free,

engineered polymerase or ligase enzymes with sequential coupling followed by 3'-deblocking steps. Initially, PEG-based watercompatible

solid supports will be used to immobilize the initiator oligo to enable easy isolation of the product. This can be later replaced by emerging membrane separation technology enabling oligo assembly at higher concentrations. Our approach uses

nucleotide triphosphate or monophosphate monomers that are easier to prepare, store, and handle, compared with the current monomers used in SPS. We envisage that our proposed methodology would enable widespread production of nucleic acids

therapeutics and vaccines, facilitating faster, low-cost, and non-toxic manufacturing of essential medicines at an industrial scale.