A Universal Nucleoside-to-Nucleoside-5'-Triphosphate Enzyme Cascade Driven by Polyphosphate

Nucleoside-5'-triphosphates (NTPs) are important building blocks for nucleic acids, but the efficiency of their chemical synthesis is limited by the low yield of the final 5'-triphosphorylation reaction. Here, we present an enzymatic cascade reaction involving just two enzymes that enable nucleosides-to-NTPs conversion for all canonical rNTPs and dNTPs. The cascade is used in a single one-pot operation driven by cost-efficient sodium polyphosphate (polyP). The broad-spectrum deoxynucleoside kinase (Dm-dNK) from Drosophila melanogaster converts nucleosides into nucleoside-5'-monophosphates (NMPs) using ATP. The polyphosphate kinase family 2, class III (EbPPK) from an Erysipelotrichaceae bacterium simultaneously recycles ATP required by Dm-dNK and converts the NMPs to NTPs in a polyP-dependent reaction. Importantly, only trace amounts of catalytic ATP (0.0001 equivalents or less) are required. In addition to canonical NTPs, the cascade can generate ribose- and base-modified NTP analogues. This is demonstrated through the synthesis of a set of eleven modified nucleotides, including the four 2'-fluoro-2'-dNTPs and nucleotides with 2,6-diaminopurine as the nucleobase. Production of an essential component for mRNA therapeutics, N1-methyl-ΨTP (m¹ΨTP), is demonstrated on a milligram scale.