A Universal Nucleoside-to-NTP 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 polyPdependent 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-or base-modified NTP analogues. This is demonstrated by synthesising the four 2′-fluoro-2′-dNTPs, as well as pseudouridine-5′-triphosphate (ΨTP) and N1-methyl-ΨTP (m 1 ΨTP), which are essential components for siRNA and mRNA therapeutics.