2''F-NAD⁺ is a broad-spectrum RNA 2'-phosphotransferase (Tpt1) poison that traps the enzyme on its abortive...

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1'' to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. A Tpt1 "poison" that arrests the reaction after step 1 could aid in the design of antifungals that interdict tRNA biogenesis. Whereas our previous studies of 2''OMeNAD+ established its efficacy in poisoning Runella slithyformis Tpt1 (RslTpt1, a bacterial enzyme), its application was limited insofar as Tpt1 enzymes from fungal pathogens were unable to utilize 2''OMeNAD⁺ for step 1 catalysis. Here we report the chemical synthesis of 2''F-NAD⁺, which proved to be a broad-spectrum poison against Tpt1 enzymes from five different taxa, including human pathogens Candida albicans and Candida auris. The resulting RNA-2'-phospho-(2''F-ADP-ribose) dead-end product remains stably trapped in a complex with Tpt1. A modified analog, 2''F-NAD-DTB, containing desthiobiotin (DTB) linked to adenine C2, provides an improved affinity-tag probe of RNA 2'-phosphate modification.