Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2'-phosphotransferase (Tpt1) 'poison' that traps the enzyme on its abortive RNA-2'-PO4-(ADP-2″OMe-ribose) reaction intermediate.

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 and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification.