Desthiobiotin (DTB)-modified and TAMRA-modified 2''OMeNAD+ are RNA 2'-phosphotransferase (Tpt1) poisons that enable affinity-tagging and fluorescence-tagging of internal RNA 2'-phosphate groups.

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. We showed previously that 2''OMeNAD⁺, a synthetic NAD⁺ analog that cannot support step 2 transesterification, is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate as an abortive product. Here we report the chemical synthesis of two novel 2''OMeNAD⁺ compounds, containing desthiobiotin (DTB) linked to adenine C2 or N6 via a diaminohexane linker. Whereas both analogs poison the RslTpt1 reaction after step 1, the 2''OMeNAD-2-DTB derivative supports a higher yield of RNA-2'-phospho-(DTB-ADP-2''OMe-ribose) product, which can be recovered by adsorption to streptavidin beads and elution with biotin. Our results recommend 2''OMeNAD-2-DTB as a novel affinity-tag probe of RNA 2'-phosphate modification. We synthesized a fluorescent derivative, 2''OMeNAD-2-TAMRA, and find that it, too, is an effective step 1 substrate for RslTpt1 that allows fluorescent labeling of an RNA 2'-phosphate.