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
Year: 2025
Type: article
Abstract: RNA 2′-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2′-PO 4 via a two-step mechanism in which (i) the 2′-PO 4 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 found that it, too, is an effective step 1 substrate for RslTpt1 that allows fluorescent labeling of an RNA 2′-phosphate.
Source: RNA
Authors: Renata Kasprzyk, Shreya Ghosh, Stewart Shuman
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4414324490