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Published on: August 22, 2019
Desthiobiotin (DTB)-modified and TAMRA-modified 2″OMeNAD+ are RNA 2'-phosphotransferase (Tpt1) poisons that enable
Renata Kasprzyk1, Shreya Ghosh2, Stewart Shuman3
1Department of Chemistry, University of Konstanz, Konstanz 78457, Germany shumans@mskcc.org renata.kasprzyk@hest.ethz.ch.
Abstract:
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 found that it, too, is an effective step 1 substrate for RslTpt1 that allows fluorescent labeling of an RNA 2'-phosphate.
Insights
Researchers developed novel desthiobiotin and fluorescent analogs of NAD+ to study RNA 2'-phosphate modification. These analogs act as affinity probes, enabling the capture and detection of RNA 2'-phospho-(ADP-ribose) intermediates formed by the Tpt1 enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- RNA 2 -phosphotransferase (Tpt1) removes internal RNA 2 -PO4 through a two-step mechanism involving NAD+.
- A previously studied analog, 2 OMeNAD+, halts the reaction after step 1, producing a detectable intermediate.
Purpose of the Study:
- To synthesize novel 2 OMeNAD+ analogs for probing RNA 2 -phosphate modification.
- To evaluate the utility of desthiobiotin (DTB) and fluorescent (TAMRA) tagged analogs as affinity probes.
Main Methods:
- Chemical synthesis of two novel 2 OMeNAD+ analogs with DTB linked to adenine.
- Assay of RslTpt1 activity using the synthesized analogs.
- Recovery of reaction products using streptavidin beads and biotin elution.
- Synthesis and testing of a fluorescent 2 OMeNAD+-TAMRA derivative.
Main Results:
- Both novel DTB-containing analogs inhibited RslTpt1 after step 1.
- The 2 OMeNAD-2-DTB analog yielded a higher amount of the RNA-2 -phospho-(DTB-ADP-2 OMe-ribose) product.
- The 2 OMeNAD-2-TAMRA analog effectively labeled RNA 2 -phosphate, enabling fluorescent detection.
Conclusions:
- The 2 OMeNAD-2-DTB analog serves as an effective affinity-tag probe for RNA 2 -phosphate modification.
- Fluorescent labeling of RNA 2 -phosphate is achievable using the 2 OMeNAD-2-TAMRA analog.
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