Kinetics of Coupling Reactions That Generate Monothiophosphate Disulfides

Impetus for the design of simple strategies for site-specific incorporation of biochemical and biophysical probes in RNAs is fueled by the increasing realization that the role of RNA and RNA-protein interactions is pervasive in various cellular and developmental processes. Advances in solid-phase oligonucleotide synthesis of small RNAs have facilitated the site-specific introduction of functional groups that lend themselves to subsequent chemical modification. For instance, a 2' amino group in an RNA oligonucleotide can be converted to a thiol (by treatment with an activated ester and a reducing agent), which in turn can be modified to incorporate a probe of choice. However, for large RNAs that cannot be synthesized chemically, a commonly employed approach has been to include guanosine-5'-monothiophosphate (GMPS) in an in vitro transcription reaction to enable enzymatic synthesis of an RNA transcript whose 5' end contains the monothiophosphate group. The nucleophilic sulfur at the 5' end can be subsequently modified to introduce a photoaffinity cross-linking agent or a spin label or an affinity tag. Despite recent reports of successful modifications of 5'-GMPS-primed RNAs, the kinetics and mechanism of the coupling reactions remain uncharacterized. In this report, Behrman and co-workers have examined the kinetics and pH-dependence of the modification reactions that generate monothiophosphate disulfide linkages using either 5'-GMPS or 5'-GMPS-primed RNA as the substrate.

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