Kinetics of coupling reactions that generate monothiophosphate disulfides: implications for modification of RNAs
1Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292, USA.
Abstract:
The inclusion of guanosine-5'-monothiophosphate (GMPS) in an in vitro transcription reaction facilitates enzymatic synthesis of an RNA transcript with a monothiophosphate group at the 5' end. A kinetic study of the modification reactions that generate monothiophosphate disulfide linkages with either 5'-GMPS alone or 5'-GMPS-primed RNA as the substrate revealed that the second-order rate constants increased as the pH was decreased. For example, when the reaction pH was lowered from 8 to 4, the k2 value for the coupling reaction between N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) and GMPS increased 67-fold from 1.84 to 123 M(-1) x s(-1). In addition to discussing a possible mechanism for coupling reactions that involve GMPS and disulfides, we also indicate conditions that are likely to be optimal for modification of the nucleophilic sulfur in 5'-GMPS-primed RNAs.
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