Kinetics of coupling reactions that generate monothiophosphate disulfides: implications for modification of RNAs.
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.
Wu
C W
CW
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292, USA.
Eder
P S
PS
Gopalan
V
V
Behrman
E J
EJ
D002474:Cell-Free System; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006157:Guanosine Monophosphate / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012333:RNA, Messenger / Q000138:chemical synthesis* / Q000737:chemistry; D013873:Thionucleotides / Q000737:chemistry*; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D004220:Disulfides; D012333:RNA, Messenger; D013873:Thionucleotides; C066524:guanosine 5'-monophosphorothioate; D006157:Guanosine Monophosphate