Kinetics of Coupling Reactions That Generate Monothiophosphate Disulfides:  Implications for Modification of RNAs

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.

Structural studies of the eIF4E–VPg complex reveal a direct competition for capped RNA: Implications for translation

“…To produce conjugates with full-length messenger RNAs (mRNAs), we had to alter our strategy in order to conjugate fulllength luciferase RNAs (∼1,800 nucleotides) to VPgΔ37(C150A/ Y64C), which yielded a species of ∼500 kDa. Using in vitro transcription, guanosine-5′-monophosphorothioate (GMPS) was incorporated into the 5′ end of luciferase transcripts and subsequently coupled to 2,2′-pyridine disulfide using standard methods (51,52). A disulfide exchange reaction of the resulting pyridyl-disulfide linkage on the 5′ end of the RNA was used for conjugation to VPgΔ37(C150A/Y64C) (53).…”

“…To generate RNA templates suitable for translation, full-length luciferase RNA (∼1,800 nucleotides) was in vitro transcribed (using the T7 Megascript Kit; Ambion) and 5′ end primed with GMPS (Axxora; Biolog Life Science Institute) (51,52). GMPS-primed RNAs were than coupled to 2,2′-pyridine disulfide (SIGMA) to produce pyridyl-disulfide linkage on the 5′ end of RNAs.…”

Staphylococcus aureus Small RNAs Possess Dephospho-CoA 5′-Caps, but No CoAlation Marks

“…5′-CoAlated dpCoA-RNA (CoA-SS-dpCoA-RNA) structurally resembles the natural substrate of CoADR except for a single-sided 3′-extension. We developed a strategy for the synthesis of CoA-SS-dpCoA-RNA, but also other dpCoA-RNA disulfides: Low-molecular-weight thiol compounds were first transformed into 2-mercaptopyridine-activated disulfides and then incubated with in vitro transcribed dpCoA-RNA to yield dpCoA-RNA disulfides ( Figure S3a ) [ 46 , 47 ]. The procedure was validated by the synthesis and analysis of 32 P-body-labeled CoA-SS-dpCoA-RNA and dpCoA-SS-dpCoA-RNA ( Figure S3b –d).…”

Cyclic Peptidomimetics as Inhibitor for miR-155 Biogenesis

“…5′-GMPS primed pre-miR-155 was first prepared by in vitro transcription as described above, except that 5′-GMPS (Biolog)/GTP/ATP/CTP/UTP (8:1:1:1:1 mM) was used . The RNA was purified by phenol/chloroform extraction and ethanol precipitation.…”

“…5′-GMPS primed pre-miR-155 was first prepared by in vitro transcription as described above, except that 5′-GMPS (Biolog)/GTP/ATP/CTP/UTP (8:1:1:1:1 mM) was used. 33 The RNA was purified by phenol/chloroform extraction and ethanol precipitation. ATTO 488 iodoacetamide (ATTO-TEC) was then conjugated onto RNA via the 5′-thiol group following a reported method.…”