Engineering disulfide cross-links in RNA using thiol-disulfide interchange chemistry.
Protocols for postsynthetic modification of 2-amino-containing oligoribonucleotides with either an alkyl-phenyl disulfide or an alkyl thiol group are described. These groups react under mild conditions to form disulfide cross-links by thiol-disulfide interchange. These reactants do not form a disulfide bond when incorporated on opposite faces of a short continuous RNA helix, but do form disulfide bonds rapidly when they are placed in proximity. Inaddition, by incorporating these groups at various positions on large RNAs by semisynthesis, the dynamics of thermal motions can be detected. Such motions arebelieved to be linked to biological function, and the protocols presented in this unit are among the few simple ways to assess such dynamics.