Interstrand disulfide cross-linking of internal sugar residues in duplex RNA

Disulfide cross-linking is being used increasingly more to study the struct ure and dynamics of nucleic acids. We have previously developed a procedure for the formation of disulfide cross-links through the sugar-phosphate bac kbone of nucleic acids. Here we report the preparation and characterization of an RNA duplex containing a disulfide interstrand cross-link. A self-com plementary oligoribonucleotide duplex containing an interstrand cross-link was prepared from the corresponding 2'-amino modified oligomer. Selective m odification of the 2'-amino group with an aliphatic isocyanate, containing a protected disulfide. gave the corresponding 2'-urea derivative in excelle nt yield. An RNA duplex containing an intrahelical, interstrand disulfide c ross-link was subsequently prepared by a thiol disulfide exchange reaction in nearly quantitative yield as judged by denaturing polyacrylamide gel ele ctrophoresis (DPAGE). The cross-linked RNA was further characterized by enz ymatic digestion and the structure of the crosslink lesion was verified by comparison to an authentic sample, prepared by chemical synthesis. The effe ct of the chemical modifications on duplex stability was determined by UV t hermal denaturation experiments. The intrahelical cross-link stabilized the duplex considerably: the disulfide cross-linked oligomer had a melting tem perature that was ca. 40 degrees C higher than that of the noncross-linked oligomer. (C) 2000 Elsevier Science Ltd. All rights reserved.