Interstrand disulfide cross-linking of internal sugar residues in duplex RNA.
Disulfide cross-linking is being used increasingly more to study the structure and dynamics of nucleic acids. We have previously developed a procedure for the formation of disulfide cross-links through the sugar-phosphate backbone of nucleic acids. Here we report the preparation and characterization of an RNA duplex containing a disulfide interstrand cross-link. A self-complementary oligoribonucleotide duplex containing an interstrand cross-link was prepared from the corresponding 2'-amino modified oligomer. Selective modification of the 2'-amino group with an aliphatic isocyanate, containing a protected disulfide, gave the corresponding 2'-urea derivative in excellent yield. An RNA duplex containing an intrahelical, interstrand disulfide cross-link was subsequently prepared by a thiol disulfide exchange reaction in nearly quantitative yield as judged by denaturing polyacrylamide gel electrophoresis (DPAGE). The cross-linked RNA was further characterized by enzymatic digestion and the Structure of the cross-link lesion was verified by comparison to an authentic sample, prepared by chemical synthesis. The effect of the chemical modifications on duplex stability was determined by UV thermal denaturation experiments. The intrahelical cross-link stabilized the duplex considerably: the disulfide cross-linked oligomer had a melting temperature that was ca. 40 degrees C higher than that of the noncross-linked oligomer.
Alefelder
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S
Department of Chemistry, University of Washington, Seattle 98195-1700, USA.
Sigurdsson
S T
ST
D002241:Carbohydrates / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D012313:RNA / Q000737:chemistry*
D002241:Carbohydrates; D004220:Disulfides; D012313:RNA