| A synthetic methodology for conformationally restraining nucleic acids using disulfide cross-links is described. This chemistry is designed to position cross-links site-specifically at sterically accessible locations on oligonucleotides so that the native helical structure remains unperturbed while providing a large increase in conformational stability. To test this hypothesis, the design, synthesis, and analysis of three different disulfide cross-linked nucleic acid constructs were studied. In the first example, analogs of a well-defined duplex structure, d(CGCGAATTCGCG);Analysis of the oxidized (disulfide cross-linked) and reduced (thiol modified) nucleic acid constructs by UV, CD, and NMR spectroscopies, and susceptibility to EcoRI cleavage indicates that the thiol-modifications are structurally non-perturbing. Optical thermal denaturation and differential scanning calorimetry measurements indicate that the cross-link(s) increase the conformational stability towards thermal-, ionic-, pH-, and concentration-induced structural changes. UV and DSC data suggest that this increase the thermal stability is entropic in origin. In all three examples, the disulfide cross-link traps a single nucleic acid conformation which will be important for applications of disulfide cross-linked oligomers in studies of nucleic acid structure and function. In addition, this methodology can be incorporated into a variety of nucleic acids secondary and tertiary structures which should prove valuable in examining the structural and thermodynamic aspects of unusual nucleic acid conformations in vitro. |
