DESIGN, SYNTHESIS, AND ANALYSIS OF DISULFIDE CROSS-LINKED DNA DUPLEXES

The design, synthesis, and analysis of analogs of d(CGCGAATTCGCG)(2) p ossessing one or two intrahelical disulfide cross-links is reported. T he cross-linked oligomers were prepared by first synthesizing duplexes where the 3'- and 5'-terminal bases of the parent sequence were repla ced with N-3-thioethylthymidine. Following deprotection and purificati on, air oxidation afforded the desired cross-linked constructs in high yield. Analysis of both the oxidized (disulfide cross-linked) and red uced (thiol modified) duplexes by UV, circular dichroism, and NMR spec troscopies along with susceptibility to EcoRI cleavage indicates that the modifications are not structurally perturbing. Optical thermal den aturation and differential scanning calorimetry measurements suggest t hat introducing disulfide cross-link(s) into d(CGCGAATTCGCG)(2) does, however, cause two fundamental changes. First, the cross-link(s) incre ase the thermal stability of the modified duplexes by changing the mol ecularity of denaturation without an increase in enthalpy. Second, the disulfide cross-link traps one of the conformations of the conformati onally heterogeneous parent molecule resulting in a conformationally h omogeneous system. Both of these features are themselves unique and wi ll be important for further applications of disulfide cross-linked oli gomers such as these in studies of nucleic acid structure and function .