Short DNA duplexes containing a 1,3-N4C-alkyl-N4C interstrand cross-link that joins the two C residues of a -CNG- sequence were prepared using either a phosphoramidite or convertible nucleoside approach. The alkyl cross-link consists of 2, 4, or 7 methylene groups. The duplexes, which contain a seven-base-pair core and A3/T3 complementary 3′-overhanging ends, were characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Ultraviolet thermal denaturation studies showed that the duplexes denature in a cooperative manner and that the length of the cross-link affects the thermal stability. Thus, the transition temperature of the ethyl cross-linked duplex, 42°C, is 16°C higher than the melting temperature of the corresponding non-cross-linked control, whereas the transition temperatures of the butyl and heptyl cross-linked duplexes, 73 and 72°C, respectively, are 46-47°C higher. The reduced molecularity of denaturation of the cross-linked duplexes versus melting of the non-cross-linked duplex most likely accounts for these differences. Examination of molecular models suggests that the ethyl cross-link is too short to span the distance between the two C residues at the site of the cross-link in B-form DNA without causing distortion of the helix, whereas less and no distortion would be expected for the butyl and heptyl cross-links, respectively. The circular dichroism spectra, which show greatest deviation in the ethyl cross-linked duplex from B-form DNA, are consistent with this expectation. Anomalous mobilities on native polyacrylamide gels of multimers produced by self-ligation of each of the cross-linked duplexes suggest that the ethyl and butyl cross-linked duplexes undergo bending deformations, whereas multimers derived from the heptyl cross-linked duplex migrated normally. The bending angle was estimated to be 20°, 13°, and 0° for the ethyl, butyl, and heptyl cross-linked duplexes, respectively. Thus, it appears that the degree of bending in these N4C-alkyl-N4C cross-linked duplexes is controlled by the length of the cross-link.
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