Interstrand Cross-Linking of Duplex DNA by Nitrous Acid: Covalent Structure of the dG-to-dG Cross-Link at the Sequence 5′-CG

It has previously been shown in synthetic oligodeoxynucleotides that the interstrand cross-linking reaction of nitrous acid with duplex DNA preferentially forms thermally- and base-stable links between deoxyguanosine residues at the duplex sequence 5′-CG. The covalent nucleus of this linkage is shown herein to result from the presence of a residue in which the original deoxyguanosyl residues on opposite strands are cross-linked through a single N² atom common to both [N²-(2- deoxyinosyl)deoxyguanosine residue]. Variation of nitrous acid concentration, reaction time, and temperature established conditions under which up to a several percent yield of cross-linked oligodeoxynucleotide was obtained. Evidence for the covalent structure of the cross-link reported herein includes comparison of spectroscopic properties (mass spectrum, ultraviolet spectra at three pH values, ¹H and NMR spectrum) of N²-(2-deoxyinosyl)deoxyguanosine isolated by enzymatic hydrolysis of cross-linked oligodeoxynucleotides to those of the same substance and its derivatives previously isolated from nitrous acid-treated calf thymus DNA. Further evidence in favor of cross-linking through N² is reported: substitution of deoxyinosine, which lacks an N² amino group, in place of deoxyguanosine at both sites in the duplex sequence 5′-CG abolishes dG-to-dG (dI-to-dI) cross-linking.