Site-specific covalent capture of human O6-alkylguanine-DNA-alkyltransferase using single-stranded intrastrand cross-linked DNA

Abstract

A methodology is reported to conjugate human O⁶-alkylguanine-DNA-alkyltransferase (hAGT) to the 3′-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA–protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2′-deoxyguanosine residue (either in a 5′→3′ or 3′→5′ fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O⁴-thymidine-alkylene-O⁴-thymidine and the (5′→3′) O⁶-2′-deoxyguanosine-alkylene-O⁴-thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5′→3′) O⁴-thymidine-alkylene-O⁶-2′-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.