title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec H4B1R6, Canada. chris.wilds@concordia.ca.
authors.forename: D K
authors.initials: DK
authors.lastname: O'Flaherty
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec H4B1R6, Canada. chris.wilds@concordia.ca.
authors.forename: C J
authors.initials: CJ
authors.lastname: Wilds
mesh_terms: D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000737:chemistry; D003849:Deoxyguanosine / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000138:chemical synthesis / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques; D013936:Thymidine / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D009943:Organophosphorus Compounds; C434331:phosphoramidite; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003849:Deoxyguanosine; D013936:Thymidine