title: Synthesis of oligonucleotides carrying fluorescently labelled O(6)-alkylguanine for measuring hAGT activity.
abstract: O(6)-alkylguanine-DNA-alkyltransferase (hAGT) activity provides resistance to cancer chemotherapeutic agents and its inhibition enhances chemotherapy. We herein present the development of a novel fluorescence assay for the detection of hAGT activity. We designed a dsDNA sequence containing a fluorophore-quencher pair, where the fluorophore was attached to an O(6)-benzylguanine. This precursor was synthesized using the Mitsunobu reaction to introduce the benzyl group. The alkyl-fluorophore group is transferred to the active site during the dealkylation, producing an increase in fluorescence which is correlated to hAGT activity. This assay can be used for the evaluation of potential inhibitors of hAGT in a straightforward manner.
authors.affiliation: Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), C/ Jordi Girona 18-26, 08034 Barcelona, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Tintoré
authors.affiliation: Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), C/ Jordi Girona 18-26, 08034 Barcelona, Spain.
authors.forename: Santiago
authors.initials: S
authors.lastname: Grijalvo
authors.affiliation: Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), C/ Jordi Girona 18-26, 08034 Barcelona, Spain.
authors.forename: Ramon
authors.initials: R
authors.lastname: Eritja
authors.affiliation: Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), C/ Jordi Girona 18-26, 08034 Barcelona, Spain. Electronic address: carme.fabrega@iqac.csic.es.
authors.forename: Carme
authors.initials: C
authors.lastname: Fàbrega
mesh_terms: D020137:Base Pair Mismatch; D057075:Enzyme Assays / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis*; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: DNA repair; Fluorescence; Mitsunobu; O(6)-alkylguanine-DNA alkyltransferase; Oligonucleotide synthesis