Synthesis of oligonucleotides carrying fluorescently labelled O6-alkylguanine for measuring hAGT activity

O6-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 O6-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.The Communities MULTIFUN (contract NMP4-LA-2011-262943), Spanish Healthcare Research Fund (grant PI06/1250) and Spanish Ministry of Economy and Competitiveness (grants CTQ-2010-20541-C03-03 and CTQ2014-52588-R) are acknowledged for financialsupport. M.T. was supported by a pre-doctoral fellowship (FPI) from MINECO. C.F is grateful to TV3 Marato 2012 for a research contract.Peer reviewe