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

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• 作者：Maria Tintoré ; Santiago Grijalvo ; Ramon Eritja ; Carme Fà ; brega ; ^{carme.fabrega@iqac.csic.es" class="auth_mail" title="E-mail the corresponding author} ; ^{recgma@cid.csic.es" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Fluorescence ; DNA repair ; Oligonucleotide synthesis ; Mitsunobu ; O6-alkylguanine-DNA alkyltransferase
• 刊名：Bioorganic & Medicinal Chemistry Letters
• 出版年：2015
• 出版时间：15 November 2015
• 年：2015
• 卷：25
• 期：22
• 页码：5208-5211
• 全文大小：738 K

文摘

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.