A high-throughput screen for detection of compound-dependent phosphodiester bond cleavage at abasic sites

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• 作者：Marc C. Rideout^a ; ^{rideout.marc@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Benjamin Liet^a ; Didier Gasparutto^b ; Nathalie Berthet^a ; ^{nathalie.berthet@ujf-grenoble.fr" class="auth_mail" title="E-mail the corresponding author}
• 关键词：DNA damage ; Elimination reactions ; Abasic site ; Artificial nuclease ; High-throughput screen
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 November 2016
• 年：2016
• 卷：513
• 期：Complete
• 页码：93-97
• 全文大小：666 K

文摘

We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA. Finally, we characterized the phosphodiester backbone cleavage at the abasic site using denaturing gel electrophoresis. This study provides a useful template for researchers seeking to rapidly identify new artificial nucleases.