Evison Benjamin J, Actis Marcelo L, Fujii Naoaki
Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Bioorg Med Chem. 2016 Mar 1;24(5):1071-8. doi: 10.1016/j.bmc.2016.01.032. Epub 2016 Jan 18.
DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP-DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP-DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.
DNA链间交联（ICL）是复制叉和转录复合物前进过程中的物理障碍。一系列诱导ICL的试剂已成功应用于癌症治疗。虽然已有研究采用紫外线A（UVA）激活的补骨脂素作为诱导ICL的模型试剂来研究ICL修复，但由于用相对较大的报告基团标记补骨脂素支架可能会干扰母体补骨脂素的生物活性，因此对该损伤的直接检测常常受到限制。在此制备了一种经过最小修饰的补骨脂素探针，其具有适合点击化学的小炔基手柄。该补骨脂素探针命名为8-炔丙氧基补骨脂素（8-POP），可在体外被UVA激活以产生ICL，这些ICL易于通过铜催化反应与叠氮化物标记的荧光报告基团进行标记后处理。改良的碱性彗星试验表明，UVA激活的8-POP能在细胞中高效产生ICL。细胞中的8-POP-DNA损伤适合原位进行点击介导的与荧光报告基团的连接，从而可通过荧光显微镜和流式细胞术对其进行检测和定量。对8-POP处理的细胞使用小分子DNA修复抑制剂可减弱8-POP-DNA损伤的去除，这证实8-POP是研究细胞ICL修复的合适探针。本研究中应用的标记后处理策略成本低廉、快速且本质上具有高度模块化，在DNA修复研究中具有多种应用潜力。
