Buchfellner Andrea, Yurlova Larisa, Nüske Stefan, Scholz Armin M, Bogner Jacqueline, Ruf Benjamin, Zolghadr Kourosh, Drexler Sophie E, Drexler Guido A, Girst Stefanie, Greubel Christoph, Reindl Judith, Siebenwirth Christian, Romer Tina, Friedl Anna A, Rothbauer Ulrich
ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University, Munich, Germany.
PLoS One. 2016 Mar 7;11(3):e0151041. doi: 10.1371/journal.pone.0151041. eCollection 2016.
Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.
聚（ADP - 核糖）聚合酶1（PARP1）是DNA修复、基因组稳定性和细胞存活中的关键因子，并且它已成为癌症治疗中高度相关的靶点。为了加深我们对PARP生物学以及PARP1靶向抗癌化合物作用机制的理解，我们制备了一种新型的PARP1亲和试剂，其在体外和活细胞中均有活性。这种PARP1生物传感器基于一种PARP1特异性单域抗体片段（约15 kDa），称为纳米抗体，它以纳摩尔亲和力识别人类PARP1的N端。在蛋白质组学方法中，固定化的PARP1纳米抗体有助于从细胞裂解物中对功能性内源性PARP1进行定量免疫沉淀。对于细胞研究，我们通过将纳米抗体编码序列与荧光蛋白序列相结合，构建了一种细胞内功能性PARP1染色体抗体。通过追踪染色体抗体信号，我们首次能够监测活细胞中内源性PARP1向DNA损伤位点的募集。此外，在用化学物质处理人类细胞后追踪染色体抗体信号的核内亚定位，能够在高内涵成像中对活性化合物进行实时分析。由于其能够在内源性PARP1酶水平作为生物传感器发挥作用，这种新型PARP1纳米抗体是用于PARP1生物学和DNA修复基础及应用研究的独特且通用的工具。
