A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells
A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells
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DOI：
10.1371/journal.pone.0151041
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发表时间：
2016-03-07
期刊：
影响因子：
3.7
通讯作者：
Rothbauer, Ulrich
中科院分区：
文献类型：
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作者：
Buchfellner, Andrea;Yurlova, Larisa;Rothbauer, Ulrich
聚（ADP-核糖）聚合酶1（PARP 1）是DNA修复、基因组稳定性和细胞存活的关键参与者，并且它成为癌症治疗的高度相关靶标。为了加深我们对PARP生物学和靶向PARP 1的抗癌化合物的作用机制的理解，我们产生了一种新的PARP 1亲和试剂，在体外和活细胞中都有活性。这种PARP 1生物传感器基于PARP 1特异性单域抗体片段（类似于15 kDa），称为纳米抗体，其以纳摩尔亲和力识别人PARP 1的N-末端。在蛋白质组学方法中，固定的PARP 1纳米抗体促进从细胞裂解物中定量免疫沉淀功能性内源性PARP 1。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来工程化细胞内功能性PARP 1染色体。通过跟踪染色体信号，我们首次能够监测内源性PARP 1在活细胞中DNA损伤位点的募集。此外，在用化学物质处理人细胞时追踪染色体信号的亚核易位使得能够在高含量成像中实时分析活性化合物。由于其在PARP 1酶的内源性水平上作为生物传感器的能力，新型PARP 1纳米抗体是PARP 1生物学和DNA修复的基础和应用研究的独特和通用工具。
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 (similar to 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.