A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

被引：33

作者：

Buchfellner, Andrea ^{[1
]}

Yurlova, Larisa ^{[1
]}

Nueske, Stefan ^{[2
]}

Scholz, Armin M. ^{[2
]}

Bogner, Jacqueline ^{[1
]}

Ruf, Benjamin ^{[1
]}

Zolghadr, Kourosh ^{[1
]}

Drexler, Sophie E. ^{[3
]}

Drexler, Guido A. ^{[3
]}

Girst, Stefanie ^{[4
]}

Greubel, Christoph ^{[4
]}

Reindl, Judith ^{[4
]}

Siebenwirth, Christian ^{[5
]}

Romer, Tina ^{[1
]}

Friedl, Anna A. ^{[3
,6
]}

Rothbauer, Ulrich ^{[7
,8
]}

机构：

[1] ChromoTek GmbH, IZB, Martinsried, Germany

[2] Univ Munich, Fac Vet Med, Livestock Ctr, Munich, Germany

[3] Univ Munich, Dept Radiat Oncol, Munich, Germany

[4] Bundeswehr Univ Munich, Inst Appl Phys & Metrol, Neubiberg, Germany

[5] Tech Univ Munich, Klinikum Rechts Isar, Dept Radiat Oncol, D-80290 Munich, Germany

[6] Helmholtz Ctr Munich, Clin Cooperat Grp Personalized Radiotherapy Head, Neuherberg, Germany

[7] Univ Tubingen, Nat & Med Inst, Reutlingen, Germany

[8] Univ Tubingen, Pharmaceut Biotechnol, Tubingen, Germany

来源：

PLOS ONE | 2016年 / 11卷 / 03期

关键词：

HUMAN POLY(ADP-RIBOSE) POLYMERASE-1; TRANSCRIPTIONALLY ACTIVE NUCLEOLI; FLUORESCENT 2-HYBRID ASSAY; DNA-DAMAGE RESPONSE; ACTINOMYCIN-D; RNA-SYNTHESIS; LIVING CELLS; CHROMATIN-STRUCTURE; ANTIBODY FRAGMENTS; MAMMALIAN-CELLS;

D O I：

10.1371/journal.pone.0151041

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

070301 [无机化学]; 070403 [天体物理学]; 070507 [自然资源与国土空间规划学]; 090105 [作物生产系统与生态工程];

摘要：

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.

引用

页数：23

共 69 条

[1]

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression [J].