title: A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.
abstract: 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.
authors.affiliation: ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Buchfellner
authors.affiliation: ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
authors.forename: Larisa
authors.initials: L
authors.lastname: Yurlova
authors.affiliation: Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University, Munich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Nüske
authors.affiliation: Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University, Munich, Germany.
authors.forename: Armin M
authors.initials: AM
authors.lastname: Scholz
authors.affiliation: ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Bogner
authors.affiliation: ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ruf
authors.affiliation: ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
authors.forename: Kourosh
authors.initials: K
authors.lastname: Zolghadr
authors.affiliation: Department of Radiation Oncology, Ludwig Maximilians University, Munich, Germany.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Drexler
authors.affiliation: Department of Radiation Oncology, Ludwig Maximilians University, Munich, Germany.
authors.forename: Guido A
authors.initials: GA
authors.lastname: Drexler
authors.affiliation: Institute for Applied Physics and Metrology, Bundeswehr University Munich, Neubiberg, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Girst
authors.affiliation: Institute for Applied Physics and Metrology, Bundeswehr University Munich, Neubiberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Greubel
authors.affiliation: Institute for Applied Physics and Metrology, Bundeswehr University Munich, Neubiberg, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Reindl
authors.affiliation: Department of Radiation Oncology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Siebenwirth
authors.affiliation: ChromoTek GmbH, IZB, Planegg, Martinsried, Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Romer
authors.affiliation: Department of Radiation Oncology, Ludwig Maximilians University, Munich, Germany.|Clinical Cooperation Group 'Personalized Radiotherapy of Head and Neck Cancer', Helmholtz Center Munich, Neuherberg, Germany.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Friedl
authors.affiliation: Natural and Medical Institute at the University of Tuebingen, Reutlingen, Germany.|Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
mesh_terms: D000918:Antibody Specificity; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006801:Humans; D047468:Immunoprecipitation; D057054:Molecular Imaging; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D061905:Single-Domain Antibodies / Q000276:immunology*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D000939:Epitopes; D061905:Single-Domain Antibodies; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases