Abstract 2754: Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme
Authors: Buchfellner Andrea , Yurlova Larisa , Dennison Stephanie , Ruf Benjamin , Rothbauer Ulrich , Romer Tina 
Published: July 2016

Abstract
DNA damage caused by normal cell activity or exogenous genotoxic agents is a constant threat to the genome. The nuclear enzyme Poly(ADP-ribose) polymerase 1 (PARP1) is rapidly activated by DNA lesions such as single-strand breaks and signals their presence by attaching ADP-ribose units to chromatin-associated proteins. To improve our understanding of PARP1 within a disease context, there is an ongoing need to develop novel PARP1 detection systems.
Here we describe the development of a VHH directed against human PARP1. The variable domain of single chain antibodies (VHH or nanobody) is a versatile research tool for a variety of applications. The high binding affinity of VHHs, their small size (15 kDa) and their robust expression in various cellular systems make them preferable to conventional antibodies. Moreover, VHH domains can be selected to recognize and bind their target structures within living cells.
This highly specific PARP1 VHH was selected by phage display using a VHH library from an immunized alpaca. We characterized its affinity, selectivity and activity both in vitro and in live cells.
To validate the in vitro interactions of this PARP1-specific VHH, we developed a PARP1 immunoprecipitation reagent by conjugating the PARP1-specific VHH to an immobilizing matrix (termed PARP1-Trap). This PARP1-Trap was shown to bind with high specificity to human PARP1 and not to other members of the PARP family. Importantly, the binding of the PARP1-Trap to PARP1 leaves the enzymatic activity of PARP1 unaffected. The epitope of the PARP1-Trap was localized to the N-terminal domain of PARP1 and consists of the three-dimensional motif of zinc fingers 2 and 3 together.
To determine whether the PARP1 VHH recognizes its target structure also in a cellular environment, the VHH was genetically fused to a fluorescent protein and expressed in living cells (termed Chromobody). The interaction of the PARP1 Chromobody with PARP1 was visualized using a protein-protein interaction assay called fluorescent two-hybrid (F2H). The F2H principle is based on a tethering strategy: a GFP-tagged protein (here GFP-PARP1) is enriched at a protein interaction platform engineered into F2H-BHK cells and serves as bait, whereas the RFP-tagged PARP1 VHH serves as a prey. Using the F2H assay, we could show that the PARP1 Chromobody is functional within living cells and specifically recognizes its antigen in a cellular environment. Moreover, by monitoring the PARP1 Chromobody signal after microirradiation, we were for the first time able to follow the recruitment of endogenous PARP1 to sites of DNA damage in living cells.
In summary, we developed a novel PARP1 VHH for both biochemical and live cell analysis of human PARP1. We anticipate that PARP1-VHH based reagents will provide new insights into the PARP1 enzyme. For example, the use of the PARP1-Trap coupled with mass spectrometry analysis may lead to the identification of hitherto unknown PARP1 interaction partners.
Citation Format: Andrea Buchfellner, Larisa Yurlova, Stephanie Dennison, Benjamin Ruf, Ulrich Rothbauer, Tina Romer. Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2754.