A Genetically\nEncoded Sensor for Real-Time Monitoring\nof Poly-ADP-Ribosylation Dynamics In Vitro and in Cells

ADP-ribosylation,\nthe transfer of ADP-ribose (ADPr) from\nnicotinamide\nadenine dinucleotide (NAD<sup>+</sup>) groups to proteins, is a conserved\npost-translational modification (PTM) that occurs most prominently\nin response to DNA damage. ADP-ribosylation is a dynamic PTM regulated\nby writers (PARPs), erasers (ADPr hydrolases), and readers (ADPR binders).\nPARP1 is the primary DNA damage-response writer responsible for adding\na polymer of ADPR to proteins (PARylation). Real-time monitoring of\nPARP1-mediated PARylation, especially in live cells, is critical for\nunderstanding the spatial and temporal regulation of this unique PTM.\nHere, we describe a genetically encoded FRET probe (pARS) for semiquantitative\nmonitoring of PARylation dynamics. pARS feature a PAR-binding WWE\ndomain flanked with turquoise and Venus. With a ratiometric readout\nand excellent signal-to-noise characteristics, we show that pARS can\nmonitor PARP1-dependent PARylation temporally and spatially in real-time.\npARS provided unique insights into PARP1-mediated PARylation kinetics\nin vitro and high-sensitivity detection of PARylation in live cells,\neven under mild DNA damage. We also show that pARS can be used to\ndetermine the potency of PARP inhibitors in vitro and, for the first\ntime, in live cells in response to DNA damage. The robustness and\nease of use of pARS make it an important tool for the PARP field.