Abstract
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.