Detecting Poly (ADP-Ribose) In Vitro and in Cells Using PAR Trackers

Abstract

ADP-ribosylation (ADPRylation) is a reversible posttranslational modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs (PAR binding zinc fingers), and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe the preparation and use of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)-optimized dimerization-dependent or split-protein reassembly PAR sensors containing a naturally occurring PAR binding domain fused to both halves of dimerization-dependent GFP (ddGFP) or split nano luciferase (NanoLuc), respectively. We also describe how these tools can be used for the detection and quantification of PAR levels in biochemical assays with extracts and in living cells. These protocols will allow users to explore the broad utility of PAR-Ts for detecting PAR in various experimental and biological systems.

Keywords: ADP-ribose (ADPR); ADP-ribosyl transferase (ART); ADP-ribosylation (ADPRylation); ADPR binding domain (ARBD); Automodification; Dimerization-dependent green fluorescent protein (ddGFP); Live cell imaging; Nano luciferase (NanoLuc); Poly(ADP-ribosyl)ation (PARylation); Posttranslational modification (PTM).