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Enzymatic Coupling of a Classical Luciferase Assay for the Quantitative Monitoring of the Protein‐Free ADP‐ribose

Historically, mechanisms of erasers of ADP‐ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP‐ribosylation reversal enzymes. Here, we developed a new NUDT5‐coupled AMP‐Glo (NCAG) assay to specifically monitor the protein‐free ADP‐ribose. We found that due to NUDT5's selectivity for protein‐free ADP‐ribose, but not protein‐bound poly‐ and mono‐ADP‐ribosylations, protein‐free poly(ADP‐ribose) chains, or NAD+, it could be used in complex solutions to target protein‐free ADP‐ribose for quantitative detection. The NCAG assay functions through a series of enzymatic reactions starting with cleavage of ADP‐ribose to AMP, followed by conversion of AMP to ATP, and finally detection via classical luciferase assay. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP‐ribosylation reversal enzymes that release protein‐free ADP‐ribose as a product. Furthermore, using the NCAG assay to monitor changes in the protein‐free ADP‐ribose can grant additional insight into ADP‐ribose recycling and reactivity.