NAD⁺ analogues for decoding the biological chemistry of PARP-mediated ADP-ribosylation

ADP-ribosylation is a NAD⁺-dependent post-translational modification that governs DNA repair, transcription, antiviral defense, and stress adaptation. Its transient and heterogeneous nature, however, has limited mechanistic understanding. Chemically modified NAD⁺ analogues have become indispensable chemical biology tools to overcome these barriers. By functionalizing the NAD⁺ scaffold at positions tolerated by PARP enzymes, these probes enable capture and enrichment of ADP-ribosylated substrates, writer-specific labeling, PAR-chain architecture control, reader identification, and real-time imaging. This Review summarizes recent progress in NAD⁺ analogue development, organized by probe function and biological application. We highlight how these chemical tools have transformed ADP-ribosylation research from descriptive detection toward mechanistic interrogation and discuss future directions for the field.