Figure 4. Screening the activity of potential analog-sensitive PARP-7 mutants with clickable NAD⁺ analogs.

(A) Chemical structures of four clickable NAD⁺ analogs used for the asPARP-7 screen. (Left) The R groups are linked at position 8 (#) of the adenine ring of NAD⁺. (Right) 8-BuT-6-Parg-NAD⁺ and 8-Bu(3-yne)T-NAD⁺ are clickable through their alkyne groups, whereas 8-oN₃-BT-NAD⁺ and 8-pN3-PAcT-NAD⁺ are clickable through their azide groups. (B) Western blots showing the wild-type and mutant PARP-7 automodification reactions performed with NAD⁺ or the clickable NAD⁺ analogs shown in (A). The MAR signals were detected using a MAR binding reagent. (C) Normalized automodification activity of wild-type and mutant PARP-7 proteins with NAD⁺ or the clickable NAD⁺ analogs shown in (A). PARP-7 automodification assays like those shown in (B) were used to determine the activity of the mutants with the NAD⁺ analogs. This screen was performed twice and yielded similar results each time; the results from one replicate are shown.