ADP-ribosylation of Fluorescent Nucleic Acids

ADP-ribosylation of Cy3-labelled DNA and RNA oligonucleotides was performed as described earlier (101 (link),130 (link)). Proteins for the assay were purified as described in the same studies. Briefly, 10 μL reactions were prepared in ADP-ribosylation buffer (20 mM HEPES–KOH (pH 7.6), 5 mM MgCl₂ and 1 mM DTT). The reactions contained 1 μM Cy3-labelled RNA or DNA oligonucleotide, 3 μM PARPs, PARP10 ART (residues 868–1025), or PARP14 WWE-ART (residues 1459–1801), and 500 μM NAD⁺. The reactions were incubated for 1 h at 37°C and stopped by adding 50 ng/μl Proteinase K and 0.15% SDS followed by incubating at 50°C for 30 min. Finally, the reactions were mixed with 2× TBE urea sample buffer (8 M urea, 20 μM EDTA (pH 8.0), 20 μM Tris–HCl (pH 7.5), and bromophenol blue) and loaded on a pre-run 15% denaturing urea polyacrylamide gel electrophoresis (PAGE) gel. The gels were run at 7 W/gel and imaged using the Molecular Imager PharosFX system (BioRad) with laser excitation for Cy3 at 532 nm.