NAD Cap Quantification by AbTir Cleavage

To measure the amount of the NAD cap before and after AbTir treatment, the NAD-capQ method was performed as previously described^{43 (link)}. Briefly, in vitro transcribed NAD-RNA (1 μg) or total RNA (500 μg) isolated from E. coli (stationary phase) or Arabidopsis (12-day-old seedlings) were subjected to AbTir or AbTir-E/A treatment at 25 °C for 16 h. Then, the RNA products were extracted with phenol/chloroform (pH 4.5), precipitated, and subjected to nuclease P1 digestion in a 20 μL reaction with 1 × nuclease P1 Reaction Buffer and 10 U/μL nuclease P1 (New England Biolabs; M0660S) at 37 °C for 2 h. Before the AbTir cleavage reaction, the isolated total RNA was treated with NAP-10 columns to get rid of any residual free NAD⁺. Following digestion with nuclease P1, 30 μL of NAD/NADH Extraction Buffer from the NAD/NADH Quantification Kit (Sigma-Aldrich, MAK037) was added to each sample. The 50 μL reactions were then used for the subsequent enzymatic cycling reaction and the colorimetric assay by following the manufacturer’s protocol from the NAD/NADH Quantitation Kit. The same RNA samples treated with the AbTir-E/A catalytic mutant served as the negative controls.