Fig. 3: NUDT2 is active on a broad range of substrates.

a EnzChek assay to quantify released phosphate from in vitro transcribed double-stranded triphosphorylated hairpin RNA (IVT4) left untreated (untreated) or incubated with recombinant NUDT2, NUDT E58A, NUDT14, or NUDT14 E121A (600 nM), as indicated. Colorimetric measurements of the samples were performed every 3 min. The line graph shows the mean of three independent experiments with the ribbon indicating ± SD. b Illustration of in vitro transcribed RNA substrates with varying starting nucleotides with or without paired 5′-overhangs. c RNA substrates depicted in (b) were incubated without (dotted line) or with (solid line) NUDT2 at a concentration of 600 nM over a time course of 2 h, and phosphate release was evaluated using the EnzChek assay. Spectrophotometric measurements to quantify phosphate release were performed every 3 min. The line graph shows the mean of three independent experiments with the ribbon indicating ± SD. d The indicated RNA was incubated with NUDT2 (600 nM) for 3 h, and the integrity was analyzed using an Agilent small RNA chip and a Bioanalyzer. e The indicated ribonucleoside triphosphates (2.5 µM) and the positive control IVT4 were incubated for 3 h with NUDT2 (600 nM), and phosphate release was determined by malachite green assay. The mean of three independent reactions ± SD is shown.