Fig. 2: Applying the MAAD reaction to RNA.

a RNA of interest (ROI) was modified with M11 to label a malononitrile tag, which can be selectively reacted with azodicarboxylate under aqueous solution. b Mass spectrometry analysis of native RNA after incubation with azodicarboxylate A1 in PBS at room temperature for 15 min. This figure was simulated from raw ESI-MS data. Original data are provided in Supplementary Fig. 8. c Mass spectrometry analysis of modified RNA after incubation with azodicarboxylate A1 in PBS at room temperature for 15 min. Modified RNA was acquired with malononitrile M11 (10 mM) in reaction buffer at 37 °C for 12 h. Peak a: 6697.8 ( + 0 M11 mod.); peak b: 6861.8 ( + 1 M11 mod.); peak c: 7061.4 ( + 1 M11 mod., +1 A1 addition); peak d: 7224.6 ( + 2 M11 mod., +1 A1 addition); peak e: 7425.9 ( + 2 M11 mod., +2 A1 addition); peak f: 7589.1 ( + 3 M11 mod., +2 A1 addition). d Biotin was incorporated into RNA via the MAAD reaction. RNA with varying degrees of M11 modification were reacted with azodicarboxylate-conjugated biotin (azo-biotin) in PBS at room temperature for 15 min. The efficiency of biotin incorporation was evaluated by a dot blot assay. Only the biotinylated products can be detected in the HRP channel, whereas all RNAs were detected by methylene blue (M. B.). e Schematic representation of POI malononitrile modification and azodicarboxylate addition. f Concentration-dependent addition of malononitrile-modified protein at 37 °C over 15 min with azo-BODIPY. Products were analyzed by in-gel fluorescence. g Schematic representation of malononitrile-modified RNA imaging. h Representative fluorescence images of HEK293T cells transfected with native RNA or M11-modified RNA in the absence or presence of azo-BODIPY (blue, Hoechst 33342; green, FAM). The experimental conditions are detailed in the Methods section. Representative image from three independent experiments with similar results. Source data are provided as a Source Data file.