“NAD-capQ” detection and quantitation of NAD caps

NAD-cap detection and quantitation assay. (A) Steps in NAD-capQ. (B) NAD-capQ analysis of RNA generated in vitro. (Red) NAD-RNA; (green) Gppp-RNA; (gray) background signal detected without nuclease P1. Error bars represent ±SD, n ≥ 3. (C) Schematic of 5′-NAD cap removal from NAD-capped RNA using pretreatment with Rai1. (D) NAD-capQ analysis with or without pretreatment of NAD-RNA with Rai1. (Dark red) Background corrected absorbance without Rai1; (light red) background corrected absorbance with Rai1. Significance as in B. (E) Thin-layer chromatography analysis revealing 80% efficiency of Rai1 hydrolysis of ³²P-5′ end labeled NAD-capped RNA spiked into 50 µg total RNA.