Analyzing RNA Decay Products by HPLC

Decay products were detected by analyzing the products of RNA decay reactions that contained 2 µM enzyme and 10 µM native or unmodified tRNA_i^Met in 100 mM NaCl, 20 mM Tris⋅Cl pH 7.5, 0.5 mM TCEP, and 5 mM ATP·MgCl₂ after incubation at 30 °C for 1 h. Reactions were stopped by addition of proteinase K to a final concentration of 0.5 mg/mL and incubation at 37 °C for 30 min to degrade proteins in the reaction. Samples were mixed with an equal volume of 2× HPLC running buffer A (50 mM potassium phosphate pH 7.0 and 10 mM tetrabutylammonium hydrogensulfate [Sigma-Aldrich]). Products were then separated by ion-pair reverse-phase HPLC (Nova-Pak C18, 60 Å, 4 mm, 3.9 × 150-mm column) by running in buffer A at a flow rate of 1 mL/min at 40 °C and eluted with 100% buffer B (buffer A containing 50% [vol/vol] acetonitrile) from 5 min to 30 min (12 (link)). UV absorbance at 260 nm was monitored to detect nucleotides or RNA products.