Double-stranded RNA sequencing via RNase III E38A digestion followed by HPLC-MS/MS analysis

Double-stranded RNA (dsRNA) is a product related impurity introduced during the production of mRNA that is challenging to detect, isolate and characterize. Ribonuclease III (RNase III) with a single amino acid substitution (E38A) has previously been reported to generate dsRNA fragments with a consistent number of nucleotides according to the divalent cation used. In this work, we investigate the utility of RNase III E38A to detect and identify potential dsRNA in RNA molecules in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS). Model dsRNA was prepared by annealing complementary strands of various lengths, followed by RNase If and RNase III E38A digestion. Digestion products were characterized by gel electrophoresis and LC-MS/MS. Using model dsRNA, we demonstrated that it is possible to sequence dsRNA by LC-MS/MS when digested by RNase III E38A.