Online Determination of mRNA Poly(A) Tail Length and Content

The determination of mRNA poly(A) attributes typically requires time-consuming and labor-intensive in-solution digestions, followed by liquid chromatography (LC), capillary electrophoresis (CE), and/or mass spectrometry (MS) analysis. In-solution digestions involve high sample consumption and pose a risk of nuclease contamination to the LC column and LC-MS instrument. In this work, we developed an online approach to determine the mRNA poly(A) tail length and content by coupling an immobilized ribonuclease (RNase) T1 cartridge online to hydrophilic interaction liquid chromatography (HILIC). We first optimized an LC setup to improve the throughput and identify potential roadblocks for the transfer of the platform technology. Three HILIC columns were evaluated for the separation of a poly(A) ladder ranging from 40 to 120 nt in length. The effect of the HILIC column temperature on the separation performance was then systematically studied between 30 and 80 °C. The poly(A) tail length was determined for various mRNA sequences and lengths. Interestingly, a bimodal poly(A) profile was observed for a catalog mRNA. A proof-of-concept study was performed to determine the poly(A) tail content in mixtures prepared with different ratios of polyadenylated and nonpolyadenylated mRNAs. The results were compared to those obtained by ion-pairing RPLC (IPRP), which highlighted the superiority of the nucleotide mapping approach in the case of mRNA samples containing many impurities.