Analytical chemistrySep 26, 2025

Measuring mRNA Tail Length and Composition in Real Time

Updated Feb 19, 2026

Abstract

An online method was developed to determine mRNA poly(A) tail length and content using an immobilized ribonuclease cartridge coupled with hydrophilic interaction liquid chromatography.

The new approach improves throughput compared to traditional methods that require extensive sample processing.
Three hydrophilic interaction liquid chromatography columns were tested for their ability to separate poly(A) fragments ranging from 40 to 120 nucleotides.
Systematic studies showed that increasing the column temperature from 30 to 80 °C affected separation performance.
A bimodal poly(A) profile was observed for one catalog mRNA, indicating variability in poly(A) tail characteristics.
A proof-of-concept study demonstrated that the new method can effectively assess poly(A) tail content in mixtures of polyadenylated and nonpolyadenylated mRNAs.

Simplified

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.

Full Text

Full text is available at the source.

View Full Text ↗

Abstract

Full Text

Related papers

What Lands in Your Inbox Each Week: