Finding New Methods for Analyzing mRNA

A recent study, published in Analytical Chemistry, features a new development in the field of messenger RNA (mRNA) technology (1). A team of researchers have developed a method to analyze mRNA poly(A) tail length and heterogeneity more efficiently, which could help evolve the landscape of therapeutic compounds that harness the potential of mRNA.

Messenger RNA has gained prominence as a powerful tool in the realm of medical treatments, with mRNA vaccines, such as those for COVID-19, demonstrating their efficacy. However, to further harness the potential of mRNA in therapeutic applications, the need for efficient analytical methods has become increasingly apparent.

This research shed light on the length and diversity of the poly(A) tail in mRNA molecules, a critical factor in mRNA stability and function. The researchers employed a multi-step approach to analyze these aspects, marking a significant milestone in the field.

First, the team used the RNase T1 enzyme to cleave the poly(A) tail from mRNA samples. The liberated poly(A) tails were then subjected to a size exclusion chromatography method, allowing for the determination of the average length of the poly(A) tail. This step provided valuable insights into the length of the poly(A) tail, which is essential for mRNA function and stability.

In addition to measuring length, the researchers were also interested in understanding the heterogeneity of the poly(A) tail. To achieve this, they employed high-resolution ion-pair reversed phase liquid chromatography (IP-RPLC). This method enabled the researchers to estimate the size heterogeneity of the poly(A) tail, even at the level of oligonucleotide variants up to 150 nucleotides long. This information is critical for understanding how the diversity of the poly(A) tail may impact mRNA function.

Both methods used ultraviolet detection. The results obtained through these techniques were further validated by conducting LC-mass spectrometry (LC–MS) analysis on the same mRNA samples, ensuring the accuracy of the findings.

The methods revealed that the poly(A) tail length and heterogeneity results obtained through the various techniques were in good agreement. This cohesiveness between methods signifies the robustness and reliability of the new analytical techniques, further cementing their importance in the study and development of mRNA-based therapeutics.

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Reference

Gilar, M.; Doneanu, C.; Gaye, M. M. Liquid Chromatography Methods for Analysis of Mrna Poly(a) Tail Length and Heterogeneity. Anal. Chem. 2023, 95 (38), 14308–14316. DOI:10.1021/acs.analchem.3c02552.