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mRNA Stability and Gene Expression

mRNA Stability and Gene Expression

The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
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Intact DNA strands can be found in fossils, while scientists sometimes struggle to keep RNA intact under laboratory conditions. The structural variations between RNA and DNA underlie the differences in their stability and longevity. Because DNA is double-stranded, it is inherently more stable. The single-stranded structure of RNA is less stable but also more flexible and can form weak internal bonds. Additionally, most RNAs in the cell are relatively short, while DNA can be up to 250 million...

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Updated: Jun 28, 2025

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

Published on: June 10, 2022

Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA.

Jonathan Currie¹, Jacob R Dahlberg², Ester Lundberg²

¹Innovation Strategies and External Liaison, Pharmaceutical Technology and Development, Operations & IT, AstraZeneca, Gothenburg, Sweden.

Journal of Pharmaceutical and Biomedical Analysis

|April 18, 2024

Summary

A new ion-pair reversed-phase liquid chromatography method effectively analyzes modified messenger RNA (mRNA) drug products. This stability-indicating method ensures quality control for therapeutic mRNA development by monitoring degradation products under stress conditions.

Keywords:

Chemical degradation Degradation study Ion-pair reversed-phase liquid chromatography Modified mRNA Nucleotides RNA ladder Ribonucleic acid Stability indicating

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Area of Science:

Pharmaceutical Science
Analytical Chemistry
Biotechnology

Background:

Modified messenger RNA (mRNA) is a rapidly advancing therapeutic drug class.
Robust analytical methods are essential for quality control in mRNA pharmaceutical development.

Purpose of the Study:

To develop and optimize a stability-indicating ion-pair reversed-phase liquid chromatography (IP-RPLC) method for characterizing modified mRNA.
To assess the method's suitability for analyzing mRNA drug products under various stress conditions relevant to pharmaceutical development.

Main Methods:

An IP-RPLC method was optimized by studying column temperature, mobile phase flow rate, and ion-pair selection.
The method was applied to separate and analyze a model RNA ladder and a modified eGFP mRNA compound (996 nucleotides).
Stability-indicating capability was demonstrated by exposing eGFP mRNA to heat, hydrolysis, and ribonuclease treatment.

Main Results:

The optimized IP-RPLC method achieved baseline separation of RNA fragments up to 1000 nucleotides.
Using 100 mM Triethylamine as the ion-pairing agent provided the highest resolution for large RNA fragments (750/1000 nucleotides) and the eGFP mRNA.
The method successfully monitored degradation products of eGFP mRNA under various stress conditions, enabling assessment of its relative stability.

Conclusions:

The developed IP-RPLC method is robust and suitable for the characterization and quality control of modified mRNA drug products.
This method provides critical insights into mRNA stability, supporting the pharmaceutical development of mRNA-based therapeutics.
The method's ability to monitor degradation products is key for ensuring the safety and efficacy of mRNA therapeutics.