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Related Experiment Video

Updated: Oct 2, 2025

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

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Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.

Irena Vlatkovic¹, János Ludwig², Gábor Boros¹

¹BioNTech SE, An der Goldgrube 12, 55131 Mainz, Germany.

|February 26, 2022

Summary

This summary is machine-generated.

A new ribozyme assay accurately measures the cap structure on in vitro-transcribed (IVT) messenger RNA (mRNA), ensuring quality for mRNA therapeutics and vaccines. This method is crucial for the development and quality control of novel mRNA-based biologics.

Keywords:

cap in vitro-transcribed (IVT) mRNA mRNA capping efficiency quality control ribozyme

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An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Published on: September 15, 2020

Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

The 5'-cap structure of in vitro-transcribed (IVT) messenger RNA (mRNA) is critical for its translation efficiency and stability.
IVT mRNA technology is rapidly advancing for applications in vaccines (e.g., COVID-19) and therapeutics (e.g., cancer immunotherapy, protein replacement).
Ensuring the correct cap structure is essential for the safety and efficacy of mRNA-based therapeutics, necessitating robust quality control methods.

Purpose of the Study:

To develop and validate a novel method for assessing the capping efficiency of IVT mRNAs.
To provide a reliable quality control tool for mRNA therapeutics in preclinical and clinical development.
To analyze how different mRNA features influence capping efficiency.

Main Methods:

Designed ribozyme assays to specifically cleave IVT mRNAs, releasing 5'-end capped or uncapped products.
Purified cleavage products using silica-based columns.
Visualized and quantified products using denaturing polyacrylamide gel electrophoresis (PAGE) and liquid chromatography-mass spectrometry (LC-MS).

Main Results:

The developed ribozyme cleavage assays accurately determined the capping efficiency of IVT mRNAs.
The method allowed for the analysis of capping efficiency across various IVT mRNA features, including cap structures, 5' untranslated regions, nucleoside modifications, and lengths.
The assays demonstrated reliability and speed for analyzing capping status.

Conclusions:

Ribozyme cleavage assays offer a fast and reliable method for analyzing mRNA capping efficiency.
This technology serves as a valuable tool for research and development in mRNA therapeutics.
The developed method is suitable for general quality control of mRNA-based biologics.