Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine
Francis Combes1, Thanh-Huong Bui1, Frida J Pettersson1
1Department of Biotechnology and Nanomedicine, SINTEF, Trondheim, Norway.
Abstract:
Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3'-5' exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.
Insights
Degradation during mRNA production impacts vaccine efficacy. A new enzymatic assay using polynucleotide phosphorylase (PNPase) quickly and sensitively measures mRNA integrity, offering a scalable alternative to existing methods.
Area of Science:
- Biotechnology
- Molecular Biology
- Analytical Chemistry
Background:
- Synthetic messenger RNA (mRNA) production and storage can generate byproducts that compromise vaccine and therapeutic integrity and functionality.
- mRNA integrity is a critical quality attribute requiring advanced analytical evaluation before clinical application.
Purpose of the Study:
- To demonstrate the impact of heat degradation on mRNA translatability.
- To introduce a novel enzymatic assay for assessing the integrity of conventional and self-amplifying mRNA.
Main Methods:
- Hybridization of oligo-T to the poly(A) tail of intact mRNA.
- Digestion of unhybridized RNA fragments using a 3'-5' exoribonuclease.
- Conversion of released adenosine-containing nucleotides to ATP and detection via luminescence.
- Development of a polynucleotide phosphorylase (PNPase)-based assay for mRNA byproduct detection.
Main Results:
- The study quantifies the effect of heat degradation on mRNA translatability.
- A sensitive luminescence-based method was established to detect mRNA byproducts.
- The novel PNPase assay provides fast and sensitive mRNA integrity evaluation across different mRNA lengths.
Conclusions:
- The developed enzymatic assay is a sensitive and scalable method for evaluating mRNA integrity.
- This PNPase-based approach offers a viable alternative to traditional chromatographic, electrophoresis, or sequencing techniques for mRNA quality control.
