Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides.
RNA Biol
; 21(1): 1-8, 2024 Jan.
Article
em En
| MEDLINE
| ID: mdl-38836544
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polirribonucleotídeo Nucleotidiltransferase
/
RNA Mensageiro
Limite:
Humans
Idioma:
En
Revista:
RNA Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Noruega