Removal of Double-Stranded RNA Contaminants During Template-Directed Synthesis of mRNA

S V Sharabrin; A A Bondar; E V Starostina; D N Kisakov; L A Kisakova; A M Zadorozhny; A P Rudometov; A A Ilyichev; L I Karpenko

doi:10.1007/s10517-024-06102-2

Removal of Double-Stranded RNA Contaminants During Template-Directed Synthesis of mRNA

Bull Exp Biol Med. 2024 Apr;176(6):751-755. doi: 10.1007/s10517-024-06102-2. Epub 2024 Jun 19.

Authors

S V Sharabrin¹, A A Bondar², E V Starostina³, D N Kisakov³, L A Kisakova³, A M Zadorozhny³, A P Rudometov³, A A Ilyichev³, L I Karpenko³

Affiliations

¹ State Research Center of Virology and Biotechnology "VECTOR", Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Koltsovo, Novosibirsk region, Russia. sharabrin.sv@gmail.com.
² Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
³ State Research Center of Virology and Biotechnology "VECTOR", Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Koltsovo, Novosibirsk region, Russia.

PMID: 38896322
DOI: 10.1007/s10517-024-06102-2

Abstract

The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.

Keywords: dsRNA; mRNA; mRNA purification.

MeSH terms

Animals
Antigens, CD / genetics
Antigens, CD / metabolism
Antigens, Differentiation, T-Lymphocyte / genetics
Antigens, Differentiation, T-Lymphocyte / metabolism
CD69 Antigens
DNA-Directed RNA Polymerases / genetics
DNA-Directed RNA Polymerases / metabolism
Interferon-alpha / biosynthesis
Interferon-alpha / genetics
Interferon-alpha / metabolism
Lectins, C-Type / genetics
Lectins, C-Type / metabolism
Mice
RNA, Double-Stranded* / genetics
RNA, Double-Stranded* / metabolism
RNA, Messenger* / genetics
RNA, Messenger* / metabolism
Viral Proteins / genetics
Viral Proteins / metabolism

Substances

RNA, Messenger
RNA, Double-Stranded
Antigens, Differentiation, T-Lymphocyte
Antigens, CD
Lectins, C-Type
DNA-Directed RNA Polymerases
Interferon-alpha
Viral Proteins
CD69 Antigens
bacteriophage T7 RNA polymerase