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Cited by 218RNA Interference and Gene Deliveryinterferon and immune responsesRNA and protein synthesis mechanisms

An origin of the immunogenicity of in vitro transcribed RNA

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Mu, Xin, Greenwald, Emily, Ahmad, Sadeem, Hur, Sun·2018·Nucleic Acids Research
DOI 10.1093/nar/gky177PMC PMC6007322

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Abstract

The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg2+ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated.

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Authors
Mu, X., Greenwald, E., Ahmad, S., & Hur, S.
Affiliations
Boston Children's Hospital (US)
Corresponding author
Hur, Sun
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2018-03-09
Volume
46
Issue
10
Pages
5239-5249
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gky177
PMC
PMC6007322
OpenAlex
W2953295872
Cited by
218
References
8
Citations by year
2026: 42 · 2025: 28 · 2024: 44 · 2023: 32 · 2022: 30 · 2021: 15 · 2020: 13 · 2019: 12 · 2018: 2
FWCI
3.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
Funders
Cancer Research Institute (R01AI106912, R01AI111784, R21AI130791)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
in vitro transcription, T7 RNA polymerase, immunogenicity, RNA therapeutics, antisense RNA, double-stranded RNA, MDA5, cytosolic pattern recognition receptors, innate immune activation, modified nucleotides, magnesium content, RNA quality control
MeSH terms
DEAD Box Protein 58, Interferon-Induced Helicase, IFIH1, Humans, Immunity, Innate, Magnesium, Nucleotides, Promoter Regions, Genetic, Receptors, Immunologic, DNA-Directed RNA Polymerases, RNA, Double-Stranded, Transcription, Genetic, Viral Proteins, Interferon-beta, HEK293 Cells

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