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Cited by 95RNA and protein synthesis mechanismsRNA Interference and Gene DeliveryMolecular Biology Techniques and Applications

pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences

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Grier, Alexandra E, Burleigh, Stephen, Sahni, Jaya, Clough, Courtnee A, Cardot, Victoire, Choe, Dongwook C, Krutein, Michelle C, Rawlings, David J +3 more·2016·Molecular Therapy - Nucleic Acids
DOI 10.1038/mtna.2016.21PMC PMC5014522
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Abstract

Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3' polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3' termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s).

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Crossref · 10.1038/mtna.2016.21

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  • The presence of the poly(A) tail on the plasmid template for mRNA production is not stable and prone to shortening in bacteria during plasmid DNA amplification.

    “However, the presence of the poly(A) tail on the plasmid template for mRNA production is not stable and prone to shortening in bacteria during plasmid DNA amplification.17”

    From Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo (2025) · cites this paper as [17]

    The Results section (‘Homopolymeric poly(A) tracts 175 bp or longer are extremely unstable in circular plasmids’; page not provided) reports shortening after bacterial transformation of circular plasmids carrying long poly(A) tracts. The claim omits important limits: 70-bp tracts remained stable, and another Results section reports that poly(A) tracts in the linear pEVL plasmid were highly stable during extended propagation, except that the ~500-bp tract tended to shorten. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • The instability of the traditional poly(A) tail during plasmid amplification in bacterial systems presents a significant challenge in mRNA production.

    “The instability of the traditional poly(A) tail during plasmid amplification in bacterial systems presents a significant challenge in mRNA production.”

    From Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo (2025) · cites this paper as [17]

    The Results section ('Homopolymeric poly(A) tracts 175 bp or longer are extremely unstable in circular plasmids'; page not provided) reports that extended poly(A)-encoding DNA tracts shortened after cloning and propagation in E. coli. The Introduction identifies obtaining defined poly(A)-tail lengths as a challenge for IVT mRNA production. The claim is broader than the evidence: a 70-bp tract remained stable, so the paper does not say that poly(A) tails generally are unstable during bacterial plasmid amplification. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Shortening of the poly(A) sequence directly affects the translation efficiency of the mRNA.

    “This instability, characterized by shortening of the poly(A) sequence, directly affects the translation efficiency of the mRNA.17”

    From Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo (2025) · cites this paper as [17]

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Authors
Grier, A. E., Burleigh, S., Sahni, J., Clough, C. A., Cardot, V., Choe, D. C., Krutein, M. C., Rawlings, D. J., Jensen, M. C., Scharenberg, A. M., & Jacoby, K.
Affiliations
  • University of Washington (US)
  • Seattle Children's Research Institute (US)
Corresponding author
Scharenberg, Andrew M
ORCID
Venue
Molecular Therapy - Nucleic Acids
Publisher
Elsevier BV
Published
2016
Volume
5
Pages
e306
ISSN
2162-2531 (ISSN-L)
DOI
10.1038/mtna.2016.21
PMC
PMC5014522
OpenAlex
W2339574870
Cited by
95
References
3
Citations by year
2026: 13 · 2025: 14 · 2024: 12 · 2023: 15 · 2022: 12 · 2021: 14 · 2020: 5 · 2019: 5 · 2018: 3 · 2017: 2
FWCI
1.42
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institute of General Medical Sciences (T32GM007266)
  • National Institute of Allergy and Infectious Diseases (U19AI096111)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Keywords
linear plasmids, in vitro transcription, mRNA synthesis, poly(A) tail, 3' polyadenylation, T7 promoter, transient expression, homopolymeric tracts

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