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Cited by 58RNA Interference and Gene DeliveryRNA and protein synthesis mechanismsRNA Research and Splicing

Design, Assembly, Production, and Transfection of Synthetic Modified mRNA

Open access
Oh, Sanders, Kessler, John A.·2018·Methods
DOI 10.1016/j.ymeth.2017.10.008PMC PMC5807177
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Abstract

Proteins are drivers of cell functions and are targets of many therapies. Exogenous protein expression techniques, therefore, have been essential for research and medicine. The most common method for exogenous protein expression relies on DNA-based viral or non-viral vectors. However, DNA-based vectors have the potential to integrate into the host genome and cause permanent mutations. RNA-based vectors solve this shortcoming. In particular, synthetic modified mRNA provides non-viral, integration-free, zero-footprint method for expressing proteins. Modified mRNA can direct cell fate specification and cellular reprogramming faster and more efficiently than other methods. Furthermore, when simultaneously express multiple different proteins, mRNA vectors allow for greater flexibility and control over stoichiometric ratios, dose titrations, and complete silencing of expressions. Additionally, modified mRNAs have been shown to be viable and safe as therapeutic agents for gene therapy and vaccine, providing an alternative approach to address diseases. Despite these advantages, technical challenge, mRNA instability, and host immunogenicity have caused significant barriers to widespread use of this technology. The comprehensive method presented here addresses all of these shortcomings. This stepwise protocol describes every step necessary for the synthesis of modified mRNA from any coding DNA sequence of interest. The meticulously detailed protocol enables the users to make alterations to each component of modified mRNA for even more significant customization, allowing the researchers to apply this technology to a wide range of uses. This non-cytotoxic synthetic modified mRNA can be used for protein expression, regulation of cell reprogramming or differentiation, and drug delivery.

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  • A poly(A) tail of 120 units is considered the standard size for mRNA vaccines.

    “A poly(A) tail of 120 units is considered the standard size for mRNA vaccines.9,20,21”

    From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [21]

    The Introduction says this protocol attaches a consistent, standardized 120-base poly(A) tail to each mRNA vector. It does not say that 120 bases is the standard size for mRNA vaccines generally; it also describes a 100–250-base range for actively translated eukaryotic mRNAs. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Oh, S., & Kessler, J. A.
Affiliations
Northwestern University (US)
Corresponding author
Oh, Sanders
ORCID
Kessler, John A.
Venue
Methods
Publisher
Elsevier BV
Published
2018-01
Volume
133
Pages
29-43
ISSN
1046-2023 (ISSN-L)
DOI
10.1016/j.ymeth.2017.10.008
PMC
PMC5807177
OpenAlex
W2768060390
Cited by
58
References
8
Citations by year
2026: 3 · 2025: 11 · 2024: 5 · 2023: 8 · 2022: 7 · 2021: 16 · 2020: 3 · 2019: 3 · 2018: 2
FWCI
0.87
Type
journal article
Language
en
Open access
Licence
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
Locations
  • Methods (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • March of Dimes Foundation
  • National Institute on Drug Abuse (T90DA022881)
  • National Institute of General Medical Sciences (T32GM008152, T32GM144295)
  • National Institute of Neurological Disorders and Stroke (R25NS070695)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
mRNA synthesis, mRNA transfection, non-viral vectors, mRNA stability, immunogenicity, cellular reprogramming, cell differentiation, mRNA vaccines, gene therapy, drug delivery
MeSH terms
Cell Differentiation, DNA, Fibroblasts, Genetic Vectors, Humans, RNA, Messenger, Transfection, Genetic Therapy, Induced Pluripotent Stem Cells, Cellular Reprogramming

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