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Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates
Open accessAbstract
A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.
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Cited by
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Karikó, K., Muramatsu, H., Ludwig, J., & Weissman, D. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr695 · Cited by 898
A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA
Baiersdörfer, M., Boros, G., Muramatsu, H., Mahiny, A., Vlatkovic, I., Sahin, U., & Karikó, K. (2019) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2019.02.018 · Cited by 569
Burd, C. G., & Dreyfuss, G. (1994) · The EMBO Journal · doi:10.1002/j.1460-2075.1994.tb06369.x · Cited by 488
The promise of mRNA vaccines: a biotech and industrial perspective
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“First, the purity of the mRNA is a crucial determinant of yields, and it is known that the DNA-dependent RNA polymerases yield smaller oligoribonucleotide impurities as a result of abortive initiation events, 11 as well as double-stranded (ds) RNA generated by self-complementary 3′ extension, 12 which can result in ty…”
Background
Metadata
- Authors
- Milligan, J. F., Groebe, D. R., Witherell, G. W., & Uhlenbeck, O. C.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 1987
- Volume
- 15
- Issue
- 21
- Pages
- 8783-8798
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2085272777
- Cited by
- 2,136
- References
- 0
- FWCI
- 18.36
- Type
- journal article
- Language
- en
- Open access
- bronze
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- Milligan, J. F., Groebe, D. R., Witherell, G. W., & Uhlenbeck, O. C. (1987). Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Research. https://doi.org/10.1093/nar/15.21.8783
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
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