A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates
We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted lucifer...
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| Published in: | BioTechniques Vol. 69; no. 4; pp. 281 - 288 |
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| Main Authors: | , , |
| Format: | Journal Article |
| Language: | English |
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London, UK
Future Science Ltd
01-10-2020
Taylor & Francis Group |
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| Abstract | We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning.
transcription reactions were performed starting with a double-stranded DNA fragment with the bacteriophage SP6 promoter. A 5′ CAP and 3′ poly (A) tail were added via enzymatic reactions to these single-stranded RNAs to produce functional mRNAs. Synthetic mRNAs were transfected into HeLa cells and expression of translated proteins determined. |
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| AbstractList | We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning.
In vitro
transcription reactions were performed starting with a double-stranded DNA fragment with the bacteriophage SP6 promoter. A 5′ CAP and 3′ poly (A) tail were added via enzymatic reactions to these single-stranded RNAs to produce functional mRNAs. Synthetic mRNAs were transfected into HeLa cells and expression of translated proteins determined. We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning. We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning. transcription reactions were performed starting with a double-stranded DNA fragment with the bacteriophage SP6 promoter. A 5′ CAP and 3′ poly (A) tail were added via enzymatic reactions to these single-stranded RNAs to produce functional mRNAs. Synthetic mRNAs were transfected into HeLa cells and expression of translated proteins determined. |
| Author | Esmond, Tashawna M Aune, Thomas M Tossberg, John T |
| AuthorAffiliation | 2Pathology, Microbiology & Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA 1Department of Medicine & Vanderbilt University Medical Center, Nashville, TN 37232, USA |
| AuthorAffiliation_xml | – name: 2Pathology, Microbiology & Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA – name: 1Department of Medicine & Vanderbilt University Medical Center, Nashville, TN 37232, USA |
| Author_xml | – sequence: 1 givenname: John T surname: Tossberg fullname: Tossberg, John T organization: Department of Medicine & Vanderbilt University Medical Center, Nashville, TN 37232,USA – sequence: 2 givenname: Tashawna M surname: Esmond fullname: Esmond, Tashawna M organization: Department of Medicine & Vanderbilt University Medical Center, Nashville, TN 37232,USA – sequence: 3 givenname: Thomas M orcidid: 0000-0002-4968-4306 surname: Aune fullname: Aune, Thomas M organization: Department of Medicine & Vanderbilt University Medical Center, Nashville, TN 37232,USA, Pathology, Microbiology & Immunology,Vanderbilt University Medical Center, Nashville, TN 37232,USA |
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| CitedBy_id | crossref_primary_10_4049_jimmunol_2001428 crossref_primary_10_1080_14760584_2024_2320327 crossref_primary_10_1093_braincomms_fcac238 crossref_primary_10_1007_s40005_022_00569_9 crossref_primary_10_3389_fimmu_2022_818023 crossref_primary_10_1016_j_jhep_2021_12_014 crossref_primary_10_1016_j_crimmu_2021_04_001 crossref_primary_10_1093_braincomms_fcae100 |
| Cites_doi | 10.3389/fimmu.2019.00594 10.1016/0092-8674(76)90128-8 10.1038/nrd.2017.243 10.1146/annurev-immunol-030409-101212 10.1038/nmeth.1177 10.1083/jcb.108.2.229 10.1021/nn305466z 10.1136/gutjnl-2019-318269 10.3389/fimmu.2018.02512 10.1128/mr.44.2.175-205.1980 10.1007/s00018-012-0990-9 10.1006/bbrc.1996.1573 10.1038/nature13590 10.1016/j.omtn.2019.04.012 10.1146/annurev.iy.07.040189.001045 10.1038/d41573-020-00073-5 10.1093/nar/14.8.3521 10.1073/pnas.95.21.12226 10.1016/j.jaut.2019.02.003 10.1016/S0378-1119(01)00350-X 10.1016/j.gene.2012.03.021 10.1002/anie.201304986 10.1146/annurev.immunol.20.083001.084359 10.1016/j.biomaterials.2017.11.034 10.1073/pnas.1106610108 10.1126/science.1178334 10.1016/j.omtm.2019.07.006 10.1128/MMBR.63.2.446-456.1999 |
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| SubjectTerms | alternative to molecular cloning cell transfection chemical DNA synthesis protein translation synthetic mRNA from DNA template |
| Title | A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates |
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