A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates

• Published in: BioTechniques Vol. 69; no. 4; pp. 281 - 288
• Main Authors: Tossberg, John T, Esmond, Tashawna M, Aune, Thomas M
• Format: Journal Article
• Language: English
• Published: London, UK Future Science Ltd 01-10-2020; Taylor & Francis Group
• Subjects: alternative to molecular cloning; cell transfection; chemical DNA synthesis; protein translation; synthetic mRNA from DNA template
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/btn-2020-0037

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.