Introduction
Ribonucleic acids (RNAs) are an important class of biomolecules. The 4 canonical bases that exist in RNA are adenine, uracil, guanosine, and cytosine; with countless modifications possible to these bases.1 RNA is essential to many biological functions of a cell, including key processes of the central dogma, such as transcription and translation. They function either as coding RNAs for protein synthesis (mRNA), or non-coding RNAs, which perform functional roles (tRNA and rRNA) as well as regulatory roles in gene expression (siRNA, microRNA).
When polymerized, RNAs exhibit diverse functions, depending on their type.
- Messenger RNA (mRNA) - single-stranded coding RNA which consists of specific nucleotide sequences transcribed from the triplet codes of DNA during transcription. The codons in mRNA are thus complementary to the original DNA template and serve as a messenger of genetic information for protein synthesis.
- Transfer RNA (tRNA) - single-stranded non-coding RNA folded into a characteristic cloverleaf shape. tRNA functions primarily as the carrier of specific amino acids, which it transfers to the growing polypeptide chain at the ribosome during the process of mRNA translation.
- Ribosomal RNA (rRNA) - non-coding RNA that functions as a primary component of ribosomes. It functions as a ribozyme that carries out protein synthesis in the cytoplasm of the cell.
Among RNAPs, the T7 RNA Polymerase (T7 RNAP) is one of the most commonly utilized RNAPs for biotechnology applications, primarily due to its high T7 promoter specificity2 and high elongation efficiency3. Its high specificity to the T7 promoter, which has the inherent advantage of high gene expression4, in addition to its non-affinity to unrelated DNAs, even closely related T3 promoters5, makes T7 RNAP the ideal RNAP for the production of therapeutic products such as mRNA vaccines.
Motivations behind our project
- The focus on mRNA technology has heightened due to its recent applications in vaccines for COVID-19 and other diseases.6,7 Conventionally, the synthesis of mRNA products such as mRNA vaccines relies on the use of specific RNA polymerases (RNAPs) for in-vitro transcriptions (IVTs).8 However, existing polymerases suffer from shortcomings, such as poor processivity that hinders the synthesis of long transcripts.
- Hence, our approach is to first develop a robust reporter system to evaluate the wild-type T7 RNAP and any variants generated. Next, our project aims to generate a diverse library of RNAP variants using different methods such as site-directed mutagenesis, ancestral reconstruction, and random mutagenesis. These variants will then be screened using our reporter system to identify any better-performing variants.
Our Approaches
Reporter System
One of the aims of our project is to develop a reporter system without capitalizing on increasing antibiotic resistance. By using a fluorescent-readout assay, variants can be better quantified, easing the identification process of better-performing variants.
Introduction to Library Preparation
Together, these techniques (site-directed mutagenesis, ancestral reconstruction, and random mutagenesis) provide a comprehensive toolkit for evolving T7 RNA polymerase, allowing for the optimization of this enzyme for various biotechnology applications.
- Site-directed mutagenesis allows for precise alterations to the T7 RNAP coding sequence. These mutations can include point substitutions, deletions, and insertions, which are introduced via PCR amplification. By designing specific primers that introduce these targeted mutations, the resulting variants can be screened to identify those that exhibit desired traits, such as improved transcriptional efficiency or stability. Furthermore, beneficial mutations can be combined to create more robust T7 RNAP variants. This strategy enhances the functionality of the enzyme and provides insights into the interactions between different mutations and their cumulative effects on performance.
- To further enhance the capabilities of T7 RNAP, ancestral reconstruction was also utilized. This technique involves inferring the sequences of ancestral forms of T7 RNAP based on existing variants. By synthesizing these ancestral proteins, this approach provides valuable insights into how changes can improve certain traits of the enzyme, guiding future engineering efforts.
- Random mutagenesis using error-prone PCR (epPCR) was also implemented, introducing a wide range of mutations throughout the T7 RNAP gene. This technique allows for the generation of a diversity of variants.
References
1 Jonkhout, N., Tran, J., Smith, M. A., Schonrock, N., Mattick, J. S., & Novoa, E. M. (2017). The RNA modification landscape in human disease. RNA (New York, N.Y.), 23(12), 1754–1769. https://doi.org/10.1261/rna.063503.117
2 Rong, M., He, B., McAllister, W. T., & Durbin, R. K. (1998). Promoter specificity determinants of T7 RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 95(2), 515–519. https://doi.org/10.1073/pnas.95.2.515
3 Borkotoky, S., & Murali, A. (2018). The highly efficient T7 RNA polymerase: A wonder macromolecule in the biological realm. International Journal of Biological Macromolecules, 118(Pt A), 49–56. https://doi.org/10.1016/j.ijbiomac.2018.05.198
4 Graumann, K., & Premstaller, A. (2006). Manufacturing of recombinant therapeutic proteins in microbial systems. Biotechnology Journal, 1(2), 164–186. https://doi.org/10.1002/biot.200500051
5 Klement, J. F., Moorefiedl, M. B., Jorgensen, E., Brown, J. E., Risman, S., & McAllister, W. T. (1990). Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site. Journal of Molecular Biology, 215(1), 21–29. https://doi.org/10.1016/S0022-2836(05)80091-9