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Bioinformatics, Computational and Systems Biology

Programmable knockdown of target genes in polycistronic mRNA using de novo-designed sRNA

(A-35) Programmable knockdown of target genes in polycistronic mRNA using de novo-designed sRNA

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row A - Poster # 35

Presenting Author(s)

Eva Soler

Undergraduate Researcher
Columbia University, United States

Co-Author(s)

JH

Jaeseung Hahn

Postdoctoral Research Associate
Columbia University, United States
KD

Krishnaveni Dole

Undergraduate Researcher
Columbia University, United States
TH

Tasfia Haque

Undergraduate Researcher
Stony Brook University, United States

Primary Investigator(s)

TD

Tal Danino

Principal Investigator
Columbia University, United States

Introduction:: In prokaryotes, small RNA (sRNA) can bind to mRNA and interfere with ribosome binding and translation. sRNA-mediated gene silencing has been extensively tested in monocistronic mRNA that encodes a single protein; however, there is a gap in knowledge about how sRNA-mediated gene silencing works with polycistronic mRNA that encodes multiple genes under separate ribosome binding sites. Understanding gene expression in the context of polycistronic mRNA can enable scientists to deduce biochemical pathways, optimize living medicines, and manipulate therapeutic microbes for targeted therapy. Therefore, we designed and constructed a plasmid for a polycistronic mRNA that encodes two fluorescent reporter genes and a fluorescent aptamer as well as a panel of sRNA plasmids to elucidate the interactions between polycistronic mRNA and sRNA and their effects on gene silencing. By measuring fluorescence signal, we identified silencing efficiency and mRNA degradation from implementing our first sRNA design for each ribosome binding site (RBS). The findings in our study to understand how sRNA works in the context of polycistronic mRNA have the potential to translate prokaryotic sRNA technologies for controlling gene expression towards biomedical applications.

Materials and Methods:: In an effort to study the gene silencing ability of small RNA (sRNA) with polycistronic mRNA, we computationally designed and implemented five different sRNAs. Each have different sequences and final structures when bound to mRNA, and bind to two different ribosome binding sites (RBS) in a bicistronic reporter mRNA expressing two fluorescent genes. The sRNAs were engineered to manipulate the structure of the RBS and prevent ribosome binding in two ways: 1) folding mRNA, such as creating hairpin-like structure, to silence a gene and avoid mRNA degradation or 2) masking the sRNA within the mRNA’s hairpin structure. The reporter plasmid was programmed and optimized to constitutively transcribe the polycistronic mRNA expressing two orthogonal fluorescent genes, mCherry and mTag, for mRNA quantification along with a fluorescent aptamer, dBroccoli with DFHBI-1T, for the quantification of mRNA degradation. Each sRNA design was incorporated into a plasmid with an IPTG-inducible promoter and transformed alongside the reporter plasmid into a host cell to observe gene silencing. By measuring fluorescence, through spectrometry and flow cytometry, we identified which genes were silenced, monitored mRNA degradation, and any unforeseen signal changes.

Results, Conclusions, and Discussions::

Fluorescence results demonstrate that the first sRNA tested was successful at silencing genes in the polycistronic mRNA without mRNA degradation. The sRNA silenced mTag and mCherry at different rates. In the reporter, mTag had a naturally higher expression since it is the first gene translated in the mRNA and doesn’t require dimerization like mCherry. For these reasons, the fluorescence signal continued to be reflected on the read after introducing the sRNA. On the other hand, mCherry also experienced silencing but not as significantly as mTag. Monitoring the net signal over time also provided more information about this phenomenon, showing that gene silencing is temporary as the fluorescent signal recovers after 4 hours.

This information has facilitated preliminary conclusions on how we can apply our designs to better control gene expression levels and optimize sRNAs to generate a personalized sRNA control system for prokaryotes. With the sRNAs currently being tested, we hope to explore different possible routes for the sRNA’s inability to knock down certain genes, and silencing genes with or without target mRNA degradations.

Gaining a deeper understanding of how artificial sRNA works in the context of polycistronic mRNA could lead to the seamless application of sRNAs to control gene expression and improve the safety and efficacy of therapeutic microbes. Ultimately, the successful understanding and application of sRNAs in polycistronic mRNA would allow for the personalized control of natural genetic systems, which has the potential to open many exciting opportunities for their application in the biomedical field. The next steps for this project involve collecting data from the rest of the sRNA designs as well as testing different combinations and dilutions of the sRNAs in a host cell to identify their efficiency thresholds and how they may complement each other. Another avenue we are excited to explore is to apply newly developed generative AI methods for protein expression and engineering to help us expand our panel of sRNAs.

Acknowledgements (Optional): :

Our research is supported by Professor Tal Danino and Post-doctoral Research Associate Jaeseung Hahn in the Department of Biomedical Engineering at Columbia University. Figures were made using BioRender and Prism 9.

References (Optional): :

[1] Hui, C.-Y., Guo, Y., Zhang, W., & Huang, X.-Q. (2018). Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in escherichia coli. AMB Express, 8(1). https://doi.org/10.1186/s13568-018-0612-5

[2]Kim, J., Zhou, Y., Carlson, P. D., Teichmann, M., Chaudhary, S., Simmel, F. C., Silver, P. A., Collins, J. J., Lucks, J. B., Yin, P., & Green, A. A. (2019). De novo-designed translation-repressing riboregulators for multi-input cellular logic. Nature Chemical Biology, 15(12), 1173–1182. https://doi.org/10.1038/s41589-019-0388-1

[3] Komarova, E. S., Chervontseva, Z. S., Osterman, I. A., Evfratov, S. A., Rubtsova, M. P., Zatsepin, T. S., Semashko, T. A., Kostryukova, E. S., Bogdanov, A. A., Gelfand, M. S., Dontsova, O. A., & Sergiev, P. V. (2020). Influence of the spacer region between the shine–dalgarno box and the start codon for fine‐tuning of the translation efficiency in escherichia coli. Microbial Biotechnology, 13(4), 1254–1261. https://doi.org/10.1111/1751-7915.13561

[4] Noh, M., Yoo, S. M., Kim, W. J., & Lee, S. Y. (2017). Gene expression knockdown by modulating synthetic small RNA expression in escherichia coli. Cell Systems, 5(4). https://doi.org/10.1016/j.cels.2017.08.016

[5]Yoo, S. M., Na, D., & Lee, S. Y. (2013). Design and use of synthetic regulatory small RNAS to control gene expression in escherichia coli. Nature Protocols, 8(9), 1694–1707. https://doi.org/10.1038/nprot.2013.105

[6]Harimoto, T., Hahn, J., Chen, YY. et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nat Biotechnol 40, 1259–1269 (2022). https://doi.org/10.1038/s41587-022-01244-y

[7] Chien, T., Harimoto, T., Kepecs, B. et al. Enhancing the tropism of bacteria via genetically programmed biosensors. Nat Biomed Eng 6, 94–104 (2022). https://doi.org/10.1038/s41551-021-00772-3