Orthogonal control of cell-free expression using photocaged nucleic acids
Giacomo Mazzotti 1 , Denis Hartmann 1 and Michael J. Booth 1,2 1 University of Oxford, UK, 2 University College London, London, UK
Cell-free systems have become a fundamental tool in synthetic biology, with applications in biology, biotechnology, and medicine. However, to offer real-world application, the activation and deactivation of these systems need to be precisely controlled. Light is the most suitable stimulus for external control because it can be applied with great spatiotemporal precision, has low toxicity, and can be easily adjusted, favouringrapid screening and optimisation. The activation of gene expression with light in cell-free systems has been achieved through different chemical and biological methods [1], but there is a lack of ways to turn the expression off, which would offer a large set of new applications. Our group has previously modified DNA templates with tightly controllable UV and blue light photocages to activate protein expression in synthetic tissues [2], pattern communication between synthetic cells and bacteria [3], and build a DNA-based logic gate [4]. In this work, by installing UV or blue light photocages on DNA templates and antisense oligonucleotides, we were able to turn gene expression on and off orthogonally [5]. We could activate cell-free DNA transcription with one wavelength and halt the translation of the corresponding protein with the other, at different timepoints. We could also orthogonally control gene knockdown by selectively degrading different mRNAs to inhibit their translation. Controlling the activation and deactivation of gene expression precisely and remotely with this tool will open new possibilities for gene circuit design and synthetic biology applications. References 1. Hartmann D., Smith J.M., Mazzotti G., Chowdry R., Booth M.J. Controlling gene expression with light: a multidisciplinary endeavour. Biochem. Soc. Tranc. 2020 , 48, 4, 1645-1659. 2. Booth M.J., Schild V.R., Graham A.D., Olof S.N., Bayley H. Light-activated communication in synthetic tissues. Sci. Adv. 2016 , 2, 4, 1-12. 3. Smith J.M., Hartmann D., Booth M.J. Engineering cellular communication between light-activated synthetic cells and bacteria. BioRxiv 2022 . 4. Hartmann D., Chowdry R., Smith J.M., Booth M.J. Blue light-activatable DNA for remote control of cell-free logic gates and synthetic cells. ChemRxiv 2022 . 5. Mazzotti G., Hartmann D., Booth M.J. Precise, orthogonal remote-control of cell-free systems using photocaged nucleic acids. ChemRxiv 2023 .
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© The Author(s), 2023
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