Light-controllable nucleic acids for control of in vitro gene expression Denis Hartmann 1 , Razia Chowdhry 1 , Giacomo Mazzotti 1 , Jefferson M. Smith 1 and Michael J. Booth 1,2 1 University of Oxford, UK, 2 University College London, UK Precise control of oligonucleotide activity represents a crucial tool in studying biological functions, ranging from in vitro applications, such as cell-free expression (CFE) [1], to whole organisms. Such control can be achieved through a variety of stimuli, including pH, redox potential, temperature and light. Light as a stimulus is particularly attractive, as it can be applied remotely as well as being largely bioorthogonal and has already found widespread use in the control of oligonucleotide function [2]. We have developed a method of tightly controlling a DNA template of interest with light for use in CFE. For this, modified nucleobases are incorporated into a T7 promoter and reacted with a photocage carrying a biotin motif. This enables binding of monovalent Streptavidin (mSA), which blocks T7 RNA Polymerase from binding. Upon irradiation, this steric block is cleaved off, enabling transcription. Based on the design of the previously reported UV-activatable nitrobenzyl-photocage reported in our group [3], we have designed a blue-light activatable photocage based on a coumarin scaffold, which enables gene control with blue light. This photocage shows excellent caging ability and rapid uncaging with blue light. We also found it to be orthogonal to UV light and have used it in conjunction with the UV-activatable DNA to generate a DNA-based, 2 wavelength-controlled AND-Gate through expression of a split enzyme in cell-free conditions. By encapsulation of this blue light-activatable DNA together with a CFE system in a lipid bilayer, we were also able to generate synthetic cells that are activatable with blue light [4]. We could also extend the functionality of this technology and employ it on antisense oligonucleotides (ASOs) by incorporation of modified nucleobases into the ASO. Modification with the UV and blue light-activatable photocages allows for binding of mSA, preventing target mRNA degradation by RNAse H prior to illumination. This allowed for tight control of gene expression and knockdown respectively in CFE using different wavelengths and also orthogonal, RNase H-mediated degradation of different target mRNAs, depending on the applied wavelength. References 1. Silverman, A. D., Karim, A. S. & Jewett, M. C. Nature Rev. Genetics , 2020 , 151–170 2. Hartmann, D., Smith, J. M., Mazzotti, G., Chowdhry, R. and Booth, M. J., Biochem. Soc. Trans. , 2020 , 48, 1645–1659. 3. Booth, M. J., Restrepo Schild, V., Graham, A. D., Olof, S. N., and Bayley, H., Sci. Adv., 2016 , 2, e1600056. 4. Hartmann, D., Chowdhry, R., Smith, J. M., and Booth, M. J., ChemRXiv , 2022 , doi:10.26434/chemrxiv-2022-p8xgb
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