Department of Chemistry, University of Oxford, Mansfield Road, OX1 3TA Oxford, U.K.
Department of Chemistry, University College London, 20 Gordon Street, WC1H 0AJ London, U.K.
J Am Chem Soc. 2023 May 3;145(17):9481-9487. doi: 10.1021/jacs.3c01238. Epub 2023 Apr 19.
Cell-free expression of a gene to protein has become a vital tool in nanotechnology and synthetic biology. Remote-control of cell-free systems with multiple, orthogonal wavelengths of light would enable precise, noninvasive modulation, opening many new applications in biology and medicine. While there has been success in developing ON switches, the development of OFF switches has been lacking. Here, we have developed orthogonally light-controlled cell-free expression OFF switches by attaching nitrobenzyl and coumarin photocages to antisense oligonucleotides. These light-controlled OFF switches can be made from commercially available oligonucleotides and show a tight control of cell-free expression. Using this technology, we have demonstrated orthogonal degradation of two different mRNAs, depending on the wavelength used. By combining with our previously generated blue-light-activated DNA template ON switch, we were able to start transcription with one wavelength of light and then halt the translation of the corresponding mRNA to protein with a different wavelength, at multiple timepoints. This precise, orthogonal ON and OFF remote-control of cell-free expression will be an important tool for the future of cell-free biology, especially for use with biological logic gates and synthetic cells.
无细胞表达将基因转化为蛋白质已经成为纳米技术和合成生物学的重要工具。通过使用多个正交波长的光来远程控制无细胞系统,将能够实现精确、非侵入性的调节,为生物学和医学开辟许多新的应用。虽然已经成功开发了 ON 开关,但 OFF 开关的开发却一直缺乏。在这里,我们通过将硝基苄基和香豆素光笼连接到反义寡核苷酸上来开发正交光控无细胞表达 OFF 开关。这些光控 OFF 开关可以由市售的寡核苷酸制成,并显示出对无细胞表达的紧密控制。使用这项技术,我们已经证明了两种不同 mRNA 的正交降解,这取决于使用的波长。通过与我们之前生成的蓝光激活 DNA 模板 ON 开关结合,我们能够使用一种波长开始转录,然后使用不同的波长在多个时间点停止相应 mRNA 到蛋白质的翻译。这种精确的、正交的无细胞表达的 ON 和 OFF 远程控制将是无细胞生物学未来的重要工具,特别是对于生物逻辑门和合成细胞的应用。