Hartmann Denis, Booth Michael J
Department of Chemistry, University of Oxford, Mansfield Road, OX1 3TA, Oxford, UK.
Department of Chemistry, University College London, 20 Gordon Street, WC1H 0AJ, London, UK.
Commun Chem. 2023 Apr 1;6(1):59. doi: 10.1038/s42004-023-00860-2.
Controlling cell-free expression of a gene to protein with non-invasive stimuli is vital to the future application of DNA nanodevices and synthetic cells. However, little emphasis has been placed on developing light-controlled 'off' switches for cell-free expression. Light-activated antisense oligonucleotides have been developed to induce gene knockdown in living cells; however, they are complicated to synthesise and have not been tested in cell-free systems. Developing simple, accessible methods to produce light-activated antisense oligonucleotides will be crucial for allowing their application in cell-free biology and biotechnology. Here, we report a mild, one-step method for selectively attaching commercially-available photoremovable protecting groups, photocages, onto phosphorothioate linkages of antisense oligonucleotides. Using this photocaging method, upon illumination, the original phosphorothioate antisense oligonucleotide is reformed. Photocaged antisense oligonucleotides, containing mixed phosphorothioate and phosphate backbones, showed a drastic reduction in duplex formation and RNase H activity, which was recovered upon illumination. We then demonstrated that these photocaged antisense oligonucleotides can be used to knock down cell-free protein synthesis using light. This simple and accessible technology will have future applications in light-controlled biological logic gates and regulating the activity of synthetic cells.
利用非侵入性刺激控制基因到蛋白质的无细胞表达对于DNA纳米器件和合成细胞的未来应用至关重要。然而，在开发用于无细胞表达的光控“关闭”开关方面，人们关注较少。光激活反义寡核苷酸已被开发用于在活细胞中诱导基因敲低；然而，它们合成复杂，且尚未在无细胞系统中进行测试。开发简单、易获得的方法来生产光激活反义寡核苷酸对于使其在无细胞生物学和生物技术中的应用至关重要。在此，我们报道了一种温和的一步法，用于将市售的光可去除保护基团（光笼）选择性地连接到反义寡核苷酸的硫代磷酸酯键上。使用这种光笼化方法，光照后，原始的硫代磷酸酯反义寡核苷酸得以恢复。含有硫代磷酸酯和磷酸混合主链的光笼化反义寡核苷酸在双链形成和RNase H活性方面显著降低，光照后恢复。然后我们证明，这些光笼化反义寡核苷酸可用于通过光照敲低无细胞蛋白质合成。这种简单且易获得的技术将在光控生物逻辑门和调节合成细胞活性方面有未来应用。
