Accessible light-controlled knockdown of cell-free protein synthesis using phosphorothioate-caged antisense oligonucleotides.
Accessible light-controlled knockdown of cell-free protein synthesis using phosphorothioate-caged antisense oligonucleotides.
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DOI：
10.1038/s42004-023-00860-2
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发表时间：
2023-04-01
影响因子：
5.9
通讯作者：
Booth, Michael J.
中科院分区：
文献类型：
--
作者：
Hartmann, Denis;Booth, Michael J.
在非侵入性刺激下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，很少有人强调开发用于无细胞表达的光控“关闭”开关。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除；然而，它们的合成很复杂，并且还没有在无细胞系统中进行测试。开发简单、容易获得的方法来生产光激活的反义寡核苷酸，对于将其应用于无细胞生物学和生物技术将是至关重要的。在这里，我们报告了一种温和的一步法，选择性地将商业上可获得的光可移除保护基团光笼连接到反义寡核苷酸的硫代硫键上。利用这种光笼方法，在光照下，原始的硫代反义寡核苷酸被改造。含有硫代和磷酸混合骨架的光笼反义寡核苷酸显示，双链形成和核糖核酸酶H活性显著降低，并在光照下恢复。然后我们证明了这些光笼化的反义寡核苷酸可以用来用光来抑制无细胞蛋白质的合成。这项简单易用的技术将在光控制的生物逻辑门和调节合成细胞的活动方面有未来的应用。光激活的反义寡核苷酸已被开发用于在活细胞中诱导基因敲除，然而，它们的合成仍然具有挑战性，在无细胞系统中的应用还没有得到充分的探索。在这里，作者报告了一种一步法，选择性地将光笼连接到反义寡核苷酸的硫代磷酸键上，这种反义寡核苷酸可以用光击倒无细胞蛋白质的合成。
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. Light-activated antisense oligonucleotides have been developed to induce gene knockdown in living cells, however, their synthesis remains challenging and application in cell-free systems is underexplored. Here, the authors report a one-step method for selectively attaching photocages onto phosphorothioate linkages of antisense oligonucleotides that can knockdown cell-free protein synthesis using light.
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