Accessible light-controlled knockdown of cell-free protein synthesis using phosphorothioate-caged antisense oligonucleotides

Journal article

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.

Authors: Hartmann, D; Booth, MJ

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Communications Chemistry
• Volume: 6
• Issue: 1
• Pages: 59-59
• Article number: 59
• Publication date: 2023-04-01
• DOI: 10.1038/s42004-023-00860-2
• EISSN: 2399-3669
• ISSN: 2399-3669
• Language: English
• Keywords: Gene knockdown; Biochemistry; Cell; Biology; Cell biology; Chemistry; Molecular biology; Sense (electronics); Cell-free protein synthesis; Gene; Protein biosynthesis; Oligonucleotide