Precise, orthogonal remote-control of cell-free systems using photocaged nucleic acids

Abstract

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, non-invasive 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.

Reversible control of gene expression by guest-modified adenosines in a cell-free system via host–guest interaction

“…[16][17][18] Thus, photo-responsive nucleic acids have been exclusively implemented for gene expression control in CFE systems, as exemplified by the photo-control of transcription activity using azobenzene derivatives 19,20 and bulky photo-cleavable moieties. [21][22][23] Although these methods allow the remote and spatiotemporal control of gene expression, light can induce a potential dysfunction of the biological components of CFE systems. 20,24 Furthermore, photochemistry-based methods present intrinsic limitations for in vivo applications because of the low permeability of light.…”