Blue light-activatable DNA for remote control of cell-free logic gates and synthetic cells

Abstract

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and drug delivery, particularly using remote, non-damaging stimuli such as visible light. Here, we have synthesised blue light-activatable DNA parts to selectively control cell-free expression. Using this approach, we were able to control cell-free RNA and protein expression with blue light. Due to the orthogonality of this blue light-activated DNA to UV irradiation, we combined it with our previously generated UV light-activated DNA to generate a dual-wavelength controlled cell-free AND-gate. This blue light-activated DNA was also used to control synthetic cells; lipid-bounded compartments that mimic cellular function. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.

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

“…We have used this light-activated DNA (LA-DNA) to spatiotemporally activate protein synthesis in synthetic tissues, 25,26 activate communication between synthetic cells and living cells, 27 and, by employing orthogonal photocages, to engineer a dual wavelength cell-free AND-gate. 28 The main disadvantage of our current approach is that it is an irreversible ON switch. Azobenzene-modified DNA promoters have been used as photoreversible switches; however, they are leaky in the 'off-state' and do not degrade the RNA already generated.…”

“…We recently reported a blue light-activatable photocaging group to control CFPS, which acts orthogonally to UV light. 28 By combining a blue light-activatable (bLA) mV DNA template with the uvLA-V3 ASO, we envisaged a system where transcription could be selectively turned ON with blue light and translation turned OFF with UV light (Fig. 3a).…”