Blue light-activatable DNA for remote controlled logic gates in synthetic cells

Abstract

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, non-damaging stimuli such as visible light. Here, we have synthesised blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated UV light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. 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 recently reported a blue-light-activatable (bLA) photocaging group to control CFPS, which acts orthogonally to UV light . 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 (Figure a).…”

“…We recently reported a blue-light-activatable (bLA) 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 (Figure 3a). The feasibility of this approach was initially indicated by the orthogonality of the UV−vis spectra of the previously prepared bLA-T7 promoter 28 compared to the spectrum of the prepared uvLA-V3 ASO (Supporting Information).…”

“…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 (Figure 3a). The feasibility of this approach was initially indicated by the orthogonality of the UV−vis spectra of the previously prepared bLA-T7 promoter 28 compared to the spectrum of the prepared uvLA-V3 ASO (Supporting Information). We initially tested this in an IVT reaction using the bLA-mV DNA template in the presence of RNase H and uvLA-V3 (Supporting Information, Figure S4).…”

“…The advantage of this approach is that it is made from commercially available modified nucleic acids and photocleavable biotin, and has a tight “off-state,” with negligible expression without illumination. We have used this light-activated DNA (LA-DNA) to spatiotemporally activate protein synthesis in synthetic tissues, , to activate communication between synthetic cells and living cells, and, by employing orthogonal photocages, to engineer a dual-wavelength cell-free AND-gate …”

“…We have used this lightactivated DNA (LA-DNA) to spatiotemporally activate protein synthesis in synthetic tissues, 25,26 to 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.…”