Orthogonal Light-Activated DNA for Patterned Biocomputing within Synthetic Cells

[Image: see text] 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, nondamaging stimuli such as visible light. Here, we have synthesized 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 ultraviolet (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.