Orthogonal Light-Activated DNA for Patterned Biocomputing within 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, 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.

Figure 1

Caged, blue light-activatable DNA for control of cell-free expression (CFE). An amine-modified T7 promoter upstream of a gene of interest was modified with a biotinylated coumarin derivative. The binding of monovalent streptavidin (mSA) provided the necessary steric bulk to repress transcription from the DNA template. Upon illumination with a 455 nm light-emitting diode (LED), this steric bulk was cleaved off and transcription activated. This blue light-activatable DNA allowed for the control of RNA and protein synthesis in a CFE system and enabled the construction of an AND gate with 2 wavelengths as inputs, which was spatiotemporally controlled in synthetic cells.

Figure 2

Synthesis of the biotinylated coumarin-based photocage and its reaction with DNA. (a) Synthetic scheme describing the synthesis of the biotinylated coumarin active carbonates. (b) Optimal conditions for the reaction of the 7-amino-C6-dT-modified ssDNA with the coumarin photocage. TBDMS = tert-Butyldimethylsilyl-, DMAP = 4-dimethylaminopyridine, DCM = dichloromethane, NBS = N-bromosuccinimide, DMF = dimethylformamide, TFA = trifluoroacetic acid, Boc = tert-butyloxycarbonyl, EDC = N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, NHS = N-hydroxysuccinimide, TBAF = tetrabutylammonium fluoride, THF = tetrahydrofuran, DMSO = dimethylsulfoxide.

Figure 3

bLA- and uvLA-mVenus DNA under different illumination conditions. (a) Preparation of caged, blue light-activatable DNA and uncaging with blue light. bLA-T7 is used as a primer in PCR to generate a modified DNA template. The addition of mSA cages the DNA, preventing transcription. Illumination with blue light then cleaves off the molecule + mSA, allowing for expression from the DNA template. (b) Agarose gel electrophoresis of NH₂-, bLA-, and uvLA-mV DNA under different illumination conditions. Modification and binding of mSA gave larger gel retention for both bLA- and uvLA-mV DNA. Illumination of bLA-mV DNA with blue light showed uncaging toward the NH₂-mV DNA template but was not affected by UV irradiation. Illumination of uvLA–DNA with UV light showed uncaging, but illumination with blue light did not. (c) Cell-free expression of NH₂-, bLA-, and uvLA-mV DNA with blue and UV irradiation. Both bLA– and uvLA–DNA had a tight off-state in the absence of irradiation. Illumination of bLA–DNA with a blue LED gave 70% recovery of expression, whereas illumination with UV yielded only a small increase in expression. For uvLA–DNA, we saw the reverse, where illumination with UV light gave a 44% recovery of expression, whereas illumination with blue light yielded no increase in the expression of mVenus. n = 4 for bLA–DNA (with bLA–DNA + UV being n = 3) and n = 3 for uvLA–DNA samples. n.s. - nonsignificant (p-value > 0.05). *p-value < 0.05. **p-value < 0.005.

Figure 4

DNA-based AND-gate controlled by two wavelengths of light. (a) Schematic representation of the LA–DNA-based cell-free AND-gate. The two inactive β-galactosidase segments, α and ω, were encoded in a UV or blue LA–DNA part. By placing both light-activatable DNAs inside a CFE system, we generated an AND-gate controlled by light, following the AND-truth table in (b). The output was fluorescence from the enzymatic hydrolysis product umbelliferone-3-carboxylic acid (UCA). (b) Light-controlled AND Truth Table. In the absence of light, no output should be detected. With either UV or blue light only, no output should be detected either, but with both wavelengths of light applied, there is output. (c) Activity of the light-activated AND gate. Split β-galactosidase activity was only reconstituted when both UV and blue light were applied (in either order). Protein Image was reproduced from the RCSB PDB (RCSB.org), PDB ID: 1DP0,^, under a CC0 1.0 Universal (CC0 1.0) Public domain dedication licence. n = 3. n.s.—nonsignificant (p-value > 0.05).

Figure 5

1- and 2-wavelength patterning of immobilized emulsion droplet synthetic cells. (a) Through application of photomasks over organogel-immobilized emulsion droplet synthetic cells, spatial and temporal control over gene expression was achieved. By applying blue light to bLA-mNeonGreen DNA-containing droplets, photopatterns of dots (b, c) and a line (d, e) was achieved. Applying UV and blue light through orthogonal line photomasks onto droplets containing the uvLA-α and bLA-ω DNA parts of split β-gal (f), two-wavelength patterning could be achieved, with fluorescence only observed in the zone where both wavelengths overlap (g, h).