Curated Optogenetic Publication Database

Abstract: Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

Abstract: Microbial biosynthesis, as an alternative method for producing quantum dots (QDs), has gained attention because it can be conducted under mild and environmentally friendly conditions, distinguishing it from conventional chemical and physical synthesis approaches. However, there is currently no method to selectively control this biosynthesis process in a subset of microbes within a population using external stimuli. In this study, we have attained precise and selective control over the microbial biosynthesis of QDs through the utilization of an optogenetically engineered Escherichia coli (E. coli). The recombinant E. coli is designed to express smCSE enzyme, under the regulation of eLightOn system, which can be activated by blue light. The smCSE enzymes use L-cysteine and Cd2+ as substrates to form CdS QDs. This system enables light-inducible bacterial biosynthesis of QDs in precise patterns within a hydrogel for information encryption. As the biosynthesis progresses, the optical characteristics of the QDs change, allowing living materials containing the recombinant E. coli to display time-dependent patterns that self-destruct after reading. Compared to static encryption using fluorescent QD inks, dynamic information encryption based on living materials offers enhanced security.

Abstract: We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

Abstract: The investigation and manipulation of cellular processes with subcellular resolution requires non-invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light-controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on-switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.

Abstract: Photon upconversion (UC) from near-infrared (NIR) light to visible light has enabled optogenetic manipulations in deep tissues. However, materials for NIR optogenetics have been limited to inorganic UC nanoparticles. Extension to organic triplet-triplet annihilation (TTA)-based UC systems would innovate NIR optogenetics toward the use of biocompatible materials placed at a desired position. Here, we report the first example of NIR light-triggered optogenetics by using TTA-UC hydrogels. To achieve triplet sensitization even in the highly viscous hydrogel matrices, a NIR-absorbing complex is covalently linked with energy-pooling acceptor chromophores, which significantly elongates the donor triplet lifetime. The donor and acceptor are solubilized in hydrogels formed from biocompatible Pluronic F127 micelles, and we find that the additional heat treatment endows remarkable oxygen-tolerant property to the excited triplets in the hydrogel. Combined with photoactivatable Cre recombinase (PA-Cre) technology, NIR light stimulation successfully performs genome engineering such as hippocampal dendritic spine formation involved in learning and long-term memory.

Abstract: We report direct visualization of the dynamic behavior of transcription factor GAL4 with photo-switching function (GAL4-VVD) in the DNA origami structure. Using high-speed atomic force microscopy (HS-AFM), we observed photo-induced complex formation of GAL4-VVD and substrate DNAs. Dynamic behaviors of GAL4-VVD such as binding, sliding, stalling, and dissociation with two substrate DNA strands, containing specific GAL4 binding sites, were observed. We also observed inter-strand hopping on two double-stranded (ds) DNAs. On a long substrate DNA strand that contained five binding sites, a series of GAL4-VVD/DNA interactions including binding, sliding, stalling, and dissociation could be identified while interacting with the surface. We also found the clear difference in the movement of GAL4-VVD between sliding and stalling in the AFM images. Detailed analysis revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping for rapidly searching specific binding sites, and then stalled to the specific sites for the stable complex formation. The results suggest the existence of the different conformational mode of the protein for sliding and stalling. This single-molecule imaging system at the nanoscale resolution provides the insight of the searching mechanism of the DNA binding proteins.

Abstract: Once materials come in contact with a biological fluid containing proteins, proteins are generally - so desired or not - attracted by a material's surface and adsorb onto it. The aim of this review is to give an overview of the most commonly used characterization methods employed to obtain a better understanding of the adsorption processes on either planar or curved surfaces. We continue to illustrate the benefit of combining different methods to different surface geometries of the material. The thus obtained insights ideally pave the way for engineering functional materials interacting in a predetermined manner with proteins.

Abstract: Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.

Abstract: Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.

Abstract: An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Abstract: Precise spatiotemporal control of physiological processes by optogenetic devices inspired by synthetic biology may provide novel treatment opportunities for gene- and cell-based therapies. An erectile optogenetic stimulator (EROS), a synthetic designer guanylate cyclase producing a blue-light-inducible surge of the second messenger cyclic guanosine monophosphate (cGMP) in mammalian cells, enabled blue-light-dependent penile erection associated with occasional ejaculation after illumination of EROS-transfected corpus cavernosum in male rats. Photostimulated short-circuiting of complex psychological, neural, vascular, and endocrine factors to stimulate penile erection in the absence of sexual arousal may foster novel advances in the treatment of erectile dysfunction.

Abstract: Genetically encoded, light-activatable proteins provide the means to probe biochemical pathways at specific subcellular locations with exquisite temporal control. However, engineering these systems in order to provide a dramatic jump in localized activity, while retaining a low dark-state background remains a significant challenge. When placed within the framework of a genetically encodable, light-activatable heterodimerizer system, the actin-remodelling protein cofilin induces dramatic changes in the F-actin network and consequent cell motility upon illumination. We demonstrate that the use of a partially impaired mutant of cofilin is critical for maintaining low background activity in the dark. We also show that light-directed recruitment of the reduced activity cofilin mutants to the cytoskeleton is sufficient to induce F-actin remodeling, formation of filopodia, and directed cell motility.

Abstract: The regulation of gene expression is crucial in diverse areas of biological science, engineering, and medicine. A genetically encoded system based on the RNA binding domain of the Pumilio and FBF (PUF) proteins was developed for the bidirectional regulation (i.e., either upregulation or downregulation) of the translation of a target mRNA. PUF domains serve as designable scaffolds for the recognition of specific RNA elements and the specificity can be easily altered to target any 8-nucleotide RNA sequence. The expression of a reporter could be varied by over 17-fold when using PUF-based activators and repressors. The specificity of the method was established by using wild-type and mutant PUF domains. Furthermore, this method could be used to activate the translation of target mRNA downstream of PUF binding sites in a light-dependent manner. Such specific bidirectional control of mRNA translation could be particularly useful in the fields of synthetic biology, developmental biology, and metabolic engineering.