Thermoplasmonic Effect Enables Indirect ON-OFF Control over the Z-E Isomerization of Azobenzene-Based Photoswitch.

Proper formulation of systems containing plasmonic and photochromic units, such as gold nanoparticles and azobenzene derivatives, yields materials and interfaces with synergic functionalities. Moreover, gold nanoparticles are known to accelerate the Z-E isomerization of azobenzene molecules in the dark. However, very little is known about the light-driven, plasmon-assisted Z-E isomerization of azobenzene compounds. Additionally, most of the azobenzene-gold hybrids are prepared with nanoparticles of small, isotropic shapes and azobenzene ligands covalently linked to the surface of nanostructures. Herein, a formulation of an innovative system combining azobenzene derivative, gold nanorods, and cellulose nanofibers is proposed. The system's structural integrity relies on electrostatic interactions among components instead of covalent linkage. Cellulose, a robust scaffold, maintains the material's functionality in water and enables monitoring of the material's plasmonic-photochromic properties upon irradiation and at elevated temperatures without gold nanorods aggregation. Experimental evidence supported by statistical analysis suggests that the optical properties of plasmonic nanometal enable indirect control over the Z-E isomerization of the photochromic component with near-infrared irradiation by triggering the thermoplasmonic effect. The proposed hybrid material's dual plasmonic-photochromic functionality, versatility, and ease of processing render a convenient starting point for further advanced azobenzene-related research and 3D printing of macroscopic light-responsive structures.

Caged Dexamethasone to Photo-control the Development of Embryos through Activation of the Glucocorticoid Receptor.

Efficient tools for controlling molecular functions with exquisite spatiotemporal resolution are much in demand to investigate biological processes in living systems. Here we report an easily synthesized caged dexamethasone for photo-activating cytoplasmic proteins fused to the glucocorticoid receptor. In the dark, it is stable in vitro as well as in vivo in both zebrafish (Danio rerio) and Xenopus sp, two significant models of vertebrates. In contrast, it liberates dexamethasone upon UV illumination, which has been harnessed to interfere with developmental steps in embryos of these animals. Interestingly, this new system is biologically orthogonal to the one for photo-activating proteins fused to the estrogen ERT receptor, which brings great prospect for activating two distinct proteins down to the single cell level.

Neural and behavioral control in Caenorhabditis elegans by a yellow-light-activatable caged compound.

Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch.

The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site-specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.

Photo Fenton RAFT Polymerization of (Meth)Acrylates in DMSO Sensitized by Methylene Blue.

A novel open-to-air photo RAFT polymerization of a series of acrylate and methacrylate monomers mediated by matching chain transfer agent irradiated by far-red light in DMSO is reported. Hydroxyl radical (•OH) generated from methylene blue (MB) sensitized decomposition of H2 O2 via photo-Fenton like-reaction is used for polymerization initiation. The "living/control" characteristic is evidenced by kinetic study, in which a pseudo first order curve and linearly increases of molecular weight with the increase of monomer conversion are observed. The living end-group fidelity is characterized by MALDI-TOF-MS and 1 H NMR results, and confirmed by successful chain extension. The temporary controllability is proved by light on/off switch experiment.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia.

We present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560-590 nm) and has a rapid response time, within a few minutes. The glutathione-consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia-responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+ ) influx into HEK293T cells expressing the hypoxia-responsive Ca2+ channel TRPA1.

Synthesis, Derivatization and Photochemical Control of an ortho-Functionalized Tetrafluorinated Azobenzene-Modified siRNA.

We report the synthesis of an ortho-functionalized tetrafluorinated azobenzene phosphoramidite for its site-specific incorporation into RNA. The tetrafluorinated azobenzene is embedded within the antisense strand of an siRNA duplex to form an ortho-functionalized tetrafluorinated azobenzene-containing siRNA (F-siRNAzo). The F-siRNAzo is inactivated via trans to cis conversion with green light (530 nm), and reactivated with blue light (470 nm) via cis to trans conversion in cell culture. The long half-life and stability of the tetrafluorinated azobenzene unit allows for reversible control of the F-siRNAzo in cell culture for up 72 hours.

Photocontrollable Probes for Mitochondrial Protein Profiling.

The mitochondrion is the core site of cell signaling, energy metabolism and biosynthesis. Here, taking advantage of activity-based probes, we synthesized two photocontrollable probes (YGH-1 and YGH-2), composed of a mitochondrial localization moiety "triphenylphosphonium", a photo-triggered group to achieve spatially and temporally controlled protein capture, and an alkyne group to enrich the labeled protein. Proteomic validation was further carried out to facilitate identification of the mitochondrial proteome in HeLa cells. The results show that half of the identified protein hits (∼300) labeled by YGH-1 and YGH-2 belong to mitochondria, and are mostly localized in the mitochondrial matrix and inner mitochondrial membrane. Our results provide a new tool for spatial and temporal analysis of subcellular proteomes.

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists.

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.

Encoding Supramolecular Chiral Self-Assembly with Photo-Controlled Circularly Polarized Luminescence by Overcrowded Alkene-Based Bis-PBI Modulators.

Developing photoresponsive circularly polarized luminescence (CPL) materials is an essential step for biosensing and biomedical applications. However, fabricating CPL assemblies rooted in the chirality amplification and transmission of the molecular building blocks, which simultaneously show photo-controllable CPL signals, remains challenging. Herein, a molecular building block containing an overcrowded-alkene core and bis-PBI (MPBI) was designed. Importantly, the enantiopure MPBI can self-assemble into well-organized nanofibers via π-π stacking interactions and enable the transmission of the intrinsic chirality, providing opposite CPL signals. The photoisomerization of MPBI induced a transformation from nanofibers to discrete nanospheres, accompanied by a gradually decreased CPL signal. The results demonstrated the development of photo-controllable CPL materials from the assembly of chiral MPBI, which provides an alternatively facile strategy to fabricate CPL-active materials and would offer opportunities for future biosensing and biomedical applications.

Systematic Interrogation of Cellular Signaling in Live Cells Using a Membrane-Anchored DNA Multitasking Processor.

Systematic interrogation of correlative signaling components in their native environment is of great interest for dissecting sophisticated cellular signaling. However, it remains a challenge because of the lack of versatile and effective approaches. Herein, we propose a cell membrane-anchored DNA multitasking processor acting as a "traffic light" for integrated analyses of cellular signal transduction. Enhanced and controllable inhibition of c-Met signaling was achieved by membrane-anchoring of DNA processors. Moreover, the multitasking capability of the DNA processor allowed the monitoring of correlative VEGF secretion induced by c-Met activity regulation directly. By exploiting versatile aptameric nucleic acids, this modular designed DNA multitasking processor dissected how cell surface receptors coordinated with related components in live cells systematically. Therefore, it provides a powerful chemical tool for both fundamental cell biology research and precision medicine applications.

Novel antibody-drug conjugate with UV-controlled cleavage mechanism for cytotoxin release.

Antibody-drug conjugates (ADCs) are being developed worldwide with the potential to revolutionize current cancer treatment strategies. However, off-target toxicity caused by the instability of linkers remains one of the main issues to be resolved. Developing a novel photocontrol-ADC with good stability and photocontrolled release seemed to be an attractive and practical solution. In this study, we designed, for the first time, a novel ultraviolet (UV) light-controlled ADC by carefully integrating the UV-cleavable o-nitro-benzyl structure into the linker. Our preliminary work indicated that the ADC exhibited good stability and photocontrollability while maintaining a targeting effect similar to that of the naked antibody. Upon irradiation with UV light, the ADC rapidly released free cytotoxins and exerted significant cytotoxicity toward drug-resistant tumor cells. Compared to those of the unirradiated cells, the EC50 values of ADCs increased by up to 50-fold. Furthermore, our research confirmed that the degradation products of unirradiated ADC, Cys-1a, were relatively less toxic, thus potentially reducing the off-target toxicity caused by nonspecific uptake of ADCs. The novel design strategy of UV light-controlled ADCs may provide new perspectives for future research on ADCs and promote the development of photocontrol systems.

Synthesis, Derivatization and Photochemical Control of ortho-Functionalized Tetrachlorinated Azobenzene-Modified siRNAs.

We report the chemical synthesis and derivatization of an ortho-functionalized tetrachlorinated azobenzene diol. A 4',4-dimethoxytrityl (DMT) phosphoramidite was synthesized for its site-specific incorporation within the sense strand of an siRNA duplex to form ortho-functionalized tetrachlorinated azobenzene-containing siRNAs (Cl-siRNAzos). Compared to a non-halogenated azobenzene, ortho-functionalized tetrachlorinated azobenzenes are capable of red-shifting the π→π* transition from the ultraviolet (UV) portion of the electromagnetic spectrum into the visible range. Within this visible range, the azobenzene molecule can be reliably converted from trans to cis with red light (660 nm), and converted back to trans with violet wavelength light (410 nm) and/or thermal relaxation. We also report the gene-silencing ability of these Cl-siRNAzos in cell culture as well as their reversible control with visible light for up to 24 hours.

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of ~100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex.

Photoswitchable Dissipative Two-Dimensional Colloidal Crystals.

Control over particle interactions and organization at fluid interfaces is of great importance both for fundamental studies and practical applications. Rendering these systems stimulus-responsive is thus a desired challenge both for investigating dynamic phenomena and realizing reconfigurable materials. Here, we describe the first reversible photocontrol of two-dimensional colloidal crystallization at the air/water interface, where millimeter-sized assemblies of microparticles can be actuated through the dynamic adsorption/desorption behavior of a photosensitive surfactant added to the suspension. This allows us to dynamically switch the particle organization between a highly crystalline (under light) and a disordered (in the dark) phase with a fast response time (crystallization in ≈10 s, disassembly in ≈1 min). These results evidence a new kind of dissipative system where the crystalline state can be maintained only upon energy supply.

Direct Observation and Analysis of the Dynamics of the Photoresponsive Transcription Factor GAL4.

Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4-VVD, and DNA using high-speed atomic force microscopy (HS-AFM) is reported. A series of different GAL4-VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter-strand jumping on two double-stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4-binding sites revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single-molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA-binding proteins.

Pulsed infrared releases Ca2+ from the endoplasmic reticulum of cultured spiral ganglion neurons.

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 µs, 200-250 Hz for 2-5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6-12°C (above a baseline of 24-30°C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca2+ concentration ([Ca2+]) of 0.20-1.4 µM, with a simultaneous reduction of [Ca2+] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca2+] continued in medium containing no added Ca2+ (±Ca2+ buffers) and low [Na+], indicating that the [Ca2+] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca2+] response was prolonged and eventually blocked by inhibition of ER Ca2+-ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP3)-mediated Ca2+ release (xestospongin C and 2-aminoethoxydiphenyl borate). The thermal sensitivity of the response suggested involvement of warmth-sensitive transient receptor potential (TRP) channels. The IR-induced [Ca2+] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK-2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with ER marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca2+] elevations is conferred by TRP channels on ER membranes, which facilitate Ca2+ efflux into the cytosol and thereby contribute to Ca2+-induced Ca2+-release via IP3 and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca2+-induced Ca2+-release. The neurons immunostained for warmth-sensitive channels, TRPV4 and TRPM2, which colocalize with endoplasmic reticulum. Pulsed infrared radiation provides a novel experimental tool for releasing intracellular Ca2+, studying Ca2+ regulatory mechanisms, and influencing neuronal excitability.

Stability and evaluation of siRNAs labeled at the sense strand with a 3'-azobenzene unit.

siRNAs bearing a 3'-azobenzene derivative on the sense strand were evaluated for their gene silencing ability in mammalian cell culture and nuclease stability in nuclease-rich media. Azobenzene can be isomerized between cis and trans isomers through the incubation of UV (cis isomer) and visible light (trans isomer). It was demonstrated that subtle differences in nuclease stability and activity were observed. These small changes can be used to photochemically fine-tune the activity of an siRNA for gene-silencing applications.

Enhancing Temporal Control and Enabling Chain-End Modification in Photoregulated Cationic Polymerizations by Using Iridium-Based Catalysts.

Gaining temporal control over chain growth is a key challenge in the enhancement of controlled living polymerizations. Though research on photocontrolled polymerizations is still in its infancy, it has already proven useful in the development of previously inaccessible materials. Photocontrol has now been extended to cationic polymerizations using 2,4,6-triarylpyrylium salts as photocatalysts. Despite the ability to stop polymerization for a short time, monomer conversion was observed over long dark periods. Improved catalyst systems based on Ir complexes give optimal temporal control over chain growth. The excellent stability of these complexes and the ability to tune the excited and ground state redox potentials to regulate the number of monomer additions per cation formed allows polymerization to be halted for more than 20 hours. The excellent stability of these iridium catalysts in the presence of more nucleophilic species enables chain-end functionalization of these polymers.

Photocontrol in Complex Polymeric Materials: Fact or Illusion?

Photoswitches: Exciting recent progress realized in the field of light-controlled polymeric materials is highlighted. It is discussed how the rational choice of azobenzene molecules and their incorporation into complex materials by making use of physical interactions can lead to genuine photocontrollable polymeric systems.