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Photoswitching FRET to monitor protein–protein interactions
摘要： FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule's photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched 'off' rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched 'on' again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein–protein interactions and fluorescent protein-based biosensors.
关键词： FRET,imaging,microscopy,photoswitchable,fluorescence
更新于2025-09-23 15:23:52
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Structure-Activity Relationships of Photoswitchable Diarylethene-Based β-Hairpin Peptides as Membranolytic Antimicrobial and Anticancer Agents
摘要： Five series (28 structures) of photoswitchable β-hairpin peptides were synthesized, based on the cyclic scaffold of the natural antibiotic gramicidin S. Cell-type selectivity was compared for all activated (diarylethene ring-open) and deactivated (closed) forms in terms of antibacterial activity (MIC against E. coli and B. subtilis), anticancer activity (IC50 against HeLa cell line), and hemolytic cytotoxicity (HC50 against human erythrocytes). Correlations between the conformational plasticity of the peptides, their hydrophobicity and their bioactivity were also analyzed. Considerable improvements in selectivity were achieved compared to the reference compound. We found a dissociation of the anticancer activity from hemolysis. Phototherapeutic indices (PTI) – HC50(closed)/MIC(open) and HC50(closed)/IC50(open) – were introduced for the peptides as safety criteria. The highest PTI for HeLa-selective toxicity were observed among analogues containing hydroxyleucine on the hydrophobic face. For one compound, high PTIs were demonstrated across a range of different cancer cell lines, including a doxorubicin-resistant one.
关键词： photoswitchable diarylethene-based β-hairpin peptides,antimicrobial,phototherapeutic indices,gramicidin S,anticancer,hemolytic cytotoxicity
更新于2025-09-23 15:21:01
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A Polymerizable Photoswitchable Fluorophore for Super-Resolution Imaging of Polymer Self-Assembly and Dynamics
摘要： Single-molecule super-resolution microscopy has become a standard imaging tool in the life sciences for visualizing nanostructures in situ, but the application of this technique in polymer science is much less explored. A key bottleneck is the lack of fluorophores and simple covalent attachment strategies onto polymer chains. Here, we report a functional diarylethene-based photoswitchable fluorophore that can be directly incorporated into polymer backbones through copolymerization, which significantly streamlines the labeling strategy, with no further postcoupling reactions or purifications needed. The attachment of fluorophores onto selectively labeled polymers enables super-resolution imaging of a series of model polymer blend systems with different nanostructures and chemical compositions. As each individual fluorophore is able to switch several times on average between its bright and dark state, multiple time-lapse images can be acquired to observe the dynamic nanostructural evolution of polymer blends upon solvent vapor annealing. With this demonstration of a universal, simplified labeling strategy and the ability to image polymer assembly under native conditions, this reported fluorophore may promote the widespread use of super-resolution microscopy in the polymer community.
关键词： diarylethene,polymer dynamics,super-resolution microscopy,photoswitchable fluorophore,polymer self-assembly
更新于2025-09-23 15:21:01
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[Advances in Experimental Medicine and Biology] || Photoresponsive Hydrogels with Photoswitchable Stiffness: Emerging Platforms to Study Temporal Aspects of Mesenchymal Stem Cell Responses to Extracellular Stiffness Regulation
摘要： An extensive number of cell-matrix interaction studies have identi?ed matrix stiffness as a potent regulator of cellular properties and behaviours. Perhaps most notably, matrix stiffness has been demonstrated to regulate mesenchymal stem cell (MSC) phenotype and lineage commitment. Given the therapeutic potential for MSCs in regenerative medicine, signi?cant efforts have been made to understand the molecular mechanisms involved in stiffness regulation. These efforts have predominantly focused on using stiffness-de?ned polyacrylamide (PA) hydrogels to culture cells in 2D and have enabled elucidation of a number of mechano-sensitive signalling pathways. However, despite proving to be a valuable tool, these stiffness-de?ned hydrogels do not re?ect the dynamic nature of living tissues, which are subject to continuous remodelling during processes such as development, ageing, disease and regeneration. Therefore, in order to study temporal aspects of stiffness regulation, researchers have developed and exploited novel hydrogel substrates with in situ tuneable stiffness. In particular, photoresponsive hydrogels with photoswitchable stiffness are emerging as exciting platforms to study MSC responses to extracellular stiffness regulation. This chapter provides an introduction to the use of PA hydrogel substrates, the molecular mechanisms of mechanotransduction currently under investigation and the development of these emerging photoresponsive hydrogel platforms.
关键词： Photoswitchable stiffness,Biophysical regulation,Photoresponsive,Cell-matrix interaction,Regenerative medicine,Polyacrylamide,Mechanotransduction,Stiffness regulation,Mesenchymal stem cells,Cell adhesion,Temporal,Hydrogel substrates
更新于2025-09-23 15:21:01
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Blue Light Switchable Cell-Cell Interactions Provide Reversible and Spatiotemporal Control Towards Bottom-Up Tissue Engineering
摘要： Controlling cell–cell interactions is central for understanding key cellular processes and bottom-up tissue assembly from single cells. The challenge is to control cell–cell interactions dynamically and reversibly with high spatiotemporal precision noninvasively and sustainably. In this study, cell–cell interactions are controlled with visible light using an optogenetic approach by expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of cells. CRY2 and CIBN expressing cells form specific heterophilic interactions under blue light providing precise control in space and time. Further, these interactions are reversible in the dark and can be repeatedly and dynamically switched on and off. Unlike previous approaches, these genetically encoded proteins allow for long-term expression of the interaction domains and respond to nontoxic low intensity blue light. In addition, these interactions are suitable to assemble cells into 3D multicellular architectures. Overall, this approach captures the dynamic and reversible nature of cell–cell interactions and controls them noninvasively and sustainably both in space and time. This provides a new way of studying cell–cell interactions and assembling cellular building blocks into tissues with unmatched flexibility.
关键词： bottom-up tissue engineering,spatiotemporal control,photoswitchable proteins,cell adhesion,cell–cell interactions
更新于2025-09-19 17:15:36
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Light-controlled switchable complexation by a non-photoresponsive hydrogen-bonded amide macrocycle
摘要： A new photoswitchable host-guest system based on a non-photoresponsive hydrogen-bonded (H-bonded) amide macrocycle and pyridinium salts was developed. The host is found to be capable of forming stable complexes with the analyzed protonated pyridine derivatives. Particularly, the guest capture and release process could be reversibly controlled to a greater extent via proton transfer by light stimulus with merocyanine 1-MEH. The present study offers a rare example of photo-responsive 2D shape-persistent H-bonded macrocyclic host-guest system, which may find applications in constructing interlocked molecular architecture.
关键词： proton transfer,pyridinium salts,photoswitchable,light stimulus,host-guest system,hydrogen-bonded amide macrocycle
更新于2025-09-19 17:13:59
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Azo-PROTAC: novel light-controlled small-molecule tool for protein knockdown
摘要： Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable Azo-PROTACs by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photo-switch ability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off property.
关键词： protein modifications,photoswitchable PROTAC,protein knockdown,ubiquitin-proteasome system.
更新于2025-09-19 17:13:59
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Photoswitchable single-walled carbon nanotubes for super-resolution microscopy in the near-infrared
摘要： The design of single-molecule photoswitchable emitters was the first milestone toward the advent of single-molecule localization microscopy, setting a new paradigm in the field of optical imaging. Several photoswitchable emitters have been developed, but they all fluoresce in the visible or far-red ranges, missing the desirable near-infrared window where biological tissues are most transparent. Moreover, photocontrol of individual emitters in the near-infrared would be highly desirable for elementary optical molecular switches or information storage elements since most communication data transfer protocols are established in this spectral range. Here, we introduce a type of hybrid nanomaterials consisting of single-wall carbon nanotubes covalently functionalized with photoswitching molecules that are used to control the intrinsic luminescence of the single nanotubes in the near-infrared (beyond 1 mm). Through the control of photoswitching, we demonstrate super-localization imaging of nanotubes unresolved by diffraction-limited microscopy.
关键词： single-molecule localization microscopy,near-infrared,super-resolution microscopy,carbon nanotubes,photoswitchable emitters
更新于2025-09-12 10:27:22
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Reversibly Photoswitchable Dual-Color Fluorescence and Controlled Release Properties of Polymeric Nanoparticles
摘要： Here, we report a novel polymeric nanoparticle prepared by the self-assembly of amphiphilic copolymers containing a ?uorescent naphthalimide (NAPH) and a photochromic spiropyran (SP), which possesses reversibly photoswitchable dual-color ?uorescence and controlled release properties. The amphiphilic copolymers were synthesized by incorporating NAPH and SP into methyl ether poly(ethylene glycol)-poly(β-amino esters) (MPEG-PAE) via quaternization. The nanoparticles would change between yellow and purple reversibly upon UV and visible light irradiation because of the photoisomerization between SP and merocyanine (MC). The corresponding ?uorescence would be switched between green and orange-red reversibly upon blue light excitation through the ?uorescence resonance energy transfer from the excited NAPH to the photoisomerized MC. Meanwhile, the prepared spherical nanoparticles could be swollen under UV irradiation as the hydrophobic SP isomerized to hydrophilic MC; the nanoparticles could also be swollen under acidic conditions because of the protonation of the amino groups of PAE. Upon UV light irradiation and acidic stimulation, the cargoes, hydrophobic Coumarin 102, encapsulated in the nanoparticles would be released. The prepared nanoparticles, which exhibit not only excellent reversible dual-color ?uorescence properties but also prominent controlled release performance, will open up new possibilities for the combined application of ?uorescence imaging and controlled release.
关键词： naphthalimide,Coumarin 102,spiropyran,polymeric nanoparticles,photoswitchable,amphiphilic copolymers,acidic conditions,quaternization,self-assembly,FRET,UV/vis light irradiation,dual-color ?uorescence,MPEG-PAE,controlled release
更新于2025-09-11 14:15:04
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A Dynamic Tetracationic Macrocycle Exhibiting Photoswitchable Molecular Encapsulation
摘要： Designing macrocycles with appropriate molecular recognition features that allow for the integration of suitable external stimuli to control host?guest processes is a challenging endeavor which enables molecular containers to solubilize, stabilize and separate chemical entities in an externally controlled manner. Herein, we introduce photo- and thermal-responsive elements into a semi-rigid tetracationic cyclophane, OPVEx2Box4+, that is composed of oligo(p-phenylenevinylene) pyridinium units and the biphenylene-bridged 4,4-bipyridinium extended viologens, and adopts a rectangle-like geometry. It transpires that the reversibility of the photoactive oligo(p-phenylenevinylene) pyridinium unit is dramatically improved when incorporated in a macrocyclic scaffold, and the configurations of the cyclophane can go back and forth between (EE)- and (EZ)-isomers upon alternating blue light irradiation and heat. When the macrocycle is found in its (EE)-configuration, it is capable of binding various π-electron-rich guests?e.g., anthracene and perylene?as well as π-electron-deficient guests?e.g., 9,10-anthraquinone and 5,12-tetracenequinone?through charge transfer and van der Waals interactions. When irradiated with blue light, the (EE)-isomer of the cyclophane can be transformed successfully to the (EZ)-isomer, resulting in the switching off of the binding affinity for guest molecules, which are bound once again upon heating. The use of light and heat as external stimuli to control host?guest interaction involving a multi-responsive host and various guests provides us with a new opportunity to design and construct more advanced molecular switches and machines.
关键词： photoswitchable,tetracationic cyclophane,host-guest interaction,molecular encapsulation,macrocycle
更新于2025-09-09 09:28:46