We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photo-physical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

Visualizing and manipulating behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labelling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self-labelling protein tags, focusing mainly on the application of HaloTag and SNAP-tag systems. We describe the latest developments in small molecule probes that enable fluorogenic (no wash) imaging and super-resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, the current technical advancement on the HaloTag and SNAP-tag systems are becoming powerful tools to enable the visualization and manipulating of biological processes, providing invaluable scientific insights that are otherwise difficult to obtain using traditional methodologies. As the multiplex self-labelling protein tag systems continue to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.

While organic donor‐acceptor (D‐A) molecules are widely employed in multiple areas, the application of more D‐A molecules could be limited because of an inherent polarity sensitivity that inhibits photochemical processes. Presented here is a facile chemical modification to attenuate solvent‐dependent mechanisms of excited‐state quenching through addition of a β‐carbonyl‐based polar substituent. The results reveal a mechanism wherein the β‐carbonyl substituent creates a structural buffer between the donor and the surrounding solvent. Through computational and experimental analyses, it is demonstrated that the β‐carbonyl simultaneously attenuates two distinct solvent‐dependent quenching mechanisms. Using the β‐carbonyl substituent, improvements in the photophysical properties of commonly used D‐A fluorophores and their enhanced performance in biological imaging are shown.

Aberrant protein aggregation leads to various human diseases, but little is known about the physical chemical properties of these aggregated proteins in cells. Herein, we developed a boron-dipyrromethene (BODIPY)-based HaloTag probe, whose conjugation to HaloTag-fused proteins allows us to study protein aggregates using both fluorescence intensity and lifetime. Modulation of BODIPY fluorophore reveals key structural features to attain the dual function. The optimized probe exhibits increased fluorescence intensity and elongated fluorescence lifetime in protein aggregates. Fluorescence lifetime imaging using this probe indicates that protein aggregates afford different viscosity in the forms of soluble oligomers and insoluble aggregates in live cells. The strategy presented in this work can be extended to enable a wide class of HaloTag probes that can be used to study a variety of physical properties of protein aggregates, thus helping unravel the pathogenic mechanism and develop therapeutic strategy.

We developed an efficient, versatile, and accessible super-resolution microscopy method to construct a nanoparticle assembly at a spatial resolution below the optical diffraction limit. The method utilizes DNA and a photoactivated DNA cross-linker. Super-resolution optical techniques have been used only as a means to make measurements below the light diffraction limit. Furthermore, no optical technique is currently available to construct nanoparticle assemblies with a precisely designed shape and internal structure at a resolution of a few tens of nanometers (nm). Here we demonstrate that we can fulfill this deficiency by utilizing spontaneous structural dynamics of DNA hairpins combined with single-molecule fluorescence resonance energy transfer (smFRET) microscopy and a photoactivated DNA cross-linker. The stochastic fluorescence blinking due to the spontaneous folding and unfolding motions of DNA hairpins enables us to precisely localize a folded hairpin and solidify it only when it is within a predesigned target area whose size is below the diffraction limit. As the method is based on an optical microscope and an easily clickable DNA cross-linking reagent, it will provide an efficient means to create large nanoparticle assemblies with a shape and internal structure at an optical super-resolution, opening a wide window of opportunities toward investigating their photophysical and optoelectronic properties and developing novel devices.

While organic donor‐acceptor (D‐A) molecules are widely employed in multiple areas, the application of more D‐A molecules could be limited because of an inherent polarity sensitivity that inhibits photochemical processes. Presented here is a facile chemical modification to attenuate solvent‐dependent mechanisms of excited‐state quenching through addition of a β‐carbonyl‐based polar substituent. The results reveal a mechanism wherein the β‐carbonyl substituent creates a structural buffer between the donor and the surrounding solvent. Through computational and experimental analyses, it is demonstrated that the β‐carbonyl simultaneously attenuates two distinct solvent‐dependent quenching mechanisms. Using the β‐carbonyl substituent, improvements in the photophysical properties of commonly used D‐A fluorophores and their enhanced performance in biological imaging are shown.

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