Biology subjects

Deo, C.

Publications and source records attributed to Deo, C..

5 recordsLinked to original sources

bioRxiv2025

Photoclickable HaloTag Ligands for Spatiotemporal Multiplexed Protein Labeling on Living Cells

Precise spatiotemporal control over fluorescence labeling is a powerful approach for selective marking and tracking of proteins of interest within living systems. Here, we report a photoclickable labeling platform based on the 2,3-diaryl-indanone epoxide (DIO) photoswitch scaffold and the self-labeling protein HaloTag. Upon illumination, the protein-bound DIO undergoes reversible photoisomerization to form a metastable oxidopyrylium ylide (PY) that reacts with ring-strained dipolarophiles via [5+2] cycloaddition, enabling covalent spatiotemporal labeling. We synthesize and characterize a library of DIO-HaloTag and DIO-SNAP- tag ligands, systematically examining the effects of linker architecture and scaffold substitution on the photoswitching and photoclick reactivity in vitro and on living cells. We identify a naphthyl-substituted DIO ligand exhibiting superior photoswitching and photoclick efficiency, allowing robust and selective labeling of HaloTag on the surface of living cells using visible light activation. Using this system, we achieve two- and three-color labeling of defined cell surface regions with excellent spatial and temporal precision, additionally allowing combinatorial labeling. Together, this work establishes a versatile framework for multiplexed, light- directed protein labeling compatible with living systems, with promising future applications in long-term tracking and cellular barcoding.

biochemistry↗

bioRxiv2025

Bridging Light and Sound: a Spironaphtopyran-Rhodamine Dyad with High-Contrast Photoswitching Between Fluorescence and Photoacoustic Signal.

Fluorescence and photoacoustic imaging are complementary modalities that provide distinct advantages for biological imaging: fluorescence microscopy offers high sensitivity and resolution, while photoacoustic imaging enables deeper penetration in complex tissue. Leveraging the strengths of both modalities through optically switchable contrast agents can offer enhanced imaging contrast and facilitate dual-modality imaging. Here, we report a photoswitchable probe capable of toggling between high fluorescence and high photoacoustic signal upon illumination, exploiting Forster Resonance Energy Transfer (FRET). We engineer novel spironaphtopyran photoswitches which undergo reversible photoisomerization between absorbing and non-absorbing states. Their photoswitching properties were systematically characterized, establishing structure-properties relationships, and providing the first photoacoustic investigation into this class of compounds. The best-performing switch was incorporated into a FRET dyad with a rhodamine fluorophore, which exhibits robust, reversible switching between fluorescent and photoacoustic-dominant states with excellent contrast in vitro, establishing a foundation for multimodal imaging probes with promising potential for dynamic correlative imaging.

biochemistry↗

bioRxiv2024

A photoswitchable HaloTag for spatiotemporal control of fluorescence in living cells

Photosensitive fluorophores, which emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features, and facilitating super-resolution microscopy techniques. While irreversibly photoactivatable fluorophores are well established, reversible reporters which can be re-activated multiple times remain scarce, and only few have been applied in living cells using generalizable protein labelling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labelling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show high performance in vitro and in living cells, with large, reversible, far-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.

biochemistry↗

bioRxiv2024

Chemigenetic far-red labels and Ca2+ indicators optimized for photoacoustic imaging.

Photoacoustic imaging is an emerging modality with significant promise for biomedical applications such as neuroimaging, owing to its capability to capture large fields of view, deep inside complex scattering tissue. However, the widespread adoption of this technique has been hindered by a lack of suitable molecular reporters for this modality. In this work, we introduce chemigenetic labels and calcium sensors specifically tailored for photoacoustic imaging, using a combination of synthetic dyes and HaloTag-based self-labelling proteins. We rationally design and engineer far-red "acoustogenic" dyes, showing high photoacoustic turn-ons upon binding to HaloTag, and develop a suite of tunable calcium indicators based on these scaffolds. These first-generation photoacoustic reporters show excellent performance in tissue-mimicking phantoms, with the best variants outperforming existing sensors in terms of signal intensity, sensitivity and photostability. We demonstrate the application of these ligands for labelling HaloTag-expressing neurons in mouse brain tissue, producing strong, specifically targeted photoacoustic signal, and provide a first example of in vivo labelling with these chemigenetic photoacoustic probes. Together, this work establishes a new approach for the design of photoacoustic reporters, paving the way towards deep tissue functional imaging.

biochemistry↗

bioRxiv2020

Bright and tunable far-red chemigenetic indicators

Functional imaging using fluorescent indicators has revolutionized biology but additional sensor scaffolds are needed to access properties such as bright, far-red emission. We introduce a new platform for chemigenetic fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. This approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in neurons.

molecular biology↗