Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

AbstractBiocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

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AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

Polymer Chemistry ◽

10.1039/d0py01085e ◽

2021 ◽

Author(s):

Nilesh Umakant Deshpande ◽

Mishika Virmani ◽

Manickam Jayakannan

Keyword(s):

Energy Transfer ◽

Confocal Microscopy ◽

Real Time ◽

Fluorescence Resonance Energy Transfer ◽

Imaging Techniques ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Intracellular Enzyme ◽

Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412.v1 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

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3D super-resolved imaging in live cells using sub-diffractive plasmonic localization of hybrid nanopillar arrays

Nanophotonics ◽

10.1515/nanoph-2020-0105 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2847-2859

Author(s):

Soojung Kim ◽

Hyerin Song ◽

Heesang Ahn ◽

Seung Won Jun ◽

Seungchul Kim ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Imaging Techniques ◽

Optical Loss ◽

Super Resolution ◽

Living Cells ◽

Live Cells ◽

Complex Biological System ◽

Observation Volume ◽

Resolution Imaging ◽

Nanopillar Arrays

AbstractAnalysing dynamics of a single biomolecule using high-resolution imaging techniques has been had significant attentions to understand complex biological system. Among the many approaches, vertical nanopillar arrays in contact with the inside of cells have been reported as a one of useful imaging applications since an observation volume can be confined down to few-tens nanometre theoretically. However, the nanopillars experimentally are not able to obtain super-resolution imaging because their evanescent waves generate a high optical loss and a low signal-to-noise ratio. Also, conventional nanopillars have a limitation to yield 3D information because they do not concern field localization in z-axis. Here, we developed novel hybrid nanopillar arrays (HNPs) that consist of SiO2 nanopillars terminated with gold nanodisks, allowing extreme light localization. The electromagnetic field profiles of HNPs are obtained through simulations and imaging resolution of cell membrane and biomolecules in living cells are tested using one-photon and 3D multiphoton fluorescence microscopy, respectively. Consequently, HNPs present approximately 25 times enhanced intensity compared to controls and obtained an axial and lateral resolution of 110 and 210 nm of the intensities of fluorophores conjugated with biomolecules transported in living cells. These structures can be a great platform to analyse complex intracellular environment.

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Probing interdomain linkers and protein supertertiary structure in vitro and in live cells with fluorescent protein resonance energy transfer

Journal of Molecular Biology ◽

10.1016/j.jmb.2020.166793 ◽

2021 ◽

pp. 166793

Author(s):

Sujit Basak ◽

Nabanita Sakia ◽

Laura Dougherty ◽

Zhuojun Guo ◽

Fang Wu ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells

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Fluorescent Protein-Based Autophagy Biosensors

Materials ◽

10.3390/ma14113019 ◽

2021 ◽

Vol 14 (11) ◽

pp. 3019

Author(s):

Heejung Kim ◽

Jihye Seong

Keyword(s):

Energy Transfer ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Treatment Strategies ◽

Resonance Energy ◽

Live Cells ◽

Spectral Profile ◽

Spatiotemporal Resolution ◽

Future Directions ◽

Complex Process

Autophagy is an essential cellular process of self-degradation for dysfunctional or unnecessary cytosolic constituents and organelles. Dysregulation of autophagy is thus involved in various diseases such as neurodegenerative diseases. To investigate the complex process of autophagy, various biochemical, chemical assays, and imaging methods have been developed. Here we introduce various methods to study autophagy, in particular focusing on the review of designs, principles, and limitations of the fluorescent protein (FP)-based autophagy biosensors. Different physicochemical properties of FPs, such as pH-sensitivity, stability, brightness, spectral profile, and fluorescence resonance energy transfer (FRET), are considered to design autophagy biosensors. These FP-based biosensors allow for sensitive detection and real-time monitoring of autophagy progression in live cells with high spatiotemporal resolution. We also discuss future directions utilizing an optobiochemical strategy to investigate the in-depth mechanisms of autophagy. These cutting-edge technologies will further help us to develop the treatment strategies of autophagy-related diseases.

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Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity

Molecules ◽

10.3390/molecules26113355 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3355

Author(s):

Matija Sambol ◽

Patricia Benčić ◽

Antonija Erben ◽

Marija Matković ◽

Branka Mihaljević ◽

...

Keyword(s):

Biological Activity ◽

Rational Design ◽

Human Cancer ◽

Photophysical Properties ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Quinone Methide ◽

Quantum Yields ◽

Photochemical Reactivity ◽

Human Cancer Cell Lines

Quinone methide precursors 1a–e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10−2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10−5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105–106 M−1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M−1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a–e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

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Super-resolution FRET measurements

Nanoscale ◽

10.1039/d1nr05769c ◽

2021 ◽

Author(s):

Fernando D Stefani ◽

Alan M. M. Szalai ◽

Cecilia Zaza

Keyword(s):

Energy Transfer ◽

Fluorescence Microscopy ◽

Biological Systems ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Forster Resonance Energy Transfer ◽

Förster Resonance Energy Transfer ◽

Dynamic Architecture ◽

Förster Resonance

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems,...

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