A Bright Surprise: Live‐Cell Labeling with Negatively Charged Fluorescent Probes based on Disulfonated Rhodamines and HaloTag

Kim, Dojin; Stoldt, Stefan; Weber, Michael; Jakobs, Stefan; Belov, Vladimir N.; Hell, Stefan W.

doi:10.1002/cmtd.202200076

Cited by 5 publications

(3 citation statements)

References 49 publications

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“…We first focused on the novel carborhodamine 59 (Scheme ), which is a fluorine-containing analog of ATTO 647N ( 105 ; Figure a). The “famous” ATTO 647N is perhaps the best dye for in vitro single-molecule imaging and has found widespread use as a label for advanced imaging experiments. ,, ATTO 647N conjugates have not been used in live-cell imaging experiments, however, due to structural constraints. A key design principle used in many ATTO dyes involves amidation of the ortho -carboxyl group on the pendant ring with 4-(methylamino)butanoic acid.…”

Section: Resultsmentioning

confidence: 99%

Optimized Red-Absorbing Dyes for Imaging and Sensing

Grimm,

Tkachuk,

Patel

et al. 2023

J. Am. Chem. Soc.

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Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone−zwitterion equilibrium constant (K L−Z ) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure−activity relationships that govern K L−Z . We discovered that the auxochrome substituent strongly affects the lactone− zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Forster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure−activity relationships that will guide the design of future probes.

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Section: Resultsmentioning

confidence: 99%

Optimized Red-Absorbing Dyes for Imaging and Sensing

Grimm,

Tkachuk,

Patel

et al. 2023

J. Am. Chem. Soc.

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“…[11,12] Of the various pairs of self-labeling protein tags and fluorescent dyes, the combination of the HaloTag protein and rhodamine dyes has been most widely utilized to date. [13][14][15][16][17][18][19] In particular, rhodamine dyes bearing a ortho-carboxy-substituted aryl group at the 9-position are in a dynamic equilibrium between a fluorescent zwitterionic form and a non-fluorescent neutral spirolactone form, the latter being able to readily permeate the lipid bilayer (Figure 1a). [20][21][22][23] Exploiting this equilibrium, Johnsson and co-workers have developed a series of HaloTag probes based on silicon-rhodamine (SiR) (Figure 1b, SiR-Halo).…”

Section: Introductionmentioning

confidence: 99%

Stereochemistry‐Dependent Labeling of Organelles with a Near‐Infrared‐Emissive Phosphorus‐Bridged Rhodamine Dye in Live‐Cell Imaging

Wu,

Taki,

Tanaka

et al. 2024

Angew Chem Int Ed

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The development of near‐infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha‐rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2‐carboxy‐3‐benzothiophenyl group at the 9‐position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X‐ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non‐specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag‐fused proteins, enabling the long‐term imaging of cell division and the 5‐color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR‐emissive properties, multi‐color time‐lapse super‐resolution 3D imaging, namely super‐resolution 5D‐imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.

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“…Charged bioactive molecules are currently used to selectively address extracellular targets: to inhibit extracellularly exposed receptors, photouncage signaling lipids locally at the plasma membrane, or bioorthogonally label cell-surface proteins – tags. This field is also still in early stages and would benefit from more systematic evaluations: e.g., Belov et al recently revealed that double sulfonation, which had been assumed to guarantee cell impermeability, actually does not reliably prevent cell entry for rhodamines . While these methods are elegant and are starting to engage properly with membrane permeability, there remains an unmet need for generalized approaches to simultaneously mask and impermeabilize molecules, such that only intracellular reactions in permeabilized cells activate them: ideally giving “impermeable, off → ON” fluorogenic probes or prodrugs.…”

Section: Introductionmentioning

confidence: 99%

Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury

Mauker,

Beckmann,

Kitowski

et al. 2024

J. Am. Chem. Soc.

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Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies in vivo. Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate damaged volumes in vivo nor image non-nucleated cell types or compartments). Here, we develop modular fluorogenic probes that reveal the whole cytosolic volume of damaged cells, with near-zero background fluorescence so that no washing is needed. We identify a specific disulfonated fluorogenic probe type that only enters cells with damaged membranes, then is enzymatically activated and marks them. The esterase probe MDG1 is a reliable tool to reveal live cells that have been permeabilized by biological, biochemical, or physical membrane damage, and it can be used in multicolor microscopy. We confirm the modularity of this approach by also adapting it for improved hydrolytic stability, as the redox probe MDG2. We conclude by showing the unique performance of MDG probes in revealing axonal membrane damage (which DNA fluorogens cannot achieve) and in discriminating damage on a cell-by-cell basis in embryos in vivo. The MDG design thus provides powerful modular tools for wash-free in vivo imaging of membrane damage, and indicates how designs may be adapted for selective delivery of drug cargoes to these damaged cells: offering an outlook from selective diagnosis toward therapy of membrane-compromised cells in disease.

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A Bright Surprise: Live‐Cell Labeling with Negatively Charged Fluorescent Probes based on Disulfonated Rhodamines and HaloTag

Cited by 5 publications

References 49 publications

Optimized Red-Absorbing Dyes for Imaging and Sensing

Optimized Red-Absorbing Dyes for Imaging and Sensing

Stereochemistry‐Dependent Labeling of Organelles with a Near‐Infrared‐Emissive Phosphorus‐Bridged Rhodamine Dye in Live‐Cell Imaging

Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury

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