Skip to main content

Optimized Xanthene Fluorophores Permit Dual-Color Live-Cell Imaging

Single-molecule localization microscopy (SMLM) is an important method for the high resolution 

investigation of microscopic cellular structures and processes. Traditional organic fluorophores used 

in SMLM utilize intense laser irradiation to induce fluorescence, but this can cause undesired 

photobleaching and can be cytotoxic. In this study,1 the Urano group sought to apply their developed 

xanthene-based fluorophores, HMSiR and HEtetTFER, in multi-color SMLM. The HMSiR 

fluorophore, a first-in-class spontaneously blinking fluorophore (near-infrared range, NIR), already 

possesses optimal SMLM characteristics because it does not require laser irradiation or additives to 

perform under physiological conditions.2 Conversely, HEtetTFER is a green-light emitting 

fluorophore that has been used with HMSiR to achieve dual-color SMLM in fixed cells,3 but due to 

 poor membrane permeability and unfavorable subcellular localization, HEtetTFER is not suitable 

for imaging live cells.


Building on their previous investigation of the reactivity of xanthene fluorophores with endogenous 

 glutathione (GSH),4 the group developed structurally optimized SMLM-compatible fluorophores,  

SiP650 and CP550, working in the NIR and green range, respectively (Figure 1). Notably, these 

structures needed to be tuned to possess the appropriate equilibrium constants between their 

fluorescent dissociated form and their non-fluorescent GSH adduct form, and with fast blinking 

kinetics. Key parameters are shown in Table 1.





HaloTag ligands of both of these fluorophores confirmed specific protein labelling and spontaneous blinking in live cells. Using HMSiR in combination with the newly developed CP550, dual-color live-cell SMLM was achieved in mammalian and bacterial cells (Figure 3). This work is important because new fluorophores are needed with various optical properties and blinking kinetics, and this approach will minimize photodamage and image buffer optimizations that are common characteristics of similar tools.


 References

1.      1.     Morozumi, A.; Kamiya, M.; Uno, S.-N.; Umezawa, K.; Kojima, R.; Yoshihara, T.; Tobita, S.; Urano, Y. J. Am. Chem. Soc. 2020, 142 (Just accepted 4/28/2020), doi: 10.1021/jacs.0c00451

2     2.     Uno, S.; Kamiya, M.; Yoshihara, T.; Sugawara, K.; Okabe, K.; Tarhan, M. C.; Fujita, H.; Funatsu, T.; Okada, Y.; Tobita, S.; Urano, Y. Nat. Chem. 2014, 6, 681.

3     3.     Uno, S.; Kamiya, M.; Morozumi, A.; Urano, Y. Chem. Commun. 2018, 54, 102.
4     4.     Umezawa, K.; Yoshida, M.; Kamiya, M.; Yamasoba, T.; Urano, Y. Nat. Chem. 2017, 9, 279.

Comments

Post a Comment

Popular posts from this blog

Photoswitchable azopyrazoles Flipping the Switches of Ion Channels

Abnormally activated Ca 2+ channels are related to many human diseases, including Stormorken Syndrome, which makes Ca 2+ release-activated Ca 2+ (CRAC) channel a very promising therapeutic target. Several small molecule therapeutics targeting CRAC channels have been developed, including the GSKs series and Synta 66 . Those compounds have relatively high specificity. Meanwhile, a controllable system that can be activated with a switch can probably serve as a convenient tool for further related research.    Photoswitchable chemistry has been applied to a lot of bioactive targets such as ion channels, receptors enzymes and nucleic acids. Recently, the Li group developed CRAC channel inhibitors that can be turned ‘on’ and ‘off’ by UV-light exposure. Scheme 1 . C onverting CRAC channel inhibitors, GSKs , into photoswitchable derivatives, piCRACs . Starting from the well-established GSK-based CRAC inhibitor ( Scheme 1 ), the authors developed a series of nitr...
1 comment
Read more

RIPK2 PROTAC Burns up the Bridges between PK and PD

GlaxoSmithKline (GSK) previously reported a series of RIPK2 PROTACs with various E3 ligase binders (Figure 1). These PROTACs can degrade RIPK2 potently and efficiently in a concentration-dependent manner (Figure1). However, their poor solubility and high microsomal clearance limited their in vivo applications. To develop a RIPK2 degrader suitable for  in vivo  applications, the GSK group identified another IAP based RIPK2 PROTAC4 that is significantly different from PROTAC2, possessing a different RIPK2 binder, IAP ligase binder and linker (Figure2). PTROTAC4 can degrade RIPK2 in a time-dependent manner and shows reduced lipophilicity and lower clearance in hepatocytes. However, the modest binding affinity between the warhead of PROTAC4 and RIPK2 requires a high dose to reach efficient RIPK2 degradation. To address this limitation, the researchers performed extensive medicinal chemistry optimization based on PROTAC 4 and identified PROTAC 6 (Figure2), which contains an...
Post a Comment
Read more

Multiple Postdoc and Technician Positions Available at Baylor College of Medicine

  Multiple postdoc and technician positions are available in Dr. Jin Wang’s group in the Department of Pharmacology and Chemical Biology at Baylor College of Medicine (BCM). BCM is located in the world largest medical center and has access to enormous biomedical resources. The research in the Wang lab is highly interdisciplinary and translational. The Wang lab has both chemistry and biology operations. For chemistry work, we have 17 chemical fume hoods, 4 ISCO combiflash chromatography systems, Agilent analytical LC-MS, Agilent preparative HPLC with auto-purification, and Genevac high throughput solvent evaporation, along with convenient access to 600 and 800 MHz NMR. For biology work, we have common equipment for biochemistry, molecular and cell biology, including tissue culture hoods, RT-PCR, plate reader, and imaging plate reader, and perform animal work to evaluate the efficacy of the experimental therapeutics developed in the group. In addition, we recently acquired a state-...
Post a Comment
Read more