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Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

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Engineered fluorogenic HaloTag ligands for turn-on labelling in live cells.

Bryan J Lampkin¹, Joshua A Kritzer¹

¹Department of Chemistry, Tufts University, 62 Talbot Rd, Medford, MA 02155, USA. joshua.kritzer@tufts.edu.

Chemical Communications (Cambridge, England)

|December 4, 2023

Summary

Researchers improved organic fluorescent dyes called benzothiadiazoles for protein labeling. These new dyes show significantly brighter fluorescence within cells by minimizing unwanted light loss.

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Area of Science:

Organic chemistry
Biochemistry
Fluorescence microscopy

Background:

Organic fluorophores are crucial in biological imaging.
Benzothiadiazoles are used as turn-on fluorescent probes for protein labeling.
Protein labeling requires probes with minimal non-radiative decay.

Purpose of the Study:

To enhance benzothiadiazole-based fluorescent dyes for protein labeling.
To investigate steric and electronic effects on non-radiative decay pathways.
To develop brighter and more specific fluorescent probes for cellular imaging.

Main Methods:

Conjugation of benzothiadiazoles to the HaloTag protein.
Analysis of non-radiative decay pathways within the HaloTag binding site.
Minimization of steric hindrance and electronic interactions affecting fluorescence.
Evaluation of dye performance in cellular environments.

Main Results:

Minimized non-radiative decay pathways in benzothiadiazole dyes.
Reduced nonspecific interactions with cellular components.
Achieved a 136-fold increase in fluorescence signal over background in cells.
Demonstrated improved turn-on characteristics of the developed dyes.

Conclusions:

Steric and electronic factors significantly influence non-radiative decay in benzothiadiazole dyes within the HaloTag protein.
Optimized benzothiadiazole dyes offer superior brightness and specificity for protein labeling.
These enhanced probes represent a significant advancement for fluorescence microscopy and cellular imaging applications.