A fluorogenic TMP-tag for high signal-to-background intracellular live cell imaging.

Area of Science:

• Chemical Biology
• Cellular Imaging
• Synthetic Biology

Background:

• Chemical tags offer modular fluorophore incorporation for live cell imaging, but suffer from background noise.
• Fluorescent proteins are limited by photophysical properties and potential toxicity.
• Existing chemical tags lack the signal-to-noise ratio required for high-resolution imaging of diverse proteins.

Purpose of the Study:

• To engineer a fluorogenic trimethoprim-based chemical tag (TMP-tag) that overcomes background noise limitations.
• To develop a tag that becomes fluorescent only upon specific binding to a target protein.
• To enable high-resolution live cell imaging of both abundant and low-abundance proteins.

Main Methods:

• Designed a trimethoprim-fluorophore-quencher molecule (TMP-Q-Atto520) utilizing proximity-induced reactivity.
• Engineered a modified E. coli dihydrofolate reductase (eDHFR) with a cysteine residue (L28C) to cleave the quencher.
• Validated the fluorogenic reaction and covalent labeling in vitro and in living cells.

Main Results:

• The eDHFR:L28C mutant rapidly and efficiently cleaved TMP-Q-Atto520, generating a fluorescent signal.
• Achieved significant fluorescence enhancement upon specific binding and covalent labeling.
• Successfully imaged both highly abundant, localized proteins and less abundant, dynamic cytoplasmic proteins in live cells.

Conclusions:

• Fluorogenic TMP-tags represent a significant advancement for high-resolution live cell imaging.
• Proximity-induced reactivity and organic chemistry offer powerful tools for synthetic biology.
• This approach enhances the ability to visualize diverse intracellular protein populations with improved clarity.