Fluorescent Affinity Probe for mTOR Protein Synthesized via Click Chemistry and Its Applications

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Mammalian target of rapamycin (mTOR) is a key regulator of cell growth and proliferation, making it a critical drug target.
• Studying endogenous mTOR activity and drug interactions in living cells presents significant challenges.
• Current methods for visualizing mTOR activity in situ are limited.

Purpose of the Study:

• To synthesize and validate a novel fluorescent probe for in situ labeling of endogenous mTOR in living cells.
• To investigate the interaction between mTOR and drugs within living cells using the developed probe.
• To overcome the limitations of existing probes for studying mTOR dynamics.

Main Methods:

• Synthesis of a rapamycin-based fluorescent probe using click chemistry (IEDDA reaction).
• Incorporation of trans-cyclooctene into rapamycin (RPT) and ATTO680 dye onto tetrazine (TA680).
• Utilized fluorescence correlation spectroscopy (FCS) and fluorescence imaging for validation.

Main Results:

• The novel probe specifically labeled endogenous mTOR in living cells via IEDDA click reaction.
• Achieved an 8-fold increase in fluorescence intensity upon probe activation (turn-on fluorescence).
• Demonstrated suitability for investigating drug-mTOR interactions in real-time.

Conclusions:

• The developed fluorescent probe offers a sensitive and specific tool for studying mTOR in living cells.
• The probe's turn-on fluorescence property enhances its utility compared to traditional probes.
• This advancement facilitates better understanding of mTOR-related diseases and drug development.