A genetically encoded cyclobutene probe for labelling of live cells

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Site-specific protein modification is crucial for biological research and therapeutic development.
• Amber nonsense codon suppression is a powerful tool for incorporating non-canonical amino acids into proteins.

Purpose of the Study:

• To establish an efficient and site-specific method for incorporating cyclobutene-containing amino acids into proteins.
• To demonstrate the utility of this method for fast and fluorescent protein labeling in various biological contexts.

Main Methods:

• Identification and engineering of an aminoacyl-tRNA synthetase/tRNA pair specific for a cyclobutene-containing amino acid.
• Utilizing amber nonsense codon suppression for site-specific amino acid incorporation.
• Employing the inverse electron demand Diels-Alder reaction between the cyclobutene moiety and tetrazine for fluorescent labeling.

Main Results:

• Successful site-specific incorporation of a cyclobutene-containing amino acid into proteins.
• Demonstration of rapid and efficient fluorescent labeling of purified proteins.
• Validation of the labeling technique in intact proteins within live cells.

Conclusions:

• The developed aminoacyl-tRNA synthetase/tRNA pair enables efficient and site-specific protein modification.
• This method provides a versatile platform for fast and fluorescent protein labeling, applicable to both purified proteins and live-cell systems.