A novel sensitive and selective ligation-based ATP assay using a molecular beacon

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Adenosine triphosphate (ATP) is a crucial molecule for cellular energy.
• Accurate quantification of ATP is essential for understanding cellular processes.
• Existing ATP assays may lack sensitivity or selectivity.

Purpose of the Study:

• To develop a novel, highly sensitive, and selective fluorometric assay for ATP detection.
• To utilize molecular beacons and T4 DNA ligase for enhanced ATP quantification.
• To apply the assay for determining cellular ATP concentrations in various cell lines.

Main Methods:

• A molecular beacon system was designed to respond to ATP presence.
• T4 DNA ligase was employed to catalyze oligonucleotide ligation in an ATP-dependent manner.
• Fluorescence changes of the molecular beacon indicated ATP concentration.

Main Results:

• The assay demonstrated high sensitivity, detecting ATP down to 0.14 nM in homogeneous solution.
• Exceptional selectivity was achieved due to the specific action of T4 DNA ligase.
• Cellular ATP levels were successfully measured in lysates from 8.0 × 10^3 cells.

Conclusions:

• The developed fluorometric ATP assay offers a sensitive and selective method for ATP quantification.
• This assay provides a valuable tool for biochemical and cellular studies.
• The method is applicable for determining ATP concentrations in biological samples.