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

Abstract

In this paper, a novel, facile fluorometric ATP assay with high sensitivity and excellent selectivity has been reported. This approach utilizes a molecular beacon, T4 DNA ligase and two short oligonucleotides. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction and the ligation product restores the fluorescence of the molecular beacon. Owing to the high sensitivity of the molecular beacon and T4 DNA ligase's high substrate dependence, this novel ATP assay demonstrates exceptional selectivity and high sensitivity down to 0.14 nM in homogeneous solution. Cellular ATP concentrations in several cell lines have been determined by measuring the lysate sample containing 8.0 × 10³ cells.