ScholarWorks@중앙대학교

초록

Background: Metal ions play a crucial role in a variety of physiological processes by serving as essential cofactors that regulate protein activity and contribute to the folding of nucleic acids and proteins. The quantification of cationic cofactor binding to nucleic acids and proteins is crucial for optimizing their function. In addition, abnormal concentrations of metal ions can trigger various diseases, which are often treated with metal-chelating drugs. Therefore, precise measurement of metal ions is essential for evaluating the effectiveness of these treatments. Results: Here, we introduce an assay platform that employs single-molecule fluorescence resonance energy transfer to quantitatively measure metal ion concentrations by observing the metal ion-induced folding of a single hairpin DNA. We successfully quantified monovalent ions, Na+ and K+, in the millimolar range and divalent ions, Mg2+ and Ca2+, in the micromolar range. Moreover, the detection range and limit of detection can be tuned by systematically modulating the thermodynamic stability of the hairpin DNA. Our assay was applied to determine the binding affinity of three widely used chelators for Mg2+ and/or Ca2+, namely, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N'-tetraacetic acid (EGTA) and 1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid (BAPTA), demonstrating the Ca2+-specific binding characteristics of EGTA and BAPTA. Additionally, our hairpin DNA was able to repetitively quantify metal ion concentrations over a 2-week period under various storage conditions. Significance: Our work represents metal ion quantification assay based on the shift of equilibrium in the folding process of hairpin DNA induced by positively charged metal ions, making it applicable to various metal ions. The strength of our assay lies in its minimal usage of hairpin DNA probes, and the recycling capacity of these probes, allowing for over 50 measurements within a period of 2 weeks. © 2025 Elsevier B.V.

키워드