Probing the stability of Hg2+-mediated self-duplexes: Effects of thymine residues position and number via a single-molecule protein nanopore sensing technique

In this study, we report for the first time that mercury metal ions (Hg²⁺) can stabilize self-duplexes that would otherwise fail to hybridize. We designed two short DNA fragments containing varying numbers of thymine residues, positioned either in the central region or at the terminal ends of the resulting double-stranded structures formed via non-canonical T-Hg²⁺-T base pairs. The stability and structural properties of these newly formed duplexes were investigated using single-molecule nanopore electrophysiology. Our results demonstrated that duplexes with metal-coordinated base pairs located in the central region could be unzipped with relative ease, whereas those with terminal T-Hg²⁺-T pairs exhibited remarkable structural rigidity, resisting modification even under high external force. We emphasize that both the position and the number of thymine residues within the DNA strands are critical key players in the hybridization efficiency and the stability of duplexes. This novel coordination chemistry approach holds significant potential for the development of highly responsive, label-free nanopore sensors for metal ions, the facilitation of strand self-recognition, and the precise detection of thymine positioning within DNA duplexes.