We have developed a label-free approach for direct detection of gene mutations using free-energy values that are derived from single-molecule force spectroscopy (SMFS)-based nucleic acid unbinding experiments. From the duplex unbinding force values acquired by SMFS, the force-loading-rate-independent Gibbs free-energy values were derived using Jarzinsky's equality treatment. Because it provides molecule-by-molecule information, this approach is a major shift compared to the earlier reports on label-free detection of DNA sequences, which are mostly based on ensemble level data. We tested our approach in the disease model framework of multiple drug-resistant tuberculosis using the nuclease-resistant and conformationally rigid locked nucleic acid probes that are a robust and efficient alternative to the DNA probes. All of the major mutations in Mycobacterium tuberculosis (MTB), as relevant to MTB's resistance to the first-line anti-TB drugs rifampicin and isoniazid, could be identified, and the wild type could be discriminated from the most prevalent mutation and the most prevalent mutation from the less occurring ones. Our approach could also identify DNA sequences (45 mer), having overhang stretches at different positions with respect to the complementary stretch. Probably for the first time, the findings show that free-energy-based detection of gene mutations is possible at molecular resolution.
Keywords: MDR-TB; free energy; gene mutation; locked nucleic acid; nucleic acid sensing; single-molecule force spectroscopy.