Additional label-free chemical resolution to single-molecule force spectroscopy

Identifying the chemical alterations and perturbations on DNA is important for understanding gene regulation, disease development, and their potential applications in diagnostics and therapeutics. Given the complex and dynamic spatial distributions of these chemical modifications, it becomes essential to investigate them at the individual DNA molecule level, which can reveal rare events and uncover crucial biological phenomena that may remain hidden in bulk studies. Here, we combine optical tweezers-based single-molecule force manipulation with single-molecule surface-enhanced Raman spectroscopy (SERS) characterization, enabling real-time, label-free, and multi-dimensional characterizations of individual metal complex-bound DNA molecules. Specifically, the molecular vibrational characteristics of a single DNA molecule were identified with exceptional sensitivity at the nanoscale during the site-specific SERS detections, while the single DNA molecule was under precise mechanical manipulation to record its real-time force responses and molecular extensions associated with conformational changes. Moreover, the binding modes and kinetics between the single DNA molecule and metal complexes, as well as the mechanical properties of the altered DNA, were quantified by the complementary single-molecule DNA-stretching measurements. Leveraging the correlative molecular control and chemical identification capabilities, this approach offers high spatial resolution and site-specific recognition accuracy to unveil the chemical alterations and perturbations induced by metal complexes on DNA at the single-molecule level.