Structural and Kinetic Profiling of Rolling Circle Amplification via Solid-State Nanopore Sensing Using miR-21 as a Model

Abstract

Rolling Circle Amplification (RCA) is a robust isothermal nucleic acid amplification technique widely used in molecular diagnostics. In this study, we combine RCA with solid-state nanopore sensing to monitor the amplification process at the single-molecule level using miR-21 as a model biomarker. This label-free platform enables detailed analysis of amplification kinetics and structural transitions over time. Changes in translocation dwell time and current blockage were evaluated across RCA incubation periods (30 min, 1 h, 2 h), revealing time-dependent increases consistent with the generation of longer and more complex DNA concatemers. These findings were validated by Urea-PAGE and atomic force microscopy (AFM), while Mfold-based secondary structure predictions further supported the evolution of more stable and folded configurations. Additionally, a custom-developed signal extraction application facilitated reproducible event classification and visualization. Overall, this integrated approach provides new insights into RCA behavior and highlights the potential of nanopore-based sensing for the development of sensitive, structure-resolved diagnostic tools. © 2025 The Authors. Published by American Chemical Society