DNA Motors Powered by Exonuclease III for Autonomous Rolling Motion and Biosensing Applications

Nucleic acid-based synthetic motors emulate key behaviors of biological machines, enabling applications in biosensing and nanoscale actuation. Among the reported synthetic motors, RNase H-powered motors offer high speed and processivity with demonstrated applications in computation and viral sensing. However, these motors rely on RNA as “fuel” source, limiting their stability. Here, we report the development of a robust, RNA-free DNA motor powered by Exonuclease III. These motors exhibit self-avoiding rolling motion driven by enzymatic hydrolysis of surface-bound DNA fuel strands, consistent with a burnt-bridge Brownian ratchet mechanism of translocation. We systematically optimized motor performance by chemically tuning the fluorescence reporter, DNA sequence composition, and surface fuel density. Fluorescence and brightfield microscopy revealed super-diffusive and Lévy-like stop-and-go dynamics under optimized conditions. Importantly, the established DNA-only architecture confers resistance to RNase degradation, and the system can be configured for motion-based biosensing via aptamer-functionalized components that selectively stall in response to viral targets. Beyond the significance of creating a chemically stable, tunable, and biosensing-compatible DNA motor platform, the work also establishes the modularity of the rolling motor platform and highlights how enzymatic diversity can expand their chemical and functional scope.