Ordered DNA Cube-Braced Hierarchical Ladder Track-Confined Efficient Circular DNA Walker for Rapid and Ultrasensitive Electrochemical Detection of UDG Activity.

Uracil-DNA glycosylase (UDG) plays a critical role in genome maintenance by specifically excising damaged uracil bases, and its rapid and sensitive detection is therefore imperative for early disease diagnosis. However, the existing UDG activity detection methods suffer from slow detection rate and limited sensitivity. Herein, we developed a novel DNA cube-braced hierarchical ladder track (DCB-HLT) as an ordered and rigid structured pathway to enhance the autonomous movement and high-efficiency recognition of the circular DNA walker (CD-walker), enabling highly accurate and sensitive electrochemical detection of UDG activity. Notably, unlike conventional DNA walkers moved on the stochastic tracks, the orderliness and controllable spatial morphology of DCB-HLT ensures a designated movement pathway for the CD-walker. This confined track significantly minimizes derailment probability and prevents invalid diffusive movement of the CD-walker, thereby facilitating productive movement with higher directionality, speed, and longevity. Moreover, benefiting from high biological stability and mechanical flexibility, the well-defined CD-walker could be endowed with extended operational lifespan and superior system robustness compared with those of traditional DNA walkers. As a proof of concept, the CD-walker based on DCB-HLT exhibited a 6-fold faster speed than that of a similar walker on a random track, a 30.8-fold greater resistance to exonucleases and a 5-fold enhanced stability in cell lysates compared to its linear counterparts. As a result, the prepared electrochemical biosensor achieved rapid and ultrasensitive detection of UDG activity with an exceptionally low detection limit of 3.98 × 10-8 U·mL-1, demonstrating great promising potential for biomedical diagnostics.