Selective Activation of Cascade Assembly Amplification for DNA Methyltransferase Detection Using a Double-Loop Interlocked DNA Circuit.

ABSTRACT

Precise and reliable monitoring of DNA adenine methyltransferase (Dam) activity is essential for disease diagnosis and biological analysis. However, existing techniques for detecting Dam activity often rely on specific DNA recognition probes that are susceptible to DNA degradation and exhibit limited target sensitivity and specificity. In this study, we designed and engineered a stable and dynamic DNA nanodevice called the double-loop interlocked DNA circuit (DOOR) that enables the sensitive and selective monitoring of Dam activity in complex biological environments. The DOOR incorporates two interlocked specialized sequences a palindromic sequence for Dam identification and an initiator sequence for signal amplification. In the presence of Dam, the DOOR is cleaved by double-stranded DNA phosphodiesterase I endonuclease, generating massive double-stranded DNA (dsDNA) units. These units can self-assemble into a long dsDNA scaffold, thereby enhancing the subsequent reaction kinetics. The dsDNA scaffold further triggers a hyperbranched hybrid chain reaction to produce a fluorescent 3D DNA nanonet, enabling more precise monitoring of the Dam activity. The DOOR device exhibits excellent sensitivity, specificity, and stability, rendering it a powerful tool for studying DNA methylation in various biological processes and diseases.