Specific Response Assembly of 3D Space-Confined DNA Nanoaggregates for Rapid and Sensitive Detection of DNA Methyltransferase

Rapid and sensitive detection of DNA adenine methyltransferase (Dam) activity is crucial for both research and clinical applications. Herein, we utilize two types of spherical nucleic acids (SNAs) to specific response assemble into 3D space-confined DNA nanoaggregates that enable the rapid and sensitive detection of Dam activity. The SNAs feature 3D order DNA scaffolds that serve as cores for anchoring signal hairpin probes (S-HPs) and target hairpin probes (T-HPs). Specifically, two distinct S-HPs are labeled with FAM fluorophores and BHQ1 quenchers and share identical hairpin sequences, while two types of T-HPs are designed with different linking sequences and specific recognition regions, resulting in the formation of two types of SNAs (SNA1 and SNA2). In the presence of Dam, the recognition region of the T-HPs is methylated and subsequently cleaved by auxiliary endonuclease, releasing the loop of the T-HP as a walking strand and exposing the linking sequence on the SNAs. Notably, the prior design of complementary linking sequences in the two types of SNAs facilitates their assembly into 3D DNA nanoaggregates, creating a confined space for walking strands to recover fluorescent signals. The 3D DNA nanoaggregate system not only provides highly ordered tracks but also enhances the spatial continuity of the walking strands, greatly improving the reaction kinetics for detecting Dam activity. This strategy enables the rapid and sensitive detection of Dam activity within 105 min, achieving a limit of detection of 2.9 × 10^-4 U mL^-1, demonstrating significant potential for advancing research in DNA methylation.