Specific Response\nAssembly of 3D Space-Confined DNA\nNanoaggregates for Rapid and Sensitive Detection of DNA Methyltransferase

Rapid and sensitive detection of DNA adenine methyltransferase\n(Dam) activity is crucial for both research and clinical applications.\nHerein, we utilize two types of spherical nucleic acids (SNAs) to\nspecific response assemble into 3D space-confined DNA nanoaggregates\nthat enable the rapid and sensitive detection of Dam activity. The\nSNAs feature 3D order DNA scaffolds that serve as cores for anchoring\nsignal hairpin probes (S-HPs) and target hairpin probes (T-HPs). Specifically,\ntwo distinct S-HPs are labeled with FAM fluorophores and BHQ1 quenchers\nand share identical hairpin sequences, while two types of T-HPs are\ndesigned with different linking sequences and specific recognition\nregions, resulting in the formation of two types of SNAs (SNA1 and\nSNA2). In the presence of Dam, the recognition region of the T-HPs\nis methylated and subsequently cleaved by auxiliary endonuclease,\nreleasing the loop of the T-HP as a walking strand and exposing the\nlinking sequence on the SNAs. Notably, the prior design of complementary\nlinking sequences in the two types of SNAs facilitates their assembly\ninto 3D DNA nanoaggregates, creating a confined space for walking\nstrands to recover fluorescent signals. The 3D DNA nanoaggregate system\nnot only provides highly ordered tracks but also enhances the spatial\ncontinuity of the walking strands, greatly improving the reaction\nkinetics for detecting Dam activity. This strategy enables the rapid\nand sensitive detection of Dam activity within 105 min, achieving\na limit of detection of 2.9 × 10<sup>–4</sup> U mL<sup>–1</sup>, demonstrating significant potential for advancing\nresearch in DNA methylation.