Construction of a hierarchical DNA circuit for single-molecule profiling of locus-specific N-methyladenosine-MALAT1 in clinical tissues.

N-methyladenosine (mA) is the most important internal methylation in eukaryotic RNAs, and it is critically implicated in diverse RNA metabolisms for cancer development. Because epigenetic modifications do not interfere with Watson-Crick base pairing and mA modification is not susceptible to chemical decorations, standard hybridization-based techniques cannot be applied for sensing mA in RNAs. Consequently, the development of new methods for accurate and sensitive profiling of locus-specific mA in RNAs remains a great challenge. Herein, we demonstrate for the first time the construction of a hierarchical DNA circuit for single-molecule profiling of locus-specific mA-metastasis-associated lung adenocarcinoma transcript 1 (mA-MALAT1) in clinical tissues. Taking advantage of high discrimination of VMC10-DNAzyme between mA and A, exponential efficiency of hierarchical DNA circuit, and ultrahigh signal-to-noise ratio of single-molecule detection, this nanodevice exhibits attomolar sensitivity with a limit of detection (LOD) of 1.8 aM for mA-MALAT1 in vitro and a dynamic range of 7 orders of magnitude. Moreover, it can discriminate 0.001% mA-MALAT1 from excess A-MALAT1, quantify mA-MALAT1 in diverse cancer cells at single-cell level, distinguish mA-MALAT1 expressions in breast cancer patients and healthy individuals, and monitor cellular mA-MALAT1 for gene therapy, offering a promising platform for epitranscriptomic research and clinical diagnostics.