Construction of a hierarchical DNA circuit for single-molecule profiling of locus-specific N6-methyladenosine-MALAT1 in clinical tissues

Abstract

N⁶-methyladenosine (m⁶A) 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 m⁶A modification is not susceptible to chemical decorations, standard hybridization-based techniques cannot be applied for sensing m⁶A in RNAs. Consequently, the development of new methods for accurate and sensitive profiling of locus-specific m⁶A 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 m⁶A-metastasis-associated lung adenocarcinoma transcript 1 (m⁶A-MALAT1) in clinical tissues. Taking advantage of high discrimination of VMC10-DNAzyme between m⁶A 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 m⁶A-MALAT1 in vitro and a dynamic range of 7 orders of magnitude. Moreover, it can discriminate 0.001% m⁶A-MALAT1 from excess A-MALAT1, quantify m⁶A-MALAT1 in diverse cancer cells at single-cell level, distinguish m⁶A-MALAT1 expressions in breast cancer patients and healthy individuals, and monitor cellular m⁶A-MALAT1 for gene therapy, offering a promising platform for epitranscriptomic research and clinical diagnostics.