Methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA methyltransferases

Abstract

DNA methylation is one important epigenetic modification that involves in regulating multiple cellular functions. DNA methyltransferases (MTases) maintain methylation patterns in genomic DNA, and their aberrant activities may lead to multiple diseases including cancers. However, the efficient sensing of multiple DNA MTases is limited by the diversity of catalytic substrates and the rareness of methylation-dependent restriction endonucleases. Herein, inspired by the intrinsic superiorities of light-up RNA aptamers and unique properties of T7-based transcription amplification, we construct a novel methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases. Taking advantage of ultrahigh specificity of MDREs toward methylated DNAs, high efficiency of bicyclic cascade amplifications and high signal-to-noise ratio of light-up RNA aptamer-fluorophore pairs, this nanosensor exhibits good specificity and high sensitivity with a detection limit of 3.77 × 10−3 U/mL for M.SssI and 7.21 × 10−5 U/mL for Dam. Furthermore, it can screen potential inhibitors and quantify M.SssI and Dam MTases activities in E. coli cells and human serum samples. Notably, this assay can be completed homogeneously within 2 h in a single tube in a label-free manner, providing a powerful platform for sensing multiple DNA MTases in biomedical research and clinical therapeutics.