Controllable assembly of dendritic DNA nanostructures for ultrasensitive detection of METTL3-METTL14 m6A methyltransferase activity in cancer cells and human breast tissues

Abstract

N6-Methyladenosine (m6A) is a reversible chemical modification in eukaryotic messenger RNAs and long noncoding RNAs. The aberrant expression of RNA methyltransferase METTL3-METTL14 complex may change the m6A methylation level and cause multiple diseases including cancers. The conventional METTL3-METTL14 assays commonly suffer from time-consuming procedures and poor sensitivity. Herein, we develop a controllable amplification machinery based on MazF-activated terminal deoxynucleotidyl transferase (TdT)-assisted dendritic DNA structure assembly for ultrasensitive detection of METTL3-METTL14 complex activity in cancer cells and breast tissues. The presence of METTL3-METTL14 complex catalyzes the formation of m6A in detection probe, effectively preventing the cleavage of methylated detection probes by MazF. The methylated detection probes with 3′-OH termini can function as the primers for template-free polymerization catalyzed by TdT on magnetic beads (MBs), producing long chains of poly-thymidine (poly-T) sequences. Then poly-T sequences hybridize with signal probes that contain poly-adenine (poly-A) sequence, inducing TdT-mediated polymerization and the subsequent hybridization with more poly-A signal probes for generating dendritic DNA nanostructures assembled on MBs. After magnetic separation and elevated temperature treatment, the signal probes are disassembled from MBs to generate a high fluorescence signal. This method possesses excellent specificity and high sensitivity with a limit of detection (LOD) of 2.61 × 10−15 M, and it can accurately quantify cellular METTL3-METTL14 complex at single-cell level. Furthermore, it can screen inhibitors, evaluate kinetic parameters, and discriminate breast cancer tissues from normal tissues.