RESUMEN
N6-methyladenosine (m6A) is an ubiquitous post-transcriptional modification catalyzed by METTL3/14 complex in eukaryotic mRNAs. The abnormal METTL3/14 complex activity affects multiple steps of RNA metabolism and may induce various diseases. Herein, we demonstrate the RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues. METTL3/14 complex can catalyze the methylation of RNA probe to prevent it from being cleaved by MazF. The intact RNA probe is recognized by the magnetic bead (MB)-capture probe conjugates to induce duplex-specific nuclease (DSN)-assisted cyclic digestion, exposing numerous shorter ssDNAs with 3'-OH end. The shorter ssDNAs on the MB surface can act as the primers to initiate terminal deoxynucleotidyl transferase (TdT)-enhanced tyramide signal amplification (TSA), forming the Cy5 fluorescence-encoded nanostructures. After magnetic separation, the Cy5 fluorescence-encoded nanostructures are digested by DNase I to release abundant Cy5 ï¬uorophores that can be simply quantified by ï¬uorescence measurement. This assay achieves good specificity and high sensitivity with a detection limit of 58.8 aM, and it can screen METTL3/14 complex inhibitors and quantify METTL3/14 complex activity at the single-cell level. Furthermore, this assay can differentiate the METTL3/14 complex level in breast cancer patient tissues and healthy volunteer tissues.
Asunto(s)
Técnicas Biosensibles , Humanos , Metilación , Sondas ARN , ARN , ADN Nucleotidilexotransferasa , ADN de Cadena Simple , Metiltransferasas/genéticaRESUMEN
The METTL3/14 complex is an important RNA N6-Methyladenosine (m6A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m6A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10-17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery.