High specific microRNA detection based on G-quadruplex sensitive fluorescence under isothermal amplification

MicroRNAs (miRNAs) are crucial for various basic cellular processes and have been regarded as an important indicator for pathogenesis and development of cancer. While miRNAs are characterized by short sequence length, large single cell copy number variation, low abundance in vivo, and high homologous sequence similarity. The development of miRNA detection methods with high sensitivity, good selectivity and low cost is the key to study the function of miRNA. In this article, a method combining isothermal chain replacement amplification and G-quadruplex (G4) specific detection is developed for highly specific and sensitive detection of miRNA. By designing proper DNA sequences, a three-stranded DNA complex was formed and produced a lot of G4 structure sequences, which can be stained by specific fluorescence dye and quantified. In the experiment, the fluorescence dye shows extremely high specificity, which brings excellent signal discrimination, so that the target miRNAs can be accurately detected. Experiments under high background noise also verified this method is highly selective for target miRNAs. This method holds great imagination in expanding the detection methods to more platforms, such as nanopore sensors, because it alters the secondary structure of DNA sequences.