Highly sensitive detection of microRNAs based on isothermal exponential amplification-assisted generation of...

It is well-known that microRNAs (miRNAs) have become an ideal class of biomarker candidates for clinical diagnosis of cancers, thus sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, as well as discovery of new targets for drugs. In this work, we have developed a sensitive method for microRNAs detection based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme, and demonstrated its practical application in biological sample of cell lysate. The assay involves a combination of polymerase strand extension, single-strand nicking and catalytic reaction of G-quadruplex/hemin complex. It is designed such that, the target miRNA initiates the efficient synthesis of two kinds of short oligonucleotide fragments in the continuous cycle of the polymerization, nicking and displacement reactions, by means of thermostable polymerase and nicking endonuclease. One fragment has the same sequence as the target miRNA, except that the deoxyribonucleotides and thymine replace the ribonucleotides and uridine in the miRNA, to activate new cyclic chain reactions of polymerization, nicking and displacement reactions as the target miRNA. The other is the signal molecule of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. With such designed signal amplification processes, the proposed assay showed a quantitative analysis of sequence-specific miRNAs in a wide range from 1 fM to 100 nM with a low detection limit of 1 fM. Moreover, this assay demonstrated excellent differentiation ability for the mismatch miRNAs targets and good performance in biological samples.