Research On Ultrasensitive Detection Of MicroRNA In Cells

MicroRNA are a type of endogenous,non-coding RNA,and their abnormal expression is related to the development of many diseases including cancers.MicroRNA has become a biomarker for the diagnosis and treatment of cancer,and it can be used as a potential target for new drugs development.Thus,the development of effective microRNA detection methods which can be applied for accurate quantification of microRNA in complex clinical samples has great potential for further applications in clinical diagnosis and biomedical research studies.Recently,enzyme-assisted cyclic signal amplification strategies have been widely used in the detection of microRNA.Duplex-specific nuclease(DSN)is a kind of nuclease that can specifically degrade DNA in double-stranded DNA and DNA-RNA duplex,but has little activity on single-stranded nucleic acid molecules and double-stranded RNAs.In addition,this enzyme is able to distinguish between fully and non-fully matched duplexes.DSN can accurately distinguish single-nucleotide mismatches of DNA-DNA double strands of 10-12 bp,it can produce cleavage activity while dsDNA is greater than 10 bp and DNA-RNA is greater than 15 bp.Currently,DSN is widely used for homogenization after full-length cDNA enrichment,transcriptome library construction based on second-generation sequencing,and also used for detection of genomic DNA homogenization,cDNA deletion and single nucleotide polymorphism(SNP),multiplex fluorescence detection of microRNA and quantitative detection of telomere ends.Pyrene is a kind of spatial-sensitive fluorescent dye that can form an excimer when two pyrene molecules are brought into close proximity.In comparison with conventional molecular beacons,pyrene excimers have a large Stokes shift(~130 nm)and they may avoid the self-quenching effect even at high concentrations.In addition,the formation of pyrene excimers is a quencher-free signal output which can efficiently eliminate the false positivity resulting from incomplete quenching.Moreover,pyrene excimers possess a much longer lifetime(more than 100 ns)at a wavelength of 485 nm compared to a typical chromophore(less than 10 ns)in biological fluids.Recently,pyrene excimers have been widely applied for the detection of metal ions,nucleic acids,proteins.In this paper,we introduced a simple fluorescence method for the sensitive detection of microRNA based on duplex specific nuclease-assisted target recycling and pyrene excimer switching.We have designed a specific probe and a hairpin probe which is labeled with pyrene molecules at both ends of the stem as reporters.In the presence of target microRNA,the hybridization of the probe with target microRNA leads to the formation of a probe-microRNA duplex whose probe strand may be digested with DSN,initiating the cyclic digestion of multiple probe strands and consequently the transformation of the reporter to a hairpin structure with the formation of pyrene excimers.The research has demonstrated for the first time the use of pyrene excimers as signal reporters for microRNA detection,compared with other microRNA detection methods,it has significant advantages of low background signal,high sensitivity,and excellent specificity.In addition,it can also be applied for accurate quantification of microRNA in cell extracts and blood samples,holding great application prospects for further applications in clinical diagnosis and biomedical research studies.