Sensitive Detection Of MiRNA Based On DNA-silver Nanoclusters Fluorescence Probe Combining With Duplex Specific Nuclease Signal Amplification

MicroRNAs(miRNAs)are a class of small noncoding ribonucleic acids molecules,consisiting of about 22 ribonucletides.They were found in eukaryotic cells.MiRNAs regulate the expression of genes in plants,animals,and humans by controlling the translation of intracellular proteins.Studies have shown that abnormal miRNA expression is closely related to the occurrence and development of tumors and cancers.Therefore,the detection of miRNA expression levels in tissues or cells can provide valuable information for biomedical research and early clinical diagnosis of related disease.However,There are some disadvantages for miRNA detection,such as poor stability,high family sequence homology,low expression levels in cells and so on.Because of this,simple and sensitive miRNA detection presents a challenge for researchers.The establishment of simple,fast,sensitive,and specific miRNA detection methods is of great significance for miRNA research and the early diagnosis of cancer or tumor.Compared with traditional organic fluorescent dyes and semiconductor quantum dots,metal nanoclusters have many advantages,such as poor photobleaching,good biological compatibility,adjustable size of clusters with luminescent wavelength,and mild synthesis conditions and so on.In this study,the dark DNA-silver nanoclusters(DNA-AgNCs)have been synthesized.The fluorescence of the dark DNA-AgNCs is extinguished and the fluorescence intensity is very low.When a G-rich sequence is near the dark DNA-AgNCs,fluorescence of DNA-AgNCs can be exited.Based on this property combing with duplex-specific nuclease signal amplification,a series of simple and rapid miRNA detection methods have been established.The main contents are as follows:(1)Detection of miRNA based on DNA-templated silver-nanoclusters probe combing with duplex-specific nuclease signal amplificationIn this experiment,a DNA probe with the phosphorylated modification at its 3' terminal.It contains two parts.One is complementary sequence with let-7a at 3' terminal of DNA probe;Another is complementary sequence with the dark DNA-AgNCs at its 5' terminal.The target,let-7a,is able to trigger subsequent DSNSA amplification,releasing large amounts of short-stranded DNA.Then,under the action of TdT and dGT,the rich G is extended at the 3'terminal of the short-stranded DNA.The short-stranded DNA hybridizes with the DNA sequence of the AgNCs,which shortens the distance between the g-rich sequence and AgNCs,and the fluorescence of AgNCs is excited.The detection limit for let-7a is 89 fmol.This method for miRNA,based on DNA-AgNCs fluoredcence probe,with simple design and low cost opens up a new way for the detection of miRNA(2)Label-free and sensitive detection miRNA based on DNA-templated silver nanoclusters fluorescence probe combing with duplex-specific nuclease signal amplification.In this experiment,we still use DNA-AgNCs as fluorescent probe.A hairpin structure probe(HP)has been designed.When the target miRNA-21 is present,it hybridizes with HP.The hairpin structure of HP is opened.With the addition of double-stranded specific nucleases(DSN),the DSNSA amplification reaction occurs,releasing a large number of rich-G sequences that can hybridize with dark AgNCs.Dark AgNCs is added to hybridize with the released sequence,which shortens the distance between the G-rich sequence and the dark AgNCs.The fluorescence of the dark AgNCs is excited.The dection limit for miRNA-21 is8.3 fmol.This method is lable-free,simpler and more sensitive.