Duplex Specific Nuclease Signal Amplification-based Nano-sensors For The Detection Of MicroRNA

MicroRNA(miRNA)is a kind of endogenous noncoding small RNA molecule.It plays an important role in all kinds of life activities,such as regulating gene expression and so on.Some researches show that many of the human diseases,including cancer,are closely related to the abnormal expression of miRNA.It has been used as a new biomarker for early diagnosis of major diseases such as cancer and so on.However,short length,low abundance in vivo and high similarity in the homologous sequences,which create challenges to the detection of miRNA.So researchers have been interested in the study of highly sensitive detection methods of miRNA.But at the same time in the pursuit of high sensitivity,the simple and low-cost method is equally important in the practical application of the method.How to establish a simple and sensitive detection method for miRNA is of great significance for the clinical diagnosis of related diseases and the drug research of miRNA as a target molecule.Duplex-specific nuclease signal amplification(DSNSA),induced by duplex-specific nuclease(DSN),is an isothermal nucleic acid amplification technology.DSN can hydrolyze DNA in RNA-DNA heteroduplexes,the RNA molecule is released and initiates another round of hybridization and DSN digestion reactions.Signal amplification is realized in this way.Nanomaterials are widely used in the field of biosensors due to theirs supper properties such as small size,larger surface area,better biocompatibility and so on.The purpose of my study is to establised simple and sensitive methods for detection of miRNA based on DSNSA and metal nanosensors.The main contents are as follows:1.Gold nanoparticles-based colorimetric determination of miRNA by combining with duplex-specific nuclease signal amplification.Single stranded DNA(ssDNA)can be adsorbed on the surface of gold nanoparticles(AuNPs)to prevent the salt-induced aggregation of AuNPs,with an accompanying color change from blue to red.Interestingly,we find that the short DNA fragment exhibits superior capability for stabilizing unmodified AuNPs against salt-induced aggregation to the longer ssDNA sequence.These findings are exploited in a simple and sensitive colorimetric assay for miRNA detection coupled to DSNSA.A ssDNA probe,which is completely complementary to the target mi RNA,is firstly designed.In the presence of target let-7a,the ssDNA probe hybridizes with let-7a to form RNA-DNA heteroduplexes.DSN selectively recognizes and cleaves the ssDNA sequence in the RNA-DNA hybrid generating very small DNA fragments.The RNA molecule is released and initiates another round of hybridization and DSN digestion reactions.In this manner,each miRNA target can specifically trigger various cycles of hybridization and DSN cleavage events to yield numerous small fragments of DNA oligonucleotides.These short cleaved DNA fragments can stabilize the AuNPs more effectively than the original ssDNA probes against the salt-induced aggregation.As a result,the red color of the AuNPs will be maintained as a function of target miRNA.In contrast,in the absence of let-7a,DSN will not hydrolyze the ssDNA.When the unmodified AuNPs is mixed with appropriate concentration of ssDNA,the amount of ssDNA is not enough to fully stabilize the AuNPs.So if concentrated NaCl solution is added to the mixture,salt-induced self-aggregation of AuNPs will occur rapidly accompanied with color change from red to blue.In this method,10 fmol miRNA can be distinguished by the nake eye.The proposed method is simple and requires no markup.It realizes the visualization and sensitive detection of miRNA under constant temperature based on DSNSA and AuNPs.2.Silver nanoclusters-based fluorescence strategy for the determination of miRNA by combining with duplex-specific nuclease signal amplification.Silver nanoclusters(AgNCs)have super fluorescence properties.Based on AgNCs and DSNSA,a simple and sensitive fluorescence assay for miRNA detection has been developed.Two special ssDNA probes are designed.The 5' terminal of DNA probe I is complementary to the target miRNA,its 3' terminal is G-rich sequence.The 5' terminal of DNA probe ? is the DNA template of dark AgNCs,its 3' terminal is DNA squences as the same as target miRNA.In the absence of target miRNA,the DNA probe I can't be hydrolyzed by DSN.Its 5' terminal hybridizes with 3' terminal of DNA probe ?,leading to a close proximity betweenthe AgNCs and G-rich squence of DNA probe I,generating higher fluorescence signal.In contrast,in the presence of target miRNA,miRNA hybridizes with 5' terminal of DNA probe I,forming RNA-DNA heteroduplexes.DSN selectively recognizes and cleaves the DNA sequence in the RNA-DNA hybrid.5' terminal of DNA probe I is hydrolyzed by DSN,leading to that the miRNA and the G-rich squence of DNA probe I is released.The released miRNA can initiate another round of hybridization and DSN digestion reactions.Repeating the cycle until all of the DNA probe I is hydrolyzed by DSN.The released G-rich sequence can't get close enough to AgNCs by hybridization,accompanying with a weak fluorescence signal.The detection limit is as low as 0.08 fmol(0.8 pmol/L)let-7a.Baesd on AgNCs sensor and DSNSA,our strategy realizes the sensitive detection of miRNA.A new nanosensor platform has been provided for miRNA detection.