Nucleic Acid Functionalized DNA Nanowires For MiRNA Analysis In Cells And In Vivo

The development of accurate methods for early disease diagnosis has been an long concerned subject.At present,some related biomolecules are often used for diseases diagnosis,such as miRNAs,which are a class of short,small and nonprotein-coding ribonucleotides and play important roles in the regulation of gene expression.A number of researches have shown that aberrant miRNA expression is strongly associated with a variety of diseases.Therefore,the development of efficient miRNA detection technologies is important for the early diagnosis of diseases.DNA nanowires are a new kind of nanostructures,which possess flexible design,high stability and low cytotoxicity.So,by combining DNA structure and nucleic acid probe,it may provide a new method for analyzing of tumor markers in cells and in vivo.To address the drawbacks of reported nucleic acid probes,such as slow response,poor stability and high false positive signals,herein,by employing nucleic acid functionalized DNA nanowires,two novel DNA nanoprobes were developed for imaging of miRNAs in tumor cells and in vivo.These studies are summarized as follows:(1)Multivalent self-assembled DNA nanoprobe for accelerated biomarker imaging in living cells and in vivoIn this work,by assembling aptamer and DNA probe on the surface of DNA nanowire,we constructed a DNA nanoprobe with spatial confinement effect for miRNA sensing.In the presence of target miRNA-21,they can trigger a strand displacement reaction and release the strand with quencher labeled,thereby restoring the fluorescent signal.The experimental results show that the nanoprobe reduces the reaction time by about 2.5 times compared with the free double-stranded probe,and the detection limit is 100 pM.Based on the excellent stability and biocompatibility of DNA nanostructures,the probe was also enabled imaging miRNA-21 in vivo.(2)Endogenous enzyme driven DNA nanoprobe for imaging miRNA in cells and in vivoTo improve the detection accuracy,an endogenous enzyme driven DNA nanoprobe was developed for imaging miRNA in cells and in vivo.The developed DNA nanoprobe was used for ratiometric fluorescence imaging of let-7a via AP site functionalized DNA nanowires and fluorescence resonance energy transfer(FRET).When both targets are present,the system generated FRET signals for accurate detection of miRNA.Based on the excellent stability of DNA structure,the imaging ability of the probe in living mice was further explored and the results showed that the nanoprobe could accurately distinguish normal tissue from tumor area.