Research On Construction And Application Of FRET Nanoflare Probe Based On DNA Nanowire

MicroRNAs(miRNAs)are a class of endogenous,and non-coding RNAs with a length of 19-23 nucleotides,which play an important role in regulating gene expression.It has been found that miRNAs are related to the occurrence and development of various human diseases.The expression profile of miRNAs can be used to identify a variety of specific diseases.Therefore,it is necessary to develop efficient methods for miRNA detection.FRET nanoflare is a new type of molecular beacon for detecting targets by combining fluorescence resonance energy transfer technology(FRET)and double fluorescent group-labeled DNA strands(Flare).FRET fluorescence signals can be used to achieve the ratio fluorescence detection of target.This method can effectively reduce the fluctuations brought by the system and avoid the generation of false positive signals.In this thesis,two imaging methods for miRNA in living cells are designed with FRET nanoflare.The details are as follows :(1)It is important to develop an accurate and efficient method to detect cancer markers.In this work,combined aptamer and hybridization chain reaction,a FRET nanoflare probe based on DNA nanowires was designed to detect miRNA in cells.When the target cells exist,aptamer can specifically recognize the target cells and trigger the hybridization chain reaction,forming the nanowires containing many double fluorescent groups labeled signal probes(F),which enter the cells through aptamer mediated.When miRNA-21 exists in the cell,F is replaced and hairpin structure is formed,thus FRET signal is generated.On the one hand,FRET signal is used to reduce the influence of system fluctuation;on the other hand,aptamer is used to recognize tumor cells and nanoflare probe is used to recognize miR-21,which significantly improves the specificity of detection.(2)Because of the low content of some target s in cells,it is necessary to use signal amplification technology to further improve the sensitivity of detection.This work combined strand displacement amplification technology,developed a DNA nanowire-based FRET nanoflare probe to detect miRNA-21 in cells.DNA nanowires are constructed by self-assembly,which contain many double fluorescent groups labeled Flares(F)and auxiliary strands,and then entered the cells via aptamer.When miRNA-21 is present in the cell,F is replaced and hairpin structure is formed to generate a FRET signal.At the same time,miRNA-21 can be released through the auxiliary strand to participate in the next round of response.In this design,a target participating in the reaction can generate multiple FRET signals.This method not only has the advantage of high specificity of the previous work,but also reduces the detection limit by two orders of magnitude,which significantly improves the detection sensitivity.