Study Of New Methods For The Imaging Of Cell Surface Membrane Proteins And Intracellular MiRNAs Based On DNA Molecular Devices

Changes of cellular components are closely related to human diseases.Therefore,it is of great significance to study the role of related components such as protein and miRNA in cell life activities.Cell imaging and cellular component analysis methods,for instance,immunofluorescence,Western blot and in situ hybridization technology,have provided the potent tools for deeply understanding the changes of cellular components.However,as the research moves,there are many challenges need to be solved.DNA is not only the genetic material of life,but also a natural nano-material which has attracted extensive attention because of the unique biochemical function and physical property.The emergence of molecular devices constructed based on DNA,such as DNA switch,DNA motor,and DNA walker,have injected new vitality for the innovation of cell imaging and analysis techniques.In this study,two type of DNA molecular devices had been constructed for different purposes of cell imaging.One is for imaging and quantification of tumor membrane protein.The other is for imaging of miRNA in single cell.The detailed contents were shown as follows.1.Nondestructive analysis of tumor-associated membrane protein in situ imaging and quantification based on DNA molecular device constructed by double-labeled deoxyribozymeThe comprehensive analysis of tumor-associated membrane proteins(TMPS)expression level and positioning is very important for profiling tumor cell.The existing analysis techniques can be basically divided into ex situ detection and in situ imaging.The most commonly used technique of the former is Western blot,while that of the latter is immunofluorescence(IF).These two types of technique are necessary to comprehensive analysis of tumor cell.However,the common techniques are limited to some extent,and the mostimportant point is that they can not comprehensively analyze the same batch of cells at the same time through experiment.Based on the above considerations,a well-designed and double labeled DNAzyme were used to construct DNA molecular device.Compared with western blot results,the fluorescence quantitative analysis results of the DNA molecular device showed good reliability,universality and sensitivity.The DNA molecular device allowed for imaging in situ and quantification of the batch of cell in same system.What is more,flow cytometer results,cell drug susceptibility results,cell growth results both show no damage to cell after analysis.2.Imaging of miRNA in single cell based on Vent and Nb.Bsm I powered DNA walkerIn situ imaging of miRNA in single cell can help us to deeply und erstand the role of miRNA in regulating loop in cell and individual differ ence in disease.However,miRNA sequences is short,low abundance,and high homologous sequences often result in severe nonspecificity.Therefor e,it remains a challenge to find in situ imaging method with high accura cy and good sensitivity.Because of favorable loading effect of old nanop article and programmability of DNA,a DNA molecular device had been c onstructed of fluorescence-labeled DNA and gold nanoparticles that allowe d for miRNA imaging in situ.Briefly,miRNA would extend along the flu orescence-modified DNA chain under the action of Vent amplification enz yme.After that a digestion site of Nb.BsmI was exposed,so Nb.BsmI ca n cut fluorescence-labeled DNA which result in strong fluorescence signal in solution.The specificity of detection was guaranteed under the action circle of recognition-amplification-digestion-recognition-digestion.The imagi ng results are in good agreement with the results of qRT-PCR.Such DN A device had provided a new method for study miRNA imaging in cell.