Study And Application Of Functional Dna Nanoprobes In Living Cells And Living Animals Imaging

Molecular probes that enable imaging of specific molecules or physiological processes in living cells and animals are indispensable tools in deciphering pathological biology and theranostics for different diseases.Fluorescence is a very practical non-invasive technology that uses super high sub-cellular resolution to visualize morphological details in vivo and perform effective medical diagnosis and treatment.In addition to high sensitivity and high spatial resolution,fluorescence also features fast imaging,low cost,and convenience.In recent years,in vivo fluorescence imaging technology has been developed,which is very important for exploring the basic biological mechanism and pathological progress,and it provides a lot of important information for the diagnosis and treatment of diseases.Cancer is one of the leading causes of death in the world.The quantitative detection of tumor markers at different stages has profound implications for identifying patients at different clinical stages and developing appropriate treatment.A number of fluorescence molecular probes targeting cancer biomarkers have been developed to target living cells or living animals,including conventional antibodies,DNA/RNA aptamers,peptide-based probes and so on.With the rapid development of DNA nanotechnology,one-,two-or three-dimensional DNA nanomaterials can be designed and widely used in the field of biochemistry.Due to its controllable size,shape and surface function,DNA nanostructure can be assembled to the defined size,and component strands can be programmed to carry functional probes or drugs throug h complementary base pairing.These characteristics make DNA nanostructures own strong transportation capabilities,and provide new prospects for the construction of drug carriers.DNA nanostructures can be designed and modified as the basic unit of biosen sors for the detection of cancer markers.Due to advantages of safety,non-toxicity and convenient synthesis,DNA nanostructures show great potential in in vivo applications,and provide an effective disease detection platform for the field of biological a nalysis.Combining main challenges in the fields of biomedical research,we have developed some new fluorescence molecular probes for the detection of biomarkers,fluorescence imaging,drug delivery,diagnosis and treatment in living cells and animals using DNA nanostructure probes as well as in vivo fluorescence imaging technology.The main contents are described as follows:In chapter 2,a novel tripartite DNA probe has been developed based on DNA-minimal Y-shaped structure to connect three different functional units.Two of these units achieve fluorescence detection of target RNA through hybrid ization chain reaction(HCR),and another unit is used to connect a probe with multiple folates for specific targeting living cells in the following.Due to DNA-minimal structure,the probe achieves simple,economical and high-yield modular synthesis,and has high sensitivity and high specificity for the detection of in vitro mi R-21,the detection limit has reached sub-picomolar.Due to the simple design of structure,sensitive and accurate detection method,and better stability in serum,the tripartite DNA probe may open the door for RNA imaging in living cells and living animals using DNA-minimal structure.In chapter 3,based on the novel tripartite DNA probe which designed in chapter2,RNA fluorescence imaging has been achieved in living mice via in vivo hybridization chain reaction(HCR)for the first time.The multiple folates linked DNA probe exhibits selective and efficient internalization into folate(FA)receptor-overexpressed cells via a caveolar-mediated endocytosis mechanism,avoiding lysosomal degradation effectively.The tripartite DNA probe with enhanced stability enables specific delivery into tumor cells and allows high-contrast imaging of mi R-21 in living mice.The tripartite DNA design may creat a new direction for RNA imaging in mammals and provides great potential for tumor biology studies and theranostics.In chapter 4,an aptamer-linked Y-shape DNA probe has been designed using catalytic hairpin assembly(CHA)strategy to amplify trace amounts of target mRNA.At the same time,we introduce the phosphorothioate-modified hairpin probes H1 and H2 for mRNA fluorescence detection in living mice.This probe owns high efficiency and high selectivity to internalize into nucleolin-overexpressed cells and enables quantitative detection of survivin mRNA in different cell lines.The phosphorothioate-modified DNA probe shows further improved stability and resistance to nucleases,making it more suitable for in vivo fluorescence imaging of target mRNA.Combining Y-shape DNA scaffold and catalytic hairpin assembly(CHA)method,the intramolecular CHA and target cyclic release have been achieved,improving the sensitivity of target detection and reducing the reacti on background in vivo.Therefore,this method provides an improved strategy to promote the application of in vivo fluorescence imaging in early clinical diagnosis and treatment.In chapter 5,multi-dimension nanovehicles have been constructed for loading siRNA which specific targeting e GFP gene.The nanovehicles consist of dozens of DNA single strands.This simple,flexible and rapid design method is more suitable for large-scale production in biomedical research on living cells and living animals.The2D/3D nanostructures achieve high-efficient delivery into living cells,making e GFP-specific siRNA enter cells and silence the corresponding gene,achieving e GFP protein knockout successfully.Compared with single-stranded DNA,rectangular and brick nanovehicles can enter cells efficiently for tumor targeted therapy,which will play an important clinical role in the fields of cancer diagnosis and treatment.