Protein Scaffolded DNA Nanotetrads For Highly Efficient And Sensitive Detection Of Biomaekers Through Hybridization Chain Reaction

Recently,the problems of disease threats,food security and environmental pollution are increasingly arresting humans' attention,especially for human health.As analytical chemistry has become the key to the research fields of life science and nanotechnology in the 21 st century,people require for a much higher demand on scientific researchers for the purpose of addressing the growing social issues.Biological markers,which can reflect pathologic process of the body,play critical roles in various aspects in recent years,including early diagnosis,disease prevention,drug target identification and prognosis of monitoring.Thus,it is necessary to build biosensors with highly specificity and sensitivity.The isothermal amplification of hybridization chain reaction(HCR),which has the advantages of simple operation,high sensitivity and powerful specificity,is widely used for the detection of biological macromolecules,such as nucleic acids,proteins and enzymes.Based on protein scaffolded DNA nanotetrads mediated HCR,we has established new biosensing methods for highly efficient and sensitive detection of biomaekers of telomerase and uracil-DNA glycosylase(UDG).Specific study of the paper was described as follows:Telomerase has a big influence on DNA replication,chromosome stability and tumorigenesis.Efficient platforms for intracellular delivery of nucleic acids are essential for biomedical imaging and gene regulation.In chapter 2,we develop a recombinant fusion streptavidin as a novel protein scaffold for DNA nanotetrads for highly efficient nucleic acid delivery and telomerase imaging in living cells via crosslinking hybridization chain reaction(cHCR).The recombinant streptavidin(SA)protein is designed to fuse with multiple SV40 NLSs and nuclear export signals and prepared through Escherichia coli expression.The recombinant NLS-SA protein allows facile assembly with four biotinylated DNA probes via high-affinity non-covalent interactions,forming a well-defined DNA tetrad nanostructure.The DNA nanotetrads are demonstrated to confer efficient cytosolic delivery of nucleic acid probes via a caveolar mediated endocytosis pathway with effective lysosomal escaping.Moreover,the nanotetrads enable efficient cHCR assembly in response to telomerase in vitro and in cellulo,affording ultrasensitive detection and spatially resolved imaging for telomerase with a detection limit as low as 90 cells/mL.The fluorescence brightness obtained in live cell imaging is found to be dynamicallycorrelated to telomerase activity and the inhibitor concentrations.Therefore,the proposed strategy may provide a highly efficient platform for nucleic acid delivery and imaging of biomarkers in living cells.Uracil-DNA glycosylase(UDG),one of the most important base excision repair enzymes,plays a vital role in removing the genotoxic uracils from DNA and maintaining the genome integrity.Traditional methods for UDG detection suffer from the lack of signal amplification,which limit the rapid and sensitive detection of low abundance of UDG.In chapter 3,we develop a novel DNA nanotetrads mediated cHCR strategy for rapid and highly sensitive detection of UDG activity.DNA nanotetrads are prepared by assembling the streptavidin protein with four biotinylated hairpin DNA probes through non-covalent high affinity interactions.Because the nanotetrad has four DNA probes on its surface,it enables cHCR to UDG response to form clumps of 3D crosslinked DNA hydrogel products,affording high sensitivity and specifity for UDG detection.Moreover,the nanotetrad allows proximity reactions of cHCR,which results in high local concentration of DNA probes,providing a simple platform for high efficiency of HCR circuits.The proposed method shows better sensitivity than reported methods with a detection limit as low as 0.0013 U/mL.Therefore,this strategy may have the potential to provide a simple,rapid and sensitive platform for UDG activity analysis,living cell imaging and related-pharmaceutical screening.