Functionalized DNA Nanomachines Based On Framework Nucleic Acid For Intracellular Imaging Of APE1

As a multifunctional characterization technology,fluorescence analysis is widely used in physics,chemistry,biology and other fields.Establishing specific recognition and response mechanism between fluorescence analysis method and target biomolecules constitutes fluorescence biosensor.According to the fluorescence signals of different intensity generated by the reaction systems with different concentrations of targets,the quantitative analysis of target can be realized.Fluorescence biosensors are widely used in disease diagnosis,food safety analysis and environmental monitoring due to their advantages of fast response speed,simple operation,high sensitivity,high spatial and temporal resolution.However,for the detection of some low abundance biomarkers（e.g.,proteins,small molecules,nucleic acids,etc.）,traditional fluorescence biosensors cannot achieve accurate and sensitive quantification.Therefore,the construction of fluorescence biosensors combined with efficient amplification methods for detection of low abundance biomarker still needs to be explored.In recent years,some enzyme-free nucleic acid amplification methods,such as catalytic hairpin assembly（CHA）,hybridization chain reaction（HCR）and entropy-driven catalytic reaction（EDC）,have attracted wide attention because of their simple operation and sensitive response.Furthermore,as a typical DNA molecular structure,framework nucleic acids（FNAs）have the advantages of high stability,modifiability,high programmability,precise assembly on the nanoscale,predictability,and control,which can be realized by precise design of multi-functional nucleic acid.Therefore,different DNA nanomachines were constructed by the multi-functional framework nucleic acid structure combined with enzyme-free nucleic acid amplification technology in this dissertation,which could be used for the cycle amplification of biological target molecules,realizing sensitive detection of tumor markers and tumor therapy.This study is mainly divided into the following two parts:1.Construction of fast-walking tetrahedral DNA walker with four arms for sensitive detection and intracellular imaging of apurinic/apyrimidinic endonuclease 1.DNA walker is a kind of DNA molecular device with programmable structure and function,which is assembled by walking arm and DNA track.Such devices can autonomously travel along preset DNA tracks,which have broad prospects for biosensor and bioanalysis applications.In early reports,single-strand DNA walkers with free swinging arms were able to walk around the tracks freely;however,they walked on DNA tracks relying on random collisions in a homogeneous solution with low walking efficiency and low nuclease resistance as well as easy derailment from the preset tracts.To address those issues,we designed a DNA walker with a tetrahedral DNA framework structure and nonplanar multisites arms to improve the walking efficiency and nuclease resistance.The DNA walker was driven by the CHA reaction under the triggering of apurinic/apyrimidinic endonuclease 1（APE1）,which could be used for evaluating APE1activity and intracellular imaging.In contrast to traditional DNA walkers,the tetrahedral DNA walker with the rigid 3D framework structure and nonplanar multi-sites walking arms endowed with high collision efficiency,showing a fast walking rate and high nuclease resistance.Impressively,the initial rate of the tetrahedral DNA walker with four arms was 4.54 times faster than that of the free bipedal DNA walker and produced a significant fluorescence recovery in about 40 minutes,achieving a sensitive detection of APE1 with a low detection limit of 5.54×10^-6 U/μL as well as ultrasensitive intracellular APE1 fluorescence activation imaging.This strategy provides a novel DNA walker for accurate identification of low-abundance cancer biomarker and potential medical diagnosis.2.Target-mediated self-assembly of DNA networks for sensitive detection and intracellular imaging of APE1 in living cells.In the last system,we designed a DNA walker for the detection and imaging of APE1,but only one purpose could be realized in the last strategy.Therefore,in order to further realize the multifunctional application,we have developed a nanoplatform that integrates multifunctional diagnosis and gene regulation to realize the function of diagnosis and treatment.Meanwhile,in view of some problems existing in the current traditional CHA reaction,such as low efficiency of random collision before reactants in homogeneous solution,long reaction time,etc.,we improved these problems through the design of DNA network self-assembly.Herein,giant DNA networks were assembled by two kinds of functionalized tetrahedral DNA nanostructures f-TDNs（f-TDN1 and f-TDN2）for sensitive detection and intracellular imaging of APE1 as well as gene therapy in tumor cells.Impressively,the reaction rate of catalytical hairpin assembly（CHA）reaction on the f-TDNs was much faster than that of conventional free CHA reaction owing to high local concentration of hairpins,spatial confinement effect and the production of giant DNA networks,which significantly enhanced fluorescence signal to achieve a sensitive detection of APE1 with the limit of 3.34×10^-8 U/μL.More importantly,the aptamer Sgc8assembled on f-TDNs could enhance the targeting activity of the DNA structure to tumor cells,allowing it to endocytose into cells without any transfection reagents,which could achieve selective imaging of intracellular APE1 in living cells.Meanwhile,the si RNA carried by f-TDN1 could be accurately released to promote tumor cell apoptosis in the presence of endogenous target APE1,realizing effective and precise tumor therapy.Benefiting from the high specificity and sensitivity,the developed DNA nanostructures provide an excellent nanoplatform for precise cancer diagnosis and therapy.