Construction Of Functional DNA Nanoprobes And Their Applications In Tumor Diagnosis

Malignant tumors are one of the major diseases threatening human health and life at present,and their morbidity and mortality are increasing year by year,which seriously affects the quality of human survival and life expectancy.The development of highly sensitive,highly specific,intelligent and safe activated probes is of great significance to achieve precise diagnosis,efficient treatment and prognostic assessment of tumors.In recent years,the rapid development and continuous integration of functional nucleic acids and DNA nanotechnology have provided new directions and ideas for tumor diagnostic strategies.On the one hand,functional nucleic acids with special biological functions,such as Aptamer,DNAzyme and molecular beacons(MB),have been developed one after another and shown flexible design,easy synthesis and modification,thus providing the important molecular tools for the construction of novel tumor diagnostic probes.On the other hand,DNA nanotechnology developed by using nucleic acid molecules as “building blocks” has shown good biocompatibility,biodegradability,structural controllability,programmability and addressability,and can integrate multiple functional units in a reasonable and orderly manner,thus providing the solid technical support for the development of safe,stable,efficient and multifunctional nucleic acid molecular probes.However,most nucleic acid probes are still facing some challenges in tumor diagnosis,such as high background signal,low sensitivity and insufficient precision.In this thesis,with the goal of developing new strategies for the accurate and effective tumor diagnosis,we selected tumor cells and intracellular biomarkers as targets based on the differences between tumor cells and normal cells,and constructed a series of in situ activated functionalized DNA nanoprobes by using functional nucleic acids and DNA nanotechnology for systematic evaluate tumor activation imaging at the cellular or living animal level.The specific work is as follows:1.Highly sensitive detection of tumor cells via split aptamer mediated proximity-induced hybridization chain reactionTo address the problems of high background signal,difficult design and low detection sensitivity of conventional aptamer probes,we developed a cell surface-specific recognition-driven in situ DNA self-assembly strategy for highly sensitive detection of tumor cells by utilizing target-induced allosteric split aptamers and proximity-induced hybridization chain reaction(HCR).This strategy relies on two split aptamer probes(Sp-a and Sp-b)with split aptamer sequences and split HCR trigger sequences and two hairpin probes(H1 and H2).In the presence of target cells,Sp-a and Sp-b would self-assemble on the cell surface leading to a change in their conformation,allowing the two split trigger sequences to form an intact trigger sequence to initiate the HCR reaction with H1 and H2,thereby generating significantly enhanced fluorescence signals for highly sensitive detection of tumor cells.Using human hepatocellular carcinoma SMMC-7721 cells as the targets,this strategy could detect as low as 18 cells/150μL binding buffer and was successfully used for the quantitative detection of target cells in 10% fetal bovine serum samples.Moreover,this strategy showed superior specificity,enabling the identification and characterization of target cells in mixed cell samples.Therefore,benefiting from the low-background split aptamer and proximity-induced HCR amplification,this strategy has achieved high sensitivity and specificity for the detection of tumor cells,promising new ideas for the development of nucleic acid molecular probes in tumor diagnostic studies.2.An endogenous miRNA-activated DNA nanomachine for intracellular miRNA imaging and gene silencingIn the previous work,we performed fluorescence imaging analysis of whole tumor cells,which could not provide insight into the pathological mechanisms and dynamic change processes of tumors at the molecular level.Based on this,we selected tumor-associated miRNA as the target,used aptamer-functionalized DNA nanowires as the tumor-specific targeting vectors,and combined with the entropy-driven catalysis(EDC)and split-DNAzyme for target m RNA cleavage to construct an endogenous miRNA-activated DNA nanomachine(EMDN)for amplified imaging of miRNAs in tumor cells and activated gene silencing therapy.When EMDN specifically enters tumor cells,the target miRNA initiates the EDC reaction of EMDN,generating significantly enhanced and amplified ratiometric fluorescent signals,and activating split-DNAzyme to recognize and cleave the target m RNA,thus enabling simultaneous diagnosis and treatment of tumor cells.In this system,since the target miRNA-triggered EDC reaction is carried out along DNA nanowires,its reaction kinetics and detection sensitivity are significantly enhanced,and the dynamic changes of specific miRNAs within tumor cells can be monitored in real time.In addition,DNA strand replacement reactions activated by endogenous miRNAs are used for the controlled release of split-DNAzyme,which is conducive to precise and effective gene therapy.Therefore,EMDN integrates tumor marker imaging and stimulus-responsive gene therapy,offering great potential for the construction of early tumor diagnosis and efficient therapeutic platforms.3.A dual endogenous stimuli-responsive framework nucleic acid nanodevice for intracellular miRNA imagingTo further improve the precision of tumor imaging,based on framework nucleic acids and HCR,we constructed a dual endogenous stimulus-responsive framework nucleic acid nanodevice(D-FNAN)for precise and highly sensitive imaging of miRNAs in tumor cells using overexpressed adenosine triphosphate(ATP)and human depurine/depyrimidine endonuclease 1(APE1)in tumor cells as the endogenous stimuli,ATP aptamer and AP sites as the stimulus-responsive components,and AS1411 aptamer as the target recognition element.When DFNAN was endocytosed into the tumor cells,the target miRNA would initiate the DNA self-assembly cascade reaction of D-FNAN under the co-activation of ATP and APE1,resulting in an amplified fluorescent signal.The results showed that D-FNAN could effectively distinguish tumor cells from normal cells,and successfully achieved the imaging of specific miRNA selectively activated by endogenous ATP and APE1 in nude mice with improved tumor imaging contrast.Therefore,this work describes a multi-stimulus-responsive framework nucleic acid nanodevice for the precise and effective tumor imaging,which is important for clinical diagnosis and therapeutic evaluation of tumors.4.A endogenous enzyme-driven framework nucleic acid nanodevice for dual imaging of intracellular miRNAsTo address problems such as the single miRNA imaging strategies in the previous two work that may not provide reliable and satisfactory pathological information,we constructed an endogenous APE1-driven framework nucleic acid nanodevice(A-FNAN)for simultaneous,highly sensitive imaging of dual intracellular miRNAs.This system employs two tumor-associated miRNAs as the target molecules with APE1 as the driver,AP sites as the specific response elements,and framework nucleic acids as the structural backbone,and further incorporates the HCR reaction.When internalized into the target cells,A-FNAN would trigger the DNA self-assembly cascade reactions in response to mi R-21 and mi R-155 driven by the tumor-specific APE1,resulting in two amplified fluorescent signals,respectively.The results showed that A-FNAN achieved simultaneous amplified imaging of two target miRNAs in the presence of APE1 in tumor cells,which was beneficial to avoid false-positive signals and improve the accuracy of relevant tumor diagnosis,and was successfully used for precise differentiation of target tumor cells from other tumor cells and normal cells.Thus,A-FNAN achieves the simultaneous detection of multiple tumor markers driven by endogenous cellular stimulator,compensating for the uncertainty of detecting a single marker,which is expected to provide a reliable basis for the development of accurate and effective tumor diagnostic platforms.