Construction And Application Of DNA Logic Sensing-Based Cancer Cell Imaging And Tumor Marker Monitoring Method

Currently,relevant biomarkers secreted or expressed by tumor cells have become novel targets for early cancer diagnosis.some tumor markers are not expressed only on specific tumor cells,and most of the currently reported biosensing strategies detect only a single tumor marker and are prone to false-positive signals,which greatly hinders early cancer diagnosis and precision therapy.DNA logic circuits can logically analyze the cellular DNA nanoflowers have the advantages of easy modification of ultra-long strand DNA,accepting a large number of various signal molecules and excellent control over the size of the assembled structures,which facilitate the design of highly sensitive biosensing systems and the introduction of signal units.These characteristics facilitate high-sensitivity biosensing and multiplexed bioimaging.In this thesis,two novel DNA logic sensing platforms were constructed for tumor selective imaging and precise and efficient gene therapy by taking advantage of the excellent structural properties of DNA nanoflowers.The research is summarized as follows.(1)A multifunctional DNA nanoflower based on AND logic gates to trigger cancer cell imaging and co-activate DNAzyme-driven gene silencing was constructed,and the RCA template was cleverly designed to contain three parts: AS1411 aptamer,AND logic gates recognizing two biomarkers APE1 enzyme and ATP,and therapeutic DNAzyme in an inactive conformation.The AS1411 aptamer,which recognizes the nucleophilin receptor of cancer cell membrane,helps to facilitate the specific uptake of DNA nanoflowers by cancer cells;the two biomarkers APE1 enzyme and ATP,which are highly expressed in tumor cells,are used as inputs and are analyzed by the AND logic gate logic calculation to precisely realize the fluorescence imaging of MCF-7 cells;the therapeutic DNAzyme in inactive conformation is also affected by the AND logic gate execution situation,and DNAzyme activity was activated when two tumor biomarkers were co-present,specifically silencing MCF-7 intracellular genes and reducing MCF-7 cell survival to 69.3% with no toxic effect on L02 cells.In addition,the expression of survivin protein was almost completely inhibited.Therefore,this DNA logic sensing platform provides a new idea for the delivery of therapeutic DNAzyme and tumor-specific imaging and gene therapy,and has a promising application in the precision diagnosis and treatment of malignant tumors.(2)In this study,novel dual-target DNA nanoflowers were constructed for targeted delivery of siRNA and imaging monitoring of the release process.A large number of siRNAs whose dangling DNA sequences bind to RCA products through base complementary pairing,wrapping them into tight flower-like nanostructures to protect siRNAs from nuclease degradation;the dual-target DNA nanoflowers contain a large number of tandemly repeated AS1411 aptamers,which can trigger receptor-mediated endocytosis and thus enhance the uptake of siRNAs without transfection agents;the construction of AND logical unit was constructed,which can intelligently respond to the cancer cell environmental factor GSH and tumor marker mi R-21,logically control the release of siRNA in tumor cells and can monitor the release process using fluorescence imaging.siRNA was precisely released into tumor cells,inhibiting the growth of MCF-7 cells,reducing their survival rate to 72.1%,and well suppressing the expression of survivin protein.Thus,this study reports that DNA nanoflowers generated by rolling loop amplification open up new ideas in the protective,precise,and visual delivery of siRNA,providing new possibilities for clinical applications of siRNA in cancer therapy.