Studies Of Cancer Diagnosis And Therapy Based On MOF-polymerase Amplification Reaction And DNA Origami Logic Chips

World Health Organization cancer experts represent,there will be about 19.2million cancer cases and 10 million cancer deaths among the worldwide in 2020,but one-third of cancers could be cured by early detection.Therefore,early detection of cancer and timely treatment is the key to improve cancer survival.In recent years,the continuous discovery of tumor markers provides a reliable basis for the early detection of cancer.However,there are differences about the types and expression levels of tumor markers in cells.To find cancer as soon as possible,it is necessary to establish some multifarious and highly sensitive analytical detection methods in cells for the analysis of different types and expression level tumor markers.In addition,among many c ancer treatments,gene therapy has been favored by the majority of researchers because of its advantages such as small side effects and no drug resistance.So,we used the MOF nanomaterials,polymerase-based amplification reaction,DNA logic computing,DNA origami in the advantages of sensitive detection and effective treatments for cancer.Through the combination of MOF nanomaterials and polymerase-based amplification reaction,DNA logic computing and DNA origami,we construct a series of new reaction and delivery platforms for the detection of cancer markers,and diagnose-responsive gene therapy drug release to enable the integration of diagnosis and gene therapy.Main sections as follows:Polymerase amplification can realize highly sensitive detection of tumor markers.Because polymerase fail to freely enter cells,it is difficult to detect and analyze tumor markers in living cells by polymerase-based amplification reactions.To solve this problem,In Chapter 2,we develop biomineralized metal-organic framework nanoparticles(MOF NPs)as a carrier system for intracellular co-delivery of?29 DNA polymerase(?29DP)and nucleic acid probes.A p H-sensitive biodegradable MOF NP of zeolitic imidazolate framework-8(ZIF-8)is utilized to encapsulate?29DP and adsorb nucleic acid probes.After uptake into cells,the encapsulated?29DP and surface adsorbed DNA probes are released and escaped from endoly-sosomes.In the presence of?29DP and circular DNA templates,the intracellular mi RNA-21 triggers a rolling circle amplification(RCA)reaction and the autonomous synthesized Mg²⁺-dependent DNAzyme cleaves the fluorogenic substrate,providing a readout fluorescence signal for the monitoring of mi RNA-21.Inspired by the study of Chapter 2,we build a DNA machine based o n a primer exchange reaction(PER)for m RNA imaging and gene therapy in Chapter 3.By using zeolitic imidazolate framework-8 nanoparticles(ZIF-8 NPs)to co-deliver the components including a primer,hairpin and strand displacing polymerase to the living cells,the PER-based DNA machine can be initiated by intracellular survivin m RNA and continuously produce Bcl-2 antisense DNA(ASD),where the Bcl-2 ASD react with molecular beacons(MBs)outputting fluorescence signal for target imaging and bind to a fragment of the Bcl-2 m RNA sequence to silence Bcl-2 gene expression.The results demonstrate that ZIF-8 NPs can not only protect the polymerases and nucleic acid probes from protease attack and nuclease degradation but also efficiently release cargos and escape from endo-lysosomes.The intracellular PER-based DNA machine has been demonstrated to be able to sensitively image survivin m RNA expression levels and selectively kill the cancer cells and has no effect on the normal cells.The PER-based DNA machine may provide a promising platform for early stage tumor diagnosis and more precise tumor therapy.At the molecular level,a great deal of research work on dynamic DNA molecular circuits is used for disease diagnosis and biomedicine.However,how to design programmable molecular circuit devices to autonomously and accurately diagnose multiple low abundance biomolecules in complex cellular environments remains a challenge.In Chapter 4,we constructed a DNAzyme logic circuit for the analysis and imaging of multiple micro RNAs in living cells using Cu/ZIF-8 NPs as the nanocarrier of the logic gate element and the Cu²⁺donor of Cu-DNAzymes.The logic gate element of the logic operation system was adsorbed on the surface of Cu/ZIF-8 NPs via electrostatic interaction.After internalization,p H-responsive ZIF-8 NPs can efficiently release the logic gate element and Cu²⁺,we realized multiple endogenous mi RNA-initiated biocomputing operations,including one YES logic gates and binary logic gates(OR and AND)in different living cells.Cu²⁺-DNAzymes logic circuits can not only operate to quickly response multiple endogenous mi RNAs in the complex cell environment,which also provides a new research method for the application of DNA biocomputing circuit in living cells.Because the DNA has the advantages of self-assembly and specific recognition,DNA based-logic circuits have aroused wide interest.However,some logic circuits lack space limitations and completely rely on diffusion between logic elements to promote the interaction between sensing elements.To address this problem,in Chapter 5 we integrate all the logic elements into a DNA origami nanostructure to form a series of mi RNAs responsive DNA origami gene logic chips(DOGLC).We created four DOGLC(AND,OR,NAND,NOR,and XOR)using mi RNAs as input,red fluorescent protein(RFP)as the output and the half adder using mi RNAs as input,red fluorescent protein(RFP)and green fluorescent protein(GFP)as the output in mammalian cell.We observed RFP or GFP significant repression and activation in the OFF and ON systems,respectively for response multiple endogenous mi RNAs to identify the normal and cancerous cells.Additionally,we selectively control cell-death pathways between target and normal cells by connecting a 3 input AND-AND DOGLC with apoptotic regulatory circuits.Thus,our synthetic DOGLC with logic operation could provide a powerful tool for future therapeutic applications.