Studies Of Smart Nanoplatforms Based On DNA Framework Materials And MnO₂/ZIF-8 For Biosensing And Therapy

The development of biosensor platform plays a key role in the identification of biomarkers and the applications of clinical diagnosis in research environments.Currently,there are many forms of nucleic acid-based biosensors,from simple DNA double-stranded structures to multidimensional nucleic acid nanostructures that can respond to stimuli.Based on their programmability,spatial organization and biocompatibility,nucleic acid nanostructures have been widely used in biomedical research,such as biosensors and imaging,disease diagnosis and treatment.However,how to achieve sensitive monitoring of intracellular biomolecular markers remains to be explored.In this dissertation,we will take the advantages of DNA nanoframe structure to realize sensitive detection and imaging of intracellular target molecules through catalytic hybridization self-assembly(CHA)of three-dimensional(3D)DNA tetrahedra.At the same time,this study will also focus on the use of DNA tweezer for intracellular biomarker imaging and gene silencing therapy,to realize the integration of diagnosis and treatment.Because of its remarkable chemical and physical properties,nanomaterials have been widely used in the detection of cancer-related markers and the diagnosis of tumors and other diseases.Using nanomaterials to carry DNA functional probes into the nanoplatform of cells can monitor the biomarkers closely associated with tumorogenesis in living cells,realize the early diagnosis and prognosis monitoring of cancer,and improve the cure rate of the disease.Among the existing treatment methods,gene therapy has been paid more attention because of its biocompatibility and no drug resistance.Based on this,we also combined the hybrid chain reaction(HCR)with manganese dioxide nanosheets,CHA and metal-organic framework materials(ZIF-8)for sensitive detection of intracellular tumor markers and gene silencing therapy.Specific reaearch contents are as follows:Based on 3D DNA framework structure due to its good biocompatibility,strong structural stability,highly programmable and perfect cell transferability.In Chapter 2,a novel three-dimensional DNA tetrahedral amplifier(DTA)was developed to rapidly and efficiently image Mn SOD m RNA in living cells by the spatially constrained catalytic hairpin assembly(CHA)in DNA nanostructures.First,four DNA strands assemble to form a DNA tetrahedron,and then two metastable DNA hairpins,H1(Cy5)and H2(Cy3),were assembled at specific locations in the DNA tetrahedron,respectively.In the presence of Mn SOD m RNA,Mn SOD m RNA can trigger CHA reaction on DTA,forming an H1-H2 double-stranded nanostructure,and obtaining a significant fluorescence resonance energy transfer(FRET)signal.First,four DNA strands assemble to form a DNA tetrahedron,and then two metastable DNA hairpins,H1(Cy5)and H2(Cy3),are assembled at specific locations in the DNA tetrahedron,respectively.In the presence of Mn SOD m RNA,Mn SOD m RNA can CHA reaction on the DTA,forming an H1-H2 double-stranded nanostructure,and obtaining a significant fluorescence resonance energy transfer(FRET)signal.Meantime,the Mn SOD m RNA released during the reaction can trigger the next CHA reaction.Because of the 3D tetrahedral space constraint effect,DTA cyclic reaction can realize the rapid and efficient amplification detection of target Mn SOD m RNA in living cells.Moreover,the DTA show excellent structural stability and non-cytotoxicity.This strategy presents a versatile method for the ultrasensitive detection of biomarkers in living system and gains a deeper development of the DNA nanostructures in biomedical functions.DNAzyme is regarded as a promising gene therapy drug.However,poor cellular uptake efficacy and low biological stability limit the utilization of DNAzyme in gene therapy.Based on this conundrum,in Chapter 3,a programmable DNA tweezer(DZNT)for delivery of DNAzyme was designed for detection of TK1 m RNA and targeted silencing of survivin m RNA.At the end of the DZNT arm,there are two functionalized single-stranded DNA(ss DNA),and each consists of two parts:the segment complementary to TK1 m RNA and the split-DNAzyme segment.The hybridization with intracellular TK1 m RNA enables the imaging of TK1 m RNA.Meanwhile,the hybridization draws the split-DNAzyme close to each other and activates DNAzyme to cleave the survivin m RNA to realize gene silenc ing therapy.The results demonstrate that the DZNT nanocarrier has excellent cell penetration,good biocompatibility and non-cytotoxicity.DZNT can image intracellular biomolecule TK1m RNA with high contrast.Furthermore,the split-DNAzyme can efficiently cleave the survivin m RNA with the aid of TK1 m RNA commonly present in cancer cells,accordingly can selectively kill cancer cells and has no harm to normal cells.Taken together,the multi-functional programmable DZNT provides a promising platform for early diagnosis of tumors and gene therapy.Chapter 3 mentioned that DNAzyme is a DNA molecule with excellent catalytic performance.However,its poor biological stability in cells and insufficient cofactors also limit its application in biology.To address t his problem,in Chapter 4,we adsorbed functionalied DNAprobes(H1/H2)on Mn O₂ to construct the Mn O₂@H1/H2nanoplatform based on hybridization chain reaction(HCR),and realized selective cell amplified imaging and self-sufficient gene silencing therapy.In the presence of GSH,Mn O₂ was reduced to Mn²⁺,and released H1/H2 DNA probes.The mi RNA-21 induced HCR achieves the amplified imaging of mi RNA-21 and generates a tandem of activated DNAzyme cascade.Activated DNAzyme realizes survivin m RNA gene silencing under the action of cofactor Mn²⁺.The results demonstrate that the Mn O₂@H1/H2 nanoplatform can simultaneously achieve high contrast amplification of mi RNA-21 imaging and selectively kill cancer cells.Mn O₂,as a transport carrier of H1/H2,not only inhibits the degradation of H1/H2 by nucleases,but also provides Mn²⁺as a cofactor for DNAzyme.Moreover,multiple imaging and synergistic gene silencing can be achieved by programming functional domains of H1/H2.Altogether,the intelligent Mn O₂@H1/H2 nanoplatform provides a prospective platform for early diagnosis and gene silencing of tumors.Effective gene silencing guided by antisense oligonucleotides(ASOs)provides a new means for cancer treatment.In Chapter 5,a ZIF-8@H1/H2 nanoplatform based on a combination of diagnosis and therapy was prepared via a one-step synthesis method for magnification imaging and gene therapy of intracellular tumor biomolecules.In order to improve its biocompatibility and homologous targeting,cancer cell membrane was coate d on the surface of ZIF-8@H1/H2 to form CM/ZIF-8@H1/H2(CMZ@H1/H2).According to the biodegradable nature of ZIF-8 in acidic environment,CMZ@H1/H2 releases H1/H2 in cells.The endogenous Mn SOD m RNA stimulated H1/H2 to catalyze hairpin assembly(CHA)to realize its sensitive imaging.Simultaneously,the blocked antisense DNA of survivin was activated and hybridized with the coding domain of survivin m RNA.The survivin gene was knocked out by intracellular RNase H.As a biosensor and delivery carrier of H1/H 2,CMZ@H1/H2 has the advantages of high intracellular transmission efficiency,low immunogenicity and excellent biocompatibility.It is expected that this multi-functional CMZ@H1/H2 platform has great potential in the diagnosis and treatment of tumors.