Studies On Functional Nucleic Acids For Cancer Diagnosis And Therapy

With the progress of life science and technology,human have pa id much more attention to the quality of life.Recently,issues in life activities-related biomedical fields,such as early diagnosis and treatment of diseases,are be coming extraordinarily important.Therefore,the scientists have made continuous exploration in the field of biomedicine,especially in the sensitive detection of disease-related biomarkers and the construction of novel biosensor system,which have become the essential research subject of the modern biomedical science.Among them,functional nucleic acids can provide new strategies for biomedical research as effective tools with special functions.Meanwhile,nanomaterials are getting more and more attention by advantages of their outstanding physical and chemical characters.Furthermore,a series of novel multifunctional nanosystems can be constructed by the combination of functional nucleic acids and nanomaterials.They can be applied in biological imaging and treatment,serve as a group of ideal biosensing tools.As a type of nucleic acid with special functions,functional nucleic acids including nucleic acid aptamers,DNAzymes,i-motif structures,antisense DNA and siRNAs are different from traditional nucleic acids as genetic information carriers.Among them,screened by SELEX technology,nucleic acid aptamer is a single-stranded RNA or DNA which can selectively and specifically recognize and bind the target.In addition,DNAzymes are a type of single-stranded DNA with catalytic activity that can be obtained through in vitro screening,and have similar functions as ribozymes,such as RNA or DNA cleavage or connection,and photo-cleavage of thymine dimers.Owing to the strong recognition and binding capabilities,high water solubility and biocompatibility,the functional nucleic acids have been widely used in the fields of biosensing,environmental analysis,drug release,cancer diagnosis and treatment.However,functional nucleic acids still have many limitations in practical applications.For example,the complex of functional nucleic acid and target is susceptible to interference in complicated systems,difficult to enter cells,easy to be degraded by nucleases,and with short circulation time in the body.Nanomaterials are materials with special properties whose geometric dimensions reach the nanometer level.Due to the characteristics of large specific surface area,strong load capacity,and high biocompatibility,nanomaterials have been widely used in the fields of biosensing and tumor theranostics.Typical representatives include mesoporous silica nanomaterials,manganese dioxide（MnO₂）nanomaterials,gold nanoparticles,upconversion nanomaterials and DNA nanomaterials.In particular,nanomaterials with magnetic properties,charging properties,fluorescent properties or photothermal effects can be used as imaging molecules or therapeutic agents for cancer diagnosis and treatment.Based on the above research background,we focus on the issue of improving the effectiveness of aptamers in complex systems and employing functional nucleic acid modified nanomaterials to tumor diagnosis and treatment.This dissertation has developed a series of biosensors based on functional nucleic acids and nanomaterials.The system is used for biological imaging and treatment.The detail for each chapter is listed as follows:（1）Aiming at the problem that the binding between the nucleic acid aptamer and the target protein in a complex system,we used the protein-aptamer template（Protein-Aptamer Template,PAT）guided cross-linking method for selective cross-linking the protein’s lysine residues and F-carboxyl group（α,α-gem-difluoromethyl carboxyl group,F-carboxyl）modified on the nucleic acid aptamers to obtain protein-nucleic acid aptamer conjugates.The experimental results prove that the nucleic acid aptamer modified by the F-carboxyl group can still specifically recognize and bind the target protein on the cell membrane and can effectively perform a specific cross-linking reaction with the target protein.This method provides a new approach for related biological research such as cell membrane protein imaging and separation.（2）Besides the challenge of being easily interfered with the binding of target proteins in complex systems,it is difficult for functional nucleic acids to enter cells.Due to the unique hydrophobicity and self-polymerization of the F bases,we integrated them into the sequence of DNA molecular and studied the cell internalization behavior of functional nucleic acids with modifications of different numbers and sites.Experiments have demonstrated that due to the pairing of hydrophobic F bases,the linear functional nucleic acid modified with single-stranded F bases has stronger cell internalization ability than hairpin functional nucleic acids that form F-F base pairs on the stem.This result confirms that the conformational switch can enhance the cell internalization ability of the functional nucleic acid modified by the F base.（3）In order to further improve the efficiency of functional nucleic acid entring into cells,we used DNAzyme amplification and catalytic hairpin assembly（Catalytic Hairpin Assembly,CHA）amplification combined strategy to construct a cascade amplification reaction-based nanoprobe（Cascade Amplification Reaction-based Nanoprobe,CAR nanoprobe）and use it for the detection of telomerase activity in living cells.The nanoprobe is composed of MnO₂ nanosheets,DNAzyme,CHA and other components.MnO₂ nanosheets can not only be used as carriers to deliver DNAzyme and CHA into cells,but also be degraded in cells,then release all DNA strands to the same place in the cell.The Mn²⁺produced by degradation can promote the lysosomal escape of the DNA chain through the ion sponge effect and at the same time act as a cofactor of the DNAzyme to realize the digestion of the DNA chain.The subsequent intracellular cascade amplification reaction can generate a strong fluorescent signal and achieve ultra-low level detection of telomerase activity.This method provides a new idea for the sensitive detection of biomolecules in cells.（4）In order to expand the application of functional nucleic acids in biomedicine,we loaded the photosensitizer PcC₄ into the mesoporous silica particles,and blocked the pores with DNA strands O1,and constructed a new nano-photosensitizer driven and activated by"dual protein target".In the presence of d NTPs,the DNA strand O1 on the surface of this novel nano-photosensitizer particle can be extended by telomerase,and a telomere repeat sequence is generated at the 3’end of the DNA strand,which is complementary to the sequence at the 5’end.The complementary hybrid strands will form a rigid hairpin-type DNA structure and detach from the surface of the mesoporous silica particles,leading to the release of PcC4.The released PcC4 binds to intracellular albumin and activates the self-quenched PcC4 to generate ROS under light conditions to achieve specific tumor imaging and photodynamic therapy.