Cell Imaging And Gene Regulation Based On Functional Nucleic Acid Nanomaterials

Cancer is a serious threat to human life and health.Thus,it is of great importance to develop nanomedicine for early diagnosis and cancer treatment.Because the nucleic acid amplification reaction is fast,efficient,and easy to design,it is widely used in the detection of tumor markers,providing a good idea for early diagnosis of cancer.However,the single-stage signal amplification method is prone to produce false negative signals.To solve the problem of low sensitivity,we construct a cascade signal amplification circuit to enhance the signal amplification ability.A concatenated CHA-HCR system was established as an isothermal enzyme-free amplification strategy for highly sensitive and selective nucleic acid assay.Target catalyzes the self-assembly of CHA hairpin substrates into ds DNA products,where the split segments of HCR trigger are successively connected to motivate the subsequent autonomous cross-opening of HCR hairpins,leading to the construction of HCR tandem copolymeric ds DNA nanowires.The resulting HCR copolymer brings a fluorophore donor/acceptor pair into close proximity that allows an efficient generation of FRET readout signal.Moreover,the optimized CHA-HCR circuit,upon the incorporation of an auxiliary sensing module,can be converted into a universal sensing platform for detecting cancerous biomarkers(e.g.,a well-known oncogene mi R-21)through a convenient easy-to-integrate procedure.The concatenated CHA-HCR amplifier enables the accurate intracellular mi RNA imaging in living cells,which is especially suitable for in situ amplified detection of lowly expressed endogenous analytes.The inherent synergistically accelerated recognition and hybridization features of CHA-HCR circuit contribute to the amplified detection of endogenous RNAs in living cells.The flexible and programmable nature of homogeneous CHA-HCR system provides a versatile and robust toolbox for a wide range of research fields,such as in vivo bioimaging,clinical diagnosis and environmental monitoring.Since the detection of biomarkers in vivo is more complex,it is necessary to design circuits with higher amplification ability to improve sensitivity.We proposed a versatile programmed autocatalytic hybridization chain reaction-catalytic hairpin assembly(AHCR-CHA),of which the initiator motivated the autonomous cross-activation of HCR and CHA accessories,leading to the assembly of copolymeric ds DNA nanowires with tremendously(exponential)amplified readout signal.The successively guaranteed recognitions and synergistically accelerated signal amplifications of HCR and CHA constitute layers enable a highly sensitive and selective detection of nucleic acid target from its gene mutants.The AHCR-CHA amplifier was systematically investigated,suggesting that the operation of the recycling DNA circuit was closely associated with the performance of each constitute.The computer-aided simulations were also applied to thoroughly analyze the dynamic processes of the AHCR-CHA amplifier via the calculation of rate constants of dominated reaction and signal leakage reactions.Furthermore,the autocatalytic circuit was demonstrated to be a versatile amplification module by integrating an auxiliary sensing module for monitoring other trace amount of analytes,e.g.,micro RNA(mi RNA).This easy-to-integrate“plug-and-play”procedure empower the universal amplified system to accurately localize intracellular mi RNA in real time.The AHCR-CHA strategy provides a universal amplifying circuit element that is simple,sensitive,rational and robust,and thus has great potential in bioanalytical and biomedical applications.Nucleic acid can be designed as a nucleic acid circuit for detection,and it can also be used as drug for cancer treatment.Since DNAzyme can efficiently cleave specific m RNAs in cells,it can be used as gene drugs to combat cancer.Si RNA can also inhibit tumors progression by significantly fluctuating the expression of specific proteins.However,these nucleic acid drugs are easily degraded.These metal-organic frameworks(MOFs)can protect the nucleic acid drug from degradation,transport the nucleic acid to the tumor site through the EPR effect,and promote the p H-responsive release of the nucleic acid,leading to the maximization of the therapeutic effect of nucleic acid drugs on the tumor.We report a self-sufficient MOFs-sustained and DNAzyme-based multimodal therapeutic nanosystem for highly efficient combined genetherapy and photodynamic therapy.The 10-23 DNAzyme-encapsulated ZIF-8nanoparticles were facilely engineered through a one-step synthesis procedure to prevent DNAzymes from enzymatic digestion and thus to facilitate their efficient delivery to tumor location.The acidic tumor microenvironment stimulates the disassembly of ZIF-8 MOF with the concomitant release of DNAzyme strands and Zn²⁺ions that respectively serve as messenger RNA-addressing motors and prerequisite DNAzyme cofactors for activating efficient gene silencing operations.The self-sufficient DNAzyme@ZIF-8 system can be extensively developed as a multi-functional therapeutic platform through the integration of other functional groups onto DNA probes,such as the photodynamic agent Chlorin e6(Ce6).The tumor acidic microenvironment-responsive release of Ce6 could be utilized as an in vivo imaging probe to monitor the biodistribution of these intravenously injected nanomaterials,and to furtherly realize the generation of ¹O₂ for photodynamic therapy under photoirradiation.The remarkably combined genetherapy and photodynamic therapy of our Ce6-DNAzyme@ZIF-8 nanoplatform is furtherly demonstrated in vivo,which plays an important role on cancer diagnosis,therapy,and posttreatment in future.In Chapter 5,we developed hypoxia addressable gene silencing via a multifunctional nanoplatform for sensitizing photochemotherapy.In this system,the p H-responsive ZIF-8 nanoparticles could effective facilitates their delivery of Ce6,DOX,and si RNA to the tumor tissue and subsequent endocytosis by tumor cells.Laser irradiation activated the generation of plentiful ROS by photosensitizer Ce6,resulting in the enhanced lysosomal escape of DOX and si RNA.It is noted that the down-regulation of HIF-1?by gene silencing could relieve the hypoxic responses,including recovering broken DNA restoration,overcoming multidrug resistance and suppressing metastasis.We demonstrated the remarkable synergistic photochemotherapy and gene therapy effects of the multifunctional CDHNs nanoplatform in vitro and in vivo,which provides new ideas for future gene silencing-based cancer treatments.