Highly Sensitive Imaging Of RNA In Living Cells And Gene Therapy Based On DNA Self-assembled Nanostructures

Cancer is also called malignant tumor which poses a serious threat to human life.Early diagnosis and precise therapy are effective means to improve the survival rate of the cancer patients,but there are huge challenges to achieve this goal.The mutation and abnormal expression level of theRNA are often closely related to the occurrence and development of many diseases,which makesRNA a clinically valuable diagnostic marker and a promising therapeutic target.The detection ofRNA still faces challenge due to the low concentration and sequence similarity.Therefore,the development of new signal amplification technology for in situ imaging intracellularRNA molecules with high-sensitivity and high-selectivity to further reflect their expression levels is of great significance for the early diagnosis of cancer.In terms of cancer therapy,precise therapy that maximizes the therapeutic effect and minimizes side effects is the hotspots of the current therapeutic research.Nucleic acids can be carriers of genetic information in organisms,and have the advantages of excellent base complementary pairing ability,sequence programmability,structural diversity and unique mechanical properties.They are simple to synthesize and easy to modify,and have been widely used in the biosensing,cell imaging,drug delivery and gene therapy.Based on the above analysis,we developed a series of new methods based on different nucleic acid signal amplification strategies to achieve sensitive in situ imaging of the intracellularRNA,providing the new ideas for early diagnosis of cancer.In addition,we also designed functionalized catenane structures to achieve highly specific gene silencing through targeted delivery of the natural DNAzymes,thereby enabling precise therapy.The studied contents are mainly as follows:1.Sustainable and cascaded catalytic hairpin assembly for amplified sensing of miRNA biomarkers in living cellsThe sensing of intracellular microRNAs（miRNAs）is of significance for early-stage disease diagnosis and therapeutic monitoring.DNA is an interesting building material that can be programed into assemblies with rigid and branched structures,especially suitable for imaging intracellular biomolecules or therapeutic drug delivery.Here,by introducing the palindromic sequences into the programmable DNA hairpins,we describe an endogenous target-responsive three-way branched and palindrome-assisted catalytic hairpin assembly（3W-p CHA）approach for imaging miRNA-155 of living tumor cells with high sensitivity.The miRNA-155 triggers autonomous assembly of the fluorescently quenched signal hairpin and two hairpin dimers formed via hybridization of their respective palindromic sequences to yield branched DNA junctions,which carry the unopened hairpins and thus provide addressable substrates for continuous assembly formation of DNA nanostructures.During the formation of the DNA nanostructures,the miRNA-155 is cyclically reused and many signal probes are unfolded to show highly intensified fluorescence for detecting miRNA-155 down to 6.9 pmol/L in vitro with high selectivity.More importantly,these probes can be transfected into live cancer cells to initiate the assembly process triggered by intracellular miRNA-155,which provides a new way for imaging highly under-expressed miRNAs in cells.Besides,this approach can also be employed to differentiate miRNA-155 expression variations in different cells,indicating its promising potentials for early-stage disease diagnosis and biological studies in cells.2.Size-selective recognition-assisted and endogenous ATP-powered cascaded strand displacement reactions for accurately imaging mature miRNAs in living cellsMiRNAs are closely associated with cells proliferation,differentiation and carcinogenesis,and monitoring miRNAs contributes to our information of the molecular mechanisms of disease progression and its regulatory roles.However,the accurate and reliable detection of mature miRNA-21 in complex physiological environments faces challenges of undesired detection accuracy that caused by their precursor microRNAs（pre-miRNAs）or the degradation of sensing probes.Here,we designed a DNA nanocage-assisted and endogenous adenosine triphosphate（ATP）-driven cascaded toehold-mediated strand displacement reaction（Cage-TSDR）,offering accurate and reliable fluorescence imaging of miRNA-21 without the interference of pre-miRNAs.Due to the appropriate sizes of well-designed nanocage,the large pre-miRNA-21 cannot enter the inner cavity of the nanocage,while the small miRNA-21 molecules are able to enter the inner cavity and initiate the toehold-mediated strand displacement reaction（TSDR）.Subsequently,the abundant ATP binds its aptamer sequences and results in the release of fuel strand,which can participate in the second TSDR to release the target miRNA-21.Then the miRNA-21 can participate in the next TSDR,generating a significantly enhanced green fluorescent signal for accurate and robust detection of miRNA-21.This platform not only achieves the size-selective monitoring of mature miRNA without the interference of pre-miRNAs,but also effectively improves cell internalization ability and enhances nuclease resistance of sensing probe.Overall,the excellent imaging performances provide promising tools for constructing accurate and robust sensing platform in clinical diagnoses and biomedical studies.3.3D DNA scaffold-assisted dual intramolecular amplifications for multiplexed and sensitive miRNA imaging in living cellsThe simultaneous live cell imaging of multiple intracellular and disease-related miRNAs with low abundances is highly important to enhance specificity and accuracy for disease diagnosis.On the basis of the improved cell internalization and accelerated reaction kinetics,we develop a 3D DNA nanoprobe that integrates intramolecular DNAzyme（intra-Dz）and catalytic hairpin assembly（intra-CHA）amplifications to simultaneously monitor multiple miRNAs in living cells.The sensing components are loaded on the DNA scaffold via the sticky-end hybridizations of the DNA sequences to increase the local concentrations of the signal probes.The miRNA-21 and miRNA-155target sequences can trigger intra-Dz and-CHA amplifications on the nanoprobes to show significantly amplified and distinct fluorescence at different wavelengths for simultaneously monitoring low levels of miRNAs.Real-time fluorescence microscopy reveals that such a 3D DNA nanoprobe design with the intra-Dz and-CHA amplifications can accelerate the reaction rate against that of the conventional free Dz and CHA because of the increased local concentrations of the sensing components.Importantly,the 3D DNA nanoprobe has desirable stability and biocompatibility and can be readily delivered into living cells to achieve multiplexed and highly sensitive sensing of intracellular miRNA-155 and miRNA-21 sequences.With the demonstration of its intracellular application,the developed 3D DNA nanoprobe thus holds promising potentials for biological studies and accurate disease diagnosis.4.Biodegradable nanoparticle-assisted and multiplexed imaging of asymmetricRNA expressions in live cells for precise cancer diagnosis and prognosisThe simultaneous imaging of the dynamic expression variations of regulatoryRNAs in cells,which remains a major challenge,has important applications in precise disease diagnosis,treatment and prognosis.Here,we describe the establishment of a biodegradable Zn O nanoparticle（Zn O NP）-assisted asymmetric amplification approach for the simultaneous imaging of microRNA-21（miRNA-21）and programmed cell death4（PDCD4）mRNA at distinct expression levels in live cells.The DNA signal probe complexes are immobilized on the Zn O NPs and readily delivered into the target cancer cells via the endocytosis pathway.The acidic microenvironment in cancer cells leads to the dissolution of the Zn O NPs to release Zn²⁺ions and the intracellular miRNA-21activates the Zn²⁺-dependent DNAzyme to cleave the substrate signal probes with the assistance of the Zn²⁺cofactor to show green fluorescence for imaging miRNA-21.Meanwhile,the PDCD4 mRNA can displace the other quenched signal probes to generate red fluorescence.Importantly,the PDCD4 mRNA sequences can be recycled and reused by using the DNAzyme-cleaved sequences as the fuel strands through two strand displacement reactions to yield amplified red fluorescence for detecting low levels of PDCD4 mRNA.Moreover,our approach can be used to evaluate the varied expression levels of miRNA-21 and PDCD4 mRNA responsive to different drugs in cells,reflecting its usefulness for precise cancer diagnosis and prognosis upon anticancer drug treatment.5.Targeted and direct intracellular delivery of native DNAzymes enables highly specific gene silencingDNAzymes exhibit high potentials as gene silencing agents for therapeutic applications.Such purposes,however,are significantly challenged by the targeted and successful delivery of unmodified DNAzymes into cells with minimal side effects.Here,we set out to formulate and demonstrate a new stimuli-responsive and constrained aptamer/DNAzyme（Apt/Dz）cantenane nanostructure for highly specific gene silencing.The rational design of the Apt/Dz cantenane nanostructure with the respective integration of the aptamer sequence and the completely closed cantenane format enables such a construct both the targeted capability and significantly improved nuclease resistance,facilitating the stable and targeted delivery of unmodified Dz into cancer cells.Moreover,the Dz enzymatic activity in the constrained structure can only be conditionally regulated by the specific intracellular mRNA sequences to silence the target gene with highly reduced side effects.Results show that the Apt/Dz cantenane nanostructure can effectively inhibit the expression of the target gene and the proliferation of cancer cells with high specificity.