Study On Highly Sensitive Imaging Of MicroRNA And Protein Delivery In Cancer Cells

In recent years,cancer has gradually become an important threat to the health of human.Early diagnosis and the effective targeting therapy of cancer are critical to human health,but remains a major challenge.MicroRNA(miRNA)plays important regulatory roles in cellular processes such as cell proliferation,migration,and apoptosis Abnormal expression of miRNAs has proved to be correlated with a series of human diseases,including cancers,viral infections,and cardiovascular and neurological diseases.Therefore,miRNAs can be used as valuable biomarkers for cancer diagnosis and prognosis.However,it is a great challenge to monitor these cancer-related miRNAs in living cells because of their natural characteristics,such as small size,highly homologous sequences among family members,and low abundance.Therefore,it is of great significance to develop novel strategies for in situ miRNA detection with ultrasensitive and highly selective.With the rapid development of nanotechnology,nanoprobes have been widely used in tumor cell imaging,early cancer diagnosis and tumor treatment.New drug release methods have received increasing attention from researchers over the past decade.Therefore,it is a hotspot to design drug carriers for targeted drug delivery and controlled release.DNA nanomaterials with the advantages of spatial addressability,programmability,good biocompatibility and easy functionalization,have been widely used in targeted drug delivery,DNA logic gates,cell imaging and so on.Based on the above analysis,we constructed a series of multifunctional bioprobe for the sensitive detection and imaging of miRNAs through various amplification methods,providing a reliable and effective tool for the early diagnosis of cancer.In addition,functionalized DNA nanomaterials were designed for the targeted drug delivery and release in a controlled manner.The studied contents are mainly as follows1.Lighting-up RNA aptamer transcription synchronization amplification for ultrasensitive and label-free imaging of microRNA in single cellsSensitive imaging of intracellular microRNAs(miRNAs)in cells is of great significance in clinical diagnoses and disease treatments,and it remains a major challenge to achieve this goal.Herein,we report a new in situ rolling circle transcription synchronization machinery(RCTsm)of lighting-up RNA aptamer strategy for highly sensitive imaging and selective differentiation of miRNA expression levels in cells Such a RCTsm approach utilizes a DNA promoter to recycle the target miRNAs to trigger the initiation of multiple RCT process for the yield of many lighting-up RNA aptamers.The malachite green dye further binds these aptamers to show significantly enhanced fluorescence for completely label-free detection of the target miRNAs with a high sensitivity in vitro with a low femtomolar detection limit.More importantly,sensitive detection of under-expressed miRNAs in cells and distinct differentiation of the miRNA expression variations in different cells can also be realized with this RCTsm approach in a washing-free format,making it a versatile and useful tool for imaging trace miRNAs in single cells with the great potential for early cancer diagnosis as well as biomedical research.2.A DNA-Fueled and Catalytic Molecule Machine Lights Up Trace Under-Expressed MicroRNAs in Living CellsThe detection of specific intracellular microRNAs(miRNAs)in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion However,because of the characteristic nature of miRNAs in terms of small sizes,low abundance,and similarity among family members,it is a great challenge to monitor miRNAs in living cells,especially those with much lower expression levels.In this work,we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells.The presence of the target miRNA releases the hairpin sequences from the dsDNA(containing the fluorescence resonance energy transfer(FRET)pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles(dsDNA-AuNPs),and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions,leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences.As a result,the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets.Besides,the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells,and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated.With the advantages of in cell signal amplification and reduced background noise,the developed method thus offers new opportunities for detecting various trace intracellular miRNA species3.Bio-cleavable nanoprobes for target-triggered catalytic hairpin assembly amplification detection of microRNAs in live cancer cellsThe monitoring and imaging of intracellular microRNAs(miRNAs)with specific sequences plays a vital role in cell biology as it can potentially elucidate many cellular processes and diseases related to miRNAs in living cells with accurate information However,the detection of trace amounts of under-expressed intracellular miRNAs in living cells represents one of the current major challenges.In an effort to address this issue,we describe the establishment of an in cell catalytic hairpin assembly(CHA)signal amplification strategy for imaging under-expressed intracellular miRNAs in this work.Gold nanoparticles functionalized with FAM-and TAMRA-labeled hairpins with disulfide bonds in the stems are readily delivered into cells via endocytosis.Glutathione with evaluated concentrations in cancer cells cleaves the disulfide bonds in the hairpins by reduction to release the hairpins,and the target miRNAs further trigger CHA between the two hairpins to form many DNA duplexes,which bring the FAM and TAMRA labels into close proximity to generate apparently enhanced fluorescence resonance energy transfer(FRET)for the sensitive monitoring of low amounts of under-expressed miRNAs in live cancer cells.Using CHA to amplify the signal output and FRET to reduce the background noise,a significantly enhanced signal-to-noise ratio,thereby high sensitivity,over conventional fluorescence imaging can be realized,making our method particularly suitable for monitoring low levels of intracellular species4.An all-sealed divalent DNA tetrahedron facilities stable,targeted and efficient intracellular delivery of native anticancer proteinProtein therapy has been increasingly evolved as one of the most promising means for cancer treatment.Yet,the protein-based therapy is significantly challenged by the successful delivery of the native protein into the target cancer cells.We address this challenge here using an all sealed divalent aptamer DNA tetrahedron nanostructure delivery platform,in which the protein drug is encapsulated inside the cavity stoichiometrically via a reversible chemical bond.The ligase-assisted seal of the nicks of the DNA tetrahedron results in a highly enhanced stability against nuclease digestion to effectively protect the protein therapeutic from degradation.In addition,the divalent aptamer sequences incorporated into the nanostructure favors its targeted and efficient delivery capability.Importantly,upon being readily delivered into the target cancer cells,the endogenous glutathione can trigger the release of the native protein therapeutic from the DNA nanostructure in a traceless fashion by cleaving the reversible chemical bond,thereby leading to effective apoptosis of the specific cancer cells.