Phenotype-Directed DNA Nanomachine for in Situ Analysis of Stem Cell-like Subpopulation in Breast Cancer

The cancer stem cell hypothesis provides a basis for prediction of the recurrence and risk of metastasis in breast cancer.However,the unique expression pattern of stemness markers and the presence of nonstem-like cancer cells with varied phenotypes have brought great challenges to the characterization of breast cancer stem cells.To address these challenges,a phenotype-directed DNA nanomachine has been designed for high-accuracy labeling and in situ analysis of the stem cell-like subpopulation in breast cancer.The key for the design is to use cell surfaceanchored inputs to activate the nanomachine,which undergoes different branch migration pathways such that the signal strand can only be brought onto the cancer cells having the stem cell-like phenotype.Highly sensitive determination and single-step isolation of the stem cell-like subpopulation were achieved by incorporating functional groups into the signal strand such that the nanomachine was successfully applied in a tumor-bearing mouse model.Overall,the approach provides for a substantial improvement in capability for the analysis of the breast cancer stem cell-like subpopulation,and it is expected that the new approach will advance the use of DNA nanomachines in cancer-related studies.

Fabrication of nanozyme@DNA hydrogel and its application in biomedical analysis

Nanozymes have received great attention owing to the advantages of easy preparation and low cost. Unlike natural enzymes that readily adapt to physiological environments, artificial nanozymes are apt to passivate in complex clinical samples （e.g., serum）, which may damage the catalytic capability and consequently limit the application in biomedical analysis. To conquer this problem, in this study, we fabricated novel nanozyme@DNA hydrogel architecture by incorporat^ng nanozymes into a pure DNA hydrogel. Gold nanoparticles （AuNPs） were adopted as a model nanozyme. Results indicate that AuNPs incorporated in the DNA hydrogel retain their catalytic capability in serum as they are protected by the hydrogel, whereas AuNPs alone totally lose the catalytic capability in serum. The detection of hydrogen peroxide and glucose in serum based on the catalysis of the AuNPs@DNA hydrogel was achieved. The detection limit of each reaches 1.7 and 38 ~M, respectively, which is equal to the value obtained using natural enzymes. Besides the mechanisms, some other advantages, such as recyclability and availability, have also been explored. This nanozyme@DNA hydrogel architecture may have a great potential for the utilization of nanozymes as well as the application of nanozymes for biomedical analysis in complex physiological samples.

Visualized and cascade-enhanced gene silencing by smart DNAzyme-graphene nanocomplex

BCL-2 gene as well as its products is recognized as a promising target for the molecular targeted therapy of tumors.However,due to certain defense measures of tumor cells,the therapeutic effect based on the gene silencing of BCL-2 is greatly reduced.Here we fabricate a smart response nucleic acid therapeutic that could silence the gene effectively through a dual-targeted and cascade-enhanced strategy.In brief,nano-graphene oxide(GO),working as a nano-carrier,is loaded with a well-designed DNAzyme,which can target and silence the BCL-2 mRNA.Furthermore,upon binding with the BCL-2 mRNA,the enzymatic activity of the DNAzyme can be initiated,cutting a substrate oligonucleotide to produce an anti-nucleolin aptamer AS1411.Nucleolin,a nucleolar phosphoprotein,is known as a stabilizer of BCL-2 mRNA.Via binding and inactivating the nucleolin,AS1411 can destabilize BCL-2 mRNA.By this means of simultaneously targeting mRNA and its stabilizer in an integrated system,effective silencing of the BCL-2 gene of tumor cells is achieved at both the cellular and in vivo levels.After being dosed with this nucleic acid therapeutic and without any chemotherapeutics,apoptosis of tumor cells at the cellular level and apparent shrinkage of tumors in vivo are observed.By labeling a molecular beacon on the substrate of DNAzyme,visualization of the enzymatic activity as well as the tumor in vivo can be also achieved.Our work presents a pure bio-therapeutic strategy that has positive implications for enhancing tumor treatment and avoiding side effects of chemotherapeutics.

Dual-enzyme-propelled unbounded DNA walking nanomachine for intracellular imaging of lowly expressed microRNA

Despite the progress on the analysis of miRNA either in vitro or in vivo,intracellular imaging of lowly expressed microRNA remains a challenge.Here we develop a novel dual-enzyme-propelled DNA walking nanomachine,which is tailored to accomplish this mission.The nanomachine is constructed with nanoparticles-loaded DNA tracks,on which the targeted miRNA working as a single-foot DNA walker can move autonomously under the catalysis of two cooperative enzymes.Cleavage of the DNA tracks like a "burnt-bridge" mechanism is thereafter triggered,resulting in an amplified fluorescent signal.After the comprehensive study and optimization of the DNA nanomachine,miR-892b,a significantly down-regulated miRNA in breast cancer cells,is selected as a model target.Sensitivity detection in vitro is achieved with a superior detection limit of 4 pM.While being delivered into cells,the DNA nanomachine is available for the imaging of the lowly expressed microRNA,which is totally missing using the conventional fluorescence in situ hybridization (FISH) method.Up-regulation or down-regulation of the miRNA by exogenous regulatory factors can be also well evaluated.This DNA nanomachine provides a competitive approach for the analysis of miRNA,and has the potential to be extended to some other biomolecules.

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage

Evaluating tumor development is of great importance for clinic treatment and therapy.It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell.So,in this work,cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids,i.e.,N-glycolylneuraminic acid(Neu5Gc)and N-acetylneuraminic acid(Neu5Ac).Specifically,PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface,followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin.Subsequently,the activated Cas12a indiscriminately cleaves DNA,resulting in the release of PbS and CdS quantum dots,both of which can be simultaneously detected by anodic stripping voltammetry.Consequently,Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL,respectively.Therefore,a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface,which can be further used as a tool to identify bladder cancer cells at different development stages.Our method to evaluate tumor development is simple and easy to be operated,so it can be potentially applied for the detection of tumor occurrence and development in the future.