Selective inhibition of c-Met signaling pathways with a bispecific DNA nanoconnector for the targeted therapy of cancer.

Hepatocyte growth factor receptor (c-Met) is a suitable molecular target for the targeted therapy of cancer. Novel c-Met-targeting drugs need to be developed because conventional small-molecule inhibitors and antibodies of c-Met have some limitations. To synthesize such drugs, we developed a bispecific DNA nanoconnector (STPA) to inhibit c-Met function. STPA was constructed by using DNA triangular prism as a scaffold and aptamers as binding molecules. After c-Met-specific SL1 and nucleolin-specific AS1411 aptamers were integrated with STPA, STPA could bind to c-Met and nucleolin on the cell membrane. This led to the formation of the c-Met/STPA/nucleolin complex, which in turn blocked c-Met activation. In vitro experiments showed that STPA could not only inhibit the c-Met signaling pathways but also facilitate c-Met degradation through lysosomes. STPA also inhibited c-Met-promoted cell migration, invasion, and proliferation. The results of in vivo experiments showed that STPA could specifically target to tumor site in xenograft mouse model, and inhibit tumor growth with low toxicity by downregulating c-Met pathways. This study provided a novel and simple strategy to develop c-Met-targeting drugs for the targeted therapy of cancer.

Glucose deprivation triggers DCAF1-mediated inactivation of Rheb-mTORC1 and promotes cancer cell survival.

Low glucose is a common microenvironment for rapidly growing solid tumors, which has developed multiple approaches to survive under glucose deprivation. However, the specific regulatory mechanism remains largely elusive. In this study, we demonstrate that glucose deprivation, while not amino acid or serum starvation, transactivates the expression of DCAF1. This enhances the K48-linked polyubiquitination and proteasome-dependent degradation of Rheb, inhibits mTORC1 activity, induces autophagy, and facilitates cancer cell survival under glucose deprivation conditions. This study identified DCAF1 as a new cellular glucose sensor and uncovered new insights into mechanism of DCAF1-mediated inactivation of Rheb-mTORC1 pathway for promoting cancer cell survival in response to glucose deprivation.

Cell Membrane-Anchored DNA Nanoinhibitor for Inhibition of Receptor Tyrosine Kinase Signaling Pathways via Steric Hindrance and Lysosome-Induced Protein Degradation.

Receptor tyrosine kinase (RTK) plays a crucial role in cancer progression, and it has been identified as a key drug target for cancer targeted therapy. Although traditional RTK-targeting drugs are effective, there are some limitations that potentially hinder the further development of RTK-targeting drugs. Therefore, it is urgently needed to develop novel, simple, and general RTK-targeting inhibitors with a new mechanism of action for cancer targeted therapy. Here, a cell membrane-anchored RTK-targeting DNA nanoinhibitor is developed to inhibit RTK function. By using a DNA tetrahedron as a framework, RTK-specific aptamers as the recognition elements, and cholesterol as anchoring molecules, this DNA nanoinhibitor could rapidly anchor on the cell membrane and specifically bind to RTK. Compared with traditional RTK-targeting inhibitors, this DNA nanoinhibitor does not need to bind at a limited domain on RTK, which increases the possibilities of developing RTK inhibitors. With the cellular-mesenchymal to epithelial transition factor (c-Met) as a target RTK, the DNA nanoinhibitor can not only induce steric hindrance effects to inhibit c-Met activation but also reduce the c-Met level via lysosome-mediated protein degradation and thus inhibition of c-Met signaling pathways and related cell behaviors. Moreover, the DNA nanoinhibitor is feasible for other RTKs by just replacing aptamers. This work may provide a novel, simple, and general RTK-targeting nanoinhibitor and possess great value in RTK-targeted cancer therapy.

Highly sensitive and ratiometric detection of nitrite in food based on upconversion-carbon dots nanosensor.

Nitrite (NO2-) is extensively found in the daily dietary environment. However, consuming too much NO2- can pose serious health risks. Thus, we designed a NO2--activated ratiometric upconversion luminescence (UCL) nanosensor which could realize NO2- detection via the inner filter effect (IFE) between NO2--sensitive carbon dots (CDs) and upconversion nanoparticles (UCNPs). Due to the exceptional optical properties of UCNPs and the remarkable selectivity of CDs, the UCL nanosensor exhibited a good response to NO2-. By taking advantage of NIR excitation and ratiometric detection signal, the UCL nanosensor could eliminate the autofluorescence thereby increasing the detection accuracy effectively. Additionally, the UCL nanosensor proved successful in detecting NO2- quantitatively in actual samples. The UCL nanosensor provides a simple as well as sensitive sensing strategy for NO2- detection and analysis, which is anticipated to extend the utilization of upconversion detection in food safety.

A sequentially triggered DNA nanocapsule for targeted drug delivery based on pH-responsive i-motif and tumor cell-specific aptamer.

Targeted drug delivery with minor off-target effects is urgently needed for precise cancer treatments. Here, a sequentially triggered strategy based on double targeting elements is designed to meet this purpose. By using an acidic pH-responsive i-motif DNA and a tumor cell-specific aptamer as targeting elements, a smart dual-targeted DNA nanocapsule (ZBI5-DOX) was constructed. ZBI5-DOX can be firstly triggered by acidic pH, and then bind to target cells via aptamer recognition and thus targeted release of the carried DOX chemotherapeutics. With this smart DNA nanocapsule, the carried DOX could be precisely delivered to target SMMC-7721 tumor cells in acidic conditions. After drug treatments, selective cytotoxicity of the DNA nanocapsule was successfully achieved. Meanwhile, the DNA nanocapsule had a specific inhibition effect on target cell migration and invasion. Therefore, this sequentially triggered strategy may provide deep insight into the next generation of targeted drug delivery.

MoS2 quantum dots as fluorescent probe for methotrexate detection.

Herein, we developed a unique fluorescence biosensor for methotrexate assay based on MoS2 quantum dots, which were synthesized in one step using sodium molybdate and cysteine as raw materials. The fluorescence of MoS2 QDs could be quenched when encountered with methotrexate, which was attributed to the inner filter effect (IFE). Furthermore, this present IFE-based method showed the linearity between the MoS2 QDs fluorescence intensity and the methotrexate concentration in the range of 0.05-1 µM with the LOD of 42 nM. The practical applicability of this strategy was successfully demonstrated by detecting methotrexate in real samples. Results indicated that the proposed method could be a promising sensing platform for methotrexate analysis.

Split-aptamer mediated regenerable temperature-sensitive electrochemical biosensor for the detection of tumour exosomes.

In this paper, a split-aptamer mediated regenerable temperature-sensitive (SMRT) electrochemical biosensor was constructed for the detection of exosomes. The split-aptamer used in this SMRT biosensor was composed of two fragments, one of which was immobilized on the surface of an electrode via sulfhydryl groups and named split-a and the other was labelled with methylene blue and named split-b. The two fragments could form sandwich structures at the electrode surface via target-induced self-assembly in the presence of target exosomes at 4 °C in PBS, and then realizing the detection of exosomes via voltammetry. In addition, due to the temperature sensitivity of the split-aptamer, the electrode could be regenerated through temperature-induced disassembly of the sandwich structures. Consequently, the SMRT biosensor realized sensitive and specific analysis of target exosomes with a limit of detection of 1.5 × 106 particles/mL and could be quickly and easily regenerated by washing with PBS at 37 °C for 30 s without any additives. This is the first study on the construction of a reproducible electrochemical biosensor using a split-aptamer for the specific detection of tumour exosomes, and may provide an innovative strategy for the economical and efficient design of regenerable electrochemical biosensors.

A label-free cyclic amplification strategy for microRNA detection by coupling graphene oxide-controlled adsorption with superlong poly(thymine)-hosted fluorescent copper nanoparticles.

Herein, based on a terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly-T-templated-copper nanoparticles (poly T-CuNPs) strategy, a simple, universal and label-free fluorescent biosensor for the detection of miRNA was constructed by employing graphene oxide (GO) and DNase I. In this strategy, GO and DNase I were used as a switch and amplifier of the signal generation pathway, respectively, and the fluorescence of poly T-CuNPs was used as the signal output. In the presence of target miRNA, the DNA dissociated from the GO surface by forming a miRNA/DNA duplex and was degraded by DNase I. The short oligos with 3'-OH, the product of DNase I degradation, could be recognized by the TdT and added to a long poly-T tail. Finally, the fluorescence signal was output through the synthesis of poly T-CuNPs. As a proof of concept, let-7a was analyzed. The method showed good sensitivity and selectivity with a linear response in the 50 pM-10,000 pM let-7a concentration range and a 30 pM limit of detection (LOD = 30 pM, R2 = 0.9954, the relative standard deviation were 2.79%-5.30%). It was also successfully applied to the determination of miRNA in spiked human serum samples. It showed good linearity in the range of 500-10000 pM (R2 = 0.9969, the relative standard deviation were 1.61%-3.85%). Moreover, both the adsorption of GO and the degradation of DNase I are DNA sequence-independent; thus, this method can be applied to the detection of any miRNA simply by changing the assisted-DNA sequence.

Imaging specific cell-surface sialylation using DNA dendrimer-assisted FRET.

Sialylation plays a vital role in multiple different physiologic processes, aberrant sialylation is highly related to disease development. Especially in cancer development, changed states of specific cell-surface sialylation implies rich cancer-related information. Therefore, it is necessary to image specific cell-surface sialylation for better understanding biological functions of sialylation. To meet this purpose, we designed a DNA dendrimer-assisted fluorescence resonance energy transfer (FRET) strategy in this work. By labeling multiple FRET donors and acceptors on the target molecules through metabolic oligosaccharide engineering (MOE) and targeted recognition of aptamer-tethered DNA dendrimer, the FRET was significantly improved. With the DNA dendrimer-assisted FRET strategy, specific imaging of cell-surface sialylation on SMMC-7721 and CEM cells were successfully achieved. The obtained FRET signal intensity was approximately four times higher than the control without the assistance of DNA dendrimer. Moreover, this method is competent to monitor changed states of PTK7-specific sialylation induced by tunicamycin. The proposed imaging strategy may provide a powerful tool to explore the physiological roles of specific cell-surface sialylation and the related mechanism of diseases.

In Situ Modulating DNAzyme Activity and Internalization Behavior with Acid-Initiated Reconfigurable DNA Nanodevice for Activatable Theranostic.

DNAzyme-mediated gene silencing was still challenged by off-target toxicity. In this study, we developed a split DNAzyme-based nanodevice (sDz-ND) that leveraged acidic tumor microenvironments to drive in situ assembly, thus modulating internalization behavior and silencing activity of DNAzymes. sDz-ND consisted of two different modules, which functionalized with split DNAzyme fragments, respectively. At psychological pH (∼7.4), the two modules were monodispersed, showing cleavage anergy and quenched fluorescence. At pH 6.3, the separated modules could cross-link with each other to form integrated sDz-ND, resulting activation of theranostic function. Meanwhile, the increased particle size and acquired multivalent effect favored 2.1-fold enhanced binding ability, which further facilitated rapid endocytosis of sDz-ND into target cancer cells, then allowing DNAzyme mediated gene silencing. The strategy provides a promising and general concept for precise tumor imaging and gene therapy.

Recognition triggered assembly of split aptamers to initiate a hybridization chain reaction for wash-free and amplified detection of exosomes.

Here, we develop a facile split aptamer-based system for the amplified, specific and wash-free detection of exosomes in situ assisted by a target-induced hybridization chain reaction (HCR). This design was successfully used to detect target exosomes in a bio-matrix and distinguish cancer patients from healthy individuals.

Recognition-Driven Remodeling of Dual-Split Aptamer Triggering In Situ Hybridization Chain Reaction for Activatable and Autonomous Identification of Cancer Cells.

Proximity-dependent hybridization chain reaction (HCR) has shown great potential in sensing biomolecules on the cell surface. However, the requirement of two adjacent bioevents occurring simultaneously limits its application. To solve the problem, split aptamers with target binding ability were introduced to combine with split triggers for initiating HCR, thus producing a novel dual-split aptamer probe (DSAP). By employing cancer-related receptors as models, in situ HCR on a cancer cell surface induced by recognition-driven remodeling of the DSAP was demonstrated. The DSAP consisted of two sequences. Each contained two segments; one derived from split aptamers and the other originated in split triggers. In the presence of target cells, split aptamers reassembled on the cell surface under the "induced-fit effect", thus forcing two split triggers close to each other. The remodeled DSAP worked as an intact trigger, which opened the H1 hairpin probe and then hybridized with the H2 hairpin probe, thus initiating HCR to produce an activated fluorescence signal. As a proof of concept, human liver cancer SMMC-7721 cells and their split ZY11 aptamer were used to construct the DSAP. Results indicated that the DSAP realized sensitive analysis of target cells, permitting the actual detection of 20 cells in the buffer. Moreover, the specific identification of target cells in mixed cell samples and the quantitative analysis of target cells in serum were also achieved. The DSAP strategy is facile and universal, which not only would expand the application range of HCR but also might be developed as a multitarget detection technique for bioanalysis.

Enzyme-free amplified detection of miRNA based on target-catalyzed hairpin assembly and DNA-stabilized fluorescent silver nanoclusters.

MicroRNAs (miRNAs) have been shown to be promising biomarkers for disease diagnostics and therapeutics. However, the rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their characters of small size, vulnerability to degradation, low abundance, and sequence similarity. Herein, we describe an enzyme-free amplification platform, consisting of a catalytic hairpin assembly (CHA) and DNA-templated silver nanoclusters (DNA/AgNCs), for miRNA analysis. In this work, two DNA hairpins (H1 and H2) were first designed for target miR-21-induced CHA, and then the fluorescence of DNA/AgNCs was quenched by BHQ1 to construct an activatable probe (AP). In the presence of target miR-21, hairpin H1 was opened by miR-21 through a hybridization reaction, and hairpin H2 was then opened by H1. During this process, miR-21 was released from H1 and participated in the next round of hybridization, triggering the CHA cycle reaction. The obtained H1-H2 products with sticky ends could react with the AP, forcing BHQ1 away from the DNA/AgNCs and thus causing the fluorescence recovery of the DNA/AgNCs. The assay for miR-21 detection demonstrated an excellent linear response to concentrations varying from 200 pM to 20 nM with the detection limit of 200 pM. The simple and cost-effective strategy holds great potential for application in biomedical research and clinical diagnostics.

Knocking Out SST Gene of BGC823 Gastric Cancer Cell by CRISPR/Cas9 Enhances Migration, Invasion and Expression of SEMA5A and KLF2.

BACKGROUND: The impact and potential molecular mechanisms of SST in the occurrence and development of GC have not been determined. MATERIALS AND METHODS: Two pairs of sgRNA and reporter were designed according to targeting sequence of SST gene for double-nicking. Plasmids were transfected into 293T for selecting sgRNA with higher cutting efficiency. The subline which has knocked-out SST gene were selected by FACS and verified by sequencing and expression level. Moreover, the migration and invasion ability was evaluated by wound healing and transwell after knocking out SST. Besides, the protein expression of SEMA5A and KLF2 were observed by Western blotting and LSCM. Last, we detected the expression levels of SST, SEMA5A, and KLF2 in GC tissues by Western blotting. RESULTS: The results revealed that the new subline 1E9, which had knocked out SST gene, was established by CRISPR/Cas9. In addition, the knockout of SST in GC cells markedly increased migration and invasion ability. The results also demonstrated that the knockout of SST increased the expression of SEMA5A and KLF2. The expression level of SST was decreased in GC tissues, and its decrease was associated with overexpression of SEMA5A and KLF2. CONCLUSION: SST plays an inhibitory role in the migration and invasion of GC cell BGC823. The protein expression levels of SEMA5A and KLF2 were enhanced in GC cells and tissues lacking SST expression.

Dual-microRNA-controlled double-amplified cascaded logic DNA circuits for accurate discrimination of cell subtypes.

Accurate discrimination between different cells at the molecular level is particularly important for disease diagnosis. Endogenous RNAs are such molecular candidates for cancer cell subtype identification. But the key is that there is often low abundance of RNAs in live cells, or some RNAs are often shared by multiple types of cells. Thus, we have designed dual-microRNA-controlled double-amplified cascaded logic DNA circuits for cancer cell subtype identification. The basic idea is to improve sensitivity by cascading DNAzyme and hybridization chain reaction (HCR), and improve accuracy by simultaneous detection of miR-122 and miR-21. The in-tube and in-cell experimental results show that the cascaded logic DNA circuits can work and serve to differentiate the liver cancer cells Huh7 from other normal cells and cancer cells. We anticipate that this design can be widely applied in facilitating basic biomedical research and accurate disease diagnosis.

One-pot synthesized Cu/Au/Pt trimetallic nanoparticles with enhanced catalytic and plasmonic properties as a universal platform for biosensing and cancer theranostics.

Cu/Au/Pt trimetallic nanoparticles (TMNPs) with enhanced catalytic activity and intense plasmonic absorption in the NIR-I biowindow (650-950 nm) were prepared using a fast, gentle and one-pot protocol. Based on these properties and assembly of thiolated-aptamers on Cu/Au/Pt TMNPs, a universal platform was developed for applications in biosensing and theranostics.

DNA nanotriangle-scaffolded activatable aptamer probe with ultralow background and robust stability for cancer theranostics.

Activatable aptamers have emerged as promising molecular tools for cancer theranostics, but reported monovalent activatable aptamer probes remain problematic due to their unsatisfactory affinity and poor stability. To address this problem, we designed a novel theranostic strategy of DNA nanotriangle-scaffolded multivalent split activatable aptamer probe (NTri-SAAP), which combines advantages of programmable self-assembly, multivalent effect and target-activatable architecture. Methods: NTri-SAAP was assembled by conjugating multiple split activatable aptamer probes (SAAPs) on a planar DNA nanotriangle scaffold (NTri). Leukemia CCRF-CEM cell line was used as the model to investigate its detection, imaging and therapeutic effect both in vitro and in vivo. Binding affinity and stability were evaluated using flow cytometry and nuclease resistance assays. Results: In the free state, NTri-SAAP was stable with quenched signals and loaded doxorubicin, while upon binding to target cells, it underwent a conformation change with fluorescence activation and drug release after internalization. Compared to monovalent SAAP, NTri-SAAP displayed greatly-improved target binding affinity, ultralow nonspecific background and robust stability in harsh conditions, thus affording contrast-enhanced tumor imaging within an extended time window of 8 h. Additionally, NTri-SAAP increased doxorubicin loading capacity by ~5 times, which further realized a high anti-tumor efficacy in vivo with 81.95% inhibition but no obvious body weight loss. Conclusion: These results strongly suggest that the biocompatible NTri-SAAP strategy would provide a promising platform for precise and high-quality theranostics.

Ultra-pH-responsive split i-motif based aptamer anchoring strategy for specific activatable imaging of acidic tumor microenvironment.

A non-blocking split i-motif based aptamer anchoring strategy was developed as a general platform for sensing weakly acidic tumor microenvironment. By rationally tuning the response range to pH 7.0-6.4 and adjusting aptamer types, the strategy achieved specific, pHe-activated imaging of different cancers in vitro and in vivo.

Label-free and sensitive microRNA detection based on a target recycling amplification-integrated superlong poly(thymine)-hosted copper nanoparticle strategy.

Poly(thymine)-hosted copper nanoparticles (poly T-CuNPs) have emerged as a promising label-free fluorophore for bioanalysis, but its application in RNA-related studies is still rarely explored. Herein, by utilizing duplex-specific nuclease (DSN) as a convertor to integrate target recycling mechanism into terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly T-CuNPs platform, a specific and sensitive method for microRNA detection has been developed. In this strategy, a 3'-phosphorylated DNA probe can hybridize with target RNA and then be cut by DSN to produce 3'-hydroxylated fragments, which can be further tailed by TdT with superlong poly T for fluorescent CuNPs synthesis. As proof of concept, an analysis of let-7d was achieved with a good linear correlation between 20 and 1000 pM (R2 = 0.9965) and a detection limit of 20 pM. Moreover, both homologous and heterologous microRNAs were also effectively discriminated. This strategy might pave a brand-new way for designing label-free and sensitive microRNA assays.

In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.