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Nanoparticles are promising for drug delivery applications, with several clinically approved products. However, attaining high nanoparticle accumulation in solid tumours remains challenging. Here we show that tumour cell-derived small extracellular vesicles (sEVs) block nanoparticle delivery to tumours, unveiling another barrier to nanoparticle-based tumour therapy. Tumour cells secrete large amounts of sEVs in the tumour microenvironment, which then bind to nanoparticles entering tumour tissue and traffic them to liver Kupffer cells for degradation. Knockdown of Rab27a, a gene that controls sEV secretion, decreases sEV levels and improves nanoparticle accumulation in tumour tissue. The therapeutic efficacy of messenger RNAs encoding tumour suppressing and proinflammatory proteins is greatly improved when co-encapsulated with Rab27a small interfering RNA in lipid nanoparticles. Together, our results demonstrate that tumour cell-derived sEVs act as a defence system against nanoparticle tumour delivery and that this system may be a potential target for improving nanoparticle-based tumour therapies.
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To explore the mechanisms and therapeutic strategies for G-quadruplex (G4) mediated diseases, it is crucial to manipulate and intervene in intracellular G4 structures using small molecular tools. While hundreds of G4 stabilizers have been developed, there is a significant gap in the availability of G4 unwinding agents. Here, we propose a strategy to disrupt G-quadruplexes by forming G-C hydrogen bonds with chemically modified cytidine trimers. We validated a good G4 unwinder, the 2'-F cytidine trimer (2'-F C3). 2'-F C3 does not inhibit cell growth nor cause severe DNA damage at a concentration below 10â µM. Moreover, 2'-F C3 does not affect gene transcription nor RNA splicing, while it significantly enhances the translation of G4-containing mRNA and upregulates RNA splicing, RNA processing and cell cycle pathways. The discovery of this G4 unwinder provides a functional tool for the chemical modulation of G4s in living cells.
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The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30-45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants.
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G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can specifically bind to G4 structures. There is increasing evidence that G4-protein interactions involve in the regulation of important cellular processes, such as DNA replication, transcription, RNA splicing, and translation. Additionally, G4-protein interactions have been demonstrated to be potential targets for disease treatment. In order to unravel the detailed regulatory mechanisms of G4-binding proteins (G4BPs), biochemical methods for detecting G4-protein interactions with high specificity and sensitivity are highly demanded. Here, we review recent advances in screening and validation of new G4BPs and highlight both their features and limitations.
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Quadruplex G , DNA/química , Replicação do DNA , RNA/químicaRESUMO
G-quadruplex (G4) structures exist in the single-stranded DNA of chromatin and regulate genome function. However, the native chromatin G4 landscape in living cells has yet to be fully characterized. Herein, a genetic-encoded live-cell G4 identifier probe (LiveG4ID) is constructed and its cellular localization, biocompatibility, and G4-binding specificity is evaluated. By coupling LiveG4ID with cleavage under targets and tagmentation (CUT&Tag), LiveG4ID-seq, a method for mapping native chromatin G4 landscape in living cells with high accuracy is established. Compared to the conventional G4 CUT&Tag method, LiveG4ID-seq can identify more chromatin G4 signals and have a higher ratio of true positive signals. Using LiveG4ID-seq, the dynamic landscape of chromatin G4 structures during the cell cycle is profiled. It is discovered that chromatin G4 structures are prevalent in the promoter regions of cell cycle-specific genes, even in the early M phase when the chromatin is condensed. These data demonstrate the capacity of LiveG4ID-seq to profile a more accurate G4 landscape in living cells and promote future studies on chromatin G4 structures.
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Cromatina , Quadruplex G , Cromatina/genética , Ciclo Celular/genética , Divisão CelularRESUMO
Pathogenic infection remains the primary threat to human health, such as the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutated variants, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we firstly describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges involved in on-site diagnostic assays and present emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.
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The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for versatile diagnostic assays that can discriminate among emerging variants of the virus. Here we report the development and performance benchmarking of an inexpensive (approximately US$0.30 per test) assay for the rapid (sample-to-answer time within 30 min) colorimetric detection of SARS-CoV-2 variants. The assay, which we integrated into foldable paper strips, leverages nucleic acid strand-displacement reactions, the thermodynamic energy penalty associated with single-base-pair mismatches and the metal-ion-controlled enzymatic cleavage of urea to amplify the recognition of viral RNAs for the colorimetric readout of changes in pH via a smartphone. For 50 throat swab samples, the assay simultaneously detected the presence of SARS-CoV-2 and mutations specific to the SARS-CoV-2 variants Alpha, Beta and Gamma, with 100% concordance with real-time quantitative polymerase chain reaction and RNA sequencing. Customizable and inexpensive paper-based assays for the detection of viruses and their variants may facilitate viral surveillance.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Colorimetria , Humanos , Nucleotídeos , SARS-CoV-2/genéticaRESUMO
Single-cell imaging has unique advantages of maintaining the inâ situ physiological state, morphology, and microenvironment, becoming a powerful tool to unravel the nature of intracellular nucleic acids. The analysis of nucleic acids unprecedentedly demands the sub-molecule details at segment or subunit, secondary structure and monomer levels, instead of just probing the sequence and the abundance of nucleic acids. Detection of nucleic acids at the sub-molecule level requires higher specificity and higher sensitivity, which becomes a new challenge in nucleic acid analysis. Herein, we summarize the recent progress in the design and the application of single-cell nucleic acid imaging methods at the sub-molecule level, including the visualization of RNA splicing variants, RNA G-quadruplexes in an individual gene, single nucleotide variation of mitochondrial DNA, and RNA m6 A methylation. Remarkably, we highlight the key strategy, "Module Assembly", for high-performance molecular recognition and demonstrate the required improvements in future research.
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Quadruplex G , Ácidos Nucleicos , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , RNA/químicaRESUMO
G-quadruplexes (G4s) are a kind of non-canonical nucleic acid secondary structures, which involve in various biological processes in living cells. The relationships between G4s and human diseases, such as tumors, neurodegenerative diseases, and viral infections, have attracted great attention in the last decade. G4s are considered as a promising new target for disease treatment. For instance, G4 ligands are reported to be potentially effective in SARS-COV-2 treatment. However, because of the lack of analytical methods with high performance for the identification of intracellular G4s, the detailed mechanisms of the biofunctions of G4s remain elusive. Meanwhile, through demonstrating the principles of how the G4s systematically modulate the cellular processes with advanced detection methods, biochemical targeting of G4s in living cells can be realized by chemical and biological tools and becomes useful in biomedicine. This review highlights recent methodological advances about intracellular G4s and provides an outlook on the improvement of the bioanalysis and biochemical targeting tools of G4s.
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G-quadruplexes (G4s), non-canonical nucleic acid secondary structure, regulate many biological functions and are considered potential molecular targets for cancer therapeutics. However, due to the lack of analytical methods, the regulating mechanism of monogenic G4s is still unclear. Here, we developed a Module Assembled Multifunctional Probes Assay (MAMPA) for visualizing endogenous G4s in individual genes in single cells. Two modular probes separately recognize G4 structures and the adjacent RNA sequences, and the module assembly enables imaging of G4s in an individual RNA with high specificity. Through imaging G4s in several individual genes, we found that G4s were steadily occupied by G4 Binding Proteins (G4BPs) in various mRNAs in every cell line and defined "Occupied G4 Ratio". We demonstrated MAMPA was suitable for most experimental situations and found that Occupied G4 Ratios had the potential to become a new parameter for the study of G4s in living cells.
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Bioensaio , Proteínas Proto-Oncogênicas c-bcl-2/genética , Análise de Célula Única , Quadruplex G , Células HeLa , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/análise , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Exosomes that carry multiple proteins from the originating cells are known as emerging biomarkers for tumor diagnostics. However, it is still technically challenging to accurately evaluate subtle differences of exosomal membrane proteins. Here, we developed a rolling circle amplification (RCA)-assisted flow cytometry approach (FCA) to simultaneously profile surface proteins and quantify exosomes. In this work, specific anti-CD63 antibody-conjugated magnetic beads were first utilized to capture exosomes. Then, the captured exosomes were bound with DNA primers, which comprise exosomal surface protein-specific recognition aptamers. The RCA reaction generates repeat DNA sequences for fluorescent probe hybridization. Finally, a conventional flow cytometer was introduced to phenotype exosomal protein markers. Such a sensitive RCA-assisted FCA displays an excellent detection limit of 1.3 × 105 exosome/mL. The variable composition of four protein markers on different cell-derived exosomes was sensitively detected through changing the protein-recognition sequence of the DNA primer, which reveals a heterogeneous pattern. Exosomes from different cell sources could be distinguished by the abundance difference of multiple surface proteins. Furthermore, the developed RCA-assisted FCA enabled quantitative analysis of blood samples from lung cancer patients, indicating its potential for early clinical diagnosis and prognosis of cancer.
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Exossomos , Proteínas de Membrana , DNA , Exossomos/genética , Citometria de Fluxo , Humanos , Proteínas de Membrana/genética , Hibridização de Ácido NucleicoRESUMO
Glioblastoma (GBM) is the most malignant brain tumor with unmet therapeutic demand. The blood-brain-barrier (BBB) and tumor heterogeneity limit the treatment effectiveness of various interventions. Here, an ultrasound augmented chemo/immuno therapy for GBM using a neutrophil-delivered nanosensitizer, is developed. The sensitizer is composed of a ZnGa2 O4 :Cr3+ (ZGO) core for persistent luminescence imaging and a hollow sono-sensitive TiO2 shell to generate reactive oxygen species (ROS) for controlled drug release. Immune checkpoint inhibitor (Anti-PD-1 antibody) is trapped in the interior of the porous ZGO@TiO2 with paclitaxel (PTX) loaded liposome encapsulation to form ZGO@TiO2 @ALP. Delivered by neutrophils (NEs), ZGO@TiO2 @ALP-NEs can penetrate through BBB for GBM accumulation. After intravenous injection, ultrasound irradiation at GBM sites initiates ROS generation from ZGO@TiO2 @ALP, leading to liposome destruction for PTX and anti-PD-1 antibody release to kill tumors and induce local inflammation, which in-turn attractes more ZGO@TiO2 @ALP-NEs to migrate into tumor sites for augmented and sustained therapy. The treatment enhances the survival rate of the GBM bearing mice from 0% to 40% and endows them with long-term immuno-surveillance for tumor recurrence, providing a new approach for precision therapy against GBM and other cancers.
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Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/terapia , Portadores de Fármacos/administração & dosagem , Glioblastoma/terapia , Inibidores de Checkpoint Imunológico/administração & dosagem , Terapia por Ultrassom/métodos , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Imunoterapia/métodos , Luminescência , Camundongos , Camundongos Nus , Nanocápsulas , Neutrófilos/metabolismo , TitânioRESUMO
Stroke is a lethal cerebral disease with severe sequelae and high mortality. Microglia, the main immune cell in the cerebrum, possess therapeutic potential for strokes as its specific anti-inflammatory phenotype can reduce inflammation and promote neuron regeneration. However, the on-demand anti-inflammatory polarization of microglia at the stroke site is uncontrollable for therapeutic application. Here, we develop a platelet hybrid microglia platform which can specifically polarize to the anti-inflammatory phenotype by ultrasound irradiation for targeted cerebrum repair after stroke. The engineered microglia have strong adherence to the injured cerebral vessels with platelet membrane fusion and realize on-demand anti-inflammatory polarization with ultrasound-responsive IL-4 liposome decoration. The intravenously injected microglia platform showed anti-inflammatory polarization at the stroke site with insonation, and accelerated the M2-type polarization of endogenous microglia for long-term stroke recovery. Satisfied prognoses were achieved with reduced apoptosis, promoted neurogenesis, and functional recovery, indicating the implications of the microglia platform for stroke therapy.
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Plaquetas/metabolismo , Inflamação/terapia , AVC Isquêmico/terapia , Microglia/metabolismo , Animais , Apoptose/fisiologia , Plaquetas/química , Engenharia Celular , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Inflamação/etiologia , Inflamação/metabolismo , Interleucina-4/química , Interleucina-4/metabolismo , AVC Isquêmico/complicações , AVC Isquêmico/metabolismo , Lipossomos/química , Lipossomos/efeitos da radiação , Masculino , Camundongos Endogâmicos C57BL , Microglia/química , Neurogênese/fisiologia , Protoporfirinas/química , Recuperação de Função Fisiológica/fisiologia , Ondas UltrassônicasRESUMO
Cancer vaccines hold great promise for improved cancer treatment. However, endosomal trapping and low immunogenicity of tumour antigens usually limit the efficiency of vaccination strategies. Here, we present a proton-driven nanotransformer-based vaccine, comprising a polymer-peptide conjugate-based nanotransformer and loaded antigenic peptide. The nanotransformer-based vaccine induces a strong immune response without substantial systemic toxicity. In the acidic endosomal environment, the nanotransformer-based vaccine undergoes a dramatic morphological change from nanospheres (about 100 nanometres in diameter) into nanosheets (several micrometres in length or width), which mechanically disrupts the endosomal membrane and directly delivers the antigenic peptide into the cytoplasm. The re-assembled nanosheets also boost tumour immunity via activation of specific inflammation pathways. The nanotransformer-based vaccine effectively inhibits tumour growth in the B16F10-OVA and human papilloma virus-E6/E7 tumour models in mice. Moreover, combining the nanotransformer-based vaccine with anti-PD-L1 antibodies results in over 83 days of survival and in about half of the mice produces complete tumour regression in the B16F10 model. This proton-driven transformable nanovaccine offers a robust and safe strategy for cancer immunotherapy.
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Antígenos/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Preparações de Ação Retardada/química , Nanosferas/química , Neoplasias/prevenção & controle , Animais , Antígenos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Concentração de Íons de Hidrogênio , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Polímeros/química , PrótonsRESUMO
The ability to 'see' genetic information directly in single cells can provide invaluable insights into complex biological systems. In this review, we discuss recent advances of in situ imaging technologies for visualizing the subtlest sequence alteration, single-nucleotide variation (SNV), at single-cell level. The mechanism of recently developed methods for SNV discrimination are summarized in detail. With recent developments, single-cell SNV imaging methods have opened a new door for studying the heterogenous and stochastic genetic information in individual cells. Furthermore, SNV imaging can be used on morphologically preserved tissue, which can provide information on histological context for gene expression profiling in basic research and genetic diagnosis. Moreover, the ability to visualize SNVs in situ can be further developed into in situ sequencing technology. We expect this review to inspire more research work into in situ SNV imaging technologies for investigating cellular phenotypes and gene regulation at single-nucleotide resolution, and developing new clinical and biomedical applications.
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Sequenciamento de Nucleotídeos em Larga Escala , Nucleotídeos/genética , Análise de Célula Única , Perfilação da Expressão Gênica , Variação Genética/genética , HumanosRESUMO
MicroRNAs (miRNAs) play a critical role in multifarious biological processes and being deemed to be important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Thus, assays for sensitive and accurate quantification of miRNAs are highly demanded. Herein, we have constructed a RNA aptamer involved cascade transcription amplification method (termed RACTA), enabling label-free, ultrasensitive, and specific detection of miRNA. Target miRNA-initiated strand-displacement amplification would allow for the production of plenty of ssDNA that triggers the subsequent transcriptional amplification of spinach RNA aptamers. Consequently, transcribed tremendous spinach aptamers activated fluorophore DFHBI (( Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1 H-imidazol-5(4 H)-one) for miRNA quantitative analysis. RACTA outperforms conventional strand displacement amplification (SDA) at both background and amplification rate due to the light-up mechanism of DFHBI dye-Spinach aptamer and cascade signal amplification of RACTA. Thus, the signal-to-noise ratio of RACTA was increased by about 20-fold compared to that of SDA. This RACTA assay could confer a highly sensitive detection of miRNA with a detection limit of 5.12 × 10-18 M and excellent specificity enabling differentiation between miRNAs and homologous families. Besides, this assay has been successfully demonstrated for quantification of miRNAs in different cell lines. Therefore, the proposed method holds great potential for miRNA biomarker based early diagnosis and prognosis monitoring.
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Aptâmeros de Nucleotídeos/genética , MicroRNAs/análise , Células Cultivadas , Amplificação de Genes , Células HEK293 , Células HeLa , Humanos , Células MCF-7RESUMO
Mitochondrial redox homeostasis, the balance between reactive oxygen species and antioxidants such as glutathione, plays critical roles in many biological processes, including biosynthesis and apoptosis, and thus is a potential target for cancer treatment. Here, we report a mitochondrial oxidative stress amplifier, MitoCAT-g, which consists of carbon-dot-supported atomically dispersed gold (CAT-g) with further surface modifications of triphenylphosphine and cinnamaldehyde. We find that the MitoCAT-g particles specifically target mitochondria and deplete mitochondrial glutathione with atomic economy, thus amplifying the reactive oxygen species damage caused by cinnamaldehyde and finally leading to apoptosis in cancer cells. We show that imaging-guided interventional injection of these particles potently inhibits tumour growth in subcutaneous and orthotopic patient-derived xenograft hepatocellular carcinoma models without adverse effects. Our study demonstrates that MitoCAT-g amplifies the oxidative stress in mitochondria and suppresses tumour growth in vivo, representing a promising agent for anticancer applications.
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Carbono/química , Ouro/química , Mitocôndrias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Estresse Oxidativo , Animais , Antineoplásicos/farmacologia , Apoptose , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CRISPR/Cas9 has already become a powerful tool for genomic manipulation, and further engineering of the system allows it to be precisely regulated in response to external signals, thus, broadening its application possibilities, such as biosensing or bioimaging. However, most stimuli-responsive CRISPR systems are built based on elaborately designed and engineered inducible Cas9 proteins, and external stimuli are still mostly limited as small molecules and light. To construct more precise and easy-to-build responsive CRISPR systems and broaden their responsive species, we seek to engineer conditional guide RNA, rather than Cas9 protein, to mediate conditional CRISPR corresponding to logic operation. Here, we construct mRNA-sensing CRISPR by gRNA reconfiguration and toehold mediated strand displacement, in which each target site could be independently controlled. We show that switches can be embedded into the gRNA and used as RNA sensors, capable of detecting multiple mRNA inputs orthogonally and providing CRISPR/Cas9 response outputs. NOR and NAND logical gates are also constructed, demonstrating its orthogonality and programmability. This strategy promises potential uses in constructing genetic circuits to detect endogenous mRNAs and initiate cellular responses.
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Sistemas CRISPR-Cas , Genoma Humano , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , Vetores Genéticos , Células HeLa , Humanos , Células MCF-7 , RNA Mensageiro/genética , Recombinação GenéticaRESUMO
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA (lncRNA) located in the cell nucleus, is a critical regulator of tumor cell migration. Antisense oligonucleotides (ASOs), which can downregulate the expression level of specific RNAs, have been used in clinical for disease treatment. Herein, we constructed MALAT1-specific ASO and nucleus-targeting TAT peptide cofunctionalized Au nanoparticles, namely, ASO-Au-TAT NPs, which stabilized the fragile ASOs, enhanced nuclear internalization, and exhibited good biocompatibility. After treatment with the ASO-Au-TAT NPs, A549 lung cancer cells showed a greatly reduced MALAT1 expression level and decreased migration ability in vitro. Moreover, the ASO-Au-TAT NPs significantly reduced metastatic tumor nodule formation in vivo. Our results demonstrate that the ASO-Au-TAT nanostructures (NSs) have great potential for treatment of cancer metastasis.
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Adenocarcinoma de Pulmão , Sistemas de Liberação de Medicamentos , Ouro , Neoplasias Pulmonares , Nanopartículas Metálicas , Oligonucleotídeos Antissenso , RNA Longo não Codificante , RNA Neoplásico , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Ouro/química , Ouro/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Metástase Neoplásica , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/química , Peptídeos/farmacologia , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Neoplásico/antagonistas & inibidores , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologiaRESUMO
The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging-associated diseases. Imaging of single-nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9-mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single-molecule resolution. Using CasPLA, we observed a mtDNA-transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy genomic loci ( KRAS gene) in the nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.