RESUMO
Molecular engineering enables the creation of aptamers with novel functions, but the prerequisite is a deep understanding of their structure and recognition mechanism. The cellular-mesenchymal epithelial transition factor (c-MET) is garnering significant attention due to the critical role of the c-MET/HGF signaling pathway in tumor development and invasion. This study reports a strategy for constructing novel chimeric aptamers that bind to both c-MET and other specific proteins. c-MET was identified to be the molecular target of a DNA aptamer, HF3-58, selected through cell-SELEX. The binding structure and mechanism of HF3-58 with c-MET were systematically studied, revealing the scaffold, recognition, and redundancy regions. Through molecular engineering design, the redundancy region was replaced with other aptamers possessing stem-loop structures, yielding novel chimeric aptamers with bispecificity for binding to c-MET and specific proteins. A chimeric bispecific aptamer HF-3b showed the ability to mediate the adhesion of T-cells to tumor cells, suggesting the prospective utility in tumor immunotherapy. These findings suggest that aptamer HF3-58 can serve as a molecular engineering platform for the development of diverse multifunctional ligands targeting c-MET. Moreover, comprehensive understanding of the binding mechanisms of aptamers will provide guidance for the design of functional aptamers, significantly expanding their potential applications.
RESUMO
Conditionally activated molecule release in live cells would provide spatiotemporal control for the study and intervention of biological processes, e.g., bioactive molecule monitoring and controlled drug release. Mitochondria are the main sites of reactive oxygen species (ROS) production in cells. Here, we report an ROS-triggered molecule release strategy in mitochondria. A molecule IRTO with dual targeting groups was designed by covalently linking IR-780 (a mitochondrial targeted heptamethine cyanine) and 4-aminobutyl-thiazole orange (NH2-TO, a nuclear dye). IRTO diffused into live cells and first accumulated in mitochondria. As the cyanine moiety reacted with mitochondrial ROS directly or with the help of mitochondrial cytochromes, NH2-TO was released, escaped from mitochondria, and finally located in the nucleus. This process could be visualized by fluorescent imaging, i.e., red fluorescence (from the cyanine moiety of IRTO) first located in mitochondria, and green fluorescence (from NH2-TO) appeared and gradually enhanced in the nucleus with the increase of incubation time. The addition of H2O2 or lipopolysaccharide (LPS, an ROS accelerator) could accelerate the release of NH2-TO, whereas N-acetyl-l-cysteine (NAC, an ROS inhibitor) and mitoquinone mesylate (MitoQ, a mitochondrial ROS scavenger) could obviously decrease the release of NH2-TO. These results suggest that IRTO could serve as a fluorescent probe for monitoring ROS in mitochondria and that IR-780 might be a promising endogenous ROS-triggered molecule release platform.
Assuntos
Peróxido de Hidrogênio , Mitocôndrias , Espécies Reativas de Oxigênio , Corantes Fluorescentes , Acetilcisteína/farmacologiaRESUMO
Cellular prion protein (PrPC) is highly expressed in a variety of tumor cells and plays a crucial role in neurodegenerative diseases. Its N-terminal domain contains a conserved octapeptide (PHGGGWGQ) repeat sequence. The number of repeats has been correlated with the species as well as the development of associated diseases. Herein, PrPC was identified to be the molecular target of a high-affinity DNA aptamer HA5-68 obtained by cell-SELEX. Aptamer HA5-68 was further optimized to two short sequences (HA5-40-1 and HA5-40-2), and its binding site to PrPC was identified to be located in the loop-stem-loop region of the head of its secondary structure. HA5 series aptamers were demonstrated to bind the octapeptide repeat region of PrPC, as well as the synthesized peptides containing different numbers of octapeptide repeats. The PrPC expression on 42 cell lines was measured by using aptamer HA5-68 as a molecular probe. The clear understanding of the molecular structure and binding mechanism of this set of aptamers will provide information for the design of diagnostic methods and therapeutic drugs targeting PrPC.
Assuntos
Aptâmeros de Nucleotídeos , Doenças Priônicas , Príons , Humanos , Proteínas Priônicas , Aptâmeros de Nucleotídeos/química , Ligação Proteica , Príons/genética , Sítios de Ligação , Doenças Priônicas/metabolismoRESUMO
The L1 cell adhesion molecule (L1CAM) plays important roles in the development and plasticity of the nervous system as well as in tumor formation, progression, and metastasis. New ligands are necessary tools for biomedical research and the detection of L1CAM. Here, DNA aptamer yly12 against L1CAM was optimized to have much stronger binding affinity (10-24 fold) at room temperature and 37 °C via sequence mutation and extension. This interaction study revealed that the optimized aptamers (yly20 and yly21) adopted a hairpin structure containing two loops and two stems. The key nucleotides for aptamer binding mainly located in loop I and its adjacent area. Stem I mainly played the role of stabilizing the binding structure. The yly-series aptamers were demonstrated to bind the Ig6 domain of L1CAM. This study reveals a detailed molecular mechanism for the interaction between yly-series aptamers and L1CAM and provides guidance for drug development and detection probe design against L1CAM.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias , Molécula L1 de Adesão de Célula Nervosa , Humanos , Aptâmeros de Nucleotídeos/química , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neoplasias/metabolismoRESUMO
Cell-SELEX is a powerful tool to generate aptamers that specifically bind the native molecules on living cells. Here, we report an aptamer ZAJ4a generated by cell-SELEX. The molecular target of ZAJ4a was pulled down by the enriched cell-SELEX pool and identified to be the receptor-type tyrosine-protein phosphatase F (PTPRF) through a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic method. ZAJ4a showed high binding affinity with nanomolar range to cancer cells expressing PTPRF. Meanwhile, PTPRF was proven to highly express on several cancer cell lines using ZAJ4a as a molecular probe and to highly express in many kinds of cancer samples using gene expression profiling interactive analysis (GEPIA2) from the TCGA and GTEx databases. These results indicate that the aptamer generated by cell-SELEX showed good specificity at the molecular level. This cell-SELEX and target identification strategies show great potential for identifying biomarkers on the cell surface.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteômica , Biomarcadores , Sondas Moleculares , Técnica de Seleção de Aptâmeros/métodosRESUMO
A photo-activated aptamer-drug conjugate, HG1-9-DNP, was developed based on an aptamer HG1-9 and a photolabile naphthalimide derivative DNP. HG1-9-DNP could be internalized into cells mediated by TfR, then photocleaved, and released a promising cytotoxic agent, DNNH, which arrested the cell cycle at the G2/M phase, resulting in high photo-induced cytotoxicity.
Assuntos
Aptâmeros de Nucleotídeos , Doxorrubicina , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Citotoxinas , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Naftalimidas , Oligonucleotídeos , Preparações FarmacêuticasRESUMO
As a kind of recognition molecule, aptamers can be inserted into some regulatory sequences for the smart response of their targets. However, the molecular engineering might lead to the change of the binding affinity. Here, we present a stable aptamer ZAJ-2c and an environmentally sensitive aptamer ZAJ-2d optimized from an original cell-binding aptamer ZAJ-2, and the molecular target was further identified as CD49c on the cell membrane. ZAJ-2c was characterized with high binding ability independent of the presence of divalent cations at a temperature range from 4 to 37 °C, showing promise for measuring the expression of CD49c on cancer cells. Moreover, ZAJ-2d had a nanomolar binding affinity in the binding buffer at 4 °C, the same as ZAJ-2c, but lost the binding ability in a PBS buffer supplemented with 5 mM EDTA at 37 °C. This aptamer variant proved to selectively capture and release the CD49c positive cells by simply adjusting the temperatures and divalent cations. This set of aptamers might provide a toolbox for monitoring and operating of a wide range of cancer cells with CD49c expression on the surface, which will be helpful for the studying the heterogeneity of rare cells.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias , Aptâmeros de Nucleotídeos/química , Cátions Bivalentes , Integrina alfa3 , Técnica de Seleção de AptâmerosRESUMO
Malignant melanoma is regarded as the most aggressive form of skin cancer, and is responsible for most death caused by skin cancer. BRAF mutations occur in approximately 40-50% of melanomas, with V600E being the most common mutation. Testing for BRAF mutations and targeted therapy have markedly improved long-term survival for patients with BRAF-mutated melanoma. Here, we report two aptamers, CH1 and CH5 generated by Cell-SELEX, against BRAF V600E-mutated human melanoma cells A375. The two aptamers exhibited high affinity to target cells with low dissociation constants (Kd) in the nanomolar range. Furthermore, the binding of two aptamers to target cells was independent of incubation temperature, and their molecular targets were demonstrated to be membrane proteins on the cell surface. We also demonstrated that aptamer CH1 was able to bind to metastatic colorectal cancer cells harboring BRAF V600E mutation, indicating a relationship between aptamer CH1 and BRAF V600E-related metastatic cancer. Owing to the structure stability and high selectivity to BRAF V600E-mutated targeting cells, aptamer CH1 holds great potential as a molecular probe for the detection of BRAF V600E-mutated metastatic melanoma.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , DNA de Cadeia Simples , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas de Membrana , Oligonucleotídeos , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Neoplasias/metabolismo , Receptores da Transferrina/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , Humanos , Ligantes , Neoplasias/patologia , Transcitose , Células Tumorais CultivadasRESUMO
A blue light activated anti-cancer prodrug, NST, was designed based on a photoactive 4-aminonaphthalimide derivative and an anticancer drug, 10-hydroxycamptothecin. NST was hard to be taken up by living cells and showed negligible dark cytotoxicity. The irradiation caused photocleavage of NST and resulted in high cytotoxicity.
Assuntos
Luz , Ftalimidas/química , Ftalimidas/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Antineoplásicos/metabolismo , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Linhagem Celular Tumoral , Humanos , Ftalimidas/metabolismo , Pró-Fármacos/metabolismoRESUMO
Herein, we report a FnCas12a/crRNA assisted Dumbbell-PCR method for the detection of isomiRs with double specificity and magnification. The single nucleotide variant of isomiRs in terminals and/or inner sequence could be discriminated by this strategy. Using this method, let-7a isomiRs in MCF-7 and MCF-7R cell lines were analyzed.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Reação em Cadeia da Polimerase/métodos , Fluorescência , Humanos , Células MCF-7RESUMO
Lycium barbarum L. is a widely used functional food and medicinal herb in Asian countries. L. barbarium polysaccharides (LBP) are considered as one of the major medicinal components of L. barbarium fruit and exhibits a wide range of biological activities. Here, we investigated the immunomodulatory effects of LBP and its uptake behaviors at the cellular level. LBP was prepared by water extraction and ethanol precipitation, and divided into two fractions based on the molecular weight distribution by ultrafiltration (LBP > 10 kDa and LBP < 10 kDa). The physicochemical properties of LBP and LBP fractions were well characterized. The LBP > 10 kDa fraction greatly enhanced the viability of macrophages RAW264.7 cells and induced cell polarization, but had weak effects to other tested tumor cell lines and normal cell line. This fraction could regulate the production of NO, TNF-α, IL-6 and ROS in RAW264.7 cells, suggesting both pro-inflammatory and anti-inflammatory effects. The dye-labeled LBP could be internalized into all tested cell lines and accumulated in lysosomes. The internalization of LBP in RAW264.7 cells is mainly through the clathrin-mediated endocytosis pathway. The Caco-2 intestinal transport experiment demonstrated that the dye labeled LBP could be transported through the Caco-2 cell monolayer (mimic intestinal epithelium) through clathrin-mediated endocytosis. These results demonstrate the immunomodulatory effects of LBP and its effective uptake by macrophages and intestine.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Fatores Imunológicos/farmacologia , Extratos Vegetais/farmacologia , Animais , Células CACO-2 , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/isolamento & purificação , Fluorescência , Humanos , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , TemperaturaRESUMO
Circulating tumor-related materials (CTRMs) shed from original or metastatic tumors, carry a lot of tumor information and are considered as important markers for cancer diagnosis and metastasis prognosis. Herein, we report a colorimetric detection strategy for CTRMs based on aptamer-based magnetic isolation and endogenous alkaline phosphatase (AP)-signal amplification. This strategy exhibited high sensitivity and selectivity toward the CTRMs that express AP heterodimers (the target of aptamer, a potential tumor marker). For clinical samples, this CTRM assay significantly discriminated colorectal cancer patients (n = 50) from healthy individuals (n = 39, p < 0.0001). The receiver operating characteristic (ROC) analysis indicated the sensitivity and specificity reached 92% and 82%, respectively, at the optimal cutoff point, the area under the curve of ROC reached 0.93, suggesting great potential for colorectal cancer diagnosis and therapeutic monitoring. Compared with CTC assays, this strategy is simple and has the potential for point-of-care testing.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Técnicas Biossensoriais/métodos , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , Colorimetria , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Cell-SELEX is an effective strategy to discover aptamers that can distinguish the molecular signatures of target cells from control cells. The molecular targets of such aptamers have the potential to be biomarkers. Here, we report target identification of aptamer wy-5a generated by cell-SELEX against a prostate cancer cell line, PC-3. This aptamer specifically binds PC-3 cells and a doxorubicin-resistant breast cell line, MCF-7R, as well as tissue sections of prostate cancer with high risk of metastasis. Prion protein was identified to be the molecular target of wy-5a by stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic method. The octapeptide repeat region of prion protein was demonstrated to be the binding site of aptamer wy-5a. The expression levels of prion protein in cancer tissues were further tested by immunohistochemical staining of tissue sections from 48 prostate cancer patients and 98 breast cancer patients. The results suggest that prion protein has the potential to be one of the referenced markers of prostate and breast cancers.
RESUMO
Dimerization of proteins occurs frequently and plays integral roles in biological processes. However, no single molecular probe is available for in situ detection of protein dimers on cells and tissues because of the difficulty of isolating complete protein dimers for probe preparation and screening, which has greatly hampered the biomedical study of protein dimers. Herein, a G-rich DNA aptamer (termed BG2) that only binds alkaline phosphatase (AP) heterodimers rather than monomers is reported. This aptamer is generated by the cell-SELEX (systematic evolution of ligands by exponential enrichment) technique and proves to fold into a duplex stabilized antiparallel G-quadruplex structure. Using BG2 as molecular probe, AP heterodimers are found to be expressed on several kinds of cancer cells. As an affinity ligand, BG2 could isolate AP heterodimers from cell lysate. BG2 is also demonstrated to be applicable for tumor imaging in mice xenografted with cells highly expressing AP heterodimers. AP isozymes are found in several tissues and blood throughout the body, but the function and tissue distribution of AP heterodimers are totally unknown; therefore, BG2 could serve as a molecular probe to uncover the mystery of AP heterodimers. The generation of aptameric probes by cell-SELEX will open up a new situation for the study of protein dimers.
RESUMO
Dead cells always accompany with live cells in vivo and in cell culture. It is important to distinguish dead cells from live cells in various biological studies. Currently, the probes for dead cells are mainly nucleic acid-intercalators, most of which have low affinity and potential toxicity to live cells. In this work, we report a novel aptameric probe (Ch4-1) for the first time, which binds cell nuclei with high affinity (apparent Kd = 6.65 ± 3.40 nM). Ch4-1 was generated by Cell-SELEX process, it was identified to target nucleoproteins in cell nuclei. As an oligonucleotide, Ch4-1 cannot penetrate the integrated cell membrane; therefore, it only binds to dead cells rather than live cells. Compared with traditional DNA-targeting nuclear dyes, Ch4-1 possesses a high affinity to the nucleus, no toxicity to live cells, and can be easily labeled with different fluorescent dyes. It was demonstrated to serve as a probe for distinguishing dead cells from live cells in apoptosis assay, as well as for the nuclear staining of tissue sections.
Assuntos
Apoptose , Aptâmeros de Nucleotídeos/química , Núcleo Celular/química , DNA/química , Neoplasias/classificação , Nucleoproteínas/análise , Animais , Morte Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias/patologia , Nucleoproteínas/química , Oligodesoxirribonucleotídeos/química , Técnica de Seleção de AptâmerosRESUMO
Ovarian cancer is regarded as the most lethal gynecologic malignancy with poor prognosis and high mortality rate. Drug-resistance was thought to be the main obstacle to improving overall survival rate of ovarian cancer. New ligands for drug-resistant ovarian cancer cells have potential for the development of diagnosis and therapy of ovarian cancer. In present work, we reported two aptamers, HF3-58 and HA5-68 generated by cell-SELEX, against a paclitaxel-resistant ovarian cancer cell line (A2780T). Both two aptamers exhibited high selectivity and strong affinity to target cells with low nanomolar dissociation constants. The binding of aptamers to target cells was independent of divalent ions, and was tolerant of incubation temperature and nucleases in serum. Molecular targets of the two aptamers were preliminarily demonstrated to be two different glycoproteins on cell surface of A2780T cells. Owing to the structure stability and high resistance to nuclease, these two aptamers had good performance in the detection of drug-resistant ovarian cancer cells in human serum.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/patologia , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismoRESUMO
Neurite outgrowth is the critical step of nervous development. Molecular probes against neurites are essential for evaluation of the nervous system development, compound neurotoxicity, and drug efficacy on nerve regeneration. To obtain a neurite probe, we developed a neurite-SELEX strategy and generated a DNA aptamer, yly12, that strongly binds neurites. The molecular target of yly12 was identified to be neural cell adhesion molecule L1 (L1CAM), a surface antigen expressed in normal nervous system and various cancers. Here, yly12 was successfully applied to image the three-dimensional network of neurites between live cells, as well as the neurite fibers on normal brain tissue section. This aptamer was also found to have an inhibitory effect on neurite outgrowth between cells. Given the advantages of aptamers, yly12 hold great potential as a molecular tool in the field of neuroscientific research. The high efficiency of neurite-SELEX suggests that SELEX against a subcellular structure instead of the whole cells is more effective in obtaining the desired aptamers.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Crescimento Neuronal/fisiologia , Linhagem Celular Tumoral , Humanos , Molécula L1 de Adesão de Célula Nervosa/química , Técnica de Seleção de AptâmerosRESUMO
Real-time monitoring of the distribution of energy released during oxidative phosphorylation (OXPHOS) in living cells would advance the understanding of metabolic pathways and cell biology. However, the relationship between intracellular temperature and ATP fluctuation during the OXPHOS process is rarely studied due to the limitation of the sensing approach. Novel fluorescent polymer probes were developed for accurate simultaneous measurements of intracellular temperature and ATP. Utilizing the fluorescence imaging techniques, it was demonstrated for the first time that the temperature in mitochondria increased 2.4 °C and the ATP fluctuation level simultaneously decreased 75% within 2 min during the OXPHOS process. Moreover, the resultant fluorescent polymer probes had good performance and properties for mitochondrial targeting, providing an effective way for investigating mechanisms by which energy is released during the OXPHOS process.
Assuntos
Trifosfato de Adenosina/análise , Ácidos Borônicos/química , Cumarínicos/química , Corantes Fluorescentes/química , Mitocôndrias/metabolismo , Compostos Organofosforados/química , Resinas Acrílicas/síntese química , Resinas Acrílicas/química , Resinas Acrílicas/toxicidade , Trifosfato de Adenosina/química , Ácidos Borônicos/síntese química , Ácidos Borônicos/toxicidade , Cumarínicos/síntese química , Cumarínicos/toxicidade , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Microscopia Confocal/métodos , Compostos Organofosforados/síntese química , Compostos Organofosforados/toxicidade , Fosforilação Oxidativa , Temperatura , TermogêneseRESUMO
Serum is a common supplement for cell culture due to it containing the essential active components for the growth and maintenance of cells. However, the knowledges of the active components in serum are incomplete. Apart from the direct influence of serum components on cultured cells, the reaction of serum components with tested drugs cannot be ignored, which usually results in the false conclusion on the activity of the tested drugs. Here we report the toxicity effect of polyamines (spermidine and spermine) on cultured cells, especially on drug-resistant cancer cell lines, which resulted from the oxidative degradation of polyamines by amine oxidases in serum supplement. Upon adding spermidine or spermine, high concentration of H2O2, an enzyme oxidation product of polyamines, was generated in culture media containing ruminant serum, such as fetal bovine serum (FBS), calf serum, bovine serum, goat serum or horse serum, but not in the media containing human serum. Drug-resistant cancer cell lines showed much higher sensitivity to the oxidation products of polyamines (H2O2 and acrolein) than their wild cell lines, which was due to their low antioxidative capacity.