RESUMEN
Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.
Asunto(s)
Anticoagulantes , Aptámeros de Nucleótidos , Trombina , Humanos , Anticoagulantes/química , Aptámeros de Nucleótidos/química , Dicroismo Circular , G-Cuádruplex , Guanina/química , Trombina/químicaRESUMEN
Low-molecular drug discovery using DNA-encoded chemical library (DEL) is a powerful technology, although improving the partitioning efficiency of affinity ligands from DEL remains a challenge. Here, we assessed the usefulness of microbead-assisted capillary electrophoresis (MACE) for partitioning peptide-oligonucleotide conjugates (POCs), in which high selection pressure is applied because of different mobility of target-modified beads and POCs during CE. Despite their different charge characteristics, all POCs were well separated from the beads. When bead extraction was performed, the tagged DNA amplification was observed only in the couple of a ligand/target, suggesting proficiently specific partitioning of peptide ligands was accomplished using MACE.
Asunto(s)
Oligonucleótidos , Péptidos , Microesferas , Péptidos/química , Oligonucleótidos/química , Electroforesis Capilar/métodos , ADN/química , ADN de Cadena SimpleRESUMEN
High-dose methotrexate (MTX) therapy is used to treat a wide variety of cancers such as leukemia and lymphoma, while the resulting high blood concentration of MTX faces a risk of life-threatening side effects, so it is essential to monitor the concentration carefully. Currently, the MTX concentration is measured using antibody-based kits in a clinical setting; however, the heterogeneity and batch-to-batch variation of antibodies potentially compromise the detection limit. Here, we developed MTX detection systems with chemically synthesizable homogeneous oligonucleotides. Microbead-assisted capillary electrophoresis (MACE)-SELEX against MTX successfully identified MSmt7 with a similar level of specificity to anti-MTX antibodies within three rounds. The 3'-end of MSmt7 was coupled to a peroxidase-like hemin-DNAzyme to construct a bifunctional oligonucleotide for MTX sensing, where MTX in 50% human serum was detected with a limit of detection (LoD) of 118 nM. Furthermore, amplifying the DNAzyme region with rolling circle amplification significantly improved the sensitivity with an LoD of 290 pM. Presented oligonucleotide-based MTX detection systems will pave the way for antibody-independent MTX detection with reliability and less cost in the laboratory and the clinic.
Asunto(s)
Aptámeros de Nucleótidos , ADN Catalítico , Humanos , Metotrexato , Reproducibilidad de los Resultados , HeminaRESUMEN
Due to the dynamic conformations of G-quadruplex structures (G4), determining the guanines that form G4 in a guanine-rich sequence is elusive. Here, we report a method for identifying deoxyguanines (dGs) forming antiparallel G4 by optical spectroscopy. The method, referred to as dG-to-deoxythymidine (dT) scanning, compares the spectra between a wild type and a single nucleobase dG-to-dT mutant at all dG positions. The most strongly involved dGs to form antiparallel G4 in the two model sequences were estimated using dG-to-dT scanning by circular dichroism (CD) and UV-Vis melting curve. This simple and robust method will facilitate understanding de novo antiparallel G4.
Asunto(s)
G-Cuádruplex , Dicroismo Circular , Guanina , TimidinaRESUMEN
Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using inâ vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells. For further improving the bioactivity of the aptamer, dimerization programed by hybridization was evaluated, resulting in the Apt14 dimer exhibited a twofold higher binding affinity and stronger inhibition compared to the monomer counterpart. Rapid identification of bioactive aptamers using MACE in combination with facile dimerization by hybridization accelerates the discovery of novel bioactive aptamers, paving the way toward replacing current monoclonal antibody therapy with the less expensive and non-immunogenic aptamer therapy.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Descubrimiento de Drogas , Técnica SELEX de Producción de Aptámeros , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Aptámeros de Nucleótidos/síntesis química , Aptámeros de Nucleótidos/química , Línea Celular , Electroforesis Capilar , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex-based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach-based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas de Química Analítica , Colorantes Fluorescentes/química , Sondas ARN/química , ARN/análisis , Aptámeros de Nucleótidos/síntesis química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Colorantes Fluorescentes/síntesis química , ARN/química , Sondas ARN/síntesis química , Relación Señal-RuidoRESUMEN
In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a "DNA Aptaomics" approach to systematically design functional aptamers as well as to obtain high affinity aptamers.
Asunto(s)
Aptámeros de Nucleótidos , Electroforesis Capilar , Secuenciación de Nucleótidos de Alto Rendimiento , Reproducibilidad de los Resultados , TrombinaRESUMEN
The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology via genetic engineering may prove to be promising for the tailored production of novel metabolites. For the transformation of microalgae, nucleic acids such as plasmid DNA (pDNA) are delivered into the cells using physical and mechanical techniques, such as electroporation, bombardment with DNA-coated microprojectiles, and vortexing with glass beads. However, owing to the electrostatic repulsion between negatively charged cell walls and nucleic acids, the delivery of nucleic acids into the microalgal cells is challenging. To solve this issue, in this study, we investigated microalgal transformation via electroporation using polyplexes with linear polyethyleneimine (LPEI) and pDNA. However, the high toxicity of LPEI decreased the transformation efficiency in Chlamydomonas reinhardtii cells. We revealed that the toxicity of LPEI was due to oxidative stress resulting from the cellular uptake of LPEI. To suppress the toxicity of LPEI, an antioxidant, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was covalently conjugated with LPEI; the conjugate was named as TEMPO-LPEI. Interestingly, with a cellular uptake tendency similar to that of LPEI, TEMPO-LPEI dramatically decreased oxidative stress and cytotoxicity. Electroporation using polyplexes of TEMPO-LPEI and pDNA enhanced the transformation efficiency, compared to those treated with bare pDNA and polyplexes of LPEI/pDNA. This result indicates that polycations conjugated with antioxidants could be useful in facilitating microalgal transformation.
RESUMEN
Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C. debaryana was encapsulated in a Ca2+-alginate gel for the culture with high cell density. We discovered that the growth of the encapsulated cells resulted in the formation of spherical palmelloid colonies with high cell density, where daughter cells with truncated flagella remained wrapped within the mother cell walls. Interestingly, gel encapsulation markedly promoted proliferation of C. debaryana cells, and the encapsulated cells reached the stationary phase earlier than that of the free-living cells. Gel encapsulation also enhanced TAG accumulation. Gene expression analysis revealed that two genes of acyltransferases, DGAT1 and DGTT3, were upregulated in the stationary phase of free-living C. debaryana. In addition, the gene expression of these acyltransferases increased earlier in the encapsulated cells than that in the free-living cells. The enhanced production of TAG by alginate gel encapsulation was not found in C. reinhardtii which is known to use a different repertoire of acyltransferases in lipid accumulation.
Asunto(s)
Chlamydomonas/crecimiento & desarrollo , Chlamydomonas/metabolismo , Microalgas/metabolismo , Triglicéridos/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Alginatos , Biocombustibles , Proliferación Celular , Chlamydomonas/citología , Chlamydomonas/genética , Clorofila/análisis , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Geles , Regulación de la Expresión Génica de las Plantas , Nitrógeno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , TranscriptomaRESUMEN
Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.
Asunto(s)
Sistemas CRISPR-Cas , Neuronas/fisiología , Optogenética/métodos , Diferenciación Celular , Células Cultivadas , ADN Complementario , Fibroblastos , Regulación de la Expresión Génica/fisiología , Humanos , Luz , Mediciones Luminiscentes , Transcripción GenéticaRESUMEN
A fingerprint-based sensing approach was used to characterize in vitro cellular senescence. Secretion profiles of cultured human fibroblasts in different senescent stages were transformed into colorimetric enzyme-activity fingerprints by applying cell culture media to a polyion complex array. Analysis of the obtained fingerprints using pattern recognition methods, such as linear discriminant analysis and hierarchical clustering analysis, revealed that the polyion complex array allows the noninvasive tracking of the replicative senescence progress even in those stages where a conventional marker such as senescence-associated ß-galactosidase is negative. This fingerprint-based approach should thus offer an effective way for the routine monitoring or screening of in vitro cell senescence studies.
Asunto(s)
Rastreo Celular/métodos , Senescencia Celular , Colorimetría/métodos , Fibroblastos/citología , Técnicas Biosensibles/métodos , Humanos , Reconocimiento de Normas Patrones Automatizadas/métodos , beta-Galactosidasa/análisisRESUMEN
Since much attention has been paid to in vivo biological functions of G-quadruplexes, structural analyses of G-quadruplexes are essential for understanding their functional mechanisms. Here, we established a simple optical-spectroscopy-based method for the estimation of G-quartet-forming guanines in parallel-type G-quadruplexes using measurements of circular dichroism and the thermal melting temperature.
Asunto(s)
G-Cuádruplex , Guanina/química , Dicroismo Circular , Análisis Espectral , TemperaturaRESUMEN
Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes. Upon application of an electric field, HL-60 cells as models of promyelocytes aggregated and floated between the saw-shaped electrodes, while UE7T-13 cells as models of BMSCs were effectively captured on the tips of the saw-shaped electrodes. After washing out the HL-60 cells from the device selectively, the purity of the UE7T-13 cells was increased from 33% to 83.5% within 5 min. Although further experiments and optimization are required, these results show the potential of the DEP device as a label-free rapid cell isolation system yielding high purity for rare and precious cells such as BMSCs.
Asunto(s)
Células de la Médula Ósea/citología , Separación Celular/métodos , Electroforesis/instrumentación , Células Madre Mesenquimatosas/citología , Células HL-60 , Humanos , Factores de TiempoRESUMEN
Wound-healing factors secreted from mesenchymal stem cells (MSCs) modulate the immune response and facilitate proliferation of neighboring cells. Although in vitro three-dimensional (3D) culture techniques have improved the therapeutic potential of MSCs, no studies have focused on the effects of cell aggregation alone. In this study, the effect of cell aggregation on the up-regulation of wound-healing proteins secretions was investigated by constructing small spheroids of human adipose-derived stem cells (hADSCs) on a micropatterned surface. These spheroids were mostly unaffected by the secondary effects of cell aggregation, such as hypoxia, low-nutrient supply, and metabolic waste accumulation. Small spheroids of hADSCs, which were of 100 µm in diameter, were successfully constructed using micropatterned surface. Expression of the wound-healing-related factors, VEGF-A and IL-8, was markedly enhanced at the gene and protein levels, whereas the enhancement of VEGF-A expression was transient and IL-8 enhancement was maintained for a long time.
Asunto(s)
Interleucina-8/metabolismo , Células Madre Mesenquimatosas/metabolismo , Esferoides Celulares/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas , Agregación Celular , Células Cultivadas , Humanos , Factores de TiempoRESUMEN
A single-round DNA aptamer selection for mammalian cells was successfully achieved for the first time using a capillary electrophoresis (CE)-based methodology called polymer-enhanced capillary transient isotachophoresis (PectI). The PectI separation yielded a single peak for the human lung cancer cell line (PC-9) complexed with DNA aptamer candidates, which was effectively separated from a free randomized DNA library peak, ensuring no contamination from free DNA in the PC-9-DNA aptamer complex fraction. The DNA aptamer candidates obtained after a single-round selection employing counter selection with HL-60 were proven to bind selectively and form kinetically stable complexes with PC-9 cells. Interestingly, most aptamer candidates showed high binding ability (Kd = 70-350 nM) with different extents of binding on the cell surface. These facts proved that a single-round selection for mammalian cells by PectI is feasible to obtain various types of aptamer candidates, which have high-affinity even for non-overexpressed but unique targets on the cell surface in addition to overexpressed targets.
Asunto(s)
Aptámeros de Nucleótidos , Electroforesis Capilar , Biblioteca de Genes , Isotacoforesis , Línea Celular Tumoral , Humanos , PolímerosRESUMEN
Human plasma proteins and even structurally similar homologous albumins were fingerprinted and discriminated by a sensor array consisting of a polyion complex library with artificial differentiation constructed by facile tuning of PEGylated polyamine functionalities.
Asunto(s)
Biblioteca de Péptidos , Mapeo Peptídico/métodos , Poliaminas/química , Polietilenglicoles/química , HumanosRESUMEN
A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two types of gel electrophoresis, to obtain an accurate distribution of protein-bound metal ions in biological samples. First, proteins are separated by PAGE without the uptake of contaminant metal ions in the separation field and dissociation of metal ions from the proteins. This is followed by another PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using a fluorescent metal probe. The former is a new technique using blue-native (BN) PAGE to electrophoretically sweep all metal contaminants by employing two kinds of chelating agents. These agents form complexes with contaminants in the gel and the separation buffer solution, which migrate towards opposite pole directions, thus lowering the contaminants to below the ppt level during separation. This is termed "Metal Ion Contaminant Sweeping BN-PAGE (MICS-BN-PAGE)". After the separation of proteins under these first metal-free conditions, the metal ions in the gel fractions are eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxide dismutase were quantitatively determined, in addition to the exchangeable albumin-bound copper ions. This system successfully provided distribution maps of protein-copper in human serum. The precise distribution of copper in human serum was investigated, and found to be different from that which is widely accepted.
Asunto(s)
Cobre/sangre , Electroforesis en Gel de Poliacrilamida/métodos , Metaloproteínas/análisis , Cobre/metabolismo , Humanos , Iones , Metales/química , Metales/metabolismo , Unión Proteica/fisiologíaRESUMEN
Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.
RESUMEN
Novel fluorescent probes have been developed for the ultratrace detection of heavy metal ions by capillary electrophoresis using laser-induced fluorescence detection. Based on a molecular design, the probes are composed of an octadentate chelating moiety, a macrocyclic DOTA (tetraazacyclododecanetetraacetic acid) and an acyclic DTPA (diethylenetriaminepentaacetic acid) frame, a spacer and a fluorophore (fluorescein). These were chosen on the basis of their ability to form kinetically inert and highly emissive complexes, and to prevent a quenching effect even with heavy and paramagnetic metal ions. Addition of a cationic polymer, polybrene, in the separation buffer provided high resolution and simultaneous detection of Ca(2+), Mg(2+), Cu(2+), Zn(2+), Ni(2+), Co(2+), Mn(2+), Cd(2+) and Pb(2+). The direct fluorescence detection of these metal ions with high sensitivity at lower ppt levels, typically 2-7 × 10(-11) M (potentially sub-ppt), was successfully achieved. While separation of anionic compounds using a counter cation ("Ion Association (IA)" mode) is typically controlled by the ion association equilibrium constants, K(ass), it was found that differences in the mobilities, µ(ep(IAC)), of the ion association complexes formed between the probe complexes and counter cations are the driving forces for separation in this new method. This suggests that each of the polybrene-probe complexes has different chemical structures among metal ions, which were able to be determined by CD spectra in this investigation. This novel separation mode was termed the "Ion Association Complex (IAC)" mode, distinct from the IA mode.
RESUMEN
Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (10(6) M(-1)) follows in the order of AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19) in solutions containing 110 mM Na(+) (pH 7.0, at 20 degrees C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (DeltaC(p)), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (-161 cal/mol K) and ATMND (-217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (DeltaG(obs)) is dissected into its component terms.