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Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.
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ADN , Guanina , Humanos , ADN/genética , Estrés Oxidativo , Especies Reactivas de Oxígeno , Técnicas de Amplificación de Ácido NucleicoRESUMEN
G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.
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Benzotiazoles , ADN , Fluorescencia , Uracil-ADN Glicosidasa , Humanos , Benzotiazoles/química , Benzotiazoles/metabolismo , Técnicas Biosensibles/métodos , Dicroismo Circular , ADN/química , ADN/metabolismo , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia , Uracil-ADN Glicosidasa/metabolismo , Uracil-ADN Glicosidasa/químicaRESUMEN
Nucleic acid modifications play important roles in biological activities and disease occurrences, and have been considered as cancer biomarkers. Due to the relatively low amount of nucleic acid modifications in biological samples, it is necessary to develop sensitive and reliable qualitative and quantitative methods to reveal the content of any modifications. In this review, the key processes affecting the qualitative and quantitative analyses are discussed, such as sample digestion, nucleoside extraction, chemical labeling, chromatographic separation, mass spectrometry detection, and data processing. The improvement of the detection sensitivity and specificity of analytical methods based on mass spectrometry makes it possible to study low-abundance modifications and their biological functions. Some typical nucleic acid modifications and their potential as biomarkers are displayed, and efforts to improve diagnostic accuracy are discussed. Future perspectives are raised for this research field.
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Ácidos Nucleicos , Espectrometría de Masas/métodos , Biomarcadores de TumorRESUMEN
Succination is a nonenzymatic and irreversible post-translational modification (PTM) with important biological significance, yielding S-(2-succino) cysteine (2SC) residue. This PTM is low in abundance and often requires a large amount of protein samples for 2SC quantification. In this work, an efficient quantification method based on ethanol/acetyl chloride chemical derivatization was developed. The three carboxyl groups of 2SC were all esterified to increase hydrophobicity, greatly improving its ionization efficiency. The sensitivity was increased by 112 times; the limit of detection was reduced to 0.885 fmol, and the protein usage was reduced by at least 10 times. The established method was used to detect the overall concentration of 2SC in fumarate accumulation cells quantitatively.
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Nucleosides have been found to suffer in-source fragmentation (ISF) in electrospray ionization mass spectrometry, which leads to reduced sensitivity and ambiguous identification. In this work, a combination of theoretical calculations and nuclear magnetic resonance analysis revealed the key role of protonation at N3 near the glycosidic bond during ISF. Therefore, an ultrasensitive liquid chromatography-tandem mass spectrometry system for 5-formylcytosine detection was developed with 300 fold signal enhancement. Also, we established a MS1-only platform for nucleoside profiling and successfully identified sixteen nucleosides in the total RNA of MCF-7 cells. Taking ISF into account, we can realize analysis with higher sensitivity and less ambiguity, not only for nucleosides, but for other molecules with similar protonation and fragmentation behaviors.
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Nucleósidos , Espectrometría de Masa por Ionización de Electrospray , Nucleósidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía LiquidaRESUMEN
Oxidative DNA damage is tightly linked to the development of multiple age-related diseases. The prominent oxidation product is 8-oxo-7,8-dihydroguanine (OG), which has been proved to be an important epigenetic-like biomarker. Quantification of the locus-specific OG frequency includes quantitative and locating information, which is of great significance for exploring the functional roles of OG in disease induction and gene regulation. Herein, an ultrasensitive quantification of OG at single-base resolution was established using real-time fluorescence quantitative polymerase chain reaction as an amplification tool. Based on the coding property of Bsu DNA polymerase that incorporates adenine on the opposite site of OG and the selectivity of the ligase for perfectly matched sequences, the difference between OG and G on the sequence could be enlarged. Well-performed Taq DNA ligase was selected out, and as low as 46.2 zmol of target DNA with an OG site and an OG frequency of 5% could be detected. G contents on a specific site were also detectable based on the similar principle, thus the OG frequency of this locus could be accurately determined by a standard addition method. This strategy was successfully applied to the evaluation of locus-specific OG in both model DNA and genomic DNA from human cervical carcinoma cell lines under multiple oxidative stress, showing the potential for functional research and dynamic monitoring of critical OG sites.
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Reparación del ADN , Guanina , ADN/genética , Daño del ADN , Guanina/análogos & derivados , HumanosRESUMEN
Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 µg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.
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Neoplasias de la Mama , Nucleósidos , Humanos , Femenino , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , ADN/química , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisisRESUMEN
Exosomes are nanosized extracellular vesicles that have a critical role in intercellular communication and tumor microenvironment regulation. Extensive research has shown that exosomal small RNAs contribute to metastasis in multiple tumor types and that abnormal epigenetic modifications in nucleic acids also have an association with diverse diseases. However, the content of modified nucleosides on exosomal small RNAs has not been quantitatively reported. Because of the trace amounts of exosomes and matrix complexity, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a powerful tool for label-free sensitive and simultaneous determinations of six important modified nucleosides on small RNAs inside exosomes. This system performed well using only approximately 107-108 particles of exosomes to obtain modified nucleoside levels between 0.001 and 0.03, and the most striking result was that the content of m6A in exosomal small RNAs was continuously higher than that in the cells being analyzed. We hope that this conclusion helps establish a greater degree of deciphering accuracy on exosomes, which has considerable application potential in the diagnosis and prognosis of diseases.
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Exosomas , ARN , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Epigénesis Genética , Exosomas/genética , Espectrometría de Masas en TándemRESUMEN
Microcrystalline cellulose (MCC) was modified using toluene-2,4-diisocyanate (TDI) in tetrahydrofuran (THF). The reaction was set up for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 24 h at 75 °C. The study was aimed at hydrophobic modification of microcrystalline cellulose (MCC) to improve its dispersion in PLA matrix. Data from the elemental analysis were used to develop a statistical model to predict the degree of substitution (DS) of the OH on the surface of the MCC using both the water contact angle (WCA) and the time of carbamation as the independent variables. Composite was fabricated at 1%, 2%, 3%, 4%, and 5% fiber loading. Fourier transformed infrared spectroscopy was used to characterize the MCC and to confirm the successful graft of TDI to the MCC surface. The morphology and elemental analysis of the modified samples were examined with SEM-EDX. The samples' wettability was analyzed with a contact angle meter to measure the water contact angle (WCA). The tensile properties of composites were analyzed on a universal testing machine. The result showed that, after 1 h of carbamation, the minimum DS recorded was 0.11, and the maximum DS after 24 h was 0.16. The SEM revealed that the modified MCC had homogeneous dispersion in the polymer matrix. At 3% fiber loading, the tensile strength (TS) and elongation were at a maximum and had improvements of 80.67% and 79.44% as compared to neat PLA. The fractured tensile surface from SEM analysis showed that surface modification enhanced fiber-matrix adhesion and significantly improved the composite's strength and toughness. The proposed model that was developed in this study had a coefficient of determination (R2) of 93% to show that the model has a near-perfect goodness of fit and can well be an effective approach to predict the DS of OH from WCA and the time of reaction at similar or the same reaction conditions.
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Propofol is a widely used intravenous anesthetic agent in sedation and general anesthesia. To improve the safety and maintain the depth of anesthesia, it is important to develop a rapid, sensitive, and reliable method to monitor the concentration of propofol in blood during anesthesia continuously. Here, we present a novel strategy based on paper spray ionization-mass spectrometry (PSI-MS) to detect propofol. Samples (in 10 µL) were mixed with methanol as protein precipitation solvent and 2,6-dimethylphenol as internal standard. Protein micro-precipitation was achieved with methanol by vortexing and centrifuging for 5 s each, and propofol was extracted to the supernatant. PSI-MS was performed in negative ionization mode, and MS signal lasted for 1 min. The analysis of a single sample was completed within 2 min. The area ratios of propofol to internal standard were calculated for quantification. Limit of detection of 5.5 ng mL-1 and limit of quantification of 18.2 ng mL-1 were achieved for propofol in whole blood. Calibration curve was linear in the range of 0.02-10 µg mL-1. The developed method was used successfully in monitoring the propofol concentration in 3 patients' whole blood during anesthesia, showing its further application in controlling and feeding-back target concentration infusion. Graphical abstract.
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Anestésicos Intravenosos/sangre , Espectrometría de Masas/métodos , Propofol/sangre , Humanos , Límite de Detección , Monitoreo Fisiológico/métodos , Papel , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Cell-free DNA (cfDNA)-based biomarkers such as mutation and methylation offer promising noninvasive strategies for disease diagnosis and prognosis. However, besides high-throughput sequencing, there has been no alternative approach to date to detect the epigenetic marks, such as 5-hydroxymethylcytosine (5hmC), in cfDNA. Here, we described a MnO2 oxidation and hydrazine-s-triazine reagent (i-Pr2N) labeling based method named 5hmC-MIQuant that achieved ultrasensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of 5hmC in low-input DNA samples. This strategy improved the detection sensitivity of 5hmC by 178 times, and the limit of detection was as low as 14 amol. With simple preparation steps, 5hmC-MIQuant could quantify the 5hmC level in as little as 340 pg genomic DNA (equivalent to 57 copies of diploid genome). cfDNA samples from human plasma were successfully analyzed using 5hmC-MIQuant. This method is promising for the identification of 5hmC function in precious samples and the 5hmC-based noninvasive disease diagnosis.
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5-Metilcitosina/análogos & derivados , Ácidos Nucleicos Libres de Células/química , 5-Metilcitosina/análisis , Animales , Línea Celular , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Humanos , Masculino , Ratones , RatasRESUMEN
A facile and rapid postsynthetic modification strategy for functionalization of covalent organic framework (COF) was developed to synthesize a tailor-made pH-responsive COF called TpPa-1@Au@GSH for highly efficient extraction of N1-methyladenosine (m1A). Glutathione (GSH) was judiciously designed as the functional group for extracting and releasing m1A by pH variations. With the aid of gold nanoparticles (Au NPs) as linkers, GSH was successfully introduced to the robust substrate TpPa-1 in only one step spending only 1 h. Owing to the several-to-one immobilization of GSH on Au NPs and the large surface area of TpPa-1, this functional COF was constructed with abundant m1A binding sites. TpPa-1@Au@GSH showed excellent selectivity for m1A extraction by capturing m1A from a mixture of 14 nucleoside analogues followed by mass spectrometry analysis. It was proved to have ultrafast adsorption ability (only 1 min incubation time), high binding capacity (5 mg g-1, m1A/TpPa-1@Au@GSH), good reusability (at least 5 times), and good storage stability (at least 8 months at room temperature). Great performance was also achieved in extracting m1A from both animal and plant biological samples. The adsorption mechanism was demonstrated to be based on the electrostatic interaction. This work proposed a new approach for m1A extraction, demonstrated the high potential of COFs in biological sample pretreatment, and offered an effective and versatile route for functionalization of COFs.
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Reliable multiple single nucleotide polymorphisms (SNPs) detection at low abundance is of great significance for disease diagnosis and biomedical research. Herein, we have developed a novel and simple method for multiple SNPs detection combining solid-phase capture by specific hybridization with online preconcentration of capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF). The method presents an excellent performance due to its favorable traits: the solid-phase short-chain hybridization ensures the high specificity of SNP detection; the effective separation ability of CGE can easily achieve multiplex detection; the simple online preconcentration significantly improves the detection sensitivity of fluorescent probe by nearly 100-fold. For a single SNP target, the assay achieves a limit of detection as low as 0.01-0.02% for three different NRAS mutations in the same codon. For multiple SNP targets, as low as 0.05% abundance can be easily realized. Our method is simple, efficient, ultrasensitive, and universal for multiple SNPs detection without complex enzymatic or chemical ligation reaction, which shows great potential in early clinical diagnosis.
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Electroforesis Capilar/métodos , Límite de Detección , Polimorfismo de Nucleótido Simple , Codón/genética , Hibridación de Ácido NucleicoRESUMEN
Snake venom is a complex mixture mainly consisting of proteins and peptides which varies with different species. These variations lead to different toxic mechanisms and different anti-venom serums for treatment and the determination of their use as drugs. Hence, it is important to develop a sensitive and reliable method to identify the species of snakes from venoms. Herein, we present a novel strategy based on the sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CESI-MS) system to characterize snake venom proteins. Through the determination of peptides, we found the characteristic peptides of 8 different snakes with high sensitivity (1 µg mL-1) and high selectivity, which provided a reliable method for the species identification and purity detection of snake venom samples.
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Venenos de Serpiente , Espectrometría de Masa por Ionización de Electrospray , Electroforesis Capilar , PéptidosRESUMEN
A G-triplex, a new kind of DNA structure, has been identified as an intermediate in the folding of G-quadruplexes. However, the studies on G-triplexes are still very limited, and the functions and applications of G-triplexes need to be further developed. In this paper, a new G-triplex sequence (5'-CTGGGAGGGAGGGA-3', G3), obtained by truncating four bases (GGGA) from the 3' end of an 18-base G-quadruplex sequence (G4), was found to significantly decrease the diffusion current of methylene blue (MB). In particular, we proved that (a) MB stabilized the structure of G3 and increased the Tm of G3 considerably based on circular dichroism; and (b) MB formed a 1:1 noncovalent complex with G3 based on electrospray ionization mass spectrometry. Moreover, molecular dynamics simulations established reliable speculation in the folding topology of G3 and interaction sites between G3 and MB. Based on the strong affinity of G3 with MB, we further developed a novel function of G3 as an electrochemical signal read-out and applied it in the fabrication of a sensitive homogeneous electrochemical aptasensor for cocaine. The features we observed in the G3/MB complex will serve as a new inspiring guideline for developing functional short G-rich ligands.
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Técnicas Biosensibles , ADN/química , Técnicas Electroquímicas , G-CuádruplexRESUMEN
Modified cytosines are important epigenetic marks that exert critical influences in a variety of cellular processes in living organisms. However, biological functions of rare modified cytosines, especially certain functions of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are still unclear due to the extremely low abundance in biological samples. In this work, a series of novel hydrazine-based reagents, which held a hydrazine group as the reaction group, a hydrophobic triazine group, and two easily charged tertiary amine groups with different alkyl chains for adjusting the hydrophobicity of the labeling reagents, were first explored to label rare modified cytosines such as 5fC and 5caC. The derivatization reaction between 5fC and the labeling reagents was extremely fast, and more than 99% derivatization efficiency could be achieved only by vortexing without additional reaction time. The detection sensitivity of 5fC increased with the increase of the hydrophobicity of the labeling reagents, the best of which was dramatically enhanced by 125-fold. The limit of detection was as low as 10 amol, realizing the most sensitive genome-wide overall quantification for 5fC. Moreover, the labeling reagents were also successfully applied for the detection of 5caC with 100-fold improvement of sensitivity. With this method, we achieved the simultaneous detection of 5fC and 5caC in different mammalian tissues using only about 600 ng of genomic DNA, which was less than one-tenth of the sample consumption for other reported methods, providing an opportunity to monitor 5fC and 5caC in precious samples and biology processes that could not be investigated before.
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Citosina/análogos & derivados , Hidrazinas/química , Indicadores y Reactivos/química , Animales , Cromatografía Líquida de Alta Presión/métodos , Citosina/análisis , ADN/química , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Ratones , Estructura Molecular , Espectrometría de Masas en Tándem , Triazinas/químicaRESUMEN
Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.
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Colorimetría/métodos , ADN/análisis , Polimorfismo de Nucleótido Simple , Alquinos/química , Armoracia/enzimología , Azidas/química , Bencidinas/química , Química Clic , Colorantes/química , Reacción de Cicloadición , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Peroxidasa de Rábano Silvestre/química , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oxidación-ReducciónRESUMEN
The enrichment of glycopeptides plays an important role in glycoproteomics. In this paper, a covalent-organic framework called TpPa-1, synthesized by the Schiff base reaction of 1,3,5-triformylphloroglucinol and paraphenylenediamine, was first successfully utilized as a hydrophilic porous material for N-linked glycopeptide enrichment. Using this material, interference from non-glycopeptides could be efficiently eliminated, which facilitated the mass spectrometry detection of glycopeptides. By capturing N-linked glycopeptides from tryptic digests of human IgG, our method was proved to have high sensitivity at the femtomole level. And it showed superior selectivity for glycopeptides even when non-glycopeptides were 1000 times more concentrated. Due to the strong covalent bonds, this material possessed good stability and could be repeatedly used for at least 10 times. The ultra-low mass density and abundant binding sites also provided it with high binding capacity (178 mg g-1, IgG/TpPa-1). Moreover, N-linked glycopeptides were easily enriched by this material from only 10 µL human serum, which demonstrated its potential in pretreatment of complex biological samples.
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Glicómica/métodos , Glicopéptidos/química , Proteómica/métodos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Espectrometría de MasasRESUMEN
G-quadruplex sequences exist in eukaryotic organisms and prokaryotes, and the investigation of the interactions between G-quadruplexes and small molecule ligands is important for gene therapy, biosensor fabrication, fluorescence imaging and so on. Here, we investigated the behaviour of methylene blue (MB), an electroactive molecule, in the presence of different intramolecular G-quadruplexes by an electrochemical method using a miniaturized electrochemical device based on its intrinsic electrochemical properties. Although the effects of MB on different intramolecular G-quadruplex structures are not obvious by circular dichroism spectroscopy, distinct differences in the binding affinities of MB with different intramolecular G-quadruplexes were quickly and easily observed by an electrochemical technique. At the same time, for the human telomerase G-rich sequence (HT), the diffusion current of MB changed sensitively under different ionic conditions due to the formation of different conformations of HT, which indicated that our electrochemical method has the potential to study the influence of metal ions on the conformations of the G-quadruplexes with simplicity, rapid response and low cost. From all these, a new stacking mechanism and rule were obtained, which were also validated by docking studies and isothermal titration calorimetry (ITC).
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G-Cuádruplex , Azul de Metileno/química , Técnicas Biosensibles , Calorimetría , Dicroismo Circular , Técnicas Electroquímicas , Humanos , Simulación del Acoplamiento Molecular , Telomerasa/químicaRESUMEN
Phytochemicals are essential secondary plant metabolites which play important roles in the areas of plant biochemistry, pharmacy and medical science because of their significant bioactivities. Conventional analysis of phytochemicals in plants needs a complex combination of different extraction and separation steps. Here a simple and universal method for profiling phytochemicals in a single plant cell was demonstrated based on high performance liquid chromatography-mass spectrometry. Single purple and colorless cell samples (about 15 µm in size) found in the outer layer of a young stem of Forsythia suspensa, just inside the cuticles were collected and transferred by glass micropipettes from cell monolayers. At least 30 peaks were separated and detected, and 24 of these peaks were identified. Apart from several common plant metabolites in high abundance, like polysaccharides and amino acids, 9 phytochemicals that have special bioactivities in this plant and in medical treatment were successfully detected. Phytochemical differences between these two kinds of cells were also distinguished which was applied to investigate the heterogeneity of cells from different parts of plants and the dependency of important plant bioprocesses on phytochemical changes.