RESUMEN
5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.
RESUMEN
Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of â¼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.
Asunto(s)
Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Desmetilación del ADN , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Células Cultivadas , Técnicas de Inactivación de Genes , RatonesRESUMEN
N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.
Asunto(s)
Adenosina/análogos & derivados , Aprendizaje Automático , Procesamiento Postranscripcional del ARN , RNA-Seq/métodos , Adenosina/química , Adenosina/metabolismo , Animales , Células HEK293 , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Motivos de Nucleótidos , ARN Mensajero/química , ARN Mensajero/metabolismo , RNA-Seq/normas , Sensibilidad y EspecificidadRESUMEN
The proposal that N6-methyl-deoxyadenosine (m6dA) acts as an epigenetic mark in mammals remains controversial. Using isotopic labeling coupled to ultrasensitive mass spectrometry, we confirm the presence of low-level m6dA in mammalian DNA. However, the bulk of genomic m6dA originates from ribo-N6-methyladenosine, which is processed via the nucleotide-salvage pathway and misincorporated by DNA polymerases. Our results argue against m6dA acting as a heritable, epigenetic DNA mark in mammalian cells.
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ADN/química , ADN/metabolismo , Desoxiadenosinas/análisis , Genómica , Marcaje Isotópico/métodos , Aminoácidos/química , Animales , Línea Celular , Metilación de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Genoma , Humanos , Espectrometría de Masas , Metiltransferasas/metabolismo , RatonesRESUMEN
RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6 A methyltransferases were identified, uncovering the specificity of m6 A deposition by structurally distinct enzymes. In order to discover additional m6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.
Asunto(s)
Drosophila , Metiltransferasas , Adenosina , Animales , Drosophila/genética , Humanos , Metiltransferasas/genética , ARN Ribosómico , ARN Ribosómico 18S/genética , CaminataRESUMEN
Hepatic induction in response to drugs and environmental chemicals affects drug therapies and energy metabolism. We investigated whether the induction is transmitted to the offspring. We injected 3-day- and 6-week-old F0 female mice with TCPOBOP, an activator of the nuclear receptor constitutive androstane receptor (CAR, NR1I3), and mated them 1-6 weeks afterward. We detected in the offspring long-lasting alterations of CAR-mediated drug disposition, energy metabolism, and lipid profile. The transmission to the first filial generation (F1) was mediated by TCPOBOP transfer from the F0 adipose tissue via milk, as revealed by embryo transfer, crossfostering experiments, and liquid chromatography-mass spectrometry analyses. The important environmental pollutant PCB153 activated CAR in the F1 generation in a manner similar to TCPOBOP. Our findings indicate that chemicals accumulating and persisting in adipose tissue may exert liver-mediated, health-relevant effects on F1 offspring simply via physical transmission in milk. Such effects may occur even if treatment has been terminated far ahead of conception. This should be considered in assessing developmental toxicity and in the long-term follow-up of offspring of mothers exposed to both approved and investigational drugs, and to chemicals with known or suspected accumulation in adipose tissue.
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Receptores Citoplasmáticos y Nucleares/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Receptor de Androstano Constitutivo , Femenino , Hígado , Ratones , Ratones Endogámicos C57BL , Fenotipo , Embarazo , Piridinas/farmacologíaRESUMEN
Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3ß and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/fisiología , Células Madre Pluripotentes/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Reprogramación Celular , Citosina/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Estratos Germinativos/fisiología , Ratones , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , TranscriptomaRESUMEN
DNA demethylation plays a central role during development and in adult physiology. Different mechanisms of active DNA demethylation have been established. For example, Growth Arrest and DNA Damage 45-(GADD45) and Ten-Eleven-Translocation (TET) proteins act in active DNA demethylation but their functional relationship is unresolved. Here we show that GADD45a physically interacts--and functionally cooperates with TET1 in methylcytosine (mC) processing. In reporter demethylation GADD45a requires endogenous TET1 and conversely TET1 requires GADD45a. On GADD45a target genes TET1 hyperinduces 5-hydroxymethylcytosine (hmC) in the presence of GADD45a, while 5-formyl-(fC) and 5-carboxylcytosine (caC) are reduced. Likewise, in global analysis GADD45a positively regulates TET1 mediated mC oxidation and enhances fC/caC removal. Our data suggest a dual function of GADD45a in oxidative DNA demethylation, to promote directly or indirectly TET1 activity and to enhance subsequent fC/caC removal.
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Proteínas de Ciclo Celular/metabolismo , Citosina/análogos & derivados , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/metabolismo , Proteínas de Ciclo Celular/genética , Citosina/metabolismo , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunoprecipitación , Oxigenasas de Función Mixta , Proteínas Nucleares/genética , Oxidación-Reducción , Unión Proteica , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Although recent methods for targeted drug delivery have addressed many of the existing problems of cancer therapy associated with undesirable side effects, significant challenges remain that have to be met before they find significant clinical relevance. One such area is the delicate chemical bond that is applied to connect a cytotoxic drug with targeting moieties like antibodies or peptides. Here we describe a novel platform that can be utilized for the preparation of drug-carrier conjugates in a site-specific manner, which provides excellent versatility and enables triggered release inside cancer cells. Its key feature is a cleavable doxorubicin-octreotide bioconjugate that targets overexpressed somatostatin receptors on tumor cells, where the coupling between the two components was achieved through the first cleavable disulfide-intercalating linker. The tumor targeting ability and suppression of adrenocorticotropic hormone secretion in AtT-20 cells by both octreotide and the doxorubicin hybrid were determined via a specific radioimmunoassay. Both substances reduced the hormone secretion to a similar extent, which demonstrated that the tumor homing peptide is able to interact with the relevant cell surface receptors after the attachment of the drug. Effective drug release was quickly accomplished in the presence of the physiological reducing agent glutathione. We also demonstrate the relevance of this scaffold in biological context in cytotoxicity assays with pituitary, pancreatic, and breast cancer cell lines.
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Doxorrubicina/administración & dosificación , Doxorrubicina/química , Octreótido/química , Péptidos/química , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos , Sistemas de Liberación de Medicamentos/métodos , Humanos , Octreótido/administración & dosificación , Péptidos/administración & dosificación , Receptores de Somatostatina/metabolismoRESUMEN
Identical molecules move with identical velocities when placed in a uniform electric field within a uniform electrolyte. Here we report that homogeneous DNA does not obey this fundamental rule. While most DNA moves with similar velocities, a fraction of DNA moves with velocities that vary within a multiple-fold range. The size of this irregular fraction increases several orders of magnitude when exogenous counterions are added to DNA. The irregular fraction decreases several orders of magnitude when DNA counterions are removed by dialysis against deionized water in the presence of a strong electric field (0.6 kV/cm). Dialysis without the field is ineffective in decreasing the size of irregular fraction. These results suggest that (i) DNA can form very stable complexes with counterions, (ii) these complexes can be dissociated by an electric field, and (iii) the observed non-uniform velocity of DNA is caused by electric-field-induced slow dissociation of these stable complexes. Our findings help to better understand a fundamental property of DNA: its interaction with counterions. In addition, these findings suggest a practical way of making electromigration of DNA more uniform: removal of strongly bound DNA counterions by electro-dialysis against deionized water.
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ADN/química , Electricidad , Secuencia de Bases , Cartilla de ADN , Electroforesis Capilar , Reacción en Cadena de la PolimerasaRESUMEN
Negatively charged DNA can form extremely stable complexes with positively charged ions. These counterions are very difficult to remove from DNA; therefore, little is known about DNA behavior in their deficiency. We investigated whether removal of counterions from the strongly bound counterion layer would elicit any novel DNA properties or behaviors. In order to remove the tightly bound counterions, we used dialysis against deionized water in the presence of a strong (0.6 kV/cm) electric field. The electric field promoted the dissociation of the DNA-counterion complexes, while dialysis facilitated irreversible partitioning of counterions and DNA. Counterintuitively, when deprived of counterions, DNA precipitated from the solution into amorphous aggregates. The aggregates remained stable even when the electric field was turned off but readily redissolved when counterions were reintroduced. The phenomenon is likely explained by attraction of like-charged DNA polyions due to entropic-stabilization of condensed counterion layers.
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ADN/química , Iones/aislamiento & purificación , ElectricidadRESUMEN
In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.
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Compensación de Dosificación (Genética) , Cromosoma X , Masculino , Femenino , Animales , Ratones , Metilación , Cromosoma X/genética , Mamíferos/genética , Estabilidad del ARNRESUMEN
Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. The discovery of N1-adenosine PARylation as a DNA modification establishes the conceptual and methodological framework to elucidate its biological relevance and extends the role of PARP enzymes.
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Poli ADP Ribosilación , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Adenosina , Adenosina Difosfato , Animales , Cromatografía Liquida , ADN/metabolismo , ADN de Cadena Simple , Desoxiadenosinas , Glicósido Hidrolasas/metabolismo , Mamíferos/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Espectrometría de Masas en TándemRESUMEN
Temperature increase in capillary electrophoresis (CE) due to Joule heating is an inherent limitation of this powerful separation technique. Active cooling systems can decrease the temperature of a large part of the capillary but they leave "hot spots" at the capillary ends which can completely ruin some CE analyses despite their short lengths. Here, we introduce a "universal method for determining electrolyte temperatures" (UMET) that can determine temperatures in both efficiently- and inefficiently-cooled parts of the capillary. UMET can be applied to all electrolytes, as it does not involve any probe; it requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. To demonstrate the universality of UMET, we measured temperatures for electrolytes of different ionic strengths as well as for different capillary diameters. We further propose a "simplified universal method for predicting electrolyte temperatures" (SUMET) which only requires one measurement of current and voltage (that can be completed in 1 min) and uses two empirical equations to predict temperatures in the efficiently- and inefficiently-cooled parts of the capillary. The equations include several instrument-specific empirical parameters that are determined using a large set of current-voltage data obtained with UMET for a range of electrolytes and different capillaries. To demonstrate the utility of SUMET, we obtained the required data set for a Beckman MDQ CE instrument and produced all required empirical parameters that enable a user of this instrument to predict the temperature for every new experimental set in a matter of minutes. We confirmed the accuracy of SUMET by measuring the temperature-sensitive dissociation rate constant of a protein-DNA complex. We foresee that UMET will be used to produce instrument-specific empirical parameters for all CE instruments and then SUMET will be routinely used for temperature prediction in CE.
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Electrólitos/química , Electroforesis Capilar/métodos , Secuencia de Bases , Cartilla de ADN , TemperaturaRESUMEN
The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K(d) values between 20 and 240nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.
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Aptámeros de Nucleótidos/química , Electroforesis Capilar/métodos , Proteínas de Escherichia coli/análisis , Oxigenasas de Función Mixta/análisis , Secuencia de Bases , Escherichia coli/enzimología , Cinética , Unión ProteicaRESUMEN
Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.
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Aptámeros de Nucleótidos/química , Proteínas/aislamiento & purificación , Aptámeros de Nucleótidos/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Sondas de Oligonucleótidos/química , Técnica SELEX de Producción de AptámerosRESUMEN
Noncovalent protein-DNA interactions are involved in many vital biological processes. In cells, these interactions may take place in the environment of an electric field which originates from the plasma and organelle membranes and reaches strengths of 1 MV/cm. Moreover, protein-DNA interactions are often studied in vitro using an electric field as strong as 1 kV/cm, for example by electrophoresis. It is widely accepted that an electric field does not affect such interactions. Here we report on the first proof that an electric field of less than 1 kV/cm can destabilize the protein-DNA complexes through increasing the monomolecular rate constant of complex dissociation.
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Proteínas de Unión al ADN/química , ADN/química , Campos Electromagnéticos , Estabilidad Proteica , TemperaturaRESUMEN
Capillary electrophoresis (CE) serves as a platform for a large family of temperature-sensitive affinity methods. To control the electrolyte temperature, the heat generated during electrophoresis is removed by actively cooling the capillary. Short parts of the capillary, particularly at its inlet, are not actively cooled, however, and the electrolyte in this part is likely to be at an elevated temperature. Owing to their relatively short lengths, the noncooled parts have never been considered as a potential source of artifacts. Here we report for the first time that electrophoresis of the sample through the short noncooled capillary inlet can lead to large systematic errors in quantitative CE-based affinity analyses. Our findings suggest that the noncooled capillary inlet region, in spite of being short, is a source of significant artifacts that must be taken into consideration by developers and users of CE-based affinity methods. We propose a simple solution for this problem: moving the sample through the noncooled inlet into the cooled region by pressure or by a low-strength electric field to save it from exposure to the elevated temperature.
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Electroforesis Capilar/métodos , Aptámeros de Nucleótidos/análisis , Electroforesis Capilar/instrumentación , TemperaturaRESUMEN
Joule heating always accompanies electrophoresis and unavoidably leads to a temperature increase of the electrolyte. The elevated temperatures are known to adversely affect the quality of separation and detection. To minimize the temperature increase in capillary electrophoresis (CE), Joule heat is removed by actively cooling the capillary. However, there are always small parts of the capillary, such as its inlet, outlet, and detection window, which are not actively cooled. The noncooled capillary inlet has been recently proven to have an elevated temperature which is high enough to significantly affect CE-based quantitative affinity analyses. The temperature difference between the cooled and noncooled regions has never been determined due to the lack of a suitable method. Here, we report on the first experimental determination of temperature in the cooled part of the capillary and the noncooled inlet region of the capillary. We found that, under typical CE conditions, with a low-conductivity run buffer, the temperature in the noncooled inlet exceeded the temperature in the cooled region by more than 15 °C. High-conductivity buffers are anticipated to have even greater temperature differences between the noncooled and cooled capillary parts. Our results strongly suggest the potential effect of the noncooled capillary regions on the quality of CE-based analyses, which cannot be ignored. The simplest way to avoid potential errors is to move the sample to the cooled region by pressure or by applying a low electric field.
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Frío , Electrólitos/química , Electroforesis Capilar/métodos , Calibración , Electroforesis Capilar/instrumentaciónRESUMEN
Electro-migration techniques, such as electrophoresis, are widely utilized in analytical sciences. If a single electrolyte is used, the field strength is typically assumed to be well-defined. Heat-associated field distortion (HAFD) has been suggested as a result of the nonuniform heat dissipation throughout the electrolyte; however, it has never been experimentally studied. Here, we experimentally demonstrated HAFD for the first time. We used capillary electrophoresis (CE) with a capillary having parts with different heat dissipation efficiencies. Our experiments showed a difference in field strength of approximately 1.5 times between the different parts of the capillary for a typical CE electrolyte. This result suggests that HAFD is a well pronounced phenomenon that can be a potential source of errors and instabilities in electro-migration experiments.