RESUMO
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.
RESUMO
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.
Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Desmetilação do DNA , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , CamundongosRESUMO
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.
Assuntos
DNA/química , DNA/metabolismo , Desoxiadenosinas/análise , Genômica , Marcação por Isótopo/métodos , Aminoácidos/química , Animais , Linhagem Celular , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Genoma , Humanos , Espectrometria de Massas , Metiltransferases/metabolismo , CamundongosRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular , Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Camadas Germinativas/fisiologia , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , TranscriptomaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citosina/análogos & derivados , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Proteínas de Ciclo Celular/genética , Citosina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunoprecipitação , Oxigenases de Função Mista , Proteínas Nucleares/genética , Oxirredução , Ligação Proteica , Proteínas Proto-Oncogênicas/genéticaRESUMO
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.
Assuntos
DNA/química , Eletricidade , Sequência de Bases , Primers do DNA , Eletroforese Capilar , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
DNA/química , Íons/isolamento & purificação , EletricidadeRESUMO
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.
Assuntos
Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases , Adenosina , Difosfato de Adenosina , Animais , Cromatografia Líquida , DNA/metabolismo , DNA de Cadeia Simples , Desoxiadenosinas , Glicosídeo Hidrolases/metabolismo , Mamíferos/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Eletrólitos/química , Eletroforese Capilar/métodos , Sequência de Bases , Primers do DNA , TemperaturaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Eletroforese Capilar/métodos , Proteínas de Escherichia coli/análise , Oxigenases de Função Mista/análise , Sequência de Bases , Escherichia coli/enzimologia , Cinética , Ligação ProteicaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas/isolamento & purificação , Aptâmeros de Nucleotídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Sondas de Oligonucleotídeos/química , Técnica de Seleção de AptâmerosRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Campos Eletromagnéticos , Estabilidade Proteica , TemperaturaRESUMO
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.
Assuntos
Eletroforese Capilar/métodos , Aptâmeros de Nucleotídeos/análise , Eletroforese Capilar/instrumentação , TemperaturaRESUMO
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.
Assuntos
Temperatura Baixa , Eletrólitos/química , Eletroforese Capilar/métodos , Calibragem , Eletroforese Capilar/instrumentaçãoRESUMO
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.
Assuntos
Eletroforese Capilar/métodos , Temperatura Alta , EletrólitosRESUMO
The development of drugs and diagnostics with desirable characteristics requires smart small-molecule ligandsligands with predefined binding parameters of interaction with the target. Here, we propose a general approach for selection of such ligands from highly diverse combinatorial libraries of small molecules by methods of kinetic capillary electrophoresis (KCE). We deduct three fundamental requirements for the combinatorial library to suit the KCE-based selection of smart ligands and suggest a universal design of the library for selecting smart small-molecule ligands: every small molecule in the library is tagged with DNA that encodes the structure of the molecule. Finally, we use several DNA-tagged small molecules, which represent a hypothetical library, to prove experimentally selection of smart small-molecule ligands by the proposed approach.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas de Química Combinatória/métodos , DNA/química , Eletroforese Capilar/métodos , Biotina/análogos & derivados , Biotina/química , Colesterol/química , Cinética , Ligantes , Reação em Cadeia da Polimerase/métodosRESUMO
N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.
Assuntos
Metilação de DNA , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Sequência de Bases , Biocatálise , DNA/genética , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Concentração Inibidora 50 , Oxigenases de Função Mista/antagonistas & inibidoresRESUMO
Base excision repair (BER) functions not only in the maintenance of genomic integrity but also in active DNA demethylation and epigenetic gene regulation. This dual role raises the question if phenotypic abnormalities resulting from deficiency of BER factors are due to DNA damage or impaired DNA demethylation. Here we investigate the bifunctional DNA glycosylases/lyases NEIL1 and NEIL2, which act in repair of oxidative lesions and in epigenetic demethylation. Neil-deficiency in Xenopus embryos and differentiating mouse embryonic stem cells (mESCs) leads to a surprisingly restricted defect in cranial neural crest cell (cNCC) development. Neil-deficiency elicits an oxidative stress-induced TP53-dependent DNA damage response, which impairs early cNCC specification. Epistasis experiments with Tdg-deficient mESCs show no involvement of epigenetic DNA demethylation. Instead, Neil-deficiency results in oxidative damage specific to mitochondrial DNA, which triggers a TP53-mediated intrinsic apoptosis. Thus, NEIL1 and NEIL2 DNA glycosylases protect mitochondrial DNA against oxidative damage during neural crest differentiation.
Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Células-Tronco Embrionárias/fisiologia , Mitocôndrias/metabolismo , Crista Neural/embriologia , Estresse Oxidativo , Animais , Diferenciação Celular , Linhagem Celular , Reparo do DNA , Camundongos , XenopusRESUMO
Here we introduce a technology for biomarker discovery in which (i) DNA aptamers to biomarkers differentially expressed on the surfaces of cells being in different states are selected; (ii) aptamers are used to isolate biomarkers from the cells; and (iii) the isolated biomarkers are identified by means of mass spectrometry. The technology is termed aptamer-facilitated biomarker discovery (AptaBiD). AptaBiD was used to discover surface biomarkers that distinguish live mature and immature dendritic cells. We selected in vitro two DNA aptamer pools that specifically bind to mature and immature dendritic cells with a difference in strength of approximately 100 times. The aptamer pools were proven to be highly efficient in flow- and magnetic-bead-assisted separation of mature cells from immature cells. The two aptamer pools were then used to isolate biomarkers from the cells. The subsequent mass spectrometry analysis of the isolated proteins revealed unknown biomarkers of immature and mature dendritic cells.
Assuntos
Aptâmeros de Nucleotídeos/química , Biomarcadores/análise , Células Dendríticas/química , Animais , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores/metabolismo , Separação Celular/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Magnetismo , Camundongos , Camundongos TransgênicosRESUMO
Application of capillary electrophoresis (CE) to temperature-sensitive biomolecular interactions requires knowledge of the temperature inside the capillary. The simplest approach to finding temperature in CE employs a molecular probe with a temperature-dependent parameter. Up until now only spectral parameters of molecular probes were utilized for temperature measurements in CE. The arbitrary nature of spectral parameters leads to several inherent limitations that compromise the accuracy and precision of temperature determination. This paper introduces the concept of finding temperature in CE through the measurement of a nonspectral parameter of the molecular probeits diffusion coefficient. Diffusion is a fundamental property of molecules that depends only on the molecular structure of the probe, the nature of the environment, and the temperature. It is ideally suited for temperature measurements in CE if an approach for measuring the diffusion coefficient in a capillary with high precision is available. This work first develops an approach for measuring the diffusion coefficient in a capillary with a relative standard deviation of as low as 2.1%. It is then demonstrated that such precise measurements of the diffusion coefficient could facilitate accurate temperature determination in CE with a precision of 1 degrees C. This new method was used to study the effect on temperature of different amounts of joule heat generated and different efficiencies of heat dissipation. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, for example, electrochemical and mass spectrometric detection.