Separation of Trp-Arg and Arg-Trp using G-quartet-forming DNA oligonucleotides in open-tubular capillary electrochromatography

DNA oligonucleotides that form intramolecular G-quartet structures were investigated as stationary phase reagents for separation of mixtures of the isomeric dipeptides Trp-Arg and Arg-Trp in open-tubular capillary electrochromatography (OTCEC). The oligonucleotides included a thrombin-binding aptamer that forms a biplanar G-quartet structure and an oligonucleotide that forms a 4-plane G-quartet structure. Fluorescence, circular dichroism and UV-visible absorbance spectroscopies were used in batch solution studies to indicate interactions between the dipeptides and the biplanar G-quartet structure. Results for OTCEC separations were compared with results obtained for capillary zone electrophoresis separations on a bare capillary. Temperature studies suggest that resolution is improved when the G-quartet structure is partially destabilized, but control experiments in which potassium chloride was not included in the mobile phase indicate that the G-quartet structure nevertheless plays a role in the separations.     

Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography

The migration of fibrinogen peptides in capillaries coated with G-quartet-forming DNA oligonucleotides in open-tubular CEC (OTCEC) was studied, in order to investigate factors affecting the retention of peptides on G-quartet DNA stationary phases. At 25 degrees C, the peptides eluted in the same order in OTCEC using a two-plane G-quartet DNA stationary phase as in CZE, including two peptides that were completely overlapped. It was found that baseline resolution of the coeluting peptides could be achieved in the OTCEC experiment, but not in CZE, at run temperatures of 35-40 degrees C. A stationary phase formed by a scrambled-sequence oligonucleotide that does not form a G-quartet did not provide any resolution of the two coeluting peptides, even at the higher temperatures, indicating that some destabilization of the G-quartet enhances resolution but that some degree of G-quartet structure is necessary. The effects of destabilization were further explored through variation of the cations (sodium or potassium) used in attachment of the G-quartet oligonucleotide to the capillary surface and in the mobile-phase buffer. Resolution was lower when a more stable, four-plane G-quartet stationary phase was used, supporting the conclusion that some flexibility in the G-quartet structure facilitates differential interactions that resolve otherwise coeluting peptides. The increase in peptide resolution upon destabilization of the G-quartet structure could prove to be an important factor in the application of G-quartet DNA stationary phases for nonaffinity-based separation of native proteins and peptides.     

Open-tubular capillary electrochromatography of bovine beta-lactoglobulin variants A and B using an aptamer stationary phase.

DNA aptamers that form a G-quartet conformation were covalently attached to a capillary surface for open-tubular capillary electrochromatographic separation of bovine beta-lactoglobulin variants A and B, which vary by 2 of their 162 amino acid residues. Separation was achieved using a 4-plane, G-quartet aptamer stationary phase with tris(hydroxymethyl)aminomethane (Tris) or phosphate buffer as the mobile phase. In control experiments, separation did not occur using either an oligonucleotide of similar base composition but which does not form a G-quartet structure, or using capillary zone electrophoresis on a bare capillary under similar experimental conditions. Separation was achieved using a capillary coated only with the covalent linker molecule. In phosphate buffer, the separations were similar for aptamer-coated and linker-only stationary phases, while in Tris buffer, retention times were almost doubled for the linker-only capillary. When Tris buffer is the mobile phase, there appears to be weaker interactions between the proteins and the stationary phase that may result in a gentler, less denaturing separation than is commonly achieved using hydrocarbon-based stationary phases.     

Capillary electrochromatographic separation of bovine milk proteins using a G-quartet DNA stationary phase

DNA oligonucleotides that form G-quartet structures were used as stationary phase reagents for separation of bovine milk proteins, including alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin. Both artificial protein mixtures and a skim milk sample were analyzed. The separations were performed using open-tubular capillary electrochromatography, in which the oligonucleotides were covalently attached to the inner surface of a fused-silica capillary. Better resolution was achieved using the G-quartet-coated capillaries than was achieved using either a bare capillary or a capillary coated with an oligonucleotide that does not form a G-quartet structure. A 4-plane G-quartet-forming stationary phase was able to resolve three peaks for alpha-casein and to detect thermal denaturation of the proteins in the milk sample. The results suggest that G-quartet stationary phases could be used to separate very similar protein structures, such as those arising from genetic variations or post-translational modifications.     

Aptamer stationary phase for protein capture in affinity capillary chromatography

The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.     

Capillary gel electrophoresis analysis of G-quartet forming oligonucleotides used in DNA-protein interaction studies

DNA-protein binding is among the most frequently studied biomolecular interactions with high importance in modern systems biology research. One interesting aspect of this rapidly developing field is the affinity capture of proteins by G-quartet forming oligonucleotides also referred to as aptamers. G-quartets are structural motifs formed by guanine-rich sequences commonly occurring in the human genome. In this paper, we describe a capillary gel electrophoresis based method to validate G-quartet formation of in-house designed oligonucleotides and discuss the effect of monovalent cation concentration on the development of this structure. The relevant aptamer was then bound to magnetic beads to form an affinity capture surface for target proteins, which were then analyzed by matrix-assisted laser desorption/ionization mass spectrometry.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

Insulin capture by an insulin-linked polymorphic region G-quadruplex DNA oligonucleotide

Insulin capture by a G-quadruplex DNA oligonucleotide containing a two-repeat sequence of the insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region is reported. The immobilized oligonucleotide was demonstrated to capture human insulin from standard solutions and from nuclear extracts of pancreatic cells with high selectivity, using affinity MALDI mass spectrometry and affinity capillary chromatography. Insulin was preferentially captured by the two-repeat ILPR oligonucleotide over another G-quadruplex-forming oligonucleotide, the thrombin-binding aptamer, as well as over a single repeat of the ILPR sequence that is not capable of forming the G-quadruplex architecture. Binding was shown to involve the beta chain of insulin. The discovery raises the possibility that insulin may bind to G-quadruplex DNA formed in the ILPR in vivo and thereby play a role in modulation of insulin gene expression, and it provides a basis for design of insulin analogues to probe this hypothesis. The availability of a DNA ligand to human insulin has analytical importance as well, offering an alternative to antibodies for in vitro or in vivo detection and sensing of insulin as well as its isolation and purification from biological samples.     

Open tubular capillary electrokinetic chromatography in etched fused-silica tubes

This review describes an open tubular approach to capillary electrochromatography (OTCEC) that first etches the inner surface of the fused-silica tube using ammonium hydrogen diflouride. This process can increase the inner surface area significantly. The new surface is then chemically modified to attach a bonded stationary phase using a silanization/hydrosilation reaction process. The surfaces are characterized spectroscopically by diffuse reflectance infrared Fourier transform and by electroosmotic flow measurements. Applications of OTCEC columns with C18, diol and chiral stationary phases are described.     

Study of physically absorbed stationary phases for open tubullar capillary electrochromatography.

A novel method based on the adsorption of positively charged compounds on the wall of a fused-silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run-to-run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590000/m and the average one above 250000/m. Stored at 2-8 degrees C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.     

丙二酰胺型大环多胺键合固定相在毛细管开管电色谱中的应用

毛细管电色谱 ( Capillary electrochromatography,简称 CEC)是近年发展起来的一种高效、快速的新型微柱分离方法 [1～ 4] .由于它在毛细管柱内填充液相色谱固定相或者在毛细管内壁键合固定相 ,且采用电渗流作为驱动力 ,因而兼有高效液相色谱和毛细管电泳的分离特点 ,已应用于复杂的药物分析 [2 ] .填充柱毛细管柱具有工艺要求高、容易产生气泡、焦耳热和价格昂贵等缺点 .开管柱电色谱( Open- tubular CEC,简称 OTCEC)是将固定相键合或涂覆在毛细管的内壁 ,避免了上述缺陷 .因此已引起高度重视 [3,4] .大环多胺的结构与冠醚类似 ,是一…     

Graphene oxide and reduced graphene oxide as novel stationary phases via electrostatic assembly for open‐tubular capillary electrochromatography

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open‐tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallyldimethylammonium chloride) (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet‐layer‐like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers, and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused‐silica capillary via electrostatic assembly.     

Aptamers as analytical reagents

Many important analytical methods are based on molecular recognition. Aptamers are oligonucleotides that exhibit molecular recognition; they are capable of specifically binding a target molecule, and have exhibited affinity for several classes of molecules. The use of aptamers as tools in analytical chemistry is on the rise due to the development of the "systematic evolution of ligands by exponential enrichment" (SELEX) procedure. This technique allows high-affinity aptamers to be isolated and amplified when starting from a large pool of oligonucleotide sequences. These molecules have been used in flow cytometry, biosensors, affinity probe electrophoresis, capillary electrochromatography, and affinity chromatography. In this paper, we will discuss applications of aptamers which have led to the development of aptamers as chromatographic stationary phases and applications of these stationary phases; and look towards future work which may benefit from the use of aptamers as stationary phases.     

Multimodal open-tubular capillary electrochromatographic analysis of amines and peptides

This study describes a comparison of different modes of open-tubular electrochromatography (OTCEC) in bare and etched capillaries. To carry out the investigation, the separation of impurities of two synthetic peptides and the separation of a mixture of five heterocyclic aromatic amines were studied. Three different types of stationary phase were evaluated: (i) fluorosurfactants (anionic and zwitterionic) adsorbed in the inner wall of the capillary (electrochromatography with dynamically modified stationary phases (DMS)CEC); (ii) physically adsorbed polymers (DMA-SO(3-) and DMA-N(+)(CH(3))(3)) and (iii) chemically modified capillaries (C(18), cholesteryl 10-undecanoate and diol). The results confirm that electrochromatography can be a viable alternative to capillary electrophoresis (CE) and liquid chromatography, more established separation techniques. It is possible to differentiate some minor species for the synthetic peptides that cannot be resolved by CE or high-performance liquid chromatography (HPLC). Moreover the separation of the amine mixture depends strongly on the stationary phase used.     

开管毛细管电色谱进展

开管毛细管电色谱是近年发展起来的一种高效、快速的新型微柱分离方法。它是在毛细管管壁涂布或键合固定相,以电渗流驱动流动相的一种色谱分离模式。该文对开管毛细管电色谱的发展、柱制备、理论进行了较为详细的综述,引用文献４７篇     

Interaction of viologens with nucleic acid G-tetrades

The interaction between the tetrade-forming oligonucleotide 5'-d(T 4G 4T 4) and monoalkylated bipyridinium salts, such as 1-ethyl-4-pyridin-4-ylpyridinium bromide, is reported. The oligomer forms tetrades in the presence of K+ ions but not with Li+. Additionally, the interaction of the thrombin-binding aptamer 5'-d(GGTTGGTGTGGTTGG) (TBA) with a dialkylated bipyridinium salt, viologen, was studied by cyclic voltammetry. This was performed either on a TiO2 electrode, derivatized with 3-aminopropyltriethoxysilane (APS), using [Fe(CN)6](4-) as a marker ion or without a marker ion on an electrostatically TiO2-bound amino-ferrocenyl derivative. Both experiments proof a strong interaction between the immobilized aptamer and the viologen. Third, the electrochemical response of the specific thrombin binding to the immobilized aptamer was studied.     

Mass spectrometric studies of alkali metal ion binding on thrombin-binding aptamer DNA

The binding sites and consecutive binding constants of alkali metal ions, (M⁺ = Na⁺, K⁺, Rb⁺, and Cs⁺), to thrombin-binding aptamer (TBA) DNA were studied by Fourier-transform ion cyclotron resonance spectrometry. TBA-metal complexes were produced by electrospray ionization (ESI) and the ions of interest were mass-selected for further characterization. The structural motif of TBA in an ESI solution was checked by circular dichroism. The metal-binding constants and sites were determined by the titration method and infrared multiphoton dissociation (IRMPD), respectively. The binding constant of potassium is 5–8 times greater than those of other alkali metal ions, and the potassium binding site is different from other metal binding sites. In the 1:1 TBA-metal complex, potassium is coordinated between the bottom G-quartet and two adjacent TT loops of TBA. In the 1:2 TBA—metal complex, the second potassium ion binds at the TGT loop of TBA, which is in line with the antiparallel G-quadruplex structure of TBA. On the other hand, other alkali metal ions bind at the lateral TGT loop in both 1:1 and 1:2 complexes, presumably due to the formation of ion-pair adducts. IRMPD studies of the binding sites in combination with measurements of the consecutive binding constants help elucidate the binding modes of alkali metal ions on DNA aptamer at the molecular level.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

吸附固定相开管毛细管电色谱方法的建立（英文）

 首次将管壁吸附作用作为开管毛细管电色谱固定相制备的推动力,成功地建立了称为“吸附固定相开管毛细管电色谱”的一种新方法。原理是：选择合适的条件,让荷正电的化合物在毛细管管壁上充分吸附,直接用吸附层作为固定相。目前,已有数类化合物被用作固定相物质,其中包括阳离子表面活性剂如十六烷基三甲基溴化铵（CTAB）、碱性蛋白质如溶菌酶和细胞色素C、碱性小肽如赖氨酸-酪氨酸和赖氨酸-丝氨酸-酪氨酸、以及碱性氨基酸如L-赖氨酸。CTAB吸附固定相用于分离电中性化合物,其它吸附固定相用于手性分离。     

吸附固定相开管毛细管电色谱方法的建立（英文）

首次将管壁吸附作用作为开管毛细管电色谱固定相制备的推动力,成功地建立了称为“吸附固定相开管毛细管电色谱”的一种新方法。原理是：选择合适的条件,让荷正电的化合物在毛细管管壁上充分吸附,直接用吸附层作为固定相。目前,已有数类化合物被用作固定相物质,其中包括阳离子表面活性剂如十六烷基三甲基溴化铵（CTAB）、碱性蛋白质如溶菌酶和细胞色素C、碱性小肽如赖氨酸-酪氨酸和赖氨酸-丝氨酸-酪氨酸、以及碱性氨基酸如L-赖氨酸。CTAB吸附固定相用于分离电中性化合物,其它吸附固定相用于手性分离。所建立的方法重现性好（迁移时间次间RSD值小于2．3％）,柱效高（最高柱效可达590000／m,平均柱效在250000／m以上）,寿命较长（2～8℃下保存,至少30d）。与已有的固定相制备方法比较,所建立的方法具有简便、快速和稳定等优点。