Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers.

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a "DNA Aptaomics" approach to systematically design functional aptamers as well as to obtain high affinity aptamers.

Purification of anionic fluorescent probes through precise fraction collection with a two-point detection system using multiple-stacking preparative capillary transient isotachophoresis.

A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.

Advanced Gel Electrophoresis Techniques Reveal Heterogeneity of Humic Acids Based on Molecular Weight Distributions of Kinetically Inert Cu2+-Humate Complexes.

Humic acids (HAs) play important roles for the fate of metal ions in the environment. Most chemical speciation models involving HAs assume heterogeneous metal ion binding. However, these models also assume that the binding affinities of metal ions with HAs are the same regardless of the molecular weight (MW) ranges of the HAs involved. Here, we develop new polyacrylamide gel electrophoresis (PAGE) techniques to investigate the MW distributions of HAs with strongly complexed Cu2+ ions. By combining contaminant metal-free and high-resolution PAGE for HAs, this work was able to provide accurate MW distributions for the complexed metal ions. The MW distribution of Cu2+ binding ability per quantity of HA indicates that strong metal-binding moieties in HAs are heterogeneous in terms of MW. Coupling of the PAGE techniques with UV-vis and excitation-emission matrix (EEM) spectrometry-parallel factor analysis (PARAFAC) methods revealed new insights into kinetically inert interactions between HAs and Cu2+ ions. By this method, we found that the protein-like fluorescence components in the high- and low-MW regions cooperatively responded through Cu2+ binding. Thus, the advanced gel electrophoresis techniques developed herein are able to shed new light on the heterogeneity of metal binding affinities of HAs in terms of MW.

A single-round selection of selective DNA aptamers for mammalian cells by polymer-enhanced capillary transient isotachophoresis.

A single-round DNA aptamer selection for mammalian cells was successfully achieved for the first time using a capillary electrophoresis (CE)-based methodology called polymer-enhanced capillary transient isotachophoresis (PectI). The PectI separation yielded a single peak for the human lung cancer cell line (PC-9) complexed with DNA aptamer candidates, which was effectively separated from a free randomized DNA library peak, ensuring no contamination from free DNA in the PC-9-DNA aptamer complex fraction. The DNA aptamer candidates obtained after a single-round selection employing counter selection with HL-60 were proven to bind selectively and form kinetically stable complexes with PC-9 cells. Interestingly, most aptamer candidates showed high binding ability (Kd = 70-350 nM) with different extents of binding on the cell surface. These facts proved that a single-round selection for mammalian cells by PectI is feasible to obtain various types of aptamer candidates, which have high-affinity even for non-overexpressed but unique targets on the cell surface in addition to overexpressed targets.

Mechanism of ion stacking in aqueous partition chromatographic processes.

It has been reported that ion enrichment phenomena are observed in liquid chromatographic processes with an aqueous mobile phase on the columns packed with nonionic materials. However, the mechanism of the ion enrichment is not at all well understood. In this study, we investigated the retention and enrichment behaviors of simple inorganic anions on a C18-bonded silica column and a cross-linked hydroxylated methacrylic polymer gel column with pure aqueous mobile phases containing various electrolytes. We show that the stacking of ionic solutes can successfully be accounted for by the ion partition model, and it takes place due to the effect of the background coion in the eluent and/or sample solution on the distribution of the ions between the bulk water and the water incorporated in the packing material, which acts as the stationary phase. Using the ion exclusion effect of fixed anionic charges on a packing material as well as the ion stacking by partition, we developed a simple and versatile method for effective enrichment of anionic solutes in aqueous solutions. The enrichment factor and the elution time of the stacked ion zone can be predicted by the ion partition model.

Surface-bubble-modulated liquid chromatography: a new approach for manipulation of chromatographic retention and investigation of solute distribution at water/hydrophobic interfaces.

In this paper, we present a new chromatographic method termed surface-bubble-modulated liquid chromatography (SBMLC), that has a hybrid separation medium incorporated with surface nanobubbles. Nanobubbles or nanoscale gas phases can be fixed at the interface between water and a hydrophobic material by delivering water into a dry column packed with a nanoporous material. The incorporation of a gas phase at the hydrophobic surface leads to the formation of the hybrid separation system consisting of the gas phase, hydrophobic moieties, and the water/hydrophobic interface or the interfacial water. One can change the volume of the gas phase by pressure applied to the column, which in turn alters the area of water/hydrophobic interface or the volume of the interfacial water, while the amount of the hydrophobic moiety remains constant. Therefore, this strategy provides a novel technique not only for manipulating the separation selectivity by pressure but also for elucidating the mechanism of accumulation or retention of solute compounds in aqueous solutions by a hydrophobic material. We evaluate the contributions of the interfacial water at the surface of an octadecyl bonded silica and the bonded layer itself to the retention of various solute compounds in aqueous solutions on the column packed with the material by SBMLC. The results show that the interfacial water formed at the hydrophobic surface has a key role in retention even though its volume is rather small. The manipulation of the separation selectivity of SBMLC for some organic compounds by pressure is demonstrated.

Determination of the cis-trans isomerization barriers of L-alanyl-L-proline in aqueous solutions and at water/hydrophobic interfaces by on-line temperature-jump relaxation HPLC and dynamic on-column reaction HPLC.

Proline cis-trans isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine cis-trans isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene-divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the cis and trans forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the cis form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water.

Molecular design of boronic acid-functionalized squarylium cyanine dyes for multiple discriminant analysis of sialic acid in biological samples: selectivity toward monosaccharides controlled by different alkyl side chain lengths.

We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with different lengths of alkyl chain residues, suitable for multiple discriminant analysis (MDA) of sialic acid (Neu5Ac) in biological samples. The SQ-BA dyes form aggregates based on hydrophobic interactions, which result in quenched fluorescence in aqueous solutions. When the boronic acid binds with saccharides, the fluorescence intensity increases as a result of dissociation to the emissive monomeric complex. We inferred that different dye aggregate structures (H-aggregates and J-aggregates) were induced depending on the alkyl chain length, so that monosaccharides would be recognized in different ways (especially, multipoint interaction with J-aggregates). A distinctive emission enhancement of SQ-BA dyes with shorter-alkyl-chains in the presence of Neu5Ac was observed (2.4-fold fluorescence enhancement; with formation constant 10(1.7) M(-1)), with no such enhancement for SQ-BA dyes with longer-alkyl-chain. In addition, various enhancement factors for other monosaccharides were observed depending on the alkyl chain length. Detailed thermodynamic and NMR studies of the SQ-BA complexes revealed the unique recognition mechanism: the dye aggregate with a shorter-alkyl-chain causes the slipped parallel structure and forms a stable 2:1 complex with Neu5Ac, as distinct from longer-alkyl-chain dyes, which form a 1:1 monomeric complex. MDA using the four SQ-BA dyes was performed for human urine samples, resulting in the successful discrimination between normal and abnormal Neu5Ac levels characteristic of disease. Thus, we successfully controlled various responses to similar monosaccharides with a novel approach that chemically modified not the boronic acid moiety itself but the length of the alkyl chain residue attached to the dye in order to generate specificity.

Multistep pH-peak-focusing liquid chromatography with a hydrophilic polymer gel column for separation of rare earth elements.

Multistep pH-peak-focusing liquid chromatography with a column packed with a hydrophilic polymer gel (a cross-linked hydroxylated methacrylic polymer gel) was developed for separation of rare earth metal ions. Metal ions in a sample solution introduced to the column are chromatographically extracted into the stationary gel phase at the top of the column equilibrated with a basic solution used as the first mobile phase containing acetylacetone and 1,10-phenanthroline by synergistic extraction effect. After the sample solution is introduced, the mobile phases are delivered into the column by stepwise gradient elution in order of decreasing pH. Each metal ion is concentrated at a pH border formed between the zones of different pH in the column and moves toward the outlet of the column with the pH border. Mutual separation of La(III), Ce(III), Nd(III), Eu(III), Y(III), Tb(III), and Yb(III) was achieved by the present method for an 1-mL sample injection with the column of which the inner volume is 11.8 mL. The multistep pH-peak-focusing liquid chromatography with a hydrophilic polymer gel column developed in this study has great potential as a useful method for the separation of rare earth metal ions on a preparatory scale.

Holo/apo conversion two-dimensional urea PAGE for speciation of Fe3+-bound transferrin in serum.

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

Determination of 90Sr in highly radioactive aqueous samples via conversion to a kinetically stable 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex followed by concentration-separation-fractionation based on capillary electrophoresis-liquid scintillation.

BACKGROUND: The Fukushima Daiichi Nuclear Power Plant accident (2011) released large amounts of radioactive substances into the environment and generated highly radioactive debris. Post-accident countermeasures are currently in the phase of fuel debris removal, which requires the analysis of radioactive contaminants in the environment and fuel. The spectra of solely ß-emitting nuclides, such as 90Sr, overlap; thus, an effective method for nuclide separation is desired. Since conventional methods for high-dose sample analysis pose substantial exposure risks and generate large amounts of secondary radioactive waste, faster procedures allowing for decreased radiation emission are highly desirable. RESULTS: In this study, we developed a 90Sr2+ quantitation technique based on liquid scintillation counting (LSC)-coupled capillary transient isotachophoresis (ctITP), along with two-point detection and relying on the rapid concentration, separation, and fractionation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-complexed 90Sr2+ in a single run. The applicability of our method for the analysis of real-world samples was verified by conducting addition-recovery experiments using a seawater reference material and radioactive liquid waste obtained from the radioactive waste treatment facility at the Japan Atomic Energy Agency. The recovery determined by LSC was 95-113%, indicating successful quantitative analysis. 90Sr recovery was determined to be 90.1% from a contaminated water sample obtained from the Fukushima Daiichi Nuclear Power Plant, which was analyzed using the standard addition of 90Sr. The sensitivity (detection limit = 0.016 Bq) of the proposed method on a radioactivity basis was equal to or higher than that of the conventional method using ion exchange-LSC (0.012-0.07 Bq). SIGNIFICANCE AND NOVELTY: Our method allows for the handling of high-dose radioactive samples at the microliter level and is substantially faster than conventional ion exchange protocols, whereas ctITP has not been used for practical applications due to inaccurate collection and lack of a suitable chemical system. The concentration-separation-fractionation protocol in ctITP is successful due to the existence of a rare inert Sr2+ complex and precise fractionation. This study establishes a pathway toward safer and more practical analysis of radionuclides.

Multistep pH-peak-focusing countercurrent chromatography with a polyethylene glycol-Na2SO4 aqueous two phase system for separation and enrichment of rare earth elements.

Multistep pH-peak-focusing countercurrent chromatography was developed for separation and enrichment of rare earth metal ions using a polyethylene glycol-Na(2)SO(4) aqueous two phase system (ATPS) and pH stepwise gradient elution. Metal ions in a sample solution are chromatographically extracted in a basic stationary phase (polymer-rich phase of the ATPS) containing a complexation ligand such as acetylacetone at the top of the countercurrent chromatography (CCC) column. After the sample solution is introduced, the mobile phases of which the pH values have been adjusted with buffer reagents are delivered into the column by stepwise gradient elution in order of decreasing pH. Each metal ion is concentrated at a pH border formed between the zones of different pH in the CCC column through extraction with a complexing agent into the stationary phase at the front side of the border (basic region) and back extraction into the mobile phase at the back side of the border (acidic region), moving toward the outlet of the column with the pH border. Mutual separations of La(III), Ce(III), Nd(III), Yb(III), and Sc(III) were achieved by the present method using five step pH gradient elution, and each rare earth metal ion was effectively enriched at each of the five pH borders. The mechanism for formation of pH profile of the column effluent and the potential of this technique for preparative scale separation are also discussed.

Separation of metalloproteins using a novel metal ion contaminant sweeping technique and detection of protein-bound copper by a metal ion probe in polyacrylamide gel electrophoresis: distribution of copper in human serum.

A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two types of gel electrophoresis, to obtain an accurate distribution of protein-bound metal ions in biological samples. First, proteins are separated by PAGE without the uptake of contaminant metal ions in the separation field and dissociation of metal ions from the proteins. This is followed by another PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using a fluorescent metal probe. The former is a new technique using blue-native (BN) PAGE to electrophoretically sweep all metal contaminants by employing two kinds of chelating agents. These agents form complexes with contaminants in the gel and the separation buffer solution, which migrate towards opposite pole directions, thus lowering the contaminants to below the ppt level during separation. This is termed "Metal Ion Contaminant Sweeping BN-PAGE (MICS-BN-PAGE)". After the separation of proteins under these first metal-free conditions, the metal ions in the gel fractions are eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxide dismutase were quantitatively determined, in addition to the exchangeable albumin-bound copper ions. This system successfully provided distribution maps of protein-copper in human serum. The precise distribution of copper in human serum was investigated, and found to be different from that which is widely accepted.

New molecular motif for recognizing sialic acid using emissive lanthanide-macrocyclic polyazacarboxylate complexes: deprotonation of a coordinated water molecule controls specific binding.

A new molecular motif--lanthanide-macrocyclic polyazacarboxylate hexadentate complexes, Ln(3+)-ABNOTA--was found to specifically bind to sialic acid with strong emission enhancement and high affinity. The selectivity toward sialic acid over other monosaccharides was one of the highest among artificial receptors. Also, the novel binding mechanism was investigated in detail; binding selectivity is controlled by interactions between sialic acid and both the central metal and a hydroxyl group produced by deprotonation of a coordinated water molecule in the Ln(3+) complex.

Surface-bubble-modulated liquid chromatography: an experimental strategy for identification of molecular processes of solute retention in reversed-phase separation systems.

Molecular level understanding of the chemistry at the aqueous/hydrophobe interface is crucial to separation processes in aqueous media, such as reversed-phase liquid chromatography (RPLC) and solid-phase extraction (SPE). Despite significant advances in our knowledge of the solute retention mechanism in these reversed-phase systems, direct observation of the behavior of molecules and ions at the interface in reversed-phase systems still remains a major challenge and experimental probing techniques that provide the spatial information of the distribution of molecules and ions are required. This review addresses surface-bubble-modulated liquid chromatography (SBMLC), which has a stationary gas phase in a column packed with hydrophobic porous materials and enables one to observe the molecular distribution in the heterogeneous reversed-phase systems consisting of the bulk liquid phase, the interfacial liquid layer, and the hydrophobic materials. The distribution coefficients of organic compounds referring to their accumulations onto the interface of alkyl- and phenyl-hexyl-bonded silica particles exposed to water or acetonitrile-water and into the bonded layers from the bulk liquid phase are determined by SBMLC. The experimental data obtained by SBMLC show that the water/hydrophobe interface exhibits an accumulation selectivity for organic compounds, which is quite different from that of the interior of the bonded chain layer, and the overall separation selectivity of the reversed-phase systems is determined by the relative sizes of the aqueous/hydrophobe interface and the hydrophobe. The solvent composition and the thickness of the interfacial liquid layer formed on octadecyl-bonded (C18) silica surfaces are also estimated from the bulk liquid phase volume determined by the ion partition method employing small inorganic ions as probes. It is clarified that various hydrophilic organic compounds as well as inorganic ions recognize the interfacial liquid layer formed on the C18-bonded silica surfaces as being different from the bulk liquid phase. The behavior of some solute compounds exhibiting substantially weak retention in RPLC or the so-called negative adsorption, such as urea, sugars, and inorganic ions, can rationally be interpreted with a partition between the bulk liquid phase and the interfacial liquid layer. The spatial distribution of solute molecules and the structural properties of the solvent layer on the C18-bonded layer determined by the liquid chromatographic methods are discussed in comparison to the results obtained by other research groups using molecular simulation methods.

Evaluation of the thermal effect on separation selectivity in anion-exchange processes using superheated water ion-exchange chromatography.

The thermal effect on retention and separation selectivity of inorganic anions and aromatic sulfonate ions in anion-exchange chromatography is studied on a quaternized styrene-divinylbenzene copolymer anion-exchange column in the temperature range of 40-120 °C using superheated water chromatography. The selectivity coefficient for a pair of identically charged anions approaches unity as temperature increases provided the ions have the same effective size, such that the retention of an analyte ion decreases with an increase in temperature when the analyte ion has stronger affinity for the ion-exchanger than that of the eluent counterion, whereas it increases when it has weaker affinity. The change in anion-exchange selectivity with temperature observed with superheated water chromatography has been discussed on the basis of the effect of temperature on hydration of the ions. At elevated temperatures, especially in superheated water, the electrostatic interaction or association of the ions with the fixed ion in the resin phase becomes a predominant factor resulting in a different separation selectivity from that obtained at ambient temperature.

Characterization of the Interfacial Liquid Layer Formed on Hydrophobic Packing Material Surfaces by Liquid Chromatographic Analysis of the Distribution of Ions and Molecules.

We determine the bulk liquid phase volumes in octadecyl-bonded silica (C18 silica) columns equilibrated with acetonitrile-water and methanol-water (0-19%(v/v)) binary mixed solvents by a liquid chromatographic method with inorganic ions used as probes. The solvent composition and the thickness of the interfacial liquid layer formed on the C18-bonded silica surface are then determined from the bulk liquid phase volume, the total liquid phase volume, the surface area of the C18 silica packing material, and the retention volumes of the isotopically labeled eluent components for the columns. We used two C18 silica packing materials having identical bonding structures but different pore sizes and surface areas. Our results show that various hydrophilic organic compounds as well as inorganic ions recognize the interfacial liquid layer as being different from the bulk phase. The behavior of the solute compounds exhibiting substantially weak retention in reversed-phase liquid chromatography or the so-called negative adsorption, such as urea, sugars, and inorganic ions, can rationally be interpreted with a partition between the bulk liquid phase and the interfacial solvation liquid layer. The structural properties of the solvent layer on the C18-bonded layer determined by liquid chromatography are consistent with the molecular dynamics simulation results that have been obtained by other researchers.

A chromatographic approach for studying the adsorption of polar small molecules on tetrabutylammonium bromide semiclathrate hydrate.

A new liquid chromatography system in which tetrabutylammonium bromide semiclathrate hydrate (TBAB-SCH) was used as the column-packing material is proposed to evaluate the adsorption mechanisms of polar small molecules on a TBAB-SCH surface. It has been revealed that the more hydrophilic molecules tend to be more strongly retained on the TBAB-SCH/hexane-diethyl ether interface, and the retentions of aromatics (ARs) and crown ethers (CEs) are differently controlled. CEs are likely retained on the TBAB-SCH interface via hydrogen-bond formation as well on water-ice, whereas the retention mechanism of ARs is unique to TBAB-SCH and has not previously been observed on water-ice. This difference between CEs and ARs may arise from differences in the types of polar groups or molecular size. This chromatographic approach will provide useful information for evaluating the adsorption mechanisms of inhibitors for clathrate hydrate agglomeration, which is a common agglomeration problem experienced by industries.