Single-molecule measurements of the binding between small molecules and DNA aptamers.

Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer-ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5'-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (ΔG(unfold)) between these two aptamers yields a ΔG of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (K(d)) of 2.0 ± 0.2 µM, a value consistent with the K(d) obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 µM) and close to that determined by affinity chromatography in the literature (6 ± 3 µM). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing.

Determination of sphingosine kinase 2 activity using fluorescent sphingosine by capillary electrophoresis.

The study of sphingosine and sphingosine-1-phosphate is now widespread due to their immense role as intra- and extracellular messenger molecules. The balance and interplay of these ceramide metabolites is dependent on the activities of kinase and phosphatase enzymes. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells; thus, they require highly sensitive techniques for quantitative analysis. In this study, we developed a quantitative assay for the determination of sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using CE-LIF. Sphingosine fluorescein was used as the substrate. The K(M) of SphK2 for sphingosine fluorescein was 2.8 ± 0.8 µM with a V(max) of 2490 ± 520 µM/min and a k(cat) of 1920 ± 402/s. The inhibition of SphK2 was also investigated using four different inhibitors for which 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole inhibitor was the most potent for the in vitro inhibition of SphK2 while N,N-dimethylsphingosine (DMS) did not inhibit but rather increased SphK2 activity. The fluorescence-based approach for the determination of the enzymatic activity of SphK2 proves to be useful for the quantitative determination of SphK2 activity in vitro and in cell lysates, and could be extended to single-cell analysis or applied in drug screening.

Metal cations for the determination of fluorescent phosphoinositides by capillary electrophoresis.

Phosphatidylinositol (PI) and its phosphorylated derivatives known as phosphoinositides (PIPs), are essential regulators of cell signaling and membrane trafficking, cytoskeletal dynamics, and nuclear functions. Disruption of PI metabolism is associated with disorders such as immune dysfunction, cardiovascular disease, and cancer; therefore, there is currently great interest in studying PIPs and their metabolic enzymes. Here, we describe a method for the separation of fluorescent PI and its seven fluorescent phosphorylated derivatives by CE-LIF. The CE method utilizes a Tris buffer and sodium deoxycholate in the presence of 30% 1-propanol and 5% of a dynamic coating reagent, EOTrol low reverse (EOTrol LR). It is simple, fast, highly sensitive, and it offers LODs in the order of 1.5 amol. The effect of cations such as lithium, sodium, potassium, cesium, barium, manganese, zinc, magnesium, calcium, spermine, and gentamicin were evaluated. Calcium and magnesium provided the best selectivity and resolution for the separation of the analytes while magnesium offered the best data reproducibility. The developed CE method would be useful in the studies of enzymatic activity in the PI and PIPs metabolic pathways using CE-based in vitro and CE cell-based assays, and/or for drug screening.

Fluorescent lipids as probes for sphingosine kinase activity by capillary electrophoresis.

Capillary electrophoresis (CE) is one among a number of highly sensitive chemical separation techniques used to characterize single or a small number of cells and to develop assays of enzymatic activity. Other commonly used techniques include mass spectrometry and electrochemistry; however, CE using laser-induced fluorescence detection (LIF) is the most sensitive of these techniques. In CE-LIF, fluorescently labeled proteins or lipids are normally separated based on their size to charge ratio in the interior of a small capillary filled with an electrolyte upon the application of an electric field. In this chapter, we describe the application of CE-LIF for the determination of the bioactivity of fluorescently lipids and sphingosine kinase activity.

Capillary electrophoresis-based methods for the determination of lipids--a review.

Capillary electrophoresis (CE) is a high-resolution technique for the separation of complex biological and chemical mixtures. CE continues to emerge as a powerful tool in the determination of lipids. Here we review the analytical potential of CE for the determination of a wide range of lipids. The different classes of lipids are introduced, and the different modes of CE and optimization methods for the separation of lipids are described. The advantages and disadvantages of the different modes of CE compared to traditional methods like gas chromatography (GC) and liquid chromatography (LC) in the determination of lipids are discussed. Finally, the potential of CE in the determination of lipids in the future is illustrated.

Determination of sphingosine kinase activity for cellular signaling studies.

Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The KM of sphingosine kinase for its fluorescently labeled substrate was 38 +/- 18 microM with a Vmax of 0.4 +/- 0.2 microM/min and a kcat of 3900 s-1. Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.

A comparison of ionic liquids to molecular organic solvents as additives for chiral separations in micellar electrokinetic chromatography.

In this study, we report the effects of adding ionic liquids (ILs), as compared to adding conventional molecular organic solvents (MOSs), to aqueous buffer solutions containing molecular micelles in the separation of chiral analyte mixtures in micellar EKC (MEKC). The molecular micelle used in this study was polysodium oleyl-L-leucylvalinate (poly-L-SOLV). The ILs were 1-alkyl-3-methylimidazolium tetrafluoroborate, where the alkyl group was ethyl, butyl, hexyl, or octyl. These ILs were chosen due to their hydrophobicity, good solvating, and electrolyte properties. Thus, it was expected that these ILs would have favorable interactions with chiral analytes and not adversely affect the background current. Common CE buffers, mixed with a molecular micelle, and an IL or a MOS, were used for these chiral separations. The buffers containing an IL in the concentration range of 0.02-0.1 v/v were found to support a reasonable current when an electric field strength of 500 V/cm was applied across the capillary. However, a current break down was observed for the buffers containing more than 60% v/v MOS on application of the above-mentioned electric field. The chiral resolution and selectivity of the analytes were dependent on the concentration and type of IL or MOS used.

Separation of fluorescent phosphatidyl inositol phosphates by CE.

Phosphatidyl inositol 4,5-bisphosphate (PIP2) and phosphatidyl inositol 3,4,5-trisphosphate (PIP3) labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) on the acyl chain or a phosphatidyl ethanolamine head group were separated by CE with LIF detection. Several methods and capillary-coating procedures were tested for the separation of these phosphatidyl inositol phosphates (PIPs) at 20 degrees C. Separation of the PIPs in less than 20 min with excellent resolution was achieved using a buffer containing sodium deoxycholate (SDC), 1-propanol, MgCl2 and the polymer coating reagent, EOTrol LR. The efficiency of the optimized method was as high as 1.3x10(5) plates. The dependence of the separation on the concentration of 1-propanol, SDC, and MgCl2 was determined. The separation of PIP2 and PIP3 was primarily due to differential binding of the lipids to Mg2+ rather than to different solubilities in the micellar phase. The role of the SDC was to prevent adsorption of the hydrophobic lipids to the capillary wall and thus enhance the efficiency. The fluorescent PIPs are of value for both in vitro and in vivo assays of phospholipid metabolism. In particular, the use of these lipids with the optimized capillary-based separation will be of utility for drug screening as well as cell-based assays.

Separation of achiral and chiral analytes using polymeric surfactants with ionic liquids as modifiers in micellar electrokinetic chromatography.

In this study, we report the use of ionic liquids as modifiers in the separation of achiral and chiral analytes in micellar electrokinetic chromatography. In this investigation, polymeric surfactants and ionic liquids were added to a low-conducting buffer solution. The polymeric surfactants used in this study were poly(sodium N-undecylelinic sulfate) and poly(sodium oleyl-l-leucylvalinate). The ionic liquids used in this study were chosen because of their high conductivity, hydrophobicity, and good solvating properties. Thus, it was expected that these ionic liquids would have the ability to assist in the separation of hydrophobic mixtures while maintaining adequate background current. Three analyte mixtures were separated using various buffer combinations of polymeric surfactant and ionic liquids. The ionic liquids were shown to improve the resolution and peak efficiency of the analytes while maintaining adequate background current.

Open-tubular capillary electrochromatography/electrospray ionization-mass spectrometry using polymeric surfactant as a stationary phase coating.

We describe the use of the polymeric surfactant poly(sodium undecylenic sulfate) (poly-SUS) as a stationary phase coating in open-tubular capillary electrochromatography (OT-CEC) coupled with electrospray ionization-mass spectrometry (ESI-MS) for the analysis of beta-blocker and benzodiazepine analytes. The production of a polymeric surfactant coating on the capillary inner wall involves (i) adsorption of the cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) to the inner surface of capillary, and (ii) adsorption of the negatively charged poly-SUS onto the cationic polymer layer via strong physical interaction of the two polymer layers. As compared with micellar electrokinetic chromatography (MEKC) coupled with ESI-MS, the main advantage of this proposed method is minimization of introduction of the monomeric or polymeric surfactant into the mass spectrometer, thus avoiding the interference of the nonvolatile micelle in ESI-MS. The effects of buffer pH and applied voltage on the separation of the analytes are also discussed. Under optimum conditions, four of the five beta-blockers and four benzodiazepines are separated.