Using Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) for Simultaneous Determination of Concentration and Equilibrium Constant

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the equilibrium constant, <i>K</i>_d, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound equilibrium fraction, <i>R</i>, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a constant concentration of L, which is as low as its reliable quantitation allows. The value of <i>R</i> is plotted as a function of the dilution coefficient, and dilution corresponding to <i>R</i> = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of <i>R</i> on the concentration of L is fitted with a function describing their theoretical dependence. Both <i>K</i>_d and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach <i>in silico</i> by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and <i>K</i>_d of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at equilibrium

Improvements to Direct Quantitative Analysis of Multiple MicroRNAs Facilitating Faster Analysis

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a “miRNA fingerprint”-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting

Surface-grafted polysarcosine as a peptoid antifouling polymer brush

Poly(N-substituted glycine) "peptoids" are a class of peptidomimetic molecules receiving significant interest as engineered biomolecules. Sarcosine (i.e., poly(N-methyl glycine)) has the simplest side chain chemical structure of this family. In this Article, we demonstrate that surface-grafted polysarcosine (PSAR) brushes exhibit excellent resistance to nonspecific protein adsorption and cell attachment. Polysarcosine was coupled to a mussel adhesive protein-inspired DOPA-Lys pentapeptide, which enabled solution grafting and control of the surface chain density of the PSAR brushes. Protein adsorption was found to decrease monotonically with increasing grafted chain densities, and protein adsorption could be completely inhibited above certain critical chain densities specific to different polysarcosine chain lengths. The dependence of protein adsorption on chain length and density was also investigated by a molecular theory. PSAR brushes at high chain length and density were shown to resist fibroblast cell attachment over a 7 week period, as well as resist the attachment of some clinically relevant bacterial strains. The excellent antifouling performance of PSAR may be related to the highly hydrophilic character of polysarcosine, which was evident from high-pressure liquid chromatography measurements of polysarcosine and water contact angle measurements of the PSAR brushes. Peptoids have been shown to resist proteolytic degradation, and polysarcosine could be produced in large quantities by N-carboxy anhydride polymerization. In summary, surface-grafted polysarcosine peptoid brushes hold great promise for antifouling applications