FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors.

Heat shock protein 90 (HSP90) is a critical target for anticancer and anti-fungal-infection therapies due to its central role as a molecular chaperone involved in protein folding and activation. In this study, we developed in vitro Förster Resonance Energy Transfer (FRET) assays to characterize the binding of C. albicans HSP90 to its co-chaperone Sba1, as well as that of the homologous human HSP90α to p23. The assay for human HSP90α binding to p23 enables selectivity assessment for compounds aimed to inhibit the binding of C. albicans HSP90 to Sba1 without affecting the physiological activity of human HSP90α. The combination of the two assays is important for antifungal drug development, while the assay for human HSP90α can potentially be used on its own for anticancer drug discovery. Since ATP binding of HSP90 is a prerequisite for HSP90-Sba1/p23 binding, ATP-competitive inhibitors can be identified with the assays. The specificity of binding of fusion protein constructs-HSP90-mNeonGreen (donor) and Sba1-mScarlet-I (acceptor)-to each other in our assay was confirmed via competitive inhibition by both non-labeled Sba1 and known ATP-competitive inhibitors. We utilized the developed assays to characterize the stability of both HSP90-Sba1 and HSP90α-p23 affinity complexes quantitatively. Kd values were determined and assessed for their precision and accuracy using the 95.5% confidence level. For HSP90-Sba1, the precision confidence interval (PCI) was found to be 70-120 (100 ± 20) nM while the accuracy confidence interval (ACI) was 100-130 nM. For HSP90α-p23, PCI was 180-260 (220 ± 40) nM and ACI was 200-270 nM. The developed assays were used to screen a nucleoside-mimetics library of 320 compounds for inhibitory activity against both C. albicans HSP90-Sba1 and human HSP90α-p23 binding. No novel active compounds were identified. Overall, the developed assays exhibited low data variability and robust signal separation, achieving Z factors > 0.5.

Nonsteady-State Electric Circuit in Electrophoresis on Paper: Thermal Consideration of Electrophoretic Lateral-Flow Assays.

Nonsteady-state behaviors are not expected in electric circuits that lack significant capacitance, inductivity, and/or active feedback. Here, we report that electrophoresis on paper─used, e.g., to electrophoretically driven lateral-flow immunoassays (LFIA)─can create a nonsteady-state electric circuit. We studied electrophoresis on 50 × 4 mm nitrocellulose membrane strips utilized in LFIA. The voltage was applied to strip termini immersed in reservoirs with a running buffer. If the electric power of this circuit exceeded approximately 0.5 W, neither the electric current nor the temperature map reached their steady states on a multiminute time scale. The current grew slowly to its maximum and then slowly decreased. The temperature map evolved slowly, with one or more hot spots appearing and disappearing gradually in different positions on the strip. The slow evolution of a temperature map led to the occurrence of a terminal hot spot in which the strip burned. No chaotic behavior was observed, i.e., time dependences of both the current and temperature map were reproducible. We analyzed major processes involved in paper-based electrophoresis and explained the nonsteady-state behavior. Unlike ordinary electric circuits with metal conductors, paper-based electrophoresis involves two slow processes: (i) intense buffer evaporation from hot spots and (ii) buffer supply from the reservoirs by an interplay of the capillary penetration and the electroosmotic flow. These processes affect heat generation and/or dissipation on the strip and, accordingly, the resistivity profile. The slow evolution of the resistivity profile is responsible for the nonsteady-state behavior. The results of our computer modeling support this explanation. The hot spots may have a destructive effect on electrophoretically driven LFIA. To avoid denaturation of immunoreagents, experimentalists should empirically confirm that spatiotemporal temperature maps are compatible with the developed assay.

Triphenylphosphine Derivatives of Allylbenzenes Express Antitumor and Adjuvant Activity When Solubilized with Cyclodextrin-Based Formulations.

Allylbenzenes (apiol, dillapiol, myristicin and allyltetramethoxybenzene) are individual components of plant essential oils that demonstrate antitumor activity and can enhance the antitumor activity of cytotoxic drugs, such as paclitaxel, doxorubicin, cisplatin, etc. Triphenylphosphine (PPh3) derivatives of allylbenzenes are two to three orders of magnitude more potent than original allylbenzenes in terms of IC50. The inhibition of efflux pumps has been reported for allylbenzenes, and the PPh3 moiety is deemed to be responsible for preferential mitochondrial accumulation and the depolarization of mitochondrial membranes. However, due to poor solubility, the practical use of these substances has never been an option. Here, we show that this problem can be solved by using a complex formation with cyclodextrin (CD-based molecular containers) and polyanionic heparin, stabilizing the positive charge of the PPh3 cation. Such containers can solubilize both allylbenzenes and their PPh3 derivatives up to 0.4 mM concentration. Furthermore, we have observed that solubilized PPh3 derivatives indeed work as adjuvants, increasing the antitumor activity of paclitaxel against adenocarcinomic human alveolar basal epithelial cells (A549) by an order of magnitude (in terms of IC50) in addition to being quite powerful cytostatics themselves (IC50 in the range 1-10 µM). Even more importantly, CD-solubilized PPh3 derivatives show pronounced selectivity, being highly toxic for the A549 tumor cell line and minimally toxic for HEK293T non-tumor cells, red blood cells and sea urchin embryos. Indeed, in many cancers, the mitochondrial membrane is more prone to depolarization compared to normal cells, which probably explains the observed selectivity of our compounds, since PPh3 derivatives are known to act as mitochondria-targeting agents. According to the MTT test, 100 µM solution of PPh3 derivatives of allylbenzenes causes the death of up to 85% of A549 cancer cells, while for HEK293T non-cancer cells, only 15-20% of the cells died. The hemolytic index of the studied substances did not exceed 1%, and the thrombogenicity index was < 1.5%. Thus, this study outlines the experimental foundation for developing combined cytostatic medications, where effectiveness and selectivity are achieved through decreased concentration of the primary ingredient and the inclusion of adjuvants, which are safe or practically harmless substances.

Fundamental Determinants of the Accuracy of Equilibrium Constants for Affinity Complexes.

The equilibrium constant of a chemical reaction is arguably the key thermodynamic parameter in chemistry; we naturally expect that equilibrium constants are determined accurately. The majority of equilibrium constants determined today are those of binding reactions that form affinity complexes, such as protein-protein, protein-DNA, and protein-small molecule. There is growing awareness that the determination of equilibrium constants for highly stable affinity complexes may be very inaccurate. However, fundamental (i.e., method-independent) determinants of accuracy are poorly understood. Here, we present a study that explicitly shows what the accuracy of equilibrium constants of affinity complexes depends on. This study reveals the critical importance of the choice of concentration of interacting components and creates a theoretical foundation for improving the accuracy of the equilibrium constants. The predicted influence of concentrations on accuracy was confirmed experimentally. The results of this fundamental study provide instructive guidance for experimentalists independently on the method they use.

Antibacterial Calcium Phosphate Coatings for Biomedical Applications Fabricated via Micro-Arc Oxidation.

A promising method for improving the functional properties of calcium-phosphate coatings is the incorporation of various antibacterial additives into their structure. The microbial contamination of a superficial wound is inevitable, even if the rules of asepsis and antisepsis are optimally applied. One of the main problems is that bacteria often become resistant to antibiotics over time. However, this does not apply to certain elements, chemical compounds and drugs with antimicrobial properties. In this study, the fabrication and properties of zinc-containing calcium-phosphate coatings that were formed via micro-arc oxidation from three different electrolyte solutions are investigated. The first electrolyte is based on calcium oxide, the second on hydroxyapatite and the third on calcium acetate. By adding zinc oxide to the three electrolyte solutions, antibacterial properties of the coatings are achieved. Although the same amount of zinc oxide has been added to each electrolyte solution, the zinc concentration in the coatings obtained vary greatly. Furthermore, this study investigates the morphology, structure and chemical composition of the coatings. The antibacterial properties of the zinc-containing coatings were tested toward three strains of bacteria-Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Coatings of calcium acetate and zinc oxide contained the highest amount of zinc and displayed the highest zinc release. Moreover, coatings containing hydroxyapatite and zinc oxide show the highest antibacterial activity toward Pseudomonas aeruginosa, and coatings containing calcium acetate and zinc oxide show the highest antibacterial activities toward Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

Covalent Conjugates of Allylbenzenes and Terpenoids as Antibiotics Enhancers with the Function of Prolonged Action.

The drug resistance of pathogenic bacteria is often due efflux pumps-specific proteins that remove foreign compounds from bacterial cells. To overcome drug resistance, adjuvants are often used that can inhibit efflux pumps or other systems that ensure the resistance of bacteria to the action of antibiotics. We assumed that a new level of effectiveness with the use of an antibiotic + an adjuvant pair could be achieved by their joint delivery into the pathogen. To test this hypothesis, we constructed a series of molecular carriers based on poly-(olygo-, dendry)mers based on cyclodextrin-grafted PEI or mannan, as well as glycol chitosan, covalently bound to antibiotic, adjuvant, and the oligosaccharide ligand to the macrophage mannose receptor (CD206), which we studied earlier and showed high efficiency and selectivity of delivery of a therapeutic "cargo" to macrophages. Moxifloxacin was used as an antibiotic, and terpenoid and allylbenzene compounds were used as adjuvants, for which we previously discovered the ability to inhibit bacterial efflux pumps. We show that: (a) the resulting structures were stable in vitro for a long time (up to 10 days); (b) they were adsorbed on bacterial cells, providing a local increase in the concentration of the antibiotic and adjuvant in pathogen cells; (c) they were internalized by bacterial cells, ensuring the accumulation of both antibiotic and adjuvant inside bacterial cells; (d) the adjuvant, after entering the bacterial cell, provided inhibition of the efflux pumps; (e) due to this action of the adjuvant, combined with the targeted delivery by the carrier, the antibiotic's half-life in rats increased by more than 2 times, the effective concentration of the drug in the blood plasma (AUC) increased up to 8-10 times; (f) a significant increase in the effectiveness of the antibacterial action against Gram+ and Gram- cells was achieved (up to 3 times). Potentially, such an approach would significantly increase the effectiveness of therapies for a number of infectious and other diseases, reduce the dosage of antibiotics, shorten the duration of treatment, and reduce the risk of developing bacterial resistance. Moreover, the use of a polymer carrier with covalently bound organic molecules of different structures will avoid problems linked to different (suboptimal) solubility and bio-distribution of the administered molecules, which would be almost inevitable when using the same compounds separately. It would be very difficult to find antibiotic/adjuvant pairs that simultaneously achieve optimal concentrations in the same target cells. In our case, terpenoids and alkylbenzenes used as adjuvants are practically insoluble as individual compounds, and their unacceptable pharmacological properties would not allow them to be used as efflux pump inhibitors.

Automated identification and tracking of cells in Cytometry of Reaction Rate Constant (CRRC).

Cytometry of Reaction Rate Constant (CRRC) is a method for studying cell-population heterogeneity using time-lapse fluorescence microscopy, which allows one to follow reaction kinetics in individual cells. The current and only CRRC workflow utilizes a single fluorescence image to manually identify cell contours which are then used to determine fluorescence intensity of individual cells in the entire time-stack of images. This workflow is only reliable if cells maintain their positions during the time-lapse measurements. If the cells move, the original cell contours become unsuitable for evaluating intracellular fluorescence and the CRRC experiment will be inaccurate. The requirement of invariant cell positions during a prolonged imaging is impossible to satisfy for motile cells. Here we report a CRRC workflow developed to be applicable to motile cells. The new workflow combines fluorescence microscopy with transmitted-light microscopy and utilizes a new automated tool for cell identification and tracking. A transmitted-light image is taken right before every fluorescence image to determine cell contours, and cell contours are tracked through the time-stack of transmitted-light images to account for cell movement. Each unique contour is used to determine fluorescence intensity of cells in the associated fluorescence image. Next, time dependencies of the intracellular fluorescence intensities are used to determine each cell's rate constant and construct a kinetic histogram "number of cells vs rate constant." The new workflow's robustness to cell movement was confirmed experimentally by conducting a CRRC study of cross-membrane transport in motile cells. The new workflow makes CRRC applicable to a wide range of cell types and eliminates the influence of cell motility on the accuracy of results. Additionally, the workflow could potentially monitor kinetics of varying biological processes at the single-cell level for sizable cell populations. Although our workflow was designed ad hoc for CRRC, this cell-segmentation/cell-tracking strategy also represents an entry-level, user-friendly option for a variety of biological assays (i.e., migration, proliferation assays, etc.). Importantly, no prior knowledge of informatics (i.e., training a model for deep learning) is required.

The Sherpa hypothesis: Phenotype-Preserving Disordered Proteins stabilize the phenotypes of neurons and oligodendrocytes.

Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are central to many physiological functions and to various pathologies that include neurodegeneration. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs that we term Phenotype-Preserving Disordered Proteins (PPDP) play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.

Polymeric Micelles Formulation of Combretastatin Derivatives with Enhanced Solubility, Cytostatic Activity and Selectivity against Cancer Cells.

Combretastatin derivatives is a promising class of antitumor agents, tubulin assembly inhibitors. However, due to poor solubility and insufficient selectivity to tumor cells, we believe, their therapeutic potential has not been fully realized yet. This paper describes polymeric micelles based on chitosan (a polycation that causes pH and thermosensitivity of micelles) and fatty acids (stearic, lipoic, oleic and mercaptoundecanoic), which were used as a carrier for a range of combretastatin derivatives and reference organic compounds, demonstrating otherwise impossible delivery to tumor cells, at the same time substantially reduced penetration into normal cells. Polymers containing sulfur atoms in hydrophobic tails form micelles with a zeta potential of about 30 mV, which increases to 40-45 mV when cytostatics are loaded. Polymers with tails of oleic and stearic acids form poorly charged micelles. The use of polymeric 400 nm micelles provides the dissolution of hydrophobic potential drug molecules. Micelles could significantly increase the selectivity of cytostatics against tumors, which has been shown using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Fourier transform infrared (FTIR) spectroscopy, flow cytometry and fluorescence microscopy. Atomic force microscopy presented the difference between the unloaded micelles and those loaded with the drug: the size of the former was 30 nm on average, while the latter had a "disc-like" shape and a size of about 450 nm. The loading of drugs into the core of micelles was confirmed by UV and fluorescence spectroscopy methods; shifts of absorption and emission maxima into the long-wavelength region by tens of nm was observed. With FTIR spectroscopy, a high interaction efficiency of micelles with the drug on cells was demonstrated, but at the same time, selective absorption was observed: micellar cytostatics penetrate into A549 cancer cells 1.5-2 times better than the simple form of the drugs. Moreover, in normal HEK293T, the penetration of the drug is reduced. The proposed mechanism for reducing the accumulation of drugs in normal cells is the adsorption of micelles on the cell surface and the preservation of cytostatics to penetrate inside the cells. At the same time, in cancer cells, due to the structural features of the micelles, they penetrate inside, merging with the membrane and releasing the drug by pH- and glutathione-sensitive mechanisms. From a methodological point of view, we have proposed a powerful approach to the observation of micelles using a flow cytometer, which, in addition, allows us to quantify the cells that have absorbed/adsorbed cytostatic fluorophore and distinguish between specific and non-specific binding. Thus, we present polymeric micelles as drug delivery systems in tumors using the example of combretastatin derivatives and model fluorophore-cytostatic rhodamine 6G.

Allylpolyalkoxybenzene Inhibitors of Galactonolactone Oxidase from Trypanosoma cruzi.

Inhibition of biosynthetic pathways of compounds essential for Trypanosoma cruzi is considered as one of the possible action mechanisms of drugs against Chagas disease. Here, we investigated the inhibition of galactonolactone oxidase from T. cruzi (TcGAL), which catalyzes the final step in the synthesis of vitamin C, an antioxidant that T. cruzi is unable to assimilate from outside and must synthesize itself, and identified allylbenzenes from plant sources as a new class of TcGAL inhibitors. Natural APABs (apiol, dillapiol, etc.) inhibited TcGAL with IC50 = 20-130 µM. The non-competitive mechanism of TcGAL inhibition by apiol was established. Conjugation of APABs with triphenylphosphonium, which ensures selective delivery of biologically active substances to the mitochondria, increased the efficiency and/or the maximum percentage of TcGAL inhibition compared to nonmodified APABs.

Electrophoretic Assembly of Antibody-Antigen Complexes Facilitates 1000 Times Improvement in the Limit of Detection of Serological Paper-Based Assay.

Serological assays detect the presence of specific antibodies in blood. There are urgent and important applications for serological point-of-care (POC) assays. However, available detection methods are either insufficiently sensitive or too complex for POC settings. Here, we demonstrate that lateral flow immunoassay (LFIA), which is arguably the simplest universal molecular detection approach, can serve as a methodological platform for highly sensitive serological POC assays if combined with a simple, fast, and inexpensive electrophoretic step. In this work, we compared such electrophoretically driven LFIA (eLFIA) with conventional LFIA for the detection of immunoglobulins G against hepatitis B and C in serum. The limit of detection of eLFIA was proven to be 1000 times lower than that of conventional LFIA and sufficiently low to support clinical serological tests. eLFIA takes less than 10 min, requires only a minor accessory powered by a small 9 V battery, and can be performed by an untrained person in the POC environment using a 3 µL specimen of finger-prick capillary blood.

Streamlined Data Processing for Determination of Equilibrium Dissociation Constants with Accurate Constant via Transient Incomplete Separation (ACTIS).

The determination of accurate equilibrium dissociation constants, Kd, of protein-small molecule complexes is important but challenging as all established methods have inherent sources of inaccuracy. Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for Kd determination using transient incomplete separation of the complex from the unbound small molecule in a pressure-driven flow inside a capillary. ACTIS is accurate, and its accuracy is invariant to variations in geometries of both the fluidic system and the flow. Furthermore, ACTIS is implemented using a simple fluidic system supporting its accuracy and providing a simple-to-follow/copy template for instrumentation. Despite the simple and robust instrumentation/acquisition, the current data processing workflow is cumbersome, time consuming, and prone to hard-to-trace human errors therefore hindering ACTIS' ability to become a practical reference method for Kd determination. This technical note describes a streamlined workflow for processing ACTIS data; the workflow is implemented as a set of open-source software tools called prACTISed (https://github.com/prACTISedProgram/prACTISed). These tools allow all steps of data processing to be performed in a fast and straightforward fashion. These practical software tools complement the simple instrumentation serving both developers and users of ACTIS.

Electrophoresis-Assisted Multilayer Assembly of Nanoparticles for Sensitive Lateral Flow Immunoassay.

Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point-of-need method. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles by electrophoresis. The nanoparticles link to immunocomplexes forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12 ng mL-1 , making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis-driven enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person.

Bulk Affinity Assays in Aptamer Selection: Challenges, Theory, and Workflow.

Selection of oligonucleotide aptamers involves consecutive rounds of affinity isolation of target-binding oligonucleotides from a random-sequence oligonucleotide library. Every next round produces an aptamer-enriched library with progressively higher fitness for tight binding to the target. The progress of enrichment can only be accurately assessed with bulk affinity assays in which a library is mixed with the target and one of two quantitative parameters, the fraction of the unbound library (R) or the equilibrium dissociation constant (Kd), is determined. These quantitative parameters are used to help researchers make a key decision of either continuing or stopping the selection. Despite the importance of this decision, the suitability of R and Kd for bulk affinity assays has never been studied theoretically, and researchers rely on intuition when choosing between them. Different approaches used for bulk affinity assays expectedly hinder comparative analyses of selections. Our current work has two goals: to give bulk affinity assays a thorough theoretical consideration and to propose a scientifically justified and practical bulk-affinity-assay approach. We postulate a formal criterion of suitability: a quantitative parameter must satisfy the principle of superposition. R satisfies this principle, while Kd does not, suggesting R as a theoretically preferable parameter. Further, we propose a solution for two limitations of R: its dependence on target concentration and narrow dynamic range. Finally, we demonstrate the use of this algorithm in both computer-simulated and experimental aptamer selection. This study sets a cornerstone in the theory of bulk affinity assays, and it provides researchers with a scientifically sound and instructive approach for conducting bulk affinity assays.

Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes.

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant (Kd) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of Kd value for a protein-aptamer complex. We conclude that "accurate constant via transient incomplete separation" (ACTIS) can serve as a reference method for affinity characterization of protein-aptamer binding in solution.

Plant Alkylbenzenes and Terpenoids in the Form of Cyclodextrin Inclusion Complexes as Antibacterial Agents and Levofloxacin Synergists.

Allylpolyalkoxybenzenes (APABs) and terpenoids from plant essential oils exhibit a range of remarkable biological effects, including analgesic, antibacterial, anti-inflammatory, antioxidant, and others. Synergistic activity with antibiotics of different classes has been reported, with inhibition of P-glycoprotein and impairment of bacterial cell membrane claimed as probable mechanisms. Clearly, a more detailed understanding of APABs' biological activity could help in the development of improved therapeutic options for a range of diseases. However, APABs' poor solubility in water solutions has been a limiting factor for such research. Here, we found that complex formation with ß-cyclodextrins (CD) is an efficient way to transform the APABs into a water-soluble form. Using a combination of spectroscopic (FTIR, NMR, UV) methods, we have estimated the binding constants, loading capacity, and the functional groups of both APABs and monoterpenes involved in complex formation with CD: ethylene, aromatic, methoxy and hydroxy groups. In the presence of a molar excess of CD (up to 5 fold) it was possible to achieve the complete dissolution of APABs and terpenoids in an aqueous medium (at 90-98% encapsulation) higher by 10-1000 times. Further, we have demonstrated that CD-APABs, if used in combination with levofloxacin (Lev), can be antagonistic, indifferent, additive, or synergistic, mostly depending on the concentration ratio: at high Lev concentration with the addition of APAB is typically neutral or even antagonistic; while at a Lev concentration below MIC, the addition of CD-APAB is either additive or synergistic (according to FICI criteria). An over three-fold increase in Lev antibacterial activity was observed in combination with eugenol (EG), as per the growth inhibition diameter measurement in agar. Interestingly, a synergistic effect could be observed with both Gram-positive and Gram-negative bacteria. So, obviously, the APAB-CD and terpenoid-CD mechanism of action is not limited to their interaction with the bacterial membrane, which has been shown earlier for CDs. Further research may open new prospects for the development of adjuvants to improve the therapeutic regimens with existing, as well as with new anti-infective drugs.

Circular Geometry in Molecular Stream Separation to Facilitate Nonorthogonal Field-to-Flow Orientation.

Molecular stream separation (MSS) is a promising complement for continuous-flow synthesis. MSS is driven by forces exerted on molecules by a field applied at an angle to the stream-carrying flow. MSS has only been performed with a 90° field-to-flow angle because of a rectangular geometry of canonic MSS; the second-order rotational symmetry of a rectangle prevents any other angle. Here, we propose a noncanonic circular geometry for MSS, which better aligns with the polar nature of MSS and allows changing the field-to-flow. We conducted in silico and experimental studies of circular geometry for continuous-flow electrophoresis (CFE, an MSS method). We proved two advantages of circular CFE over its rectangular counterpart. First, circular CFE can support better flow and electric-field uniformity than rectangular CFE. Second, the nonorthogonal field-to-flow orientation, achievable in circular CFE, can result in a higher stream resolution than the orthogonal one. Considering that circular CFE devices are not more complex in fabrication than rectangular ones, we foresee that circular CFE will serve as a new standard and a testbed for the investigation and creation of new CFE modalities.

Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis.

Partitioning of protein-DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here, we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE), which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of the protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinders must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for large-size protein targets while NECEEM should be the method of choice for small-size protein targets.

Template Instrumentation for "Accurate Constant via Transient Incomplete Separation".

Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for finding the equilibrium dissociation constant Kd of a protein-small molecule complex based on transient incomplete separation of the complex from the unbound small molecule in a capillary. This separation is caused by differential transverse diffusion of the complex and the small molecule in a pressure-driven flow. The advection-diffusion processes underlying ACTIS can be described by a system of partial differential equations allowing for a virtual ACTIS instrument to be built and ACTIS to be studied in silico. The previous in silico studies show that large variations in the fluidic system geometry do not affect the accuracy of Kd determination, thus, proving that ACTIS is conceptually accurate. The conceptual accuracy does not preclude, however, instrumental inaccuracy caused by run-to-run signal drifts. Here we report on assembling a physical ACTIS instrument with a fluidic system that mimics the virtual one and proving the absence of signal drifts. Furthermore, we confirmed method ruggedness by assembling a second ACTIS instrument and comparing the results of experiments performed with both instruments in parallel. Despite some unintentional differences between the instruments (caused by tolerances in sizes, positions, etc.) and noticeable differences in their respective separagrams, we found that the Kd values determined for identical samples with these instruments were equal. Conclusively, the fluidic system presented here can serve as a template for reliable ACTIS instrumentation.