Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry.

G-quadruplex-forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we couple on-line kinetic capillary electrophoresis with mass spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes in solution. We show that peaks shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA-metal affinity interactions and G-quadruplex formation; and ion mobility mass spectrometry (IM-MS) provides information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes. KCE-MS separates a thrombin-binding aptamer d[GGTTGGTGTGGTTGG] from mutated sequences based on affinity to potassium, and reveals the apparent equilibrium folding constant (K F≈150 µm), folding rate constant (k on≈1.70×10(3) s(-1) m(-1)), unfolding rate constant (k off≈0.25 s(-1)), half-life time of the G-quadruplex (t 1/2≈2.8 s), and relaxation time (τ≈3.9 ms at physiological 150 mm [K(+)]). In addition, KCE-MS screens for a GQ-stabilizing/-destabilizing effect of DNA binding dyes and an anticancer drug, cisplatin.

Real-time monitoring of protein conformational dynamics in solution using kinetic capillary electrophoresis.

Conformational analysis: Capillary electrophoresis (CE) allows for the rapid separation of slowly interconverting protein conformers. Kinetic analysis (k(open), k(closed), and K(d)) of electropherograms in the presence and absence of effector ligands allows the measurement of kinetic and thermodynamic constants associated with conformational changes and ligand binding.

Comparative study of three methods for affinity measurements: capillary electrophoresis coupled with UV detection and mass spectrometry, and direct infusion mass spectrometry.

We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and ß-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

Sequential-dissociation kinetics of non-covalent complexes of DNA with multiple proteins in separation-based approach: general theory and its application.

Binding of multiple proteins to DNA is crucial in many regulatory cellular processes. The kinetics of assembly and disassembly of DNA-multiple protein complexes is very difficult to study in detail due to the lack of suitable experimental approaches. A separation-based approach has been recently proposed to resolve disassembly kinetics of such complexes. While conceptually simple, the separation-based approach generates experimental data with very complex patterns. The analysis of these patterns is a challenging problem on its own. Here we report on a mathematical approach that can extract a solution for the experimental data obtained in separation-based analysis of sequential dissociation of a DNA complex with multiple proteins. This case describes the dissociation of proteins one-by-one from the complex. Generally speaking, a mathematical solution of such problems requires calculations of multiple integrals. Our approach reduces this procedure to taking double integrals and constructing their superposition. We tested this approach with the experimental data obtained for three-step sequential dissociation of complexes of DNA with two protein copies.

Separation-based approach to study dissociation kinetics of noncovalent DNA-multiple protein complexes.

Noncovalent binding of DNA with multiple proteins is pivotal to many regulatory cellular processes. Due to the lack of experimental approaches, the kinetics of assembly and disassembly of DNA-multiple proteins complexes have never been studied. Here, we report on a first method capable of measuring disassembly kinetics of such complexes. The method is based on continuous spatial separation of different complexes. The kinetics of multiple complex dissociation processes are also spatially separated, which in turn facilitates finding their rate constants. Our separation-based approach was compared with a conventional no-separation approach by using computer simulation of dissociation kinetics. It proved to be much more accurate than the no-separation approach and to be a powerful tool for testing hypothetical mechanisms of the disassembly of DNA-multiple proteins complexes. An experimental implementation of the separation-based approach was finally demonstrated by using capillary electrophoresis as a separation method. The interaction between an 80 nucleotide long single-stranded DNA and single-stranded DNA binding protein was studied. DNA-protein complexes with one and two proteins were observed, and rate constants of their dissociation were determined. We foresee that a separation approach will be also developed to study the kinetics of the formation of DNA-multiple protein complexes.

Revealing equilibrium and rate constants of weak and fast noncovalent interactions.

Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k(on), k(off), and K(d) of weak (K(d) > 1 µM) and fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor. Conceptually, an equilibrium mixture (EM) of a ligand (L), target (T), and a complex (C) is prepared. The mixture is introduced into the beginning of a capillary reactor with aspect ratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > K(d) > 80 µM) and fast (0.25 s ≥ τ ≥ 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and α- and ß-cyclodextrins. The affinity of the drugs was significantly higher for ß-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation.

Electric field destabilizes noncovalent protein-DNA complexes.

Noncovalent protein-DNA interactions are involved in many vital biological processes. In cells, these interactions may take place in the environment of an electric field which originates from the plasma and organelle membranes and reaches strengths of 1 MV/cm. Moreover, protein-DNA interactions are often studied in vitro using an electric field as strong as 1 kV/cm, for example by electrophoresis. It is widely accepted that an electric field does not affect such interactions. Here we report on the first proof that an electric field of less than 1 kV/cm can destabilize the protein-DNA complexes through increasing the monomolecular rate constant of complex dissociation.

Non-orthogonal micro-free flow electrophoresis: from theory to design concept.

Micro-free flow electrophoresis (microFFE) is a technique that facilitates continuous separation of molecules in a shallow channel with a hydrodynamic flow and an electric field at an angle to the flow. We recently developed a general theory of microFFE that suggested that an electric field non-orthogonal to the flow could improve resolution. Here, we used computer modeling to study resolution as a function of the electric field strength and the angle between the electric field and the hydrodynamic flow. In addition we used our general theory of microFFE to investigate other important influences on resolution, which include the velocity of the hydrodynamic flow, the height of the separation channel, and the magnitude and direction of the electroosmotic flow. Finally, we propose four designs that could be used to generate non-orthogonal electric fields and discuss their relative merits.

MASKE: macroscopic approach to studying kinetics at equilibrium.

The kinetics of biomolecular interactions at equilibrium is typically studied by "microscopic" methods that monitor concentration fluctuations of molecules in an "observation" volume in which the number of molecules is so small that the equilibrium is statistically impossible. Here, we introduce a "macroscopic" method for studying kinetics of biomolecular interactions at equilibrium which does not rely on monitoring the fluctuation of concentrations. We termed this method MASKE: a "macroscopic approach to studying kinetics at equilibrium". Conceptually, in MASKE, two equilibrium reaction mixtures, "unlabeled" and "labeled", are both prepared with two reactants and their complex; in the labeled mixture, one reactant is labeled for detection. A "macroscopic" amount of the labeled mixture is introduced into a long and narrow reactor filled with the unlabeled mixture, and a differential mobility of the reactant versus the complex is then induced by an external action along the reactor. The kinetics of complex formation and dissociation is then studied from the label-propagation pattern. In this work, we developed the theory of MASKE and experimentally proved it with a capillary as a reactor, a fluorophore as a label, and an electric field as a differential mobility inducer. Two pairs of molecules interacting with significantly different rate constants were used in this proof-of-principle work.

Non-orthogonal-to-the-flow electric field improves resolution in the orthogonal direction: hidden reserves for combining synthesis and purification in continuous flow.

There is a pressing need for continuous purification of products of synthesis conducted in continuous-flow microreactors. An existing technique, micro free-flow electrophoresis (microFFE), could fulfill this niche if its resolving power for similar molecules was improved. MicroFFE continuously separates ions in the hydrodynamic flow by an electric field orthogonal to the flow. Here, we prove theoretically from first principles that the resolving power of microFFE can be greatly improved by the use of a nonorthogonal to the flow field. This result may be decisive in starting practical attempts to combine synthesis in continuous-flow microreactors with continuous-flow purification by microFFE.

Transverse diffusion of laminar flow profiles--a generic method for mixing reactants in capillary microreactor.

The capillary is an attractive format for integrated microanalyses, which start with the injection of separate reactants into the capillary and their mixing inside the capillary. Due to the nonturbulent nature of flow inside the capillary, mixing reactants in a generic way is a challenging task. Three approaches have been suggested as a solution: mixing by electrophoresis, mixing by longitudinal diffusion, and, most recently, mixing by transverse diffusion of laminar flow profiles (TDLFP). This is the first review on TDLFP, describing: (i) the physical basis of the method, (ii) its theory, (iii) analytical and numerical solutions for the calculation of concentration profiles of mixed reactants, (iv) up-to-date applications, and (v) problems to be solved and future directions.

Kinetic capillary electrophoresis-based affinity screening of aptamer clones.

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K(d) values for the in-capillary reaction of an aptamer and a target and that the obtained K(d) values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.

Mathematical model for mixing reactants in a capillary microreactor by transverse diffusion of laminar flow profiles.

Transverse diffusion of laminar flow profiles (TDLFP) was recently suggested as a generic approach for mixing reactants inside a capillary microreactor. Conceptually, solutions of reactants are injected inside the capillary by high pressure as a series of consecutive plugs. Because of the laminar nature of the flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between the plugs. After the injection, the reactants are mixed by transverse diffusion across the longitudinal interfaces. TDLFP-based mixing is still in its infancy as only the principle was proved. Here, we develop the theory of TDLFP and introduce a dimensionless parameter, York number, which can be used in predicting the quality of TDLFP-based mixing. The theory uses a single simplifying assumption that the longitudinal diffusion is negligible; this assumption is readily satisfied. We then develop a numerical model of TDLFP and use it to simulate the concentration profiles of three reactants mixed by TDLFP in the capillary. The correlation between the York number and quality of mixing is analyzed. Two ways of improving the quality of TDLFP-based mixing are suggested and studied: (i) increasing the longitudinal interface between the plugs by a long last plug of a solvent and (ii) "shaking" the injected reactants by a series of alternating negative and positive pressure pulses. The developed theory and computational simulation of TDLFP will stimulate the practical use of capillary microreactors.

"Inject-mix-react-separate-and-quantitate" (IMReSQ) method for screening enzyme inhibitors.

Many regulatory enzymes are considered attractive therapeutic targets, and their inhibitors are potential drug candidates. Screening combinatorial libraries for enzyme inhibitors is pivotal to identifying hit compounds for the development of drugs targeting regulatory enzymes. Here, we introduce the first inhibitor screening method that consumes only nanoliters of the reactant solutions and is applicable to regulatory enzymes. The method is termed inject-mix-react-separate-and-quantitate (IMReSQ) and includes five steps. First, nanoliter volumes of substrate, candidate inhibitor, and enzyme solutions are injected by pressure into a capillary as separate plugs. Second, the plugs are mixed inside this capillary microreactor by transverse diffusion of laminar flow profiles. Third, the reaction mixture is incubated to form the enzymatic product. Fourth, the product is separated from the substrate inside the capillary by electrophoresis. Fifth, the amounts of the product and substrate are quantitated. In this proof-of-principle work, we applied IMReSQ to study inhibition of recently cloned protein farnesyltransferase from parasite Entamoeba histolytica. This enzyme is a potential therapeutic target for antiparasitic drugs. We identified three previously unknown inhibitors of this enzyme and proved that IMReSQ could be used for quantitatively ranking the potencies of inhibitors.

Diffusion as a tool of measuring temperature inside a capillary.

Application of capillary electrophoresis (CE) to temperature-sensitive biomolecular interactions requires knowledge of the temperature inside the capillary. The simplest approach to finding temperature in CE employs a molecular probe with a temperature-dependent parameter. Up until now only spectral parameters of molecular probes were utilized for temperature measurements in CE. The arbitrary nature of spectral parameters leads to several inherent limitations that compromise the accuracy and precision of temperature determination. This paper introduces the concept of finding temperature in CE through the measurement of a nonspectral parameter of the molecular probeits diffusion coefficient. Diffusion is a fundamental property of molecules that depends only on the molecular structure of the probe, the nature of the environment, and the temperature. It is ideally suited for temperature measurements in CE if an approach for measuring the diffusion coefficient in a capillary with high precision is available. This work first develops an approach for measuring the diffusion coefficient in a capillary with a relative standard deviation of as low as 2.1%. It is then demonstrated that such precise measurements of the diffusion coefficient could facilitate accurate temperature determination in CE with a precision of 1 degrees C. This new method was used to study the effect on temperature of different amounts of joule heat generated and different efficiencies of heat dissipation. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, for example, electrochemical and mass spectrometric detection.

Plug-plug kinetic capillary electrophoresis: method for direct determination of rate constants of complex formation and dissociation.

We present a method for direct determination of rate constants of complex formation, k(on), and dissociation, k(off). The method is termed plug-plug kinetic capillary electrophoresis (ppKCE). To explain the concept of the method, we consider the formation of a noncovalent complex C between molecules A and B; A is assumed to migrate slower in electrophoresis than B. In ppKCE, a short plug of A is injected into a capillary, followed by a short plug of B. When a high voltage is applied, the electrophoretic zone of B moves through that of A, allowing for the formation of C. When the zones of A and B are separated, C starts dissociating. The features of the resulting electropherogram are defined by both binding and dissociation. We developed a unique mathematical approach that allows finding k(on) and k(off) from a single electropherogram without nonlinear regression analysis. The approach uses algebraic functions with the only input parameters from electropherograms being areas and migration times of electrophoretic peaks. In this work, we explain theoretical bases of ppKCE and prove the principle of the method by finding k(on) and k(off) for a protein-ligand complex. The unique capability of the method to directly determine both k(on) and k(off) along with its simplicity make ppKCE highly attractive to a broad community of molecular scientists.

Selection of smart aptamers by methods of kinetic capillary electrophoresis.

We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.

Kinetic capillary electrophoresis (KCE): a conceptual platform for kinetic homogeneous affinity methods.

We propose kinetic capillary electrophoresis (KCE) as a conceptual platform for the development of kinetic homogeneous affinity methods. KCE is defined as the CE separation of species that interact during electrophoresis. Depending on how the interaction is arranged, different KCE methods can be designed. All KCE methods are described by the same mathematics: the same system of partial differential equations with only initial and boundary conditions being different. Every qualitatively unique set of initial and boundary conditions defines a unique KCE method. Here, we (i) present the theoretical bases of KCE, (ii) define four new KCE methods, and (iii) propose a multimethod KCE toolbox as an integrated kinetic technique. Using the KCE toolbox, we were able to, for the first time, observe high-affinity (specific) and low-affinity (nonspecific) interactions within the same protein-ligand pair. The concept of KCE allows for the creation of an expanding toolset of powerful kinetic homogeneous affinity methods, which will find their applications in studies of biomolecular interactions, quantitative analyses, and selecting affinity probes and drug candidates from complex mixtures.