The invention discloses a Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) method and NECEEM-based practical applications. The NECEEM method is a homogeneous technique, which, in contrast to heterogeneous methods, does not require affixing molecules to a solid substrate. The method of the invention facilitates 3 practical applications. In the first application, the method allows the finding of kinetic and thermodynamic parameters of complex formation. It advantageously allows for revealing two parameters, the equilibrium dissociation constant, K.sub.d, and the monomolecular rate constant of complex decay, k.sub.off, in a single experiment. In the second practical application, the method of this invention provides an approach for quantitative affinity analysis of target molecules. It advantageously allows for the use of affinity probes with relatively high values of k.sub.off. In the third practical application, the method of this invention presents a new and powerful approach to select target-binding molecules (ligands) from complex mixtures. Unique capabilities of the method in its third application include but not limited to: (a) the selection of ligands with pre-determined ranges of kinetic and thermodynamic parameters of target-ligand interactions, (b) the selection of ligands present in minute amounts in complex mixtures of biological or synthetic compounds such as combinatorial libraries of oligonucleotides, and (c) the selection of ligands for targets available in very low amounts. In particular, the method of this invention provides a novel approach for the selection of oligonucleotide aptamers. The NECEEM-based method can be used for discovery and characterization of drug candidates and the development of new diagnostic methods.

Description of the Invention

SUMMARY OF THE INVENTION

Introduction

The present invention provides a Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM)-based method for determining and using equilibrium and/or kinetic parameters of complex formation between two components, such as of a bimolecular interaction. In a preferred embodiment, the method is a homogeneous method. In another embodiment, the method can be used to screen for selecting components of a bimolecular interaction that have specified kinetic or binding parameters, such as in drug screening. In another embodiment, the invention can be used to determine the concentration of one or more of the components of the complex. In another embodiment, the method of the invention can be used to determine the thermodynamic parameters of a bimolecular interaction.

In one embodiment, the method of the invention allows for finding K.sub.d, k.sub.off, and/or k.sub.on for complex formation from a single electropherogram. In one embodiment, the invention provides a method for finding one, two or all three of said parameters. In one embodiment, the method is a homogenous method. In another embodiment, the components are L and T and the complex is LT. In yet another embodiment of the method, an equilibrium mixture of said components and complex is subjected to capillary electrophoresis under non-equilibrium conditions. The components and complex are separated by size and charge and detected at a detection point by a detector. The mode of detection can depend on the properties of the components and complex and how or whether the components are labeled. For instance, in one embodiment, one of the components can be fluorescently labeled. In another embodiment, the components and complex can be detected using their native light absorption or fluorescence or electrochemical properties or any combination of them.

The detector can be selected from a variety of types of detectors. In one embodiment, the detector is a UV absorbance detector, which is standard on commercial CE instruments. Many instruments also have diode array detectors available. Alternative detector modes include fluorescence laser-induced fluorescence, and electrochemical detection.

As the components and complex pass through the detector, the time of passage can be recorded to form an electropherogram containing peaks corresponding to the components and complex and exponential curves corresponding to the decay of the complex. However, other methods for recording time of passage of components, complex and rate of decay could also be used. For instance, a detector can be employed that images a large portion or whole length of the capillary. In one embodiment, the peaks and area under the curves in the electropherograms can be used to determine relative amounts of the detected component(s) and/or complex, and can be used to form calibration curves and peaks when known amounts of component(s) and/or complex are present. In another embodiment, these calibration curves and peaks can then be used to determine the concentrations or relative amounts of the components and/or complex in mixtures where these concentrations or relative amounts are not known. In another embodiment the concentration of a component can be determined by first establishing the equilibrium dissociation constant of the bimolecular interaction of the components and using the dissociation constant to determine the unknown concentration of the component. In one embodiment, the complex only slightly decays, considerably decays or completely decays during NECEEM.

In one embodiment of the invention, only one of the components is detected. In another embodiment both components are detected.

In one embodiment, the CE is coupled directly to another device, such as a thermocycler or a mass spectrometer. The hyphenation of CE and mass spectrometers is frequently used to give structural information on the resolved peaks. In another embodiment, the detectors can be interfaced with data acquisition devices to process results.

The peaks and curves of the resulting electropherograms can be used to determine the kinetic parameters of the complex or bimolecular interaction.

In one embodiment the equilibrium mixture is prepared in an electrophoresis run buffer. In another embodiment, the run buffer is free of said components and complex. In another embodiment the run buffer is optimized to separate the complex from said components in capillary electrophoresis. In another embodiment the run buffer contains a mediator that enhances electrophoretic separation of the complex from components.

In one embodiment the method of the invention can be used to determine the temperature of said capillary. This can be done by measuring the equilibrium and/or kinetic parameters of complex formation at different temperatures of said equilibrium mixture and/or said capillary. Thermodynamic parameters, such as enthalpy, the change of entropy and activation energies of the formation and decay of said complex can be determined. Calibration parameters can be determined at known temperatures and can be used to determine unknown temperatures.

In yet another embodiment, the invention provides a method selecting a ligand, L, that binds the target, T, with specified binding parameters, K.sub.d, k.sub.on, and/or k.sub.off of the formation of complex between the ligand and target using the aforementioned method for determining said kinetic parameters. In one embodiment, the ligand can be selected from a sample comprising a plurality of ligands with different binding parameters. In another embodiment, more than one ligand can be selected. In yet another embodiment, the method comprises: (a) preparing and equilibrating, e.g. incubating, a mixture comprised of said sample and target, wherein the concentration of said target and the time of said equilibration are defined by the desired values of: (i) the equilibrium dissociation constants of said complex and (ii) the bimolecular rate constant of the formation of said complex; (b) subjecting said equilibrium mixture to capillary electrophoresis, such as by injecting a plug of said equilibrium mixture into a capillary filled with the buffer solution free of the components of said sample, wherein said capillary is a part of the capillary electrophoresis instrument, wherein said buffer solution is the electrophoresis run buffer; wherein said run buffer is optimized to separate said sample from said target; wherein such run buffer is optimized not to separate the components of said sample and applying voltage to the ends of said capillary and subjecting the components of said equilibrium mixture to capillary electrophoresis; (c) collecting fractions eluting from said capillary in different time windows, wherein said time window defines the values of said binding parameters. In one embodiment, the buffer solution is free of said target. In another embodiment, said buffer solution contains said target. In one embodiment, the fraction is collected in a specific time window in said electrophoresis. In another embodiment, said time window excludes the electrophoretic peak of said sample, yet in another embodiment, the time window includes the electrophoretic peak of said sample.

In yet another embodiment, said time window includes the electrophoretic peak of said complex.

In one embodiment, the sample is a biological sample. In another embodiment, the sample is a combinatorial library, such as a library of oligonucleotides. In another embodiment, aptamers are selected from said library.

In yet another embodiment, the run buffer contains a mediator, which enhances electrophoretic separation of the components of said sample from said complex.

In another embodiment, the method of the invention is applied to the sample that was pretreated prior to the preparation of the equilibrium mixture, such as by the enrichment of the sample with the ligands using another binding assay. In one embodiment, the sample is a library of oligonucleotides and the binding assay is a heterogeneous method of enriching the population of oligonucleotides ligands in the library.

In another embodiment, the method of the invention is applied to a mixture of targets. In another embodiment, the complexes of the ligands and targets have different migration times in capillary electrophoresis. In another embodiment, the complexes are collected in different time windows in capillary electrophoresis. In yet another embodiment, the complexes are identified using another analytical method, for example, but not limited to one of the following: immunoassay, liquid chromatography, affinity chromatography, capillary affinity electrophoresis, and mass spectrometry.

In one embodiment, the migration time of the target is determined in a separate capillary electrophoresis run.

In yet another embodiment the capillary in the aforementioned methods of the invention is a channel of a microfabricated device.

In another embodiment the target is a protein, for example protein farnesyltransferase.

In one embodiment of the methods of the invention, the inner surface of the capillary is coated.

In one embodiment of the methods of the invention a mediator added to the buffer to enhance separation is a nucleic-acid binding protein, such as a single-stranded DNA binding protein.

Further Embodiments

In one embodiment, the method is realized in the following way. The complex-forming components are allowed to react and form an equilibrium mixture. This can be done either outside or inside the capillary. If the equilibrium mixture is prepared outside the capillary, a plug of the equilibrium mixture is introduced into the capillary and subjected to capillary electrophoresis under non-equilibrium conditions to permit complex decay and separation of the components and complex. The migration of one or more components and the complex is monitored. In one embodiment, the migration of the components and complex are detected at a detection point to generate an electropherogram that includes peaks and curves, the areas under which represent the amounts of components and/or complex that have passed through said detection point in a certain time interval. In a preferred embodiment, this single electropherogram may contain enough data to obtain all the kinetic parameters. However, a person skilled in the art would appreciate that any detector monitoring system can be used that enables the determination of amounts (actual or relative) of the components and complex, and rate of decay over time. In a preferred embodiment, the value of K.sub.d can be calculated from the areas under electrophoretic peaks and curves using one of the following two equations -- see Original Patent.  The method is applicable to components of different nature and origin. For example, a component can be an organic molecule, protein, peptide, enzyme, nucleic acid, aptamer, organelle, cell, virus, particle, or other reagent separable by capillary electrophoresis. If necessary, the component may be pretreated using different procedures such as, but not limited to: lysis, freeze-thaw, centrifugation, enrichment or fractionation. The components can be detected using light absorption, fluorescence, electrochemical properties, radioactivity, mass or charge properties. If the component is not detectable it can be labeled with a tag that facilitates one of the listed above modes of detection. The electrophoresis parameters are optimized to facilitate separation of the components from the complex. This optimization can include modifications to the voltage, temperature, buffer composition (including separation-enhancing mediators), buffer pH, capillary dimensions (including length, inner and outer diameters, material the capillary is made of, capillary pretreatment such as siliconization. If kinetic parameters are measured at different temperatures, then thermodynamic parameters, such as reaction enthalpy, the change of entropy, and activation energies of the formation and decay of the complex can be determined by a person skilled in the art of CE. These thermodynamic parameters can further serve as an indicator of temperature in an electrophoresis device, in which temperature is not controlled.

In another embodiment, the method of the invention allows for the determination of an unknown concentration of target (T) molecules using CE and affinity probe (L) whose complexes with the target molecules decay partially or completely during the CE process. First, the K.sub.d value of complex formation between T and L is determined as described in the previous paragraph, using known concentrations of T and L. Then, an equilibrium mixture comprised of an unknown concentration of T and a known concentration of L is subjected to CE under non-equilibrium conditions optimized by the operator to separate the complex LT from L. The electropherogram that may contain peaks of L and LT and a curve corresponding to the decay of LT are analyzed to determine the unknown concentration of T -- see Original Patent.  If necessary, T may be pretreated using different procedures such as, but not limited to: lysis, freeze-thaw, centrifugation, enrichment or fractionation. L can be detected using light absorption, fluorescence, electrochemical properties, radioactivity, mass or charge properties. If L is not detectable it can be labeled with a tag that facilitates one of the above listed modes of detection. The electrophoresis parameters are optimized to facilitate separation of L from LT. This can include modifications to the voltage, temperature, buffer composition (including separation-enhancing mediators), buffer pH, capillary dimensions (length, inner and outer diameters), capillary material, or capillary pretreatment such as siliconization. Alternatively to measuring K.sub.d, a calibration curve A.sub.L/(A.sub.LT+A.sub.decay) vs. [T] can be built. The method can be used as a diagnostic tool to measure the concentration of T present in a patient or biological sample.

In another embodiment, the method of the invention allows for screening and selecting target (T) binding molecules (L), in a fashion that overcomes some of the previously listed problems with using CE. In particular, the method of the invention allows for: (i) selecting L with a specified range of K.sub.d, k.sub.off, and k.sub.on values and/or (ii) selecting L when L constitutes only a very small fraction of the total sample and/or (iii) selecting L when T is only available in very small amounts. In one embodiment, the method is realized as follows. First, an equilibration mixture comprised of a sample and T is prepared outside or inside the capillary. The concentration of T and the time of equilibration are defined by the operator depending on the desired values of K.sub.d and k.sub.on. In the initial selection, ligands with K.sub.d<K.sub.d.sup.max=[T].sub.1 and k.sub.on>k.sub.on.sup.min=1/[T].sub.1t.sub.eq1 are selected. To achieve this, the equilibrium mixture contains a concentration of T equal to [T].sub.1 and is equilibrated for time equal to t.sub.eq1. In the following step the ligands with K.sub.d>K.sub.d.sup.min=[T].sub.2 and k.sub.on<k.sub.on.sup.max=1/[T].sub.2t.sub.eq2 are selected. To achieve this, the equilibrium mixture contains a concentration of T equal to [T].sub.2 and is equilibrated for time equal to t.sub.eq2. In general, [T].sub.2<[T].sub.1 and t.sub.eq2<t.sub.eq1. If the equilibrium mixture is prepared outside the capillary, a plug of the mixture is introduced into the capillary and subjected to capillary electrophoresis under non-equilibrium conditions. The electrophoresis conditions are optimized to separate the sample from T but not to separate the components of the sample. Fractions eluting from said capillary are collected at different time windows, which define the values of K.sub.d, k.sub.on, and k.sub.off of the collected ligands. T can be an organic molecule, protein, peptide, enzyme, nucleic acid, aptamer, organelle, cell, virus, particle, or other reagent separable by capillary electrophoresis. L can be any chemical entity that binds the target with the required specificity and affinity. L may be a component of a biological sample, patient sample, combinatorial library or other complex mixture. If necessary, T and the sample may be pretreated using different procedures such as, but not limited to: purification, enrichment, fractionation, lysis, freeze-thaw, and centrifugation. Advantageously, T does not need to be detectable. L and the other components of the sample can be detected using light absorption, fluorescence, electrochemical properties, radioactivity, mass or charge properties. If L is not detectable it can be labeled with a tag that facilitates one of the above listed modes of detection. The electrophoresis parameters are optimized to facilitate separation of L from LT. This optimization can include modifications to the voltage, temperature, buffer composition (including separation-enhancing mediators), buffer pH, capillary dimension (length, inner or outer diameter) capillary material, or capillary pretreatment such as siliconization. When the sample is a combinatorial library of oligonucleotides, the method of the invention can be used to select aptamers that bind T with specific K.sub.d, k.sub.on and k.sub.off values. PCR amplification of collected fractions can be used to amplify collected aptamers. For example, the method of the invention was used to select aptamers to protein farnesyltransferase. When selecting aptamers from oligonucleotide libraries, a single-stranded DNA-binding protein can be used to facilitate the separation of single stranded oligonucleotides from the aptamer-target complexes. When the sample is a combinatorial library containing potential therapeutic agents, the method of the invention can be used to select drug candidates or diagnostic probes that bind the therapeutic target with specific K.sub.d, k.sub.on and k.sub.off values. When the sample is a biological sample, the method of the invention can be used to select natural agents capable of binding T with specific K.sub.d, k.sub.on and k.sub.off values. The method of the invention can be applied to individual or multiple targets. To characterize the selected L, other analytical methods, such as immunoassay, liquid chromatography, affinity chromatography, capillary affinity electrophoresis, PCR, or mass spectrometry can follow the method of the invention. To further improve the efficiency of such combined methods an analytical device can be directly attached to the CE instrument. In a further embodiment, the method of the invention can be performed under equilibrium conditions when the electrophoresis buffer contains T. To conclude, the method of the invention advantageously allows blind selection of ligands when the concentrations of the target and ligands are below the limit of detection. The invention can be utilized even if only single molecules of ligand or target are present, as their detection is not required. This flexibility is essential for selecting ligands with desirable K.sub.d, k.sub.on and k.sub.off values as well as for selecting ligands when T is only available in small amounts or when L represents only a small fraction of the total sample, such as aptamers selected from a combinatorial library (a candidate aptamer may constitute as low as 10.sup.-13 of the sample (Gold, J. Biol. Chem. 270, 1995, 13581).

Claim 1 of 18 Claims

1. A homogeneous method for selecting a fraction of ligands from a mixture of potential ligands having similar electrophoretic mobility and different binding parameters wherein said selected ligands bind to a target with a desired range of ligand-target complex-formation binding parameters, said complex-formation binding parameters being selected from the equilibrium dissociation constant of the complex, the bimolecular rate constant of the formation of the complex and the unimolecular rate constant of the dissociation of the complex, the method comprising: a) selecting the desired range of the ligand-target complex-formation binding parameters; b) using the desired range of the ligand-target complex-formation binding parameters to determine an incubation time, target concentration, and time window for collecting a fraction containing ligands that bind to the target within the desired range of ligand-target complex-formation binding parameters; c) preparing a sample comprising ligands, target and complexes by incubating the mixture of the ligands and the target, for the incubation time and the target concentration determined in b) where the sample is incubated inside or outside a capillary that is part of a capillary electrophoresis instrument and the capillary is filled with an electrophoresis run buffer solution free of the ligands or ligand-target complexes prior to introduction of the sample; d) if the sample is incubated outside the capillary, introducing the sample into the capillary; e) subjecting the sample to capillary electrophoresis under non-equilibrium conditions optimized to separate the ligands from the complexes and not to separate the ligands from each other; and f) collecting the fraction containing ligands that bind to the target within the desired range of ligand-target complex-formation binding parameters eluting from the capillary in the time window determined in b) said fractions comprising the selected ligands in the form of separated intact ligand-target complexes and/or ligands dissociated from the complexes.
 

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