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WO1999045374A2 - Procedes de purification et de detection par electrophorese d'affinite reversible - Google Patents

Procedes de purification et de detection par electrophorese d'affinite reversible Download PDF

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Publication number
WO1999045374A2
WO1999045374A2 PCT/US1999/004849 US9904849W WO9945374A2 WO 1999045374 A2 WO1999045374 A2 WO 1999045374A2 US 9904849 W US9904849 W US 9904849W WO 9945374 A2 WO9945374 A2 WO 9945374A2
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Prior art keywords
analyte
electrophoretic medium
varied
affinity ligand
electric field
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PCT/US1999/004849
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English (en)
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WO1999045374A3 (fr
Inventor
Ezra S. Abrams
Philip W. Hammond
Andrew R. Muir
T. Christian Boles
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Mosaic Technologies
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Application filed by Mosaic Technologies filed Critical Mosaic Technologies
Priority to EP99909853A priority Critical patent/EP1068518A2/fr
Priority to AU28963/99A priority patent/AU2896399A/en
Priority to JP2000534862A priority patent/JP2002506204A/ja
Priority to CA002322975A priority patent/CA2322975A1/fr
Publication of WO1999045374A2 publication Critical patent/WO1999045374A2/fr
Publication of WO1999045374A3 publication Critical patent/WO1999045374A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • Zonal electrophoresis, and particularly gel electrophoresis is one of the best known methods for separation, purification and characterization of charged molecules, particularly macromolecules such as proteins or nucleic acids (Freifelder, Physical Biochemistry, 2nd ed. , (1982) pp. 276-310, Freeman, San Francisco) .
  • Electrophoresis can be used to separate molecules based on their size, charge, conformation, and many combinations of these properties.
  • electrophoresis In most electrophoresis applications, charged molecules migrate through a supporting medium under the influence of an electric field. Most frequently, electrophoresis is carried out using a linear constant voltage gradient of fixed orientation (two fixed electrodes, constant voltage) . However, for very large DNA molecules (i.e., in the size range of 30 to 2000 kb) , the polymeric chain orients with the field and snakes through the gel rendering the sieving action of the electrophoretic medium ineffective. In order to separate large DNA molecules, workers have developed applications in which the field orientation is varied cyclically, as in "field inversion gel electrophoresis" (Carle, et al .
  • the supporting medium acts to suppress convection and diffusion, and can be sieving or nonsieving.
  • affinity electrophoresis the support medium is also modified with chemical groups (i.e., ligands) that interact specifically or nonspecifically with one or more desired analytes and, thus, help to accomplish the separation of analyte and non-analyte sample components during purification by influencing its mobility.
  • Affinity electrophoresis has been used to measure the binding affinity of proteins (Horejsi and Kocourek, Biochim . Biophys . Acta (1974), 336:338-343 and Chu et al . , " . Med . Chemistry (1992), 35:2915-2917).
  • vinyl-adenine modified polyacrylmide media has been used to enhance resolution of nucleic acids in capillary electrophoresis (Baba et al . , Analytical Chemistry (1992), 64:1920-1924) .
  • An affinity electrophoresis process in which the direction of the electric field is varied in a cyclical manner while synchronously changing one, or more, properties of the electrophoretic medium between two states.
  • the property or properties which are being varied favor specific reversible binding of sample analytes to affinity ligands which are immobilized within the medium.
  • the property or properties which are being varied disfavor the binding of sample analytes to the immobilized affinity ligands.
  • the process provides a convenient method to obtain high resolution separations.
  • an apparatus for separating a target analyte from a test sample is described.
  • the apparatus combines an electrophoretic medium having an immobilized affinity ligand, an electrode system having one, or more, electrodes, capable of generating an electric field which can change in orientation, and a means of changing one, or more, properties of the electrophoretic medium between two states.
  • the property or properties which are being varied favor specific reversible binding of sample analytes to affinity ligands which are immobilized within the medium.
  • the property or properties which are being varied disfavor the binding of sample analytes to the immobilized affinity ligands.
  • the means of changing a property in the electrophoretic medium can be a device which changes the temperature of the electrophoretic medium.
  • the means of changing one, or more, properties of the electrophoretic medium can be manually or automatically changing the electrophoresis buffer.
  • Figures 1A, IB, and 1C show the separation of 5 ' -
  • Fluorescein-TGA GGC TTT CTG TTA TGG TAC-3 ' (SEQ ID NO: 1) on an electrophoretic medium having a covalently bound complementary nucleic acid strand from a non-complementary, fluorescently labeled nucleic acid.
  • Figures 2A, 2B, 2C and 2D show the separation of E. coli Rnase P RNA from 16S Hha RNA and 16S Alu RNA on an electrophoretic medium having a covalently bound nucleic acid sequence that is complementary to a sequence in E. coli Rnase P RNA.
  • Figures 3A and 3B show the separation of E. coli Rnase
  • the invention disclosed herein is directed to an electrophoretic process of separating sample components, and an apparatus designed to carry out the process, that combines the following features: 1) an electrophoretic medium that contains one or more immobilized affinity ligands; 2) use of an electric field that changes in orientation at least once during the process; and 3) a change in at least one other medium property that affects the ability of the affinity ligand(s) to form a specific binding complex with the analyte(s) , said change in medium property (or properties) occurring synchronously with the change in field orientation, thereby allowing electrophoretic separation of analyte and non-analyte components of the sample.
  • the process is a general method for performing repetitive cycles of affinity separation for purification of specific analytes in a biological or test sample.
  • the analyte molecules are purified (e.g., isolated or separated) from non-analyte sample components.
  • Each cycle is characterized by two electrophoretic steps.
  • the first step is carried out using a first field orientation and first medium condition (also referred to herein as "state"), said condition allowing formation of specific binding complexes between sample analytes and affinity ligands in the medium.
  • the next step is carried out under a second field direction and second medium condition which is obtained by varying one or more property of the electrophoretic medium from the first medium condition.
  • the second condition is designed to disrupt formation of specific binding complexes between sample analytes and affinity ligands in the medium.
  • all sample components are moved to new locations within the medium.
  • partial, or complete, separation of specific analytes from other sample components has occurred, and both fractions have been moved to new locations within the medium.
  • a single cycle may provide sufficient purification of analyte for many applications. If additional purification is desired, the purification cycle can be repeated. For example, during the second cycle, purification proceeds as in the first cycle. However, the starting materials for the second purification cycle are the partially fractionated products of the first purification cycle which are now at new locations in the electrophoretic medium. The locations of analyte and non-analyte fractions from the first cycle may or may not overlap, depending on the extent of purification achieved in the previous cycle. In either case, during the second and subsequent cycles, the two fractions are further separated.
  • an arbitrarily high number of affinity purification cycles can be performed on a single electrophoresis unit, in an automated fashion, with continuous removal of non-binding sample components during each cycle.
  • a low number of cycles for example 1 to 10 cycles, may provide the necessary purification.
  • many cycles for example 10 to several thousand, may be required to separate the components.
  • the repetitive nature of the process allows extremely efficient electrophoretic purification of the analyte molecules. For instance, if the purification efficiency of each cycle is 10-fold (e.g., 90% of non-analyte sample components removed per cycle) , four cycles would yield a purification of 10, 000-fold.
  • a key advantage of the invention is that the cyclic purification process can be carried out in a single device, such as an electrophoretic gel. This simplifies the purification process by eliminating preparation, loading, and fraction collection from multiple columns.
  • the process can be performed in an automated fashion using equipment with few (or no) moving parts.
  • the test sample can be any sample, from any source in which analyte molecules are mixed with non-analyte molecules.
  • An analyte molecule is any molecule of interest that can form a binding complex with an affinity ligand.
  • samples from biological sources containing cells obtained using known techniques, from body tissue (e.g., skin, hair, internal organs), or body fluids (e.g., blood, plasma, urine, semen, sweat) .
  • Other sources of samples suitable for analysis by the methods of the present invention are microbiological samples, such as viruses, yeasts and bacteria: plasmids, isolated nucleic acids and agricultural sources, such as recombinant plants.
  • test sample is treated in such a manner, known to those of skill in the art, so as to render the analyte molecules contained in the test sample available for binding.
  • a cell lysate can be prepared, and the crude cell lysate (e.g., containing the target analyte as well as other cellular components) can be analyzed.
  • the target analyte can be partially isolated (rendering the target analyte substantially free from other cellular components) prior to analysis. Partial isolation can be accomplished using known laboratory techniques. For example, DNA, RNA and proteins can be isolated from a variety of biological samples using TRI reagent (see Sigma catalogue, p. 1545, catalogue numbers T9424, T3809, and T3934, see also Chomczynski, et al . , Biochem . (1987), 162:156; Chomczynski, Biotechniques (1993), 15:532) in conjunct with Southern blotting (DNA) , Northern blotting (RNA) and Western blotting (proteins) procedures.
  • TRI reagent see Sigma catalogue, p. 1545, catalogue numbers T9424, T3809, and T3934, see also Chomczynski, et al . , Biochem . (1987), 162:156; Chomczynski, Biotechniques (1993), 15:532
  • Antibodies can be isolated by binding to Protein A immobilized on a solid support (see Surolia, et al . , Trends Bioch . Sci . (1981), 7:74 and Sigma catalogue p. 1462, catalogue number PURE-1) .
  • a nucleic acid analyte can also be amplified (e.g., by polymerase chain reaction or ligase chain reaction techniques) prior to analysis.
  • An affinity ligand is any molecule that can form a specific binding complex with an analyte and can be immobilized within a suitable electrophoretic medium. Methods for determining the thermal stability of binding complexes and, in particular, hybridization complexes are well known in the literature. Wetmur,
  • D and D' are an affinity ligand and an analyte, such as a first nucleic acid and a second nucleic acid containing a region complementary to the first nucleic acid sequence
  • B is the analyte/affinity ligand complex product
  • k 2 and k r are the kinetic rate constants for the analyte/affinity ligand complex formation and dissociation, respectively.
  • the reverse reaction is most relevant to the consideration of spontaneous dissociation of the analyte/affinity ligand complex, and the rate constant for dissociation, k r , is the critical variable that needs to be minimized to facilitate binding between the analyte and affinity ligand.
  • dissociation can be reduced by lowering the assay temperature; this will decrease the dissociation constant.
  • Ea is the activation energy for dissociation and R is the universal gas constant .
  • Ea can be calculated from the base sequence of the nucleic acid sequence used to form the analyte/affinity ligand complex. Wetmur, Cri tical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Use of the Arrhenius equation for this calculation is described by Tinocco, et al . , Physical Chemistry: Principles and Applications in Biological Sciences, Prentice Hall (pub.), Englewood Cliffs, NJ, pp. 290-294 (1978) .
  • an effective dissociation constant can be estimated using a temperature gradient procedure.
  • the melting behavior of an immobilized analyte/affinity ligand complex within an electrophoresis gel can be measured using a temperature gradient which increases laterally across the gel.
  • the temperature, Td, at which 50% of the complex has dissociated during the time of electrophoresis, ta can be used to estimate the dissociation constant.
  • equation (3) can be used to calculate k r at other lower temperatures that might be suitable for the first medium condition wherein conditions are selected to allow the formation of specific binding complexes between sample analytes and affinity ligands. These calculated values of k r can then be used with the first order rate law to calculate the fraction of analyte/affinity ligand complex remaining at a given assay temperature ta and electrophoresis time ta:
  • Equation (6) can be used to estimate the change in k r needed to increase B/Bo (decrease analyte/affinity ligand complex dissociation) by any specified amount. Once the desired value of k r is known, equation (3) can be used to calculate the change in temperature needed to achieve the k r value . It should be noted that the gradient gel procedure only provides an estimate of the actual analyte/affinity ligand complex Td and k r , since displaced analytes can rebind to uncomplexed immobilized affinity ligands.
  • an affinity ligand is a single-stranded nucleic acid, which can bind by hybridization, for example, to an analyte that contains a complementary nucleic acid sequence.
  • the single strand nucleic acid affinity ligand can be complementary to the entire analyte nucleic acid sequence or to a portion thereof.
  • Single-stranded nucleic acids can also be used for isolation of duplex nucleic acids by triplex formation (Hogan and Kessler, U.S. Patent No. 5,176,966 and Cantor, et al . , U.S. Patent No. 5,482,836, the teachings of which are incorporated herein by reference) .
  • Double- stranded nucleic acids can also serve as useful affinity ligands for nucleic acid binding proteins, or for nucleic acid analytes that bind to the ligand by triplex or tetraplex formation.
  • the conditions under which a single strand nucleic acid will bind to another nucleic acid to be immobilized in a gel can be estimated using the procedure outlined above for estimating the stability of analyte/affinity ligand complexes.
  • the melting temperature (Tm) of the two nucleic acids provides a reasonable framework for estimating the temperate at which an nucleic acid analyte will hybridize to a nucleic acid affinity ligand.
  • the Td is lower than the Tm by about 15 to 25°C and, therefore, the temperature at which the gel should be run to facilitate specific hybridization between the analyte and affinity ligand should be about 15 to 25°C or more below the Tm.
  • the Tm of a pair of nucleic acids is typically determined by monitoring a physical property, such as UV absorption, of a solution of the two nucleic acids in the electrophoresis buffer while uniformly varying the property of the solution that will be cyclically varied during the electrophoresis separation.
  • a physical property such as UV absorption
  • the temperature can be slowly decreased while monitoring the UV absorption.
  • the nucleic acids are single stranded.
  • complementary bases As the temperature decreases complementary bases pair off and hydrogen bond. This hydrogen bonding causes a change in UV absorption.
  • the transition between the hydrogen bonding state and the non-hydrogen bonding state occurs over a narrow temperature range. The midpoint of this temperature range is the Tm for the two nucleic acids.
  • the ionic strength or pH of the buffer can be varied in a uniform manner while holding the temperature constant and monitoring the UV absorption.
  • Nucleic acids form duplexes more readily in higher ionic strength and lower temperature conditions.
  • “Stringency conditions” for hybridization is a term of art which refers to the conditions of temperature and buffer concentration (ionic strength) which permit hybridization of a particular nucleic acid to a second nucleic acid in which the first nucleic acid may be perfectly complementary to the second, or the first and second may share some degree of complementarity which is less than perfect.
  • certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
  • high or moderate stringency conditions can be determined empirically. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined. Binding conditions for triplexes and tetraplexes can be estimated in a similar manner.
  • Nucleic acid aptamers can also be used as affinity ligands in the process of the present invention.
  • Aptamers can be selected against many kinds of analytes, including proteins, small organic molecules, and carbohydrates (reviewed in Klug and Famulok, Molecular Biology Reports (1994), 20:97-107).
  • selection of aptamer ligands offers a simple and flexible mechanism for obtaining affinity ligands against virtually any target molecule .
  • ligands include proteins or polypeptides which can bind to specific analytes.
  • An especially useful class of protein ligands are antibody molecules, which can be elicited against a wide range of analytes by immunization methods.
  • Antibodies ligands can be monoclonal or polyclonal .
  • a fragment of an antibody can be an affinity ligand.
  • receptor proteins may be useful as ligands for purification and detection of analytes that bind to or are bound by them.
  • Carbohydrates have been successfully used as affinity ligands for electrophoretic purification of lectins (Horejsi and Kocourek, Biochim . Biophys . Acta (1974), 336:338-343), and may be useful for purification and detection of molecules that bind to specific carbohydrates or glycoproteins .
  • Binding or non-binding conditions of proteins, aptamers and lectins for specific ligands can be estimated using the procedure outlined above for estimating the stability of analyte/affinity ligand complexes.
  • equilibrium dialysis experiments can provide a rational method of predicting the stability of analyte/affinity ligand complexes.
  • the dissociation constant of a protein for a particular ligand can be determined in the electrophoresis buffer at several different pHs , temperatures or ionic strengths.
  • suitable media fall into two classes.
  • the first includes media composed of gel-forming materials like crosslinked polyacrylamide and agarose .
  • the second class includes media composed of solutions of linear noncrosslinked polymers such as polyacrylamide, poly (hydroxyethylcellulose) , and poly (ethyleneoxide) .
  • the latter category is commonly used for capillary electrophoresis applications.
  • Immobilization of ligands can be accomplished by direct attachment to the polymeric components of the medium. Such attachment can be mediated by formation of covalent bonds between the ligand and the polymer.
  • Noncovalent binding between the ligand and polymer substituents can also be used.
  • strong noncovalent binding provided by the widely-used biotinstreptavidin and digoxigenin-antidigoxigenin systems can be used to attach ligands to appropriately modified polymeric media. Covalent attachment is preferred. Direct connection between the polymeric medium and the ligand is not strictly required.
  • ligands can be attached to particulate supports, such as microspheres, and the particulate supports can be immobilized within the polymer medium by physical entrapment (Cantor, et al . , U.S. Patent No. 5,482,863, the teachings of which are incorporated herein by reference in their entirety) .
  • the particles may be macroscopic, microscopic, or colloidal in nature, (see Polyciences, Inc., 1995-1996 particle Catalog, Warrington, PA) .
  • ligands can be attached to highly branched soluble polymers. Due to their branched shape, such ligand-polymer complexes display extremely large effective hydrodynamic radii and, therefore, will not migrate in the electric field in many kinds of polymeric media of appropriately small pore size. Thus, they can be entrapped within the media in the same fashion as particulate supports.
  • Absolute immobilization of the ligand within the medium is not required for all embodiments of the invention. For many applications, it is sufficient that the mobility of the analyte is changed upon formation of a binding complex with the ligand. This condition can be satisfied by coupling the ligand to a medium component that has extremely low electrophoretic mobility. However, for efficient purification the change in mobility should be as large as possible. Therefore, media utilizing true immobilization of the ligand within the medium will be preferred for use in this invention. Commonly used gel media useful for the present invention include acrylamide and agarose gels. However, other materials may be used.
  • Examples include modified acrylamides and acrylate esters (for examples see Polysciences, Inc., Polymer & Monomer catalog, 1996-1997, Warrington, PA), starch (Smithies, Biochem . J. (1959), 71:585; product number S5651, Sigma Chemical Co., St. Louis, MO), dextrans (for examples see Polysciences, Inc., Polymer & Monomer Catalog, 1996-1997, Warrington, PA), and cellulose-based polymers (for examples see Quesada, Current Opinions in Biotechnology (1997), 8:82-93). Any of these polymers can be chemically modified to allow specific attachment of ligands (including nucleic acids, proteins, peptides, organic small molecules, and others) for use in the present invention.
  • ligands including nucleic acids, proteins, peptides, organic small molecules, and others
  • composite media containing a mixture of two or more supporting materials.
  • An example is the composite acrylamide-agarose gel. These gels typically contain from 2-5% acrylamide and 0.5%- 1% agarose. In these gels the acrylamide provides the chief sieving function, but without the agarose, such low concentration acrylamide gels lack mechanical strength for convenient handling. The agarose provides mechanical support without significantly altering the sieving properties of the acrylamide. In such cases, the nucleic acid can be attached to the component that confers the sieving function of the gel, since that component makes most intimate contacts with the solution phase nucleic acid target . For capillary electrophoresis (CE) applications it is convenient to use media containing soluble polymers .
  • CE capillary electrophoresis
  • soluble polymers examples include linear polymers of polyacrylamide, poly (N,N-dimethylacrylamide) , poly (hydroxyethylcellulose) , poly (ethyleneoxide) and poly (vinylalcohol) as described in Quesada, Current Opinion in Biotechnology (1997), 8:82-93). Solutions of these polymers can also be used to practice the methods of the present invention.
  • ligands can be coupled to agarose, dextrans, cellulose, and starch polymers using cyanogen bromide or cyanuric chloride activation.
  • Polymers containing carboxyl groups can be coupled to ligands that have primary amine groups using carbodiimide coupling.
  • Polymers carrying primary amines can be coupled to aminecontaining ligands with glutaraldehyde or cyanuric chloride.
  • Many polymers can be modified with thiol -reactive groups which can be coupled to thiol -containing ligands. Many other suitable methods are known in the literature.
  • An electrode system is a system that, in conjunct with a power supply, produces and electric field gradient.
  • An electric field gradient is the voltage drop across the gel created by the electrode system (see Giddings, Unified Separation Science (1991), John Wiley & Sons, New York, p. 155-170) .
  • the orientation of the electric field gradient used for electrophoresis determines the geometry of the separation between analyte and non-analyte sample components. Many field geometries can be used. For instance, with a conventional two-electrode apparatus, a one-dimensional separation can be achieved simply by switching the polarity of the two electrodes, as practiced in field inversion gel electrophoresis (Carle et al .
  • analytes only migrate under conditions which disfavor binding to the ligands, whereas the non-analyte sample components would migrate under both sets of conditions.
  • forward the direction of net analyte movement during the purification process be called "forward". If the purification cycle is designed so that the duration of reverse field orientation is longer than the duration of forward field orientation, analyte molecules will be moved forward during each purification cycle, but non-analyte sample components will only enter the gel only transiently since they are efficiently removed from the gel by the long period of reverse field orientation.
  • Two dimensional electrode arrangements as used in pulsed field (Schwartz and Cantor, Cell (1984), 37:67) and CHEF applications (CHEF gels, U.S. Patent No. 5,549,796; Bio-Rad Life Science Research Products Catalog (1997), pp. 175-182), allow the separation process of the present invention to be performed in two dimensions. In principle, the addition of another set of electrodes operating in a third dimension could add additional separation capability if desired.
  • the state of instrumentation and methodology for performing one and two dimensional electrophoretic separations is well advanced. At least one commercially available device (CHEF gel apparatus, Bio-Rad Life Science Research Products Catalog, 1997, pp. 175-182) , offers the capability of performing two-dimensional electrophoretic separations with programmable automated control of field orientation and pulse duration.
  • the electrophoretic medium can be reversibly cycled between at least two different user-defined states by varying one or more property of the electrophoretic medium (e.g., temperature, pH or ionic strength) .
  • one state the ligand has a relatively high affinity for the analyte of interest.
  • the ligand has relatively low binding affinity with the analyte.
  • non-analyte sample components have low affinity for the ligand in both states.
  • variation in medium state and the orientation of the electric field are co-regulated, so that electrophoresis of non-analyte materials occurs under all conditions and field orientations, but electrophoresis of analytes occurs only under a limited set of conditions and field orientations.
  • analytes and non-analyte molecules will have different net mobilities for each cycle, and their separation in the medium will increase with each cycle.
  • Changing the medium temperature is one preferred means for modulating analyte-ligand binding affinity, since temperature can be varied with little or no manipulation of the electrophoresis medium, and since a great deal of instrumentation for temperature control is commercially available.
  • other medium properties may be used as well.
  • a non-limiting list of possible properties which are known to affect noncovalent chemical associations include changes in medium pH, changes in the ionic strength of the medium, and other changes in chemical composition of medium.
  • the affinity ligand is an nucleic acid and the analyte is a sample nucleic acid that has at least one region complementary to the affinity ligand nucleic acid.
  • the binding between analyte and ligand can be effectively modulated by changing the gel temperature. For example, at temperatures above the Td of the ligand-analyte complex, binding affinity will be low. Similarly, at temperatures below the Td, binding affinity will be substantially higher.
  • thermocycler Processes and means for cycling electrophoretic media between two temperatures are well known to those skilled in the art.
  • temperature-controlled equipment for performing vertical or horizontal format electrophoresis are commercially available (Bio-Rad Life Science Research Products Catalog (1997), pp. 127-133, 175-182; Pharmacia Biotech BioDirectory (1997), pp. 345, 309, 334) .
  • temperature control is achieved by circulation of water (or suitable coolant) through the instrument.
  • temperature cycling can be achieved by the switching coolant source between two regulated reservoirs set at the desired temperatures.
  • the medium is in thermal contact with a programmable thermocycler which relies on the Petier effect for heating and cooling.
  • thermocyclers can be obtained from MJ Research, Watertown MA.
  • electrophoresis unit with a Peltier heating/cooling devise can be obtained from Pharmacia (Pharmacia Biotech BioDirectory (1997), pp. 334) .
  • Another method of modulating the analyte-ligand binding affinity is by changing the ionic strength of the electrophoresis buffer.
  • the ionic strength of the buffer that will facilitate binding is dependent on the type of analyte and the affinity ligand. In general, a buffer that has a higher ionic strength facilitates binding. Buffers that have ionic strengths of about 100 mM to 1 M are preferred during the state in which the analyte is bound to the affinity ligand. Buffers that have ionic strengths of about 10 mM or less are preferred during the state in which the analyte is not bound to the affinity ligand. Equilibrium dialysis or hybridization experiments can be used to provide a rational for predicting the stability of a particular analyte/affinity ligand binding complex at a particular ionic strength.
  • a denaturing buffer contains chemicals (hereinafter "denaturants" ) which disrupt the binding of the analyte to the affinity ligand.
  • denaturants chemicals which disrupt the binding of the analyte to the affinity ligand.
  • formamide or urea can be a component of the denaturing buffer.
  • the amount of denaturant required will depend on the type of target molecule, the type of affinity binding interaction, field strength, ionic strength, and temperature of electrophoresis.
  • the denaturing buffer can have a very broad concentration range of formamide or urea.
  • Formamide can be used in concentrations up to 95% (volume/volume)
  • urea can be used at concentrations up to 8M. Equilibrium dialysis or hybridization experiments can also be used to provide a rational for predicting the stability of the analyte/affinity ligand binding complex in a particular denaturing buffer.
  • the analyte-ligand binding affinity can also be modulated by changing the pH of the buffer.
  • nucleic acids will hybridize to a complementary nucleic acid affinity ligand at or near neutral pH (e.g., pH 6-8).
  • DNA affinity ligands hybridized to DNA targets can be disrupted by acidic pH (e.g., below pH 5) or basic pH (e.g., above pH 11) .
  • Changing the pH of the electrophoresis buffer is a preferred method of disrupting the binding of protein analytes to an affinity ligand. Equilibrium dialysis experiments can be used to estimate the pH range for binding and dissociation of a particular protein to an analyte.
  • switching medium conditions should switch the analyte between completely bound and completely unbound states.
  • This clean distinction between bound and unbound analyte states can be achieved with single-stranded nucleic acid analyte-ligand systems, as exemplified in later sections of this disclosure.
  • absolute two-state behavior is not required for successful application of the invention.
  • substantially means that the mobility change observed in a ligand-containing medium is greater than the mobility change observed for a similar medium lacking the ligand, or alternatively, a medium containing a ligand which is chemically similar to the original ligand but which is incapable of forming specific binding complexes with analyte.
  • a substantial change in analyte mobility even weak specific ligand/analyte interactions can be used successfully to practice the present invention, since an arbitrarily large number of purification cycles can be repeated in an automated fashion. In these cases, each cycle gives a small but finite separation between analyte and non-analyte sample components, and the purification cycle is repeated until the required level of separation is achieved.
  • the invention can be used to purify analytes for subsequent characterization and other preparative purposes, In addition, the invention can be used for detection of analytes.
  • the invention is powerful because it allows the potential for performing many repetitive cycles of affinity purification using a single automated programmable device with inexpensive, easy to prepare affinity media.
  • elution of purified product could be accomplished electrophoretically using a variation of methods disclosed in Gombocz et al .
  • Other elution methods are possible and are well known to those skilled in the art.
  • the invention is powerful because it allows removal of non-analyte sample components which can contribute to unfavorable levels of background.
  • the general format of these assays involves the following steps :
  • step 1 total RNA is prepared from the blood sample (Chomczynski, et al . , Anal . Biochem . (1987), 162:156; Chomczynski, Biotechniques (1993), 15:532; TRI Reagent BD, Sigma (1999), catalog no. T3809, p. 1545).
  • step 2 RNA is subjected to the method of the present invention using an affinity medium specific for binding bacterial ribosomal RNA.
  • step three the output of the purification process is tested for the presence of RNA using dyes which fluoresce brightly when bound to nucleic acids, for example, Acridine Orange, SYBR green II, TO-TO or YO-YO.
  • step 1 Detection by copurification of enzymatic label
  • total RNA is prepared from the blood sample, and the bacterial ribosomal RNA analyte within the sample is bound to an enzymatic reporter molecule, such as alkaline phosphatase, horseradish peroxidase, or luciferase by means which are not disrupted during the purification process of step 2.
  • an enzymatic reporter molecule such as alkaline phosphatase, horseradish peroxidase, or luciferase by means which are not disrupted during the purification process of step 2.
  • RNA analyte attachment of the RNA analyte to the enzyme reporter is mediated by a nucleic acid sandwich hybridization probe (Ranki and Soderland, U.S. Patent No. 4,486,539; Engelhardt and Rabbani , U.S. Patent No. 5,288,609, the teachings of which are incorporated herein by reference in their entirety) to which the enzyme is conjugated.
  • the assay of step 3 detects the enzymatic reporter molecule using chromogenic, chemifluorescent , or chemiluminescent substrates.
  • step 1 total RNA is prepared from the blood sample, and the bacterial ribosomal RNA analyte within the sample is bound to an amplifiable reporter molecule, such as a substrate of Q-beta replicase, for example the MDV-1 substrate of Q-beta replicase (Lizardi et al . Bio/Technology 6:1197-1202, 1988), by means which are not disrupted during the purification process of step 2.
  • an amplifiable reporter molecule such as a substrate of Q-beta replicase, for example the MDV-1 substrate of Q-beta replicase (Lizardi et al . Bio/Technology 6:1197-1202, 1988
  • attachment of the RNA analyte to the enzyme reporter is mediated by a nucleic acid sandwich hybridization probe, to which the amplifiable reporter is conjugated.
  • the assay of step 3 detects the amplifiable reporter molecule using an appropriate enzymatic
  • Example 1 Separation of nucleic acid samples
  • the ligand is an nucleic acid GTA CCA TAA CAG CAA GCC TCA (SEQ ID NO: 2) covalently immobilized in an standard polyacrylamide gel using the methods of U.S. patent applications Serial Nos. 08/812,105 and 08/971,845, the teachings of which are incorporated herein by reference in their entirety.
  • Figures 1A and IB show the gel after the end of steps 1 and 2 in the first cycle.
  • step 1 the gel was maintained at 45oC. At this temperature, binding of the ligand to complementary nucleic acids was prevented, and the electric field, applied for 43 minutes at 100 V, had the polarity indicated in Figure 1.
  • step two the gel was maintained at 25°C, a temperature which allows binding of the ligand to complementary nucleic acids. The electric field, applied for 50 min at 100 V, had the opposite polarity to that used in step 1.
  • the mobility of the two fluorescently labeled nucleic acids was similar, as shown in Figure 1A.
  • Figure IB a separation of the two nucleic acids was observed.
  • Figure 1C shows the same gel after a total of three cycles. Each cycle consisted of steps 1 and 2 (down/hot and up/cold) as described for Figures 1A and IB, above. After the third cycle, the separation between the complementary and noncomplementary nucleic acids was increased. In particular, the complementary nucleic acid moved further down the gel, while the noncomplementary nucleic acid moved in the reverse direction. GENERATION OF RNA ANALYTES USED IN EXAMPLES 2 AND 3
  • RNA analytes were used for the experiments shown in Figures 2 and 3. All three were generated by in vitro transcription reactions using T7 polymerase (enzyme from Boehringer Mannheim catalog #881,767; other reaction components from Promega kit catalog #P1420) with fluorescently tagged ribonucleotide triphosphates as label (fluorescein labeled nucleotides from Boehringer Mannheim catalog #1,685,61 9).
  • T7 polymerase enzyme from Boehringer Mannheim catalog #881,767; other reaction components from Promega kit catalog #P1420
  • fluorescently tagged ribonucleotide triphosphates as label
  • fluorescein labeled nucleotides from Boehringer Mannheim catalog #1,685,61 9
  • E. coli RNase P RNA 377 nucleotides in length, specific RNA analyte.
  • Plasmid pSCH038 was obtained from the American Type Culture Collection (ATCC87435) .
  • the plasmid is a PCRII plasmid vector (Invitrogen, Carlsbad, California, catalogue number K2050-01) containing the E. coli 16S ribosomal RNA (rRNA) sequence from positions 674 to 1411.
  • the complete 16S molecule is 1541 nucleotides in length.
  • the vector contains a promoter site for T7 R ⁇ A polymerase to the 5 ' side of insert so that in vitro transcription with that enzyme will produce R ⁇ A with the same sense as native 16S rR ⁇ A.
  • Two 5 mg aliquots of plasmid were digested with the restriction enzymes Hha I or Alu I .
  • the digested plasmid samples were used as templates for in vitro transcription with T7 polymerase and fluorescein-labeled nucleotides. After synthesis the DNA templates were removed by digestion with Dnase I, and the transcripts were purified from unincorporated nucleotides using Pharmacia G25 spin columns .
  • the first 68 nucleotides of both transcripts are derived from the cloning vector PCRII: there are 68 nucleotides between the T7 initiation site and the 16S insert. Alu I cleaves between nucleotides 860 and 861 of the 16S rRNA sequence. Therefore, the length of the transcript generated from the Alu 1 cleaved template, 16S Alu, is 255 nucleotides.
  • the length of the 16S Hha transcript is 502 nucleotides.
  • E. coli Rnase P RNA Naturally occurring E. coli Rnase P RNA is 377 nucleotides in length (Altman, et al . , In tRNA : Structure, Biosynthesis, and Function (1995), p. 67-78, editors Soil and RajBhandary, American Society of Microbiology, Washington, D.C.) .
  • a PCR fragment containing this sequence was generated from E. coli genomic DNA.
  • the amplification primer on the 5 ' side of the gene contained a T7 RNA polymerase promoter.
  • the resulting 396 bp DNA amplification product included a T7 promoter immediately 5' of the Rnase P RNA gene sequence.
  • This DNA was used as a template in in vitro transcription reactions using T7 polymerase to generate fluorescently-labeled Rnase P RNA, as described above.
  • the in vitro transcript produced from this template was identical in sequence to the published sequence of E. coli Rnase P RNA.
  • the ligand is an nucleic acid 5 ' -CCA TCG GCG GTT TGC TCT CTG TTG-3 ' (SEQ ID NO: 4) covalently immobilized in an standard polyacrylamide gel using the methods of U.S. patent applications Serial Nos. 08/812,105 and 08/971,845.
  • This ligand is complementary to a sequence contained within E. coli Rnase P RNA. The ligand was attached via its 5' terminus, and was present in the gel at a concentration of 10 mM (strands) .
  • the gel contained 5% polyacrylamide (29:1, monomer :bisacrylamide) , 45 mM Tris-Borate pH 8.3, and 2 mM EDTA (0.5XTBE buffer) .
  • the gel was run in a temperature-controlled minigel apparatus (Penguin vertical gel box, Owl Scientific, Woburn, MA) . Gel temperature was controlled by pumped water from external recirculating water baths. For all electrophoresis steps, the applied field was 200 volts.
  • the left lane was loaded with fluorescein-labeled E. coli Rnase P RNA.
  • the right lane was loaded with a mixture of all three fluorescein-labeled transcripts, Rnase P, 16S Hha, and 16S Alu.
  • the Figure 2A shows the pattern of fluorescent products after electrophoresis down through the gel for 10 minutes at 54°C, a gel temperature which should disrupt binding of the gel ligand to the specific analyte, Rnase P RNA.
  • the gel was imaged directly without removing the glass plates using a Molecular Dynamics Fluorimager.
  • the gel was replaced in the gel box and equilibrated at 41°C, a temperature which should allow hybridization of the nucleic acid ligand to the Rnase P RNA.
  • the gel ligand is not complementary to the 16S transcripts. At this temperature, the samples were electrophoresed upward for 20 minutes. The field was shut off and the gel temperature was changed to 59°C.
  • Lane 1 contained E. coli Rnase P RNA; lane 2, 16S Alu transcript; lane 3, 16S Hha and 16S Alu; lane 4, 16S Hha and Rnase P RNA; lane 5, 6.3 mg total unlabeled RNA from E. coli; lane 6, Rnase P RNA, 16S Hha, 16S Alu, and 6.3 mg total unlabeled RNA from E. coli.
  • the gel was equilibrated to 50°C, a temperature which should disrupt hybridization of ligand to the specific analyte, Rnase P RNA, and samples were electrophoresed down for 10 minutes.
  • An image of the gel after step 1 is seen in Figure 3A.
  • the gel was equilibrated at 41°C and electrophoresed up for 40 minutes. At this temperature, the Rnase P RNA can hybridize with the gel ligand.
  • An image of the gel after step 2, shown in Figure 3B demonstrates the complete removal of the 16S Hha and Alu transcripts from the gel.
  • the specific retention of Rnase P RNA analyte in the gel was not affected by an excess of heterogeneous unlabeled RNA in lane 6, further demonstrating the high specificity of analyte capture.

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Abstract

L'invention concerne un procédé d'électrophorèse d'affinité dans lequel on fait varier le sens de l'électrophorèse de manière cyclique tout en changeant de manière synchronisée une ou plusieurs propriétés du milieu électrophorétique entre deux états, lesdits états étant caractérisés comme favorisant ou défavorisant la liaison réversible spécifique de substances à analyser d'échantillons à des ligands d'affinité immobilisés dans le milieu. Le procédé obtenu permet une séparation extrêmement efficace et pratique des substances à analyser spécifiques à détecter ou à purifier, avec des matériels et un appareil simples.
PCT/US1999/004849 1998-03-03 1999-03-03 Procedes de purification et de detection par electrophorese d'affinite reversible WO1999045374A2 (fr)

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JP2000534862A JP2002506204A (ja) 1998-03-03 1999-03-03 可逆的アフィニティー電気泳動を用いる精製および検出プロセス
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US6586177B1 (en) 1999-09-08 2003-07-01 Exact Sciences Corporation Methods for disease detection
WO2005047881A3 (fr) * 2003-11-05 2005-09-22 Exact Sciences Corp Techniques de separation repetitive par affinite et leurs utilisations
US7452668B2 (en) 1997-05-16 2008-11-18 Exact Sciences Corporation Electrophoretic analysis of molecules using immobilized probes
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