Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-20
2003-01-28
Romeo, David S. (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S069100, C435S325000, C435S320100, C530S350000
Reexamination Certificate
active
06511815
ABSTRACT:
FEDERALLY SPONSORED RESEARCH
N.A.
FIELD
The field of the invention relates to the detection of molecules which bind to steroid hormone receptors by measuring the fluorescence polarization emission from a molecule. In particular, fluorescence-emitting compounds are used in competitive assays to measure binding of molecules to steroid hormone receptors such as estrogen receptor.
BACKGROUND OF THE INVENTION
The use of labeled oligonucleotides as probes in molecular analysis has been an important technique in molecular biology. Oligonucleotides have been labeled with radioisotopes, enzymes or fluorescent molecules. Because of the relatively low molecular weights of oligonucleotides, and the common availability of instrumentation for their automated synthesis, oligonucleotides are often used in blot-hybridization procedures or in gel-retardation assays for the detection and qualitative evaluation of macromolecules with which they may associate. These macromolecules may be either proteins, RNA molecules or DNA molecules.
In a standard blot-hybridization procedure, the target macromolecule is separated by electrophoresis in a gel matrix, commonly agarose or polyacrylamide. It is then transferred to a membrane in such a way as to preserve its relative spatial positioning within the gel matrix and fix it stably to the membrane. Alternatively, the macromolecule may be attached to the membrane without prior electrophoresis. The presence of the macromolecule on the membrane is determined by binding to it a labeled oligonucleotide and subjecting the complex to autoradiography or, if the oligonucleotide is labeled with radioisotope, by scintillation counting.
In a standard gel retardation assay an oligonucleotide that has been labeled with radioisotope or other detectable moiety is electrophoresed in a gel matrix, commonly made of agarose or acrylamide, under non-denaturing conditions. The labeled oligonucleotide is also associated with a protein that may bind to the oligonucleotide and the mixture is electrophoresed on a gel, commonly in a neighboring lane, for comparing with the unassociated oligonucleotide. Because of its higher molecular weight and lower net negative charge, the protein will exhibit lower mobility in the gel than the unassociated oligonucleotide. If the oligonucleotide forms a stable complex with the protein, it will also exhibit a lower mobility than that of the unassociated oligonucleotide. Comparison of the mobility of the oligonucleotide mobility in the presence and absence of the protein allows qualitative determination of whether a complex forms between the two macromolecules. These basic methods are used for a very large variety of determinations in basic genetic research, genetic engineering, the medical sciences, and agricultural testing.
Additionally, fluorescence-labeled compounds other than oligonucleotides are used in assays to measure binding of molecules to biological substances. For example, the observation and measurement of hormone binding to hormone receptors is a technique currently in demand. Typically, a steroid hormone such as estradiol binding to a hormone receptor such as estrogen receptor is performed using radioactive hormone and receptor protein. The labeled hormone is added in excess to the receptor in a buffer and allowed to reach equilibrium. The bound hormone is then separated from the free hormone and quantitated. The binding affinity between the hormone and the receptor can be mathematically determined.
Furthermore, an assay has been described in a publication that does not require a separation step. It is based on a modified fluorescent hormone (Hwang et. al. Biochemistry 31:11536-45, 1992). The assay uses the observation that when the hormone and receptor bind, the fluorescence intensity of the hormone decreases proportional to binding. Free hormone has a high fluorescence intensity and the bound hormone has a low fluorescence intensity. However, several components of the binding mixture can affect the intensity other than the receptor. The polarity of the solvent and non-specific binding molecules can have significant affects on the intensity, which can be incorrectly interpreted as hormone/receptor binding.
Another detection method described in the early 1900's utilizes fluorescence polarization. Fluorescence polarization assay techniques are based on the principle that a fluorescently labeled compound will emit fluorescence when excited by plane polarized light, having a degree of polarization inversely related to its rate of rotation. If the labeled molecule remains stationary throughout the excited state it will emit light in the same polarized plane; if it rotates while excited, the light emitted is in a different plane.
For example, when a large labeled molecule is excited by plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained (by its size) from rotating between light absorption and fluorescent light emission. When a smaller molecule is excited by plane polarized light, its rotation is much faster than the large molecule and the emitted light is more depolarized. The emitted light has a degree of polarization that is inversely proportional to the molecular rotation. Therefore, small molecules have low polarization values and large molecules have high polarization values.
Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation or electrophoresis. Assays can be performed in real time, directly in solution and do not require an immobilized phase. For example, fluorescence polarization has been used to measure enzymatic cleavage of large fluorescein labeled polymers by proteases, DNases and RNases.
SUMMARY
The present invention is an easy method for detecting and quantitating a complex between molecules, such as hormones and nucleic acids interacting with steroid hormone receptors. The invention utilizes the measurement of fluorescence polarization of an intrinsic fluorescence-emitting compound such as a fluorescence-emitting hormone as well as emission from a fluorophore covalently coupled to an oligonucleotide. The fluorescence-emitting compound and the fluorescent oligonucleotide produce a low polarization quantitation when not bound to a complementary molecule and a high polarization quantitation when bound.
A method is provided for measuring competitive binding activity of molecules to steroid hormone receptors, comprising: mixing a fluorescence-emitting compound that binds to the steroid hormone receptors in a solution containing the steroid hormone receptors. Measuring the fluorescence polarization of the solution. Then, incubating the solution with at least one molecule that may compete with the compound for interaction with the steroid hormone receptors. Subsequently, measuring the fluorescence polarization of the solution. Finally, comparing the fluorescence polarization measurements to quantify any competitive interaction.
A method is provided for measuring binding activity of molecules to steroid hormone receptors, comprising: mixing a fluorescence-emitting compound that binds to the steroid hormone receptors at a first domain and a fluorescence-labeled nucleic acid that binds to the steroid hormone receptors at a second domain in a solution containing the steroid hormone receptors. Measuring the fluorescence polarization of each fluorescence emission from the solution. Then, incubating the solution with at least one molecule that may compete for interaction with at least one domain. Subsequently, measuring the fluorescence polarization of each fluorescence emission of the solution. Finally, comparing the fluorescence polarization measurements to quantify any interaction.
Also, a kit is provided utilizing the methods listed above for identifying natural and non-natural molecules which bind to human steroid hormone receptors, for use in treating related diseases. The kit comprises instructions for utilizing fluorescence polarization to identify the molecules;
Bolger Randall E.
Burke Thomas J.
Checovich William J.
Ervin Kerry M.
Lowery Robert G.
Marks Andrew S.
Murphy Joseph F.
PanVera LLC
Romeo David S.
Vertex Pharmaceuticals Incorporated
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