Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-18
2001-12-04
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C530S350000, C536S026600
Reexamination Certificate
active
06326142
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the use of measurement of the polarization of the fluorescence emission from a labelled macromolecule in order to assess the binding of the labelled macromolecule to a second macromolecule. Thus, the invention is directed to analytic methods for determining such binding in a quantitative fashion. The invention is also directed to apparatus for conducting binding analyses using measurement of fluorescence polarization.
2. Description of the Related Art
The use of labeled oligonucleotides as probes in macromolecular analysis is 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 bag 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 labelled with radioisotope or other detectable moiety is electrophoresed in a gel matrix, commonly made of agarose or acrylamide, under non-denaturing conditions. The labelled 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 less 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.
The present invention relates to a method for detecting and quantitating complexation between nucleic acids and proteins or other macromolecules which comprises the measurement of the polarization of fluorescence of an extrinsic fluorophore covalently coupled to an oligonucleotide. The oligonucleotide can be an oligodeoxyribonucleotide, an oligonribonucleotide or a co-polymer of both. The nucleotide bases can be derivatized, as can the backbone chain. The oligonucleotide can be single-, double- or triple-stranded. The length of the oligonucleotide is determined by the specific experiment being conducted, but is preferably less than 40 residues long, more preferably being less than 25 residues long and most preferably 8-10 residues long.
The fluorophore is incorporated into the oligonucleotide at any position using standard automated DNA synthesis techniques and fluorescently labeled amino-linker compounds (e.g. those available from Clontech, Inc., La Jolla, Calif.). Alternately, unlabeled amino-linker compounds can be incorporated and subsequently labeled with the fluorescent compound. A variety of fluorophores may be used, including fluorescein, eosin, coumarin and dansyl derivatives. The fast rotation about the short axis of the oligonucleotide results in a low value of the fluorescence polarization of the probe covalently coupled to the oligonucleotide. This value is obtained by exciting the fluorescently labeled oligonucleotide with the appropriate wavelength of plane polarized exciting light and monitoring the emission of light polarized in the planes parallel and perpendicular to the plane of polarization of the exciting light. The fluorescence polarization is calculated as:
p=(I
∥
−I
L
)/(I
∥
+I
L
)
where I
∥
is the intensity of the emitted light polarized in a plane parallel to the plane of polarization of the exciting light and I
L
is the intensity of the emitted light polarized in a plane perpendicular to the plane of polarization of the exciting light. The anisotropy of the emission is
2/3[1/(1/p−1/3)]
This value is analogous to the polarization yet linear with respect to the total fluorescence emission intensity.
Fluorescence polarization is the basis for a series of patented assays for the clinical detection of drugs, steroids and other antigens (U.S. Pat. Nos. 4,269,511; 4,516,856; 4,585,362; 4,668,640; 4,784,961 4,902,630; 4,952,691; 5,066,426; European patent application 86102035.2). Although there are a number of variations, change in the fluorescence polarization of fluorescein upon changing its interaction with specific or non-specific antibodies when the drug of interest is present in the assay solution. If the presence of the drug results in the dissociation of the fluorescein-antibody complex, for example, then the fluorescence polarization will exhibit a large decrease. The sensitivity of these assays is less than 10 pM. Urios and Cittanova (
15
) describe the use of fluorescently labeled Fab fragments of antibodies to perform fluorescence polarization measurements. The sensitivity of their assay is 2.5 &mgr;M. The use of a fluorescent-labeled oligonucleotide probe was reported by Murakami et al (
17
). Their reported sensitivity was 100 nanomolar. Giedroc et al. (
16
) used fluorescence anisotropy in evaluating the presence of single stranded DNA molecules. Their assays were conducted at oligonucleotide concentrations of 2 to 8 &mgr;M.
SUMMARY OF THE INVENTION
One object of the present invention is to provide a method by which the binding of two macromolecules to each other can be detected in the solution phase by measuring the polarization of the fluorescent emission from a probe moiety. Preferably, the probe is attached to one macromolecule and binding is determined directly by an increase in the fluorescence polarization. A second object of the present invention is to provide an apparatus suitable for performing fluorescence polarization measurements with very high sensitivity so as to be able to perform such measurements quantitatively on very small samples. In preferred embodiments of the apparatus, detection of the polarization of the fluorescent emission of as little as one femtomole of fluorophore can be detected.
A third object of the present invention is to provide diagnostic methods which are based upon measuring the binding of a polynucleotide to a second polynucleotide, or alternatively to a protein, by means of quantitation of the polarization of the emission from a fluorophore covalently attached to either said polynucleotide.
REFERENCES:
Matthews, Analytical Biochemistry 19, 1-25 (1988).*
Heyduk et al., Proc Natl Acad Sci, 87:1744-48, Mar. 1990.
Houtteman Scott W.
Marks, Esq. Andrew S.
PanVera Corporated
Walker, Esq. Shelby J.
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