Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-31
2001-09-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C536S022100, C536S024300, C536S024310, C536S024330, C536S025320
Reexamination Certificate
active
06294333
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to biopolymer binding assays, and more particularly to methods for assaying binding between nucleic acids and peptides or proteins using fluorescent intensity data.
BACKGROUND OF THE INVENTION
Protein-nucleic acid complexes are known to play an important role in a variety of biological processes. See, e.g., Hill et al. “Fluorescence Approaches to Study of Protein-Nucleic Acid Complexation,” 278 Methods in Enzymology 390 (1997). For example, DNA-binding proteins are known to play an important role in gene regulation. Genes are typically regulated at the transcriptional level by DNA-binding proteins, which are referred to as transcription factors. Transcription factors regulate gene expression by specifically binding to a target nucleic acid sequence in promoter DNA.
Due to the biological importance of protein-nucleic acid interaction, a variety of methods for studying protein-nucleic acid binding characteristics have been proposed. See, e.g., Hill et al. and the references cited therein.
U.S. Pat. No. 5,783,384 to Verdine discloses methods for determining the affinity of a DNA-binding protein for a target nucleic acid sequence. Verdine teaches methods comprising providing a reversible bond between a DNA-binding protein and a target nucleic acid sequence, and determining the relative strength of the reversible bond (and thus the affinity of the protein for the nucleic acid) by breaking it under supervised conditions. The more stringent the conditions necessary to break the bond, the higher the affinity of the protein for the nucleic acid. Verdine does not disclose or suggest fluorescence-based binding assays.
U.S. Pat. No. 5,445,935 to Royer discloses fluorescence-based methods for studying protein-oligonucleotide binding; however, the teachings of the patent are solely limited to fluorescent anisotropy techniques. Basically, anisotropy measures rotational diffusion events of free DNA or protein-bound DNA, as well as the local motions of a fluorophore attached to the DNA via a linker arm. Free DNA rotates quickly, depolarizes the light more readily and exhibits a low anisotropy value. In contrast, protein-bound DNA rotates slowly relative to the lifetime of the fluorophore, depolarizing the light only slightly and thus exhibiting a relatively high anisotropy value.
However, there are significant drawbacks to anisotropy-based assays. The degree of change in anisotropy as a function of binding is not as predictable as the proponents of anisotropy-based methods assert. Interpretation of anisotropy data to conform inconsistent data to theoretical expectations can require more effort than is desirable in an analytical method, particularly when the method is to be automated.
Radioactive labeling remains the most popular method for analyzing protein-nucleic acid interaction, despite being relatively slow, a health and environmental hazard, and relatively labor-intensive. Conventional radioactive labeling methods typically require radioactively end-labeling DNA probes with
32
P using specialized enzymes. Purification of labeled DNA from unincorporated
32
P involves polyacrylamide gel electrophoresis, overnight elution, gel filtration and concentration steps. Since the half-life of
32
P is only 14 days, radio-labeling is required approximately every three weeks for each probe. Moreover, protein-
32
P-DNA complexes need to be separated from unbound
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P-DNA by native polyacrylamide gel electrophoresis. Gels are then dried and analyzed by autoradiography or phosphoimaging.
Thus, a need has existed in the art for a simple, effective and rapid method for analyzing peptide-nucleic acid and protein-nucleic acid interaction.
All references cited herein, including U.S. Pat. No. 5,846,729 and U.S. patent applications Ser. Nos. 08/807,901 and 08/870,370 (respectively filed Feb. 27, 1997 and Jun. 6, 1997), are incorporated herein by reference in their entireties.
SUMMARY OF THE INVENTION
The invention provides a method for assaying binding between at least one fluorophore-labeled compound and at least one unlabeled compound. The method comprises detecting a quenching effect on fluorescence emitted by said at least one fluorophore-labeled compound resulting from said binding, wherein said binding is specific and other than nucleobase to nucleobase. The method is preferably conducted without separating complexes of said at least one fluorophore-labeled compound and said at least one unlabeled compound from said at least one fluorophore-labeled compound prior to said quenching effect detecting, aid without providing a signal quenching agent to quench said emitted fluorescence.
REFERENCES:
patent: 4743535 (1988-05-01), Carrico
patent: 4761382 (1988-08-01), Woodhead et al.
patent: 5187106 (1993-02-01), Fritzsche et al.
patent: 5445935 (1995-08-01), Royer
patent: 5747247 (1998-05-01), Kowalczykowski et al.
patent: 5756292 (1998-05-01), Royer
patent: 5783384 (1998-07-01), Verdine
patent: 5846729 (1998-12-01), Wu et al.
patent: 94/14980 (1994-07-01), None
patent: 98/04923 (1998-02-01), None
patent: 98/26093 (1998-06-01), None
Kadonaga et al., “Isolation of cDNA Encoding Transcription Factor Sp1 and Functional Analysis of the DNA Binding Domain,” Cell, 51:1079-1090, Dec. 24, 1987.
Bohmann et al., “Human Proto-Oncongen c-jun Encodes a DNA Binding Protein with Structural and Functional Properties of Transcription Factor AP-1,” Science, 238:1386-1392, Dec. 1987.
Sturm et al., “The Ubiquitous octamer-binding protein Oct.-1 contains a POU domain with a homeo box subdomain,” Genes & Development, 2:1582-1599, 1988.
Dalrymple et al., “DNA sequence of the herpes simplex virus type 1 gene whose product is responsible for transciptional activation of immediate early promoters,” Nucleic Acids Research, vol. 13, No. 21, pp. 7865-7879, 1985.
Wilson et al., “The VP16 Accessory Protein HCF Is a Family of Polypeptides Processed from a Large Precursor Protein,” Cell, 74:115-125, Jul. 16, 1993.
Brown et al., “Fluorescence spectroscopy as a tool to investigate protein interactions,” Current Opinion in Biotechnology, 1997, 8:45-49.
Hill et al., “Fluorescence Approaches to Study of Protein-Nucleic Acid Complexation,” Methods in Enzymology, 278:390-416, 1997.
Daksis Jasmine I.
Erikson Glen H.
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Horlick Kenneth R.
Ingeneus Corp.
Siew Jeffrey
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