Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-16
2002-02-19
Horlick, Kenneth R. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C530S300000, C530S350000
Reexamination Certificate
active
06348317
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
BACKGROUND OF THE INVENTION
DNA-binding proteins contain domains with small structural motifs designed to discriminate specific DNA sequences through an ensemble of non-covalent interactions. C.O. Pabo, et al.,
Ann. Rev. Biochem
. 61:1053-1095 (1992). Positioning of the &agr;-helix in the major groove of DNA is a common motif found in both non-sequence specific and sequence specific DNA binding proteins and yields a defined arrangement of non-covalent contacts between amino acid side chains of the protein and DNA bases and backbone structures. A. J. Doherty, et al.,
Nucl. Acids Res
. 24:2488-2497 (1996); A. Revzin, et al.,
The Biology of Non-Specific DNA-Protein Interactions
(1990); R. S. Spolar, et al.,
Science
362:777-784 (1994); D. P. Mack, et al.,
Biochemistry
29:6561-6567 (1990). Typically, in protein structural motifs such as the helix-turn-helix (“HTH”), R. G. Brennan, et al.,
J. Biol. Chem
. 264:1903-1906 (1989); E. R. P. Zuiderweg, et al.,
FEBS
174:243-247 (1984); E. R. P. Zuiderweg, et al.,
Proc. Natl. Acad. Sci. USA
80:5837-5841 (1983); S. C. Harrison, et al.,
Ann. Rev. Biochem
. 59:933-969 (1990), zinc-finger, A. Klug, et al.,
Trends Biochem. Sci
. 12:464-469 (1987); J.M. Berg,
Ann. Rev. Biophys. Biophysic. Chem
. 19:405-421 (1990); N. P. Pavletich, et al.,
Science
252:809-817 (1991); J. E. Coleman,
Ann. Rev. Biochem
. 61:897-946 (1992), or leucine zipper, E. K. O'Shea, et al.,
Science
254:539-544 (1991); E. K. O'Shea, et al.,
Science
243:538-542 (1989), the smallest DNA recognition element is an a-helix which binds to the DNA major groove. The remainder of the protein typically contains structural motifs, which orient this recognition element within the major groove, provide non-specific DNA-binding affinity, and enable additional interactions such as dimerization or ligand binding.
Single zinc fingers (“ZF”) have been studied for their ability to fold independently and bind native recognition sequences of the wild type protein. B. E. Bernstein, et al.,
Biochemistry
33:4460-4470 (1994); A. D. Frankel et al.,
Proc. Natl. Acad. Sci. USA
84:4841-4845 (1987); B. A. Krizek, et al.,
J. Am. Chem. Soc
. 113:4518-4523 (1991); M. S. Lee, et al.,
Science
245:635-637 (1989). Most proteins that recognize particular DNA sequences via zinc finger motifs have multiple zinc fingers in the DNA-binding domain (“DBD”). J. Miller, et al.,
EMBO J
. 4:1609-1614 (1985). The contribution to sequence recognition of each zinc finger within the DNA binding domain of a protein can vary significantly. D. J. Whyatt, et al.,
EMBO J
. 12:4993-5005 (1987). For example, in the glucocorticoid and estrogen receptor DNA-binding domains, the N-terminal finger contains all the interactions for sequence specificity, whereas the C-terminal zinc finger further stabilizes DNA binding by interacting non-specifically. Finger swapping studies, in which the N- and C-terminal zinc fingers of the estrogen receptor (“ER”) and glucocorticoid receptor (“GR”) where switched, clearly show that sequence recognition was a function of the N-terminal zinc finger. S. Green, et al.,
Nature
325:75-78 (1987).
These studies have made it possible to produce short (less than 50 amino acid) single zinc finger peptides that bind to specific DNA sequences with moderately high affinity. T. K. Archer, et al.,
Proc. Natl. Acad. Sci. USA
87:7560-7564 (1990). Thus, it is possible to synthetically generate peptides that bind under normal physiological conditions to specific DNA sequences, N. Y. Sardesai, et al.,
J. Biol. Inorg. Chem
. 2:762-771 (1997); M. G. Oakley, et al.,
Biocon. Chem
. 5:242-247 (1994); J. A. Shin, et al.,
Nucl. Acids Res
. 19:5233-5236 (1991); K. S. Graham, et al.,
J. Biol. Chem
. 265:16534-16540 (1990); M. G. Oakley, et al.,
Science
248:847-850 (1990); J. P. Sluka, et al.,
Science
238:1129-1132 (1987), opening the door for the production of a variety of different types of chemical reagents, which probe or modify DNA at specific sequences.
SUMMARY OF THE INVENTION
In one embodiment, the present invention is a method of identifying the presence or absence of a DNA molecule that contains a specific DNA sequence. The method comprises the steps of mixing a test sample with a peptide/dye conjugate comprising a covalently linked peptide and a cyanine dye wherein this peptide binds to a specific DNA sequence and wherein the peptide/dye conjugate will significantly fluoresce if the peptide is bound to a specific DNA sequence and measuring fluorescence, wherein specific fluorescence of above background level indicates that the conjugate is bound to the specific DNA sequence.
In a preferred form of the present invention, the dye is an intercalating dye and selected from the group of cyanine dyes.
In a particularly advantageous form of the invention, the dye is chosen from thiazole orange and oxazole yellow.
In another embodiment, the present invention is a method of cleaving a DNA molecule, wherein the DNA molecule comprises a specific DNA sequence. The method comprises the steps of mixing a test sample with a peptide/dye conjugate comprising a covalently linked peptide and a dye, wherein the peptide/dye is bound to the specific DNA sequence and wherein the peptide/dye conjugate will cleave the DNA if bound and if one applies a triggering event. One then applies the trigger event and the specific DNA sequence is cleaved.
In a preferred form of this embodiment, the triggering event is the administration if light.
In another embodiment, the present invention is a composition comprising a dye and a peptide, wherein the peptide binds to a specific DNA sequence and the dye is capable of fluorescence only when the conjugate is bound to a specific DNA sequence.
In another embodiment, the present invention is a composition comprising a covalently linked dye and peptide, wherein the peptide binds to a specific DNA sequence and the dye is capable of cleavage only when the conjugate is bound to a specific DNA sequence and only in the presence of a triggering event.
It is an object of the present invention to detect specific DNA sequences.
It is another object of the present invention to cleave specific DNA sequences.
It is another object of the present invention to provide a peptide/dye conjugate.
Other objects, advantages and features of the present invention will become apparent after one has evaluated the specification claims, and drawings.
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patent: 5534424 (1996-07-01), Uhlen et al.
patent: 5593867 (1997-01-01), Walker et al.
patent: 5656426 (1997-08-01), Law et al.
patent: 5830665 (1998-11-01), Shuber et al.
B.M. Sutherland, et al., “Promoter-Specific Synthetic Photoendonuclease; Rose Bengal-Labeled T7 RNA Polymerase,” Biochemistry 32:1788-1794, 1993.
M. Thompson and N.W. Woodbury, “Fluorescent and Photochemical Properties of a Single Zinc Finger Crosslinked with a Fluorescent DNA-binding Probe,” ACS Western Regional Meeting, Oct. 6-8, 1999.
M. Thompson and N.W. Woodbury, “Fluorescent and Photochemical Properties of a Single Zinc Finger Conjugated to a Fluorescent DNA-Binding Probe,”Biochemistry39(15):4327-4338, 2000.
Thompson Martin
Woodbury Neal W.
Horlick Kenneth R.
Quarles & Brady LLP
The Arizona Board of Regents
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