Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-04-06
2002-05-14
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120
Reexamination Certificate
active
06387703
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of screening for compounds that modulate gene expression, particularly those which lower gene expression, and compositions useful in such methods.
BACKGROUND OF THE INVENTION
Methods are known which lower or abolish gene expression. For example, gene knockouts may be performed to abolish gene expression. Conditional lethal mutants may also be created to abolish gene expression and identify essential genes (see, for example, de Lorenzo, V. et al.,
Gene
123:17-24 (1993); Neuwald, A. F. et al.,
Gene
125: 69-73(1993); and Takiff, H. E. et al.,
J. Bacteriol
. 174:1544-1553(1992). Chemical mutagenesis is yet another way to make such mutants Beckwith,
J., Methods in Enzymology
204: 3-18(1991). Ribozymes provide another way to lower gene expression levels by damaging the gene or transcript. It has also been reported that the hammerhead RZ with abasic residues can be activated by addition of the base in trans. A drawback to these methods of preparing gene knockouts or lowering gene expression is that they are often time consuming and difficult to reproduce.
A SELEX generated RNA aptamer that binds malachite green has been reported. This complex, when illuminated at 630 nm, results in cleavage of the RNA by free radical generation as shown by abolishing marker gene activity.
The present invention provides improved methods for modulating, lowering and/or abolishing gene expression in a sequence-directed manner.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
SUMMARY OF THE INVENTION
The invention provides a method for modulating gene expression comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting an organism with the binding site and binding agent; and detecting modulation of gene expression.
The invention also provides a preferred method, wherein after the contacting step the binding site and binding agent enter the cell and contact an RNA target or DNA target.
A further preferred method comprises the step of having the binding site base pair with an RNA target molecule.
The invention also provides a method wherein the modulation of gene expression is lowering or inhibiting gene expression.
Moreover, the invention provides a method wherein the binding site is selected from the group consisting of a site that binds: malachite green, a photoreactive dye, a free radical generating compound, an iron salt, an iron ion, a radioactive iron radionuclide, isofuran blue, an isosulfan blue derivative, reactive sulfonyl chloride of isosulfan blue, hypocrellins and hypericin, tetrabromorhodamine, Rose Bengal photooxidizing dye, a FotoFenton reagent, 2-mercaptopyridine N-oxide, phenanthrolines, a spontaneous nitric oxide donors, DEANO, spermine NONOate, S-nitrosoglutathione, SNAP, SIN-1, M-7904, 2-hydroxyacetophenone oxime and Fenton's reagent.
Further, the invention provides a method wherein the binding site is selected from the group consisting of a site that: binds a metal ion, creates a coordination complex with a metal ion or other atom or molecule, and is an iron response element.
Also provided is a method wherein the polynucleotide binding agent is selected from the group consisting of: malachite green, a photoreactive dye, a free radical generating compound, an iron salt, an iron ion, a radioactive iron radionuclide, isofuran blue, an isosulfan blue derivative, reactive sulfonyl chloride of isosulfan blue, hypocrellins and hypericin, tetrabromorhodamine, Rose Bengal photooxidizing dye, a FotoFenton reagent, 2-mercaptopyridine N-oxide, phenanthrolines, a spontaneous nitric oxide donors, DEANO, spermine NONOate, S-nitrosoglutathione, SNAP, SIN-1, M-7904, 2-hydroxyacetophenone oxime and Fenton's reagent.
A method is also provided for altering the structure of a polynucleotide comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting a polynucleotide with the binding site and binding agent; and detecting an alteration in the structure of the polynucleotide.
A method is provided wherein the alteration in the structure is a cleavage of the polynucleotide phosphate backbone.
Another method is provided for modulating the function of a polynucleotide comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting a polynucleotide with the binding site and binding agent; and detecting modulation of the function.
The invention provides a method for modulating gene expression comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting an organism with the binding site and binding agent; irradiating the binding agent with electromagnetic radiation; and detecting modulation of gene expression.
A preferred method is also provided wherein the contacting step also comprises contacting the binding site agent with a metal ion, metal salt or metal oxide.
Another preferred method is provided wherein the polynucleotide binding agent is malachite green and the electromagnetic radiation has a wavelength of about 630 nanometers (herein “nm”).
A kit is provide by the invention comprising at least on compartment containing an isolated polynucleotide binding agent binding site and a polynucleotide binding agent.
A preferred embodiment of the invention is a kit wherein the polynucleotide binding agent is selected from the group consisting of: malachite green, a photoreactive dye, a free radical generating compound, an iron salt, an iron ion, a radioactive iron radionuclide, isofuran blue, an isosulfan blue derivative, reactive sulfonyl chloride of isosulfan blue, hypocrellins and hypericin, tetrabromorhodamine, Rose Bengal photooxidizing dye, a FotoFenton reagent, 2-mercaptopyridine N-oxide, phenanthrolines, a spontaneous nitric oxide donors, DEANO, spermine NONOate, S-nitrosoglutathione, SNAP, SIN-1, M-7904, 2-hydroxyacetophenone oxime and Fenton's reagent.
Another preferred embodiment of the invention is a kit wherein the polynucleotide binding agent is malachite green.
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
“Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence and includes, but is not limited to organisms as defined elsewhere herein.
“Flanking sequence” is a polynucleotide sequence on either side (5′ or 3′) of and fused to the PBA binding site that is complementary, in whole or part to a polynucleotide sequence in the target polynucleotide.
“Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
“Organism(s)” means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chiamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Stre
Fedon Jason C.
Gimmi Edward R.
Kinzig Charles M.
Loeb Bronwen M.
SmithKline Beecham Corporation
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