Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-31
2001-02-06
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C436S501000
Reexamination Certificate
active
06183956
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application is related to the patent application entitled “High throughput assays for detection of mRNA in cells,” Ser. No. 09/052,841 filed Mar. 31, 1998, herein incorporated by reference.
FIELD OF THE INVENTION
The field of the invention relates to high-throughput in vitro assays for identifying modulators of transcription activity and RNA expression in solid phase assays. New assays, related compositions, apparatus and integrated systems are provided.
BACKGROUND OF THE INVENTION
Gene regulatory processes are fundamental in most, if not all, forms of disease, as well as in all of developmental biology. Accordingly, a primary goal of modern medicine is to understand gene regulation and to identify specific modulators of gene expression. These modulators serve as antineoplastic agents, antiviral agents, antifungal agents, and the like, for the treatment of a very wide variety of diseases.
Assays for monitoring gene expression are well known, including northern blotting, RT-PCR, RNase and S1 protection, reporter gene expression (e.g., chloramphenicol transferase (CAT) assays), etc. General texts that describe assays for monitoring gene expression include Sambrook et al.,
Molecular Cloning—A Laboratory Manual
(2nd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (“Sambrook”) and
Current Protocols in Molecular Biology,
F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1997) (“Ausubel”). Specialized apparatuses for performing these assays are commercially available.
In addition to these standard methods typically used to monitor gene expression, specialized formats for detection of nucleic acids are also available. For example, Coutlee et al. (1989)
Analytical Biochemistry
181:153-162 describe non-isotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody which binds DNA:RNA hybrids. In these assays, hybridization of an RNA target with a biotinylated DNA probe is performed, followed by incubation of the hybridized target-probe duplex on an anti-biotin plate, reaction of the resulting bound duplex with a &bgr;-galactosidase labeled monoclonal antibody specific for RNA-DNA hybrids, and addition of a fluorescent substrate. In another example, a “sandwich” hybridization method is described for non-isotopic detection of RNA using oligonucleotides (Ishii & Ghosh (1993)
Bioconjugate Chem.
4:34-41). In these assays, the RNA target is hybridized to a first complementary oligonucleotide, which is linked to a bead. The RNA target is then hybridized to a second complementary oligonucleotide conjugated to alkaline phosphatase. The RNA target is detected by providing a chemiluminescent alkaline phosphatase substrate.
Other investigators have also reported immunological detection of DNA:RNA hybrids, including Bogulavski et al. (1986)
J. Immunol. Methods
89:123-130; Prooijen-Knegt (1982)
Exp. Cell Res.
141:397-407; Rudkin (1976)
Nature
265:472-473, and Stollar (1970) PNAS 65:993-1000. Similarly, detection of DNA:DNA hybrids and RNA:RNA hybrids has also been described. See, Ballard (1982)
Mol. Immunol.
19:793-799; Pisetsky and Caster (1982)
Mol. Immunol.
19:645-650, and Stollar (1970)
PNAS
65:993-1000. Stollar (1970) and Rudkin (1976) (both supra) showed that DNA:RNA hybrids in solution can be captured on plastic or nitrocellulose supports coated with an anti-poly(A)-poly(dT) polyclonal antibody. A monoclonal antibody against DNA:RNA heteroduplexes and RNA:RNA hybrids, which does not recognize DNA duplexes, single-stranded DNA or single-stranded RNA, has been prepared and characterized. See, e.g., Bogulavski et al. (1986)
J. Immunol. Methods
89:123-130; Viscidi et al. (1988)
J. Clin. Microbiol.
41:199-209, and Kiney et al. (1989)
J. Clin. Microbiol.
27:6-12. This antibody was used for the detection of DNA:RNA hybrids immobilized by binding of the DNA component of the duplex to a nylon bead, or to avidin latex. Immunoassays for detecting nucleic acids have been adapted to in vitro qualitative detection of human papillomavirus (HPV), e.g., for use in detecting cervical abnormalities. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).
One strategy for identifying pharmaceutical lead compounds is to develop an assay that provides appropriate conditions for monitoring the activity of a therapeutic target for a particular disease. This assay is then used to screen large numbers of potential modulators of the therapeutic target in the assay. For example, libraries of chemical compounds can be screened in solid phase assays using robotic components. Although the immunoassay-based and sandwich-based nucleic acid detection strategies described above have been useful for detecting DNAs, RNAs, and DNA:RNA hybrids, they have not been adapted to high throughput gene expression monitoring assays that could be used for screening for pharmaceutical lead compounds. Such high throughput assays for expression monitoring and pharmaceutical compound screening are desirable. This invention provides these assays, as well as other features which will become apparent upon review.
SUMMARY OF THE INVENTION
High throughput in vitro assays for detecting modulators of RNA expression are provided. Inhibitors and activators of RNA expression can be screened using such assays, as can modulators that alter transcriptional activation. Solid phase high throughput assays are provided, as are related assay compositions, integrated systems for assay screening, and other features that will be evident upon review.
In one aspect, high throughput in vitro RNA detection assays are provided. The selected RNA in the sample is contacted with oligonucleotides that are complementary to the selected RNA. Single-stranded RNA in the sample is cleaved, typically with RNases such as RNase A. The sample is then incubated with a recognition agent that binds to RNA duplexes. The recognition agent is detected either directly or indirectly, indicating the level of expression of the selected RNA.
In another aspect, high throughput in vitro expression assays are provided. In such assays, the transcription mixtures contain an expression cassette with a promoter operably linked to a DNA encoding a selected G-less or A-less RNA. The single-stranded RNA in the sample is cleaved at guanine residues, typically by RNase T1, or at adenine residues, typically by RNase U2. The sample is then incubated with a recognition reagent that captures the RNA. The selected RNA is then detected either directly or indirectly, indicating the level of expression of the selected RNA.
In one embodiment, the selected RNA is transcribed from an expression cassette with a promoter operably linked to a DNA encoding a selected RNA. In one embodiment, expression of the selected RNA is induced by providing a transcriptional activating molecule that induces transcription of the DNA encoding the selected RNA. In another embodiment, the sample is treated with a compound suspected of having the ability to modulate expression or transcription activation of the selected RNA. In another embodiment, the recognition reagent is an antibody that binds to DNA:RNA duplexes. In another embodiment, a second recognition reagent is used to detect the selected RNA.
Kits, compositions, and integrated systems for performing the assays are also provided.
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Schiffman, “HPV Testing Helps Reduce Fasle-Positive Pap Smear Interpretations”,CB. Gyn. News, p. 10 (Jul. 1993).
Clinical Lab Products, p. 30 (Oct. 1992).
Burton W. Blais, “Transcriptional Enhancement of theListeria monocytogenesPCR and Simple Immunoenzymatic Assay o
Flores Osvaldo A.
Sivaraja Mohanram
Strulovici Berta
Marschel Ardin H.
Townsend and Townsend / and Crew LLP
Tularik, Incorporated
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