WO1999050460A1 - HIGH THROUGHPUT ASSAY FOR DETECTION OF mRNA IN CELLS - Google Patents

Abstract

High throughput assays for detecting RNA in cells and modulators of RNA transcription and expression are provided. In the methods, a cell having the selected RNA is contacted with an oligonucleotide comprising a region complementary to the selected RNA, thereby forming an RNA duplex in the cell, single-stranded RNA is cleaved in the cell, and, the RNA duplex is contacted with a recognition reagent, thereby detecting the selected RNA. Related assays for measuring effects of potential modulators following incubation of the modulators with the cell are provided. Kits, compositions and integrated systems for performing the assays are also provided.

Description

HIGH THROUGHPUT ASSAY FOR DETECTION OF mRNA IN CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to USSN 09/052,842, filed March 31, 1998, and is related to USSN 09/052,995, filed March 31, 1998 herein both incorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The field of the invention relates to high throughput assays for identifying modulators of transcription activity and RNA expression in cells. 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 and control 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 wide variety of diseases.

Assays for monitoring gene expression are well known, including northern blotting, RT-PCR, gel mobility shift assays, footprinting analysis, reporter gene expression (e.g. , chloramphenicol transf erase (CAT) assays), etc.

General texts which 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, 2

Inc. and John Wiley & Sons, Inc. , (supplemented through 1998) ("Ausubel"). Specialized apparatus for performing these assays are commercially available.

In addition to these standard methods typically used for gene expression monitoring, specialized antibody based formats for diagnostic 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 -galactosidase labeled monoclonal antibody specific for RNA-DNA hybrids and addition of a fluorescent 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)

Nαtwre 265:472-473, and Stollar (1970) RN4S 65:993-1000. Similarly, detection of DΝA-DΝA hybrids and RΝA-RΝA 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) RN4S 65:993-1000. Stollar (1970) and Rudkin (1976) (both supra) showed that DΝA-RΝA 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 DΝA-RΝA heteropolymers and RΝA- RΝA hybrids which does not recognize DΝA duplexes, single-stranded DΝA or single-stranded RΝA has been prepared and characterized. See, e.g., Bogulavski et al. (1986) J. Immunol. Methods 89: 123-130; Viscidi et al. (1988) J. Clin.

Microbial. 41:199-209 and, Kiney et al. (1989) J. Clin. Microbiol. 27:6-12. This antibody was used for the detection of DΝA-RΝA hetero-duplexes immobilized by binding of the DΝA 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 DΝA-RΝA hybrids are available, e.g. , from Digene Diagnostics, Inc. (Beltsville, MD). 3

One strategy for identifying pharmaceutical lead compounds is to develop an assay which provides appropriate conditions for monitoring the activity of a therapeutic target for a particular disease and to screen large numbers of potential modulators of the therapeutic target in the assay. For example, large libraries of chemical compounds can be screened in liquid or solid phase assays using robotic components. Although the immunoassay -based nucleic acid detection strategies described above have been useful for detecting DNAs, RNAs, and DNA-RNA heteroduplexes, they have not been adapted to high throughput gene expression monitoring assays which could be used for screening for pharmaceutical lead compounds. In addition, high throughput immunoassays for nucleic acid detection which operate in vivo or in situ (e.g. , in isolated cells) are not available, making it difficult to assess the activity of potential gene expression modulators in cells. Accordingly, high throughput assays for expression monitoring and pharmaceutical compound screening, including cell-based assays, 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 vivo assays for detecting modulators of RNA expression are provided, as are related compositions, integrated systems and kits.

Inhibitors and activators of RNA expression can be screened using such assays, as can modulators that alter transcription, RNA expression, transcription activation and transcriptional repression. Solid phase cell based high throughput assays for screening modulators are provided, as are related assa^ compositions, integrated systems for assay screening, and other features that will be evident upon review.

High throughput cell-based expression assays are provided. In the assays, cells that contain a selected RNA or DNA encoding a selected RNA are contacted with one or more oligonucleotides. Single-stranded RNA in the cell is typically cleaved, e.g. , with RNases such as RNase A. The cell is then incubated with a recognition agent such as an antibody that binds to RNA duplexes. The recognition reagent is detected either directly or indirectly, providing a measure of the level of expression of the selected RNA. 4

In one embodiment, expression of the selected RNA is induced by providing a transcription activating molecule that induces transcription of the DNA encoding the selected RNA. In another embodiment, the cell is treated with a compound suspected of having the ability to modulate expression or transcription activation of the selected RNA. The modulator can be added to the system in conjunction with induction of transcriptional activation. In these assay formats, the cell is typically fixed to a solid support such as a microtiter plate prior to analysis of RNA expression levels in the cell. The recognition reagent is optionally an antibody that binds DNA-RNA or RNA-RNA duplexes. Kits, compositions, and integrated systems for performing the assays are also provided.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 is a schematic of an exemplar assay of the invention. Fig. 2 is a data graph from an assay showing induction of IL-8 by

IL-1 and TNF.

Fig. 3 is a graph showing dose response of IL-8 expression induced by IL-1. Definitions The following terms are defined for purposes of this application.

A "ribonucleic acid" or "RNA" is a polymer comprising ribonucleotide monomer units. The polymer can be a naturally occurring ribonucleotide polymer such as mRNA, rRNA or tRNA. The polymer optionally comprises non-naturally occurring nucleotides, e.g. , synthetic monomer units in the polymer chain. The RNA can be single or double stranded. A "region" of the RNA is any sequence or subsequence of the RNA, including the full-length of the RNA.

An "RNA duplex" is a double stranded nucleic acid comprising at least one RNA strand. The duplex can be an RNA-RNA strand, an RNA-DNA strand (also referred to as an DNA-RNA hybrid or DNA-RNA heteroduplex) or can comprise a strand comprising artificial nucleotides. An RNA homoduplex is a base-paired double- stranded RNA. An RNA heteroduplex comprises an RNA 5 strand and a strand comprising DNA nucleotide monomers. All or a region of the duplex may be double stranded. Typically, at least 10 nucleotides of the duplex will be double-stranded. More typically, at least about 40 nucleotides are double- stranded (this is optionally accomplished by binding adjacent oligonucleotides to an RNA).

A "coding DNA molecule" is a DNA molecule that encodes an RNA molecule. The coding DNA molecule will typically comprise a promoter operably linked to a sequence that, when transcribed, provides a selected RNA. A "recognition reagent" is a reagent that is directly or indirectly detectable and that binds, directly or indirectly, to the indicated molecule (e.g. ,

RNA duplex, homoduplex or heteroduplex). A typical recognition reagent in the context of the invention is an antibody that specifically binds nucleic acid duplexes.

A "transcriptional activating" molecule is a molecule that stimulates or induces transcription of a selected RNA under specified conditions, e.g. , in a cell.

A "modulator of transcription activity" is a compound that increases or decreases transcription of a coding DNA in a selected system. A "potential modulator of transcription" is a compound that is to be assessed for its ability to increase or decrease transcription of a coding DNA in a selected system. A

"modulator of RNA expression" is a compound which increases or decreases the level of RNA in a selected system. A "potential modulator of RNA expression" is a compound which is to be assessed for the ability to increase or decrease the level of RNA in a selected system. The level of RNA in the cell can be a result of promoter control, signal transduction systems regulating transcription and transcription factors, splicing of nuclear RNA into mRNA, RNA degradation pathways, RNA termination, polyadenylation and the like. Samples or assays that are treated with a potential modulator are optionally compared to control samples without the test compound, to examine the extent of inhibition or activation of transcription, or the extent or inhibition or activation of expression. For example, control samples (untreated with test inhibitors or activators) are assigned a relative transcription or expression activity value of 100. Inhibition of transcription or 6 expression is achieved when the transcription or expression activity value of the test sample relative to the control is about 75, preferably 50, more preferably 25. Activation is achieved when the transcription or expression activity value of the test sample relative to the control is about 125, 150, or more preferably 200, in whatever units are appropriate to the label and assay system under consideration.

"Permeabilizing the cell" refers to the process of degrading the cell membrane (e.g. , with a chaotropic agent that denatures proteins such as guanidine isothiocyanate or a detergent or an alcohol) to make it porous to a selected compound or component. "Transcription" refers to the process by which an RNA molecule is polymerized using a coding DNA template that encodes the RNA.

"Expression" refers to the relative level of an RNA in a cell, which is the result of transcription, splicing, RNA polyadenylation, degradation, and the like. A "detectable moiety" or "label" is a composition detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes and their substrates (e.g. , as commonly used in an ELISA, e.g. , alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label or detectable moiety is typically bound, either covalently, through a linker or chemical bound, or through ionic, van der Waals or hydrogen bonds to the molecule to be detected.

A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

The phrase "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of 7 transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

DETAILED DESCRIPTION

High throughput methods, compositions, kits and integrated systems for detecting RNA levels in cells and the effect of potential modulators of RNA levels are provided. The assays of the invention provide at least two immediately useful properties. First, the assays can be used to detect RNA levels in cells, serving as a replacement for standard molecular tools such as northern analysis, in situ hybridization, RT-PCR and the like. Thus, the assays provide broadly applicable tools for assessing gene expression in a high throughput format.

Second, the assays provide for the identification of modulators of RNA expression levels. These modulators are valuable for in vitro modification of signal transduction, transcription, splicing, RNA degradation, and the like, e.g. , as tools for recombinant methods, cell culture modulators, etc. More importantly, these modulators provide lead compounds for drug development for a variety of conditions, including as antibacterial, antifungal, antiviral, antineoplastic, inflammation modulatory, or immune system modulatory agents. Accordingly, the assays are of immediate value for their ability to identify lead compounds for pharmaceutical or other applications. The assays are particularly well suited to high throughput automation, making them especially valuable for their ability to identify lead compounds.

Indeed, because RNA expression is fundamental in all biological processes, including cell division, growth, replication, differentiation, repair, infection of cells, etc., modulators identified by the assays of the invention are leads for a variety of conditions, including neoplasia, inflammation, allergic hypersensitivity, metabolic disease, genetic disease, viral infection, bacterial infection, fungal infection, or the like. In addition, transcription modulators which specifically target critical genes in undesired organisms such as viruses, fungi, agricultural pests, or the like, can serve as fungicides, bactericides, herbicides, insecticides, etc. Thus, the range of conditions that RNA expression modulators 8 are applicable to includes conditions in humans and other animals, and in plants, e.g. , for agricultural applications.

Automation of cell-based assays for the high throughput screening assays of the invention is facilitated by consideration of desired assay parameters. First, it is desirable to have high enough sensitivity to detect as little as 10⁵ mRNA molecules per cell. It will be appreciated that many prior art assays for detecting rare RNAs rely upon RT-PCR amplification of rare mRNA to enhance the amount of corresponding nucleic acid (RT-PCR provides many DNA copies of the mRNA for detection and analysis) present in the cell. PCR can result in loss of quantitation and adds additional steps to the assay, such as thermocycler steps.

PCR also destroys cells (i.e. , due to thermal lysis of the cells), making in situ analysis impossible. Another disadvantage of PCR is that there is often a loss of fidelity during reverse transcription and amplification, resulting in unwanted artifacts. Second, the assay should be as simple as possible, with a minimum number of manipulations and relatively short incubations, to facilitate robotic automation of the assay. Third, the assay should be general in format so that it can be used to monitor the expression of essentially any RNA without needing to substantially change the format of the assay to adapt it to monitoring a particular selected RNA or modulator. Fourth, the assay should be inexpensive to perform, as high throughput formats can provide thousands of separate assays per day, making cost an important consideration for development of high throughput assays. The assays of the invention meet these design parameters.

Further in this regard, several aspects of the discovery were surprising. First, the assay can be performed in a very short time frame relative to other polynucleotide assay formats which detect RNA expression; once cells are adhered to a plate (this typically takes about two hours) and any incubation with a modulator is performed (this typically takes about 0.5-15 hours), an entire assay can be performed in under three hours. Second, the assay is extremely sensitive relative to previously described formats and requires only minimal quantities of the necessary reagents and does not require expansion of the RNA to be detected

(prior art methods often rely on RT-PCR to expand RNA for detection). For example, it was possible accurately to detect IL-8 mRNA in 10,000 cells or less 9

(IL-8 is a moderate abundance mRNA, present at about 10-100 copies per cell under the conditions of the assay). Third, because of the high sensitivity and short incubation times of the assays, it is very economical to run the assays in an automated high-throughput format. Finally, the assays are very robust, with insignificant variation seen between control reactions, minimizing the need for repetitive control and calibration steps. RNA Detection Assays and Assays for Potential Modulators

In one aspect, the invention provides methods of detecting a selected RNA in a cell. In the methods, a cell which expresses the selected RNA is contacted with an oligonucleotide which has a region complementary to the selected RNA. The oligonucleotide enters the cell (typically following permeabilization of the cell membrane, e.g. , with a mild detergent or alcohol or a mild chaotropic agent), and forms an RNA duplex in the cell. Single stranded RNA in the cell is cleaved to reduce background in subsequent assays steps. The RNA duplex is then detected by contacting the RNA duplex with a recognition reagent such as an antibody which binds RNA homo or hetero duplexes. The recognition reagent is directly or indirectly detectable. This method is broadly applicable to the detection of RNA levels which result from transcription and processing of the RNA. Indeed, it will be appreciated that the assays herein can be used to detect the presence or absence of an RNA. This feature is particularly useful in the context of screening for potential modulators of RNA transcription or expression. It will be appreciated that the goal of many modulator screening protocols is to identify modulators which block expression of an RNA which would otherwise be expressed, in which case the absence of a selected RNA following modulator incubation may be a goal of the assay. Similarly, in other instances, an increase in RNA expression may be a goal of the screening protocol using the assay. In either case, it can be desirable to screen for specific modulators, i.e. , modulators which increase or decrease the level of a selected RNA such as a pathogenic RNA, but which have no effect on the level of expression of other RNAs. 10

Accordingly, in one aspect, the invention provides methods of measuring expression of a selected RNA in the presence of a potential nucleic acid transcription activity modulator in a cell. In the methods, a cell comprising a DNA encoding the selected RNA is incubated in the presence of a potential transcription activity modulator. An oligonucleotide is introduced into the cell, e.g. , following a brief incubation with a mild detergent such as triton X-100. The oligonucleotide comprises a region complementary to a region of the selected RNA. Typically, single-stranded RNA is cleaved in the cell to eliminate background problems (long RNAs often form regions of secondary structure in solution which can be recognized improperly as a duplex by, e.g. , an antibody recognition reagent; in addition, antibody recognition reagents can bind single- stranded RNA to some extent). The cell is incubated with a recognition reagent which binds to RNA duplexes (e.g. , an anti-RNA duplex antibody). The recognition reagent is directly or indirectly detectable and the level of expression of the selected RNA is determined by detecting the amount of recognition reagent bound to the RNA duplex in the cell.

Where desired, the assay can be refined by the use of positive or negative controls, and/or by comparison to the results observed for other RNAs. For example, a "positive" control assay will utilize a modulator which is known to increase the expression of a selected RNA (e.g. , a hormone for a hormone responsive promoter, Tat for a Tat responsive promoter, etc.). Such positive controls are used to infer that the components of the assay are functioning properly. A negative control can include a modulator known to decrease expression of a particular RNA, such as α-amanitin, which decreases transcription from polll promoters. Such negative controls can be used to determine base-line background signal levels in the assay. Comparison of the results of multiple assays can be used to find modulators which are specific for a single selected RNA, or for a single type of organism. For example, the effects of a modulator on expression of a selected RNA from a gene comprising a human promoter can be compared to the effects observed from a bacterial promoter to find modulators that selectively modulate expression of the bacterial promoter— a desirable feature, 11 e.g. , where the assay is used in a screening protocol to identify, e.g. , antibacterial agents.

Selected RNAs

Selected RNAs to be detected in the assays of the present invention are expressed from any of a variety of operably linked transcription elements.

The operably linked transcriptional elements can be from a native gene or a heterologous nucleic acid, e.g. , a promoter from a therapeutic target operably linked to a reporter nucleic acid. Transcription of the selected nucleic acid can be induced by addition of a direct or indirect transcriptional activator, or basal levels of transcription can be measured. A modulator is optionally added, optionally in conjunction with a transcriptional activator.

As is evident to one of skill in the art, the selected RNA can be any of a large number of RNAs. As described above, the selected RNA can be a operably linked to a native or heterologous promoter of choice. Examples of selected RNAs which are standardized and easily detectable (e.g. , in reporter constructs), referred to as "reporter RNAs" include A-less and G-less RNAs (as discussed below, certain RNases used to eliminate background in the assays specifically cleave A or G), IL-8 RNA, and other RNAs that have features allowing easy detection (e.g. , binding to a standard set of oligonucleotides). Optionally, the expression from the promoter of choice is induced by transcriptional or expression activating factors. Many transcriptional and expression activating factors act in a cascade, e.g. , they induce expression of genes, which gene products subsequently induce the expression of more genes. Transcriptional and expression activating factors, as described below, therefore also may serve as selected RNAs in the assays of the invention.

Expression and transcriptional activators

Transcription of the selected nucleic acid can be induced by addition of a direct or indirect transcriptional activator, or basal levels of transcription can be measured. A modulator is optionally added to the assays of the invention, optionally in conjunction with a transcriptional activator.

The transcriptional requirements for a number of genes are known, making it possible to induce expression of a wide variety of RNAs using available 12 techniques. For example, hormones, cytokines, chemicals, ions (e.g. , Ca^{+ +}) or the like can be incubated with an in vitro transcription reaction to induce transcription of a variety of genes. Similarly, a reaction can contain genes expressed from a selected pathogen (virus, bacteria, spore, plasmodium, protozoa, or the like) or pathogenic gene or protein (e.g. , that encode Tat or Rev from

HIV) , which typically induces transcription of selected cellular or pathogenic organism genes. Because these pathogenic agents often express RNAs encoded in the pathogenic genome, these pathogenic RNAs can be detected using the assays herein. Example transcriptional and expression activators, that may be added to the cell to induce transcription or expression (or which are, themselves, encoded by a selected detectable RNA), include factors that modulate cell growth, differentiation, regulation, or the like. Expression and transcriptional activators are found in prokaryotes, viruses, and eukaryotes, including fungi, plants, and animals, including mammals, providing a wide range of therapeutic targets. It will be appreciated that expression and transcriptional activators regulate transcription by many mechanisms, e.g. , by binding to receptors, stimulating a signal transduction cascade, regulating expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind to promoters and enhancers, unwinding DNA, splicing pre-mRNA, polyadenylating RNA, and degrading RNA. Expression activators include cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products; e.g., interleukins (e.g. , IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-/3, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA- 4/VCAM-l, ICAM-l/LFA-1, and hyalurin/CD44; signal transduction molecules and corresponding oncogene products, e.g. , Her-2/neu Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, e.g. , p53, Tat; steroid hormone receptors such as those for estrogen, progesterone, testosterone, aldosterone, and corticosterone; heat shock proteins; the LDL receptor; uncoupling proteins, and telomerase.

Similarly, expression of genes (or activation of expression of genes) in infectious organisms can be detected, including infectious fungi, e.g. , 13

Aspergillus, Candida species; bacteria, particularly E. coli, which serves a model for pathogenic bacteria, as well as medically important bacteria such as Staphylococci (e.g aureus), Streptococci (e.g. pneumoniae), Clostridia (e.g. p erf ring ens) , Neisseria (e.g gonorrhoea), Enterobacteriaceae (e.g. coli), Helicobacter (e.g pylori), Vibrio (e.g. cholerae), Capylobacter (e.g. jejuni),

Pseudomonas (e.g aeruginosa), Haemophilus (e.g. influenzae), Bordetella (e.g. pertussis), Mycoplasma (e.g. pneumoniae), Ureaplasma (e.g. urealyticum) , Legionella (e.g. pneumophila) , Spirochetes (e.g. Treponema, Leptospira and Borrelia), Mycobacteria (e.g. tuberculosis, smegmatis), Actinomyces (e.g. (israelii), Nocardia (e.g. asteroides), Chlamydia (e.g. trachomatis) , Rickettsia,

Coxiella, Ehrilichia, Rochalimaea, Brucella, Yersinia, Fracisella, and Pasteurella; protozoa such as sporozoa (e.g. Plasmodia), rhizopods (e.g. Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas Giardia, etc.); viruses such as ( + ) RNA viruses (examples include Poxviruses e.g. , vaccinia, Picornaviruses, e.g. /?o/t^'o; Togaviruses, e.g. rubella; Flaviviruses, e.g. HCV; and Coronaviruses),

( - ) RNA viruses (examples include Rhabdoviruses, e.g. VSV; Paramyxovimses, e.g. RSV; Orthomyxovimses, e.g. influenza; Bunyaviruses and Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e. Retroviruses, e.g. especially HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B virus. Other assays are designed to be relevant to non-medical uses, such as assays for inhibitors of transcription in crop pests e.g. , insects, fungi, weed plants, and the like. Coding DNAs

Coding DNAs which encode selected RNAs are optionally from a natural source or can be recombinant. The DNAs can be recombinant DNAs purified and transfected into cells upon which the assays is to be performed, e.g. , plasmids comprising a promoter of interest linked to a reporter sequence, or an expressed nucleic acid linked to a well known heterologous promoter. It will be appreciated that either configuration can be desirable, depending on the application. For example, the assay for a reporter RNA can be optimized for use with particular oligonucleotides, facilitating development of assays which detect expression of the RNA from any selected promoter. Similarly, a well understood 14 promoter can be used to direct expression of a variety of RNAs to test for effects of modulators on RNA stability, splicing or the like.

A wide variety of molecular and biochemical methods are available for making coding DNAs and selected RNAs. Examples of appropriate molecular techniques for generating recombinant nucleic acids, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques. Methods in Enzymologv volume 152 Academic Press, Inc. , San Diego, CA (Berger); as well as in Sambrook, and Ausubel (both supra). Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp. , Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc. , GIBCO BRL Life Technologies, Inc. (Gaithersberg, MD), Fluka

Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, CA, Applied Biosystems (Foster City, CA), Digene Diagnostics, Inc. (Beltsville, MD) as well as many other commercial sources known to one of skill. Oligonucleotide Synthesis and Selection

In the assays of the invention, oligonucleotides are used to bind to selected RNAs to form RNA duplexes. Most commonly, these nucleic acids are DNA or RNA oligonucleotides which are made synthetically. Synthetic oligonucleotides are typically synthesized chemically according to common solid phase phosphoramidite triester methods described, e.g. , by Beaucage and

Caruthers (1981), Tetrahedron Letts. , 22(20): 1859-1862, e.g., using an automated synthesizer, as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res. , 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. In other embodiments, the nucleic acids are made recombinantly according to standard techniques, described, e.g. , in Berger, Sambrook and Ausubel, all supra. 15

Oligonucleotides are typically selected to have particular hybridization characteristics with the selected RNA and to form a duplex with the RNA. Most typically, oligonucleotides are selected to be fully complementary to the selected RNA, although a portion of the oligonucleotide can be non- complementary (e.g. , a portion may act as a labeling or cloning element instead of participating directly in hybridization, or a single oligonucleotide can be used to detect multiple closely related RNAs in separate assays to reduce individual assay costs). The oligonucleotides are optionally selected to have melting temperatures near the temperature of the assay, to prevent unwanted detection of non-specific hybridization. However, a surprising aspect of the present invention is the discovery that it is not necessary to perform the assays at high stringency, as background has been determined often not to be a problem, even at low or moderate stringency. The ability to perform the assay at relatively low temperatures (e.g. , room temperature to about 45 °C; in preferred embodiments assays are conducted at about 39°C) simplifies automation of the assay, and is, therefore, a desirable feature of the invention.

Accordingly, while it is not necessary to optimize hybridization conditions, it is possible to decrease background in some cases by selecting oligonucleotides and assay conditions so that the oligonucleotide hybridizes to the RNA under stringent conditions, and then performing hybridization steps under stringent conditions (note that, as described below, this problem can also be reduced by appropriate oligonucleotide selection). For example, some oligonucleotides may self-hybridize or hybridize to RNAs other than the appropriate target. In these cases, more stringent hybridization conditions are selected for the assay. "Stringent hybridization" in the context of these nucleic acid hybridization experiments are sequence dependent, and are different under different environmental parameters. Generally, highly stringent hybridization conditions are selected to be about 5°-15° C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and ph. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target

RNA sequence hybridizes to a perfectly matched oligonucleotide probe. Very stringent conditions are selected to be nearly equal to the T_m for a particular probe 16

(e.g. , 0°-5° C below the melting temperature). It is expected that one of skill is thoroughly familiar with the theory and practice of nucleic acid hybridization and selection of complementary oligonucleotides. Gait, ed. Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford (1984); W.H.A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K.L. Dueholm J. Org. Chem. 59, 5767-5773

(1994); S. Agrawal (ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993) Laboratory Techniques in biochemistry and molecular biology— hybridization with nucleic acid probes, e.g., part I chapter 2 "overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York provide a basic guide to nucleic acid hybridization.

Most typically, although oligonucleotides may be of any length, they are often between 8 and 100 nucleotides in length, and preferably between about 10 and 35 nucleotides in length, often about 30 nucleotides in length. The oligonucleotides are optionally selected so that there is minimal complementarity between known genes unrelated to the target RNA and the oligonucleotide to reduce unwanted background hybridization. The oligonucleotides are optionally selected to reduce secondary structure formation within the oligonucleotide, or to reduce binding to other oligonucleotides in the assay. Self-complementary oligonucleotides have poor hybridization properties, because the complementary portions of the self hybridize (i.e., form hairpin structures); in addition, regions of self complementarity can be recognized by recognition reagents that bind duplex RNA. The oligonucleotides are also selected so that the oligonucleotides do not hybridize to each other, thereby preventing duplex formation of the oligonucleotides in solution, and possible concatenation of the oligonucleotides, either of which can lead to unwanted binding of the recognition reagent.

Oligonucleotides are optionally selected so that they have roughly the same thermal melting temperature (T_m). Oligonucleotides are typically selected to bind to adjacent sites on a selected RNA to protect large regions of the selected RNA to enhance subsequent detection (in addition, large duplex RNAs are less likely to be inadvertently washed out of the cell during subsequent washing steps).

One of skill will recognize that there are a variety of possible ways of performing the above selection steps, and that variations on the steps are 17 appropriate. Most typically, selection steps are performed using simple computer programs to perform the selection as outlined above; however, all of the steps are optionally performed manually. One available computer program for primer selection is the MacVector™ program from Kodak. Another program useful for primer selection is the MFOLD program (Genetics Computer Group, Madison WI) which predicts secondary structure of, e.g. , single-stranded nucleic acids. In addition to programs for primer selection, one of skill can easily design simple programs for any of the desired selection steps. Cell Culture and Selection The culture of cells used in the assays of the present invention, including cell lines and cultured cells from tissue or blood samples is well known in the art. Freshney (Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley-Liss, New York (1994)) and the references cited therein provides a general guide to the culture of cells. Culture of plant cells is described in Payne et al. (1992) Plant cell and tissue culture in liquid systems John Wiley & Sons,

Inc. New York, NY. Additional information on cell culture is found in Ausubel, Sambrook and Berger, supra. Cell culture media are described in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. Additional information is found in commercial literature such as the Life Science Research Cell Culture catalogue (1998) from Sigma-Aldrich, Inc (St

Louis, MO) and, e.g., the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, MO). Cells can be grown in bulk flasks and added to the substrate (e.g. , microtiter plate) or can be grown directly on the substrate (e.g. , in the wells of the microtiter plate, depending on the intended application and available equipment.

Selection of cells is based upon the intended application. Where modulation of expression of a gene in a particular cell is a target of the assay, the particular cell, or a related cell culture form of the cell is typically the target. In some embodiments, adherent cells which will adhere during culture to the substrate of the container in which the cells are placed are preferred. Many examples of adherent cell types are known, including epithelial and endothelial cell types. Cells which are not naturally adherent can often be made adherent by 18 chemically modifying the substrate (e.g. , treating the substrate with silane to provide OH groups, or with amine reagents to provide amine groups) or by expressing cell surface receptor molecules on the cell (e.g. , recombinantly) and providing an appropriate ligand fixed on the substrate. Especially preferred cells for use in the present invention include mammalian epithelial cells.

Cell Permeabilization and fixation

Methods of fixing and permeabilizing cells are known and described, e.g. , in Sambrook, Ausubel and Berger, all supra. Further information on permeabilizing and fixing cells is described in Polak and Van Noorden (1997) Introduction to Immunocytochemistry second edition, Springer Verlag, NY and in

Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals a combined handbook and catalogue Published by Molecular Probes, Inc. , Eugene, OR. A large variety of fixatives, fixation conditions, and permeabilization agents are known in the art, and other methods of fixing or permeabilizing cells in the assays present invention will be apparent to one of ordinary skill upon review of this disclosure.

In brief, cells can be permeabilized by exposure to a mild chaotropic agent such as a mild detergent (e.g. , SDS, Triton X-100, or the like) a mild organic solvent (e.g. , toluene, guanidine HCL, or Urea at a low concentration), an alcohol, or the like. Triton is a preferred detergent for permeabilization. Typically, these agents are added in low concentrations where lysis of the cell is not desired; in higher concentrations, these agents can lyse cells. Cells are also optionally permeabilized by exposure to electric current, high-velocity microprojectile bombardment, exposure to polyethylene glycol, treatment with mild acids or bases or the like. All of these procedures are well known to one of skill and optimization of permeabilization for a particular application is easily performed simply by titrating the concentration of permeabilization agent in cells in parallel experiments. For purposes of the present invention, the concentration of cell permeabilization agents is adjusted so that the cell membrane becomes porous, but the cell remains intact (e.g., as viewed by microscopy, trypan blue exclusion or the like). 19

A variety of fixatives can be used to fix cells adhered on the solid substrates of the invention. One common class of fixatives useful in the present invention is cross linking fixatives, including formalin (an aqueous solution of formaldehyde), paraformaldehyde, glutaraldehyde and combinations of formalin or paraformaldehyde and glutaraldehyde. These fixatives form hydroxy-methylene bridges between reactive end groups of adjacent protein chains. In solution, pH affects the characteristics of the fixative. For example, when using formalin at low pH there is a preponderance of the more reactive ⁺CH₂(OH) carbonium ions, while at higher pH the more reactive CH₂(OH)₂ groups predominate. Unbuffered formalin is a very strong fixative; thus, although simple solutions of formal saline

(10% aq. commercial formalin containing .9% sodium chloride) can be used, formalin or paraformaldehyde buffered at a pH of about 7 are often more useful in the present invention. Other useful cross-linking fixatives include diethyl pyrocarbonate (DEPC) and parabenzoquinone. Cells can also be fixed with precipitant fixatives such as alcohol or acetone, sometimes in combination with cross-linking fixatives (cells are also optionally permeabilized by using precipitant and other fixatives; accordingly, fixation and permeabilization can be performed in a single step) . Combination fixatives which include Picric acid (typically in combination with formalin and acetic acid) are also useful (two common combination fixatives are "Bouin's" and

"Zamboni's" fixatives. See, Polak and Van Noorden (1997), supra at pages 13- 16 and the references cited therein for a description of cell fixing methods, including cross linking fixatives, precipitant fixatives and combination fixatives. Single-Stranded RNA Cleavage In the assays of the invention, single stranded RNA in the cell is digested to reduce background in the assays. This is performed using an RNase enzyme, or using chemical reagents. Typically, the selected RNA is in a duplex with another nucleic acid, which protects the RNA from degradation by the RNase. Alternatively, the selected RNA lacks a particular nucleotide residue that is recognized by the RNase, e.g. , G, A, etc. In such a case the selected RNA is referred to as, e.g. , a G-less or an A-less RNA. 20

Suitable RNases are commercially available and known to those of skill in the art. RNases are derived from prokaryotes (e.g. , bacterial RNase I and III), viruses, and eukaryotes, and have non-specific or specific endonuclease activity. RNases that recognize single-stranded RNA substrates are preferred; however, RNases that recognize double-stranded RNAs (but not, e.g. , RNA heteroduplexes) are also useful in the present invention. Examples of suitable RNases include RNase A, which cleaves after pyrimidines; RNase B, a carbohydrate modification of RNase A; RNase S, a proteolytic fragment of RNase A; pancreatic RNase, a mixture of RNase A and B; RNase Tl, which cleaves after guanine residues (from Aspergillus oryzae or Ustilago sphaerogena); RNase T2, which cleaves non-specifically (from Aspergillus oryzae); RNase Phy M, which cleaves after adenine and uridine residues (from Physarum polysephalum); RNase U2, which cleaves after A residues (from Ustilago sphaerogena); B. cereus RNase, which cleaves after adenine and cytidine residues; and RNase VI , which cleaves double stranded RNA (from cobra venom). Preferred embodiments include RNase A and RNase Tl . Many of these RNAse enzymes are commercially available from SIGMA.

Recognition Reagents In the assays of the present invention, recognition reagents are used to bind to RNA homo- or hetero-duplexes. Most commonly, the recognition reagent will be an antibody which recognizes RNA duplexes. As discussed above, Coutlee et al. (1989) Analytical Biochemistry 181 : 153-162; 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, Stollar (1970) PNAS 65:993-

1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41: 199-209, and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA-RNA hybrids are available, e.g. , from Digene Diagnostics, Inc.

(Beltsville, MD). Accordingly, such antibodies are commercially available. 21

In addition to available antibodies, one of skill can easily make antibodies specific for RNA duplexes using existing techniques, or modify those antibodies which are commercially or publicly available. In addition to the art referenced above, general methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art. See, e.g. , Paul (ed) (1993)

Fundamental Immunology, Third Edition Raven Press, Ltd. , New York Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding (1986) Monoclonal

Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281 ; and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K_D of at least about .1 μM, preferably at least about .01 μM or better, and most typically and preferably, .001 μM or better.

As used herein, an "antibody" refers to a protein consisting of one or more polypeptide substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG,

IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen 22 recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2, a dimer of Fab which itself is a light chain joined to V_H- C_H1 by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')₂ dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology , W.E. Paul, ed. , Raven Press, N.Y.

(1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies include single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.

Indeed, the RNA duplex binding site from an antibody can be fused by recombinant methods to non-immunoglobulin protein sequences to form a recognition reagent which binds to an RNA duplex. An "RNA duplex-binding site" or "binding portion" with reference to an antibody or antibody fusion molecule refers to the part of an immunoglobulin molecule that participates in antigen (i.e. , RNA duplex) binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" which are interposed between more conserved flanking stretches known as "framework regions" or

"FRs" . Thus, the term "FR" refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an 23 antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding "surface" . This surface mediates recognition and binding of the target RNA. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs" and are characterized, for example by Kabat et al. Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987).

As used herein, the terms "immunological binding" and "immunological binding properties" refer to the non-covalent interactions of the type which occur between a recognition reagent and an RNA duplex for which the reagent is specific. The strength or affinity of binding interactions can be expressed in terms of the dissociation constant (Kj) of the interaction, wherein a smaller K_<* represents a greater affinity. Binding properties of selected reagents can be quantified using methods well known in the art. One such method entails measuring the rates of RNA duplex-recognition reagent complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (K^) and the "off rate constant" (K_off) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K_off/K™ enables cancellation of all parameters not related to affinity and is thus equal to the dissociation constant K_<*. See, generally, Davies et al. Ann. Rev. Biochem. , 59: 439-473 (1990). It will be appreciated that molecules other than immunoglobulins and derivatives also make appropriate recognition reagents, and can be made by one of skill. In general, molecules which recognize RNA duplexes (and which, preferably, do not recognize DNA homoduplexes) are suitable. Molecules which bind RNA duplexes include antibodies, nucleic acid binding proteins, nucleic acids and the like. Recognition reagents which bind to RNA duplexes and not to DNA homoduplexes are preferred because nuclear DNA is typically present in the cell in the assays of the invention; accordingly, binding of a recognition reagent to DNA 24 duplexes may cause background problems. Additional appropriate reagents can be identified, e.g. , by screening available combinatorial chemical (e.g. , peptide) libraries to find library members which preferentially bind RNA duplexes.

The recognition reagents are either directly labeled, i.e. , comprise or react to produce a detectable label, or are indirectly labeled, i.e. , bind to a molecule comprising or reacting to produce a detectable label. Labels can be directly attached to or incorporated into the recognition reagent by chemical or recombinant methods.

In one embodiment, a label is coupled to a molecule such as an antibody comprising the RNA duplex recognition domain through a chemical linker. Linker domains are typically polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. In some embodiments, proline residues are incorporated into the linker to prevent the formation of significant secondary structural elements by the linker. Preferred linkers are often flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein comprising the RNA recognition domain. In one embodiment, the flexible linker is an amino acid subsequence comprising a proline such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about 100. In other embodiments, a chemical linker is used to connect synthetically or recombinantly produced recognition and labeling domain subsequences. Such flexible linkers are known to persons of skill in the art. For example, poly(ethelyne gly col) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages. As described, recognition reagents of the invention are optionally made via recombinant ligation of nucleic acids encoding the constituent parts of the encoded fusion protein (e.g. , RNA recognition domain, linker and a label such as a phosphatase, peroxidase or other enzyme) and expression of the resulting construct. Instructions sufficient to direct one of skill through such cloning exercises are found in Sambrook, Berger and Ausubel, all supra, and again, many appropriate recognition reagents, including reagents comprising label domains, are available. 25

Labeling Strategies

The detectable labels in the present invention which are attached to the recognition reagent can be primary labels (where the label comprises an element which is detected directly or which produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden (1997) Introduction to Immunocytochemistry second edition, Springer Verlag, NY and in Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals a combined handbook and catalogue Published by Molecular Probes, Inc. , Eugene,

OR. Primary and secondary labels can include undetected elements as well as detected elements. Useful primary and secondary labels in the present invention can include spectral labels such as fluorescent dyes (e.g. , fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green^™, rhodamine and derivatives (e.g. , Texas red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes^™, and the like), radiolabels (e.g. , ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g. , horse-radish peroxidase, alkaline phosphatase etc.), spectral colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The label may be coupled directly or indirectly to a component of the detection assay (e.g. , the recognition reagent acid) according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

In general, a detector which monitors a particular probe or probe combination is used to detect the recognition reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill. Commonly, an optical image of a substrate comprising bound labeling nucleic acids is digitized for subsequent computer analysis. 26

Preferred labels include those which utilize 1) chemiluminescence (using horseradish peroxidase and/or alkaline phosphatase with substrates that produce photons as breakdown products as described above) with kits being available, e.g. , from Molecular Probes, Amersham, Boehringer-Mannheim, and Life Technologies/ Gibco BRL; 2) color production (using both horseradish peroxidase and/or alkaline phosphatase with substrates that produce a colored precipitate [kits available from Life Technologies/Gibco BRL, and Boehringer-Mannheim]); 3) hemifluorescence using, e.g. , alkaline phosphatase and the substrate AttoPhos [Amersham] or other substrates that produce fluorescent products, 4) Fluorescence (e.g. , using Cy-5 [Amersham], fluorescein, and other fluorescent tags); 5) radioactivity. Other methods for labeling and detection will be readily apparent to one skilled in the art.

One particularly preferred example of detectable secondary labeling strategies utilizes an antibody which recognizes RNA duplexes linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Preferred enzymes that can be conjugated to recognition reagents of the invention include, e.g. , β-galactosidase, luciferase, horse radish peroxidase, and alkaline phosphatase. The chemiluminescent substrate for luciferase is luciferin. One embodiment of a chemiluminescent substrate for /3-galactosidase is

4-methylumbelliferyl-j3-D-galactoside. Embodiments of alkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP), which is detected with a spectrophotometer; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TR phosphate, which are detected visually; and 4-methoxy-4-(3-phosphonophenyl) spiro[l,2-dioxetane-3,2'-adamantane], which is detected with a luminometer. Embodiments of horse radish peroxidase substrates include 2,2'azino-bis(3-ethylbenzthiazoline-6 sulfonic acid) (ABTS), 5- aminosalicylic acid (5 AS), o-dianisidine, and o-phenylenediamine (OPD), which are detected with a spectrophotometer; and 3,3,5,5'-tetramethylbenzidine (TMB), 3,3'diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and 4-chloro-l- naphthol (4C1N), which are detected visually. Other suitable substrates are known to those skilled in the art. The enzyme-substrate reaction and product detection 27 are performed according to standard procedures known to those skilled in the art and kits for performing enzyme immunoassay s are available as described above.

Most typically, RNA expression is measured by quantitating the amount of label fixed to the solid support in the cell by binding of the recognition reagent. Typically, presence of a modulator during cell incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a cell type and culture condition (e.g. , presence of transcriptional activator) . Means of detecting and quantitating labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is optically detectable, typical detectors include microscopes, cameras, phototubes and photodiodes and many other detection systems which are widely available. Modulators

Essentially any chemical compound can be used as a potential modulator in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions, or formulated in a liposomal delivery vesicle are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g. , in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involve providing a combinatorial library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve 28 as conventional "lead compounds" or can themselves be used as potential or acmal therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e. , the number of amino acids in a polypeptide compound) . Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010, 175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al. , Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al. , J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with -D-glucose scaffolding (Hirschmann et al. , J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et a , J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al. , Science 261: 1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al. , Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/ 10287), carbohydrate libraries (see, e.g., Liang et al , Science, 274: 1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and 29 metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519, 134; morp olino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,

Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J. , Asinex, Moscow, Ru, Tripos, Inc. , St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek

Biosciences, Columbia, MD, etc.).

As noted, the invention provides solid phase cell based assays in high throughput format. Control reactions which measure the expression level of the selected RNA in a cell which does not include a transcription modulator are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in a preferred embodiment, the methods of the invention include such a control reaction. For each of the assay formats described, "no modulator" control reactions which do not include a modulator provide a background level of expression from a given coding DNA.

In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. First, a known transcriptional activator (or other factor which increases RNA expression) can be incubated with cells in one sample of the assay, and the resulting increase in transcription can be detected by measuring the resulting increase in RNA according to the methods herein. For example, Tat or a plasmid expressing Tat can be transduced into a cell comprising a selected nucleic acid operably linked to a tat-responsive promoter (e.g., an HIV LTR promoter) and expression of the encoded selected RNA monitored. Second, a known inhibitor of transcription such as α-amanitin (a strong inhibitor of the pol II transcription complex) can be added, and the resulting decrease in transcription similarly detected. It will be appreciated that modulators 30 can also be combined with transcriptional activators or inhibitors to find modulators which inhibit transcriptional activation or transcriptional repression. As applied to the preceding two examples, an modulator which specifically inhibits Tat is very valuable for its ability to repress HIV replication; accordingly, an assay for Tat modulators comprises adding Tat and a potential modulator to a cell.

Similarly, an inhibitor of α-amanitin repression is very useful in counteracting the effects of α-amanitin activation (e.g. , due to wild mushroom poisoning); accordingly, an assay for α-amanitin modulators comprises adding α-amanitin and a potential modulator to a cell. Any modulator provided as described above can be incubated with a cell for any selected length of time prior to detecting RNA expression. Typical incubation times are about 0.5 hours to about 15 hours, more typically about 5 to 10 hours. One feature of the assay is that time-dependent RNA expression for a selected modulator can easily be determined by performing a time titration with the modulator by incubating the modulator and the cell for selected time intervals.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds is possible using the integrated systems of the invention.

Compositions, Kits and Integrated Systems

The invention provides compositions, kits and integrated systems for practicing the assays described herein. For example, an assay composition having a cell comprising a RNA duplex bound to recognition reagent, optionally an RNA expression or transcription modulator and a label bound to recognition reagent is provided by the present invention. As discussed above the duplex RNA typically comprises at least one and often several hybridized complementary nucleic acid 31 oligonucleotides (DNA or RNA). Typically, in the solid phase assays, the cell is adhered to a solid substrate, thereby immobilizing the cell and other assay components on the solid substrate. Additional assay components as described above are also provided. Solid substrates useful in the present invention include membranes

(e.g. , nitrocellulose or nylon), a microtiter dish (e.g. , PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g. , glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper. Most commonly, the assay will utilize 96, 384 or 1536 well microtiter plates.

The invention also provides kits for practicing the methods noted above, the kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high throughput method of screening for an RNA expression or transcription modulator, one or more containers or compartments (e.g. , to hold nucleic acids, cells, modulators, or the like), a control activity modulator (e.g. , α-amanitin which blocks polll transcription), a robotic armature for mixing kit components or the like.

The invention also provides integrated systems for high throughput screening of potential modulators for an effect on RNA expression. The systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a cell adhered to the well, typically comprising a label (e.g. , on the recognition reagent) detected by the label detector.

A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass. ; Orca, Hewlett-Packard, Palo Alto,

Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature 32 and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.

Any of the assays for compounds that modulate or mimic RNA expression levels as described herein, are amenable to high throughput screening. High throughput screening systems are commercially available (see, e.g., Zymark

Corp. , Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc. , Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high thruput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel x86 or pentium chip- compatible DOS™, OS2™ WINDOWS™, WINDOWS NT™ or WINDOWS95™ based machines), MACINTOSH™, or UNIX based (e.g., SUN™ work station) computers. One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the 33 invention are easily used for viewing any sample, e.g. , by fluorescent or dark field microscopic techniques.

Exemplar Assay: In Situ mRNA Detection Using Anti RNA-DNA Antibody Fig. 1 depicts an exemplar assay for detecting mRNA using an

RNA-DNA antibody. Cells expressing an RNA of interest, such as human endothelial cells (e.g. , ECV304) expressing a selected RNA such as IL-8 mRNA (IL-8 is a cytokine which is a mediator of, e.g. , inflammation) are incubated in the wells of a microtiter plate such as a 96 or 384 well microtiter plate, e.g. , a black tissue culture treated plate (available from Polyfiltronics) . The cells adhere to the walls due to natural anchoring processes. One or more compounds of interest is added to one or more of the wells on the microtiter plate (e.g. , in a PBS buffer at .2% DMSO). The cells are incubated for 1-2 hours. Production of the mRNA of interest is then optionally stimulated (IL-8 mRNA production is stimulated in human endothelial cells by addition of IL-1). The cells are then incubated for a selected period of time, e.g. , for about 8-24 hours.

The cells are optionally fixed. A number of fixatives are appropriate; in one preferred embodiment, 4% formaldehyde is used to fix the cells. The fixed cells are washed with a mild detergent such as 0.5 % Triton X- 100 in 2X SSC (see, Sambrook for a description of SSC buffer) to permeabilize the cells. The resulting fixed cells are washed with a solution comprising oligonucleotides which are at least partially complementary to the RNA of interest (e.g. , complementary to IL-8 mRNA). Typically, the oligonucleotides are fully complementary to the RNA of interest. In one illustrated embodiment, more than one oligonucleotide sequence is used, such that the oligonucleotides bind to continuous regions of the RNA of interest, thereby forming an extended duplex region on the RNA of interest by binding to adjacent regions of the mRNA.

An RNAse which cleaves single-stranded RNAs, such as RNAseA, is added to the cells, thereby cleaving unduplexed RNAs in the cell. An antibody comprising a label which recognizes duplexed RNA (e.g. , RNA-DNA heteroduplexes) is then added to the cells. The antibody binds to any duplexed RNA in the cell. Excess antibody, RNAse and the like is washed from the cell 34

(e.g. , using 2X SSC) and the remaining labeled antibody is quantitated. In an illustrated embodiment, the label is an alkaline phosphatase moiety and the label is quantitated by adding alkaline phosphatase substrate and measuring the resulting colorimetric reaction.

EXAMPLES The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters, which are changed or modified to yield essentially similar results.

Example 1 : High throughput assay for direct detection of IL8 mRNA in cells

The following assay was used to examine modulation of IL-8 mRNA expression in vivo. A potential activator or inhibitor compound was first added to the cell, followed by treatment with IL-1, an IL-8 transcription activator.

The level of IL-8 mRNA expression was then assessed to determine the effect of the potential inhibitor or activator.

First, about 30,000 human endothelial cells (ECV304) were plated on black tissue culture treated plates from Polyfiltronics, using standard culture conditions. After 4-5 hours the cells adhered to the bottom of the plate. The potential modulator compound in PBS buffer with 0.2 % DMSO was added to the culture medium, and the cells were incubated for 1-2 hours.

Cytokine IL-1 was then added to the culture medium to induce IL8 mRNA production. The cells were incubated for about 10 hours. This incubation time typically yielded maximal IL8 mRNA expression. The culture medium was discarded, and 100 μL of 4 % formaldehyde in 2X SSC buffer was added to fix the cells to the plate. The cells were then incubated for 15 minutes.

The cells were washed 2 times with 2X SSC buffer, and 0.5 % Triton-XlOO in 2X SSC buffer was added to permeabilize the cell membrane. The cells were incubated for 10 minutes. 35

The cells were washed 2 times with 2X SSC buffer, and 100 μL of IL8 oligonucleotides in 2X SSC buffer were added. 2 pmoles each of 20 oligonucleotides were added and incubated at 37 °C for 45 minutes.

10 μL of RNase A in water (10 μg/well) was added to the cells. This RNase A treatment specifically cuts single-stranded RNA, while the IL8

RNA-DNA hybrid is completely protected. This treatment reduces the background significantly. The cells were incubated for 30 minutes at RT.

100 μL of Digene Detection Reagent I was added to the cells. This reagent contains the Anti RNA-DNA hybrid antibody that is conjugated to Alkaline Phosphatase. The cells were incubated for 40 minutes at RT.

The plates containing the cells are washed 5x with Wash buffer (0.5 X SSC + Calf Thymus DNA 0.02 mg/ml). Alkaline Phosphatase substrate CSPD in Sapphire II (from Tropix Inc.) was added to the cells and the luminescence was counted to detect the level of IL-8 expression. An assay showing induction of IL-8 by IL-1 and TNF is shown in

Fig. 2. The assay was performed essentially as above, with the addition of 20μl of human TNF (Biosource International; catalogue number: PHC3013) or human IL-1-beta Biosource International (catalogue number: PHC0813(cy03)) (cytokines at a final concentration of 13.5 ng/ml in PBS/1 % FBS) per microtiter well. Plates were stored overnight at 37 °C. Plates were assayed the following morning

(typically at least 16, but not more than 18 hours after induction). An assay showing dose response of IL-8 expression induced by IL-1 is shown in Fig. 3.

Accordingly, the disclosures and descriptions herein are intended to be illustrative, but not limiting, of the scope of the invention which is set forth in the following claims. All patents and publications cited herein are incorporated in their entirety for all purposes, as though each were individually indicated to be incorporated by reference.

Claims

36 WHAT IS CLAIMED IS: 1. A method of detecting a selected RNA in a cell, the method comprising: (i) contacting a cell comprising the selected RNA with an oligonucleotide comprising a region complementary to the selected RNA, thereby forming an RNA duplex in the cell; (ii) cleaving single-stranded RNA in the cell; and, (iii) contacting the RNA duplex with a recognition reagent, wherein the recognition reagent is directly or indirectly detectable, thereby detecting the selected RNA.

2. The method of claim 1, wherein expression of the selected RNA is induced in the cell by providing a transcription activating molecule to the cell, which transcription activating molecule induces transcription of a coding DNA molecule in the cell, which coding DNA encodes the selected RNA.

3. The method of claim 2, wherein the transcription activating molecule is IL-1.

4. The method of claim 2, wherein the cell is contacted with a potential modulator of transcriptional activation of the coding DNA.

5. The method of claim 1, wherein the cell is contacted with a potential modulator of expression of the selected RNA to the cell.

6. A method of measuring expression of a selected RNA in the presence of a potential nucleic acid transcription activity modulator in a cell, the method comprising: (i) incubating a cell comprising a DNA encoding the selected RNA in the presence of a potential transcription activity modulator; (ii) introducing an oligonucleotide into the cell, wherein the oligonucleotide is complementary to a region of the selected RNA, forming an RNA duplex; 37 (iii) cleaving single-stranded RNA in the cell; (iv) incubating the cell with a recognition reagent, wherein the reagent binds to RNA duplexes and wherein the recognition reagent is directly or indirectly detectable; and, determining the level of expression of the selected RNA by detecting the amount of recognition reagent bound to the RNA duplex in the cell.

7. The method of claim 1 or 6, wherein the selected RNA is not amplified by RT-PCR.

8. The method of claim 1 or 6, wherein the selected RNA is IL-8 RNA.

9. The method of claim 1 or 6, wherein the oligonucleotide is a DNA oligonucleotide.

10. The method of claim 1 or 6, wherein the oligonucleotide is an RNA oligonucleotide.

11. The method of claim 1 or 6, wherein the oligonucleotide is a DNA oligonucleotide and the recognition reagent is an antibody that binds DNA- RNA duplexes.

12. The method of claim 1 or 6, wherein the recognition reagent is an antibody comprising a label.

13. The method of claim 1 or 6, wherein the recognition reagent is an antibody comprising an alkaline phosphatase label and the method comprises adding an alkaline phosphatase substrate to the antibody.

38 14. The method of claim 1 or 6, wherein the recognition reagent is an antibody comprising a horse radish peroxidase label and the method comprises adding a horse radish peroxidase substrate to the antibody.

15. The method of claim 1 or 6, wherein the cell is fixed on a solid support.

16. The method of claim 1 or 6, wherein the cell is fixed on a solid support using formaldehyde.

17. The method of claim 1 or 6, wherein the solid support is selected from the group consisting of a bead, a membrane, and a 96- well plate.

18. The method of claim 1 or 6, wherein the oligonucleotide is introduced into the cell by permeabilizing the cell and contacting the resulting permeabilized cell with the oligonucleotide.

19. The method of claim 1 or 6, wherein the single-stranded RNA is cleaved in the cell by contacting the single stranded RNA with an RNase enzyme.

20. The method of claim 1 or 6, wherein the single-stranded RNA is cleaved in the cell by contacting the single stranded RNA with RNase A.

21. The method of claim 1 or 6, the method comprising incubation of the cell with a chaotropic agent.

22. The method of claim 1 or 6, further comprising washing unbound recognition reagent from the cell, wherein the unbound recognition reagent is not bound to the duplex.

39 23. The method of claim 1 or 6, wherein the cell is incubated with the recognition reagent for less than one hour.

24. The method of claim 6, the method comprising repeating steps (i)-(iv) in parallel in a microtiter plate format.

25. The method of claim 6, the method comprising repeating steps (i)-(iv) in parallel in a microtiter plate format, wherein at least about 1,000 different potential activity modulators are tested for an effect on the level of expression of the selected RNA.

26. The method of claim 6, the method comprising repeating steps (i)-(iv) in parallel in a microtiter plate format, wherein at least about 1,000 different potential activity modulators are tested for an effect on the level of expression of the selected RNA in one day.

27. The method of claim 6, the method comprising repeating steps (i)-(iv) in parallel in a microtiter plate format, wherein between at least about 100 and at least about 6,000 different potential activity modulators are tested for an effect on the level of expression of the selected RNA in one day.

28. The method of claim 6, the method comprising repeating steps (i)-(iv) in parallel in a microtiter plate format, wherein at least about 6,000 different potential activity modulators are tested for an effect on the level of expression of the selected RNA in one day.

29. The method of claim 1 or 6, wherein the method is performed in a high throughput integrated system comprising an automatic pipetting station, a robotic armature and a robotic controller.

30. A composition comprising a fixed intact cell comprising an RNA duplex bound to an antibody. 40

31. The composition of claim 30, wherein the fixed cell is adhered to a solid substrate.

32. The composition of claim 30, wherein the fixed cell is fixed using formaldehyde.

33. The composition of claim 30, wherein the RNA duplex is an RNA heteroduplex comprising a plurality of oligonucleotides hybridized to a selected RNA.

34. The composition of claim 30, wherein the antibody comprises an alkaline phosphatase moiety.

35. The composition of claim 30, wherein single stranded RNA in the cell is cleaved with an RNAse.