Use of ribozymes for functionating genes Number:6,803,194 from the United States Patent and Trademark Office (PTO) owispatent

Title: Use of ribozymes for functionating genes

Abstract: Double stranded DNAs, expression vectors and methods for their use are provided in which the intracellular expression of the double stranded DNAs is used to alter the phenotype of a target cell so that the function of a target nucleic acid that includes a nucleotide sequence encoding a motif of interest can be determined using a combinatorial ribozyme library. The members of the library are catalytic RNAs that disrupt the expression of the transcription product of the target nucleic acid. Disruption of transcription product expression results in an altered cell phenotype which is used to determine the function of the target nucleic acid. The specific phenotype or response may be associated with normal cellular processes, or it may contribute to the generation of pathogenesis involved in disease development. The compositions find use in high-throughput screens to assign gene functions. When associated with a pathogenic phenotype, these genes or their gene products can constitute therapeutic targets for treatment of diseases. The complete sequence of the gene containing the target nucleic acid need not to be known for the method to be used successfully.

Patent Number: 6,803,194 Issued on 10/12/2004 to Keck,   et al.

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Inventors:	Keck; James G. (Redwood City, CA); Wong; Justin G. P. (Oakland, CA)
Assignee:	HK Pharmaceuticals, Inc. (San Diego, CA)
Appl. No.:	09/601,970
Filed:	August 9, 2000
PCT Filed:	February 12, 1999
PCT No.:	PCT/US99/03166
PCT Pub. No.:	WO99/41371
PCT Pub. Date:	August 19, 1999

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Current U.S. Class:	435/6 ; 435/325; 435/375; 435/455; 435/471; 435/7.1; 435/91.1; 435/91.31; 435/91.32; 435/91.33; 435/91.4
Field of Search:	435/6,7.1,91.1,91.31,91.4,91.32,91.33,325,375,455,471

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References Cited [Referenced By]

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U.S. Patent Documents

	4987071		January 1991		Cech et al.
	5037746		August 1991		Cech et al.
	5093246		March 1992		Cech et al.
	5116742		May 1992		Cech et al.
	5180818		January 1993		Cech et al.
	5190931		March 1993		Inouye
	5217879		June 1993		Huang et al.
	5217889		June 1993		Roninson et al.
	5272065		December 1993		Inouye et al.
	5354678		October 1994		Lebkowski et al.
	5354855		October 1994		Cech et al.
	5457281		October 1995		Bridges et al.
	5496698		March 1996		Draper et al.
	5504200		April 1996		Hall et al.
	5589362		December 1996		Bujard et al.
	5591610		January 1997		Cech et al.
	5599706		February 1997		Stinchcomb et al.
	5631236		May 1997		Woo et al.
	5667969		September 1997		Sullenger et al.
	5670488		September 1997		Gregory et al.
	5679523		October 1997		Li et al.
	5686279		November 1997		Finer et al.
	5837531		November 1998		Dedieu et al.
	5856188		January 1999		Hampel et al.
	6025192		February 2000		Beach et al.
	6040174		March 2000		Imler et al.
	6130092		October 2000		Lieber et al.
	6133028		October 2000		Imler et al.
	6204052		March 2001		Bout et al.
	6255071		July 2001		Beach et al.
	6391311		May 2002		Ferrara et al.
	6410011		June 2002		Branellec et al.
	6455283		September 2002		Ferrara et al.
	2002/0094324		July 2002		Branellec et al.

Foreign Patent Documents

	4424762		Jul., 1995		DE
	0707071		Apr., 1996		EP
	2319773		Jun., 1998		GB
	9201786		Feb., 1992		WO
	9205286		Apr., 1992		WO
	9413833		Jun., 1994		WO
	9420618		Sep., 1994		WO
	9426877		Nov., 1994		WO
	9428152		Dec., 1994		WO
	9514091		May., 1995		WO
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	9601314		Jan., 1996		WO
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Primary Examiner: Celsa; Bennett
Attorney, Agent or Firm: Seidman; Stephanie L. Heller Ehrman White & McAuliffe, LLP

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Parent Case Text

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This application is a 371 PCT/US99/03166 Feb. 12, 1999 which is a CIP of Ser. No. 09/023,992 Feb. 13, 1998 now abandoned.

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Claims

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What is claimed is:

1. A method of assigning a function to a target nucleic acid comprising a nucleotide sequence encoding a motif of interest, said method comprising the steps of: growing a host cell culture comprising host cells that express ribonucleic acid molecules encoded by a combinatorial ribozyme library, wherein: each said ribonucleic acid molecule comprises a binding region complementary to a transcription product of said target nucleic acid, and a catalytic domain that cleaves a sequence within said transcription product of said target nucleic acid that encodes said motif of interest so that expression of said transcription product is disrupted and an altered host cell is produced; a function of the target nucleic acid or encoded product is unknown; the combinatorial ribozyme library comprises nucleic acids encoding a plurality of the ribonucleic acid molecules each containing a recognition sequence for one of a plurality of degenerate sequences based upon a consensus nucleotide sequence encoding the motif; detecting phenotypic changes in the altered host cell; and correlating the phenotypic changes in said altered host cell, as compared to a control host cell, with the identity of said target nucleic acid by isolating DNA from said altered host cell and determining a specific ribozyme sequence contained therein that contains a binding sequence that is complementary to a transcription product of the target nucleic acid, whereby a function for said target nucleic acid is assigned based upon said phenotypic changes in said altered host cell.

2. The method according to claim 1, wherein said function is a physiological function.

3. The method according to claim 1, wherein said function is enzyme activity.

4. The method according to claim 1, wherein said function is protein synthesis.

5. The method according to claim 1, wherein said function is biological factor expression.

6. The method according to claim 1, wherein said function is membrane permeability.

7. The method according to claim 1, wherein said function is a regulatory effector function.

8. The method according to claim 7, wherein said regulatory effector function affects induction of a physiological function.

9. The method according to claim 1, wherein said function is altered directly.

10. The method according to claim 1, wherein said host cell culture comprises a plurality of mammalian cells, bacterial cells, invertebrate cells or plant cells.

11. The method according to claim 1, wherein said motif is a zinc finger motif, a receptor protein kinase motif, or an integrin motif.

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Description

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INTRODUCTION

1. Technical Field

The present invention is related to methods and compositions for identifying a gene or genes associated with the generation of a specific cellular phenotype or a specific cellular response using combinatorial libraries of catalytic RNA directed against RNA sequences encoding structural or functional polypeptide motifs. The invention is exemplified by use of a combinatorial ribozyme library to target sequences in mRNAs encoding zinc finger, protein kinase and integrin motifs.

2. Background

Properly functioning cells are necessary for any organism, including humans, to thrive; improperly functioning cells may contribute to the development of pathogenic or disease states in a given individual, including generation of cancers, autoimmune diseases, innate immunodeficiencies, neurologic diseases, and inborn errors of metabolism. In addition, even properly functioning cells may contribute to pathogenic states, including susceptibility to infectious agents, atopic/allergic pathogeneses, and pathogenic states associated with allograft transplantation. In both of the above cases, inappropriate expression, regulation, or function of a specific gene product or gene products within a cell may lead to the improper behavior of that cell within the context of its normal function in an organism. Often, the activity of a single gene product, such as a protein or polypeptide, will affect the expression, regulation, or function of other gene products within the same cell or within neighboring cells. Aberrant expression, regulation, or function of these aggregated gene products may then result in the development of specific disease phenotypes or syndromes.

Approaches that have been used to identify genes which are potentially involved in a disease development process include identification of genes which are mutated in certain diseases and differential display of actively expressed transcripts in normal versus pathologic cells. These approaches have given rise to a rapid increase in the number of DNA sequences associated with various pathologic states. These sequences include not only full length genes, but also cDNA sequences comprised of partial gene sequences or ESTs. Although sequences identified by these processes are associated with a pathologic state, it is difficult to ascertain a priori whether a given gene is directly involved in the disease development process, or whether its expression occurs in a secondary fashion after the pathogenic process has already begun.

Involvement of particular genes as causative agents in the disease development process can be confirmed by a number of methods. Confirmation of the role of particular genes in the disease development process using partial cDNA sequences is more difficult to assess, however, because many of the methods used require knowledge of the full gene sequence. Thus, while the number of potentially novel genes has expanded exponentially, identification of the functions ascribed to most of these genes and gene sequences, as well as their prospective roles in disease development has lagged far behind.

One way to establish the causative effect of a gene or gene sequence in the development of a specific cellular phenotype or response is to interfere with the expression or function of that gene or gene product, and then to determine the resulting effect on that cellular phenotype or response. Methods utilized to interfere with gene expression in vivo involve gene targeting by homologous recombination in embryonic stem cells, re-implantation of the stem cells, gestation of the embryos, and isolation of animals bearing diallellic deletions in the gene of interest, so called "transgenic technology". The development of transgenic technology has been an important advance in the tools available for studying the function of genes at the organismal level. Because this procedure can take up to a year to complete, however, it is not an efficient process for the high-throughput evaluation of genes or gene products as causative agents and as potential therapeutic targets. Methods utilized to interfere with gene expression in vitro include gene deletion or inactivation by homologous recombination or triplex technology, RNA transcript inactivation or cleavage by antisense or ribozyme technology, and protein inactivation or down-regulation by antipeptide antibody fragments or expression of randomized peptides. A limitation to utilizing systems expressing randomized peptides, antisense RNA molecules, or anti-peptide antibodies to identify gene functions and/or signaling pathways in cells is that these compounds do not act catalytically as is the case for ribozymes and therefore, relatively high intracellular concentrations may be necessary to affect a cellular function or phenotype.

Ribozymes are RNA molecules that act as enzymes and can be engineered to cleave other RNA molecules. Thus, ribozymes perform functions in the cell that are very different from ordinary RNA, in that, after binding selectively to their specific mRNA target, they act catalytically to cut, or cleave, target RNA molecules at specific sites. If an mRNA target in a cell is destroyed, the particular protein for which that mRNA molecule carries information is not produced. The ribozyme itself is not consumed in this process, and can act catalytically to cleave multiple copies of mRNA target molecules. One way to use ribozymes to identify the function of novel gene sequences is to introduce a pool of ribozymes with degenerate target recognition sites into cells in order to reduce or eliminate the expression of a gene or gene product involved in the generation of a specific cellular phenotype or response. In this strategy, ribozymes bearing the appropriate recognition sequences eliminate or reduce expression of the target gene, while ribozymes not bearing the appropriate recognition sequences do not. Loss of a specific cellular phenotype or response associated with elimination or reduction in expression of a target gene indicates involvement of that particular gene in the development of that particular phenotype or response.

Of the estimated 100,000 expressed genes in a mammalian cell, approximately one-third are likely to be necessary for normal cell respiration, metabolism, or viability. A totally degenerate ribozyme library would by necessity include ribozymes directed against these "housekeeping genes" as well as against genes involved in disease processes. Cleavage of housekeeping RNAs results in compromised cellular viability, so no information can be gained from a great number of the ribozyme sequences in such a library. This problem reduces the efficiency of using totally degenerate ribozyme libraries to identify and assign a function to novel genes or gene sequences with respect to a disease development process. Another major limitation to this system is the need to synthesize and express a completely randomized library of nucleic acids and to screen the library for functional activity. The minimal targeting or recognition sequence of a ribozyme is generally 12 nucleotides and a totally random library would contain 4.sup.12 or approximately 16 million ribozymes. Due to the large number of permutations of the ribozyme binding sequences, a specific targeting approach is essential. It is therefore of interest to develop a high throughput ribozyme based screening system that limits the potential target sequences for evaluation to those which have an increased probability of being associated with a molecular pathway that is related to a disease or phenotype.

Relevant Literature

An RNA molecule not naturally occurring in nature having enzymatic activity independent of any protein is disclosed in U.S. Pat. No. 4,987,071 General rules for the design of hammerhead ribozymes that cleave target RNA in trans are described in Haseloff and Gerlach, (1988) Nature 334:585-591. Miniribozymes are disclosed in Uhlenbeck, (1987) Nature 328:596-603. Methods for optimizing cleavage of a target RNA by a ribozyme are described in U.S. Pat. No. 5,496,698. Reporter gene suppression by engineered hammerhead ribozymes in mammalian cells is described in Cameron and Jennings, (1989) Proc. Natl. Acad. Sci. (USA) 86:9139-9143. Ribozyme expression from a retroviral vector is described in Sullenger and Cech, (1993) Science 262:1566-1569. The expression of hammerhead ribozymes operatively linked to a T7 promoter is described in Chowrira et al., (1994) J. Biol. Chem. 269:25856-25864. Co-localizing ribozymes with substrate RNAs to increase their efficacy as gene inhibitors is described in Sullenger, (1995) Appl. Biochem. Biotechnol. 54:57-61. Screening of retroviral cDNA expression libraries is described in Kitamura, et al., (1995) Proc. Nat. Acad. Sci. (USA) 92:9146. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library is described in Lieber and Strauss, (1996) Mol. Cell. Biol. 15:540-551. Approaches for the identification and cloning of differentially expressed genes is discussed in Soares, (1997) Curr. Opin. Biotechnol. 8:542-546. The development of high-throughput screen is discussed in Jayawickreme and Kost, (1997) Curr. Opin. Biotechnol. 8:629-634. The high throughput screen for rarely transcribed differentially expressed genes is described in von Stein et al., (1997) Nucleic Acids Res. 25:2598-2602. High-throughput genotyping is disclosed in Hall, et al., (1996) Genome Res 6:781-790. Methods for screening transdominant intracellular effector peptides and RNA molecules are disclosed in WO97/27212 and WO97/27213.

SUMMARY OF THE INVENTION

Methods and compositions for their use therein, are provided for determining and validating a link between a target nucleic acid which includes a nucleotide sequence that encodes a motif of interest and and a diseases and/or phenotype using a combinatorial ribozyme library. Ribo-nucleotide members of the ribozyme library include a binding region which is complementary to a transcription product of the target nucleic acid and a catalytic domain which cleaves a sequence within a transcription product of the target nucleic acid coding for the motif of interest so that expression of the transcription product is disrupted. The method includes the steps of designing a combinatorial ribozyme library by analyzing a consensus nucleotide sequence encoding a protein motif and synthesizing embers of a library of sense strands of DNA which, when expressed as RNA constitute the members of a ribozyme library; annealing the sense strands to antisense strands to form double stranded DNAs, introducing the double stranded DNAs, which optionally include a means for determining directionality of expression, into expression vectors; contacting a host cell culture containing one or more host cells with the expression vector(s) under conditions such that the expression vectors transfect or infect the host cells; growing the host cells to express the ribozyme(s); analyzing the phenotype of, or a suitable detectable marker in, the resultant transfected or infected host cells to identify any altered host cell by virtue of an alteration in phenotype or marker as compared to unmodified host cells; isolating altered host cells; and correlating the phenotype of altered host cells with the identity of the target nucleic acid encoding the motif of interest by isolating DNA from the isolated altered host cells and determining the specific ribozyme sequence contained in the isolated DNA which is complementary to sequences in the target nucleic acid so as to assign a function to the product coded for by the target nucleic acid. The ribozyme libraries and subject methods can be used, for example, for functionating a gene encoding a protein that contains a motif of interest, such as a gene involved in apoptosis, drug susceptibility, cell cycle regulation, cell differentiation or transformation of a host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general structure of the members of a combinatorial ribozyme library annealed to an mRNA encoding the minimal recognition sequence of the reverse translated zinc finger motif (SEQ ID NO:43), C-X-X-C (X=any amino acid). Upper strand (SEQ ID NO:1) is the targeted mRNA with the ribozyme cleavage site indicated. The lower stand (SEQ ID NO:2) is a hammerhead ribozyme annealed to the mRNA target. (N=any nucleotide).

FIG. 2 shows the nucleotide sequence of oligonucleotides encoding an anti-EGFP hammerhead ribozyme (SEQ ID NOS:3-22).

FIG. 3 demonstrates the isolation of cells expressing a selectable marker associated with a ribozyme-expressing construct from Jurkat T-cell cultures transduced with a library of ribozymes. The selectable marker is the cell surface molecule Lyt-2 (CD8a). Cells expressing the Lyt-2 marker are isolated from the rest of the population using a fluorescence activated cell sorter. The X axis depicts marker expression. The Y axis depicts cell number. The histogram in FIG. 3A shows the profile of marker expression in transduced cultures. The histogram in FIG. 3B shows the same histogram with an expanded Y axis to reveal the marker positive population shifting rightward in the histogram. Marker positive cells isolated by flow cytometric cell sorting were grown in culture, and marker expression was re-analyzed in the enriched cultures. The histogram in FIG. 3C shows results from this re-analysis. All cells in the enriched cultures express the marker, demonstrating the ability to isolate a stable population of cells expressing a library of pooled ribozymes using this method.

FIG. 4 demonstrates the effect of expressing a library of ribozymes on the induction of a cellular protein by cells in a culture. Loss of the ability to induce the protein exemplifies the loss of a cellular response in ribozyme-expressing cells. The X axis depicts expression of the induced protein. The Y axis depicts cell number. The histogram in FIG. 4A shows the profile of induced protein expression in normal cultures (stippled lines) or in cultures expressing a library of pooled ribozymes (solid lines). The histogram in FIG. 4B shows the same histogram with an expanded y-axis to reveal the leftward shifting population of cells, corresponding to those cells which have lost the ability to induce the protein. Cells from the leftward part of the histogram in FIGS. 3A and B were isolated by flow cytometric cell sorting, grown in culture, and induction of the cellular protein was re-analyzed. The histogram in FIG. 4C demonstrates that the subpopulation of cells which have lost the responsive phenotype (represented by the left-hand peak of the histogram) can be enriched from cultures expressing several different ribozyme species represented in the original pooled library.

BRIEF DESCRIPTION OF PREFERRED EMBODIMENT(S)

In the present invention, a combinatorial ribozyme library designed for a target nucleic acid, DNA or RNA, that contains a nucleotide sequence encoding a motif of interest is developed and used as a means of assigning a function to the target nucleic acid. The term "ribozyme" is intended to mean a synthetic RNA molecule that acts as an enzyme and has been engineered to cleave other RNA molecules; after binding selectively to a specific RNA target molecule, it acts catalytically to cut, or cleave, a specific RNA target molecule in a region encoding a motif such as a zinc finger, a protein kinase or an integrin. Ribonucleotide members of the ribozyme library include a binding region which is complementary to a transcription product of the target nucleic acid and a catalytic domain which cleaves a sequence within a transcription product of the target nucleic acid coding for the motif of interest so that expression of the transcription product is disrupted. The binding region generally flanks the catalytic domain. The ribozyme library is introduced into a viral vector such as a retrovirus vector or a plasmid vector which is then used to infect or transfect a host cell culture that is grown to express the ribozyme library; depending upon the system used, the vector can be incorporated into the host cell genome or can be episomal. Optionally, the DNA of the vector is supercoiled. The host cell culture includes at least one host cell and can contain a plurality of host cells. The host cell generally is a mammilian cell but can be a lower or higher plant cell, an invertebrate cell or a bacterial cell. The expression of the ribozyme in the host cell alters the phenotype of the host cell so that a function for the product encoded by the target nucleic acid can be assigned based upon the change in phenotype. The term "function" is intended to mean a detectable or measurable event. The target nucleic acid encodes an expression product that is directly or indirectly involved in a measurable function or phenotype in a host cell containing the target nucleic acid. Generally the expression product is a protein, including signaling molecules and structural proteins. The term "motif" intended to mean a conserved or partially conserved sequence shared by a functionally or structurally related class or family of proteins. The term "phenotype" is intended to mean a characteristic of a specific cell or cell population and includes physical functions such as membrane permeability, physiological functions which include those affected directly or indirectly by regulatory effectors, and biochemical and biological characteristics and functions such as protein synthesis and enzyme activity. The host cell exhibiting an altered phenotype is identified using and isolated using any of a variety of standard techniques. DNA coding for the ribozyme is identified in the DNA isolated from the host cell, conveniently by PCR amplification of the mRNA or genomic DNA coding for the ribozyme using a primer pair derived from vector sequences flanking the ribozyme insert. The PCR product is then sequenced to obtain the sequence of the ribozyme-coding sequence, which not only identifies the biologically active ribozyme, but also the identity of the target nucleic acid.

There are several advantages to the subject invention. By targeting the combinatorial ribozyme library to conserved or partially conserved motifs associated with known functions or properties of proteins or polypeptides containing such motifs, the number of ribozymes that need to be constructed and analyzed is significantly reduced (less than about 100,000) in comparison to a random library (over 16 million). The ability to eliminate the step of amplifying plasmid DNA in bacteria such as E. coli is a major cost saving advantage as well as a time saving advantage over existing technologies: removal of the E. coli amplification can subtract several labor intensive days from the entire process. Furthermore, the subject process lends itself to automation when implemented in a matrix format or a 96-well or similar multi-well format. The simultaneous construction, delivery and expression of multiple members of a combinatorial ribozyme library and their analysis offers the advantage that a large number of ribozymes can be expressed conveniently in host cell cultures, thereby enabling the identification of genes and determining the function of genes by a manageable high throughput screening process in a relatively short period of time. Furthermore, the combinatorial ribozyme library can be constructed with synthetic oligonucleotide DNA which offers the additional advantage that directionality is conveniently achieved by incorporating unique restriction enzyme sites at both ends of each of the oligonucleotides used to prepare the double-stranded DNA coding for these molecules so that double-stranded DNA is ligated to the delivery vector in the correct orientation for expression. This overcomes the problem that if the same restriction enzyme site, blunt ends or restriction enzyme sites comprising compatible cohesive ends are used for the ligation, theoretically about 50% of all the constructs would be ligated in the incorrect orientation. Other advantages of the subject invention include the capability to regulate the magnitude and timing of nucleic acid expression and high throughput delivery. Operatively linking the oligonucleotide DNAs encoding the combinatorial ribozyme library to a regulatable promoter provides temporal and/or cell type specific control throughout the screening assay. Additionally, the magnitude of ribozyme expression can be modulated using promoters that differ in their transcriptional activity.

Ribozyme technology in particular offers several advantages over other methods used to determine which genes are relevant to a disease because as used in the subject invention they are selective for a specific target motif sequence and act catalytically, rather than in a stoichiometric manner. Thus, a single ribozyme molecule can cleave and inactivate up to 100 RNA transcripts, while a single antisense or antipeptide molecule will only inactivate one RNA transcript or one polypeptide. These properties can be used to identify the role of a target genetic sequence and to characterize its cellular function and the function of its encoded product. In the disclosed invention, it is not necessary to develop conformational models of the target nucleic acids to identify regions which are particularly accessible. Such models typically are developed using computer-assisted predictions of possible thermodynamically stable secondary structures. The need for such computer generated models is avoided by creating a combinatorial ribozyme library targeted to nucleic acids encoding a motif of interest. Additionally, sustained expression of ribozyme activity can be achieved by utilizing plasmid or viral based expression constructs driven by cellular promoters in order to constitutively express high levels of ribozymes directed against the target of interest, ensuring sufficient levels of cellular genes are inactivated to cause a detectable change in cellular phenotype or response.

Another advantage to using ribozyme molecules for inactivation of cellular RNA transcripts is that recognition of an mRNA target by the ribozyme molecule requires the complementary base-pairing of only 12-14 nucleotides. Knowledge of the entire sequence of the gene of interest is therefore not necessary. This characteristic, together with the aforementioned ability of ribozymes to function catalytically makes them useful for identifying the roles of genes where only partial sequences are known, as well as the roles of genes where the full length sequence is known.

By constructing combinatorial ribozyme libraries bearing recognition sites derived from DNA or RNA sequences encoding known protein functional motifs, the likelihood that a ribozyme in the library will cleave a transcript involved in a "functional" gene is greatly increased. An additional advantage to this strategy is that more than one combinatorial library can be introduced into host cells simultaneously, allowing isolation of genes containing combinations of specific motifs, which contributes to specificity of the ribozyme for a particular gene. For example, one can isolate with equal ease "genes which are transmembrane protein receptors with intracellular tyrosine kinase domains and SH2 regions" as well as "all genes with kinase function."

The combinatorial ribozyme library is designed by analyzing a consensus nucleotide sequence coding for a protein motif of interest. Motifs of interest are identified by use of scientific literature; public and/or private databases; and other sources (e.g., Prosite: http://expasy/hcuge.ch/) that contain information regarding the relatedness of various proteins based on amino acid sequence homology. Proteins with one or more shared function or class tend to contain similar amino acid patterns or motifs that are common for each class of protein. For example receptor tyrosine kinases, enzymes involved in the transfer of phosphate to tyrosine residues on protein substrates, often contain the amino acid sequence: G-X-H-X-N-[LIVM]-V-N-L-L-G-A-C-T (SEQ ID NO:23) wherein X=any amino acid, and [ ]=containing only one of the amino acids listed within the brackets. Examples of tyrosine kinases that contain this sequence are platelet-derived growth factor, macrophage colony stimulating factor receptor (fns oncogene), stem cell factor receptor (kit oncogene), and vascular endothelial growth factor (VEGF) receptors Flt-1 and Flk-1/KDR. These molecules have been demonstrated to participate in various signal transduction pathways.

The subject invention is designed to identify molecules, previously known or unknown, to have comparable roles in the function of a host cell(s) and to be specifically associated with disease states or phenotypes. Other examples of conserved motifs that are contained in functionally related classes of proteins that are critical for cell function are proteases. For instance, caspase-1, known as interleukin-1 beta converting enzyme (ICE), represents a family of proteases (caspase-1 to 12) involved in apoptosis which has the consensus motifs K-P-K-[LIVMF](4)-Q-A-C-[RQG]-G (SEQ ID NO:24) and H-X(2,4)-[SC]-X(4)-[LIVMF](2)-[ST]-H-G (SEQ ID NO:25). For abbreviations, see supra. Caenorhabditis elegans, ced-3, and Drosophila ICE also contain these motifs.

Other motifs are shared by proteins that have a common structural relationship. For example, the zinc finger motif has been found in a variety of DNA-binding proteins. One zinc finger is known as the C3HC4 domain and has the consensus sequence: C-X-H-X-[LIVMFY]-C-X(2)-C-[LIVMYA] (SEQ ID NO:26). This motif is found in a diverse range of proteins including the BRCA1 protein that is associated with breast cancer, protein RAG-1 that is involved in rearrangement of immunoglobulin and T-cell receptor genes and in RO/SS-A which is associated with lupus and Sjogren's syndrome. Another example is a portion of the integrin family that has the conserved sequence: G-X-[GNQ]-X(1,3)-G-X-C-X-C-X(2)-C-X-C (SEQ ID NO:27). The integrins are involved in cell to cell and cell to matrix adhesion: cellular functions that may be important in metastasis and tumor invasion.

Motifs found in protein kinases, integrins, caspases and zinc-finger domains have been described. The combinatorial ribozyme library, however, can be designed to target the mRNA encoding any protein for which a conserved sequence can be identified. These include enzymes such as proteases, structural proteins and signaling molecules.

Different regions within the same motif can be targeted. In addition, if a family or class of proteins contains more than one motif, multiple motifs also can be targeted. The targeted motifs are not limited to those found in proteins with known mammalian regulatory functions but also can be motifs that have only been identified in other organisms such as yeast, Drosophila, Caenorhabditis elegans. Therefore, human genes critical to disease processes or phenotypes that encode proteins containing motifs similar to those in genes in lower eukaryotes can be identified.

In general, motifs that are derived from highly conserved sequences, are not desirable in making a combinatorial ribozyme library, as the sequence would be present in every potential target. By highly conserved is meant that all amino acids found in a contiguous sequence of amino acids found in a motif are identical. An optimal situation is where several conserved sequence possibilities exist, all of which can contribute to a conserved motif. By conserved is meant that amino acid sequences in a motif are at least 80% and more preferred at least 90% identical. This increases the target specificity of the combinatorial ribozyme pool. In this case, individual ribozymes contained within the library specifically target the production of functionally unique molecules. Ribozymes can be designed to motifs of any length. As the length of a motif increases, different ribozymes can be targeted to nucleotides encoding contiguous conserved or partially conserved amino acid sequences throughout the length of the motif. Generally, a combinatorial ribozyme library is designed to target an RNA encoding a partially conserved amino acid sequence found in a motif of interest. By partially conserved is meant that the amino acid sequences found in a motif are at least 60% identical.

When designing the combinatorial ribozyme library, all combinations of nucleotide sequences that give rise to the chosen motif based on codon degeneracy and usage and the location of the ribozyme cleavage sites are taken into consideration. The target-binding nucleotides of the combinatorial ribozyme library are therefor degenerate. This insures that the ribozyme library can target all possible permutations of the targeted sequence. For expression, both sense and antisense sequences are prepared: the sense strands are annealed to the corresponding antisense strands to form double stranded DNA molecules. When transcribed in a host cell culture, the sense DNA produces RNA which is complementary to an mRNA sequence encoding a motif of interest and contains a catalytic domain designed to leave the mRNA sequence. Each member of a ribozyme library includes two stretches of antisense oligonucleotides, each preferably between 5-9 nucleotides (nt) long and optimally 6 to 8 nucleotides long, to bind to the mRNA, with the sequence forming the catalytic domain or catalytic core in between. The bases immediately adjacent to either side of the catalytic core in the sense strands constitute the ribozyme binding sequence when expressed as RNA that is complementary to a mRNA sequence. The mRNA target contains a consensus cleavage site for the ribozyme. For hammerhead ribozymes the triplet GUC is best but the sequence NUN (N=any nucleotide) also can be targeted. If the catalytic domain is derived from a hairpin ribozyme, the triplet GUC is also preferred (Kashani-Sabet and Scanlon, (1995) Cancer Gene Therapy 2:213-223; Perriman, et al., (1992) Gene (Amst.) 113:157-163; Ruffner, et al., (1990) Biochemistry 29:10695-10702); Birikh, et al., (1997) Eur. J. Biochem. 245:1-16; Perrealt, et al., (1991) Biochemistry 30:4020-4025). Generally, the entire ribozyme-mRNA binding sequence is about 10 to 30 nucleotides in length with 11-17 nucleotides being preferred