TARGETED CLEAVAGE OF RNA USING RIBONUCLEASE P TARGETING AND CLEAVAGE SEQUENCES

Abstract: It has been discovered that any RNA can be targeted for cleavage by RNase P from prokaryotic or eukaryotic cells using a suitably designed oligonucleotide ("external guide sequence", or EGS) to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNase P in vitro. The EGS hydrogen bonds to the targeted RNA to form a partial tRNA like structure including the aminoacyl acceptor stem, the T stem and loop, and part of the D stem. An EGS can be modified both by changes in sequence and by chemical modifications to the nucleotides. The EGS can be a separate molecule or can be combined with an RNase P catalytic RNA sequence to form a single oligonucleotide molecule ("RNase P internal guide sequence" or RIGS). Methods are also disclosed to randomly select and to express a suitable EGS or RIGS in vivo to make a selected RNA a target for cleavage by a host cell RNase P or introduced RIGS, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease- or disorder-causing genes in vivo.

Description: TARGETED CLEAVAGE OF RNA USING RIBONUCLEASE P TARGETING AND CLEAVAGE SEQUENCES Background Of The Invention This invention is in the general area of genetic engineering of nucleic acid sequences, especially chemically modified external guide sequences and catalytic RNA sequences linked to guide sequences. There are several classes of ribozymes now known which are involved in the cleavage and/or ligation of RNA chains. A ribozyme is defined as an enzyme which is made of RNA, most of which work on RNA substrates. Ribozymes have been known since 1982, when Cech and colleagues (Cell 31: 147-157) showed that a ribosomal RNA precursor in Tetrahymena, a unicellular eukaryote, undergoes cleavage catalyzed by elements in the RNA sequence to be removed during the conversion of the rRNA precursor into mature rRNA. Another class of ribozyme, discovered in 1983, was the first to be shown to work in trans, that is, to work under conditions where the ribozyme is built into one RNA chain while the substrate to be cleaved is a second, separate RNA chain. This ribozyme, called Ml RNA, was characterized in 1983 by Altaian and colleagues as responsible for the cleavage which forms mature 5' ends of all transfer RNAs (tRNAs) in E. coli. Analogous RNA-containing enzymes concerned with tRNA synthesis have since been found in all cells in which they have been sought, including a number of human cell lines, though the relevant eukaryotic RNAs have not yet been shown to be catalytic by themselves in vitro. The discovery and characterization of this catalytic RNA is reviewed by Sidney Altaian, in "Ribonuclease P: An Enzyme with a Catalytic RNA Subunit" in Adv. Enzymol. 62: 1-36 (1989). The activity was first isolated from E. coli extracts, and subsequently determined to be a ribonucleoprotein having two components, an RNA component called Ml and a protein component called C5. The RNA cleaved substrates in a true enzymatic reaction, as measured using Michaelis-Menten kinetics. Ml was determined to be solely responsible for substrate recognition and C5 was determined to alter k ^ , but not K M , as reported by Guerrier- Takada et al , Cell 35: 849 (1983) and McClain et al , Science 238: 527 (1987). Sequencing showed that Ml RNA is 377 nucleotides long, M r approximately 125,000, and that the protein consists of 119 amino acids, M r approximately 13,800, as reported by Hansen et al , Gene 38: 535 (1987). Cleavage of precursor tRNA molecules by the RNA component of eubacterial RNase P is described by Guerrier-Takada et al. , Cell 35, 849 (1983) and reviewed by Altaian, Adv. Enzymol. 62: 1 (1989). U.S. Patent No. 5,168,053 entitled "Cleavage Of Targeted RNA By RNase P" to Altaian et al. , discloses that it is possible to target any RNA molecule for cleavage by bacterial RNase P by forming a nucleotide sequence part of which is complementary to a targeted site and which includes a terminal 3'-NCCA, wherein the sequence is designed to hybridize to the targeted RNA so that the bacterial RNase P cleaves the substrate at the hybrid base-paired region. Specificity is determined by the complementary sequence. The sequence is preferably ten to fifteen nucleotides in length and may contain non-complementary nucleotides to the extent this does not interfere with formation of several base pairs by the complementary sequence which is followed by NCCA at the 3' end. As described in WO 92/03566 to Yale University, ribonuclease P (RNase P) from E. coli can cleave oligoribonucleotides that are found in hydrogen-bonded complexes that resemble the aminoacyl stem and include the 5' leader sequence of tRNA precursors, -NCAA. Human RNase P cannot cleave in vitro the 5' proximal oligoribonucleotide in the simple complexes cleaved by RNase P from E. coli, but can do so when the 3' proximal oligoribonucleotide is bound to an external guide sequence (ΕGS) to form a structure resembling portions of a tRNA molecule. The ΕGS can include a complementary sequence to a target substrate of at least eleven nucleotides, seven bases which hydrogen bond to the targeted sequence to form a structure akin to the aminoacyl acceptor stem of a precursor tRNA, and four nucleotides which base pair with the targeted sequence to form a structure akin to the dihydroxyuracil stem. WO 92/03566 does not disclose EGS for prokaryotic RNase P with fewer than seven complementary nucleotides. WO 93/22434 to Yale University discloses an EGS for human RNase P. As described in WO 93/22434, an EGS for human RNase P consists of a sequence which, when in a complex with the target substrate molecule, forms a secondary structure resembling that of a tRNA cloverleaf, or a substantial part of it, and that results in cleavage of the target RNA by RNase P. The sequence of the EGS of WO 93/22434 is derived from any tRNA except that the D stem and aminoacyl stem are altered to be complementary to the target substrate sequence. WO 93/22434 also discloses EGS with either the anticodon loop and stem or the extra loop deleted, and EGS where the sequence of the T loop and stem are changed. WO 93/22434 does not disclose eukaryotic EGS comprising only a region complementary to the target RNA and a region forming a structure similar to only the T stem and loop of tRNA. Neither WO 92/03566 nor WO 93/22434 discloses EGS having chemically modified nucleotides. It is therefore an object of the present invention to provide methods and compositions for specifically cleaving targeted RNA sequences using linked catalytic RNA and minimal guide sequences. It is another object of the present invention to provide chemically modified external guide sequences for RNase P with enhanced resistance to nuclease degradation. It is another object of the present invention to provide a method for selecting external guide sequences, and linked catalytic RNA and guide sequences, that cleave a target RNA with increased efficiency. It is a further object of the present invention to provide methods and compositions for specifically cleaving RNA, both in vitro and in vivo within eukaryotic cells, for the treatment of disease conditions which involve RNA transcription or translation, such as diseases caused by RNA and DNA viruses and expression of excessive or pathogenic proteins from mRNA, or of excessive or pathogenic RNA, itself. Summary Of The Invention Any RNA can be targeted for cleavage by RNase P, using a suitably designed oligonucleotide ("external guide sequence") to form a hybrid with tae target RNA, thereby creating a substrate targeted for cleavage by RNase P. The EGSs contain sequences which are complementary to the target RNA and which forms secondary and tertiary structure akin to portions of a tRNA molecule. A eukaryotic EGS must contain at least seven nucleotides which base pair with the target sequence 3' to the intended cleavage site to form a structure like the amino acyl acceptor stem, nucleotides which base pair to form a stem and loop structure similar to the T stem and loop, followed by at least three nucleotides that base pair with tae target sequence to form a structure like the dihydroxyuracil stem. The EGS can be made more resistant to nuclease degradation by including chemically modified nucleotides or nucleotide linkages. The external guide sequence and the RNase P catalytic RNA can be used together as separate molecules. Alternatively, the two sequences can be combined into a single oligonucleotide molecule possessing both targeting and catalytic functions. Such a combined oligonucleotide, termed an RNase P internal guide sequence (RIGS), increases the kinetic efficiency of cleavage by reducing the number of reactants and by keeping tae targeting and catalytic elements in close proximity. Chemically modifying tae nucleotides and phosphate linkages of EGS molecules and RIGS molecules make tae oligonucleotides more resistant to nuclease degradation. Methods are also disclosed to select RIGS molecules and EGS molecules having increased substrate affinity and utility in vivo to cleave or target cleavage of a selected RNA, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease- or disorder-causing genes in vivo. As described in the examples, an RIGS was constructed by linking a guide sequence to Ml RNA (M1GS RNA). M1GS RNA can act as a sequence-specific endonuclease and can cleave target RNAs taat base pair with the guide sequence just as group I introns do. A custom- designed M1GS RNA cleaves the mRNA that encodes thymidine kinase (TK) of human herpes simplex virus 1 (HSV-1) in vitro. When this M1GS RNA is expressed in mammalian cells in tissue culture, it reduces the level of expression of TK by decreasing tae amount of the target TK mRNA. Brief Description Of The Drawings Figure 1 is the proposed secondary structure of the chimeric substrate used in the selection procedure (Sequence ID No. 1). The italicized sequence is from CAT mRNA and Λe remaining sequence, aside from changes made to assure hydrogen bonding specifically to CAT mRNA, is based on tae sequence of E. coli tRNA T r . The nine nucleotides that were randomized are each indicated by N. Some of the ΕGS analogs for various parts of a tRNA are also depicted: acceptor stem, T loop, anticodon loop, and D-loop. Figure 2 depicts tae general scheme for in vitro selection and amplification of chimeric substrates with enhanced efficiency for directing human RNase P cleavage. "Reverse transcriptase" denotes taat the DNA polymerase capability of reverse transcriptase was used to create double- stranded template DNA from the overlapping DNA oligonucleotides SΕC- 1A (Sequence ID No. 2) and SΕC-1B (Sequence ID No. 3). "RNA-PCR" refers to reverse transcription coupled to PCR. Figure 3 is the sequence and secondary structures of the precursor to tRNA Tyr (Sequence ID No. 4) and a complex of a substrate and ΕGSΔ(1-18) (Sequence ID No. 5), which is derived from tRNA Tyr but lacks the first eighteen nucleotides from tae 5' end of Λe mature tRNA Tyr . pTyr: E. coli tRNA Tyr precursor; pAva: substrate (target) RNA wiΛ 5' leader sequence and Λe first fourteen nucleotides of E. coli tRN A Tyr (Sequence ID No. 6). Figures 4A, 4B, 4C, 4D, 4Ε, and 4F are Λe proposed secondary structures of complexes of CAT mRNA and various EGS. 4A and 4D depict complexes between Λe complementary region CAT mRNA (Sequence ID No. 7) and EGS CAT (Sequence ID No. 8). 4B and 4E are complexes of CAT mRNA (Sequence ID No. 7) and EGS 9 (Sequence ID No. 9). 4C and 4F are complexes of CAT mRNA (Sequence ID No. 7) and EGS CAT ΔAC (Sequence ID No. 10). Hollow arrows denote sites of cleavage by human RNase P. In Figures 4 A, 4B and 4C solid arrowheads denote Λe sites of cleavage by RNase Tl , and arrows denote sites of cleavage by RNase T2. In Figures 4D, 4E and 4F, sites of cleavage by cobra venom nuclease are indicated by solid arrows. Figure 5 shows rates (nmol/min) of human RNase P cleavage of CAT mRNA directed by twelve individual EGS RNAs. Nine individual EGS RNAs: EGS 1 (Sequence ID No. 17), EGS 4 (Sequence ID No. 19), EGS 5 (Sequence ID No. 20), EGS 6 (Sequence ID No. 21), EGS 8 (Sequence ID No. 22), EGS 9 (Sequence ID No. 9), EGS 12 (Sequence ID No. 26), EGS 14 (Sequence ID No. 27), EGS 18 (Sequence ID No. 31), were prepared by in vitro selection. EGS 19 and EGS CAT ΔAC (Sequence ID No. 10) were prepared by in vitro mutagenesis. Results are presented as initial rates (nmol/mol) of cleavage of substrate by RNase P during Λe linear phase of each reaction. Figures 6 A, 6B, and 6C are sequence and proposed secondary structures of EGS for herpes simplex virus Λymidine kinase mRNA (Sequence ID No. 35). Figure 6A is an EGS (Sequence ID No. 36) forming an aminoacyl acceptor stem, T loop and stem, variable loop and stem, anticodon loop and stem, and D stem. Figure 6B is an EGS (Sequence ID No. 37) wherein a G in Λe T loop is substituted for a C in Λe EGS of Figure 6A. Figure 6C is an EGS (Sequence ID No. 38) where Λe anticodon loop and stem of Λe EGS of Figure 6 A is deleted. Figure 7 is a graph of percent TK mRNA expression for cell lines: CL-CAT, CL-109, CL-104, and CL-112. Figure 8 is Λe sequence and structure of HI RNA (Sequence ID No. 11) from Altaian et al , Genomics 18: 418-422 (1993). Figure 9 is a schematic representation of complexes Λat are formed by MIGS RNA (Sequence ID No. 51 and Sequence ID No. 52) and two substrates (Sequence ID No. 53 and Sequence ID No. 54). Figure 10 is a diagram of TK and CaT RNA substrate RNAs. The two 13-nt segments highlighted in Λe TK RNA and CAT RNA sequences (Sequence ID No. 55 and Sequence ID No. 56) are complementary to Λe guide sequences in M1TK13 and M1CAT13 RNAs, respectively. The cleavage sites are indicated by arrows. Figure 11 is a schematic representation of Λe tk46 substrate sequence (Sequence ID No. 57) and Λe structure of RNA enzymes used (Sequence ID No. 58, Sequence ID No. 59, Sequence ID No. 60, Sequence ID No. 61, Sequence ID No. 62, Sequence ID No. 63, Sequence ID No. 64, and Sequence ID No. 65). The arrow marks Λe expected site of cleavage. The sequences shown in [Λe MIGS RNAs] bold type are Λe guide sequences Λat contain Λe 3' CCA sequence and Λe sequence complementary to Λe TK sequence. Figure 12 is a schematic representation of retroviral vectors (ΔMITK, MITK, and NB2) containing MIGS RNAs or an EGS sequence complementary to Λe influenza viral protein PB2. Figure 13 is a graph of levels of expression (percent of control expression) of TK mRNA and protein in CRE cells and in oΛer cell lines. Values are averages of results from four independent experiments. Results from Λe four experiments varied wiΛin 5% in absolute terms. Solid bars: TK mRNA; open bars; TK protein. Detailed Description Of The Invention Guide Sequence As used herein, a guide sequence (GS) is an oligonucleotide Λat targets a substrate RNA molecule for cleavage by a catalytic RNA having Λe activity of RNase P catalytic RNA. A guide sequence may be a separate molecule, termed an external guide sequence, or combined in a single molecule with catalytic RNA. Such a combined molecule is referred to herein as an RNase P internal guide sequence (RIGS). A. Eukaryotic RNase P Targeting Sequence. A guide sequence for human RNase P consists of a sequence which, when in a complex wiΛ Λe target substrate molecule, forms a secondary structure resembling Λat of a tRNA cloverleaf or a part of it. As used herein, Λe term "resembling a precursor tRNA" means a complex formed by Λe GS with target RNA substrate to resemble a sufficient portion of Λe tRNA secondary and tertiary structure to result in cleavage of Λe target RNA by RNase P. The sequence of Λe GS can be derived from any tRNA except Λat the D stem and aminoacyl stem have to be altered to be complementary to Λe target substrate sequence. These altered stems are referred to as recognition arms. The recognition arm corresponding to Λe aminoacyl stem is referred to as Λe A recognition arm and Λe recognition arm corresponding to Λe D stem is referred to as Λe D recognition arm. The remaining portion of Λe guide sequence, which is required to cause RNase P catalytic RNA to interact wiΛ Λe GS/target sequence complex, is herein referred to as RNase P binding sequence. The presence of a 3' -CCA on an EGS enhances Λe efficiency of in vitro reaction with Λe human RNase P by about 35%. The anticodon loop and stem and extra loop can separately be deleted and Λe sequence of Λe T loop and stem can be changed wiΛout decreasing Λe usefulness of Λe guide sequence and, in Λe case of Λe anticodon stem and loop deletion, increases Λe efficiency of Λe reaction by about ten fold. Changes in oΛer parts of an EGS can increase its efficiency about one hundred fold. The desired secondary structure is determined using conventional Watson-Crick base pairing schemes to form a structure resembling a tRNA, Λat is, having structure as described below. The specific sequence of Λe hydrogen bonded regions is not as critical, as long as Λe desired structure is formed. All tRNAs, including tRNAs from a wide variety of bacteria and eukaryotes, conform to Λe same general secondary structure. This is typically written in the form of a cloverleaf, maintained by hydrogen-bonded base pairing between short complementary regions. The four major arms are named for their structare or function: The acceptor arm consists of a 3' terminal CCA QH plus a variable fourth nucleotide extending beyond Λe stem formed by base-pairing Λe 5' and 3' segments of the molecule. The oΛer arms consist of base-paired stems and unpaired loops. The "T" arm is named for the presence of the ribothymidine nucleotide and contains seven unpaired bases in Λe loop. The anticodon arm always contains Λe anticodon triplet in Λe center of Λe loop and consists of seven unpaired bases. The D arm is named for Λe presence of Λe base dihydrouridine in Λe loop, anoΛer of the chemically modified bases in tRNA, and includes between eight and twelve unpaired bases. Positions are numbered from 5' to 3' according to Λe most common tRNA structure, which has 76 residues. The overall range of tRNA lengΛs is from 74 to 95 bases. The variation in lengΛ is caused by differences in Λe structure of two of Λe arms, Λe D arm and Λe extra or variable arm, which lies between Λe T and anticodon arms, which can contain between three and five bases, or between 13 and 21 bases wiΛ a stem of about five bases. The base pairing Λat maintains Λe secondary structure is virtually invariant: Λere are always seven base pairs in Λe acceptor stem, five in Λe T arm, five in Λe anticodon arm, and three or four in Λe D arm. As used herein, a hybrid structure, consisting of an EGS hydrogen bonded to an RNA substrate, having secondary structure resembling a precursor tRNA under conditions promoting cleavage by RNase P of the substrate at Λe nucleotide at Λe 5' end of Λe base-paired region, preferably includes a D stem, an aminoacyl stem, and a T loop and stem, where Λe sequence of Λe latter may be changed compared to Λe sequence and detailed structure found in the parent molecule. A few nucleotides are always found in Λe same positions in 90 to 95% of tRNAs, with some additional nucleotides being semiconserved or semivariant. This is not an absolute requirement in Λe GS, as long as the sequence is complementary to Λe target and forms Λe secondary structure characteristic of Λe tRNA. In fact, Λe sequence forming Λe aminoacyl stem and D loop and stem are changed in Λe GS to be complementary to Λe target RNA. The base paired double-helical stems of Λe secondary structure are maintained in Λe tertiary structure, creating two double helices at right angles to each oΛer. The acceptor stem and Λe T stem form one continuous double helix with a single gap; Λe D stem and Λe anticodon stem form anoΛer continuous double helix, also wiΛ a gap. Many of Λe invariant and semi-invariant bases are involved in Λe tertiary structare. The complementary sequences will generally consist of eleven nucleotides, or, under certain conditions may consist of as few as seven nucleotides, in two blocks which base pair with Λe target sequence and which are separated by two unpaired nucleotides in Λe target sequence, preferably UU, wherein Λe two blocks are complementary to a sequence 3' to Λe site targeted for cleavage. B. Prokaryotic RNase P Targeting Sequence. The requirements for a GS functional wiΛ prokaryotic RNase P are less stringent Λan Λose for a eukaryotic GS. The critical elements of a prokaryotic GS are (1) nucleotide sequence which specifically binds to Λe targeted RNA substrate to produce a short sequence of base pairs 3' to Λe cleavage site on Λe substrate RNA and (2) a terminal 3'-NCCA, where N is any nucleotide, preferably a purine. The sequence generally has no fewer Λan four, and more usually six to fifteen, nucleotides complementary to Λe targeted RNA. It is not critical Λat all nucleotides be complementary, although Λe efficiency of the reaction will vary wiΛ the degree of complementarity. The rate of cleavage is dependent on Λe RNase P, the secondary structure of Λe hybrid substrate, which includes Λe targeted RNA and the presence of Λe 3'-NCCA in Λe hybrid substrate. Ribonuclease P Ribonuclease P is an enzyme consisting of protein and RNA subunits Λat cleaves tRNA precursors to generate Λe 5' termini of tRNAs. This essential enzymatic activity has been found in all cell types examined, boΛ prokaryotic and eukaryotic. During Λe studies on recognition of substrate by RNase P, it was found taat E. coli RNase P can cleave synthetic tRNA-related substrates Λat lack certain domains, specifically, the D, T and anticodon stems and loops, of Λe normal tRNA structure. A half-turn of an RNA helix and a 3' proximal CCA sequence contain sufficient recognition elements to allow Λe reaction to proceed. The 5' proximal sequence of Λe RNA helix does not have to be covalently linked to 3' proximal sequence of Λe helix. The 3' proximal sequence of Λe stem can be regarded as a "guide sequence" because it identifies Λe site of cleavage in Λe 5' proximal region through a base- paired region. RNase P from E. coli and human cells have similar but not identical biochemical properties. Their RNA components have similar secondary structures. However, Λe substrate range of human RNase P is much narrower Λan Λat of Λe E. coli enzyme. For example, alΛough E. coli RNase P can cleave a synΛetic tRNA-related substrate Λat lacks Λree specific domains of Λe normal tRNA structure, Λe human enzyme and Λe structurally similar enzyme from Λe yeast, S. cerevisiae, cannot cleave Λe same substrate. However, Λe E. coli RNase P can cleave a synΛetic tRNA-related substrate Λat is also cleaved by Λe human RNase P. Altaian et al, Genomics 18: 419-422 (1993), describes several mammalian RNase P catalytic RNAs and identifies common features and differences. As used herein, unless otherwise specified, RNase P refers to Λe RNase P in the cell in which the RNA to be cleaved is located, wheΛer endogenous, added to Λe cell, or as used in vitro. Many of Λe techniques described herein are known to Λose skilled in Λe art, as are meΛods for making, and sources of, reagents. The teachings of any references cited herein wiΛ respect to meΛods and reagents are specifically incorporated herein, as well as for Λe purpose of demonstrating Λe scope and level of skill in Λe art. It is not necessary to provide RNase P activity if Λe cleavage is to occur in bacterial cells or intracellularly in Λe nucleus since all eukaryotic cells contain RNase P in Λeir nuclei. RNase P must be supplied if cleavage is to occur in Λe cytoplasm of eukaryotic cells. As used herein for ease of convenience, RNase P refers to Λe ribonucleoprotein consisting of prokaryotic or eukaryotic analogues of Λe E. coli C5 protein and Ml RNA, regardless of source, wheΛer isolated, or produced by chemical synΛesis. The RNA subunit of RNase P also can be transcribed from a gene. The eukaryotic RNase P RNA subunit is referred to as HI RNA. The RNA subunit need not necessarily manifest catalytic activity in Λe absence of protein subunits in vitro. A. Endogenous RNase P. The sequence and proposed secondary structure of HI RNA, Λe RNA component of human RNase P, was reported by Altaian et al. (1993), Λe teachings of which are generally known in Λe art. The sequence and proposed structure of HI RNA is shown in Figure 8 (Sequence ID No. 11). The sequence and proposed secondary structure of Ml RNA, the RNA component of E. coli RNase P, was reported by James et al , Cell 52: 19 (1988), Λe teachings of which are generally known in Λe art. The sequence of Ml RNA is included as Sequence ID No. 40. Because of Λe similarity in secondary structure and substrate specificity among the RNase P's of diverse origin, it is possible to use an EGS designed to maximize efficiency of cleavage for the RNase P in question using techniques described herein to target any RNA in any cell, even Λough Λe catalytically active RNA subunits may have distinctly different sequences. See Altaian, Ann. Rev. Enzymology 62: 1-39 (1989); Altaian, J. Biol. Chem. 265: 20053-20056 (1990). Secondary structure is defined by intramolecular associations of complementary sequences. Base pairs can be canonical, A/U and G/C, or non-canonical, G/U, A/G, etc. B. Exogenous RNA Having Catalytic Activity. An EGS can also be used in combination with an RNA sequence Λat demonstrates enzymatic activity in Λe presence or absence of a protein. That RNA sequence can be represented by a molecule like Λe entire HI or Ml RNA molecule, any functionally equivalent molecule of prokaryotic or eukaryotic origin or derivation, or any portion Λereof shown to have catalytic activity, eiΛer alone or in combination wiΛ a protein. Such a catalytic RNA is referred to herein as RNase P catalytic RNA and its sequence is referred to as an RNase P catalytic sequence. An RNA as described above is considered an RNase P catalytic RNA regardless of source, wheΛer isolated, produced by chemical synΛesis, or transcribed from a gene. As noted above, an EGS effective to convert a targeted sequence into a substrate for human RNase P, will also be effective in making Λe substrate a target for procaryotic RNase P. RNase P catalytic RNA can be derived from naturally occurring RNase P catalytic RNAs, for example, by deleting portions and by making nucleotide substitutions. Such derived catalytic RNAs need only retain enough of the catalytic activity of naturally occurring RNase P catalytic RNA to cleave target RNA. A preferred meΛod of generating RNase P catalytic sequences is by in vitro evolution as described below. There are two principle situations in which RNase P catalytic RNA or RNase P is utilized in combination wiΛ EGS: in vitro in Λe absence of cells or cellular RNase P and in circumstances wherein Λe RNA to be cleaved is located in a portion of a cell not containing endogenous RNase P. In the latter case, Λe genes encoding Λe analogs of Ml RNA and C5 protein, as defined above, or Λe human or oΛer eukaryotic equivalents Λereof, are introduced into Λe cell at Λe desired location for cleavage using a suitable vector or oΛer meΛod known to Λose skilled in Λe art for introduction and expression of a gene in a cell. RNase P Internal Guide Sequences A guide sequence and Λe catalytic RNA subunit of an RNase P can be linked to form a single oligonucleotide molecule possessing boΛ Λe targeting function of an EGS and cleavage function of RNase P catalytic RNA. Such a combination, in a single oligonucleotide molecule, is referred to as an RNase P internal guide sequence (RIGS). An RIGS can be used to cleave a target RNA molecule in Λe same manner as EGS. RIGSs can be formed by linking a guide sequence to an RNase P catalytic sequence by any suitable means. For example, an EGS and RNase P catalytic RNA can be prepared as separate molecules which are Λen covalently linked in vitro. Alternatively, a complete RIGS can be synΛesized as a single molecule, eiΛer by chemical synΛesis, or by in vt ' trσ or in vivo transcription of a DNA molecule encoding linked GS and RNase P catalytic sequence. The linkage between Λe GS and RNase P domains of an RIGS can have any form Λat allows Λe domains to cleave a target RNA. For example, Λe two domains could be joined by an oligonucleotide linker. Preferably, Λe linker will be composed of an ordinary nucleotides joined by phosphodiester bonds. The GS and RNase P catalytic sequence components can be joined in eiΛer order, wiΛ Λe RNase P catalytic sequence linked to eiΛer Λe 3' end or 5' end of Λe GS component. RIGSs can be used for cleavage of target RNA boΛ in vitro and in vivo. In vitro, Λe RIGS can function wiΛout RNase P protein components in vitro, alΛough activity of Λe RIGS can be increased by Λe addition of RNase P protein components. In vivo, endogenous RNase P proteins will stimulate activity of the RIGS. The activity of boΛ prokaryotic- and eukary otic-based RIGSs are expected to be enhanced by the presence of eiΛer prokaryotic or eukaryotic RNase P protein components. Method For Producing EGSs And RIGSs Having Enhanced Efficacy EGSs and RIGSs having enhanced binding affinity as measured by decreased energy of binding can be designed by in vitro evolution. Such a method can be used to identify RNA molecules wiΛ desired properties from pools of molecules Λat contain randomized sequences. As demonstrated more clearly in the examples, appropriately modified, Λese meΛods can be used for Λe isolation of efficient EGSs and RIGSs. These new EGSs, when complexed with an exemplary target RNA, CAT mRNA substrate (Sequence ID No. 7), allow cleavage of Λe target by human RNase P at rates similar to Λose achieved wiΛ natural substrate. The general selection scheme is depicted in Figure 2. In each round of selection, the pool of RNAs is digested wiΛ human RNase P, or with Λe RIGS, and Λe cleaved products are isolated by electrophoresis and Λen amplified to produce progeny RNAs. One of Λe template- creating oligonucleotides is used as Λe 5' primer for Λe polymerase chain reaction (PCR) in order to allow restoration of Λe promoter sequence and Λe leader sequence of Λe chimeric RNA for Λe next cycle of selection. The stringency of selection is increased at each cycle by reducing Λe amount of enzyme and the time allowed for Λe cleavage reaction, such Λat only Λose substrates Λat are cleaved rapidly by Λe enzyme are selected. In Λe first Λree rounds of selection, RNA substrates are digested wiΛ an appropriate amount of human RNase P, for example, 3.6 units, or Λe equivalent activity of Λe RIGS. One unit of human RNase P is defined as Λat amount of enzyme Λat cleaves 1 pmol of precursor to tRNA Tyr from E. coli in 30 min at 37°C. For assays in subsequent rounds of selection, Λe amount of enzyme is reduced, and Λe incubation time is shortened so Λat less Λan 20 percent of Λe substrate is cleaved. Cleavage products are separated from uncleaved substrates by electrophoresis and RNA extracted. The purified cleavage product RNAs are reverse transcribed and amplified by PCR. The double-stranded DNA generated by PCR regains Λe promoter sequence and Λe leader sequence from Λe sequence in Λe primer, and is Λen used as a template for transcription of RNA for Λe next round of selection. After eight cycles of selection, Λe resulting pool of double-stranded DNAs is cloned into an appropriate vector and sequenced. In order to test Λe abilities of ΕGSs or RIGSs derived from Λe individual variants, sequences corresponding to Λe GS segment of each chimeric tRNA are amplified by PCR, and RNAs transcribed wiΛ an appropriate RNA polymerase. RNA cleavage by Λe selected ΕGS or RIGS is Λen assayed. Sequences in common in Λe most active ΕGS and RIGSs are Λen determined and new ΕGS and RIGSs designed. As described in Λe examples, simulation of evolution in vitro was used to select ΕGSs Λat bind strongly to a target substrate mRNA and Λat increase Λe efficiency of cleavage of Λe target by human ribonuclease P to a level equal to Λat achieved wiΛ natural substrates. The most efficient ΕGSs from tRNA precursor-like structures wiΛ Λe target RNA, in which Λe analog of Λe anticodon stem has been disrupted, an indication Λat selection for Λe optimal substrate for ribonuclease P yields an RNA structure different from Λat of present-day tRNA precursors. Chemically Modified ΕGS And RIGS AlΛough chemically unmodified oligoribonucleotides can function as effective ΕGS or RIGS in a nuclease-free environment, Λe shorter half-life in serum and inside cells reduces Λeir effectiveness as Λerapeutics. Chemical modifications can be made which greatly enhance Λe nuclease resistance of EGS and RIGS wiΛout compromising Λeir biological function of inducing or catalyzing cleavage of RNA target. For example, one or more of Λe bases of an EGS or RIGS construct can be replaced by 2' meΛoxy ribonucleotides or phosphoroΛioate deoxyribonucleotides using available nucleic acid synΛesis meΛods. SynΛesis meΛods are described by, for example, Offensperger et. al. , EMBO J. 12: 1257-1262 (1993); PCT WO 93/01286 by Rosenberg et al. (synΛesis of sulfurthioate oligonucleotides); Agrawal et al. , Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988); Sarin et al , Proc. Natl. Acad. Sci. USA 85: 7448-7794 (1989); Shaw et al , Nucleic Acids Res 19: 747- 750 (1991) (synΛesis of 3' exonuclease-resistant oligonucleotides containing 3' terminal phosphoroamidate modifications); all of which are hereby incorporated herein by reference. It is well documented in Λe current literature Λat degradation of oligonucleotide analogues is mainly attributable to 3'-exonucleases. Several studies have also demonstrated Λat various 3 '-modifications can greatly decrease Λe nuclease susceptibility of Λese analogues. Thus, anoΛer meΛod to reduce susceptibility to 3' exonucleases is introduction of a free amine to a 3' terminal hydroxyl group of Λe EGS or RIGS molecule, as described, for example, by Orson et al. , Nucl. Acids Res. 19: 3435-3441 (1991). Furthermore, cytosines Λat may be present in Λe sequence can be meΛylated, or an intercalating agent, such as an acridine derivative, can be covalently attached to a 5' terminal phosphate to reduce Λe susceptibility of a nucleic acid molecule to intracellular nucleases. Examples of Λis are described in Maher et al. , Science 245: 725-730 (1989) and Grigoriev et al , J. Biol. Chem. 267: 3389-3395 (1992). Another class of chemical modifications is modification of Λe 2' OH group of a nucleotide 's ribose moiety, which has been shown to be critical for Λe activity of Λe various intracellular and extracellular nucleases. Typical 2' modifications are Λe synΛesis of 2'-O-MeΛyl oligonucleotides, as described by Paolella et al, EMBO J. 11: 1913-1919 (1992), and 2'- fluoro and 2'-amino-oligonucleotides, as described by Pieken et al , Science 253: 314-317 (1991), and Heidenreich and Eckstain, J. Biol. Chem 267: 1904-1909 (1992). Portions of EGS and RIGS molecules can also contain deoxyribonucleotides. Such substitations improve nuclease resistance by eliminating Λe critical 2' OH group. Application Of EGS And RIGSs As Laboratory Or Clinical Reagents External guide sequences and RIGSs have applications as in vitro reagents, in a similar fashion to restriction enzymes, and as Λerapeutic agents, for cleavage and inactivation of specific host cell RNA or RNA coded for by pathogenic organisms such as bacteria or viruses, as demonstrated by the following examples. As used herein, an EGS or RIGS is an oligonucleotide molecule. It is to be understood, however, Λat for Λerapeutic purposes, a DNA molecule encoding an EGS molecule or encoding an RIGS molecule could be utilized. Accordingly, unless oΛerwise specified, administration of an EGS or RIGS encompasses boΛ Λe RNA molecule Λat hydrogen bonds to a target nucleic acid sequence Λat is cleaved, as well as a DNA molecule encoding Λe RNA molecule, which is expressed under conditions wherein Λe RNA molecule functions as an EGS or RIGS. An external guide sequence can be brought into contact wiΛ any RNA having sequences complementary to Λe EGS, in Λe presence of RNase P, and Λe RNA will be cleaved at Λe targeted site. In Λis manner, Λe activity of endogenous RNase P in any cell, such as Λe RNase P of human cells, can be directed to destroy specific messenger, viral or oΛer RNAs by Λe use of an appropriate EGS RNA. A. Reagents For In vitro Applications. DNA restriction endonucleases are invaluable reagents for Λe molecular biologist. Patterns of restriction fragment sizes are used to establish sequence relationships between DNA molecules, and large DNAs can be cleaved to give fragments of sizes useful for genetic engineering, sequencing, and studying protein binding. RNA processing enzymes can be utilized under conditions such Λat Λey also cleave RNA with considerable sequence specificity. Specific ribozymes can be prepared by combining Λe specific guide sequence with RNase P or functional equivalents Λereof . In Λe preferred embodiment, Λe external guide sequence and Λe RNase P catalytic RNA are separate; alternatively, the two sequences can be joined eiΛer directly or via a linker. The linker can be any molecule Λat can be covalently bound to oligonucleotides. Numerous linkers are known to Λose of skill in Λe art. A preferred linker is an oligonucleotide because it allows direct synthesis of Λe complete RIGS. B. Therapeutics. 1. Determination and Preparation of Complementary Sequences. Any cellular gene product expressed as RNA, including proteins encoded by mRNA and structural RNAs Λemselves, can be targeted for inactivation by RNase P, or directly by an RIGS using sequences engineered to include appropriate regions of sequence and/or - structare for binding to Λe targeted RNA and Λe desired site of cleavage. The cellular gene product could be a product of an oncogene wiΛ an altered sequence, such as Λe ras gene product; where Λe product is not a normal cell component, a viral protein, such as one encoded by an essential gene for HIV replication; or a bacterial protein. In many cases, Λe critical genes an infective or paΛological agent have been isolated and sequenced. Appropriate complementary sequences can be synthesized using standard techniques, reagents, and equipment based on Λese known sequences. 2. Preparation of an appropriate pharmaceutical composition for delivery of the EGS or RIGS to the targeted RNA. There are two primary mechanisms for delivering Λe EGS or RIGS to intracellular RNA Λat has been targeted for cleavage: diffusion and via a vector. As discussed above, any RNA Λat is important in a disease process can be targeted and appropriate complementary sequences made synΛetically or by copying cloned sequence. For example, cancer regulatory genes can be targeted. Since RNase P is predominantly found in Λe nucleus of eukaryotic cells, Λe infectious agents most likely to be inhibited by administration of appropriate EGS to Λe infected cells are Λose in which critical RNA sequences are transcribed in Λe nucleus. Important examples of Λe viral agents Λat replicate in Λe cell nucleus include herpes viruses, including herpes simplex virus, varicella-herpes zoster virus, cytomegalovirus, and Epstein-Barr virus; hepatitis B virus; adenoviruses; paramyxo viruses such as measles; and Λe retroviruses, such as human immunodeficiency virus, HIV I, HIV II, HIV III and HTLV-1. RIGSs should cleave target RNA in any area of Λe cell since Λe catalytic activity is self-contained. 3. Vector-mediated delivery of EGS and RIGSs. Preferred vectors are viral vectors such as Λe retroviruses which introduce EGS and RIGS directly into Λe nucleus where it is transcribed and released into Λe nucleus. Under Λe appropriate conditions, Λe EGS or RIGS will hybridize to Λe targeted RNA and Λe endogenous RNase P or RIGS will cleave Λe hybridized RNA at Λe 5' side of Λe hybrid region. MeΛods for using retroviral vectors for gene Λerapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286, and PCT application PCT/US89/03794 and PCT/US89/00422, Λe teachings of which are incorporated herein. Defective retroviral vectors, which incorporate Λeir own RNA sequence in Λe form of DNA into Λe host chromosome, can be engineered to incorporate EGS and RIGSs into Λe host, where copies will be made and released into Λe cytoplasm to interact with Λe target nucleotide sequences. EGS and RIGSs are particularly useful as a means of targeted Λerapy into hematopoietic cells of patients suffering from virus-induced disease of Λose cells, such as AIDS. The most efficacious meΛodology presently available for the introduction of specific genetic sequences into human cells involves Λe use of RNA-containing retroviruses which serve as vehicles or vectors for high efficiency gene transfer into human cells. RNase P-based Λerapy can also be used as a means of preventing Λe spread of HIV-1 and or providing a HIV-1 resistant population of T-cells Λat will be able to confer immune function to infected individuals. Patients who have been recently diagnosed as having antibodies to HIV-1, but who do not yet show AIDS symptomatology, are Λe preferred candidates for therapy. This procedure necessitates removal of some of Λe patient's bone marrow stem cells and subsequent partial cytoblation. The removed cells can be treated in Λe laboratory wiΛ appropriate EGS or RIGS compositions, using appropriate viral vectors, such as defective viral vectors, and Λen restored to Λe same individual. The treated cells will develop in Λe patient into mature hematopoietic cells, including T-cells. These T-cells will have normal immune function and, most importantly, will be intracellularly immunized to prevent Λeir destruction by any HIV-1 still present in Λe patient. Bone marrow stem cells and hematopoietic cells are relatively easily removed and replaced and provide a self-regenerating population of cells for Λe propagation of transferred genes. RNase P-based Λerapeutics allow the selective inactivation of oΛer unwanted genes in cells, such as activated oncogenes, involved in Λe causation and maintenance of cancer cells. In contrast to Λe approaches presently in use which are aimed at preventing or limiting infection wiΛ HIV, it should be possible to use RNase P-based technology to treat, and possibly to cure, HIV infection, and related diseases of white blood cells which are subject to transformation by retroviral vectors carrying EGS or RIGS. Particular examples of diseases Λat may be treated using EGS and RIGS include not only HTLV-1, but also various retroviral-induced leukemias resulting from chromosomal translocations Λat produce chimeric RNAs which produce proteins Λat are unique to Λose cells and Λat can act as growΛ stimulators or oncogenes. OΛer types of transformed tissues Λat might be treatable include all cancer cells carrying identified oncogenes of known sequence. 4. Topical and other EGS and RIGS compositions for local administration. EGS and RIGS may also be administered topically, locally or systemically in a suitable pharmaceutical carrier. Remington 's Pharmaceutical Sciences, 15Λ Edition by E.W. Martin (Mark Publishing Company, 1975), Λe teachings of which are incorporated herein by reference, discloses typical carriers and methods of preparation. EGS and RIGS may also be encapsulated in suitable biocompatible microcapsules, microparticles or microspheres formed of biodegradable or non- biodegradable polymers or proteins or liposomes for targeting to phagocytic cells. Such systems are well known to Λose skilled in Λe art and may be optimized for use wiΛ Λe appropriate EGS and RIGSs. Therapeutically Λe oligoribonucleotides are administered as a pharmaceutical composition consisting of an effective amount of Λe EGS or RIGS to inhibit transcription of a targeted RNA and a pharmaceutically acceptable carrier. Examples of typical pharmaceutical carriers, used alone or in combination, include one or more solid, semi-solid, or liquid diluents, fillers and formulation adjuvants which are non-toxic, inert and pharmaceutically acceptable. Such pharmaceutical compositions are preferable in dosage unit form, Λat is, physically discreet units containing a predetermined amount of Λe drug corresponding to a fraction or multiple of Λe dose which is calculated to produce Λe desired Λerapeutic response. It is essential Λat Λe oligonucleotides be delivered in a form which prevents degradation of all of the oligonucleotide before it reaches Λe intended target site. A preferred embodiment is an EGS or an RIGS administered as a viral vector, encoding Λe EGS or RIGS, or in a liposome, such Λat an effective amount of EGS or RIGS is delivered. Generally Λese will produce a concentration between 1 μM and 1 mM at Λe site of Λe cells to be treated. Such compounds and compositions can be formulated as a topical composition, for example, for application to a viral lesion such as Λat produced by herpes simplex virus. These will generally contain between 1 μM and 1 mM oligonucleotide per unit of carrier, or produce a concentration between 1 μM and 1 mM at Λe site of Λe cells to be treated. Oral compositions, although not preferred, are in Λe form of tablets or capsules and may contain conventional excipients. AnoΛer preferred composition is a polymeric material applied locally for release of EGS or RIGS. Still anoΛer preferred composition is a solution or suspension of Λe EGS or RIGS in an appropriate vector in combination wiΛ conventional pharmaceutical vehicles are employed for parenteral compositions, such as an aqueous solution for intravenous injection or an oily suspension for intramuscular injection. For clinical applications, Λe dosage and Λe dosage regimen in each case should be carefully adjusted, utilizing sound professional judgment and consideration of Λe age, weight and condition of Λe recipient, Λe route of administration and Λe nature and gravity of Λe illness. The present invention, EGS and RIGSs, will be further understood by reference to Λe following non-limiting examples. Example 1: Cleavage of tRNA precursor fragments by human RNase P in the presence of EGS. An EGS Λat can target RNA for cleavage by human RNase P was prepared using a small RNA fragment, pAva (Sequence ID No. 6, Figure 3) which contains a 5' precursor sequence and Λe first fourteen nucleotides from Λe 5' terminus of a tRNA. The leader sequence of E. coli tRNA Tyr precursor can be cleaved correctly when anoΛer piece of RNA, for example, EGSΔ1-18 (Sequence ID No. 5), which lacks Λe first eighteen nucleotides of Λe 5' terminus of mature tRNA Tyr but retains Λe remaining 3' proximal sequence, is hybridized to Λe target RNA. Human RNase P was partially purified from HeLa cells using Λe meΛod of Bartkiewicz et al. , Genes and Development 3: 488-499 (1989). The substrates were prepared by in vitro transcription in Λe presence of [α- 32 P]GTP. [α:- 32 P]GTP labelled pAva (Sequence ID No. 6) I RNA (28 nt) was mixed wiΛ unlabelled EGS RNA and Λe mixture was incubated at 37°C in 50 mM Tris-Cl (pH 7.5), 100 mM NH 4 C1, and 10 mM MgCl 2 wiΛ enzyme for 30 min. Labelled pAva (Sequence ID No. 6) I RNA alone was also incubated wiΛ or wiΛout enzyme. Analysis by gel electrophoresis shows Λat Λe EGS plus enzyme resulted in cleavage of Λe RNA. The 3' proximal oligonucleotide is Λe external guide sequence. Because Λe lengths of Λe leaders and Λeir sequences, as well as Λe sequences of Λe mature domain, are not conserved among different precursor-tRNAs, Λe main determinants for human RNase P cleavage must be in some of Λe conserved structural features of various tRNAs. This general idea is borne out by Λe fact Λat several oΛer EGSs Λat did not mimic exactly Λe structure of parts of a tRNA did not target complementary RNAs. Examples of such changes include changes in Λe number of possible base pairs in Λe D or amino acyl stems, changes in positions 8 and 9 of Λe mature tRNA sequence, and a change from cytosine to uracil at position 57. However, an EGS Λat lacked Λe anticodon stem and loop, or Λe variable stem and loop, resulted in efficient cleavage, indicating Λat Λese parts of Λe EGS, separately, were not essential for recognition of Λe target complex by Λe enzyme. Accordingly, if an mRNA, raΛer Λan part of a precursor tRNA sequence, is incorporated into Λe double-stranded stem region of a putative target complex, and Λe resulting hybrid contains Λe structural features required of a substrate for human RNase P activity, Λe mRNA will be cleaved by human RNase P. Example 2: Specific cleavage of CAT mRNA in vitro by human RNase P using an external guide sequence. Examples provided below show Λe efficiency of cleavage of Λe mRNA for chloramphenicol acetyltransferase (CAT) by human RNase P. The sequence of Λe 5' oligoribonucleotide, as well as Λat of Λe EGS, depends on Λe choice of target site in Λe mRNA. The presence of Λe appropriately designed EGS efficiently reduces CAT enzymatic activity in vivo, as well as promotes cleavage of CAT mRNA in vitro, indicating Λat Λis meΛod should be of general use for gene inactivation. An EGS custom-designed for Λe mRNA for chloramphenicol transacetylase (CAT), shown in Figure 4A, EGS CAT , Sequence ID No. 8), can direct Λe specific cleavage of CAT mRNA by human RNase P in vitro or in vivo cells in tissue culture. However, Λe cleavage reaction is inefficient compared to cleavage of natural tRNA precursor substrates. The proposed secondary structure of a complex of CAT mRNA (Sequence ID No. 7) and EGS CAT (Sequence ID No. 8) resembles Λe tRNA cloverleaf structure, but it includes sequences not normally found in Λe tRNA from which it was originally derived, namely, tyrosyl tRNA (tRNA Tyr ) (Sequence ID No. 6) of Escherichia coli. To ensure that appropriate tertiary interactions Λat facilitate Λe process of enzyme- substrate recognition exist in Λe complex, parts of Λe EGS Λat participate in tertiary interactions in Λe analogous tRNA structures were changed in two ways. First, four nucleotides in Λe equivalent of Λe T loop and five in Λe equivalent of Λe variable loop were randomized by incorporation of equimolar quantities of Λe deoxyribonucleotides dA, dG, dC and T into a DNA template to yield an initial population of 2.6 x 10 5 sequence variants. Second, during each round of selective amplification, random mutations were introduced by performing polymerase chain reaction (PCR) at an error rate of approximately 0.1 percent per nucleotide incorporated, using the meΛod of A. Beaudry and G.F. Joyce, Science 257: 635 (1992). The mRNA for Λe gene for chloramphenicol acetyltransferase (CAT) can be easily manipulated on plasmids and Λe enzymatic activity is readily expressed in tissue culture cells so it was used as a target substrate. As demonstrated below, an EGS can target CAT mRNA (Sequence ID No. 7) for specific cleavage by human RNase P. Figure 4 A shows a complex in which an EGS, EGS CAT (Sequence ID No. 8), could base-pair wiΛ nucleotides 67 to 79 of CAT mRNA (Sequence ID No. 7), where Λe first nucleotide of Λe translation initiation codon is numbered 1, and direct human RNase P to cleave Λat mRNA at nucleotide 67. The EGS CAT (Sequence ID No. 8) construct was derived from Λe E. coli tRNA Tyr (Sequence ID No. 6) gene where Λe first eighteen nucleotides from Λe 5' terminus have been deleted and Λe sequences on Λe D-loop and acceptor-loop have been changed to make base-pairs with CAT mRNA. The EGS CAT (Sequence ID No. 8) fused upstream wiΛ a T7 promoter was cloned into a pUC19 vector. The EGS CAT RNA (Sequence ID No. 8) was prepared through in vitro transcription wiΛ T7 RNA polymerase. The Ht/zdlll-βαmHI fragment of CAT gene (pCAT™, Promega) was cloned in pGem-2. The plasmid was truncated wiΛ EcσRI and a 260 nucleotide-long transcript was obtained by in vitro transcription wiΛ T7 RNA polymerase in Λe presence of [o-- 32 P]GTP. The EGS sequence was synΛesized by polymerase chain reaction, using Λe E. coli tRNA Tyr gene as template, wiΛ oligonucleotide GCCAAACTGAGCAG ACTC (Sequence ID No. 12) and GCGCggtaccAAAAATGGTGAGG CATGAAGG (Sequence ID No. 13). The bold letters in Λe oligonucleotide sequences indicate Λe bases needed to make base pairs to CAT mRNA, the underlined letters indicate Λe sequence complementary to Λe transcription termination signal, and Λe lower case letters shows an extra linker sequence. The sequence GCGC at Λe 5' end of Λe second oligonucleotide are extra nucleotides. The PCR fragment was digested wiΛ H dlll and cloned into pUC19 wiΛ a T7 promoter upstream from Λe EGS sequence. The EGS CAT RNA (Sequence ID No. 8) was transcribed wiΛ T7 RNA polymerase after the plasmid was linearized wiΛ Dral. A mixture of unlabelled and [α- 32 P]GTP labelled CAT mRNA fragment, 0.2 pmole in total, was mixed wiΛ Λe EGS CAT RNA (Sequence ID No. 8) in amounts of 4 pmole, 1 pmole, 1 pmole, 0.4 pmole and 0.2 pmole. Each mixture was incubated at 37 °C in 50 mM Tris-Cl (pH 7.5), 100 mM NH 4 C1, and 25 mM MgCl 2 with enzyme for one hour. The reaction was stopped by addition of an equal volume of dye solution wiΛ excess EDTA and Λen subjected to a 5% polyacrylamide-7 M urea gel. CAT mRNA alone was incubated without and wiΛ enzyme and loaded on Λe gel. Primer extension analysis determining Λe precise site of EGS CAT -directed cleavage by human RNase P was conducted as follows. A reverse transcription reaction was performed on Λe uncleaved and cleaved CAT mRNA using an oligodeoxyribonucleotide GGCCGTA ATATCCAGCTGAACGG (Sequence ID No. 14), complementary to nucleotides 129 to 107 of CAT mRNA. The reaction was incubated in 100 mM Tris-Cl (pH 8.3), 10 mM KC1, 6 mM MgCl 2 , 10 mM DTT and 2 units AMV reverse transcriptase at 46 °C for 2 hours. Labelled G, A, U, C were used as reference analyses of DNA sequences corresponding to Λe CAT mRNA template. The precise site of cleavage of CAT mRNA was determined by primer extension analysis using an oligodeoxyribonucleotide primer complementary to nucleotides 129 to 107 of Λe RNA, showing Λat Λe cleavage occurs between nucleotides 66 and 67, as expected. The results of Λe EGS CAT RNA-directed cleavage of CAT mRNA were analyzed by gel electrophoresis. In Λe presence of EGS CAT (Sequence ID No. 8) molecules, CAT mRNAs were cleaved to give rise to two products wiΛ the expected size. Analysis of Λe products of Λe reaction showed Λat Λe end groups contained 5' phosphoryl and 3' hydroxyl termini, Λe same as Λose normally generated by RNase P. The results show conclusively Λat the specific cleavage of CAT mRNA is due to an EGS-directed RNase P hydrolytic reaction. For up to five-fold molar excess of EGS CAT RNA to mRNA, Λe cleavage efficiency is proportional to Λe amount of EGS CAT added. However, more Λan ten-fold excess of EGS CAT molecules caused a decrease in Λe cleavage efficiency. One explanation for Λis is Λat EGS CAT alone inhibits Λe enzymatic activity by competing wiΛ Λe mRNA-EGS complex for Λe enzyme. The reaction proceeds in a linear fashion for more Λan 3 hours at 37 °C. Denaturation and reannealing of Λe oligonucleotides in Λe target complex did not improve Λe efficiency of cleavage. The reaction has an absolute requirement for Mg 2+ wiΛ an optimal concentration of 25 mM, in contrast to Λat wiΛ tRNA Tyr precursor as substrate, which has an optimal Mg 2+ concentration of between 2 and 10 mM. Example 3: Inhibition of Expression of CAT activity in green monkey CV-1 cells by EGS CAT . In order to test wheΛer Λe EGS can function in vivo, Λe EGS CAT sequence (Sequence ID No. 8) was inserted downstream of a mouse U6 snRNA gene promoter in a BLUESCRIPT™ (Stratagene, La Jolla, CA) vector forming pEGS CAT . The EGS CAT sequence (Sequence ID No. 8) can be transcribed by RNA polymerase III and Λe transcription can terminate at a T 5 cluster following Λe EGS sequence in eiΛer SI 00 extract or living cells. Green monkey fibroblast cells CV-1 were cotransfected wiΛ pCAT and pEGS CAT plasmids. After transfection wiΛ pCAT, which encodes Λe CAT gene, and pEGS CAT , cells were harvested and CAT activity was assayed. CV-1 cells were maintained in Dulbecco's modified Eagle medium Λat contained 10% fetal calf serum. One day prior to transfection cells were split 1: 10 and plated in 60 mm Petri plates. Two hours prior to transfection cells were fed wiΛ 4.5 ml of fresh medium wiΛ 10% fetal calf serum. Transfection was performed by Λe calcium phosphate precipitation procedure using 2.5 μg of pCAT DNA and various amounts of pEGS CAT DNA, ranging from one to 6.25 μg. Twenty-four hours after transfection, cells were harvested and cell extracts were assayed for CAT activity. The extract from cells cotransfected wiΛ EGS CAT construct apparently decreased Λe conversion of chloramphenicol to its acetylated forms. The degree of inhibition was measured quantitatively by counting of Λe spots excised from a TLC plate. EGS CAT cotransfection produced greater Λan 50% inhibition compared to Λe control wiΛ no EGS CAT cotransfection. There was substantial loss of ability to inhibit CAT expression when a higher ratio of pEGS CAT to pCAT was introduced. Similar experiments yielding approximately 70% of CAT activity have also been performed wiΛ human cells in tissue culture. Example 4: Preparation of EGS with altered sequence that enhance degradation of target RNA by RNase P. As explained in detail below, two classes of EGS were designed, as shown in Figures 4A, 4B, 4C, 4D, 4E, and 4F. The first class involves deletions of large segments of Λe EGS as suggested in Example 1 and described in Examples 2 and 3. For example, it has been found Λat Λe anticodon loop and stem can be deleted, as shown in EGS CAT ΔAC (Sequence ID No. 10) of Figure 4C and 4F, and Λe EGS can still promote cleavage by RNase P of a target RNA (CAT mRNA containing Sequence ID No. 7 in Figures 4C and 4F). The anticodon loop and part of Λe variable loop can alternatively be deleted from Λe EGS, and Λe EGS will promote cleavage wiΛ greater efficiency Λan Λe parent EGS molecule. The most efficient EGS of Λis deletion class was Λe one in which Λe anticodon stem and loop was deleted. This EGS CAT ΔAC (Sequence ID No. 10) promoted cleavage by human RNase P of a target mRNA (CAT mRNA) at a rate 10-fold higher Λan Λe parent EGS. The deletion of both variable and anticodon stems and loops, however, does not yield a more efficient EGS, alΛough Λe variable loop can consist of only one or two nucleotides and still be highly efficient. The second class of EGSs have changes in boΛ Λe equivalent of Λe T loop, Λe variable loop, and Λe anticodon stem of Λe tRNA-like segment of Λe EGS. Three such EGSs, described below, are EGS 6, EGS 8 and EGS 9 (Sequence ID No. 9, Figures 4B and 4E). As shown below, EGS 9 is Λe most efficient of Λe EGSs in Λese examples and directs cleavage by human RNase P of a target RNA (CAT mRNA) at a rate approximately fifty to one hundred fold greater Λan Λe parent EGS. These results apply to relative rates of cleavage at a particular site in a target mRNA. The absolute rates of cleavage at any particular site still depend on access of Λe EGS to Λat particular site. Example 5: Preparation of RNAs with randomized nucleotides. A. Preparation of Chimeric Covalently Linked mRNA-EGS Substrate. The procedure to select for EGSs Λat are more efficient in guiding RNase P to Λe target CAT mRNA involves Λe synΛesis of a population of chimeric, covalently linked mRNA-EGS substrates which are Λen run Λrough cycles of in vitro mutation and selection for molecules which can serve as substrates for RNase P. Double-stranded DNA templates were made by annealing of and enzymatically extending two overlapping synΛetic oligonucleotides: TAATACGACTCACTATA GAACATTTTGAGGCATTTCAGTCAGTTGGCCAAACTGAGCAGAC (SEC-1A, Sequence ID No. 2) and TGGTGAGGCATGAAGGNN NNGAACCTTCNNNNNGCAGATTTAGAGTCTGCTCAGTTTGGCC (SEC-IB, Sequence ID No. 3), where Λe complementary sequences are underlined and Λe randomized nucleotides (N) were introduced during Λeir machine synΛesis by incorporating equimolar quantities of four nucleotides. These sequences create a chimeric tRNA gene which contains sequences from CAT mRNA and tRNA Tyr from E. coli as well as nine nucleotides (N) Λat are randomized. A promoter for T7 bacteriophage RNA polymerase is included in SEC-IA (Sequence ID No. 2). The extension was carried out with AMV reverse transcriptase at 46 °C for two hours. Variant RNA pools were prepared by transcription wiΛ T7 polymerase in 40 mM Tris.Cl, pH 7.9, 6 mM MgCl 2 10 mM ditaioΛreitol 2 mM spermidine 1 mM NTPs containing 20 μCi [α- 32 P]GTP at 37 °C. B. The Selection Procedure. The general selection scheme is described above. One of Λe template-creating oligonucleotides (SEC-IA, Sequence ID No. 2) was also used as Λe 5' primer for Λe polymerase chain reaction (PCR) in order to allow restoration of Λe T7 promoter sequence and Λe leader sequence of Λe chimeric RNA for Λe next cycle of selection. The stringency of selection was increased at each cycle by reducing Λe amount of enzyme and Λe time allowed for Λe cleavage reaction, such Λat only Λose substrates Λat were cleaved rapidly by Λe enzyme were selected. In the first Λree rounds of selection, RNA substrates were digested wiΛ 3.6 units of human RNase P, purified Λrough Λe glycerol gradient step described by Yuan and Altaian, Proc. Natl. Acad. Sci. USA 89: 8006-8010 (1992) and Bartkiewicz et al , Genes and Dev. 3: 488 ' (1989)) in 50 mM Tris-Cl (pH 7.5), 10 mM MgCl 2 , and 100 mM NH 4 C1 at 37°C for 2 hours. One unit of human RNase P is defined as that amount of enzyme that cleaves 1 pmol of precursor to tRNA T r from E. coli in 30 min at 37 °C. For assays in subsequent rounds of selection, Λe amount of enzyme was reduced, and Λe incubation time was shortened so Λat less Λan 20 percent of Λe substrate was cleaved. Cleavage products were separated from uncleaved substrates by electrophoresis on an eight percent polyacrylamide-7 M urea gel. RNA was extracted from Λe gels by Λe crush and soak meΛod. The purified cleavage product RNAs were reverse transcribed and amplified by PCR with SEC-IA (Sequence ID No. 2) and SEC-IC (TGGTGAGGCATGAAGG, Sequence ID No. 15) as primers wiΛ a Perkin Elmer RNA PCR kit. The double-stranded DNA generated by PCR regained Λe T7 promoter sequence and Λe leader sequence from Λe sequence in Λe primer SEC-IA, and it was Λen used as a template for transcription of RNA for Λe next round of selection. C. Characterization of the selected RNAs and EGS RNA derived from them. After eight cycles of selection Λe resulting pool of double- stranded DNAs were cloned into Λe BLUESCRIPT™ vector (Stratagene, La Jolla, CA) vector. Eighteen plasmid DNAs were sequenced using Sequenase 2.0 (U.S. Biochemicals, Cleveland, OH). In order to test the abilities of EGSs derived from Λe individual variants selected above for CAT mRNA targeting, sequences corresponding to Λe EGS segment of each chimeric tRNA were amplified by PCR using primers SEC-IA (Sequence ID No. 2) and SEC-IT (TAATACGACTCACTATAGGCCAACTGAGCAGAC, Sequence ID No. 16), which contains a promoter sequence for T7 polymerase, and RNAs were transcribed wiΛ T7 RNA polymerase. EGS-directed CAT mRNA cleavage was assayed in 10 μl of 50 mM Tris-Cl, pH 7.5, 10 mM MgCl 2 100 mM NH 4 C1 containing 0.25 pmol (1000 cpm) of substrate RNA and 1 or 5 pmol of EGS RNAs. Reaction mixtures were incubated at 37 °C for 30 min wiΛ 10 units of RNase P from HeLa cells, followed by electrophoresis in 5% polyacrylamide/7 M urea gels. The gels showed a species of RNA migrating in Λe position expected for cleavage of substrate RNA in Λose lanes where Λe newly selected EGSs have been included in Λe reaction mixtures. D. Sequence Analysis of Randomized EGSs. Sixteen individual clones were sequenced. The sequence of Λe anticodon stem/loop, Λe variable (V) loop, and Λe T stem/loop are shown in Figure 6. From Λe sequence Λat had been randomized in Λe T-loop, two particular sequences were most frequently selected: UUCGUGC, found in seven clones, and UUCGCCC, also found in seven clones. The T loop sequences of Λe two remaining clones contained single transition mutations of Λe two major sequences, UUCGUCC and UUCACCC. By contrast, no significant sequence-related bias was seen in Λe sequence of five nucleotides in Λe variable loop. In addition to Λe sequences in Λe T and variable loops Λat were selected from Λe totally randomized sequence, a considerable number of mutations were introduced into Λe EGS in Λe chimeric substrates as a consequence of Λe conditions for PCR. Some of Λese mutations were beneficial and, Λerefore, Λe sequences Λat included Λem were selected and accumulated. In almost all of Λe individual selected clones, Λe integrity of base-pairing in Λe anticodon stem was disrupted. Table 1 shows Λe partial sequences of Λe anticodon loop/stem region, Λe variable (V) loop, and Λe T stem/loop region of Λe parent chimeric mRNA-EGS CAT substrate (P), nucleotides 28 through 88 of Sequence ID No. 1. Table 1 also shows partial sequences of EGS segments of some individual chimeric mRNA-EGS substrates, nucleotides 1 Λrough 60 of Sequence ID No. 9 and Sequence ID Nos. 17 Λrough 31, which were obtained as a result of Λe in vitro selection procedure. Nucleotides Λat differ from Λe parent chimeric mRNA-EGS CAT substrate sequence are given in bold letters and are underlined. Hyphens indicate deletions. The remaining part of Λe sequence of Λe mRNA-EGS chimeric substrates is shown in Figure 4A. Numbering of Λe partial sequences is not uniformly consecutive, since some clones did not have appropriate inserts and only sixteen selected sequences are listed. Table 1: Substrate # D stem/ Anticodon V loop T stem/ AA stem (ID No. ) loop stem/loop loop 19 30 47 52 69 SCE-1 GGCCAAACUGA GCAGACUCUAAAUCUGC NNNNN GAAGGUUCNNNNCCUUC AUGCCUCACCA (28-88 of 1) #1 (17) GGCCAAACUGA GCAGACUCUAAAUCGGC CCUUC GAAGGUUCGCCCCCUUC AUGCCUCACCA #2 (18) GGCCAAACUGA GCAGACUCUAAAUCUGC ACGAGA GAAGGUUCGUGCCCUUC AUGCCUCACCA #4 (19) GGCCAAACUGA GCAGACUCUAAACUGGC CUAAC GAAGGUUCGCCCCCUUC AUGCCUCACCA #5 (20) GGCCAAACUGA GCAGACUCUAAAU-UGC CCAAC GAAGGUUCACCCCCUUC AUGCCUCACCA #6 (21) GGCCAAACUGA GCAGACUCCAAAUC--C ACCAA GAAGGUUCGUGCCCUUC AUGCCUCACCA #8 (22) GGCCAAACUGA GCAGACUCUAAA-CUCC UCCCA GAAGGUUCGUGCCCUUC AUGCCUCACCA #9 GGCCAAACUGA GCAGACUCUAAAUC-GC AAACG GAAGGUUCGUGCCCUUC AUGCCUCACCA (1-60 of :9) #10 (23) GGCCAAACUGA GCAGACUCUAAAUCGGC CUACG GAAGGUUCGCCCCCUUC AUGCCUCACCA #11 (24) GGCCAAACUGA GCAGACGCUAAAUCUAC CCCGU GAAGGUUCGUCCCCUUC AUGCCUCACCA #12 (25) GGCCAAACUGA GCAGACUCUAAAUUUGC CACCA GAAGGUUCGCCCCCUUC AUGCCUCACCA #13 (26) GGCCAAACUGA GCAGACUC-AAAUCUGGC CAUUC GAAGGUUCGCCCCCUUC AUGCCUCACCA w #14 (27) GGCCAAACUGA GCAGACUCUAAAUC-GC AGUGU GAAGGUUCGUGCCCUUC AUGCCUCACCA -e- #15 (28) GGCCAAACUGA GCAGACUCUAAAUCAGC GCGUG GAAGGUUCGUGCCCUUC AUGCCUCACCA #16 (29) GGCCAAACUGA GCAGACUCUAAAUCGGC CGCAC GAAGGUUCGCCCCCUUC AUGCCUCACCA #17 (30) GGCCAAACUGA GCAGACACUAAAUUUGC ACGAG GAAGGUUCGCCCCCUUC AUGCCUCACCA #18 (31) GGCCAAACUGA GCAGACCCUAAAUCUGC CCCCG GAAGGUUCGUGCCCUUC AUGCCUCACCA E. Synthesis and analysis of individual EGS RNAs based on selected chimeric substrates. The best chimeric substrate selected was clone 9 whose corresponding RNA sequence is partially shown in sequence 9 (nucleotides 1 through 60 of Sequence ID No. 9) in Table 1. The clone 9 chimeric substrate was cleaved about 5.5 times more efficiently Λan Λe parent, non-randomized, chimeric substrate, mRNA-EGS CAT chimera. Using Λe sequences of Λe selected chimeric substrates, nine individual corresponding EGS RNAs were synthesized: EGS-1 (Sequence ID No. 17), EGS-4 (Sequence ID No. 19), EGS-5 (Sequence ID No. 20), EGS-6 (Sequence ID No. 21), EGS-8 (Sequence ID No. 22), EGS-9 (nucleotides 1 Λrough 60 of Sequence ID No. 9), EGS-12 (Sequence ID No. 25), EGS-14 (Sequence ID No. 27) and EGS-18 (Sequence ID No. 31), in order to probe Λe function of Λese selected EGSs in directing RNase P to Λe target CAT mRNA. Each of Λe individual EGS RNAs was mixed wiΛ 32 P-labeled CAT mRNA and Λe mixtures were Λen exposed to RNase P. Every selected EGS RNA increased Λe initial rate of Λe cleavage reaction, as measured during Λe linear phase of Λe reaction, over Λat wiΛ EGS CAT , and cleavage occurred at Λe expected site in Λe target mRNA, as demonstrated by Figure 5. The most dramatic improvement in rates occurred wiΛ Λe EGS sequence based on clone 9, EGS 9 (nucleotides 1 Λrough 60 of Sequence ID No. 9), which directed cleavage of Λe CAT mRNA at an overall rate more Λan 30 times faster Λan Λat observed wiΛ EGS CAT in Λe complex. The Λree most efficient EGSs tested, derived from clone 6, clone 8 and clone 9, all had a common sequence, UUCGUGC, in Λe T loop. F. Secondary structural analysis of mRNA-EGS complexes. The proposed secondary structure of Λe complex of CAT mRNA and EGS 9 (Sequence ID No. 9) (Figure 4B) can be compared wiΛ Λe parent CAT mRNA-EGS CAT complex (Figure 4A). The structures of CAT mRNA-EGS complexes were confirmed in part by partial digestion with RNases Tl and T2 under conditions Λat allowed formation of Λe mRNA-EGS complex and identification of single- stranded regions in RNA. Partial digestion of CAT mRNA-EGS complexes wiΛ RNases Tl and T2 (Pharmacia) were performed in RNase P assay buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , and 100 mM NH 4 C1). The reaction mixture contained substrate RNA labeled wiΛ [ 32 P] at its 5' terminus (2000 cpm), 0.2 mg/ml of rat 5S RNA as carrier, and three different concentrations, 2 x 10 "4 , 1 x 10 "3 and 5 x 10 ~3 units/ml, of RNase Tl or RNase T2. Reactions were incubated at room temperature for 5 min. The samples were analyzed by PAGE in 12.5% sequencing gels. Double-stranded regions were identified by digestion wiΛ cobra venom nuclease (Pharmacia, Alameda, CA). Conditions for digestion wiΛ cobra venom nuclease were as described above for RNase Tl and T2 except Λat incubation was at 37 °C. The sites of cleavage by Λe various nucleases in CAT mRNA- EGS CAT complex are indicated by solid arrows in Figures 4A and 4D. The tRNA domain in Λis structure is very similar to Λat found in a natural tRNA. The results obtained wiΛ cobra venom nuclease indicate Λat Λe first few nucleotides in Λe analog of Λe D loop are involved in a tertiary interaction, presumably wiΛ nucleotides in Λe variable loop (Figure 4D). This interaction is eiΛer absent or much less extensive in Λe complex wiΛ EGS 9 (Sequence ID No. 9) (Figure 4E) and, indeed, Λe same region in Λe analog of Λe D loop is susceptible to attack by RNases Tl and T2, an indication Λat it is in a single-stranded conformation (Figure 4B). This result, togeΛer wiΛ Λe appearance of new sites of susceptibility to attack by cobra venom in Λe anticodon loop of EGS 9 confirm Λat Λis EGS endows Λe complex of Λe EGS and CAT mRNA wiΛ new tertiary interactions Λat enhance Λe rate of cleavage by RNase P. The results of cleavages by nucleases also confirmed Λat Λe anticodon stem in EGS 9 was disrupted as a result of a single nucleotide deletion. EGSs 9 and 14 are not similar to each oΛer in terms of Λeir efficiency in directing RNase P to a target substrate. The only difference in Λeir nucleotide sequences is in Λe variable loop, as shown by Table 1. This difference alone must account for Λe relative inefficiency of EGS 14 in terms of targeting ability. Furthermore, digestion wiΛ RNase Tl of Λe complex Λat contained EGS 9 revealed strong protection of Λe last nucleotide, G, in Λe variable loop from attack by RNase Tl, as shown by Figure 4B. The role of Λis G may be similar to Λat played by nucleotide 57 in tertiary interactions in tRNA molecules; namely, Λe G may form hydrogen bonds wiΛ a nucleotide in Λe CAT mRNA sequence to ensure folding of Λe "tRNA" domain of Λe mRNA sequence. G. Anticodon stem and loop of an EGS decrease cleavage rate of RNase P and are dispensable. An independent study of recognition of precursor tRNA substrates by human RNase P showed Λat Λe anticodon stem and loop form a dispensable structural feature in Λe recognition of substrates by human RNase P. To determine wheΛer Λe anticodon stem and loop may act in a negative fashion on Λe overall rate of cleavage of target RNA by RNase P, two more EGS RNAs were constructed, one being a deletion mutant Λat lacked Λe equivalent of Λe anticodon stem and loop, EGS CAT ΔAC (Sequence ID No. 10), as compared to Λe parent EGS CAT (Figure 4A), and Λe oΛer, EGS 19, being a derivative of EGS 9 in which Λe structure of Λe anticodon stem was restored. DNA coding for EGS CAT ΔAC was synΛesized by PCR wiΛ pEGS CAT DNA, described by Yuan et al. , Proc. Nat. Acad. Sci. USA 89: 8006 (1992), as template. Oligonucleotide EC-1ΔAC (GCCAAACTG ACGTCATCGACTTCG, Sequence ID No. 32) and M13 reverse primer (AACAGCTATGACCATG, Sequence ID No. 33) were used as primers. The DNA generated by PCR was digested wiΛ H dIII and Λen inserted into pUC19 downstream from a T7 RNA polymerase promoter sequence. EGS CAT ΔAC RNA (Sequence ID No. 10) was prepared by transcription in vitro after Λe new plasmid DNA had been linearized wiΛ Dral. DNA Λat coded for EGS 19 was synΛesized by PCR procedure in a manner similar to Λat used for Λe synΛesis of DNA for EGS 9, wiΛ oligonucleotides SEC-IC (Sequence ID No. 15) and SEC-1I (GTAATACGACTCACTATAGGCCAAACTGAGCAGACTCTAAATCT_ GCAAACGGAAGGTTC, Sequence ID No. 34): Λe newly inserted T residue in SEC- II is underlined. The DNA was transcribed in vitro wiΛ T7 RNA polymerase to give EGS 19 RNA. EGS 19 (Sequence ID No. 39) RNA differs from EGS 9 RNA only in Λe additional U Λat restores Λe structure of Λe anticodon stem. In EGS CAT ΔAC (Sequence ID No. 10), Λe lengΛ of EGS CAT was reduced by 25 % : Λe shorter deletion mutant directed cleavage of target RNA about six times more efficiently Λan Λe parent EGS, as shown by Figure 5. Restoration of Λe anticodon-stem structure, as in EGS 19, reduced Λe rate of cleavage of Λe target RNA wiΛ EGS 19 to four times lower Λan Λat wiΛ EGS 9. These results, togeΛer wiΛ Λe measurement of Λe rates of reaction with EGSs selected in vitro, indicate a significant inverse correlation between Λe efficiency of an EGS in Λe cleavage reaction and Λe existence of an anticodon stem in EGS RNA. H. Stability of EGS-mRNA complexes. The stability of EGS-mRNA complexes was measured based on boΛ Λe binding constants between Λe mRNA and each EGS and Λe dependence on Mg 2+ ions of Λe cleavage reaction, presuming Λat relatively high concentrations of Mg 2+ ions were needed to stabilize relatively unstable complexes. The dissociation constants (Kj) of mRNA- EGS complexes were measured directly by a gel mobility shift assay in polyacrylamide gels Λat contained 10 mM Mg 2+ ions according to Λe meΛod of Pyle et al , Proc. Natl. Acad. Sci. USA 87: 8187-8191 (1990). A fragment of CAT mRNA, 160 nucleotides in lengΛ, was prepared by transcription wiΛ T7 RNA polymerase in Λe presence of [α- 32 P]GTP. 10 μ\ EGS RNA in 2X binding buffer was heated at 80 °C for 4 min before it was mixed wiΛ an equal volume of 2 nM CAT mRNA fragment in water. IX binding buffer contains 50 mM Tris-Cl (pH 7.5), 10 mM MgCl 2 , 100 mM NH 4 C1, 3% glycerol, 0.05% xylene cyanol. The mixtures were incubated at 37 °C for 20 min and immediately separated on 5% polyacrylamide gels at 9 watts. The electrophoresis buffer consisted of 36 mM Tris base, 64 mM HEPES, 0.1 mM EDTA, 10 mM MgCl 2 (pH 7.5 wiΛout any adjustment). Quantitation of free target RNA and of Λe complex was performed wiΛ a Betascope (Betagen, WalΛam, MA). The free energies of binding were determined from Λe equation ΔG° = -RT ln(l/K d ), where R = 0.00198 kcal/mol and T = 310.15°K. The dissociation constants for selected EGSs are shown in Table 2. Table 2: Dissociation constants of EGSs. Substrates K d K m V raax V max /K m (nM) (nM) (nmol/min) xlO "5 xlO 6 pTyr 10 2-9 2^ CAT mRNA + EGS CAT 880 120 2.9 0.24 (Sequence ID No. 8) + EGS CAT ΔAC 20 150 11.4 0.76 (Sequence ID No. 10) + EGS-5 210 125 16.3 1.30 (Sequence ID No. 20) + EGS-8 25 125 21.3 1.70 (Sequence ID No. 22) + EGS-9 78 125 30.0 2.40 (nucleotides 1-60 of Sequence ID No. 9) + EGS-19 710 130 12.5 0.96 Table 2 shows Λe kinetic parameters of EGS-directed cleavage of CAT mRNA in vitro by RNase P from HeLa cells. K d refers to measurements of Λe dissociation constant for binding of EGS to CAT mRNA. The oΛer parameters were determined in standard assays of enzyme kinetics. N^ is Λe value obtained wiΛ 0.5 ml (0.6 units) of human RΝase P. pTyr refers to Λe precursor to tRΝA Tyr from E. coli. The results shown in Table 2 indicate Λat Λe K d values of in vitro selected EGSs are 4 to 40 times lower Λan Λat of Λe parent EGS. Thus, Λe selected EGSs had higher affinity for Λe target RNA Λan did EGS CAT . The chimeric substrate derived from clone 9 was cleaved by RNase P at a rate only about 1.5 times faster Λan was Λe target in Λe mRNA-EGS 9 complex, an indication Λat Λe ability of Λe EGS to bind tightly to Λe target RNA in solution must be a critical determinant in Λe efficiency of Λe substrate complexes. The differences in K d values between complexes wiΛ EGS CAT (Sequence ID No. 8) and Λe selected EGSs correspond to Λe contribution of -1 to -2.4 kcal/mol to Λe free energy of binding (ΔG°) wiΛ selected EGSs (ΔG°) is -8.5 kcal/mol for Λe complex wiΛ EGS CAT , -10.1 for Λe complex with EGS 9 (Sequence ID No. 9) and -10.9 for Λe complex wiΛ EGS CAT ΔAC (Sequence ID No. 10), Λus revealing new interactions in Λe selected EGS-mRNA complexes. Deletion of the anticodon stem from EGS CAT resulted in the K,, for EGS CAT ΔAC being 44 times lower, and restoration of Λe stem of EGS 9 (EGS 19) resulted in a K d Λat was ten times higher Λan Λat for EGS 9 and was close to Λat of EGS CAT (Table 2). Thus, Λe intact anticodon stem stabilized a conformation of Λe EGS Λat could bind as strongly to Λe target RNA as does an EGS wiΛ no organized anticodon stem. Accordingly, Λe enhancement of Λe ability of Λe selected EGSs for targeting RNA can be assigned, in part, to Λe increase in Λe strengΛ of Λeir binding to target RNA. Cleavage of mRNA directed by Λe original EGS CAT requires Mg 2+ ions wiΛ an optimum concentration of 25 mM. By contrast, Λe reaction wiΛ certain selected EGSs, namely, EGS 6 (Sequence ID No. 21) and EGS 9 (nucleotides 1 Λrough 60 of Sequence ID No. 9), proceeds optimally in 2 to 10 mM Mg 2+ ions. This latter concentration is close to Λe optimal concentration of Mg 2+ ions for processing of tRNA precursors by human RNase P, reported by Doersen et al. , J. Biol. Chem. 260: 5942 (1985). Since high concentrations of Mg 2+ ions are especially effective in Λe neutralization of repulsion between adjacent regions of Λe phosphate backbone and Λe stabilization of RNA folding, Λe results indicate Λat Λe selected EGSs can achieve Λe appropriate folded structures in Λe complex with target RNAs wiΛout Λe aid of high concentrations of Mg 2+ ions. A kinetic analysis was performed to determine Λe Michaelis constant (K m ) and Λe maximum velocity (V ^ ) of Λe enzymatic reactions. The cleavage of Λe precursor to tRNA Tyr from E. coli and of CAT mRNA in mRNA-EGS complexes was assayed at various substrate concentrations boΛ above and below the K m for Λese substrates. Aliquots were withdrawn from reaction mixtures at regular intervals and analyzed on polyacrylamide-urea gels. Values of K m and V^ were obtained from Lineweaver-Burk double-reciprocal plots. The effective concentrations of substrate used were calculated as Λe concentration of Λe complex of target mRNA wiΛ EGS as determined from Λe Kj values shown in Table 2. The K m for the precursor to tRNA Tyr (pTyr) is 10 nM wiΛ human RNase P, whereas Λe K m value of Λe complex of mRNA- EGS^ 1' is twelve-fold higher (Table 2). The K m for all Λe selected EGSs tested was the same as Λat of EGS CAT . However, Λe maximum velocities of Λe reactions wiΛ selected EGSs were up to ten times higher Λan Λat wiΛ Λe original EGS. Thus, Λe value of V ^ /IC.,, for selected EGSs was increased. The value of V ^ /K ^ of EGS 9, was for example, ten-fold higher Λan Λat of EGS CAT and was very close to Λat of Λe tRNA T r precursor. When Λe anticodon stem of EGS 9 was restored, however, as it was in EGS 19, V, ^ fell about 2.5-fold, suggesting Λat Λe rate of release of Λe product was specifically reduced by physical interactions of Λe enzyme wiΛ an intact anticodon stem. These data show Λat Λe enhanced abilities for targeting of Λe selected EGSs, as measured in Λe overall rate of Λe cleavage reaction, were due to boΛ enhanced affinity of binding to substrate RNAs and to increases in Λe velocity of Λe enzymatic reaction. Example 6: Inhibition of viral mRNA expression with human ribonuclease P. Herpes simplex virus was used to demonstrate Λat EGS can be used to target a viral gene in vivo to inhibit viral replication. Herpes simplex viruses are DNA-containing viruses Λat infect cells, induce synΛesis of messenger RNAs, which are transcribed to produce enzymes related to DNA synΛesis and breakdown: including Λymidine kinase, DNA polymerase and a DNA exonuclease, and viral DNA and viral structural proteins are made and assembled into infectious viral particles. The structure and organization of Λe herpes simplex virus genome is known, for example, as reported by Roizman, Cell 16: 481-494 (1979). The nucleotide sequence of Λe Λymidine kinase gene of herpes simplex type 1 was described by Wagner et al. , Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981). An EGS, shown in Figure 6 A, was designed to target Λe mRNA encoding Λymidine kinase (TK). The target site for RNase P cleavage is about 25 nucleotides downstream from Λe TK translation initiation site. An EGS forming an approximately Λree-fourΛs-like tRNA was designed and shown in vitro to cleave Λe TK sequence at Λe proposed cleavage site. Two oΛer EGSs, which contain a single point mutation in Λe T-loop (C to G) or deletion of Λe anti-codon region, shown in Figures 6B and 6C, respectively, were constructed based on Λe results observed in Example 5. Cell lines and EGS expression vectors were Λen constructed. Five cell lines were constructed by transfecting plasmid DNAs into human 143TK-cells, which can be obtained from Λe American Type Culture Collection, Rockville, MD. Plasmid pFL116 was constructed using pGEM-7Z (Promega, Wisconsin) and incorporating a gene for neomycin resistance (Neo), which is commercially available from Clontech or Stratagene, CA. EGS DNA (Figures 6A, 6B, and 6C) was digested wiΛ Kpnl and inserted into Λe plasmid pmU6(-315/l), described by Yuan et al. , Proc. Natl. Acad. Sci. USA 89: 8006-8010 (1992) and Das et al , EMBO 7: 503-512 (1988), at Λe Pstl (blunted) IKpnl site. This plasmid contains Λe promoter for Λe gene for U6 small nuclear RNA, a very strong promoter, and a signal for termination of transcription (T cluster) by RNA polymerase II. EGS plasmids were designated pFL104, pFL109, and pFL112, respectively. The plasmid based on sequence 9 in Table 1 was used as a control. The cells were stably transfected using a calcium phosphate precipitation meΛod described by Wigler et al. , Proc. Natl. Acad. Sci. USA 76: 1373-1376 (1979), wiΛ the pFLllό to yield CL116, plasmids EGS 9 and pFLllό to yield CLCAT, plasmids pFL104 and pFLllό to yield CL104, plasmids pFL109 and pFLllό to yield CL109, and plasmids pFL112 and pFLllό to yield CL112, followed by neomycin selection. Cells were cloned, expanded and RNA isolated. BoΛ total and cytoplasmic RNA was isolated and the RNase protection meΛod described in "Molecular Cloning: A Laboratory Manual, Second Edition" Sambrook et al. , (Cold Spring Harbor Laboratory Press, 1989) at pages 7.71-7.78. This is an extremely sensitive assay where digestion of RNA: RNA hybrids formed using radiolabelled probe is used to assess which cell clones express Λe EGS. The results indicated Λat Λe EGSs are expressed in boΛ nuclei and cytoplasm. Cells were Λen infected wiΛ herpes simplex virus using a multiplicity of infection (MOI) of 1 to 1.5, specifically, 1 to 1.5 million viral particles/ 1 million cells, in order to resemble a natural infection wiΛ virus. RNA was harvested at 4, 8 and 12 hours post-infection. The internal control probe was used to detect Λe mRNA levels of HSV α47 and late genes U s 10 and U s ll. The probe is selected to assure Λe detection of a high level of viral mRNA expression over Λe entire cycle of viral infection. The results are shown in Figure 7. TK mRNA expressed was decreased 0% in Λe control CL-CAT, 30% in CL-109, 45% in CL-104, and 65% in CL-112. Example 7: RNase P Internal Guide Sequences. A. Construction of plasmids, catalytic RNAs and RNA substrates for studies in vitro. DNA templates for transcription in vitro of RNA substrates tk7, tk46, and cat7 were constructed by annealing Λe T7 promoter containing oligonucleotide, OliT7 (5'-TAATACGACTCACTATAG-3') (Sequence ID No. 41) wiΛ oligo-nucleotides Olitk7 (5'-CGCAGAC GGTCCTATAGTGAGTCGTATTA-3') (Sequence ID No. 42), OliTK46 (5'-ACCGCCGCAGCCTGGTCGAACGCAGACGCGTGTTGATGGCAG GGGTCTATAGTGAGTCGTATTA-3') (Sequence ID No. 43), and Olicat7 (5-ATGCCTCGGTCCTATAGTGAGTCGTATTA-3') (Sequence ID No. 44), respectively. Plasmids pTK117 and pTK146 are derivatives of pUC19 and were described by Guerrier-Takada and Altman, Proc. Natl. Acad. Sci. USA 89: 1266-1270 (1992). The DNA sequences coding for Ml RNA (pTK117) and mutant Ml RNA wiΛ a deletion from nucleotides 167 to 377 (pTK146), are under Λe control of Λe T7 RNA polymerase promoter. The DNA templates for M1TK19, M1TK16, M1TK13, M1TK10, M1TK5, and M1CAT13 were constructed by Λe polymerase chain reaction (PCR) wiΛ Λe gene for Ml RNA as found in plasmid pTK117 wiΛ OHT7 as Λe 5' primer oligonucleotide and 3' primers Λat contained Λe appropriate guide sequences. The 3' primers were OHTK19 (5'-GTGGTGTCTGCGTTCGACCAGGCTATGAC CATG-3') (Sequence ID No. 45), OliTKlό (5'GTGGIGTCTGCGTTC GACCAGTATGACCATG-3') (Sequence ID No. 46), OliTK13 (5'-GTGGTGTCTGCGTTCTATGACCATG-3') (Sequence ID No. 47), OliTKlO (5'-GTGGΪGTCTGCGTTCTATGACCATG-3') (Sequence ID No. 48), OliTK5 (5'-GTGGTGTCTGTATGACCATG-3') (Sequence ID No. 49), and OliCAT13 (5'-G GGTGAGGCATTTCAGTTATGAC CATG-3') (Sequence ID No. 50). The 3' proximal sequences of 10 no serve as Λe primers for Λe PCR wiΛ Λe pUC19 sequence. The underlined sequences and Λe bold sequences correspond to Λe 3'CCAC sequence and Λe guide sequences, respectively. The DNA template for ΔM1(167-377)TK13 RNA was constructed by PCR with Λe sequence for Ml RNA in pTK146 and primers OHT7 and OUTK13. The DNA template for EGS TK16 was constructed by PCR wiΛ Λe 5' primer OliT7 and Λe 3' primer OUTK16 for DNA template pUCT7, which was derived from pUC19 by insertion of a T7 promoter sequence into Λe Bamϊ site. The CAT mRNA fragment of 550 nt was synΛesized wiΛ T7 RNA polymerase and EcoRI-digested pCAT-1 plasmid DNA (Promega Inc.) while the TK mRNA fragment of 450 nt was synΛesized from plasmid pTKlOl DNA in which Λe sequence for TK mRNA is under control of Λe T7 promoter. B. Assays for cleavage by MIGS RNA. RNA enzyme (20 nM) and substrate (50 nM), eiΛer uniformly labeled wiΛ [o.- 32 P]GTP or 5' end-labeled with [7- 32 P]ATP, were incubated for 30 mins at 37°C or 50°C in buffer A (50 mM Tris, pH 7.5, 100 mM NH 4 C1, 100 mM MgCl 2 ) or buffer B (50 mM Tris, pH 7.5, 100 mM NH 4 C1) Λat contained MgCl 2 at various concentrations. Reactions were stopped by the addition of 8 M urea and Λe cleavage products were Λen separated on eiΛer 15% or 20% polyacrylamide gels Λat contained 8 M urea. C5 protein and human RNase P protein were purified from E. coli and HeLa cells, respectively, as described previously by Vioque et al , J. Mol. Biol. 202: 835-848 (1988), and Bartkiewicz et al , Genes & Dev. 3: 488-499 (1989). The RNase P holoenzyme from E. coli was assembled by mixing Ml RNA and C5 protein at a molar ratio of 1:20. Assays to determine kinetic parameters under single- and multiple-turnover conditions were performed as described previously by Guerrier-Takada et al , Cell 35: 849-857 (1983), and Liu and Altman, Cell 11: 1083-1100 (1994). Cleavage was assayed at various concentrations of substrate, in 2- to 20-fold excess over enzyme concentration, boΛ above and below Λe K m for Λe substrate. Aliquots were wiΛdrawn from Λe reaction mixtures at regular intervals and Λe cleavage products were separated in polyacrylamide-urea gels, quantitation was carried out wiΛ a phosphorimager (Molecular Dynamics). The values of K m and k cat were obtained from Lineweaver- Burk double reciprocal plots. In single turnover experiments, trace amounts of substrates were used and Λe concentrations, 1 nM, were much lower Λan Λe K m ( > 80 nM). The concentration of enzyme ranged from 5 nM to 200 nM. The observed rate of cleavage (k„ bs ) was determined, and Λe value of k^/K m was obtained from Λe equation k cat /K,-. = k obs /tE], where [E] is Λe concentration of Λe enzyme. C. Viruses, cells and antibodies. The properties of HSV-l(F), a prototype of human herpes simplex virus 1 have been described by Ejercito et al. , J. Gen. Virol. 2: 357-364 (1968). The retroviral vector LXSN, retroviral packaging cell lines, PA317 (amphotropic) and ι/*CRE (ectopic), and Λeir maintenance and propagation were described by Miller and Rosman, BioTechniques 1: 980-990 (1989), and Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85: 6460-6464 (1988). The rabbit polyclonal antibody against Λymidine kinase of HSV-1 (F) was described by Liu and Summers, Virology 163: 638-642 (1988), and Λe mouse monoclonal antibody MCA406 against HSV-1 ICP35 protein was described by Liu and Roizman, J. Virol 65: 206-212 (1991), and purchased from Harlan Bioproducts for Sciences Inc. (Indianapolis, IN). The secondary antibodies used in Λe Western blots, anti-rabbit or anti-mouse IgGs conjugated with horseradish peroxidase, were purchased from Vector Laboratories Inc. and Bio-Rad Inc., respectively. D. Construction of plasmids for studies in vivo. The promoter sequence for U6 small nuclear RNA and a signal for Λe termination of transcription (T cluster) by RNA polymerase III, described by Yuan et al. (1992), were inserted into Λe EcøRI site for Λe retroviral vector LXSN, described by Miller and Rosman (1989), to create pRVO. Retroviral constructs PB2, MITK and ΔMITK were constructed by placmg Λe DNA sequence Λat coded for Λe ΕGS wiΛ Λe ability to target Λe mRNA for influenza viral protein PB2, mlTK13 RNA and ΔM1(167-377)TK13 RNA under Λe control of Λe U6 promoter in plasmid pRVO, respectively. Plasmid pTK129 and pTK141 were constructed, respectively, by placing the Bglϊl-Mlul fragment (87 nt) of Λe HSV-l(F) BamHI Q fragment and BamHl-Accl fragment (181 nt) of Λe HSV-1 (F) BamHI Z fragment under the control of a phage T3 RNA polymerase promoter. These two fragments correspond to Λe sequences Λat encode Λe 5' sequence of TK mRNA and of Λe overlapping transcripts for the HSV-1 α.47, UslO, and Us 11 genes, respectively, as described by McMeoch et al , J. Gen. Virol. 69: 1531-1574 (1988). E. Construction of cell lines. The protocols were modified from Miller and Rosman (1989). In brief, cells were transfected wiΛ retroviral vector DNAs wiΛ Λe aid of a mammalian transfection kit purchased from Stratagene Inc. (La JoUa, CA). Forty-eight hours post transfection, neomycin (Gibco-BRL) was added to the culture medium at a final concentration of 600 μg/ml. Cells were subsequently selected in Λe presence of neomycin for two weeks and neomycin resistant cells were cloned and allowed to proliferate in neomycin-containing medium, as described by Sambrook et al. (1989). The newly constructed cell lines, NB2, ΔMITK, and MITK, and a control cell line in which cells were transfected wiΛ LXSN vector DNA, were indistinguishable in terms of cell growΛ and viability for up to two monΛs. Finally, aliquots of Λese cells were eiΛer frozen for long-term storage in liquid nitrogen or used immediately for further studies in vivo. F. Viral infection and preparation of RNA and protein extracts. Approximately 10 6 cells in a T25 flask were eiΛer mock- infected or infected with HSV-1 in 1.5 ml of Medium 199 (M199; GIBCO) supplemented wiΛ 1 % fetal calf serum. After 2 hours of exposure of cells to virus at 37°C, Λe medium was replaced wiΛ Dulbecco's modified Eagle's medium supplemented wiΛ 5% fetal bovine serum. The cells were incubated for 11 hours before harvesting for isolation of viral mRNA and/or protein. RNA and protein extracts were prepared from cells Λat had eiΛer been mock-infected wiΛ HSV-1, as described previously by Jenkins and Howett, J. Virol. 52: 99-107 (1984), and Liu and Roizman (1991). G. RNase protection assay for viral mRNA expression. The RNA probes used to detect TK mRNA and Λe transcripts of Λe α47, UslO, and Us 11 genes were synΛesized in vitro wiΛ T3 RNA polymerase (Promega, Madison, WI) from Λe DNA templates pTK129 and pTK141, respectively, Λat had been linearized wiΛ Eagl. RNase protection assays were performed as described previously by Yuan et al. (1992). The protected RNA products were separated in 8 M urea 8% polyacrylamide gels and quantitated wiΛ a phosphorimager. quantitation was performed in Λe linear range of RNA detection. H. Εlectrophoretic separation and staining with antibodies of poly peptides from infected cells. Denatured, solubilized polypeptides from cell lysates were separated on SDS-9% [v/v] polyacrylamide gels. The separated polypeptides were transferred electrically to nitrocellulose membranes and allowed to react in an enzyme-linked immunoassay wiΛ antibodies against eiΛer mouse or rabbit IgG Λat had been conjugated wiΛ horseradish peroxidase after reaction wiΛ antibodies against HSV-1 TK or ICP35. The membranes were subsequently stained wiΛ Λe color-development substrate from peroxidase substrate kit purchased from Vector Laboratories Inc. or reacted wiΛ the chemiluminescent substrate in a LumiGLO™ chemiluminescence kit (Kirkegaard and Perry Laboratories Inc.) and subsequently subjected to exposure to X-ray film. Finally, Λe amounts of TK and ICP35 protein on Λe membrane were quantitated by scanning Λe films wiΛ a densitometer (Bio-Rad, Inc.). Quantitation was performed in Λe linear range of protein detection. I. Cleavage of model substrates by MIGS RNA in vitro. The HSV-1 TK gene has been well characterized and is reviewed by Roizman and Sears, "Virology", 2nd edition, edited by Fields et al , pages 1795-1841 (Raven Press, New York, 1990). AlΛough Λe TK gene product is not essential for viral replication in tissue culture cells, because it is so well studied we have used it as a model target for gene inactivation by MIGS RNA. DNA encoding a guide sequence (TK 13) that contains a sequence of 13 nt complementary to Λe 5' terminal sequence of mRNA for HSV-1 TK protein was covalently linked to Λe 3' end of DNA Λat encoded Ml RNA (Figure 9). The RNA transcript of this construct, M1TK13, cleaved target RNA, tk7, Λat contains 7 nt of Λe 5' sequence of TK mRNA and an unrelated sequence of 5 nt Λat serves as a leader sequence (Sequence ID No. 52). Cleavage in the target occurs at position 5, yielding two cleavage products of 5 nt and 7 nt in lengΛ, respectively. M1TK13 remains unchanged during Λe reaction, as expected of a true enzyme, since only labeled, full-lengΛ M1TK13 RNA is detected after incubation of Λe reaction mixture. Attachment of guide sequences to Λe 3' end, raΛer Λan to Λe 5' end, of Ml RNA in Λe construction of MIGS RNA is preferred because Λe presence of a 3' terminal CCA sequence in Λe guide sequence is important for maximum efficiency of cleavage. Furthermore, additional sequences downstream from Λe CCA sequence lower Λe rate of cleavage of substrates. MIGS RNA only cleaves substrates Λat are complementary to Λe guide sequence. MITK 13 RNA can cleave substrate tk7 but not substrate cat7, which contains a sequence of 7 nt from Λe mRNA for chloramphenicol acetyltransferase (CAT). However, M1CAT13 RNA, in which Λe guide sequence contains a sequence of 13 nt complementary to CAT mRNA, can efficiently cleave cat7 but not tk7. Therefore, MIGS RNA appears to act as a sequence-specific endonuclease, recognizing its substrates through specific base-pairing between Λe GS and Λe target sequence, as has also been shown by Frank et al. , Biochemistry 33: 10800-10808 (1994), wiΛ different constructs. To determine wheΛer longer RNAs can be specifically cleaved by these new RNA enzymes, uniformly [ 32 P] -labeled fragments of Λe TK (450 nts) and CAT (550 nts) mRNA sequences (Figure 10) were incubated wiΛ eiΛer M1TK13 RNA or M1CAT13 RNA. Sequence-specific and efficient cleavage of Λese RNA substrates by the appropriate RNA enzymes resulted. A set of RNA enzymes, designated M1TK19, M1TK16, M1TK13, M1TK10 and M1TK5, were constructed in which Λe GSs contained sequences of 19, 16, 13, 10, 5 nucleotides complementary to Λe TK mRNA sequence, respectively (Figure 11), in order to relate Λe lengΛ of Λe target helix to efficiency of cleavage. The substrate tk46, which contains a TK mRNA sequence of 46 nt, was cleaved by all Λe constructs and Λe cleavage site was determined to be at position G21, as expected (Figure 11). Further kinetic analysis of cleavage of substrate tk46 by M1TK16, M1TK13, M1TK10, and M1TK5 RNAs was also performed and the results are shown in Table 3. Table 3 : Kinetic parameters of reactions catalyzed by various MIGS RNA constructs Enzyme K 2 (μM) k t (min 1 ) cat m k ca m M1TK16 0.08 0.14 1.6 2.4 M1TK13 0.09 0.21 2.3 2.4 M1TK10 0.15 0.10 0.7 0.7 M1TK5 0.22 0.03 0.1 0.1 Values of k^ increased in Λe rank order from M1TK5 to M1TK16 RNA. Values of K m decreased progressively wiΛ increasing lengΛ of Λe duplex. The overall K m of Λe reaction undoubtedly involves boΛ Λe substrate binding to the guide sequence (helix formation) and docking of Λe double helical segment in the active site of Ml RNA, a complex process. There was one exception to Λe rule of increasing k ∞ . wiΛ lengΛ of guide sequence: k^, decreased from M1TK13 to M1TK16. This last result can be explained if release of product becomes Λe rate-limiting step in Λe reaction governed by M1TK16 RNA under single- and multiple-turnover conditions as described by Fersht, "Enzyme structure and mechanism", 2nd edition (W.H. Freeman and Co., New York, 1985). Further analysis of Λe reaction wiΛ M1TK13 showed Λat about 5 pmol of tk46 were cleaved by 1 pmol of M1TK13 in 30 minutes, mdicating Λat Λe RNA enzyme turns over 5 times during Λe incubation period. This result is consistent wiΛ Λe value of k^ measured by classical Michaelis- Menten kinetic analysis (see Table 3). MIGS RNAs act more efficiently Λan does Ml RNA in Λe classic cleavage reaction in trans. MITK 16 cleaves tk46 at least 10 times faster Λan does Ml RNA when using a separate guide sequence, TK16, under equivalent experimental conditions. Moreover, MIGS RNA cleaves its substrates more efficiently in buffers that more closely mimic physiological conditions in terms of magnesium ion concentration (10 mM MgCl 2 ) than does Ml RNA with a separated guide sequence in buffers Λat contain 100 mM MgCl 2 , an indication Λat Λe high concentrations of Mg 2+ ions are needed to mediate binding of Λe substrate to Ml RNA alone, as indicated by Kazakov and Altman, Proc. Natl. Acad. Sci. USA 88: 9193-9197 (1991), and Smith and Pace, Biochemistry 32: 5273-5281 (1993). Further analysis of Λe cleavage reaction of MIGS RNA indicated Λat Λe cleavage proceeds optimally at a temperature of 62°C, at concentrations of monovalent cations of 100 mM, and at concentrations of Mg 2+ ions of 60-100 mM. To prove Λat Λe catalytic activity of MIGS RNA resides in Λe sequence Λat encodes Ml RNA, a set of MITK RNAs was constructed in which each RNA had a deletion in a different region of Λe Ml RNA sequence but in which each had Λe same guide sequence, TK13. The various Ml RNA deletion mutants, for example, Δ 167-377, Δl-163, and Δ65, lack Λe catalytic activity needed to process pre-tRNAs, as shown by Guerrier-Takada et al , Science 286: 1578-1584 (1989) and Guerrier-Takada and Altman (1992). MIGS RNA constructs wiΛ Λese deletion mutants and the linked TK13 sequence were unable to cleave tk46. Stimulation of the activity of MIGS RNA by proteins. C5 protein, Λe protein subunit of RNase P from E. coli, increases Λe rate of cleavage of natural substrates by Ml RNA as described by Guerrier-Takada et al. (1983) and Reich et al , Science 239: 178-181 (1988). C5 protein also stimulates Λe cleavage by MIGS RNA by a factor of 30 or more. Furthermore, cleavage by MIGS RNA can be stimulated at least five-fold by a partially purified preparation of human RNase P. This enhancement in rate was anticipated as it had been previously shown Λat protein from a crude preparation of human (HeLa cells) RNase P can enhance Λe cleavage of ptRNA by Ml RNA as described by Gold and Altman, Cell 44: 243-249 (1986). The rate stimulation cannot be ascribed to residual human RNase P activity as it, alone, cannot cleave substrate tk46 in complexes wiΛ catalytically inactive ΔM1(167-377)TK13 RNA. The last result is consistent wiΛ observations Λat a simple stem-loop structure can serve as a substrate for Ml RNA but not for eukaryotic RNase P. This was shown specifically wiΛ human and X. laevis RNase P by Yuan and Altman, Science 263: 1269-1273 (1994), and Carrara et al , Proc. Natl. Acad. Sci. USA. No stimulation of cleavage of substrate tk46 by MITK 13 RNA was observed when fractions devoid of human RNase P activity were used. Accordingly, it is expected Λat when MIGS RNA constructs are present in mammalian cells, Λeir activity should be enhanced by endogenous proteins. J. Expression in vivo of MIGS RNA. To express MIGS RNA in mammalian cells, retroviral constructs, MITK and ΔMITK, were generated by cloning genes for M1TK13 and ΔM1(163-377)TK13 into Λe retroviral vector LXSN under Λe control of a mouse U6 snRNA promoter, described by Das et al. , EMBO J. 1: 503-512 (1988), and Yuan et al. (1992) (Figure 12). An additional retroviral DNA construct, NB2, containing Λe U6 promoter and an external guide sequence (NB2) Λat targets Λe mRNA Λat encodes Λe PB2 protein of human influenza virus, was used as a control. The targeted cleavage site of TK mRNA expressed in cells infected wiΛ HSV-1 appears to be modifiable by dimeΛyl sulfate (DMS) in vivo, based on Peattie and Gilbert, Proc. Natl. Acad. Sci. USA 11: 4679-4682 (1980), Inoue and Cech, Proc. Natl. Acad. Sci. USA 82: 648- 652 (1985), Climie and Friesen, J. Biol. Chem. 263: 15166-15175 (1988), and Ares and Igel, Genes & Dev. 4: 2132-2145 (1990). This is an indication Λat Λis site might be accessible for binding to MIGS in vivo. Amphotropic packaging cells (PA317) were transfected wiΛ retroviral vector DNAs to produce retroviruses Λat encoded Λe genes for MIGS RNA. Subsequently, esotropic packaging cells (-ψCKE) were infected with Λese retroviruses, and cells expressing Λe retroviruses and ribozymes were cloned. Stable expression of MIGS RNAs was demonstrated by an RNA protection assay with a probe Λat was complementary to Λe sequence of Ml RNA and RNA isolated from Λese cell lines. Furthermore, RNA extracted from MlTK-expressing cells can cleave substrate tk46 in vitro while RNA extracted from Λe parental CRE cells, cells Λat expressed NB2 RNA and ΔMITK RNA do not show cleavage activity. These results demonstrate Λat Λe MITK RNA Λat is expressed in cultured cells was intact and catalytically active. K. Inhibition of expression of TK of HSV-1 in cells that express MIGS RNA. Cells were infected wiΛ herpes simplex virus 1 (HSV-1) at a multiplicity of infection (MOI) of 0.05 to 0.1. Levels of TK mRNA in Λe infected cells were determined by an RNase protection assay wiΛ an RNA probe (TK probe) Λat contained a sequence of 87 nt complementary to Λe 5' proximal sequence of TK mRNA. An RNA probe (o-47), containing a sequence of 181 nt complementary to Λe overlapping regions of ----47, Us 10, and Us 11 mRNAs encoded by HSV-1, was used to determine Λe levels of Λese latter mRNAs. The levels of Λe latter RNAs were used as internal controls for quantitation of expression of TK mRNA. Figure 13 graphically summarizes the results of Λe RNase protection experiments wiΛ both Λe TK and o-47 probes. A reduction of about 80 + 5%, averaged over four experiments, in Λe level of TK mRNA expression was observed in cells Λat expressed MITK RNA while cells Λat expressed ΔMITK RNA only exhibited a reduction of 9 ± 3%, averaged over four experiments. Thus, it appears Λat cleavage of TK mRNA by MITK RNA did indeed occur inside cells, wiΛ a subsequent reduction in Λe level of TK mRNA Λat could be translated. No products of Λe cleavage of TK mRNA were detected in our RNase protection assays presumably because Λese RNAs, which lack eiΛer a cap structure or a poly A sequence, are rapidly degraded by intracellular RNases. Protein extracts of Λe infected cells were analyzed for Λe presence of Λe TK polypeptide. Polypeptides were transferred to two identical membranes and one was stained with a TK-specific antibody (anti-TK), described by Liu and Summers (1988), while Λe oΛer was stained wiΛ a monoclonal antibody against Λe capsid protein ICP35 of HSV-1 (anti-ICP35), described by Liu and Roizman (1991). The expression of ICP35 serves as an internal control for Λe quantitation of expression of TK. The results of four independent experiments are summarized in Figure 13: a reduction of at least 76 ± 5%, averaged over four experiments, in Λe level of TK protein was observed in cells Λat expressed MITK RNA while a reduction of only 10 ± 4%, averaged over four experiments, was seen in cells Λat expressed ΔMITK RNA. The low level of inhibition found in cells Λat expressed ΔMITK RNA was presumably due to an antisense effect. These examples show Λat when Ml RNA is converted to an RIGS (MIGS RNA), it cleaves one particular substrate in a reaction governed by sequence specificity. A custom-designed RIGS specific for Λymidine kinase sequence (MITK RNA), cleaves Λe TK mRNA in vitro and can be stably expressed in mammalian cells and can reduce Λe level of expression of TK by > 75 % when Λese cells are infected wiΛ HSV- 1. The reduction in Λe level of expression of TK Λat can be ascribed to Λe antisense effect of Λe (internal) guide sequence is no more Λan 15% of Λe total inhibitory effect. Moreover, Λe high degree of sequence specificity, which is governed by a guide sequence Λat hybridizes to a complementary sequence in Λe substrate, makes our construct suitable for use as a tool for targeting RNAs in vivo. The optimal lengΛ of an antisense sequence for in vivo targeting is about 13 nucleotides, as discussed by Stein and Cheng, Science 261: 1004-1012 (1993). The extent of inactivation that we observed was very similar to Λat achieved when endogenous RNase P is used as Λe catalytic agent to cleave complexes of target TK mRNA and separate EGSs expressed from synthetic genes Λat have been stably incorporated into human cells in tissue culture. The activity of MIGS RNA was stimulated in vitro by C5 protein and mammalian proteins. Gold and AlΛian (1986) suggested Λat C5 protein and protein subunits of human RNase P might bind to homologous sequences and similar structures Λat are found in boΛ Ml RNA and HI RNA, Λe RNA component of RNase P from HeLa cells. The cleavage reactions by MIGS RNA in cells Λat contain proteins Λat bind RNase P catalytic RNA are expected to proceed at rates higher than Λose observed in vitro. In particular, higher rates are expected in Λe presence of additional, non-specific, RNA-binding proteins, RNA chaperons, which are known to stimulate Λe activity of oΛer RNA enzymes as indicated by Tsuchihashi et al. , Science 262: 99-102 (1993), Coetzee et al. , Genes & Dev. 8: 1575-1588 (1994), Bertrand and Rossi, EMBO J. 13: 2904-2912 (1994), and Herschlag et al , EMBO J. 13: 2913-2924 (1994). SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Yuan, Yan Guerrier-Takada, Cecilia Altman, Sidney Liu, Fenyong (ii) TITLE OF INVENTION: TARGETED CLEAVAGE OF RNA USING RIBONUCLEASE P TARGETING AND CLEAVAGE SEQUENCES (iii) NUMBER OF SEQUENCES: 65 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Patrea L. Pabst (B) STREET: 2800 One Atlantic Center 1201 West Peachtree Street (C) CITY: Atlanta (D) STATE: Georgia (E) COUNTRY: USA (F) ZIP: 30309-4530 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/207,547 (B) FILING DATE: 07-MAR-1994 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Pabst, Patrea L. (B) REGISTRATION NUMBER: 31,284 (C) REFERENCE/DOCKET NUMBER: YU112 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (404) -873-8794 (B) TELEFAX: (404) -873-8795 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GAACAUUUUG AGGCAUUUCA GUCAGUUGGC CAAACUGAGC AGACUCUAAA 50 UCUGCNNNNN GAAGGUUCNN NNCCUUCAUG CCUCACCA 88 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : TAATACGACT CACTATAGAA CATTTTGAGG CATTTCAGTC AGTTGGCCAA 50 ACTGAGCAGA C 61 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : TGGTGAGGCA TGAAGGNNNN GAACCTTCNN NNNGCAGATT TAGAGTCTGC 50 TCAGTTTGGC C 61 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GAAUACACGG AAUUGGUGGG GUUCCCGAGC GGCCAAAGGG AGCAGACUCU 50 AAAUCUGCCG UCAUCGACUU CGAAGGUUCG AAUCCUUCCC CCACUGCCA 99 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : GCCAAAGGGA GCAGACUCUA AAUCUGCCGU CAUCGACUUC GAAGGUUCGA 50 AUCCUUCCCC CACCACCAUC A 71 (2) INFORMATION FOR SEQ ID NO: 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 : GAAUACACGG AAUUGGUGGG GUUCCCGA 28 (2) INFORMATION FOR SEQ ID NO:7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 : AUGGAACAUU UUGAGGCAUU UCAGUCAGUU UAA 33 (2) INFORMATION FOR SEQ ID NO: 8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : GGCCAAACUG AGCAGACUCU AAAUCUGCCG UCAUCGACUU CGAAGGUUCG 50 AAUCCUUCAU GCCUCACCAU 70 (2) INFORMATION FOR SEQ ID NO: 9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : GGCCAAACUG AGCAGACUCU AAAUCGCAAA CGGAAGGUUC GUGCCCUUCA 50 UGCCUCACCA U 61 (2) INFORMATION FOR SEQ ID NO:10: " (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGCCAAACUG ACGUCAUCGA CUUCGAAGGU UCGAAUCCUU CAUGCCUCAC 50 CAU 53 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 341 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (x) PUBLICATION INFORMATION: (A) AUTHORS: Altman et al . (C) JOURNAL: Genomics (D) VOLUME: 18 (F) PAGES: 418-422 (G) DATE: 1993 (K) RELEVANT RESIDUES IN SEQ ID NO: 1 : FROM 1 TO 341 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCCAAACTGA GCAGACTC 18 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCGCGGTACC AAAAATGGTG AGGCATGAAG G 31 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGCCGTAATA TCCAGCTGAA CGG 23 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TGGTGAGGCA TGAAGG 16 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TAATACGACT CACTATAGGC CAACTGAGCA GAC 33 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GGCCAAACUG AGCAGACUCU AAAUCGGCCC UUCGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GGCCAAACUG AGCAGACUCU AAAUCUGCAC GAGAGAAGGU UCGUGCCCUU 50 CAUGCCUCAC CA 62 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGCCAAACUG AGCAGACUCU AAACUGGCCU AACGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GGCCAAACUG AGCAGACUCU AAAUUGCCCA ACGAAGGUUC ACCCCCUUCA 50 UGCCUCACCA 60 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GGCCAAACUG AGCAGACUCC AAAUCCACCA AGAAGGUUCG UGCCCUUCAU 50 GCCUCACCA 59 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GGCCAAACUG AGCAGACUCU AAACUCCUCC CAGAAGGUUC GUGCCCUUCA 50 UGCCUCACCA 60 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGCCAAACUG AGCAGACUCU AAAUCGGCCU ACGGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GGCCAAACUG AGCAGACGCU AAAUCUACCC CGUGAAGGUU CGUCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GGCCAAACUG AGCAGACUCU AAAUUUGCCA CCAGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GGCCAAACUG AGCAGACUCA AAUCUGGCCA UUCGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GGCCAAACUG AGCAGACUCU AAAUCGCAGU GUGAAGGUUC GUGCCCUUCA 50 UGCCUCACCA 60 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GGCCAAACUG AGCAGACUCU AAAUCAGCGC GUGGAAGGUU CGUGCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGCCAAACUG AGCAGACUCU AAAUCGGCCG CACGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: GGCCAAACUG AGCAGACACU AAAUUUGCAC GAGGAAGGUU CGCCCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GGCCAAACUG AGCAGACCCU AAAUCUGCCC CCGGAAGGUU CGUGCCCUUC 50 AUGCCUCACC A 61 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GCCAAACTGA CGTCATCGAC TTCG 24 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AACAGCTATG ACCATG 16 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GTAATACGAC TCACTATAGG CCAAACTGAG CAGACTCTAA ATCTGCAAAC 50 GGAAGGTTC 59 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: ATGGCTTCGT ACCCCTGCCA TCAACACGCG TCTGCGTTCG ACCAGGC 47 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GGTTAACGTC GGACAGACTC TAAATCTGTT GCGGTCTCCG CGCGCAGGTT 50 CAAATCCTGC CGCAGACGTT T 71 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGTTAACGTC GGACAGACTC TAAATCTGTT GCGGTCTCCG CGCGCAGGTT 50 GAAATCCTGC CGCAGACGTT T 71 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GGTTAACGTC GGTGCGGTCT CCGCGCGCAG GTTCAAATCC TGCCGCAGAC 50 GTTT 54 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: tRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GGCCAAACUG AGCAGACUCU AAAUCUGCAA ACGGAAGGUU CGUGCCCUUC 50 AUGCCUCACC AU 62 (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 377 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: GAAGCUGACC AGACAGUCGC CGCUUCGUCG UCGUCCUCUU CGGGGGAGAC 50 GGGCGGAGGG GAGGAAAGUC CGGGCUCCAU AGGGCAGGGU GCCAGGUAAC 100 GCCUGGGGGG GAAACCCACG ACCAGUGCAA CAGAGAGCAA ACCGCCGAUG 150 GCCCGCGCAA GCGGGAUCAG GUAAGGGUGA AAGGGUGCGG UAAGAGCGCA 200 CCGCGCGGCU GGUAACAGUC CGUGGCACGG UAAACUCCAC CCGGAGCAAG 250 GCCAAAUAGG GGUUCAUAAG GUACGGCCCG UACUGAACCC GGGUAGGCUG 300 CUUGAGCCAG UGAGCGAUUG CUGGCCUAGA UGAAUGACUG UCCACGACAG 350 AACCCGGCUU AUCGGUCAGU UUCACCU 377 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: TAATACGACT CACTATAG 18 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CGCAGACGGT CCTATAGTGA GTCGTATTA 29 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: ACCGCGCAGC CTGGTCGAAC GCAGACGCGT GTTGATGGCA GGGGTCTATA 50 GTGAGTCGTA TTA 63 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: ATGCCTCGGT CCTATAGTGA GTCGTATTA 29 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: GTGGTGTCTG CGTTCGACCA GCTATGACCA TG 32 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GTGGTGTCTG CGTTCGACCA GTATGACCAT G 31 (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GTGGTGTCTG CGTTCGACTA TGACCATG 28 (2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GTGGTGTCTG CGTTCTATGA CCATG 25 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GTGGTGTCTG TATGACCATG 20 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GTGGTGAGGC ATTTCAGTTA TGACCATG 28 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ( i) SEQUENCE DESCRIPTION: SEQ ID NO:51: AUGCCUCACC AC 12 (2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CGCAGACACC AC 12 (2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: GGACCGAGGC AU 12 (2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GGACCGUCUG CG 12 (2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: GUCUGCGUUC GA 12 (2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: GAGGCAUUUC AGU 13 (2) INFORMATION FOR SEQ ID NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: GACCCCTGCC ATCAACACGC GTCTGCGTTC GACCAGGCTG CGCGGT 46 (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: AGCCUGGUCG AACGCAGACA CCAAAA 26 (2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: AGCCUGGUCG AACGCAGACA CCAC 24 (2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: CUGGUCGAAC GCAGACACCA C 21 (2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: GUCGAACGCA GACACCAC 18 (2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO-.62: GAACGCAGAC ACCAC 15 (2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single

Claims: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CAGACACCAC 10 (2) INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: GUCGAACGCA GACACCAC 18 (2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: CUGGUCGAAC GCAGACACCA C 21 We claim: 1. An isolated oligonucleotide molecule comprising an external guide sequence wherein Λe external guide sequence comprises a recognition sequence complementary to a targeted sequence in a target RNA molecule, and an RNase P binding sequence, wherein Λe RNase P binding sequence comprises a nucleotide sequence base pairing wiΛ itself to form a structure similar to Λe T stem and loop of a precursor tRNA, wherein Λe RNase P binding sequence does not form structure similar to all or portions of Λe variable stem, Λe variable loop, Λe anticodon stem and Λe anticodon loop of a precursor tRNA, and wherein Λe external guide sequence promotes RNase P catalytic RNA-mediated cleavage of Λe target RNA molecule. 2. The oligonucleotide molecule of claim 1 further comprising an RNase P catalytic sequence, wherein Λe oligonucleotide molecule cleaves Λe target RNA. 3. The oligonucleotide molecule of claim 2 wherein Λe RNase P catalytic sequence is an HI sequence or an Ml sequence. 4. The oligonucleotide molecule of claim 2 wherein Λe recognition sequence comprises an A recognition arm and a D recognition arm, wherein Λe A recognition arm is located at Λe 3' end of Λe RNase P binding sequence and Λe D recognition arm is located at Λe 5' end of Λe RNase P binding sequence. 5. The oligonucleotide molecule of claim 4 wherein Λe RNase P catalytic sequence is linked to Λe 3' end of Λe A recognition arm. 6. The oligonucleotide molecule of claim 4 wherein Λe RNase P catalytic sequence is linked to Λe 5' end of Λe D recognition arm. 7. The oligonucleotide molecule of claim 4 wherein Λe RNase P binding sequence consists of a nucleotide sequence base pairing wiΛ itself to form a structure similar to Λe T stem and loop of a precursor tRNA. 8. The oligonucleotide molecule of claim 2 wherein Λe RNase P catalytic sequence comprises Λe sequence of a naturally occurring RNase P catalytic RNA. 9. The oligonucleotide molecule of claim 2 wherein Λe oligonucleotide molecule is selected by randomizing a section of Λe sequence of Λe starting oligonucleotide molecule; selecting for a subpopulation of Λe randomized sequences for Λeir ability to efficiently cleave Λe target RNA; amplifying Λose sequences cleaving more efficiently Λan Λe starting oligonucleotide molecule; and repeating Λe selection and amplification steps. 10. A composition for promoting cleavage of a target RNA wherein Λe composition comprises Λe oligonucleotide molecule of claim 2 in a pharmaceutically acceptable delivery system. 11. The composition of claim 10 wherein Λe pharmaceutically acceptable delivery system is selected from Λe group consisting of liposomes, virosomes, microspheres and microcapsules. 12. The composition of claim 10 wherein Λe pharmaceutically acceptable delivery system is selected from Λe group consisting of carriers suitable for topical, subcutaneous, parenteral, and enteral administration. 13. A composition for promoting cleavage of a target RNA wherein Λe composition comprises an engineered expression vector encoding Λe oligonucleotide molecule of claim 2. 14. The composition of claim 13 wherein Λe engineered expression vector is a viral vector selected from Λe group consisting of retroviral vectors, adeno-associated viral vectors and Epstein-Barr viral vectors. 15. A meΛod for cleaving a target RNA comprising bringing into contact, under conditions Λat promote RNase P cleavage, Λe target RNA and an oligonucleotide molecule comprising an external guide sequence wherein Λe external guide sequence comprises a recognition sequence complementary to a targeted sequence in a target RNA molecule, and an RNase P binding sequence, wherein Λe RNase P binding sequence comprises a nucleotide sequence base pairing with itself to form a structure similar to Λe T stem and loop of a precursor tRNA, wherein Λe RNase P binding sequence does not form structure similar to all or portions of Λe variable stem, Λe variable loop, Λe anticodon stem and Λe anticodon loop of a precursor tRNA, and wherein Λe external guide sequence promotes RNase P catalytic RNA-mediated cleavage of Λe target RNA molecule. 16. The meΛod claim 15 wherein Λe oligonucleotide molecule further comprises an RNase P catalytic sequence, wherein Λe oligonucleotide molecule cleaves the target RNA. 17. The meΛod of claim 16 wherein Λe step of bringing into contact is accomplished by administering to a patient or cells from a patient Λe oligonucleotide molecule, and wherein Λe oligonucleotide molecule is in a pharmaceutically acceptable delivery system. 18. A meΛod for selecting a population of RNase P internal guide sequences Λat cleave a target RNA wiΛ increased efficiency over a starting RNase P internal guide sequence comprising randomizing a section of Λe starting RNase P internal guide sequence; selecting for a subpopulation of Λe randomized sequences for Λeir ability to efficiently cleave Λe target RNA; amplifying Λose sequences cleaving more efficiently Λan Λe starting RNase P internal guide sequence; and repeating Λe selection and amplification steps. 19. An isolated oligonucleotide molecule comprising an external guide sequence wherein Λe external guide sequence comprises a recognition sequence complementary to a targeted sequence in a target RNA molecule, and an RNase P binding sequence; wherein Λe external guide sequence promotes RNase P catalytic RNA-mediated cleavage of Λe target RNA molecule, and wherein at least one nucleotide in Λe oligonucleotide molecule is selected from Λe group consisting of chemically modified nucleotides and chemically unmodified deoxyribonucleotides. 20. The oligonucleotide molecule of claim 19 wherein Λe recognition sequence comprises an A recognition arm and a D recognition arm, wherein Λe A recognition arm is located at Λe 3' end of Λe external guide sequence and Λe D recognition arm is located at Λe 5' end of Λe external guide sequence. 21. The oligonucleotide molecule of claim 20 wherein Λe A recognition arm comprises a nucleotide sequence including at least seven nucleotides complementary to and base pairing wiΛ Λe substrate immediately 3' to a site in Λe substrate to be cleaved to form a structure similar to an aminoacyl acceptor stem, Λe RNase P binding sequence comprises a nucleotide sequence base pairing wiΛ itself to form a structure similar to Λe T stem and loop of a precursor tRNA, and Λe D recognition arm comprises a nucleotide sequence including at least Λree nucleotides complementary to and base pairing wiΛ Λe substrate. 22. The oligonucleotide molecule of claim 21 wherein Λe RNase P binding sequence comprises a nucleotide sequence base pairing wiΛ itself to form a structure similar to Λe T stem and loop of a precursor tRNA, and a nucleotide sequence forming structure similar to all or portions of Λe variable stem, the variable loop, the anticodon stem and Λe anticodon loop of a precursor tRNA. 23. A composition for promoting cleavage of a target RNA molecule wherein Λe composition comprises Λe oligonucleotide molecule of claim 19 in a pharmaceutically acceptable delivery system. 24. The composition of claim 23 wherein Λe pharmaceutically acceptable delivery system is selected from Λe group consisting of liposomes, virosomes, microspheres and microcapsules. 25. The oligonucleotide molecule of claim 19 wherein one or more of Λe 2' hydroxyl groups of Λe ribonucleotides are replaced wiΛ a chemical group selected from Λe group consisting of hydrogen, an O-alkyl group, an amino group, and fluorine, wherein one or more of Λe phosphate linking groups are replaced with a linking group selected from Λe group consisting of meΛyl phosphonate and phosphoroΛioate, and wherein replacement of one or more of the 2' hydroxyl groups increases resistance of Λe external guide sequence to nucleases. 26. The oligonucleotide molecule of claim 19 wherein one or more of Λe 2' hydroxyl groups of Λe ribonucleotides are replaced wiΛ hydrogen or a meΛoxy group; and wherein one or more of the phosphate linking groups are replaced wiΛ phosphoroΛioate. 27. The oligonucleotide molecule of claim 19 further comprising an RNase P binding sequence, wherein Λe oligonucleotide molecule cleaves Λe target RNA. 28. An DNA molecule Λat encodes an RNA molecule comprising an external guide sequence wherein Λe external guide sequence comprises a recognition sequence complementary to a targeted sequence in a target RNA molecule, and an RNase P binding sequence, wherein the RNase P binding sequence comprises a nucleotide sequence base pairing wiΛ itself to form a structure similar to Λe T stem and loop of a precursor tRNA, wherein Λe RNase P binding sequence does not

Inventors: ALTMAN SIDNEY, GUERRIER-TAKADA CECILIA, LIU FENYONG, YUAN YAN, ALTMAN, SIDNEY, GUERRIER-TAKADA, CECILIA, LIU, FENYONG, YUAN, YAN
Applicants: ALTMAN SIDNEY, GUERRIER TAKADA CECILIA, LIU FENYONG, UNIV YALE, YUAN YAN, ALTMAN, SIDNEY, GUERRIER-TAKADA, CECILIA, LIU, FENYONG, YALE UNIVERSITY, YUAN, YAN
Application year: 1995
Main classification: C12N15
IPC codes: A61K9, A61K31, A61K35, A61K38, A61K48, A61P31, A61P35, C07H21, C12N9, C12N15, C12Q1