CA2208097A1 - Vs ribozymes - Google Patents

Abstract

Ribozyme able to cleave a separate substrate RNA molecule, said ribozyme having three base-paired regions generally, but not limited to, in a proposed "I" configuration, wherein "upper" and "lower" based-paired regions comprise between about 4 and 80 bases inclusive of which at least about 50 % are paired with each other, and wherein the "connecting" region between said upper and lower base paired regions comprises between about 4 and 20 bases inclusive of which at least about 50 % are paired.

Description

WO96/19577 PCT~B95/00141 VS RIBOZYMES

Backaround o~ The Invention This invention relates to rihozymes.
The following is a brief description o~ publications concerning ribozymes, and in particular, VS ribozymes.
None are admitted to be the prior art to the pending claims, and all are incorporated by re~erence herein.
Si~ basic varieties of naturally-occurring enzymatic nucleic acids are known presently. Each can catalyze the hydrolysis of RN7~ phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the cllaracteristics o~ these ribozymes.
In general, enzymatic nucleic acids act by ~irst binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enz~natic portion of the molecule that acts to cleave the target RNA. Thus, the enzym~tic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut tho target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded pro~ein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new'targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule generally simply binds to a nucleic acid target to block its translation) since the concentration o~ ribozyme necessary to affect a SU~SrITUTE S~EET (RULE 263 PC~/IBg5/00141 W~6119577 therapeutic treatment is lower than that of an ~ntisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of S target RNA. In addition, the ribozyme is a highly specific inh.ibitor, with the specificity of inhibi~ion depending not only on the base-pairing mechanisrn of bindiny to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-sub.stitutions, near the site of cleavage can completelyeliminate catalytic activity o~ a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992 Proc. Natl. Acad. Sci. USA 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
~ small number of RNAs isolated from a variety of natural sources have been found to possess a self-cleavage activity that is involved in processing multimeric transcripts into monomers, apparently as part of the replication cycle. Several different RNA sequences and secondary structures appear to be capable of such activity. These include the hammerhead, ~ound in several plant viral satellite RN~s, a viroid RNA, and the transcript of a nuclear satellite DNA of a newt (revi~wed by Symons, 1992 Annu. Rev. Biochem. 61, 641); the hairpin (or paper-clip) in the minus strand of the satellite of tobacco ringspot virus and related viruses (Buzayan et al., 1986 Nature 323, 3q9; Feldstein et al., l990 Proc.
30 ~atl. Acad. sci. USA. 87, 2623); the genornic and antigenomic RNAs o~ hepatitis delta virus (HDV; Sharmeen et al ., 1988 J. Virol . 62 , 2674 ; Kuo et al ., l988 ~.
Virol. 62, 4439; Perrotta and Been, 1991 Nature 350, 434);
and Varkud Satellite (VS) RNA in the mitochondria of certain Neurospora isolates (Saville and Collins, 1990 Cell 61, 685).

SUBSTITUTE S~EET (RULE 26 _ _ _ _ W O 96119577 PCT~B95/00141 In their natural contexts the ribozymes mentioned above, as well as others such as Group I (Cech, 1990 ~7nu.
~ev. Biochem. 59, 543 ) and Group II introns (~lichel et al., 1989 Gene 82, 5), perform intramolecular sel~-cleavag~ and, in some cases, ligation reactions.
Structure-function studies of Group I introns (Zaug alld Cech, 1986 Science 231, ~70; Szostak, 1986 Nature 322, 83) and later hammerhead (Uhlenbeck, 1987 ~at ure 3 2 8, 5 9 6 ), hairpin ~Feldstein et al., 1990 supra; Hampel et ~1., 1990 Nucleic Acids Res. 18, 299), and HDV (Perrotta and Been, 19 9 2 Bl ochemi s try 3 1, 16; Branch and Robertson, 1991 Proc .
~at . Acad. sci . USA 88, 10163) ribozymes have been facilitated by altering these RNAs to perEorm intermolecular trans-cleavage reactions. In a ~rans-1~ cleavage reaction one RNA, the substrate, contains the site to be cleaved; a separate RNA, the ribozyme, provides the sequences required to catalyze the cleavage. One naturally-occurring trans-acting ribozyme has been discovered: the RNA component of RNase P, which cleaves pre-tRNA precursors in trans (Guerrier-Takada et al., 1983 Cell 35, 849). Trans-cleavage reactions of most riboz~nes have been designed such that binding of the substrate occurs via formation of multiple Watson-Crick base pairs with the ribozyme. Non-Watson-Crick and tertiary interactions are also involved in substrate bincling and may be essential Eor proper binding ~Pyle et al., 1992 Nature 358, 123; Dib-Haij et al., 1993 Nucl. Acids Res.
21, 1797: Smith et al ., 1992 J. Biol . Chem. 267, 2429;
Guerrier-Takada and Altman, 1993 Biochemistry 32, 7152).
With hammerhead, hairpin and Group I riboz~nes it has been ~ound that very ~ew specific nucleotides in the substrate are required for trans cleavage, provided that the adjacent region(s) are complementary to the binding site on the ribozyme. This property has allowed the ~ 35 engineering of ribozymes that can c].eave sequences other than those recognized by the naturally-occurriny riboz~ne.
Some engineered ribozymes also ~unction in vivo in non-native host cells, which has raised the possibility oE

SUBSTITUrE ~EET (RULE 26~
.

W O 96/19577 PCTnB95/00141 their use as therapeutic agents in dominant inherited disorders and against retroviruses and RNA viruses (reviewed by Castanotto et al., 1992 Critical Reviews i~
Eukaryotic Gene Expression 2, 331) .

SummarY Of The InventLon This invention concerns novel catalytic nucleic acid which performs the same type of RNA cleavage as hammerhead, hairpin, and HDV ribozymes, leaving products with 2',3' cyclic phosphate and 5' OH termini ~Saville and ~0 Collins, 1990 supra), but it is different in sec~uence, secondary structure, choice of cleavage site, and functional properties from trans-cleaving ribozymes kn,o-~in the art (Collins and Olive, 1993 Biochemistry 32, 2795;
Guo et al., 1993 Mol. Biol., 232, 351).
This invention features the construction an(~ use of enzymatic nucleic acid molecules, for example, those derived from ~eurospora Varkud Satellite (VS) RNA, that can catalyze a trans-cleavage reaction, wherein a separate substrate RNA is cleaved at a specific target site. The minimal substrate may form a stable hairpin stem-loop base-paired structure (Fig. 6). Substrate recognition ~y the catalytic nucleic acid involves multiple, including tertiary interactions. The catalytic nucleic acid includes an RNA target binding domain which interacts with nucleotides of the target RNA (preferably with b~ses 3' of the cleavage/ligation site), and an en~ymatic portion twhich may include a part or all of the RNA s~lbstrate binding portion) having the enzymatic activity. The nucleic acid binds to the target RNA, preferably, w:ith bases 3' of the cleavage/ligation site and causes cleavage of the RNA substrate at that cleavage site. Thus, in one aspect, the invention features a nucleic acid molecule that catalyzes the cleavage of a separate double-stranded ~JA target molecule i.n a sequence-specific manner.

SUBSrITUTE S~EET (RULE 26) WO96119577 PCTnB95/00141 By ~trans-cleavage" is meant that the ribozyme is able to act in ~rans to cleave another RNA molecule whic11 is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecular cle~vage reaction.
By "base-pair" is meant a nucleic acid that can fonn hydrogen or other bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional t~es (for example Hoogsteen type) of interactions.
The en2ymatic RN~ molecules o~ this invention can be designed to cleave RNA (minimum length of between ~-20 nt) having only a pre~erence for at least one nucleotide immediately 5' to the cleavage site and the availability of an adjacent 2' hydroxyl group for cleavage to occur.
Tho 2'-hydroxyl group is generally provided by the substrate R~A molecule. Thus, these enzymatic RNA
molecules provide signi~icant in vitro and in vivo activities which can be used for diagnostic and therapeutic procedures.
Thus, in a first aspect, the invention features a ribozyme able to cleave a separate substrate RNA molecule.
The ribozyme has three base paired regions generally in an "I" con~iguration. The upper and lower based paired regions of the proposed "I" include between abou~ lO and ~0 bases inclusive, of which at least about 50~ are paired with each other. The connecting region of the proposed "I"
betweon said upper and lower base paired regions includes between about 8 and 20 bases inclusive, of which at least about 50% are paired.
~ 30 By ~ribozyme~ is meant any enzymatic nucleic acid molecule, usually containing at least some ~ ribonucleotides, which is active to cleave an RNA mo].ecule without ~orming a covalent bond with that substrate.
Thus, the molecule generally lacks any nucleophilic ~ttac~ing group that is able to cause cleavage o~ the SU~SI ITUTE ~EET (RULE 26) CA 02208097 l997-06-l8 W O96/19577 PCTnB95/00141 substrate and form a covalent bond wi~h that substrate (at least in a transient form).
A "separate RNA molecule" is one that is not covalently bonded with the ribozyme, and may cont.ain non-5 ribonucleotides within its length. It is pre~erably anaturally occurring RN~ molecule, such as a viral mRNA, or pathogenic RNA molecule.
The proposed "I" configuration is shown generall~ in the figures 5B through 8. This structure may cont:ain other nucleic acid chains attached to dif~erent portions of the ~I", but those in the art will recognize that it is advantageous to have as few of these extra cl1ains as possible so that secondary structure interactions are reduced and so that the size of the molecule is maintained as small as possible. The proposed "I~' has an "upper" and 'lower" region as describe above and these are connected by an intermediate ("connecting') region. Togeth~r these reyions provide enzymatic activity to the ribozyme. While base pairing in these regions is important, those in the art will recognize that other types of pairing interactions, e.g., Hoogsteen pairing, are also useful in this invention. These regions may, as noted, includo unpaired regions at the ends of the paired regions, or even within or intermediate these paired regions so long as enzymatic activity is not eliminated. By 50~ base-pairing is meant that along a length of the region at least half of the bases in the region interact with other bases to hold the ribozyme in the generally an "I" shape.
In pre~erred embodiments, there is at least 70 or even 80 base pairing, as is illustrated in the attached figures.
The proposed "I" configuration is meant to be a non-limiting structure. Those with ordinary skill in the art will rf-cognize modifications (insertions, deletions, base-substitutions and/or chemical modifications) to the proposed "I" structure can be readily generatef1 using techniques known in the art. Additionally, structures SUBSTITUTE S~EET (RULE 26~

WO96119577 PCT~B9~/00141 distinct from the proposed "I" con~iguration can be readily generated by those skilled in the art and are within the scope of this invention.
In other preferred embodiments, the "connecting"
region ~urther includes a single-strand region oE between about 3 and 7 bases inclusive, e.g., the single-strand region is adjacent the "upper" base-paired region as shown in ~igures 6-8 the "upper" region includes a "left" and ~right" hand portion each between at least about 6 and 30 bases inclusive; and the "lower'~ region also includes a ~le~t" and "right" hand portion each between at least about 6 and 30 bases inclusive. Such regions are delineated by the "connecting" region noted above and as showrl in the figures.
In yet other preferred embodiments, the "lower" region and/or the "connecting" regions includes at least one bulged nucleotide (e.g., A), that is an unpaired base, wh~ch may be available for interaction with proteins; the "upper" base-paired region includes bases unpaired with other bases in the "upper" base paired region which are available to base pair with a substrate RNA, e.g., as shot~n in the figures 8 and 9, where the bases w11ich are unpaired include at least 3 bases. In addition, the substrate for the ribozyme has a base-paired region of at least 2 base pairs, e.g., the substrate ha~s the sequence 3' G~NN ~' where cleavage by the ribozyme is between each N (each N independently is any base; throughout the document the term N or N' is independently any base or base equivalent).
In fur~her preferred embodiments, the '~lower" base-paired region has unpaired bases at its 5' end, available to base pair with a substrate RN~; the ribozyme contacts the RN~ substrate only 3' of the cleavage site; the RNA
substrate is a double-stranded RNA, and the nucleic acid mol~cule is able to contact the double-strancled RN~
substrate only 3' of the cleavage site and cause cleavage SU~STITUTE SI~EET (RULE 26) _ _ . . . . _ W O96/19577 PCT~B95/00141 o~ the RNA substrate at the cleavage site; the RNA
substrate is a single-stranded RNA, and the ribozyme is able to contact the single-stranded RN~ substrate only 3' of the cleavage site and cause cleavage of the RNA
5 substrate at the cleavage site.
In a most preferred embodiment, ~he ribozyme is derived from Neurospora VS RNA. That is, the ribozyme has the essential bases of the VS RN~ molecule held together in a suitable configuration as described above so that RNA
substrates can be cleaved at th~ cleavage site. Such essential bases and configuration are determined as described below; those in the art will recognize ~ha~ it is now ro~tine to determine such parameters. One e~ample of such a ribozyme is that having about 80 - 90~ the sequence shown in the figures 5-8.
In other em~odiments, the ribozyme is enzymatically active to cut an RNA duplex having at least two base-pairs; the ribozyme is enzymatically active to cut 5' to the sequence, 5'NAGNnGUCNm 3'(see ~ig. 6B), where eac:h N
is independently any nucleotide base, n and m are independently an integer between 3 and 20 inclusive, and the sequence forms at least two intramolecular base-pairs;
the RNA substrate binds the ribozyme at a site distant from the cleavage site; the ribozyme is a circular molecule, where the circuLar molecule contacts a separate RN~ substrate and causes cleavage of the RNA substrate at a cleavage site; and the ribozyme includes RNA.
In other aspects, the invention features a cell including nucleic acid encoding the ribozyme above, an expression~ vector having nucleic acid encoding this ribozyme in a manner which allows expression of ~he rlbozyme within a cell, and a cell including such an expression vector. Other aspects also include an expression vector where the ribozyme encoded by the vector is capable of cleaving a separate RNA substrate nlolecule SU{~STITUTE S~IEET (RULE 26~

WO9611g577 PCT~B95100141 selected from a group consistiny of viral RNA, messenger E~NA, pathogenic RNA and cellular RNA.
In further related aspects, t11e invention features a method for cleaving a single-stranded RN~ substrate at a 5 cleavage site by causing base-pairing of the RNA substrate with a nucleic jacid molecule only 3~ of the cleavage site (Figure 7). Such a method includes contactiny the RN~
substrate with a nucleic acid molecule having an RNA
substrate cleaving enzymatic activity which cleaves a separate RNA substrate at a cleavage site. T~lis nuclei.c acid molecule includes an RNA substrate binding portion, which base pairs with the RNA substrate only 3~ of the cleavage site, and an enzymatic portion (which may include a part or all of the RN~ substrate binding portion) having ~he enzymatic activity. The nucleic acid molecule is able to base pair with the RNA substrate only 3' of the cleavage site, and causes cleavage of the RNA substrate at the cleavage site. The contacting is per~ormed under conditions in which the nucleic acid molecule causes cleavage of the RNA substrate at the cleavage site.
In preferred embodiments of the above aspects, the nucleic acid molecule is derived from Neurospora VS RNA;
the nucleic acid molecule is active to cleave 5~ to the RN~ duplex substrate (Fig. 6) of sequence 5'-AAGGGCGUCGUCGCCCCGA, or ~'-NNNNNNNNNNNNNNNNNNN, where each N independently can be any specified nucleotide base, where the sequence forms at least 2 base-pair duplex structure; the nucleic acid molecule is RNA; the nucleic acid is a mixture of ribo and deoxyribonucleotides; the nucleic acid contains at least one nucleotide-containing modificatilons of sugar, phosphate and/or base or combinations thereof; the nucleic acid molecule may or contain abasic and/or non-nucleotide substitutions; the nucleic acid molecule contacts the target RNA sequence;
the nucleic acid molecule is circular; and the nucleic acid molecule is active to cut a single-stranded ~IA lFig.

SU~SrITUTE SI~EET (RULE 26) WO96/19577 PCTnB9SI~0141 7) 5' to the sequence AAGGGCG or NNNNNNN or ~AGGGCGUCGUC
or N~JNNNNNNNNNN where each N independently can be any specified nucleotide base, where the sequence ~orms at least 2 base-pairs with a complementary sequence in the 5' region o~ the enzymatic nucleic acid moleculo, where the substrate RNA has at least one nucleotide 5' of the cleavage site.
By "derived" is meant that the enzymatic portion of the proposed "I" ribozyme is essentially the se~uence shown in Fig. SA and 6A.
In yet another preferred embodiment, the nucleic acid molecule derived from Neurospora VS RNA contacts a separate RNA duplex substrate molecule via base-paired interactions (Fig. 8 and 9) and causes cleavage of the duplex substrate RNA at the cleavage site. ~rhis interaction improves the specificity of the RN~ cleavage ~eaction.
In another aspect, the invention features synthesis and assembly of enzymatic nucleic acid in one or rnore pieces, where the nucleic acid contacts a .separate suhstrate RNA molecule and cleaves the substrate RNA at the cleavage site.
In yet another aspect, the invention features a circular nucleic acid molecule having an enzymatic activity which cleaves a separate RN~ substrate at a cleavage site. The circular nucleic acids can be constructed using one of the methods described in the art (e.g., Been et al., WO 93/14218; Puttaraju et al., 1993 Nucleic A,cids Res. 21, 4253, Blumenfeld et al., WO
93/05~.57).
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

S(~SrITUTE SffEET (RULE 26) -CA 02208097 1997-06-lX

PCT~B95100141 Descri~tion of the Preferred Embodiments The drawings will first brie~ly be described.

Dra~,Jinqs Figure 1 is a diagrammatic representation o~ a hammerhead ribozyme domain known in the art. Stem II can be ~ 2 base-pair long, or can ev~n lack base pairs and consist of a loop region.
Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Fig-lre 2b is a diagrammatic representation o~ the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596) into a substrate and enzyme portion; Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (198~, Nature , 334, 585) into two portions; and Figure 2d is a similar diagram showing the hammerhead divided by Je~fries and Symons (1989, Nucleic. Acids.
~es., 17, 1371) into two portions.
Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is pro~ided with a least 4 base pairs (i.e., n is 1, 2, 3 or ~) and helix 5 can be optionally provided o~ length 2 or more bases (preferably 3 - 20 bases, i.e., m is from 1 -20 or more). Helix 2 and helix ~ may he covalently linked by one or more bases (i.e., r is 2 1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 -20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as SUBSrITUTE SHEET (RULE 26) W Og6/19577 PCTnB95100141 specific bases in the structure, but those in the art will recognize that one or more may be modi~ied chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without signi~icant effect.
~ielix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting ]oop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" :is 2 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H , refers to bases A, U
or C. Y refers to pyrimidine bases. " " refers to a chemical bond.
Figure 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art (Perrotta and Been, 1991 supra) .
Figure 5 ~ is a representation of the general structure of the self-cleaving Neurospora VS RNA domain.
B is a line diagram representing the "I" ribozyme motif.
Tho figure shows the "Upper" and the "Lower" base-paired regions linked by the "connecting" region. IV (left) and V ~right) shows the left and the right handed regions ithin the "upper" region, respectively. II (left) and VI
(right) shows the left and the right handed regions within the "lo~er" region, respectively).
Figure 6 is a diagrammatic representation of a trans-cleaving VS RNA enzyme catalyzed cleavage of a double-stranded duplex RNA . A) Stem I is an intramolecular he:Lix formed within the substrate RN~. Stems II through VI are in~ramolecular helices formed within the ribozyme. B) schematic ~epresentation of minimal substrate sequerlce requirement for cleavage by the "I" ribozyme. N,. refers to any base. N' refers to any base that is complementary to N. Y, refers to a pyrimidine.

Figure 7 is a diagrammatic representation o~ a trans-cleaving vs RNA enzyme catalyzed cleavage of a sin~le-SUBSrl~UTE S~EET (RULE 26) WO96119577 PCT~B95/00141 stranded RNA. A) Stem I is an intermolecular helix formed between the substrate RNA and the ribozyme. Stems II
through VI are intramolecular helices formed within the ribozyme.B) An alternate strategy to facilitate cleavage of a single-stranded R~A by the "I" ribozyme.
Figure 8 is a diagrammatic representation of the VS
self-cleaving RNA. Base-paired interactions between nucleotides in the loop l (G630, U631 and C632) with complementary nucleotides in loop 5 (C69g, A698 and G697) 10 is shown as bold lines.
Figure 9 is an enlarged view of the interaction between loop l and loop V. A) shows base-pairincr of G630 with C699, U631 with A698 and C632 and G697. B) shows bas~-paired interaction between nucleotides in loop l with nucleotides in loop V, where N can be any base (e.g., A, U, G, C) and N' can be any base that is complementary to N.
By "complementary" is meant a nucleotide sequence that can form hydrogen bond(s) with other nucleotide sequence by either traditional Watson-Crick or other non-traditional types (for example Hoogsteen type) of base-paired interactions.
Figure lO shows the time course of double-.stranded (ds) RNA cleavage by the VS RNA. A plot of fraction of substrate RNA cleaved as a function of time is shown.
Figure ll shows the rate of RNA cleavage by the VS
ribozyme as a function of ribozyme concentration.

Figure 12 shows the effect of temperature variation on ~ the RNA cleavage reaction catalyzed by the vS ribo~yme.
Figure 13 shows the effect of pH on RNA cleavage reaction catalyzed by the VS ribozyme.

SUBSrlTUTE SHEET ~RULE 26) CA 02208097 l997-06-l8 W O96119577 PCT~B95/~0141 Figure 14 shows the effect of spermidine concentration on the RNA cleavage reaction catalyzed by the VS ribozyme.
Figure 15 shows the ef~ect of Mg2~ concentration on ~ cleavage reaction catalyzed by the VS ribozy~e.
Figure 16 shows the kinetics of RNA cleavage reaction catalyzed by the VS ribozyme. A) E~fect of ribozyme concentration on the trans-cleavage reaction under optimum reaction conditions. B) Effect of substrate R~A
concentration on the trans-cleavage reaction under opt:imum reaction conditions.
Figure 17 shows enhancement of RN~ cleavage react:ion catalyzed by the VS ribozyme. Numbers 0, 5, and 30 min refers to the length of pre-incubation of VS RNA with 100 mM viomycin prior to the initiation of RNA catalysiC:. -viomycin refers to RNA catalysis in the absence ofviomycin.
Figure 18 shows viomycin-dependent reduction in the concentration of magnesium chloride required for catalysis.
Tarqe~ sites Targets for useful ribozymes can be deternlined as disclosed in Draper et al . WO 93/23S69, Sullivan et al., wo 9~/02595 as well as by Draper et al., "~lethod and reagent for treatment of arthritic conditions U.S.S.N.
08/152,487, filed 11/12/93, and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples, not limiting to those in the art. Ribozymes to such targets are designed generally as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.

SUBSTITUTE S~EET tRULE 26) WO9611gS77 PCTnB9Sl00141 Ribozyme activity can be optimized by chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases, modifications which enhance their efficacy in cells, and removal of helix-containing bases to shorten RNA synthesis times and reduce cllemical requirements. See e.g., Eckstein et al ., International Publication No. WO 92/07065; Perrault 1990 et al ., Nature 344:565; Pieken et al., 1991 Sciellce 253:314; Usman and Cedergren, 1992 Trends in ~ioc}lem. Sci.
17:334; Usman et al ., International Publication No.
wo 93/15187; and Rossi e~- al ., International Publication No. WO 91/03162, as well as Usman, N. et al. US Patent ~ppli cation 07/829,729, and Sproat, B. Europeall Patent Application 9Z110298. 4 ;Chowrira and Burke, 1992 supra;
Chowrira et al., 1993 J Biol. Chem. 268, 19458, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.
R7 bozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The RN~ or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their incorporation in biopolymers.
Sullivan, et al., supra, describes the general methods for delivery of enzymatic R~A molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted ~ to, encaps~lation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodeyradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. ~lternatively, the SU~SI ITUTE ~lEET ~RULE 26) CA 02208097 l997-06-l8 W O 96/19577 PCTnB95/00141 RN~/vehicle combination is locally dalivered by di1~ect injection or by use o~ a catheter, infusion pump or stent.
Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill ~orm), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et al., supra and Draper, et al., supra which have ~een incorporated by reference herein.
~ nother means o~ accumulating high concentrations of a ribozyme(s) within cells is to incorporate the riboz~e-enco~ing sequences into a DNA expression ~ector.
Transcription of the ribozyme sequences are driven ~rom a promoter ~or eukaryotic RNA polymerase I (pol I), RNA
polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depencl on the nature of the gene regulatory sequences (enhancers, silencers, etc. ) present nearby. Prokaryotic RNA
polymerase promoters are also used, providing ~hat the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein, O. and Moss, B., 1990, 25 Proc. Natl. Acad. sci. U S A, 87, 6743-7; Gao, X. and Huang, L., 1993, Nucleic Acids Res., 21, 2867-72; Lieber, A., et al., 1993, Methods Enzymol., 217, 47-66; Zhou, Y., et al., 1990, Mol . Cell . Biol ., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g.
Kashani-Sapet, M., et al .,, 1992 , Antisense Res. Dev. 2, 3-15: Ojwang, J. O., et al. ., 1992, Proc. Natl. Acad.
sci. U S A 89, 10802-6; Chen, C. J., et al.,, 1992, Nucleic Acids ~es., 20, 4581-9; Yu, M., et al., 1993, 35 Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L~Huillier, P.
J., et al., 1992, EMBO ~., 11, 4~11-8; Lisziewicz, J., et al., 1993, Proc. Natl. Acad. Sci. U. S. A., 90, 8000-SUBSTITUTE ~IEET (RULE 26) W O 96119577 PCTnB95/00141 4)).The activity o~ such ribozymes can b~ augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT W094/02~95, both hereby incorporated in their totality by re~erence herein; Ohkawa et al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30, Ventura et al., 1993 Nucleic Acids Res ., 21, 3249-55; Chowrira et al., 1994 ~. Biol .
Cl~em. 269, 25~56). The above ribozyme transcription units tO can be incorporated into a variety of vec~ors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral R~A vectors (such as retroviral and alpha virus vectors).
In a preferred embodiment of the invention, a transcription unit expressing an ~ ribozyme tha~ cleaves target RNA is inserted into a plasmid DMA vector or an adenovirus or adeno-associated DNA viral or retroviral vector. Viral vec~ors have been used to transfer genes to the lung and these vectors lead to transient gene expression (Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi . ~iotech. 3, 533) and both vectors lead to ~ransient gene expression. The adenovirus vector is delivered as recombinant adenoviral particles. DNA may be delivered alono or complexed with vehicles (as described for RNA above).The DNA, DNA~vehicle complexes, or the recombinant adenovirus particles are locally administered to the site o~ treatment, e.g., through the use of an injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

In another aspect of the invention, ribozymes that cleave target molecules are expressed from transcription units inserted into ~NA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors.
Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, SVBSTITUTE S~IEET (RULE 26) CA 02208097 1997-06-lX

W O96/19577 PCT~B95/~0141 retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of e~pressing the ribozymes are locally delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary.
Once expressed, the ribozymes cleave the target mRNA.
Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.
Thus, ribozymes of the present invention that cleave target m~NA and thereby inhibit and/or reduce taxget activity have many potential therapeutic uses, and there are reasonable modes of delivering the riboz~les in a number of the possible indications. Developmollt of an effective ribozyme that inhibits specific ~unction are described in the art.
By "inhibit" is meant that the activity or level of target RNA is reduced below that observed in the absence of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule c~ble to bind to the same site on the RN~, but unable to cleave that R~A.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
Exam~les The following materials and methods were usecl in the following examples:

SllBSTITUTE StlEET (RULE 26) -W O96/l9S77 PCT~B95/00141 Clones and site-~irected muta~enesis.
Clone G11 has been described previously (Guo et al., ; 19g3 J. Mol. Biol. 232, 351) and contains bases 617 to 881 of VS RNA in vector pTZ19R. Mutations were made in clone 5 G11 or DG11 (from which the ScaI, AvaI, AcyI sites in the vector had been destroyed to facilitate future subcloning by retaining only a uni~ue site for each enzyme within thi vs sequence). Substitutions on the 5' or 3' side o~ a helix were made by oligonucleotide directed mutagenesis (Kunkel et al., 1987 in Me~ods Enzymol. eds. Wu and Grossman, vol. 154, pp. 367, Academic Press, San Diego, CA.); compensatory mutants were also made this way unless a unique restriction site separated the 5' and 3' mutations, in which case recombinant DNA techniaues were us~d to combine the two mutations into a single clone.
Usually two separate isolates of each mutant were identified and sequenced from the T7 promoter to the SspI
site which was the 3' end of the run-off transcripts used to measure cleavage rates.
Measurement of self-cleavaae rates:
RNAs were synthesized by T7 transcription from plasmid templatos linearized with SspI (VS RNA nt 783). Uncleaved precursor RNAs were obtained ~rom wild type an~ active mutctnts using decreased magnesium concentrations during transcription (Collins and Olive, 1993 ~iochemistry 32, 2795). Transcription reactions were extracted once each with phenol/Chloro~orm.Isoamyl Alcohol (CIA) and once with CIA and precipitated with ethanol. RNAs (approximately 50 n~) were dissolved in water, preincubated at 37~C, and mi~ed with one fifth v~lume o~ 5X buffer (final concentxations: 50 mM Tris-HCl pH 8.0, 50 mM KCl, 2 n~
spermidine, 10 mM MgCl2). Aliquots were removed at various times, the precursor and product RNAs separated hy electrophoresis and quantitated using a PhosphorImager as described previously (Collins and Olive, supra) . First-SUBSTITUTE S~EET (RULE 26) W O96/19577 PCTnB95/~0141 2~
order self-cleavage rates were determined from the s].opes of plots of ~raction of uncleaved RNA versus time.
Site-Directed muta~enesis:
~pplicant constructed site-directed base substi~ution mutants that would be predicted to disrupt helices by changing one or more bases on the 5' or 3' side of predicted helices. Compensatory mutations that would restore a helix, but using a different base pair, were also constructed. Self-cleavage rates were measured for wild-type, the 5' and 3' mutants, and the compensatory mutant, denoted 5'3'. The data for representative mutants are shown in Table 2.
DNA tem~lates and svnthesis of RNAs:
Fragments of VS DNA were cloned into vectors E~TZl~,R or l9R (Pharmacia). Clone Gll (see Guo et al., 1993 supra) contains VS nts 617 to 881 numbered as in Saville and Collins (199~ Cell 61, 685); the cleavage site is between nucleotides G620 and A621. substrate RNAs were transcribed (see below) ~rom Gll or its derivatives which had been linearized at the AvaI site (nucleotide 639) or the SspI site (nucleotide 783) to make RN~s designated Gll/Ava and Gll/Ssp, respectively. These RNAs begin with nine vector nts (5'gggaaagcu; see Figure 5) fo]lowed by VS
sequence. A site-directed mutant of Gll, clone 621U which contains a single A to U substitution immediately ~ollowing the self-cleavage site, was also used.
Clone A-3 contains VS sequences downstream of the AvaI
site (nts. 640-881) in a derivative oE pTZ19R that lacks the XbaI and SphI sites in the multiple cloning site (constructed for reasons unrelated to the project described here). Transcripts of clone A-3 digested with SspI (VS nucleotide 783) begin with 9 vector nucleotides (5'GGGAAAGCU) ~ollowed by 144 nucleotides of VS RNA; this RNA is designated the Ava ribozyme, or Rz.

SU~SI ITUTE StlEET (RULE 26) WO96119577 PCTnB95/00141 RNAs were prepared by in vi~ro Bacteriophage T7 RNA
polymerase transcription ~rom linearized plasmid DN~s.
Trar~scription reactions (usually 300 ~l) containe~ lO to 20 ~Ig o~ appropriately linearized template, l ~ o~ each MTP (Pharmacia), 5 m~l dithiothreitol, lX T7 polymerase bu~fer (Bethesda Research Laboratories: 40 mM Tris-HCl pH
8.0; 8 mM MgC12; 25 n~I NaCl; 2 mM spermidine-HCl~), 300 U
RNAguard (Pharmacia), lS0 to 200 Units T7 RNA polymerase (Bethesda Research Laboratories) for 2 hrs at 37~C.
Radioacti~e transcrlpts were prepared as above except an additional 30 mCi of ~a-32P] GTP (or, ~or speci~ic experiments, ATP or UTP) was added. Samples were subsequently treated with DNase I (Pharmacia; S U/~g DNA
template) ~or 15 minutes, then EDT~ was added to lO ~I.
RN~s were extracted with phenol: chloro~orm: isoamyl alcohol, chloroform:isoamyl alcohol (CIA) and ethanol precipitated in the presence of 0.3 M sodium acetate, pH
5.2.
Precipitated RNAs were dissolved in water and two volumes o~ sequencing dye (95% ~ormamide, 0.5X T~E, 0.1%
xylene cyanol, 0.1% bromphenol blue), heated at 75~C for 3 min, and fractionated by electrophoresis on denaturing polyacrylamide gels (40:1 acrylamide:bis-acrylamide) of appropriate concentration containing 8.3 M urea and lX TBE
(135 m~I Tris, 45 mM boric acid, 2.5 mM EDTA). RNAs were visualized either by autoradioyraphy or UV shadowing.
B~nds o~ interest were excised, eluted overnight at 4~C in water and filtered to remove residual polyacrylamide.
RNAs were precipitated with ethanol in the presence of 0.3 M sodium acetate and dissolved in water. Concentrations were determined spectrophotometrically, assuming l ~D260 to correspond to an ~NA concentration of 40 mg/ml.
End-labelinq o~ RNAs:
RNAs were labeled at 5' termini using T4 polynucleotide kinase and ~g_32p] ATP or at 3' termini using T4 RNA ligase and S~[32p~ pCp. End-labeled RNAs SllBSTlTUTE SHEET (RULE 26) W O 96/19577 PCT~B95/~)0141 were fractionated on denaturing polyacrylamide gels and detected by autoradiography.
In order to remove 5' triphosphates prior to 5' end labeling, some RNAs were treated with 1 U calf intest:inal alkaline phosphatase (Boehringer ~Iannheim) in a lO ~Ll reaction containing 50 mM Tris-HCl pH 8.0, O.l ~ EDT,~ at 55~C for 30 min. Reactions were termi.nated by e~traction with phenol:CIA and CIA.
Txans-cleavaqe reactions:
Trans-cleavage of substrate RNA (S) by the Ava ribozyme (Rz) was carried out following pre-incu}~ation of gel-purified S and Rz in the appropriate lX reaction solution for 2 min. Reactions were initiated by addition of ribozyme to substrate in a final volumè of 20 l~l. In a typical reaction, lO aliquots of l.5 ~ll were removed at specified times, terminated by addition of 13.5 ~Ll of stop mix (70% formamide, 7 mM EDTA, 0.4x TBE, 0.07~ xylene cyanol, and 0.07~ bromphenol blue) and stored on ice.
Samples were fractionated by electrophoresis on denaturing 20~ po].yacrylamide gels.

The effects of temperature, pH, MgCl2 and spermidine-(HCl)3 on the trans-cleavage reaction were analyzed by incubating equimolar concentrations of Rz and S (0.05 ~M
each) in solutions described in the figure legends.
final study of the effects of MgCl2 under otherwise "optimized" conditions was performed at 30~C, 50 m~I Tris-HCl pH 8.0, 2 mM spermidine, 25 mM KCl.
Experiments to establish single-turnover conditions (Fi~. 10) ~ere performed at 30~C in 50 mM Tris-HCl pH 7.l, 25 mM MgCl2, 25 mM KCl, 2 mM spermidine. Analyses of the effect of pH under single turnover conditions (l~ig. 13) were performed as above, except the concentrations of Rz and S were 5 ~IM and 0.13 ~M, respectively. 50 mM Tris-I~Cl was used for pHs 7.1 to 8.9; 16.5 mM PIPES/44 m~irrris was used for pH 6.

SU~STITUTE SHEET (RU~E 26) -Amounts of substrate and products were quantitated using a PhosphorImager and ImageQuant version 3.0 software (Molecular Dynamics, Sunnyvale, CA, USA) .
Estimates o~ initial cleavage rates were derived from plots of fraction of substrate cleaved vs. time using Grafit software tErithacus Software Ltd, Staines, U.KJ.
Up to 9O~ o~ the substrate could be cleaved in ~0 minutes at approximately equimolar concentration of ribozyme, with the curve indicating the presence o~ approximately 10~
unr~active starting material. Curves were not adjusted to lOO~ completion, and the nature of the unreactive substrate has not been characterized further.
Exam~le 1: Mutational analvsis of the sel~-cleavina V5 RNA
t5 As a starting point ~or structure precliction, applicant used the MFOLD program of Zuker and collaborators (Zuker, 19 89 Sci ence 244, 48) to obtain five ma~or families o~ thermodynamically reasonable models ~or the minimal self-cleaving RNA. The models di~fered in the number or length of helices and/or the predicted pairing partners for a given region of the sequence, ancl ranged from the structure predicted to be most stable to sub-optimal foldings 10% less stable than the lowest free energy structure. Structures witllin this range o~ ~re~
energy have been found to predict the majority of helices in other RNAs (Jaegar et al., 1989 Proc. Natl. Acad. sci.
USA ~6, 7706). These various structural models were tested by making use of site-directed mutagenesis.
Of the various models evaluated, that shown in Fig. 5A
was the most consistent with the data from the cleavage activity of all of the mutants. In general, mutal:ions on the 5' or 3' side of predicted helixes II througll VI
inactivated the ribozyme or decreased activity well below that o~ the wild type sequence. Compellsatory 3~ substitutions that restored a helix, but with a diE~erent base sequence, restored activity usually to that of wild SUBSrlTUTE StlEET (RULE 26~

_ _ _ , . _ .

W O 96/19577 PCTnB95100141 type or greater, but always to a level at leas~ gxeater than that of the individual 5' or 3' mutants. These data sho~,Jed that regions of each of these helices perform :roles that are not se~uence-specific but are presumably involved in proper folding o~ the RNA.
In some cases, mutations on the S' and 3' side did not reduce activity to the same extent. For example, mutant Va5~ shows essentially no activity, but Va3' retains more than hal~ the activity of wil~ type. It may be that: the particular substitutions chosen did not disrupt the helix e~ually well or that one of the bases makes a specific contribution to local or tertiary structure (Cech, 1988 Gene 73, 259).
At some positions activity could not be restored by the compensatory substitutions attempted, even though restoration was possible at other positions in the same helix. This was especially common at predicted base pairs adjacent to natural disruptions in a helix, such as the unpaired adenosines at positions 652 and 718 (Table 2;
ZO e.g., positions IIc and IIIc). Mutant G653C showed no actitJity, as did each of the three substitutions at: the predicted complementary position C771; the double mutant G653C:C771G showed some restoration of activity, but ~Jas still 10-fold slower than wild type (mutant IIc).
Similarly, the A661:U717 pair immediately above unpaired A718 could not be replaced by a U:~ (mutant IIIc), even though the next pair, C662:G716, could be substituted by a G:C (rnutant IIIb). Deletion of either unpaired adenosine also decreased activity, severely so in the case of A652.
These observations suggest that specific local struct:ures may be espbcially important in these areas, or that some of these bases may be involved in alternative and/or additional interactions.
The structure and sequence re~uirements of ~Ielix ~
appear to be more complex than implied by the model in Fig. 5. Although several base substit:utions decreased SUBSTITUTE ~HEET (RULE 26) PCT~B95/00141 WO g611~577 2~
activity severely (e.g., mutants Ia5', Ic3~), other mutations that might be expected to have an equally disruptive effect on the helix (mutants Ia3', Ib3', Ic5') decreased activity only slightly. We have not ~ound any positions at which the compensatory substitution.s that we have tried restored activity much above the level of the individual mutants. This may result in part ~rom the stem o~ ~ive G-C pairs, possibly extended by non-Watson-Crick interactions, which would be predicted to be very stable.
This existence and stability o~ helix I is sup~orted by chemical structure probing and difficulties in sequencing this region. Taken together, these observations suggest that certain bases in helix I may be involved in alternative secondary structures or tertiary interactions that are crucial for activity.
Based on the above data, applicant has constructed a model for the secondary structure of the VS self-cleaving R~A, which contains the minimal contiguous region o f VS
R~ required for self-cleavage. In five of the six holices pxoposed in the model, site directed base substitution mutations that disrupt the helix decrease or eliminate activity. Compensatory substitutions restore activity, usually to wild type level or even greater.
These data provide strong support Eor a sequence-independent, presumably structural, role for portions ofthese five helices.
Several observations suggest that the formation o~ the active structure is more complicated than implied above.
While site directed mutants of helices II through VI
indicate that portions of these helices play a sequence-independen~ structural role, mutants in helix I show a more complex pattern. ~iutations at certain positions in helix I in~ctivated the ribozyme but compensatory substitutions did not restore activity. Furthermore, there is evidence from site-directed mutagenesis and compensating substitutions for a tertiary interaction tllat SUB5TITUl E SHEET (RULE 263 W O96/19577 PCT~B95/(~0141 requires the unwinding of at least the top base pair in helix I (G628:C632), to allow an interaction with loop V
(see Figure 8 and 9). Taken together, these observations suggest that a substantial con~ormational change may c~ccur in helix I under native conditions. The model predicts that VS RNA contains some structural features foun~ or predicted in other RNAs. The GU~ tetraloop capping heli~
VI is an example of a GNRA loop that is common in rRNAs (Woese et al ., 1990 Proc. natl . Acad. sci . USA 87, 8467) and contains internal hydrogen bond and stac]cing interactions that stabilize the loop structure (Heus and Pardi, 1991 Science 253, 191; Santa-Lucia et al., 1992 Science 256, 217).
The secondary structure of VS RNA is different from the hammerhead and hairpin ribozymes in that, although a short helix upstream of the site of cleavage could form in VS RNA, it is not required for activity (Guo et al., 1993 supra) as it is in these two ribozymes (Foster and Symons, 1987 Cell 50, 9;Berzal-Herranz et al ., 1993 EM~O. ~. 12 , 2567). Also, VS RNA does not contain the set of bases known to be important for activity of hammerhead (Symon, 1992 Ann. ~ev. Biochem. 61, 641) or hairpin (Berzal-Herranz et al., supra) ribozymes. Like vS RNA, the HDV
ribozyme (Been, 1994 TI~S 19, 251) re~uires only a single nucleotide upstream of the cleavage site, and a GC-rich helix is found downstream of the cleavage site in both ribozymes. Beyond these similarities, however, the secondary structures have nothing in common.
ExamDle 2: Trans-cleavaae reaction catalvzed bv the VS
RNA.
The trans-reaction described below was constru~ted using various restriction fragments of VS DN~ cloned ln a T7 promoter vector to construct pairs of non-overlapping regions of VS ~JA. One member of each pair, the substrate (S), contained the expected cleavage site, following nucleotide G620 (numbered as in Saville and Collir-s, 1990 SUBSTITUTE SltEET (RULE 26~

-CA 02208097 1997-06-lX

W O96/19577 PCTnB95/00141 supra): the other, the enzyme or ribozyme ~RzJ, contained the remainder of the VS sequence, terminating a~ the SspI
site at nucleotide 783. In preliminary experimerlts these transcripts were mi.xed at approximately 1:1 ratio and incubated under conditions known to support self-cleavage (Collins and Olive, 1993 supra). Most combinatioIIs showed little or no cleavage; however, almost complete cleavage of a 32 nucleotide substrate RNA that terminates at the ~aI site (nucleotide 639) was observed during a one hour inctlbation with a ribozyme that begins at the AvaI site and ends at the Ssp~ site ~nucleotide 783), no cleavage was observed in the absence of ribozyme. Tho elec~rophoretic mobility of the two cleavage products were appro~imately those expected for cleavage a~ter nucleotide 620, which is the site o~ intramolecular self-cleavage or VS RNA. Applicant chose to examine this trans-cleavage reaction in further detail.
Exam~le 3- Trans-cleavaqe Qccurs at the same si~e as sel~-cleavaae To determine the precise site of cleavage, Gll~Ava substrate, Pl and P2 were labeled at their 5' ends and sequenced by partial enzymatic digestion using RNases Tl or U2. Cleavage products o~ a mutant substrate containing a single base substitution 3' o~ the cleavage site (A621U) were also characterized to resolve possible ambiguities due to anomalous migration of some bands. Because the substrate and Pl are identical in sequence from the 5' end to the cleavage site, all R~ase sequenciny bands comigrated, as expected. Full length P1 comigrated with the 13 nucleotide RNase Tl fragment o~ Gll/Ava that terminates ,at G620, which is the site of intramolec~llar self-cleavage in VS RNA. Also the 3' end of P1 was found to be guanosine 2'3' cyclic phosphate, indicating that both the location and chemical pathway of trans cleavage are the same as in the self-cleavage reaction.

SU~STITUTE S~EET (RULE 26) W O96119577 PCT~B95/~0141 As expected ~rom the finding of a cyclic phosphate at the 3' end of Pl, a 5' hydroxyl group was found at the 5' end o~ P2, as evidenced by its end-labeling by [g_32p] ATP
and T4 polynucleotide kinase without prior phosphatase 5 treatment. Alkaline hydrolysis ladders of 5' end-labeled b P2 contained only 18 of the expec~ed 19 bands. This is the result of a compression artifact involving the formation of a very stable stem-loop structure in the longer RNAs; this is described in detail below.
10 Nonetheless, the 5' terminal nucleotides of P2 derived from cleavage of G11/Ava S and the A621U mutant were A and U, respectively, confirming that cleavage occurred between nts 620 and 621, as in the self-cleavage reaction.
~xam~le 4: ~linimal lenath of the substxate RNA
To determine the minimal se~uence required downstream of the cleavage site, applicant used essentially the approach described by Forster and Symons (1987 su~ra). 5' end-labeled G11/Ssp RNA was partially hydro]yzed by treatment at high pH, then incubated with or without the 20 ribozyme. Incubation in the absence of ribozyme confirmed applicant's previous finding that full length G11/SSD RNA
and deletion derivatives lacking ten or fewer nucleotides at the 3' end can self-cleave (Guo et al., 1993 supra) .
Incubation with the ribozyme resulted in the 25 disappearance, or at least decrease in intensity, of bands corresponding to RNAs terminating at nucleotide 639 or longer. A few RNAs were not cleaved to completion under these conditions, indicating that they are relatively poorer substrates. The minimal length substrate 30 terminates at residue 639, which by coincidence correspondslprecisely to the RNA used in Fig. 6, which was synthesized by runoff transcription o~ a template linearized at the AvaI site. Thus only 19 nucleotides downstream of the cleavage site are required for trans-35 cleavage by the Ava ribozyme.

SUBSTITUTE S~EET (RULE 26) PCT~B95/00141 WO961195~7 A parallel experiment using 3' end-labeled RNA showed t~lat only a single nucleotide upstream o~ the cleavage site is required for trans-cleavage. Taken together with the results from 5' end labeled RN~, these data show that ~- 5 the minimum contiguous region of the native RNA required for ~rans-cleavage consists of one nucleotide upstream o~
the cleavage site and l9 nucleotides downstream.
Exam~le S: The minimzl substrate RNA consists mostlv of a hair~in 10Q~
RNA structure prediction using the MFO~D program of Zuker and collaborators (Zuker, 1989 supra) suggests that the most thermodynamically reasonable structure of the s~Ibstrate RNA would be the hairpin-containing structure drawn in Fig. 6. During the characterization of the trans-cleavage products applicant noted several observations that were consistent with such a structure. P2 migrated faster than expected relative to size markers for a l9 nucleotide RNA, suggesting that it contained a structure that was not fully denatured even in a gel containing 8.3 M urea. Certain guanosine (523-625, 627 and 633) and adenosine (621 and 622) residues in S and P2 were cleaved weakly or not at all by RNases Tl and~or U2, even though sequencing reactions were performed under putatively denaturing conditions of 50~C, 1 mM EDTA and 7 M urea.
Only 18 of the expected l9 bands were observed in the 5' end-labeled partial alkaline hydrolysis products of P2.
~xamvle 6: Biochemical Characteristics of the Trans-5leavaae Reaction Conditions:
Applicant has investigated the effects of several variables t~at would be expected to affect RNA s~ructure - and that have been found to affect the cleavage rates of other ribozymes. An equimolar ratio of S and Rz (0.05 ~IM
each) for most initial investigations was used; more detailed analysis specifically under either steady-state or single-turnover conditions is described below.

SU~STITUTE ~EET ~RUL~ 26) W O 96/19577 PCTnB95/00141 Cleavage rate increased with temperature unti:L an optimum was reached around 30~C, and then decreased sharply above 40~C (Fig. 12). No reaction was observed in the absence o~ a divalent cation, and reaction rate increased with increasing MgCl2, reaching a maximum around 100 mM, when magnesium was the only cation present. To determine whether some of the MgC12 was acting simply as a structural counterion, the e~fects of spermidine (Fig.
14), NaC1, and KCl were investigated in the presence of a subsaturating concentration of MgC12 (10 n~i). In the presence of 10 mM MgC12, spermidine at 1 mM or greater enhanced the rate of cleavage nearly 10-fold compared to the same reaction without spermidine (Fig. 14). Low concentrations of KCl (< 100 mM) also stimulated the reaction rate up to about 10 fold. Perhaps surprisingly, NaCl had almost no effect. These observations are ~imilar to the e~fects of cations observed previously on the :rate of self-cleavage of vs RNA (Collins and Olive, 1993 supra).
The rate of reaction showed only a small pH
dependence: the nearly 100-fold increase in the hydroxide concentration between pH 7.1 and 8.9 resulted in only a 2-fold increase in rate (Fig. 13). The effect o~ pH
specifically under single turnover conditions is clescribed below.
~ inally, the e~fect of MgCl2 was re-assayed under "optimized" reaction conditions containing 50 mM Tris, pH
8.0, 2 mM spermidine, 25 m~I KCl, and incubated at 30~C
(Fig. 15). Under these conditions 10 mM MgCl2 allowed the same rate of cleavage as a reaction containing 70 mM MgCl2 under suboptimal conditions. Thus, the combined effects o~ temperature, pH, and cations other than mc~gnesium enhanced cleavage substantially. However, no reaction was observed in the absence of MgC12, indicating that neit:her spermidine nor KCl can replace magnesium in cleavage.

SU~SfITUTE SH~ET (RULE 26) W O 96/19577 PCTnB95/00141 Ef~ects of ~I under sinale turnover conditions:
The trans-cleavage reaction rate showed only a small - pH dependence at equimolar concentrations of ribozyme and substrate ~Fig. 13). However, these experiments wore per~ormed at subsaturating concentrations of MyCl~ and they were probably not under single turnover conditions.
Consequently it was possible that some step in the reaction other than the actual cleavage step itself may have been the rate limiting step, thereby maslcing the effect of increased hydroxide ion concentration. To investigate this possibility, single turnover conditions were established empirically under optimized reaction conditions-by measuring the initial rates o~ trans-cleavage of 0.13 mM substrate by increasing concentrations of ribozyme. The initial rate of cleavage increased with ribozyme concentration up to about 2.5 rn~I, and subsequently leveled off, suggesting that the reaction was approaching single turnover conditions (Fig. 11). The cleavage rate as a function of concentration o~ ~gC12 was ~0 re-investigated using 0.13 ~M S and 5 ~I Rz and Eound to be essentially the same shape as in Fig. 15; a concentration of 25 mM MgC12 was chosen to ensure that magnesium was not limiting. Trans-cleavage reactions using 0.13 ~ substrate and 5 ~IM ribozyme over a range of pH showed only a minor enhancement in reaction rate.
Steadv-state reaction kinetics:
To determine if the Ava ribozyme i5 capable of multiple turnover, Rz was incubated with approximately a 20-fold molar excess o~ S (Fig. 16). If each ribozyme molecule cleaved only a single substrate, a maximum of 1~20th of ~ could be cleaved. In contrast, we observed that cloavage continued at a constant rate until about 40%
of S was cleaved, and then decreased slowly as the concentration of available uncleaved S decreased. This indicated that the Ava ribozyme behaved like a true enzyme, in that it was capable oE multiple ro~lnds oE

SUBSTITUTE SHEET (RULE 26~

WO 961195M PCT~B9S/0~141 cleavage. Also, as expected of an enzyme, the initial rate of cleavage was directly proportional to the concentration of the ribozyme under conditions of substrate excess (Fig. 16).
The trans-cleavage reaction exhibits a saturat:ion curve with respect to substrate concentration that is typical of Michaelis-~enten kinetics (Fig. 16B). ~ KM of 0.13 ~LM and kCat of 0.7 min~l were obtained ~rom these data. These values have been observed to vary by aboul~ a factor of about two when experiments were repeated with different batches of ribozyme over a period of two years.
Applicant has modified the natural intramolecu].ar self-cleavage reaction of VS RNA by constructing a ribozyme containing 144 nucleotides of VS R~A that is capable of an intermolecular trans-cleavage reaction.
This ribozyme acts as a true enzyme in cleavi.ng a 32 nucleotide substrate RNA. In the presence of excess substrate, the initial rate of cleavage is proport:ional to ribozyme concentration, and a single ribozyme molecule c:an cleave multiple substrate molecules. The ribozyme is specific in cleaving a single phosphodiester bvnd, the same one as cleaved in the natural self-cleavage reaction.
The trans-cleavage reaction exhibits Michaelis-Menten kinetics, with Km ~ 0.13 ~IM and kCat ~ 0.7 min~~-. Fedor and Uhlenbeck (1990 P~oc. Natl. Acad. Sci. USA 87, 168) have noted that KCat values in the range of 1 min~l and Km values in the nanomolar range are characteristic of many diverse ribozymes.
The shortest contiguous region of VS RNA that 3Q functions as a substrate for the ribozyme described here contains a single nucleotide upstream of the cleavage site and lg nucleotides downstream. Applicants previous characterization of the intramolecular self-cleavage reaction also showed that only a single nucleotide is required upstream of the cleavage site IGuo et al., 1993);
in this respect, VS is similar to HDV ribozymes which also SU~3STITUTE SI~EET (RULE 26) WO96119577 PCTnB95/00141 require only a sinyle upstream nucleotide for self- or trans-cleavage (Been, 1994 supra) . The substrate consists mostly o~ a stem-loop structure ~lanked by three nucleotides on the 5' and 3' ends, some of which may be involved in non-Watson-Crick structure (Fig. 6). This conclusion is based on minimum free energy predic~ions, aberrant electrophoretic mobility and the pa~tern o~
accessibility to single-strand-specific nucleases.
Disruption o~ some base pairs in the stem by certain single base substitutions has little or'no effect on self-cleavage. However, at some positions the identity of one of the bases in a particular pair is critical: even when the compensating substitution is made in the complementary position to restore the helix, cleavage is not restored.
1~ Applicant believes that specific bases at specific positions are more important than simply the presence o~ a stem-loop s~ructure.
The stem-loop structure of the vs substrate RNA leaves no long regions available for Watson-Crick pairing with ZO the ribozyme. The secondary structure of the minimal self-cleaving VS RNA has been determined and a working model for the structure of the ribozyme has been proposed (Fig. 5). The ribozyme has no long (i.e., more ~han 5 nucleotides) single-stranded regions. This is in contrast to most trans-acting ribozymes derived from hammerhead, hairpin, HDV and Group I intron RNAs, which have been designed to lnteract with single-stranded regions of their substrates via formation of one or two intermolecular helices flanking the site to be cleaved.
In addition to base-pairing, tertiary interactions are known or suspected to contribute to substrate bil1ding oE
several ribozymes (Pyle et al., 1992 Nature 350, 628). In ~act, tertiary interactions alone are sufficient to allow very weak (KM ~O.l ~LM) but specific binding of the Pl stem-loop of a Group I intron to its catalytic core (Doudna and Szostak, 1989 Nature 339, 519). RNase ~ also SU~SrITUTE SHEET (RULE 26~

W O96/19577 PCT~B9S/1~0141 recognizes substrates that contain substantial secondary structure and have very limited potential ~or Watson-Criclc pairi.ng with the ribozyme (Guerrier-Takada and Altman, 1993 ~iochemistry 32, 7152).
we noted in our previous characterization of the VS
RNA se].~-cleavage reaction that the cleavaye rate was essentially unaffected by pH (Collins and Olive, 1993 supra). Consistent with this observation, the trans-cleavage reaction described here also showed little, if any, pH dependence, even when examined under single turnover conditions. These observations differ from results examining the rate of the chemical cleavage step o~ hammerhead ribozymes (Dahm et al., 1993 ~iochemistry 3~, 13040), RN~se P (Guerrier-Takada et al., 1986 Biocl~emistry 25, 1509; Smith and Pace, 1993 ~iochemistry 32, 5~.73; Beebe and Fierke, 1994 Biochemistry 33, 10294) and Totrah~nena Group I intron (Herschlag et al., 1993 ~iochemistry 32, 8312). For these ribozymes, the rate of the cleavage step was found to increase with increasing pH. Failure to observe pH dependence in VS RNA could mean that OH- is not involved in the cleavage reaction, that the reaction proceeds via a novel mechanism or, more likely, that the vS trans reaction is not limited by the rate of the chemical cleavage step under these conditions.
but rather by some step that precedes actual cleavage.
One interesting candidate for such a rate-limiting step would be a conformational change in the substrate and/or ribozyme following binding. At saturating ribozyme concentration, the pseudo-first-order rate constant ~or trans-cleavage of S (~0.6 min~1) is about 10-fold higher than the rate of sel~-cleavage of G11 RNA under similar conditions (Collins and Olive, 1993 supra). Since we envision that the trans-cleavage reaction recreates essentially the same RNA conformation as in the self-3~ cleavage reaction, the higher rate suggests that thecleavable conformation may be more easily attained whell S

SUBSTITUTE SffEET ~RULE 26) W O96/19577 PCTnB95/00141 (stem-loop I; Fig. 5) is not constrained by covalent attachment to the ribozyme core. In support of this idea, we have also found that rate of self-cleavage of ~11 RNA
can be increased several fold by increasing the distance r 5 between stem-loop I and the ribozyme core. These observations are consistent with the idea of at least one confoxmational change involving the substrate stem-loop occurring during the reaction.

The temperature optimum of the trans-cleavage reaction 10 is substantially lower than for the self-cleavage reaction (30~C vs -45~C) and activity drops off much more sharply a~ higher temperatures (Collins and Olive, 1993 supra) .
The retention of activity at higher temperature.s in the self-cleavage reaction indicates that the active site o~
15 the ribozyme does not begin to denature until at least 45~C. The lower optimum temperature of the trans-cleavage reaction may reflect decreased ~inding of the substrate at higher temperatures.
The observation that the VS ribozyme can recognize a 20 substrate that contains a stable secondary structure may be useful from the perspective of ribozyme engineering.
Among the limitations to modifyin~ hammerhead, hairpin or Group I intron ribozymes to cleave non-native target RN~s is the requirement that the target site be in a single-25 stranded region to allow recognition via base pairing with the ribozyme. Because the cleavage site for the VS
ribozyme is adjacent to a stable secondary structure, the VS ribozyme may have unique properties that can be adapted to cleaving certain RNAs that are not accessible to the 30 action of other ribozymes.
~xam~le 7: Antibiotic-mediated enhancement of RNA
~ Cleava~e reaction catalvzed bv the VS ribozvme Several examples of inhibition of the function of a ribozyme or RNA-protein interaction have sho~n tllat 3~ certain antibiotics can interact specifically with RN~

SUBSTITUTE St~EET (RULE 26) W O96/19577 PCTnB95/00141 (Yarus, 1988 Science 240, 1751; Schroeder et al., 1993 Science 260, 1443). Small peptide antibiotics like viomycin ~las been shown to inhibit reactions of certain R~ and RN~-protein complexes (Liou and Tana]ca, .l976 BBr~C
71, 477; Wank et al., 1993 J. Mol. ~iol. 236, lOO.l).
~ pplicant has found that certain peptide antibiotics ( e . g., viomycin) enhance RN~ cleavage reactions catalyzed by the VS ribozyme. ~ntibiotics decrease, at least by an order of magnitude, the concentration of metal ions rQ~uired for ribozyme activity. Additionally, viomycin facilitates inter-molecular interactions between VS RNA
molecules.
Referring to Fig. 17, vS RN~ are pre-incubated ~ith 100 mM viomycin ~or 0, 1, 15 and 30 min prior to adding ~ho reaction buffer (40 mM Tris-HCl pH 8.0;50 m~l~CCl and 10 m~ MgC12). The reaction is carried out at 37~C and aliquots are taken out at regular intervals of time and the reaction is stopped by adding an equal volumQ of formamide stop buffer. The reaction products are reso]ved on denaturing polyacrylamide gels. A plot o~ ~raction of substrate cleaved as a function of time is plotted. The fraction of RNA cleavage increased with an increase in the time of preincubation. The antibiotic-mediated enhancement in rates of cleavage is observed in solutions that already contains optimal concentrations of magnesium and KCl.
Referring to Figure 18, antibiotic-mediated lowering of the re~uirement of divalent cation (Mg2+) is discussed.
RMA cleavage reaction catalyzed by the VS ribozyme is assayed u~der varying concentrations of magnesium chloride. VS RNA are pre-incubated with 75 mM viomycin for 30 min in tl~e presence of 4~ mM Tris-HCl. Reaction was initiated at 37~C by adding varying concentrations of llgC12. A plot of rate (min~l) as a function of time is shown. The presence of viomycin appears to signiEicantly lowQr the requirement of MgCl2 in the reaction.

SUBSl ITUTE S~EET (RULE 26) W O 96/19577 PCT~B95/00141 Sequences listed in Figures 6-9 are meant to be non-limiting Those skilled in the art will recognize that variants (base-substitutions, deletions, insertions, mutati-ons, chemical modifications) of the VS ribozyme can be readily generated using techniques known in the art, and are within the scope of the present invention.
Diaanostic uses Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells, or to detect specific RNA molecules, such as virus RNA. The close relationship between ribozyme activi.ty and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RN~. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vi tro, as well as in cells and tissues. Cleavage of target R~As with ribozymes may be used to inhibit gene expression and define the role (essentially) of speci~ied gene products in the progression of disease. In this manner, other genotic targets may be defined as important mediators of the disease. These experiments will lead to better treatment o~ the disease progression by affording the possibility o~ combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled ~ith known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include det~ction of the presence of mRMA associa~ed with a related condition. Such RN~ is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RMA are used for SUBSrITUTE SHEET (RULE 263 W O 96/19577 PCTnB95/1~0141 the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA ln the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two riboz~nes, two substrates and one unknown sam~le which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavaye fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expres;sion of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRMA whose protein product is implicated in the development of the phenotype is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RN.~ levels will be adequate and will decrease the cost of tl1e initial diagnosis. Higher mutant form to wild-type ratios will ~e correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

SU~STITUTE ~EET (RULE 26) CA 02208097 l997-06-l8 W Og6119577 PCTnB95/00141 TABLE I

~ Characteristics of Ribozymes Group I Introns Size: -200 to >1000 nucleotides.
Requires a U in the target sequence immediately :~' of the cleavage site.
Binds 4-6 nucleotides at 5' side of cleav2ge site.
Over 75 known members of this class. Found in Tetrahymen2 thermophila rRNA, fungal mitochondria, chloropiasts, phage T4, blue-green algae, and others.
RNAseP F~NA (M1 RNA) Size: ~290 to 400 nucleotides.
RNA portion oF a ribonucleoprotein enzyme. Cleaves tRNA
precursors to form mature tRNA.
Roughly 10 known members of this group all are bacterial in origin.
Hammerhead Ribozym~
Siz~: -13 to 40 nucl20tides.
Re~uires the target sequence UH immediately ~' of the cleavage sit2.
Binds a variable number nucleotides on both sides of the cleavage site.
14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the in,ectious agent (rigures 1 and 2) Hairpin Ribozyme Size: -aO nucleotides.
Requires the target sequence GUC immedialely 3' Gf the cle-vaae sit2.
Binds 4-6 nucleotides at 5' side of the cleavase site and a v_riable number to the 3' side of the cleavage site.
Only 3 known member of this class. Found in three plant pa~hogen (satellite RNAs of the tobacco ringspot virus, ~rabis mosaic virus and chicory yellow mottle virus) which uses RNA as tne infeclious agent (Figure 3).
Hepatitis Delta Virus (HDV) Ri~ozyme Size: ~0 - 6~ nucleotides (at present).
Cleavage of target RNAs recently demonstrated.
Sequence requirements not fully determined.
Binding sites and structural requirements not fully delermined, although no sequences 5' of cleavage site are required.
Only 1 known member of this class. Found in humar, HDV ('igure 4).
Ne~rospora VS RNA Ribozyme Size: -144 nucleotides SU~STITUTE SHEET (RULE 26) W O 96/19577 PCT~B95N0141 Found in Neurospora VS RNA (Figure ~A).

SUBSTITUTE S~EET (RULE 26 W O 9611gS77 41 1~-111b5S~14l ~ Ta~le II. Effect of base-substitutions on th~ rate of self-cleavage of the VS
RNA
Helix 8ase- k Substitution -- G11 wildtype 1.002 la 5' G624CtG625C 0.02 la 3' C634G/C635G 0.64 la 5'3' <0.01 Ib 5' C626G 1.21 Ib 3' G633C 0.74 Ib 5'3' 0.31 Ic 5' G627C 0.64 Ic 3' C632G c0.01 Ic 5'3' <0.01 lla 5' G650C 0.12 lla 3' C773G 0.29 lla 5'3' 1.27 llb 5' G655C <0.01 ilb 3' C769G 0.18 llb 5'3' 1.32 llc 5' G653C <0.01 .
Ilc 3' C771G cO.0-i llc 5'3' 0.09 Illa 5' U659A ~0.01 Illa 3' A720U 0.05 Illa 5'3' 1.19 Illb 5' C662G 0.23 - Illb 3' G716C 0.21 Illb 5'3' 0.94 Illc5' A661U/C662G 0.06 Illc3' G716C/U717A 0.02 Illc 5'3' 0.08 SUB~ITUTE ~iEET[RUEE263 W O 96/19577 PCTnB95/aO141 . .
Illd 5' C665G0.01 Illd 3' G711C F3 Ilid 5'3' 0.01 IV 5'U670A/C672G 0.~4 IV 3'G678C/A681U <0.01 IV 5'3' 0.88 Va 5'A690U/C692G 0.07 Va 3'G704C/U706A 0.78 Va 5'3' 1.48 Vb4 5' U695G0.06 Vb4 3' A701C0.04 Vb4 5'3' 1.67 Vc 5'A693U/G694C ND5 Vc 3'C702G/U703A ND5 Vc 5'3' 0.31 Vla 5'G722C/C723G c0.01 Vla 3'G762C/C763G <0.01 Vla 5'3' 075 Vlb 5'G727C/U728A <0.01 Vlb 3'A759U/C760G <0.01 Vlb 5 3 0 94 Vlc 5'A735U/U737A 0.25 Vlc 3'A748U/U750A 0.~8 Vlc 5'3' 1.15 652~A <0.01 A652G ~0.01 . . 718~A 0.15 rate constant of the mutant divided by the rate constant of wild-type G 1 1 .
Z the rate constant for the G11 varied from ~ 0.06 to 0.08 min~l .
3 cleavage rate not measured accuratley, but similar to wild type.
4 these mutants were made in a variant of G11 that contained r, o different base pairs in helix V ( mutant Vc). Rates are normalized using mutant Vc as the revelant wild type.
5 cleav2ge rate not determined.
SUBSTITUrE S~EET (RULE 26)

Claims (28)

Claims

1. Ribozyme able to cleave a separate substrate RNA
molecule, said ribozyme having three base-paired regions generally in an "I" configuration, wherein the upper and lower based-paired regions comprising between 4 and 80 bases inclusive of which at least 50% are paired with each other, and wherein a connecting region between said upper and lower base paired regions comprises between 4 and 20 bases inclusive of which at least about 50% are paired with each other.

2. The ribozyme of claim 1, wherein said connecting region further comprises a single-stranded region of between 1 and 7 bases inclusive.

3. The ribozyme of claim 2, wherein said single-stranded region is adjacent said upper base-paired region.

4. The ribozyme of claim 1, wherein said upper region comprises a left and right hand portion each between at least 3 and 30 bases inclusive.

5. The ribozyme of claim 1, wherein said lower region comprises a left and right hand portion each between at least 3 and 30 bases inclusive.

6. The ribozyme of claim 1, wherein said lower region comprises at least one bulged base.

7. The ribozyme of claim 1, wherein said connecting region comprises at least one bulged base.

8. The ribozyme of claim 1, wherein said upper base paired region comprises bases unpaired with other bases in said upper base-paired region which are available to base pair with a substrate RNA.

9. The ribozyme of claim 8, wherein said bases which are unpaired comprise at least 3 bases.

10. The ribozyme of claim 1, wherein the substrate for said ribozyme comprises a base paired region comprising at least 2 base pairs.

11. The ribozyme of claim 10, wherein said substrate comprises the sequence 3'GANN 5' wherein cleavage by said ribozyme is between each said N, and wherein each N independently is any base.

12. The ribozyme of claim 1, wherein said lower base-paired region comprises unpaired bases at its 5' end, available to base pair with a substrate RNA.

13. The ribozyme of claim 1, wherein said ribozyme contacts said RNA substrate only 3' of the cleavage site,

14. The ribozyme of claim 1, wherein said RNA substrate is a double-stranded RNA, wherein said nucleic acid molecule is able to contact said double-stranded RNA
substrate only 3' of the cleavage site and cause cleavage of said RNA substrate at the cleavage site.

15. The ribozyme of claim 1, wherein said RNA
substrate is a single-stranded RNA, and wherein said ribozyme is able to contact said single-stranded RNA
substrate only 3' of the cleavage site and cause cleavage of said RNA substrate at the cleavage site.

16. The ribozyme of claim 1, wherein said nucleic acid molecule is derived from Neurospora VS RNA.

17. The ribozyme of claim 1, wherein said ribozyme is enzymatically active to cut an RNA duplex having at least two base-pairs.

18. The ribozyme of claim 1, wherein said ribozyme is enzymatically active to cut 5' to the sequence, NAGNnGUCNm, wherein each N is independently any nucleotide base, wherein n and m are independently an integer between 3 and 20 inclusive, and wherein said sequence forms at least internal two base-pairs.

19. The ribozyme of claim 1, wherein, said RNA
substrate binds said ribozyme at a site distant from said cleavage site.

20. The ribozymes of claim 1, wherein said ribozyme is a circular molecule, wherein said circular molecule contacts a separate RNA substrate and causes cleavage of said RNA substrate at a cleavage site.

21. The ribozyme of claim 1, wherein said ribozyme comprises ribonucleotides.

22. A cell comprising nucleic acid encoding the ribozyme of claim 1.

23. An expression vector comprising nucleic acid encoding the ribozyme of claim 1, in a manner which allows expression of said ribozyme within a cell.

24. A cell including an expression vector of claim 23.

25. An expression vector of claim 23, wherein the ribozyme encoded by said vector is capable of cleaving a separate RNA substrate molecule selected from a group consisting of viral RNA, messenger RNA, pathogenic RNA and cellular RNA.

26. The ribozyme of claim 1, wherein the activity of the said ribozyme is increased by a cofactor.

27. The ribozyme of claim 26 wherein said cofactor is selected from the group consisting of antibiotics and peptides.

28. Method for cleaving a separate RNA molecule comprising, contacting said molecule with a ribozyme of claim 1.