WO1996019577A1 - Ribozymes vs - Google Patents

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Publication number
WO1996019577A1
WO1996019577A1 PCT/IB1995/000141 IB9500141W WO9619577A1 WO 1996019577 A1 WO1996019577 A1 WO 1996019577A1 IB 9500141 W IB9500141 W IB 9500141W WO 9619577 A1 WO9619577 A1 WO 9619577A1
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Prior art keywords
ribozyme
rna
cleavage
substrate
base
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PCT/IB1995/000141
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English (en)
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Richard Collins
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Richard Collins
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Publication date
Application filed by Richard Collins filed Critical Richard Collins
Priority to AU17168/95A priority Critical patent/AU1716895A/en
Priority to JP8519614A priority patent/JPH10510715A/ja
Priority to EP95909083A priority patent/EP0795016A1/fr
Publication of WO1996019577A1 publication Critical patent/WO1996019577A1/fr
Priority to MXPA/A/1997/004726A priority patent/MXPA97004726A/xx

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes

Definitions

  • This invention relates to ribozy es .
  • enzymatic nucleic acids act by first binding to a target RN ⁇ . Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic 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 the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RN ⁇ to search for another target and can repeatedly bind and cleave new 'targets.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology
  • ribozyme since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
  • concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
  • This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
  • RNA sequences and secondary structures appear to be capable of such activity. These include the hammerhead, found in several plant viral satellite RN ⁇ s, a viroid RNA, and the transcript of a nuclear satellite DNA of a newt (reviewed 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 Na ture 323, 349; Feldstein et al .
  • RNA component of RNase P which cleaves pre-tRNA precursors in trans (Guerrier-Takada et al..
  • ribozymes With hammerhead, hairpin and Group I ribozymes it has been found that very few 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 engineering of ribozymes that can cleave sequences other than those recognized by the naturally-occurring ribozyme. Some engineered ribozymes also function in vivo in non- native host cells, which has raised the possibility of their use as therapeutic agents in dominant inherited disorders and against retroviruses and RNA viruses (reviewed by Castanotto et al . , 1992 Cri tical Revi ews in Eukaryotic Gene Expression 2, 331) .
  • 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 Collins, 1990 supra) , but it is different in sequence, secondary structure, choice of cleavage site, and functional properties from trans-cleaving ribozymes known in the art (Collins and Olive, 1993 Biochemistry 32, 2795; Guo et al . , 1993 Mol . Biol . , 232, 351) .
  • This invention features the construction and use of enzymatic nucleic acid molecules, for example, those derived from Neurospora 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 by 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 bases 3 ' of the cleavage/ligation site) , and an enzymatic portion (which may include a part or all of the RNA substrate binding portion) having the enzymatic activity.
  • the nucleic acid binds to the target RNA, preferably, with bases 3 ' of the cleavage/ligation site and causes cleavage of the RNA substrate at that cleavage site.
  • the invention features a nucleic acid molecule that catalyzes the cleavage of a separate double-stranded RNA target molecule in a sequence-specific manner.
  • trans-cleavage is meant that the ribozyme is able to act in trans to cleave another RNA molecule which is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecular cleavage reaction.
  • base-pair is meant a nucleic acid that can form hydrogen or other bond(s) with other RN ⁇ sequence by either traditional Watson-Crick or other non-traditional types (for example Hoogsteen type) of interactions.
  • the enzymatic RNA molecules of this invention can be designed to cleave RNA (minimum length of between 8-20 nt) having only a preference for at least one nucleotide immediately 5' to the cleavage site and the availability of an adjacent 2' hydroxyl group for cleavage to occur.
  • the 2 ' -hydroxyl group is generally provided by the substrate RNA molecule.
  • the invention features a ribozyme able to cleave a separate substrate RNA molecule.
  • the ribozyme has three base paired regions generally in an "I" configuration.
  • the upper and lower based paired regions of the proposed "I” include between about 10 and 80 bases inclusive, of which at least about 50% are paired with each other.
  • the connecting region of the proposed "I” between said upper and lower base paired regions includes between about 8 and 20 bases inclusive, of which at least about 50% are paired.
  • ribozyme any enzymatic nucleic acid molecule, usually containing at least some ribonucleotides, which is active to cleave an RNA molecule without forming a covalent bond with that substrate.
  • the molecule generally lacks any nucleophilic attacking group that is able to cause cleavage of the substrate and form a covalent bond with that substrate (at least in a transient form) .
  • a "separate RNA molecule” is one that is not covalently bonded with the ribozyme, and may contain iion- ribonucleotides within its length. It is preferably a naturally occurring RN ⁇ molecule, such as a viral mRNA, or a pathogenic RNA molecule.
  • the proposed “I” configuration is shown generally in the figures 5B through 8.
  • This structure may contain other nucleic acid chains attached to different portions of the “I”, but those in the art will recognize that it is advantageous to have as few of these extra chains 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. Together these regions 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, include 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.
  • 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.
  • the "connecting" region further includes a single-strand region of between about 3 and 7 bases inclusive, e . g. , the single-strand region is adjacent the "upper” base-paired region as shown in figures 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 “left” 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 shown in the figures .
  • the "lower" region and/or the “connecting" regions includes at least one bulged nucleotide (e.g., A) , that is an unpaired base, which 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 shown in the figures 8 and 9, where the bases which are unpaired include at least 3 bases.
  • the substrate for the ribozyme has a base-paired region of at least 2 base pairs, e.g., the substrate has the sequence 3 ' G ⁇ NN 5 ' where cleavage by the ribozyme is between each M (each N independently is any base; throughout the document the term N or N' is independently any base or base equivalent) .
  • 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 molecule is able to contact the double-stranded RN ⁇ substrate only 3' of the cleavage site and cause cleavage of 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 RNA substrate only 3 ' of the cleavage site and cause cleavage of the RNA substrate at the cleavage site.
  • the 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 the cleavage site. Such essential bases and configuration are determined as described below; those in the art will recognize that it is now routine to determine such parameters.
  • One example of such a ribozyme is that having about 80 - 90% the sequence shown in the figures 5-8.
  • 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 ' AGN n GUCN m 3 ' (see Fig.
  • each 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
  • the ribozyme includes RNA.
  • the invention features a cell including nucleic acid encoding the ribozyme above, an expressiorii vector having nucleic acid encoding this ribozyme in a manner which allows expression of the ribozyme 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 RN ⁇ substrate molecule selected from a group consisting of viral RN ⁇ , messenger RNA, pathogenic RNA and cellular RNA.
  • the invention features a method for cleaving a single-stranded RN ⁇ substrate at a cleavage site by causing base-pairing of the RNA substrate with a nucleic ⁇ acid molecule only 3' of the cleavage site ( Figure 7) .
  • Such a method includes contacting 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.
  • This nucleic 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 the 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 performed under conditions in which the nucleic acid molecule causes cleavage of the RNA substrate at the cleavage site.
  • 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 5 ' -NNNNNNNNNNNNNNNNNNN, where each N independently can be any specified nucle ⁇ tide 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 modifications 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-
  • each N independently can be any specified nucleotide base, where the sequence forms at least 2 base-pairs with a complementary sequence in the 5' region of the enzymatic nucleic acid molecule, where the substrate RNA has at least one nucleotide 5' of the cleavage site.
  • derived is meant that the enzymatic portion of the proposed "I” ribozyme is essentially the sequence shown in Fig. 5A and 6A.
  • 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. This interaction improves the specificity of the RN ⁇ cleavage reaction.
  • the invention features synthesis and assembly of enzymatic nucleic acid in one or more pieces, where the nucleic acid contacts a separate substrate RNA molecule and cleaves the substrate RNA at the cleavage site.
  • the invention features a circular nucleic acid molecule having an enzymatic activity which cleaves a separate RNA 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 Nucl ei c Afids Res . 21, 4253, Blu enfeld et al . , WO 93/05157) .
  • Figure 1 is a diagrammatic representation of a hammerhead ribozyme domain known in the art.
  • Stem II can be ⁇ 2 base-pair long, or can even lack base pairs and consist of a loop region.
  • Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art
  • Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, JVature, 327, 596) into a substrate and enzyme portion
  • Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Na ture , 334, 585) into two portions
  • Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Sy ons (1989, Nucl eic . Acids . Res . , 17, 1371) into two portions.
  • FIG 3 is a diagrammatic representation of the general structure of a hairpin ribozyme.
  • Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i . e . , m is from 1 - 20 or more) .
  • Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is > 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.
  • each N and N' independently is any normal or modified base and each . dash represents a potential base-pairing
  • 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 specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop.
  • the connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is > 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 A 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. The 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 within the "upper” region, respectively.
  • II (left) and VI (right) shows the left and the right handed regions within the "lower” region, respectively) .
  • FIG. 6 is a diagrammatic representation of a trans- cleaving VS R A enzyme catalyzed cleavage of a double- stranded duplex R ⁇ A.
  • A) Stem I is an intramolecular helix formed within the substrate R ⁇ A.
  • Stems II through VI are intramolecular helices formed within the ribozyme.
  • ⁇ refers to any base.
  • ⁇ ' refers to any base that is complementary to ⁇ .
  • Y refers to a pyrimidine.
  • FIG. 7 is a diagrammatic representation of n trans- cleaving VS R ⁇ enzyme catalyzed cleavage of a single- stranded RNA.
  • 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.
  • Figure 8 is a diagrammatic representation of the VS self -cleaving RNA. Base-paired interactions between nucleotides in the loop 1 (G630, U631 and C632) with complementary nucleotides in loop 5 (C699, A698 and G697) is shown as bold lines.
  • Figure 9 is an enlarged view of the interaction between loop 1 and loop V.
  • A) shows base-pairin ⁇ f of G630 with C699, U631 with A698 and C632 and G697.
  • B) shows base-paired interaction between nucleotides in loop 1 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.
  • 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 10 shows the time course of double- tranded (ds) RNA cleavage by the VS RNA. A plot of fraction of substrate RNA cleaved as a function of time is shown.
  • Figure 11 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 ribozyme.
  • Figure 13 shows the effect of pH on RNA cleavage reaction catalyzed by the VS ribozyme.
  • Figure 14 shows the effect of spermidine concentration on the RNA cleavage reaction catalyzed by the VS ribozyme.
  • Figure 15 shows the effect of Mg 2+ concentration on RNA cleavage reaction catalyzed by the VS ribozyme.
  • Figure 16 shows the kinetics of RNA cleavage reaction catalyzed by the VS ribozyme.
  • Figure 17 shows enhancement of RN ⁇ cleavage reaction 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 catalysis.
  • - viomycin refers to RNA catalysis in the absence of viomycin.
  • Figure 18 shows viomycin-dependent reduction in the concentration of magnesium chloride required for catalysis .
  • Targets for useful ribozymes can be determined as disclosed in Draper et al . WO 93/23569, Sullivan et al . , WO 94/02595 as well as by Draper et ai . , "Method 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 . /19577 PCI7IB95/00141
  • 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 chemical requirements.
  • Eckstein et ai . International Publication No. WO 92/07065; Perrault 1990 et al . , Na t ure 344:565; Pieken et al . , 1991 Sci ence 253:314; Usman and Cedergren, 1992 Trends in Bioche . Sci . 17:334; Usman et al . , International Publication No.
  • Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposo es, or otherwise delivered to target cells.
  • the RN ⁇ or RNA complexes can be locally administered to relevant tissues ex vi vo , or i n vi vo through injection, aerosol inhalation, infusion pump or stent, with or without their incorporation in biopolymers .
  • Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres .
  • ribozymes may be directly delivered ex vi vo to cells or tissues with or without the aforementioned vehicles.
  • the RN ⁇ /vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), 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 been incorporated by reference herein.
  • RNA polymerase I RNA polymerase I
  • RNA polymerase II RNA polymerase II
  • RNA polymerase III RNA polymerase 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 depend on the nature of the gene regulatory sequences (enhancers, silencers, etc. ) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein, O. and Moss, B., 1990, 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) .
  • ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet, M., et al . , , 1992, Antisense Res. Dev. ⁇ _ 2, 3-15; Ojwang, J. 0. , et a .. , 1992, Proc. Natl. Acad. Sci. U S A ⁇ , 89, 10802-6; Chen, C. J., et al . , , 1992, Nucleic Acids Res., 20, 4581-9; Yu, M., et al . , 1993, Proc. Natl. Acad. Sci.
  • ribozyme transcription units can be incorporated into a variety of vectors 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 RNA vectors (such as retroviral and alpha virus vectors) .
  • plasmid DNA vectors such as adenovirus or adeno-associated vectors
  • viral RNA vectors such as retroviral and alpha virus vectors
  • a transcription unit expressing an "I" ribozyme that cleaves target RNA is inserted into a plasmid DNA vector or an adenovirus or adeno-associated DNA viral or retroviral vector.
  • Viral vectors 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 . Bio tech . 3, 533) and both vectors lead to transient gene expression.
  • the adenovirus vector is delivered as recombinant adenoviral particles.
  • DNA may be delivered alone or complexed with vehicles (as described for RNA above) .
  • the DN ⁇ , DNA/vehicle complexes, or the recombinant adenovirus particles are locally administered to the site of treatment, e.g., through the use of an injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.
  • ribozymes that cleave target molecules are expressed from transcription units inserted into DNA 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, retrovirus, adenovirus, or alphavirus .
  • the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and persist in target cells.
  • 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.
  • 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.
  • ribozymes of the present invention that cleave target mRNA and thereby inhibit and/or reduce target activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits specific function are described in the art.
  • inhibitor 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 R ⁇ A molecule able to bind to the same site on the RNA , but unable to cleave that R ⁇ A.
  • vectors any nucleic acid- and/or viral- based technique used to deliver a desired nucleic acid.
  • R ⁇ As were synthesized by T7 transcription from plasmid templates linearized with Sspl (VS R ⁇ A nt 783) .
  • Uncleaved precursor R ⁇ As were obtained from wild type and active mutants using decreased magnesium concentrations during transcription (Collins and Olive, 1993 Biochemis try 32, 2795) .
  • Transcription reactions were extracted once each with phenol/Chloroform.Isoamyl Alcohol (CIA) and once with CIA and precipitated with ethanol.
  • CIA phenol/Chloroform.Isoamyl Alcohol
  • R ⁇ As (approximately 50 nM) were dissolved in water, preincubated at 37°C, and mixed with one fifth volume of 5X buffer (final concentrations: 50 mM Tris-HCl pH 8.0, 50 mM KCl, 2 mM spermidine, 10 mM MgCl2 ) • Aliquots were removed at various times, the precursor and product R ⁇ As separated by electr ⁇ phoresis and quantitated using a Phosphorlmager as described previously (Collins and Olive, supra ) . First- order self-cleavage rates were determined from the slopes of plots of fraction of uncleaved RNA versus time.
  • Applicant constructed site-directed base substitution 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.
  • RNAs were transcribed (see below) from Gil or its derivatives which had been linearized at the Aval site (nucleotide 639) or the Sspl site (nucleotide 783) to make RNAs designated
  • RNAs begin with nine vector nts (5 'gggaaagcu; see Figure 5) followed by VS sequence.
  • Clone A-3 contains VS sequences downstream of the Aval site (nts. 640-881) in a derivative of pTZ19R that lacks the Xba l ' and Sphl sites in the multiple cloning site (constructed for reasons unrelated to the project described here) .
  • Transcripts of clone A-3 digested with Sspl (VS nucleotide 783) begin with 9 vector nucleotides ( 5 'GGGAAAGCU) followed by 144 nucleotides of VS RN ⁇ ; this RNA is designated the Ava ribozyme, or Rz .
  • RNAs were prepared by in vi tro Bacteriophage T7 RNA polymerase transcription from linearized plasmid DN ⁇ s .
  • Transcription reactions (usually 300 ⁇ l) contained 10 to 20 ⁇ g of appropriately linearized template, 1 mil of each NTP (Pharmacia) , 5 mM dithiothreitol, IX T7 polymerase buffer (Bethesda Research Laboratories: 40 M Tris-HCl pH 8.0; 8 mM MgCl2_ 25 mM NaCl; 2 mM sper idine-HCli) , 300 U RNAguard (Pharmacia) , 150 to 200 Units T7 RNA polymerase (Bethesda Research Laboratories) for 2 hrs at 37°C.
  • Radioactive transcripts were prepared as above except an additional 30 mCi of [a- 3 P] GTP (or, for specific experiments, ATP or UTP) was added. Samples were subsequently treated with DNase I (Pharmacia; 5 U/ ⁇ g DNA template) for 15 minutes, then EDTA was added to 10 m . RN ⁇ s were extracted with phenol: chloroform: isoa yl alcohol, chloroform: isoamyl alcohol (CIA) and ethanol precipitated in the presence of 0.3 M sodium acetate, pH 5.2.
  • RNAs were dissolved in water and two volumes of sequencing dye (95% formamide, 0.5X TBE, 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-acrylamid ) of appropriate concentration containing 8.3 M urea and IX TBE (135 M Tris, 45 mM boric acid, 2.5 mM EDTA) . RNAs were visualized either by autoradiography or UV shadowing.
  • RNAs were precipitated with ethanol in the presence of 0.3 M sodium acetate and dissolved in water. Concentrations were determined spectrophotometrically, assuming 1 OD260
  • R ⁇ As were labeled at 5' termini using T4 polynucleotide kinase and [g-- 32 ?] ATP or at 3 ' termini using T4 R ⁇ A ligase and 5' [ 32 P] pCp. End-labeled R ⁇ s were fractionated on denaturing polyacrylamide gels and detected by autoradiography.
  • RNAs were treated with 1 U calf intestinal alkaline phosphatase (Boehringer Mannheim) in a 10 ⁇ l reaction containing 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA at 55°C for 30 min. Reactions were terminated by extraction with phenol:CIA and CIA.
  • Trans-cleavaoe reactions Trans-cleavage of substrate R ⁇ A (S) by the Ava ribozyme (Rz) was carried out following pre-incubation of gel-purified S and Rz in the appropriate IX reaction solution for 2 min. Reactions were initiated by addition of ribozyme to substrate in a final volume of 20 ⁇ l . In a typical reaction, 10 aliquots of 1.5 ⁇ l were removed at specified times, terminated by addition of 13.5 ⁇ l of stop mix (70% formamide, 7 mM EDTA, 0.4x TBE, 0.07% xylene cyanol, and 0.07% bro phenol blue) and stored on ice. Samples were fractionated by electrophoresis on denaturing 20% polyacrylamide gels.
  • Example 1 Mutational analysis of the self-cleaving VS B ⁇
  • applicant used the MFOLD program of Zuker and collaborators (Zuker, 1989 Science 244, 48) to obtain five major families of thermodynamically reasonable models for the minimal self-cleaving RNA .
  • the models differed in the number or length of helices and/or the predicted pairing partners for a given region of the sequence, and ranged from the structure predicted to be most stable to sub- optimal foldings 10% less stable than the lowest free energy structure. Structures within this range of free energy have been found to predict the majority of helices in other R ⁇ As (Jaegar et al . , 1989 Proc . Na tl . Acad. Sci . USA 86, 7706) . These various structural models were tested by making use of site-directed mutagenesis.
  • Fig. 5A was the mos It consistent with the data from the cleavag "e activity of all of the mutants.
  • mutations on the 5' or 3' side of predicted helixes II through VI inactivated the ribozyme or decreased activity well below that of the wild type sequence.
  • Compensatory substitutions that restored a helix, but with a di Cferent base sequence restored activity usually to that of wild type or greater, but always to a level at least greater than that of the individual 5' or 3 ' mutants.
  • regions of each of these helices perform roles that are not sequence-specific but are presumably involved in proper folding of the RNA.
  • mutant Va5 ' shows essentially no activity, but Va3 ' retains more than half the activity of wild type. It may be that the particular substitutions chosen did not disrupt the helix equally well or that one of the bases makes a specific contribution to local or tertiary structure (Cech, 1988 Gene 73, 259) .
  • Helix I The structure and sequence requirements of Helix I appear to be more complex than implied by the model in
  • VI is an example of a GNRA loop that is common in rRNAs
  • VS RNA 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 EMBO . J. 12, 2567) . Also, VS RNA does not contain the set of bases known to be important for activity of hammerhead (Symon, 1992 Ann . Rev . Biochem . 61, 641) or hairpin (Berzal- Herranz et al .
  • the HDV ribozyme (Been, 1994 TIBS 19, 251) requires 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.
  • Example 2 Trans-cleavaoe reaction catalyzed bv the VS RNA.
  • the trans-reaction described below was constructed using various restriction fragments of VS DNA cloned in a T7 promoter vector to construct pairs of non-overlapping regions of VS RNA.
  • the substrate (S) contained the expected cleavage site, following nucleotide G620 (numbered as in Saville and Collins, 1990 supra ) ; the other, the enzyme or ribozyme (Rz), contained the remainder of the VS sequence, terminating at the SspJ site at nucleotide 783.
  • these transcripts were mixed at approximately 1:1 ratio and incubated under conditions known to support self-cleavage (Collins and Olive, 1993 supra) .
  • Example 3 Trans-cleava ⁇ e occurs at the same site as self- cleavaoe
  • Gll/Ava substrate, PI and P2 were labeled at their 5' ends and sequenced by partial enzymatic digestion using RNases Tl or U2.
  • Cleavage products of a mutant substrate containing a single base substitution 3' of the cleavage site (A621U) were also characterized to resolve possible ambiguities due to anomalous migration of some bands. Because the substrate and PI are identical in sequence from the 5' end to the cleavage site, all RNase sequencing bands comigrated, as expected.
  • Pi Full length Pi comigrated with the 13 nucleotide RNase Tl fragment of Gll/Ava that terminates • at G620, which is the site of intramolecular self-cleavage in VS RNA. Also the 3' end of Pi 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. /19577
  • Example 4 Minimal length of the substrate RNA To determine the minimal sequence required downstream of the cleavage site, applicant used essentially the approach described by Forster and Symons (1987 supra) . 5' end-labeled Gll/Ssp RNA was partially hydrolyzed by treatment at high pH, then incubated with or without the ribozyme. Incubation in the absence of ribozyme confirmed applicant's previous finding that full length Gll/Ssp RNA and deletion derivatives lacking ten or fewer nucleotides at the 3' end can self-cleave (Guo et al . , 1993 supra) .
  • Example 5 The minimal substrate R ⁇ A consists mostly of a hairoin loop R ⁇ A structure prediction using the MFOLD program of Zuker and collaborators (Zuker, 1989 supra) suggests that the most thermodynamically reasonable structure of the substrate RNA would be the hairpin-containing structure drawn in Fig. 6.
  • P2 migrated faster than expected relative to size markers for a 19 nucleotide R ⁇ , suggesting that it contained a structure that was not fully denatured even in a gel containing 8.3 M urea.
  • the trans-cleavage reaction rate showed only a small pH dependence at equi olar concentrations of ribozyme and substrate (Fig. 13) .
  • these experiments were performed at subsaturating concentrations of MgCl2 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 masking the effect of increased hydroxide ion concentration.
  • single turnover conditions were established empirically under optimized reaction conditions - by measuring the initial rates of 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 mM, and subsequently leveled off, suggesting that the reaction was approaching single turnover conditions (Fig. 11) .
  • the cleavage rate as a function of concentration of MgCl2 was re-investigated using 0.13 ⁇ M S and 5 ⁇ M Rz and found to be essentially the same shape as in Fig. 15; a concentration of 25 mM MgCl2 was chosen to ensure that magnesium was not limiting.
  • Trans-cleavage reactions using 0.13 ⁇ N substrate and 5 JI ribozyme over a range of pH showed only a minor enhancement in reaction rate.
  • the trans-cleavage reaction exhibits a saturation curve with respect to substrate concentration that is typical of Michaelis-Menten kinetics (Fig. 16B) .
  • a KM of 0.13 ⁇ M and k at of 0.7 min -1 were obtained from these data. These values have been observed to vary by about 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 intramolecular self-cleavage reaction of VS RNA by constructing a ribozyme containing 144 nucleotides of VS RNA that is capable of an intermolecular trans-cleavage reaction.
  • This ribozyme acts as a true enzyme in cleaving a 32 nucleotide substrate RNA. In the presence of excess substrate, the initial rate of cleavage is proportional to ribozyme concentration, and a single ribozyme molecule can cleave multiple substrate molecules.
  • the ribozyme is specific in cleaving a single phosphodiester bond, the same one as cleaved in the natural self-cleavage reaction.
  • Fedor and Uhlenbeck (1990 Proc . Na tl . Acad . Sci . USA 87, 168) have noted that K C at values in the range of 1 min ⁇ l and K m values in the nanomolar range are characteristic of many diverse ribozymes.
  • VS RNA that functions as a substrate for the ribozyme described here contains a single nucleotide upstream of the cleavage site and 19 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 (Guo et al . , 1993); in this respect, VS is similar to HDV ribozymes which also require only a single upstream nucleotide for self- or trans-cleavage (Been, 1994 supra) .
  • the substrate consists mostly of a stem-loop structure flanked 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 predictions, aberrant electrophoretic mobility and the pattern of accessibility to single-strand-specific nucleases . Disruption of 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. Applicant believes that specific bases at specific positions are more important than simply the presence of a stem-loop structure.
  • the stem-loop structure of the VS substrate RNA leaves no long regions available for Watson-Crick pairing with the ribozyme.
  • the secondary structure of the minimal self-cleaving VS R ⁇ A 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 than 5 nucleotides) single-stranded regions. This is in contrast to most trans-acting ribozymes derived from hammerhead, hairpin, HDV and Group I intron R ⁇ As, which have been designed to interact with single-stranded regions of their substrates via formation of one or two intermolecular helices flanking the site to be cleaved.
  • tertiary interactions are known or suspected to contribute to substrate binding of several ribozymes (Pyle et ai., 1992 Na ture 350, 628) . In fact, tertiary interactions alone are sufficient to allow very weak (K M >0.1 ⁇ M) but specific binding of the PI stem-loop of a Group I intron to its catalytic core (Doudna and Szostak, 1989 Na ture 339, 519) . R ⁇ ase P also recognizes substrates that contain substantial secondary structure and have very limited potential for Watson-Crick pairing with the ribozyme (Guerrier-Takada and Altman, 1993 Biochemistry 32, 7152) .
  • the temperature optimum of the trans-cleavage reaction is substantially lower than for the self-cleavage reaction (30°C vs «45°C) and activity drops off much more sharply at higher temperatures (Collins and Olive, 1993 supra ) .
  • the retention of activity at higher temperatures in the self-cleavage reaction indicates that the active site of the ribozyme does not begin to denature until at least 45°C.
  • the lower optimum temperature of the trans-cleavage reaction may reflect decreased binding of the substrate at higher temperatures.
  • VS ribozyme can recognize a substrate that contains a stable secondary structure may be useful from the perspective of ribozyme engineering .
  • Group I intron ribozymes to cleave non-native target RN ⁇ s is the requirement that the target site be in a single- 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 action of other ribozymes.
  • Example 7 Antibiotic-mediated enhancement of RNA Cleavage reaction catalyzed bv the VS ribozvme
  • peptide antibiotics e.g., viomycin
  • Antibiotics decrease, at least by an order of magnitude, the concentration of metal ions required for ribozyme activity.
  • viomycin facilitates inter-molecular interactions between VS RNA molecules .
  • VS RN ⁇ are pre-incubated with 100 mM viomycin for 0, 1, 15 and 30 min prior to adding the reaction buffer (40 mM Tris-HCl pH 8.0,-50 mM KCl and 10 mM MgCl2) •
  • 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 volume of formamide stop buffer.
  • the reaction products are resolved on denaturing polyacrylamide gels.
  • a plot of fraction 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.
  • RNA cleavage reaction catalyzed by the VS ribozyme is assayed upder varying concentrations of magnesium chloride.
  • VS RNA are pre-incubated with 75 mM viomycin for 30 min in the presence of 40 mM Tris-HCl. Reaction was initiated at 37°C by adding varying concentrations of MgCl2- A plot of rate (min -1 ) as a function of time is shown. The presence of viomycin appears to signi icantly lower the requirement of MgCl2 in the reaction. 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,
  • 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 R ⁇ A.
  • the close relationship between ribozyme activity and the structure of the target R ⁇ A 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 R ⁇ 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 specified gene products in the progression of disease.
  • ribozymes of this invention include detection of the presence of mR ⁇ associated with a related condition. Such R ⁇ is detected ' by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
  • ribozymes which can cleave only wild-type or mutant forms of the target R ⁇ are used for 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 in the sample.
  • 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.
  • each analysis will require two ribozymes, two substrates and one unknown sample 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 cleavage 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 expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.
  • the expression of mRNA v/hose 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 RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
  • RNAseP RNA 1 RNA
  • Size -290 to 400 nucleotides.
  • RNA portion of a ribonucleoprotein enzyme Cleaves tRNA precursors to form mature tRNA.
  • Hairpin Ribozyme Size -50 nucleotides.
  • RNA RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus which uses RNA as the infectious agent ( Figure 3).
  • HDV Hepatitis Delta Virus

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Abstract

Ribozyme capable de couper une molécule séparée d'ARN substrat, ladite ribozyme ayant généralement trois régions à paires de bases, mais n'étant pas limitée à ces trois régions, dans une configuration proposée en 'I' dans laquelle les régions à paires de bases 'supérieure' et 'inférieure' comprennent entre environ 4 et 80 bases dont au moins environ 50 % sont appariées les unes avec les autres, et dans laquelle la région de liaison entre lesdites régions à paires de bases supérieure et inférieure comporte entre environ 4 et 20 bases dont au moins environ 50 % sont appariées.
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US6054576A (en) * 1997-10-02 2000-04-25 Ribozyme Pharmaceuticals, Inc. Deprotection of RNA
US6316612B1 (en) 1997-08-22 2001-11-13 Ribozyme Pharmaceuticals, Inc. Xylofuranosly-containing nucleoside phosphoramidites and polynucleotides
US6673611B2 (en) 1998-04-20 2004-01-06 Sirna Therapeutics, Inc. Nucleic acid molecules with novel chemical compositions capable of modulating gene expression

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US7803308B2 (en) 2005-12-01 2010-09-28 Molecular Imprints, Inc. Technique for separating a mold from solidified imprinting material
US7670530B2 (en) 2006-01-20 2010-03-02 Molecular Imprints, Inc. Patterning substrates employing multiple chucks

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BEATTIE, T. ET AL.: "A secondary-structure model for the self-cleaving region of Neurospora VS RNA.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 92 (10). 4686-4690, 9 May 1995 (1995-05-09) *
COLLINS, R. & OLIVE, J.: "Reaction conditions and kinetics of self-cleavage of a ribozyme derived from Neurospora VS RNA", BIOCHEMISTRY, vol. 32, 23 March 1993 (1993-03-23), EASTON, PA US, pages 2795 - 2799 *
GUO, H. ET AL.: "NUCLEOTIDE SEQUENCE REQUIREMENTS FOR SELF-CLEAVAGE OF NEUROSPORA VS RNA.", J MOL BIOL 232 (2). 1993. 351-361 *
OLIVE, J. ET AL.: "Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics", EMBO JOURNAL, vol. 14, no. 13, 3 July 1995 (1995-07-03), EYNSHAM, OXFORD GB, pages 3247 - 3251 *
SAVILLE, B. & COLLINS, R.: "A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria", CELL, vol. 61, 18 May 1990 (1990-05-18), NA US, pages 685 - 696 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6316612B1 (en) 1997-08-22 2001-11-13 Ribozyme Pharmaceuticals, Inc. Xylofuranosly-containing nucleoside phosphoramidites and polynucleotides
US6489465B2 (en) 1997-08-22 2002-12-03 Ribozyme Pharmaceuticals, Inc. Xylofuranosly-containing nucleoside phosphoramidites and polynucleotides
US6797815B2 (en) 1997-08-22 2004-09-28 Sirna Therapeutics, Inc. Xylofuranosly-containing nucleoside phosphoramidites and polynucleotides
US6054576A (en) * 1997-10-02 2000-04-25 Ribozyme Pharmaceuticals, Inc. Deprotection of RNA
US6162909A (en) * 1997-10-02 2000-12-19 Ribozyme Pharmaceuticals, Inc. Deprotection of RNA
US6673918B2 (en) 1997-10-02 2004-01-06 Sirna Therapeutics, Inc. Deprotection of RNA
US6673611B2 (en) 1998-04-20 2004-01-06 Sirna Therapeutics, Inc. Nucleic acid molecules with novel chemical compositions capable of modulating gene expression

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