Patent Application: US-86631601-A

Abstract:
this invention relates to novel chemically - modified nucleic acid molecules having specific formulae that exhibit increased resistance to nucleases and increased binding affinity to target nucleic acid molecules . the invention further relates to methods of modulating gene expression using the novel chemically modified nucleic acid molecules , and compositions and cells comprising said molecules .

Description:
fig1 shows examples of chemically stabilized ribozyme motifs . hh rz , ( seq id no : 6 ) represents hammerhead ribozyme motif ( usman et al ., 1996 , curr . op . struct . bio ., 1 , 527 ); nch rz ( seq id no : 8 ) represents the nch ribozyme motif ( ludwig & amp ; sproat , international pct publication no . wo 98 / 58058 ); g - cleaver , ( seq id no : 10 ) represents g - cleaver ribozyme motif ( kore et al ., 1998 , nucleic acids research 26 , 4116 - 4120 , eckstein et al ., international pct publication no . wo 99 / 16871 ). n or n , represent independently a nucleotide which can be same or different and have complementarity to each other ; ri , represents ribo - inosine nucleotide ; arrow indicates the site of cleavage within the target . position 4 of the hh rz and the nch rz is shown as having 2 ′- c - allyl modification , but those skilled in the art will recognize that this position can be modified with other modifications well known in the art , so long as such modifications do not significantly inhibit the activity of the ribozyme . fig2 shows an example of the amberzyme ribozyme motif ( seq id no : 11 ) that is chemically stabilized ( see for example beigelman et al ., international pct publication no . wo 99 / 55857 ). fig3 shows an example of the zinzyme a ribozyme motif ( seq id no : 13 ) that is chemically stabilized ( see for example beigelman et al ., beigelman et al ., international pct publication no . wo 99 / 55857 ). fig4 shows an example of a dnazyme motif ( seq id no : 15 ) described by santoro et al ., 1997 , pnas , 94 , 4262 . synthesis of nucleic acids greater than 100 nucleotides in length can be difficult using automated methods , and the therapeutic cost of such molecules can be prohibitive . in this invention , small nucleic acid motifs (“ small refers to nucleic acid motifs less than about 100 nucleotides in length , preferably less than about 80 nucleotides in length , and more preferably less than about 50 nucleotides in length ; e . g ., antisense oligonucleotides , hammerhead or the nch ribozymes ) are preferably used for exogenous delivery . the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of rna structure . exemplary molecules of the instant invention are chemically synthesized , and others can similarly be synthesized . oligonucleotides ( eg ; antisense , geneblocs ) are synthesized using protocols known in the art as described in caruthers et al , 1992 , methods in enzymology 211 , 3 - 19 , thompson et al ., international pct publication no . wo 99 / 54459 , wincott et al ., 1995 , nucleic acids res . 23 , 2677 - 2684 , wincott et al ., 1997 , methods mol . bio ., 74 , 59 , brennan et al ., 1998 , biotechnol bioeng ., 61 , 33 - 45 , and brennan , u . s . pat . no . 6 , 001 , 311 . all of these references are incorporated herein by reference . the synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups , such as dimethoxytrityl at the 5 ′- end , and phosphoramidites at the 3 ′- end . in a non - limiting example , small scale syntheses are conducted on a 394 applied biosystems , inc . synthesizer using a 0 . 2 μmol scale protocol with a 2 . 5 min coupling step for 2 ′- o - methylated nucleotides and a 45 sec coupling step for 2 ′- deoxy nucleotides . table ii outlines the amounts and the contact times of the reagents used in the synthesis cycle . alternatively , syntheses at the 0 . 2 μmol scale can be performed on a 96 - well plate synthesizer , such as the instrument produced by protogene ( palo alto , calif .) with minimal modification to the cycle . a 33 - fold excess ( 60 μl of 0 . 11 m = 6 . 6 μmol ) of 2 ′- o - methyl phosphoramidite and a 105 - fold excess of s - ethyl tetrazole ( 60 μl of 0 . 25 m = 15 μmol ) can be used in each coupling cycle of 2 ′- o - methyl residues relative to polymer - bound 5 ′- hydroxyl . a 22 - fold excess ( 40 μl of 0 . 11 m = 4 . 4 μmol ) of deoxy phosphoramidite and a 70 - fold excess of s - ethyl tetrazole ( 40 μl of 0 . 25 m = 10 μmol ) can be used in each coupling cycle of deoxy residues relative to polymer - bound 5 ′- hydroxyl . average coupling yields on the 394 applied biosystems , inc . synthesizer , determined by calorimetric quantitation of the trityl fractions , are typically 97 . 5 - 99 %. other oligonucleotide synthesis reagents for the 394 applied biosystems , inc . synthesizer include ; detritylation solution is 3 % tca in methylene chloride ( abi ); capping is performed with 16 % n - methyl imidazole in thf ( abi ) and 10 % acetic anhydride / 10 % 2 , 6 - lutidine in thf ( abi ); and oxidation solution is 16 . 9 mm i 2 , 49 mm pyridine , 9 % water in thf ( perseptive ™). burdick & amp ; jackson synthesis grade acetonitrile is used directly from the reagent bottle . s - ethyltetrazole solution ( 0 . 25 m in acetonitrile ) is made up from the solid obtained from american international chemical , inc . alternately , for the introduction of phosphorothioate linkages , beaucage reagent ( 3h - 1 , 2 - benzodithiol - 3 - one 1 , 1 - dioxide , 0 . 05 m in acetonitrile ) is used . deprotection of the antisense oligonucleotides is performed as follows : the polymer - bound trityl - on oligoribonucleotide is transferred to a 4 ml glass screw top vial and suspended in a solution of 40 % aq . methylamine ( 1 ml ) at 65 ° c . for 10 min . after cooling to − 20 ° c ., the supernatant is removed from the polymer support . the support is washed three times with 1 . 0 ml of etoh : mecn : h2o / 3 : 1 : 1 , vortexed and the supernatant is then added to the first supernatant . the combined supernatants , containing the oligoribonucleotide , are dried to a white powder . the method of synthesis used for rna and chemically modified rna including certain enzymatic nucleic acid molecules follows the procedure as described in usman et al ., 1987 , j . am . chem . soc ., 109 , 7845 ; scaringe et al ., 1990 , nucleic acids res ., 18 , 5433 ; and wincott et al ., 1995 , nucleic acids res . 23 , 2677 - 2684 wincott et al ., 1997 , methods mol . bio ., 74 , 59 , and makes use of common nucleic acid protecting and coupling groups , such as dimethoxytrityl at the 5 ′- end , and phosphoramidites at the 3 ′- end . in a non - limiting example , small scale syntheses are conducted on a 394 applied biosystems , inc . synthesizer using a 0 . 2 μmol scale protocol with a 7 . 5 min coupling step for alkylsilyl protected nucleotides and a 2 . 5 min coupling step for 2 ′- o - methylated nucleotides . table ii outlines the amounts and the contact times of the reagents used in the synthesis cycle . alternatively , syntheses at the 0 . 2 μmol scale can be done on a 96 - well plate synthesizer , such as the instrument produced by protogene ( palo alto , calif .) with minimal modification to the cycle . a 33 - fold excess ( 60 μl of 0 . 11 m = 6 . 6 μmol ) of 2 ′- o - methyl phosphoramidite and a 75 - fold excess of s - ethyl tetrazole ( 60 μl of 0 . 25 m = 15 μmol ) can be used in each coupling cycle of 2 ′- o - methyl residues relative to polymer - bound 5 ′- hydroxyl . a 66 - fold excess ( 120 μl of 0 . 11 m = 13 . 2 μmol ) of alkylsilyl ( ribo ) protected phosphoramidite and a 150 - fold excess of s - ethyl tetrazole ( 120 μl of 0 . 25 m = 30 μmol ) can be used in each coupling cycle of ribo residues relative to polymer - bound 5 ′- hydroxyl . average coupling yields on the 394 applied biosystems , inc . synthesizer , determined by colorimetric quantitation of the trityl fractions , are typically 97 . 5 - 99 %. other oligonucleotide synthesis reagents for the 394 applied biosystems , inc . synthesizer include ; detritylation solution is 3 % tca in methylene chloride ( abi ); capping is performed with 16 % n - methyl imidazole in thf ( abi ) and 10 % acetic anhydride / 10 % 2 , 6 - lutidine in thf ( abi ); oxidation solution is 16 . 9 mm i 2 , 49 mm pyridine , 9 % water in thf ( perseptive ™). burdick & amp ; jackson synthesis grade acetonitrile is used directly from the reagent bottle . s - ethyltetrazole solution ( 0 . 25 m in acetonitrile ) is made up from the solid obtained from american international chemical , inc . alternately , for the introduction of phosphorothioate linkages , beaucage reagent ( 3h - 1 , 2 - benzodithiol - 3 - one 1 , 1 - dioxide 0 . 05 m in acetonitrile ) is used . deprotection of the rna is performed using either a two - pot or one - pot protocol . for the two - pot protocol , the polymer - bound trityl - on oligoribonucleotide is transferred to a 4 ml glass screw top vial and suspended in a solution of 40 % aq . methylamine ( 1 ml ) at 65 ° c . for 10 min . after cooling to − 20 ° c ., the supernatant is removed from the polymer support . the support is washed three times with 1 . 0 ml of etoh : mecn : h20 / 3 : 1 : 1 , vortexed and the supernatant is then added to the first supernatant . the combined supernatants , containing the oligoribonucleotide , are dried to a white powder . the base deprotected oligoribonucleotide is resuspended in anhydrous tea / hf / nmp solution ( 300 μl of a solution of 1 . 5 ml n - methylpyrrolidinone , 750 μl tea and 1 ml tea · 3hf to provide a 1 . 4 m hf concentration ) and heated to 65 ° c . after 1 . 5 h , the oligomer is quenched with 1 . 5 m nh 4 hco 3 . alternatively , for the one - pot protocol , the polymer - bound trityl - on oligoribonucleotide is transferred to a 4 ml glass screw top vial and suspended in a solution of 33 % ethanolic methylamine / dmso : 1 / 1 ( 0 . 8 ml ) at 65 ° c . for 15 min . the vial is brought to r . t . tea · 3hf ( 0 . 1 ml ) is added and the vial is heated at 65 ° c . for 15 min . the sample is cooled at − 20 ° c . and then quenched with 1 . 5 m nh 4 hco 3 . for purification of the trityl - on oligomers , the quenched nh 4 hco 3 solution is loaded onto a c - 18 containing cartridge that had been prewashed with acetonitrile followed by 50 mm teaa . after washing the loaded cartridge with water , the rna is detritylated with 0 . 5 % tfa for 13 min . the cartridge is then washed again with water , salt exchanged with 1 m nacl and washed with water again . the oligonucleotide is then eluted with 30 % acetonitrile . inactive hammerhead ribozymes or binding attenuated control ( bac ) oligonucleotides can be synthesized by substituting a u for g 5 and a u for a 14 ( numbering from hertel , k . j ., et al ., 1992 , nucleic acids res ., 20 , 3252 ). similarly , one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control . the average stepwise coupling yields are typically & gt ; 98 % ( wincott et al ., 1995 nucleic acids res . 23 , 2677 - 2684 ). those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 well format , with the ratio of chemicals being used in the reaction adjusted accordingly . alternatively , the nucleic acid molecules of the present invention can be synthesized separately and joined together post - synthetically , for example by ligation ( moore et al ., 1992 , science 256 , 9923 ; draper et al ., international pct publication no . wo 93 / 23569 ; shabarova et al ., 1991 , nucleic acids research 19 , 4247 ; bellon et al ., 1997 , nucleosides & amp ; nucleotides , 16 , 951 ; bellon et al ., 1997 , bioconjugate chem . 8 , 204 ). the nucleic acid molecules of the present invention are modified extensively to enhance stability by modification with nuclease resistant groups , for example , 2 ′- amino , 2 ′- c - allyl , 2 ′ flouro , 2 ′- o - methyl , 2 ′- h ( for a review see usman and cedergren , 1992 , tibs 17 , 34 ; usman et al ., 1994 , nucleic acids symp . ser . 31 , 163 ). ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography ( hplc ; see wincott et al ., supra , the totality of which is hereby incorporated herein by reference ) and are re - suspended in water . methods for the delivery of nucleic acid molecules are described in akhtar et al ., 1992 , trends cell bio ., 2 , 139 ; and delivery strategies for antisense oligonucleotide therapeutics , ed . akhtar , 1995 which are both incorporated herein by reference . sullivan et al ., pct wo 94 / 02595 , further describes the general methods for delivery of enzymatic rna molecules . these protocols can be utilized for the delivery of virtually any nucleic acid molecule . nucleic acid molecules can 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 . alternatively , the nucleic acid / vehicle combination is locally delivered by direct injection or by use of an infusion pump . other routes of delivery include , but are not limited to oral ( tablet or pill form ) and / or intrathecal delivery ( gold , 1997 , neuroscience , 76 , 1153 - 1158 ). other approaches include the use of various transport and carrier systems , for example , through the use of conjugates and biodegradable polymers . for a comprehensive review on drug delivery strategies including cns delivery , see ho et al ., 1999 , curr . opin . mol . ther ., 1 , 336 - 343 and jain , drug delivery systems : technologies and commercial opportunities , decision resources , 1998 and groothuis et al ., 1997 , j . neurovirol ., 3 , 387 - 400 . more detailed descriptions of nucleic acid delivery and administration are provided in sullivan et al ., supra , draper et al ., pct wo93 / 23569 , beigelman et al ., pct wo99 / 05094 , and klimuk et al ., pct wo99 / 04819 all of which have been incorporated by reference herein . the molecules of the instant invention can be used as pharmaceutical agents . pharmaceutical agents prevent , inhibit the occurrence , or treat ( alleviate a symptom to some extent , preferably all of the symptoms ) of a disease state in a patient . the negatively charged polynucleotides of the invention can be administered ( e . g ., rna , dna or protein ) and introduced into a patient by any standard means , with or without stabilizers , buffers , and the like , to form a pharmaceutical composition . when it is desired to use a liposome delivery mechanism , standard protocols for formation of liposomes can be followed . the compositions of the present invention can also be formulated and used as tablets , capsules or elixirs for oral administration ; suppositories for rectal administration ; sterile solutions ; suspensions for injectable administration ; and the other compositions known in the art . the present invention also includes pharmaceutically acceptable formulations of the compounds described . these formulations include salts of the above compounds , e . g ., acid addition salts , for example , salts of hydrochloric , hydrobromic , acetic acid , and benzene sulfonic acid . a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration , e . g ., systemic administration , into a cell or patient , preferably a human . suitable forms , in part , depend upon the use or the route of entry , for example oral , transdermal , or by injection . such forms should not prevent the composition or formulation from reaching a target cell ( i . e ., a cell to which the negatively charged polymer is desired to be delivered to ). for example , pharmacological compositions injected into the blood stream should be soluble . other factors are known in the art , and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect . the term “ systemic administration ” as used herein , refers to in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body . administration routes which lead to systemic absorption include , without limitations : intravenous , subcutaneous , intraperitoneal , inhalation , oral , intrapulmonary and intramuscular . each of these administration routes expose the desired negatively charged polymers , e . g ., nucleic acids , to an accessible diseased tissue . the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size . the use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug , for example , in certain tissue types , such as the tissues of the reticular endothelial system ( res ). a liposome formulation which can facilitate the association of drug with the surface of cells , such as , lymphocytes and macrophages is also useful . this approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells , such as cancer cells . the terms “ pharmaceutically acceptable formulation ” or “ pharmaceutically acceptable carrier ” as used herein , refers to a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity . non - limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include : peg conjugated nucleic acids , phospholipid conjugated nucleic acids , nucleic acids containing lipophilic moieties , phosphorothioates , p - glycoprotein inhibitors ( such as pluronic p85 ) which can enhance entry of drugs into various tissues , for example the cns ( jolliet - riant and tillement , 1999 , fundam . clin . pharmacol ., 13 , 16 - 26 ); biodegradable polymers , such as poly ( dl - lactide - coglycolide ) microspheres for sustained release delivery after implantation ( emerich , d f et al , 1999 , cell transplant , 8 , 47 - 58 ) alkermes , inc . cambridge , mass . ; and loaded nanoparticles , such as those made of polybutylcyanoacrylate , which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms ( prog neuropsychopharmacol biol psychiatry , 23 , 941 - 949 , 1999 ). other non - limiting examples of delivery strategies , including cns delivery of the nucleic acid molecules of the instant invention include material described in boado et al ., 1998 , j . pharm . sci ., 87 , 1308 - 1315 ; tyler et al ., 1999 , febs lett ., 421 , 280 - 284 ; pardridge et al ., 1995 , pnas usa ., 92 , 5592 - 5596 ; boado , 1995 , adv . drug delivery rev ., 15 , 73 - 107 ; aldrian - herrada et al ., 1998 , nucleic acids res ., 26 , 4910 - 4916 ; and tyler et al ., 1999 , pnas usa ., 96 , 7053 - 7058 . all these references are hereby incorporated herein by reference . the invention also features the use of the composition comprising surface - modified liposomes containing poly ( ethylene glycol ) lipids ( peg - modified , or long - circulating liposomes or stealth liposomes ). nucleic acid molecules of the invention can also comprise covalently attached peg molecules of various molecular weights . these formulations offer a method for increasing the accumulation of drugs in target tissues . this class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system ( mps or res ), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug ( lasic et al . chem . rev . 1995 , 95 , 2601 - 2627 ; ishiwata et al ., chem . pharm . bull . 1995 , 43 , 1005 - 1011 ). such liposomes have been shown to accumulate selectively in tumors , presumably by extravasation and capture in the neovascularized target tissues ( lasic et al ., science 1995 , 267 , 1275 - 1276 ; oku et al ., 1995 , biochim . biophys . acta , 1238 , 86 - 90 ). the long - circulating liposomes enhance the pharmacokinetics and pharmacodynamics of dna and rna , particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the mps ( liu et al ., j . biol . chem . 1995 , 42 , 24864 - 24870 ; choi et al ., international pct publication no . wo 96 / 10391 ; ansell et al ., international pct publication no . wo 96 / 10390 ; holland et al ., international pct publication no . wo 96 / 10392 ; all of which are incorporated by reference herein ). long - circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes , based on their ability to avoid accumulation in metabolically aggressive mps tissues such as the liver and spleen . all of these references are incorporated by reference herein . the present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent . acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art , and are described , for example , in remington &# 39 ; s pharmaceutical sciences , mack publishing co . ( a . r . gennaro edit . 1985 ) hereby incorporated by reference herein . for example , preservatives , stabilizers , dyes and flavoring agents can be provided . these include sodium benzoate , sorbic acid and esters of p - hydroxybenzoic acid . in addition , antioxidants and suspending agents can be used . a pharmaceutically effective dose is that dose required to prevent , inhibit the occurrence , or treat ( alleviate a symptom to some extent , preferably all of the symptoms ) of a disease state . the pharmaceutically effective dose depends on the type of disease , the composition used , the route of administration , the type of mammal being treated , the physical characteristics of the specific mammal under consideration , concurrent medication , and other factors which those skilled in the medical arts will recognize . generally , an amount between 0 . 1 mg / kg and 100 mg / kg body weight / day of active ingredients is administered dependent upon potency of the negatively charged polymer . the nucleic acid molecules of the invention and formulations thereof can be administered orally , topically , parenterally , by inhalation or spray or rectally in dosage unit formulations containing conventional non - toxic pharmaceutically acceptable carriers , adjuvants and vehicles . the term parenteral as used herein includes percutaneous , subcutaneous , intravascular ( e . g ., intravenous ), intramuscular , or intrathecal injection or infusion techniques and the like . in addition , there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier . one or more nucleic acid molecules of the invention can be present in association with one or more non - toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants , and if desired other active ingredients . the pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use , for example , as tablets , troches , lozenges , aqueous or oily suspensions , dispersible powders or granules , emulsion , hard or soft capsules , or syrups or elixirs . compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents , flavoring agents , coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations . tablets contain the active ingredient in admixture with non - toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets . these excipients can be for example , inert diluents , such as calcium carbonate , sodium carbonate , lactose , calcium phosphate or sodium phosphate ; granulating and disintegrating agents , for example , corn starch , or alginic acid ; binding agents , for example starch , gelatin or acacia , and lubricating agents , for example magnesium stearate , stearic acid or talc . the tablets can be uncoated or they can be coated by known techniques . in some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period . for example , a time delay material such as glyceryl monosterate or glyceryl distearate can be employed . formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent , for example , calcium carbonate , calcium phosphate or kaolin , or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium , for example peanut oil , liquid paraffin or olive oil . aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions . such excipients are suspending agents , for example sodium carboxymethylcellulose , methylcellulose , hydropropyl - methylcellulose , sodium alginate , polyvinylpyrrolidone , gum tragacanth and gum acacia ; dispersing or wetting agents can be a naturally - occurring phosphatide , for example , lecithin , or condensation products of an alkylene oxide with fatty acids , for example polyoxyethylene stearate , or condensation products of ethylene oxide with long chain aliphatic alcohols , for example heptadecaethyleneoxycetanol , or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate , or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides , for example polyethylene sorbitan monooleate . the aqueous suspensions can also contain one or more preservatives , for example ethyl , or n - propyl p - hydroxybenzoate , one or more coloring agents , one or more flavoring agents , and one or more sweetening agents , such as sucrose or saccharin . oily suspensions can be formulated by suspending the active ingredients in a vegetable oil , for example arachis oil , olive oil , sesame oil or coconut oil , or in a mineral oil such as liquid paraffin . the oily suspensions can contain a thickening agent , for example beeswax , hard paraffin or cetyl alcohol . sweetening agents and flavoring agents can be added to provide palatable oral preparations . these compositions can be preserved by the addition of an anti - oxidant such as ascorbic acid . dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent , suspending agent and one or more preservatives . suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above . additional excipients , for example sweetening , flavoring and coloring agents , can also be present . pharmaceutical compositions of the invention can also be in the form of oil - in - water emulsions . the oily phase can be a vegetable oil or a mineral oil or mixtures of these . suitable emulsifying agents can be naturally - occurring gums , for example gum acacia or gum tragacanth , naturally - occurring phosphatides , for example soy bean , lecithin , and esters or partial esters derived from fatty acids and hexitol , anhydrides , for example sorbitan monooleate , and condensation products of the said partial esters with ethylene oxide , for example polyoxyethylene sorbitan monooleate . the emulsions can also contain sweetening and flavoring agents . syrups and elixirs can be formulated with sweetening agents , for example glycerol , propylene glycol , sorbitol , glucose or sucrose . such formulations can also contain a demulcent , a preservative and flavoring and coloring agents . the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension . this suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above . the sterile injectable preparation can also be a sterile injectable solution or suspension in a non - toxic parentally acceptable diluent or solvent , for example as a solution in 1 , 3 - butanediol . among the acceptable vehicles and solvents that can be employed are water , ringer &# 39 ; s solution and isotonic sodium chloride solution . in addition , sterile , fixed oils are conventionally employed as a solvent or suspending medium . for this purpose any bland fixed oil can be employed including synthetic mono - or diglycerides . in addition , fatty acids such as oleic acid find use in the preparation of injectables . the nucleic acid molecules of the invention can also be administered in the form of suppositories , e . g ., for rectal administration of the drug . these compositions can be prepared by mixing the drug with a suitable non - irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug . such materials include cocoa butter and polyethylene glycols . nucleic acid molecules of the invention can be administered parenterally in a sterile medium . the drug , depending on the vehicle and concentration used , can either be suspended or dissolved in the vehicle . advantageously , adjuvants such as local anesthetics , preservatives and buffering agents can be dissolved in the vehicle . dosage levels of the order of from about 0 . 1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above - indicated conditions ( about 0 . 5 mg to about 7 g per patient per day ). the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration . dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient . it is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed , the age , body weight , general health , sex , diet , time of administration , route of administration , and rate of excretion , drug combination and the severity of the particular disease undergoing therapy . for administration to non - human animals , the composition can also be added to the animal feed or drinking water . it can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet . it can also be convenient to present the composition as a premix for addition to the feed or drinking water . the nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect . the use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects . in yet another aspect the invention features an expression vector comprising nucleic acid sequence encoding at least one of the catalytic nucleic acid molecule of the invention , in a manner which allows expression of that nucleic acid molecule . the expression vector comprises in one embodiment ; a ) a transcription initiation region ; b ) a transcription termination region ; c ) a gene encoding at least one said nucleic acid molecule ; and wherein said gene is operably linked to said initiation region and said termination region , in a manner which allows expression and / or delivery of said nucleic acid molecule . in another preferred embodiment the expression vector comprises : a ) a transcription initiation region ; b ) a transcription termination region ; c ) an open reading frame ; d ) a gene encoding at least one said nucleic acid molecule , wherein said gene is operably linked to the 3 ′- end of said open reading frame ; and wherein said gene is operably linked to said initiation region , said open reading frame and said termination region , in a manner which allows expression and / or delivery of said nucleic acid molecule . in yet another embodiment the expression vector comprises : a ) a transcription initiation region ; b ) a transcription termination region ; c ) an intron ; d ) a gene encoding at least one said nucleic acid molecule ; and wherein said gene is operably linked to said initiation region , said intron and said termination region , in a manner which allows expression and / or delivery of said nucleic acid molecule . in another embodiment , the expression vector comprises : a ) a transcription initiation region ; b ) a transcription termination region ; c ) an intron ; d ) an open reading frame ; e ) a gene encoding at least one said nucleic acid molecule , wherein said gene is operably linked to the 3 ′- end of said open reading frame ; and wherein said gene is operably linked to said initiation region , said intron , said open reading frame and said termination region , in a manner which allows expression and / or delivery of said nucleic acid molecule . antisense molecules can be modified or unmodified rna , dna , or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis ( wu - pong , nov 1994 , biopharm , 20 - 33 ). the antisense oligonucleotide binds to target rna by watson crick base - pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating rnase h enzyme . antisense molecules can also alter protein synthesis by interfering with rna processing or transport from the nucleus into the cytoplasm ( mukhopadhyay & amp ; roth , 1996 , crit . rev . in oncogenesis 7 , 151 - 190 ). in addition , binding of single stranded dna to rna can result in nuclease degradation of the heteroduplex ( wu - pong , supra ; crooke , supra ). to date , the only backbone modified dna chemistry which will act as substrates for rnase h are phosphorothioates , phosphorodithioates , and borontrifluoridates . recently it has been reported that 2 ′- arabino and 2 ′- fluoro arabino - containing oligos can also activate rnase h activity . a number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides , secondary structure , and / or rnase h substrate domains ( woolf et al ., international pct publication no . wo 98 / 13526 ; thompson et al ., international pct publication no . wo 99 / 54459 ; hartmann et al ., u . s . ser . no . 60 / 101 , 174 which was filed on sep . 21 , 1998 ) all of these are incorporated by reference herein in their entirety . in addition , antisense deoxyoligoribonucleotides can be used to target rna by means of dna - rna interactions , thereby activating rnase h , which digests the target rna in the duplex . antisense dna can be expressed via the use of a single stranded dna intracellular expression vector or equivalents and variations thereof . single stranded dna can be designed to bind to genomic dna in a sequence specific manner . tfos are comprised of pyrimidine - rich oligonucleotides which bind dna helices through hoogsteen base - pairing ( wu - pong , supra ). the resulting triple helix composed of the dna sense , dna antisense , and tfo disrupts rna synthesis by rna polymerase . the tfo mechanism can result in gene expression or cell death since binding can be irreversible ( mukhopadhyay & amp ; roth , supra ). the 2 - 5a system is an interferon mediated mechanism for rna degradation found in higher vertebrates ( mitra et al ., 1996 , proc nat acad sci usa 93 , 6780 - 6785 ). two types of enzymes , 2 - 5a synthetase and rnase l , are required for rna cleavage . the 2 - 5a synthetases require double stranded rna to form 2 ′- 5 ′ oligoadenylates ( 2 - 5a ). 2 - 5a then acts as an allosteric effector for utilizing rnase l which has the ability to cleave single stranded rna . the ability to form 2 - 5a structures with double stranded rna makes this system particularly useful for inhibition of viral replication . ( 2 ′- 5 ′) oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of rna cleavage ( torrence , supra ). these molecules putatively bind and activate a 2 - 5a dependent rnase , the oligonucleotide / enzyme complex then binds to a target rna molecule which can then be cleaved by the rnase enzyme . several varieties of naturally - occurring enzymatic rnas are presently known , for example see table i . in addition , several in vitro selection ( evolution ) strategies ( orgel , 1979 , proc . r . soc . london , b 205 , 435 ) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages ( joyce , 1989 , gene , 82 , 83 - 87 ; beaudry et al ., 1992 , science 257 , 635 - 641 ; joyce , 1992 , scientific american 267 , 90 - 97 ; breaker et al ., 1994 , tibtech 12 , 268 ; bartel et al ., 1993 , science 261 : 1411 - 1418 ; szostak , 1993 , tibs 17 , 89 - 93 ; kumar et al ., 1995 , faseb j ., 9 , 1183 ; breaker , 1996 , curr . op . biotech ., 7 , 442 ; santoro et al ., 1997 , proc . natl . acad . sci ., 94 , 4262 ; tang et al ., 1997 , rna 3 , 914 ; nakamaye & amp ; eckstein , 1994 , supra ; long & amp ; uhlenbeck , 1994 , supra ; ishizaka et al ., 1995 , supra ; vaish et al ., 1997 , biochemistry 36 , 6495 ; all of these are incorporated by reference herein ). each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans ( and thus can cleave other rna molecules ) under physiological conditions . the enzymatic nature of an enzymatic nucleic acid molecule can allow the concentration of enzymatic nucleic acid molecule necessary to affect a therapeutic treatment to be lower . this reflects the ability of the enzymatic nucleic acid molecule to act enzymatically . thus , a single enzymatic nucleic acid molecule is able to cleave many molecules of target rna . in addition , the enzymatic nucleic acid molecule 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 . single mismatches , or base - substitutions , near the site of cleavage can be chosen to greatly attenuate the catalytic activity of a enzymatic nucleic acid molecule . nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate rna molecules in a nucleotide base sequence - specific manner . such enzymatic nucleic acid molecules can be targeted to virtually any rna transcript , and achieve efficient cleavage in vitro ( zaug et al ., 324 , nature 429 1986 ; uhlenbeck , 1987 nature 328 , 596 ; kim et al , 84 proc . natl . acad . sci . usa 8788 , 1987 ; dreyfus , 1988 , einstein quart . j . bio . med ., 6 , 92 ; haseloff and gerlach , 334 nature 585 , 1988 ; cech , 260 jama 3030 , 1988 ; and jefferies et al ., 17 nucleic acids research 1371 , 1989 ; santoro et al ., 1997 supra ). because of their sequence specificity , trans - cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human disease ( usman & amp ; mcswiggen , 1995 ann . rep . med . chem . 30 , 285 - 294 ; christoffersen and marr , 1995 j . med . chem . 38 , 2023 - 2037 ). enzymatic nucleic acid molecules can be designed to cleave specific rna targets within the background of cellular rna . such a cleavage event renders the rna non - functional and abrogates protein expression from that rna . in this manner , synthesis of a protein associated with a disease state can be selectively inhibited ( warashina et al ., 1999 , chemistry and biology , 6 , 237 - 250 ). chemically synthesizing nucleic acid molecules with modifications ( base , sugar and / or phosphate ) that prevent their degradation by serum ribonucleases can increase their potency ( see e . g ., eckstein et al ., international publication no . wo 92 / 07065 ; perrault et al ., 1990 nature 344 , 565 ; pieken et al ., 1991 , science 253 , 314 ; usman and cedergren , 1992 , trends in biochem . sci . 17 , 334 ; usman et al ., international publication no . wo 93 / 15187 ; and rossi et al ., international publication no . wo 91 / 03162 ; sproat , u . s . pat . no . 5 , 334 , 711 ; and burgin et al ., supra ; all of these describe various chemical modifications that can be made to the base , phosphate and / or sugar moieties of the nucleic acid molecules herein ). modifications which enhance their efficacy in cells , and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired . ( all these publications are hereby incorporated by reference herein ). there are several examples in the art describing sugar , base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy . for example , oligonucleotides are modified to enhance stability and / or enhance biological activity by modification with nuclease resistant groups , for example , 2 ′- amino , 2 ′- c - allyl , 2 ′- flouro , 2 ′- o - methyl , 2 ′- h , nucleotide base modifications ( for a review see usman and cedergren , 1992 , tibs . 17 , 34 ; usman et al ., 1994 , nucleic acids symp . ser . 31 , 163 ; burgin et al , 1996 , biochemistry , 35 , 14090 ). sugar modification of nucleic acid molecules have been extensively described in the art ( see eckstein et al ., international publication pct no . wo 92 / 07065 ; perrault et al . nature , 1990 , 344 , 565 - 568 ; pieken et al . science , 1991 , 253 , 314 - 317 ; usman and cedergren , trends in biochem . sci ., 1992 , 17 , 334 - 339 ; usman et al . international publication pct no . wo 93 / 15187 ; sproat , u . s . pat . no . 5 , 334 , 711 and beigelman et al ., 1995 , j biol . chem ., 270 , 25702 ; beigelman et al ., international pct publication no . wo 97 / 26270 ; beigelman et al ., u . s . pat . no . 5 , 716 , 824 ; usman et al ., u . s . pat . no . 5 , 627 , 053 ; woolf et al ., international pct publication no . wo 98 / 13526 ; thompson et al ., u . s . ser . no . 60 / 082 , 404 which was filed on apr . 20 , 1998 ; karpeisky et al ., 1998 , tetrahedron lett ., 39 , 1131 ; earnshaw and gait , 1998 , biopolymers ( nucleic acid sciences ), 48 , 39 - 55 ; verma and eckstein , 1998 , annu . rev . biochem ., 67 , 99 - 134 ; and burlina et al ., 1997 , bioorg . med . chem ., 5 , 1999 - 2010 ; all of the references are hereby incorporated in their totality by reference herein ). such publications describe general methods and strategies to determine the location of incorporation of sugar , base and / or phosphate modifications and the like into ribozymes without inhibiting catalysis , and are incorporated by reference herein . in view of such teachings , similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention . while chemical modification of oligonucleotide internucleotide linkages with phosphorothioate , phosphorothioate , and / or 5 ′- methylphosphonate linkages improves stability , too many of these modifications can cause some toxicity . therefore when designing nucleic acid molecules the amount of these internucleotide linkages should be minimized . the reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules . nucleic acid molecules having chemical modifications that maintain or enhance activity are provided . such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid . thus , in a cell and / or in vivo the activity can not be significantly lowered . therapeutic nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target rna has been inhibited long enough to reduce the levels of the undesirable protein . this period of time varies between hours to days depending upon the disease state . nucleic acid molecules are preferably resistant to nucleases in order to function as effective intracellular therapeutic agents . improvements in the chemical synthesis of rna and dna ( wincott et al ., 1995 nucleic acids res . 23 , 2677 ; caruthers et al ., 1992 , methods in enzymology 211 , 3 - 19 ( incorporated by reference herein ) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above . use of the nucleic acid - based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies ( e . g ., multiple antisense or enzymatic nucleic acid molecules targeted to different genes , nucleic acid molecules coupled with known small molecule inhibitors , or intermittent treatment with combinations of molecules ( including different motifs ) and / or other chemical or biological molecules ). the treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules . therapeutic nucleic acid molecules ( e . g ., enzymatic nucleic acid molecules and antisense nucleic acid molecules ) delivered exogenously are optimally stable within cells until translation of the target rna has been inhibited long enough to reduce the levels of the undesirable protein . this period of time varies between hours to days depending upon the disease state . these nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents . improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above . in one embodiment , nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided . such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acid . thus , in a cell and / or in vivo the activity of the nucleic acid can not be significantly lowered . as exemplified herein such enzymatic nucleic acids are useful in a cell and / or in vivo even if activity over all is reduced about 10 fold ( burgin et al ., 1996 , biochemistry , 35 , 14090 ). such enzymatic nucleic acids herein are said to “ maintain ” the enzymatic activity of an all rna ribozyme or all dna dnazyme . by “ enhanced enzymatic activity ” is meant to include activity measured in cells and / or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability . in this invention , the product of these properties is increased or not significantly ( less that 10 fold ) decreased in vivo compared to an all rna ribozyme . one of the most challenging tasks in drug discovery is the choice of a therapeutic target . historically , traditional biochemical and other studies have offered limited information in this regard . however , recent advances in genomics offer the potential to revolutionize both the speed and certainty of therapeutic target identification . progress in characterizing the genes in the human genome has been very rapid , and it is now estimated that the entire complement of genes in the human genome can be sequenced before the end of this century . however , this mass of information is coming to the scientific world without a road map . converting pure gene sequence information into a functional understanding of their role in human disease is proving to be a much more difficult problem . even after a group of genes is associated with a particular disease , the process of validating which genes are appropriate for use as therapeutic targets is often slow and costly . most companies with genomics activities now have access to myriad partial or full sequences , but do not possess adequate technologies to determine which of those sequences is an appropriate therapeutic target . as a result , only a few genes have been unequivocally identified as the causative agent for a specific disease . the nucleic acid molecules of the present invention can inhibit gene expression in a highly specific manner by binding to and causing the cleavage of the mrna corresponding to the gene of interest , and thereby prevent production of the gene product ( christoffersen , nature biotech , 1997 , 2 , 483 - 484 ). appropriate delivery vehicles can be combined with these nucleic acid molecules ( including polymers , cationic lipids , liposomes and the like ) and delivered to appropriate cell culture or in vivo animal disease models as described above . by monitoring inhibition of gene expression and correlation with phenotypic results , the relative importance of the particular gene sequence to disease pathology can be established . the process can be both fast and highly selective , and allow for the process to be used at any point in the development of the organism . the novel chemical composition of these nucleic acid molecules can allow for added stability and therefore increased efficacy . the following are non - limiting examples demonstrating the utility of the nucleic acid molecules of the instant invention . those in the art will recognize that certain experimental conditions such as temperatures , reaction times , media conditions , transfection reagents and rna assays are not meant to be limiting and can be readily modified without significantly altering the protocols . geneblocs ( seq id nos : 1 - 4 , table iii ) were designed , synthesized , and were tested in the rat corneal model of vegf - induced angiogenesis ( nucleic acid res ., vol . 27 : 2569 , 1999 ). briefly , a filter paper disk soaked in vegf ( 1 μl of a 30 μm solution in 82 mm tris - hcl , ph 6 . 9 ) was implanted in a stromal pocket in the eye of an anesthesized male sprague - dawley rat , 1 mm from the edge of the corneal limbus . after implantation of the disk , vehicle control ( 600 nl sterile water ), mismatch control ( seq id no : 2 or seq id no : 4 ) ( 10 μg in 600 nl sterile water ), or active genebloc ( seq id no : 1 or seq id no : 3 ) ( 10 μg in 600 nl sterile water ) was administered by intraconjunctival injection adjacent to the disk implant site , 1 mm from the edge of the corneal limbus . further control treatments included implantation of tris - soaked disks and injection of vehicle , and for specificity control basic fgf - soaked disks and injection of active genebloc . five days after surgical implantation of the disks , animals were euthanized and cornea were digitally imaged for quantitation of neovascular surface area using computerized morphometry . animal housing and experimentation adhered to standards outlined in the 1996 guide for the care and use of laboratory animals ( national research council ). male sprague dawley rats ( 250 - 300 g ) were anesthetized with ketamine ( 50 mg / kg ), xylazine ( 10 mg / kg ), and acepromazine ( 0 . 5 mg / kg ) administered intramuscularly ( im ). the level of anesthesia was monitored every 2 - 3 min by applying hind limb paw pressure and examining for limb withdrawal . atropine ( 0 . 4 mg / kg , im ) was also administered to prevent potential corneal reflex - induced bradycardia . for corneal implantation , 0 . 57 mm diameter nitrocellulose disks , prepared from 0 . 45 μm pore diameter nitrocellulose filter membranes ( millipore corporation ), were soaked for 30 min in 1 μl of 30 μm vegf 165 in 82 mm tris hcl ( ph 6 . 9 ) in covered petri dishes on ice . the rat corneal model used in this study was a modified from koch et al . supra and pandey et al ., supra . briefly , corneas were irrigated with 0 . 5 % povidone iodine solution followed by normal saline and two drops of 2 % lidocaine . under a dissecting microscope ( leica mz - 6 ), a stromal pocket was created and a presoaked filter disk ( see above ) was inserted into the pocket such that its edge was 1 mm from the corneal limbus . immediately after disk insertion , the tip of a 40 - 50 μm od injector was inserted within the conjunctival tissue 1 mm away from the edge of the corneal limbus that was directly adjacent to the vegf - soaked filter disk . six hundred nanoliters of test solution ( ribozyme , attenuated control or sterile water vehicle ) were dispensed at a rate of 1 . 2 μl / min using a syringe pump ( kd scientific ). the injector was then removed , serially rinsed in 70 % ethanol and sterile water and immersed in sterile water between each injection . once the test solution was injected , closure of the eyelid was maintained using microaneurism clips until the animal began to recover gross motor activity . following treatment , animals were warmed on a heating pad at 37 ° c . nucleic acid molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of specific rnas in a cell . the close relationship between for example ribozyme activity 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 rna . by using multiple ribozymes described in this invention , one can map nucleotide changes which are important to rna structure and function in vitro , as well as in cells and tissues . cleavage of target rnas with ribozymes can be used to inhibit gene expression and define the role ( essentially ) of specified gene products in the progression of disease . in this manner , other genetic targets can be defined as important mediators of the disease . these experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies ( e . g ., multiple nucleic acid molecules targeted to different genes , nucleic acid molecules coupled with known small molecule inhibitors , or intermittent treatment with combinations of nucleic acid molecules and / or other chemical or biological molecules ). other in vitro uses of nucleic acid molecules of this invention are well known in the art , and include detection of the presence of rnas related to various conditions . such rna is detected by determining the presence of a cleavage product after treatment with for example , an enzymatic nucleic acid molecule using standard methodology . in a specific example , ribozymes which can cleave only wild - type or mutant forms of the target rna 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 . 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 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 whose protein product is implicated in the development of a 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 . potential uses of sequence - specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of rna that dna restriction endonucleases have for the study of dna ( nathans et al ., 1975 ann . rev . biochem . 44 : 273 ). for example , the pattern of restriction fragments can be used to establish sequence relationships between two related rnas , and large rnas can be specifically cleaved to fragments of a size more useful for study . the ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of rnas of unknown sequence . applicant has described the use of nucleic acid molecules to down - regulate gene expression of target genes in bacterial , microbial , fungal , viral , and eukaryotic systems including plant , or mammalian cells . all patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains . all references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually . one skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those inherent therein . the methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention . changes therein and other uses will occur to those skilled in the art , which are encompassed within the spirit of the invention , are defined by the scope of the claims . it will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention . thus , such additional embodiments are within the scope of the present invention and the following claims . the invention illustratively described herein suitably can be practiced in the absence of any element or elements , limitation or limitations which is not specifically disclosed herein . thus , for example , in each instance herein any of the terms “ comprising ”, “ consisting essentially of ” and “ consisting of ” can be replaced with either of the other two terms . the terms and expressions which have been employed are used as terms of description and not of limitation , and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof , but it is recognized that various modifications are possible within the scope of the invention claimed . thus , it should be understood that although the present invention has been specifically disclosed by preferred embodiments , optional features , modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art , and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims . in addition , where features or aspects of the invention are described in terms of markush groups or other grouping of alternatives , those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the markush group or other group . requires a u in the target sequence immediately 5 ′ of the cleavage site . additional protein cofactors required in some cases to help folding and over 300 known members of this class . found as an intervening sequence complete kinetic framework established for one ribozyme [ iii , iv , v , vi ]. studies of ribozyme folding and substrate docking underway [ vii , viii , ix ]. the small ( 4 - 6 nt ) binding site may make this ribozyme too non - specific for targeted rna cleavage , however , the tetrahymena group i intron has been used to repair a “ defective ” β - galactosidase message by the ligation of new β - galactosidase sequences onto the defective message [ xii ]. reaction mechanism : possible attack by m 2 + - oh to generate cleavage rnase p is found throughout the prokaryotes and eukaryotes . the rna important phosphate and 2 ′ oh contacts recently identified [ xvi , xvii ] trans cleavage of target rnas recently demonstrated [ xviii , xix ]. [ xx , xxi ] in addition to rna cleavage and ligation . important 2 ′ oh contacts beginning to be identified [ xxiii ] trans cleavage of hairpin target rnas recently demonstrated [ xxv ]. reaction mechanism : attack by 2 ′- oh 5 ′ to the scissile bond to generate only 1 known member of this class . found in neurospora vs rna . binds a variable number nucleotides on both sides of the cleavage site . reaction mechanism : attack by 2 ′- oh 5 ′ to the scissile bond to generate 14 known members of this class . found in a number of plant pathogens complete kinetic framework established for two or more ribozymes [ xxix ]. binds 4 - 6 nucleotides at the 5 ′- side of the cleavage site and a variable reaction mechanism : attack by 2 ′- oh 5 ′ to the scissile bond to generate 3 known members of this class . found in three plant pathogen ( satellite rnas of the tobacco ringspot virus , arabis mosaic virus and chicory essential structural features largely defined [ xxxi , xxxii , xxxiii , xxxiv ] reaction mechanism : attack by 2 ′- oh 5 ′ to the scissile bond to generate o xli nly 2 known members of this class . found in human hdv . circular form of hdv is active and shows increased nuclease stability [ xlii ] i michel , francois ; westhof , eric . slippery substrates . nat . struct . biol . 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( 1995 ), 83 ( 4 ), 529 - 38 . xxi griffin , edmund a ., jr . ; qin , zhifeng ; michels , williams j ., jr . ; pyle , anna marie . group ii intron ribozymes that cleave dna and rna linkages with similar efficiency , and lack contacts with substrate 2 ′- hydroxyl groups . chem . biol . ( 1995 ), 2 ( 11 ), 761 - 70 . xxii michel , francois ; ferat , jean luc . structure and activities of group ii introns . annu . rev . biochem . ( 1995 ), 64 , 435 - 61 . xxiii abramovitz , dana l . ; friedman , richard a . ; pyle , anna marie . catalytic role of 2 ′- hydroxyl groups within a group ii intron active site . science ( washington , d . c .) ( 1996 ), 271 ( 5254 ), 1410 - 13 . xxiv daniels , danette l . ; michels , william j ., jr . ; pyle , anna marie . two competing pathways for self - splicing by group ii introns : a quantitative analysis of in vitro reaction rates and products . j . mol . biol . 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