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WO1995006764A2 - Oligonucleotides ayant une activite de segmentation de l'arn - Google Patents

Oligonucleotides ayant une activite de segmentation de l'arn Download PDF

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Publication number
WO1995006764A2
WO1995006764A2 PCT/IB1994/000288 IB9400288W WO9506764A2 WO 1995006764 A2 WO1995006764 A2 WO 1995006764A2 IB 9400288 W IB9400288 W IB 9400288W WO 9506764 A2 WO9506764 A2 WO 9506764A2
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Prior art keywords
substituted
oligonucleotide
diol
rna
formula
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PCT/IB1994/000288
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English (en)
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WO1995006764A3 (fr
Inventor
Larry W. Mclaughlin
Dong-Jing Fu
Fritz Benseler
Gerd Kotzorek
Janos Ludwig
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Vpi Holdings Ltd.
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Priority to AU76231/94A priority Critical patent/AU7623194A/en
Publication of WO1995006764A2 publication Critical patent/WO1995006764A2/fr
Publication of WO1995006764A3 publication Critical patent/WO1995006764A3/fr

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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    • 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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • 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
    • C12N2310/121—Hammerhead
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3183—Diol linkers, e.g. glycols or propanediols
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3527—Other alkyl chain
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    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon
    • C12N2310/3533—Halogen
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon
    • C12N2310/3535—Nitrogen

Definitions

  • the present invention relates to oligonucleotides, including oligonucleotides with RNA cleavage activity.
  • RNA is known to have endoribonuclease activity.
  • RNA molecules called ribozymes have been used to bind to target RNA molecules and catalyze their cleavage, thus blocking the activity of the target RNA.
  • the so-called hammerhead ribozymes have been widely studied.
  • the recognition site and catalytic site of these oligoribonucleotides are well characterized and ribozymes containing a recognition sequence specific for any desired target RNA which contains a specified triplet can be constructed. These compounds can therefore be considered as potential therapeutic agents with possibly higher biological activity than the simple antisense
  • Eckstein et al. introduced 2'-amino or 2'-F substituents into the pyrimidine positions or the hammerhead ribozyme. Pieken, W.A.,
  • adenosines A 6 , A 9 or A 15.1 , A 14 , A 13 is replaced by 2'-F. Replacement of more 2'-OH-s, however, is not allowed with this substituent.
  • McLaughlin et al. proposed a role for the 2'-OH as of G 5 or G 8 . They interact with H 2 O molecules bound in the first co-ordination sphere of the Mg 2+ cofactor. Fu, D.J. and
  • oligonucleotides selected from the following general sequences of Formulas I through V:
  • A is adenosine or 2'-deoxyadenosine
  • C is cytidine or 2'-deoxycytidine
  • G is guanosine or 2'-deoxyguanosine
  • U is uridtne or 2'-deoxyuridine
  • a is either A as defined above or substituted deoxyadenosine according to formula Ia below, and
  • g is either G as defined above or substituted deoxyguanosine according to formula Ib below,
  • the C2' stereogenic center is of either S or R configuration, according to the Cahn-lngold-Prelog nomenclature, and preferably is of the R configuration, and the substituent R is selected from -CF 2 H, -CF 2 , -CCl 2 H, -CCI 3 , CBr 2 H, CBr 3 , Cl 2 H, Cl 3 , - CONH 2 , -CONHR', -CONR'R", -NHCOH, -NHCOR", -N(R')COR", -SH, -SR', -NH 2 . -NHR', -NHR'R", -COOR', and -NHCOOR', or
  • R' and R" are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or
  • P 1 is a) A-U, b) U-A, c) G-C, or d) C-G,
  • P 2 is a) A-U, b) U-A, c) G-C, or d) C-G,
  • P 3 is a) A-U, b) U-A, c) G-C, or d) C-G,
  • P 4 is a) A-U, b) U-A,c) G-C, or d) C-G,
  • W is either i) a tetranucleotide loop sequence composed of C, G, A and U residues, for example the tetranucleotide IIa has been used for general sequence IV when P 1 is C-G, P 2 is A-U, P 3 is G-C and
  • P4 is G-C 5'-C-C-G-A-3' IIa or the nucleotide loop sequence IIb has been used when, P 1 is C-G, P 2 is C-G, P 3 is G-C and P 4 is G-C
  • m diol bridges are connected together, and one or more of the connections may be through phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted
  • n may be independently selected for each bridge from 1 to 10.
  • n is two or three in each bridge and there are four to six bridges;
  • m is 4 to 6.
  • oligonucleotide of Formulas I, II, III, IV or V more than one a and/or g group of the oligonucleotide is of the formula la or formula lb as defined above, and more preferably each a and g group of the oligonucleotide is other than A or G, i.e. each a and g group of the oligonucleotide is of the formula la or lb as such formulas are defined above.
  • the present invention provides in another aspect provides oligonucleotides selected from the general sequences Formulas IA- VA:
  • A is adenosine or 2'-deoxyadenosine
  • C is cytidine or 2'-deoxycytidine
  • G is guanosine or 2'-deoxyguanosine
  • U is uridine or 2'-deoxyuridine
  • P 1 is a) A-U, b) U-A, c) G-C, or d) C-G,
  • P 2 is A-U, b) U-A, c) G-C, or d) C-G,
  • P 3 is a) A-U, b) U-A, c) G-C, or d) C-G,
  • P 4 is a) A-U, b) U-A, c) G-C, or d) C-G
  • W is 1) the diol bridges iii connected with phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted phosphoramidate, or methylphosphonate derivative linkages m(Z) iii in which each diol bridge (Z) is of formula iv - [O-(CH2) n ] - iv n is 1 - 10,
  • m diol bridges are connected together, one or more of the connections may be through phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted
  • W is 2) the tetranucleotide IIa, 5'-C-C-G-A-3' IIa or W is 3) the tetranucleotide IIb 5--G-U-U-A-3' IIb.
  • n may be independently selected for each bridge from 1 to 10.
  • n is two or three in each bridge and there are four to six bridges;
  • m is 4 to 6.
  • the invention also provides novel methods, including a method of inhibiting expression of susceptible single-stranded RNA that comprises contacting said susceptible RNA with an expression inhibition effective amount of an oligonucleotide of Formula l-V or IA- VA.
  • the invention provides those
  • Figure 1 shows the nucleotide sequence of a hammerhead ribozyme.
  • Figure 2 shows an example of therapeutic target interaction between the myconcogene mRNA and an oligonucleotide of the invention of the type described in Example 1.
  • Figure 3 is the 31 P-NMR spectrum of the 1 ,3-propanediol linker synthesized as the 4,4 , -dimethoxytrityl- ⁇ -cyanoethyl-phosphoramidite derivative of Example 8.
  • Figure 4 shows the results of polyacrylamide gel
  • Figure 5 is a copy of a typical autoradiogram used to monitor the cleavage of the 12-mer substrate in Example 8.
  • Groups X and Y of Formulas I-V and IA-VA above are target specific recognition sequences made up of any deoxyribonucleosides N depending on the target RNA sequence. That is, X and Y each will be deoxyribonucieoside sequences complementary, at least in part, to the sequence of a single-stranded RNA to be cleaved by the particularly oligonucleotide of the invention. X and Y may be of the same or different length. There is no need for the molecule to be symmetrical. Each of X and Y may be 4 to 25 nucteotides long, preferably 6 to 20 nucleotides, containing one or more of A, G, C and U nucleotides.
  • X or Y may include one or more stabilizing modifications, i.e. structural features that inhibit degradation of the oligonucleotide relative to a sequence that lack such features.
  • stabilizing modifications i.e. structural features that inhibit degradation of the oligonucleotide relative to a sequence that lack such features.
  • two or three natural 3'-5' phosphodiester linkages present at least on the 3' end of X may be modified in an attempt to protect the oligonucleotide from attack by 3'-exonucleases.
  • 3'-5' phosphodiester linkage may be replaced by phosphorothiate linkages such as thiophosphodiester linkages.
  • Suitable sequences of X and Y for a particular target single-stranded RNA can be readily ascertained by those skilled in the art .
  • the sequence of a target RNA i.e. an RNA to be cleaved by an oligonucleotide of the present invention, can be determined by known means such as by sequencing the
  • sequences of X and Y are selected to complement at least a substantial number of the nucleotides of the target RNA, e.g. where the nucieotides of X and Y complement at least about 60% of the nucieotides of the target RNA, more preferably at least about 75%, still more preferably at least about 90-96 or 98% and even more preferably all of the nucieotides of X and Y
  • the 2'-R substituent of oligonucleotides. of Formulas I-V and IA- VA is a non-nucleophilic group which is preferably, but not
  • the substituent ideally has both H-bond donor and acceptor abilities.
  • a 2'-COOH substituent which can chelate with Mg 2+ , is advantageous as -COOH containing amino acid side chains can be important for Mg 2+ binding in enzymes.
  • modifications according to the invention which involve the 2'-R substitution of nucleotides in the catalytic/cleavage region of the oligonucleotide can provide a desirable increase in stability against degradation and can increase catalytic activity due to improved Mg 2+ binding.
  • Oligonucleotides according to the invention with diol bridges can be made by machine more easily and more cheaply than conventional ribozymes.
  • Preferred alkyl groups of the oligonucleotides of Formulas l-V include those groups having from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1 to about 6 carbon atoms.
  • Methyl, ethyl, propyl and butyl including isopropyl and branched butyl groups such as sec-butyl and f-butyl are particularly preferred alkyl groups.
  • alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members. Straight or branched chain noncyclic alkyl groups are generally more preferred than cyclic groups.
  • Preferred alkenyl and alkynyl groups of oligonucleotides of Formulas l-V and IA-VA have one or more unsaturated linkages and from 2 to about 12 carbon atoms, more preferably 2 to about 8 carbon atoms, still more preferably 2 to about 6 carbon atoms, even more preferably 2, 3 or 4 carbon atoms.
  • alkenyl and alkynyl as used herein refer to both cyclic and noncyclic groups, although straight or branched noncyclic groups are generally more preferred.
  • Preferred alkoxy groups of the oligonucleotides of the invention include groups having one or more oxygen linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms, even more preferably 1 , 2, 3 or 4 carbon atoms.
  • Preferred thioalkyl groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Particularly preferred are thioalkyl groups having 1 , 2, 3 or 4 carbon atoms.
  • aminoalkyl groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably one to about 8 carbon atoms, still more preferably 1 to about 6 carbon atoms, even more preferably 1 , 2, 3 or 4 carbon atoms.
  • Secondary and tertiary amine groups are generally more preferred than primary amine moieties.
  • Substituted moieties of oligonucleotides of the invention may be substituted at one or more available positions by one or more suitable groups such as, e.g., halogen such as fluoro, chloro, bromo and iodo; cyano; hydroxyl; alkyl groups inctuding those groups having 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms, preferably noncyclic alkyl groups including branched chain groups such as isopropyl and t-butyl; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon or from 2 to about 6 carbon atoms; thioalkyl groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; alkoxy groups having those having one or more oxygen linkages and from 1 to about 12 carbon atoms or 1 to about 6 carbon atoms; and
  • the oligonucleotides of the present invention may be used as intermediates for further modification to improve their ease of up-take by the cell (in comparison to unmodified oligonucleotides of the invention and known ribozymes), for example by the attachment of carrier molecules.
  • the embodiments having diol bridges are considered to be particularly useful as intermediates for this purpose.
  • the invention provides methods for inhibiting expression of susceptible mRNA comprising contacting the RNA an expression inhibition effective amount of an oligonucleotide of the invention.
  • Inhibition effective amounts of particular oligonucleotide can be readily determined, e.g., by the methods of E xample 8 which follows.
  • the oligonucleotides of the present invention thus are potential antagonists of a wide range of therapeutic targets which involve over-expression of products.
  • a product e.g. an enzyme or a protein
  • a gene is causative of an illness or
  • the oligonucleotides of the present invention will have utility as anticancer and antiviral agents as well as anti-inflammatory and anti-ulcer agents.
  • the stability of the oligonucleotides of the present invention indicates that they can be used in nanomblar amounts. This offers a significant improvement over known ribozymes which have to be used in relatively large amounts to compensate for their intracellular degradation by nucleases.
  • the oligonucleotides of the invention may achieve true catalytic activity, i.e. they will not be destroyed in the cleavage reaction.
  • oligonucleotides Formulas l-V and IA-VA will be useful as therapeutic for the treatment of mammals, including humans, particularly for the treatment of mammals having cancer cells susceptible to one or more of the oligonucleotides.
  • the invention provides a method for treatment of susceptible cancer cells, e.g., a solid or disseminated tumor, in mammals including humans, the method comprising administration to the mammal of an antitumor effective amount of one or more oligonucleotides of the invention, once or several times a day or other appropriate schedule, orally, rectally, parenterally, topically, etc.
  • a mammal such as a human that has a susceptible viral infection is administered an antiviral effective amount of one or more oligonucleotides of the invention, once or several times a day, or other appropriate schedule, by a suitable route of administration such as orally or parenterally, particularly intravenously.
  • a suitable route of administration such as orally or parenterally, particularly intravenously.
  • an anti-inflammatory or anti-ulcer effective amount of one or more oligonucleotides of the invention to a mammal in need thereof, particularly a human in need thereof, according to an appropriate schedule and route of administration such as orally or parenterally.
  • the oligonucleotides of the present invention may be suitably administered to a subject as a pharmaceutically acceptable salt
  • Such salts can be prepared in a number of ways.
  • salts can be formed from an organic or inorganic acid, e.g. hydrochioride, sulfate, hemisuifate, phosphate, nitrate acetate, oxalate, citrate, maleate, etc.
  • pharmaceutically acceptable salts include those formed form alkali metals, e.g. a sodium salt.
  • an oligonucleotide of the invention is typically administered to a subject in aqueous or non-aqueous sterile injection solutions as are known in the art.
  • the therapeutic of the invention may be administered to a subject such as a human in discrete units such as capsules or tablets each containing a predetermined amount of the therapeutic, as a solution or a suspension in an aqueous or non-aqueous liquid, as an oil/water liquid emulsion, in powdered carriers such as lactose or sucrose, etc.
  • Oligonucleotides of Formulas l-V and IA-VA will also be useful in screening methods of the invention, including methods that enable identification of therapeutic agents which inhibit RNA cleavage.
  • Such therapeutics will have significant utility in treatment of subjects, particularly mammals such as humans, that fail to express adequate amounts of particular proteins due to abnormal RNA degradation.
  • Such therapeutics can be identified by a method comprising steps of:
  • RNA molecule(s) (a) contacting targeted single-stranded RNA molecule(s) with a compound suspected of inhibiting degradation (e.g., cleavage) of said RNA molecule;
  • step (b) adding to the mixture obtained in said step (a) one or more oligonucleotides of Formulas l-V or IA-VA as defined above under conditions suitable for said oligonucleotides to cleave said RNA molecule, and wherein the oligonucleotide can cleave the RNA molecule in the absence of said suspected compound; and
  • step (c) assaying the mixture obtained from step (b), such as by northern blot analysis, to determine cleavage of said RNA molecule, wherein lack of cleavage of said RNA molecule relative to a control indicates that the compound inhibits degradation of the RNA
  • test compound suspected of inhibiting RNA degradation will be added to the target single-stranded sequence, i.e. the "substrate" sample, prior to mixing with the oligonucleotide of the invention.
  • the control sample for the above method will be obtained by following the same protocol as for the test compound, but with the omission of adding the test compound to the RNA.
  • nucleosides (A, G, C and U) were obtained from Miiligen (New
  • triethylenegiyycol and 1,3-propanediol prepared as the corresponding 4,4'-dimethoxytrityl- ⁇ -cyanoethyl phosphoramidite derivatives, were synthesized according to known methods.
  • the 31 P-NMR resonances of the three phosphoamidite linker derivatives were 148.6, 148.5 and 147.2 ppm, respectively.
  • a typical 31 P-NMR spectrum is illustrated in Fig. 3 for the propanediol linker.
  • Some of the 1 ,3-propanediol linker was also obtained from Glen Research (Sterling, VA).
  • Oligonucleotides were synthesized using an Applied Biosystems 381A DNA synthesizer. High performance liquid chromatography (HPLC) was carried out on an ODS-Hypersil column (0.46 ⁇ 25 cm, Shandon Southern, England) using a Beckman EPLC system. 1 H NMR spectra were obtained at 300 MHz or 500 MHz on Varian XL-300 or 500 multinuclear spectrometers. 31 P NMR spectra were obtained at 121 MHZ on the Varian XL-300. Absorption spectra were recorded by a Perkin-Elmer Lambda 3B UV/Vis spectrophotometer. Nuclease S1 is a product of United States Biochemical Corporation (Cleveland, Ohio). RNase T2 was obtained from Sigma (St. Louis, MO).
  • N represents any deoxyribonucleotide recognition sequence specific for the target RNA
  • N.N represents a thiophosphodiester linkage replacing a natural
  • A is deoxyadenosine
  • G is deoxyguanosine
  • P 1 in this case is C - G and P 2 is A - U,
  • a is 2'substituted deoxyadenosine according to formula Ia as identified and defined above
  • g is 2'substituted deoxyguanosine according to formula Ib as identified and defined above.
  • W is 3'-G-G-A-G-C-C-C-S'
  • N represents any deoxyribonucleotide recognition sequence specific for the target RNA
  • N.N represents a thiophosphodiester linkage replacing a natural 3'-5' phosphodiester linkage between two nucieotides
  • A is deoxyadenosine
  • G is deoxyguanosine
  • P 1 in this case is C - G and P 2 is C - G,
  • a is 2'substituted deoxyadenosine according to formula Ia as identified and defined above
  • g is 2'substituted deoxyguanosine according to formula Ib as identified and defined above
  • oligonucleotide having the following sequence in which N, N.N, A, C, G, U, a and g are as defined for
  • N, N.N, A, C, G, U, a and g are as defined for Example 1 and the diol bridges are connected with phosphodiester or substituted neutral phosphotoreceptor derivative linkages.
  • A is adenosine
  • G is guanosine
  • A is adenosine
  • G is guanosine
  • FIG. 2 A therapeutic target interaction between mRNA and an oligonucleotide of the invention is shown in Figure 2.
  • the -myc oncogene mRNA secondary structure is shown from nucleotide 1 to nucleotide 900. Translation starts at nucleotide 421 and the triplet cleavage site is positions 433 to 435 (GUU).
  • Native hammerhead has the following structure
  • loop L as indicated in the above structure is replaced completely or partially with simple synthetic organic linkers.
  • Complexes have been prepared in which loop L comprises a four base-pair stem and a loop comprising linkers based on hexaethylene giycol 8(1), bis(triethylene giycol) phosphate 8(2), tris(propanediol) bisphosphate 8(3), bis(propanediol) phosphate 8(4) and propanediol 8(5), the structures of such complexes specified below.
  • hammerhead-like catalysts composed of as few as twenty two nucleosides.
  • a series of complexes may be generated that vary in the length of the synthetic linker, which in turn varies the spacing of the tethered nucleoside residues Ag and G 10 -
  • the linkers differ in the presence or absence of one or more negatively charged
  • polyacrylamide gel electrophoresis indicated the presence of a single species and the mobility of the various RNA/linker sequences was directly related to the number of nucleoside residues present. The presence of additional phosphodiester residues in the linkers increased the distance of migration.
  • Cleavage activity of the oligonucleotide complexes identified above and specified in the below Table was measured under single turnover conditions with a large excess of the ribozyme-like catalyst (i.e., complex) in order to ensure complexation of the substrate sequence as detailed more fully below.
  • First order rate constants characterizing the cleavage reaction were calculated from the halflife of the complexes and were corrected for the extent of cleavage at too as specified below:
  • the number of base pairs in the stem of loop L can be reduced from four to two with the use of the neutral hexaethyleneglycol linker without any change in the cleavage rate (compare 8(6) with 8(1)), but the similar sequence in 8(7) with two negatively charged
  • phosphodiester residues is less active.
  • Two additional complexes containing the hexaethyleneglycol linker, one with a single base pair 8(8), and one without any base pair 8(11) in the stem are also active catalysts, but the cleavage rates are reduced by 10- and 200-fold, respectively. The reduced cleavage rates with these two complexes suggests that the presence of a base pair, or similar structure, at the base of the stem is critical for formation of the active complex.
  • Sequences comprising a phosphodiester-containing linker and either a single C-G base pair (8(9) or 8(10)), or no base pair (8(12), 8(13) & 3(14)) in the stem are alt less active than the corresponding
  • the neutral giycol linker can be employed to replace all of the nucleosides GUUA of loop L and two of the base pairs of the stem without any observable loss of cleavage activity. Some loss of activity is observed as three or all four of the base pairs of the stem are eliminated, but these complexes remain active RNA-cieaving
  • oligonucleotides identified above were synthesized from 1 umol of bound nucleoside on wide-pore silica supports using
  • RNA was separated and dissolved in 0.1 to 0.5mL of sterile water. Purification was carried out in 2mm thick 20% polyacrylamide gels containing 7M urea. After electrophoresis at 23 Watts for approximately 16h, the oligonucleotides were visualized by UV shadowing, excised from the gel and the RNA extracted
  • RNA sequences were analyzed by analytical means.
  • the gel is, illustrated in Fig. 4.
  • the lane numbers and complex numbers are as follows: 1 - 8(2); 2 - 8(1); 3 - 8(3); 4 - 8(4); 5 - 8(5); 6
  • RNA fragments eluted as single peaks from a reversed-phase column ODS-Hypersil eluted with 50mM triethyiammonium acetate pH 7.0 and a gradient or acetonitrile.
  • Nucleotide (or nucleoside) composition and the integrity of the nucleoside 3'-5' phosphodiester linkage was determined after S1 nuclease (or S1 nuclease and calf intestinal alkaline pbosphatase) hydrolysis.
  • linkers could be confirmed by treatment of the sequence with RNase T2 followed by calf intestinal alkaline phosphatase. Under these conditions the linker remained bound to the nucleoside 5'-hydroxyl through a phosphodiester linkage.
  • Standards could be prepared by coupling the DMT -phosphoramidrte linker to the appropriate (usually G, but C of sequences 8(7) - 8(10)) followed by ammonia, TBAF and acid deprotection. Standards were generally used without purification.
  • the 12-mer substrate was 5'-end labeled with [Y- 32 P] ATP as follows: A 100 uL reaction mixture containing 2 A ⁇ units of 24-mer (about 0.1 mM), 40mM Tris.HCl, pH 8.0, 10 mM MgCI 2 , 10 mM dithiothreitot, 0.2 mM Na 2 EDTA, 0.1 mM ATP, 300-600 uCi of [Y- 32 P] ATP, and 20 units of T4 potynucleotide kinase was incubated for 60 min at 37°C. The product was isolated by absorption on a C18 Sep-Pak cartridge.
  • the cartridge was washed with water and then with 40-50% aqueous methanol to elute the product.
  • the labeled 12-mer was repurified by electrophoresis in a 20% polyacryiamide/7 M urea gel.
  • the product band was excised, and eiectrophoretically extracted with 0.1 M ammonium acetate, pH 7.0, and desalted with a C18 Sep-Pak cartridge.
  • the specific activity of the 12-mer was typically 0.01 uCi/pmol.
  • Fig. 5 is a copy of a typical autoradiogram used to monitor cleavage of the 12-mer substrate.
  • the upper band in each case is the 12-mer substrate and the lower band is the 5-mer product.
  • Four radioactive alignment markers are also present After autoradiography, the substrate and product bands were excised, lyophilized to dryness, and the

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Abstract

L'invention se rapporte à de nouveaux oligonucléotides, notamment aux oligonucléotides ayant une activité de segmentation de l'ARN.
PCT/IB1994/000288 1993-09-03 1994-09-05 Oligonucleotides ayant une activite de segmentation de l'arn WO1995006764A2 (fr)

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Cited By (18)

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WO1996000232A1 (fr) * 1994-06-24 1996-01-04 Gene Shears Pty. Ltd. RIBOZYMES PORTANT DES BRAS D'HYBRIDATION, DES TIGES ET DES BOUCLES OPTIMISES, RIBOZYMES ENCAPSULES DANS L'ARNt ET COMPOSITIONS DERIVEES
EP0837933A1 (fr) * 1995-06-07 1998-04-29 Commonwealth Scientific And Industrial Research Organisation Minizymes et miniribozymes optimises et utilisation de ces derniers
US5840876A (en) * 1995-04-20 1998-11-24 Ribozyme Pharmaceuticals, Inc. 2'-O-alkylthioalkyl and 2'-C-alkylthioalkyl-containing nucleic acids
US6127159A (en) * 1997-06-06 2000-10-03 The Board Of Trustees Of The Leland Stanford Junior University Mitofusin genes and their uses
WO2001012787A2 (fr) * 1999-08-12 2001-02-22 City Of Hope Ribozymes chimeres adn/arn contenant du propanediol
WO2003050241A2 (fr) 2001-12-12 2003-06-19 The Government Of The United States Of America As Represented By The Secretary, Department Of Healthand Human Services Procedes d'utilisation d'inhibiteurs de l'adenosine extracellulaire et d'inhibiteurs de recepteur de l'adenosine aux fins d'amelioration de reponse immune et d'inflammation
US6953680B2 (en) 1999-10-06 2005-10-11 The Board Of Trustees Of The Leland Stanford, Junior University Mitofusins, Fzo Homologs and functional derivatives thereof
US6969589B2 (en) 2001-03-30 2005-11-29 Perlegen Sciences, Inc. Methods for genomic analysis
EP1958964A2 (fr) 2004-02-24 2008-08-20 The Government of the United States of America, as represented by The Secretary, Department of Health and Human Services RAB9A, RAB11A, et modulateurs correspondants liés à une maladie infectieuse
EP1970058A1 (fr) 1998-08-18 2008-09-17 The Regents of the University of California Office of Technology Transfer Antagonistes EGF-R pour le traitement de l'hypersécretion de mucus par voie respiratoire
EP1982720A1 (fr) 2002-05-20 2008-10-22 The Regents of the University of California Ribaviren pour utilisation pour traiter le cancer
WO2009042686A1 (fr) 2007-09-27 2009-04-02 Perlegen Sciences, Inc. Procédés d'analyse génétique
EP2311530A2 (fr) 2004-10-27 2011-04-20 Vanderbilt University Genes mammaliens intervenant dans une infection
US8106013B2 (en) 2006-05-19 2012-01-31 Georgia Tech Research Corporation ABC transporter ligand GATX1
US8324158B2 (en) 2006-07-14 2012-12-04 Georgia Tech Research Corporation Methods for inhibiting CLC-2 channel with GATX2
WO2016187217A2 (fr) 2015-05-18 2016-11-24 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions permettant de traiter des troubles associés au vieillissement
US10202615B2 (en) 2010-12-10 2019-02-12 Vanderbilt University Mammalian genes involved in toxicity and infection
US10265372B2 (en) 2014-08-12 2019-04-23 The Regents Of The University Of California Molecular composition for enhancing and rejuvenating maintenance and repair of mammalian tissues

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WO1994013793A1 (fr) * 1992-12-04 1994-06-23 Apollon, Inc. Composes et methodes de traitement des leucemies
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WO1993015187A1 (fr) * 1992-01-31 1993-08-05 Massachusetts Institute Of Technology Nucleoenzymes
WO1994013789A2 (fr) * 1992-12-04 1994-06-23 Ribonetics Gmbh Oligonucleotides presentant une activite de clivage de l'arn
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Cited By (29)

* Cited by examiner, † Cited by third party
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AU703359B2 (en) * 1994-06-24 1999-03-25 Gene Shears Pty. Limited Ribozymes with optimized hybridizing arms, stems, and loops,tRNA embedded ribozymes and compositions thereof
US6107078A (en) * 1994-06-24 2000-08-22 Gene Shears Pty Limited Ribozymes with optimized hybridizing arms, stems, and loops, tRNA embedded ribozymes and compositions thereof
WO1996000232A1 (fr) * 1994-06-24 1996-01-04 Gene Shears Pty. Ltd. RIBOZYMES PORTANT DES BRAS D'HYBRIDATION, DES TIGES ET DES BOUCLES OPTIMISES, RIBOZYMES ENCAPSULES DANS L'ARNt ET COMPOSITIONS DERIVEES
US5840876A (en) * 1995-04-20 1998-11-24 Ribozyme Pharmaceuticals, Inc. 2'-O-alkylthioalkyl and 2'-C-alkylthioalkyl-containing nucleic acids
EP0837933A4 (fr) * 1995-06-07 2003-05-21 Commw Scient Ind Res Org Minizymes et miniribozymes optimises et utilisation de ces derniers
EP0837933A1 (fr) * 1995-06-07 1998-04-29 Commonwealth Scientific And Industrial Research Organisation Minizymes et miniribozymes optimises et utilisation de ces derniers
US6127159A (en) * 1997-06-06 2000-10-03 The Board Of Trustees Of The Leland Stanford Junior University Mitofusin genes and their uses
US6284507B1 (en) 1997-06-06 2001-09-04 The Board Of Trustees Of The Leland Stanford Junior University Mitofusin genes and their uses
EP1970058A1 (fr) 1998-08-18 2008-09-17 The Regents of the University of California Office of Technology Transfer Antagonistes EGF-R pour le traitement de l'hypersécretion de mucus par voie respiratoire
WO2001012787A2 (fr) * 1999-08-12 2001-02-22 City Of Hope Ribozymes chimeres adn/arn contenant du propanediol
JP2003507037A (ja) * 1999-08-12 2003-02-25 シティ・オブ・ホープ プロパンジオールを含有するキメラｄｎａ／ｒｎａリボザイム
US6379931B1 (en) 1999-08-12 2002-04-30 City Of Hope Chimeric DNA/RNA ribozymes containing propanediol
WO2001012787A3 (fr) * 1999-08-12 2001-06-21 Hope City Ribozymes chimeres adn/arn contenant du propanediol
AU781297B2 (en) * 1999-08-12 2005-05-12 City Of Hope Chimeric DNA/RNA ribozymes containing propanediol
US6953680B2 (en) 1999-10-06 2005-10-11 The Board Of Trustees Of The Leland Stanford, Junior University Mitofusins, Fzo Homologs and functional derivatives thereof
US6969589B2 (en) 2001-03-30 2005-11-29 Perlegen Sciences, Inc. Methods for genomic analysis
US11031098B2 (en) 2001-03-30 2021-06-08 Genetic Technologies Limited Computer systems and methods for genomic analysis
WO2003050241A2 (fr) 2001-12-12 2003-06-19 The Government Of The United States Of America As Represented By The Secretary, Department Of Healthand Human Services Procedes d'utilisation d'inhibiteurs de l'adenosine extracellulaire et d'inhibiteurs de recepteur de l'adenosine aux fins d'amelioration de reponse immune et d'inflammation
EP1982720A1 (fr) 2002-05-20 2008-10-22 The Regents of the University of California Ribaviren pour utilisation pour traiter le cancer
EP1958964A2 (fr) 2004-02-24 2008-08-20 The Government of the United States of America, as represented by The Secretary, Department of Health and Human Services RAB9A, RAB11A, et modulateurs correspondants liés à une maladie infectieuse
EP2311530A2 (fr) 2004-10-27 2011-04-20 Vanderbilt University Genes mammaliens intervenant dans une infection
US8106013B2 (en) 2006-05-19 2012-01-31 Georgia Tech Research Corporation ABC transporter ligand GATX1
US8324158B2 (en) 2006-07-14 2012-12-04 Georgia Tech Research Corporation Methods for inhibiting CLC-2 channel with GATX2
EP2772553A1 (fr) 2007-09-27 2014-09-03 Genetic Technologies Limited Procédés d'analyse génétique
WO2009042686A1 (fr) 2007-09-27 2009-04-02 Perlegen Sciences, Inc. Procédés d'analyse génétique
US10202615B2 (en) 2010-12-10 2019-02-12 Vanderbilt University Mammalian genes involved in toxicity and infection
US10265372B2 (en) 2014-08-12 2019-04-23 The Regents Of The University Of California Molecular composition for enhancing and rejuvenating maintenance and repair of mammalian tissues
WO2016187217A2 (fr) 2015-05-18 2016-11-24 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions permettant de traiter des troubles associés au vieillissement
EP3892315A1 (fr) 2015-05-18 2021-10-13 The Board of Trustees of the Leland Stanford Junior University Méthodes et compositions permettant de traiter des troubles associés au vieillissement

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