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WO2000011013A1 - Nucleomonomeres modifies et leurs procedes d'utilisation - Google Patents

Nucleomonomeres modifies et leurs procedes d'utilisation Download PDF

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WO2000011013A1
WO2000011013A1 PCT/US1999/019029 US9919029W WO0011013A1 WO 2000011013 A1 WO2000011013 A1 WO 2000011013A1 US 9919029 W US9919029 W US 9919029W WO 0011013 A1 WO0011013 A1 WO 0011013A1
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oligomer
odn
cells
duplex
oligomers
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Barry Gold
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University of Nebraska Lincoln
University of Nebraska System
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C12N2310/152—Nucleic acids forming more than 2 strands, e.g. TFOs on a single-stranded target, e.g. fold-back TFOs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • modified nucleomonomers are provided which are incorporated into antisense oligomers. Such oligomers demonstrate increased duplex DNA stability when hybridizing to target nucleic acid sequences. Methods employing the modified nucleomonomers of the invention are also provided.
  • Antisense oligodeoxynucleotides are capable of blocking expression of target genes in cells. Antisense technologies offer new therapies for the treatment of human diseases . Gene expression is inhibited ODNs bind to a complementary messenger RNA sequence and prevents its translation. Antisense effects have been reported using ODNs in mammalian cells in tissue culture and in in vivo studies (Weiss, B. , editor, Antisense Oligodeoxynucleotides and Antisense RNA novel pharmacological and therapeautic agents, CRC Press, Boca Raton, Florida 1997) . The efficacy of antisense techniques is currently being investigated in several human diseases including hypertension, Burkitt's lymphoma, and infection by human immunodeficiency virus (Weiss et al . , supra) .
  • ODNs can nonspecifically activate the SP1 transcription factor of a cell (Perez, J. R. et al. Proc . Natl . Acad. Sci . 1994, 91, 5957- 5961) .
  • ODNs can inhibit viral infection by non- antisense methods that may include interference with absorption, penetration or uncoating (Azad, R. F.; Driver, V. B.; Tanaka, K. ; Crooke, R. M.
  • ODNs can affect cell proliferation and differentiation (Kamano, H. et al . Biochem. Int . 1992, 26, 537-543) . ODNs can be degraded both intra- and extracellularly (Agrawal, S . ; Temsamani, J. ; Tang, J. Y. Proc . Natl . Acad. Sci . U. S.A . 1991 88, 7595-7599), and these breakdown products may, in part, be responsible for the observed non-antisense effects, particularly when the ODN is used at high concentrations.
  • ODN' s which are physiologically stable, non-toxic, able to penetrate cells, and maintain stringent base-pairing fidelity for unique DNA sequences (Wagner, R. W. Nature 1994, 372, 333-335) .
  • the present invention provides modified nucleomonomers and compositions containing the same for inhibiting or regulating expression of target genes and nucleic acids
  • the modified nucleomonomers of the invention are selected from those having the structural formulas (I) and (II) shown below:
  • m and n are 1, p is 3 and R is OH.
  • a modified nucleomonomer wherein X is -C ⁇ C-(CH 2 ) m - and m is 1.
  • the novel nucleomonomers of the invention are incorporated into oligomer analogues.
  • Such oligomers have the ability to form DNA triplexes and regulate gene expression.
  • Any target sequence may be chosen for regulation provided that the sequence incorporates one of the base analogs of the invention.
  • Suitable sequences for targeting include those from pathogenic bacteria, fungi and viruses, oncogenes, growth hormones, and enzymes.
  • the oligomers of the invention may be incorporated into pharmaceutically acceptable carriers. Such pharmaceutical compositions are useful in the treatment of disease.
  • the oligomers of the invention may also be used in diagnostic applications to detect target sequences in biological samples.
  • Nucleomonomer means a moiety comprising (1) a base covalently linked to (2) a second moiety. Nucleomonomers include conventional and chemically modified nucleosides and nucleotides. Nucleomonomers can be linked to form oligomers that bind to target or complementary base sequences in nucleic acids in a sequence specific manner.
  • a "second moiety” as used herein includes a sugar moiety, such as deoxyribose or ribose.
  • Base as used herein includes those moieties which contain not only the known purine and pyrimidine heterocycles but also the modified pyrimidines of the invention.
  • Purines include adenine, guanine and xanthine .
  • Pyrimidines include uracil and cytosine and their analogs
  • nucleoside means a base covalently attached to a sugar or sugar analog.
  • nucleotide means nucleoside having a phosphate group or phosphate analog.
  • Linkage means a phosphodiester moiety (--O--P(O) (O)--O--) that covalently couples adjacent nucleomonomers.
  • substitute linkage means any analog of the native phosphodiester group that covalently couples adjacent nucleomonomers.
  • Substitute linkages include phosphodiester analogs, e.g. such as phosphorothioate and methylphosphonate, and nonphosphorus containing linkages, e.g. such as acetals and amides .
  • Oligomers are defined herein as two or more nucleomonomers covalently coupled to each other by a linkage or substitute linkage moiety. Thus, an oligomer can have as few as two nucleomonomers (a dimer) . Oligomers can be binding competent and, thus, can base pair with cognate single-stranded or double-stranded nucleic acid sequences. Oligomers (e.g. dimers-hexamers) are also useful as building blocks for longer oligomers as described herein.
  • oligomer includes oligonucleotides, oligonucleosides, polydeoxyribo-nucleotides (containing 2 ' -deoxy-D-ribose or modified forms thereof), i.e., DNA, polyribonucleotides (containing D-ribose or modified forms thereof) , or RNA, and any other type of polynucleotide which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base. Oligomer as used herein is also intended to include compounds where adjacent nucleomonomers are linked via amide linkages as previously mentioned (Nielsen, P.
  • oligomers containing the modified nucleomonomers of the present invention are believed to be primarily a function of the base alone. Because of this, elements ordinarily found in oligomers, such as the furanose ring and/or the phosphodiester linkage can be replaced with any suitable functionally equivalent element. "Oligomer” is thus intended to include any structure that serves as a scaffold or support for the bases wherein the scaffold permits binding to target nucleic acids in a sequence-dependent manner.
  • Oligomers that are currently known can be defined into four groups that can be characterized as having (i) phosphodiester and phosphodiester analog (phosphorothioate, methylphosphonate, etc) linkages, (ii) substitute linkages that contain a non-phosphorous isostere (formacetal, riboacetal, carbamate, etc), (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose in place of ribose or deoxyribose and (iv) nucleomonomers linked via amide bonds or acyclic nucleomonomers linked via any suitable substitute linkage.
  • the oligomers of the invention can be formed using the modified nucleomonomers of the invention or conventional nucleomonomers and synthesized using standard solid phase (or solution phase) oligomer synthesis techniques, which are now commercially available.
  • the oligomers of the invention can be synthesized by a method comprising the steps of: synthesizing a nucleomonomer or oligomer having a protecting group and a base and a coupling group capable of coupling to a nucleomonomer or oligomer; coupling the nucleomonomer or oligomer to an acceptor nucleomonomer or an acceptor oligomer; removing the protecting group; and repeating the cycle as needed until the desired oligomer is synthesized.
  • the oligomers of the present invention can be of any length including those of greater than 40, 50 or 100 nucleomonomers. In general, preferred oligomers contain 2-30 nucleomonomers. Lengths of greater than or equal to about 8 to 20 nucleomonomers are useful for therapeutic or diagnostic applications. Short oligomers containing 2, 3, 4 or 5 nucleomonomers are specifically included in the present invention and are useful as starting materials for longer oligomers. Oligomers having a randomized sequence and containing about 6 or 7 nucleomonomers are useful as primers for cloning or amplification protocols that use random sequence primers, provided that the oligomer contains residues that can serve as a primer for polymerases or reverse transcriptases .
  • Oligomers can contain conventional phosphodiester linkages or can contain substitute linkages such as phosphoramidate linkages. These substitute linkages include, but are not limited to, those wherein a moiety of the formula --0--P (O) (S) --0-- ("phosphorothioate"), --O--P(S) (S)--O-- ("phosphorodithioate”) , --O--P(O) (NR' 2 )--X--, --O--P(O) (R')--O--, --O--P(S) (R 1 ) --0-- ("thionoalkylphosphonate") , --P(O) (OR”) --X--, --O--C(O) --X--, or
  • oligomers of the present invention include phosphodiester, phosphorothioate, methylphosphonate and thionomethylphosphonate linkages. Phosphorothioate and methylphosphonate linkages confer added stability to the oligomer in physiological environments. While not all such linkages in the same oligomer need be identical, particularly preferred oligomers of the invention contain uniformly phosphorothioate linkages or uniformly methylphosphonate linkages.
  • blocking group refers to a substituent other than H that is conventionally attached to oligomers or nucleomonomers, either as a protecting group, a coupling group for synthesis, P0 3 "2 , or other conventional conjugate such as a solid support.
  • blocking group is not intended to be construed solely as a protecting group, according to common terminology, but also includes, for example, coupling groups such as a hydrogen phosphonate or a phosphoramidite .
  • Protecting group means any group capable of preventing the O-atom or N-atom to which it is attached from participating in a reaction or bonding. Such protecting groups for 0- and N-atoms in nucleomonomers are described and methods for their introduction are conventionally known in the art. Protecting groups also prevent reactions and bonding at carboxylic acids, thiols and the like.
  • Coupling group as used herein means any group suitable for generating a linkage or substitute linkage between nucleomonomers such as a hydrogen phosphonate and a phosphoramidite.
  • Conjugate means any group attached to the oligomer at a terminal end or within the oligomer itself.
  • Conjugates include solid supports, such as silica gel, controlled pore glass and polystyrene; labels, such as fluorescent, chemiluminescent , radioactive, enzymatic moieties and reporter groups; oligomer transport agents, such as polycations, serum proteins and glycoproteins, polymers and the like.
  • Pi bond as used herein means an unsaturated covalent bond such as a double or triple bond. Both atoms can be carbon or one can be carbon and the other nitrogen, for example, phenyl, propynyl, cyano and the like. Pharmaceutically acceptable salts.
  • Such “salts” are preferably metal or ammonium salts of the oligomers of the invention and include alkali or alkaline earth metal salts, e.g., the sodium, potassium, magnesium or calcium salt; or advantageously easily crystallizing ammonium salts derived from ammonia or organic amines, such as mono-, di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl) -amides, lower alkylenediamines or lower (hydroxyalkyl or arylalkyl) -alkylammonium bases, e.g.
  • ethylamine diethylamine, triethylamine, dicyclohexylamine, triethanolamine, ethylenediamine, tris- (hydroxymethyl) -aminomethane or benzyl -trimethylammonium hydroxide.
  • the oligomers of the invention form acid addition salts, which are preferably therapeutically acceptable inorganic or organic acids, such as strong mineral acids, for example hydrohalic, e.g., hydrochloric or hydrobromic acid; sulfuric, phosphoric; aliphatic or aromatic carboxylic or sulfonic acids, e.g., formic, acetic, propionic, succinic, glycollic, lactic, malic, tartaric, gluconic, citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4-hydroxybenzoic, salicylic, 4-amino salicylic, methanesulfonic, ethanesulfonic, hydroxy ethanesulfonic, benzenesulfonic, sulfanilic or cyclohexylsulfamic acid and the like.
  • hydrohalic e
  • a "positive modification” is any modification of the nucleomonomer of formula (1) or (2) above which results in increased binding affinity.
  • a "negative modification” is any nucleomonomer modification of an oligomer comprising a base of formula (1) or (2) which results in a decrease in binding affinity or use of a substitute linkage which may result in a decrease in binding affinity.
  • Transfection refers to any method that is suitable for enhanced delivery of oligomers into cells.
  • Subject as used herein means an animal, including a mammal, particularly a human.
  • Figure 1 shows the synthesis of modified deoxyuridine and deoxycytosine phosphoramidites .
  • Figure 2 illustrates the structure of the universal support utilized to synthesize the oligomers of the invention.
  • the complementary strand of the duplex has been removed for viewing purposes .
  • ODNs oligonucleotides
  • Modifications to the bases, deoxyribose ring, and phosphate backbone have been generated in order to satisfy some of these criteria (Milligan, J. F. ;
  • phosphorothioate-based ODNs are complex diastereomeric mixtures, cause toxicity due to mechanisms which are poorly understood, and most importantly, form base pairs with reduced stability (Kibler-Herzog, L.; Zon, G. ; Uzanski, B . ; Whittier, G. ; Wilson, W. D. Nucl . Acids Res . 1991, 19, 2979-2986) .
  • modified deoxynucleosides are provided. Representative structures are set forth in formulas (I) and (II) above. Such modified deoxynucleotides may be conveniently synthesized from known starting materials according to the synthetic scheme depicted in Figure 1. Briefly, the syntheses of 5P ⁇ NH2-dU and 5P ⁇ OH-dU were accomplished by coupling 5-iodo-2 ' -deoxyuridine with 1- aminoprop-2-yne (protected as the N-phthalimide) or 1- hydroxyprop-2-yne (protected as the benzoyl ester) in the presence of [ (PPh) 3 ] 4 Pd (0) as previously described (Heystek, L.
  • the compounds of the invention have the following formulae:
  • m and n are 1, p is 3 and R is OH.
  • oligomers of the invention demonstrate significant single-stranded or double-stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association, these oligomers are useful in diagnosis and therapy of diseases that are associated with expression of one or more genes, which includes many diverse pathological conditions, as noted below.
  • Therapeutic applications employing the oligomers to specifically inhibit the expression of genes (or inhibit translation of RNA sequences encoded by those genes) that are associated with either the establishment or the maintenance of a pathological condition are also contemplated to be within the scope of the present invention.
  • RNAs that can be targeted include those that encode enzymes, hormones, serum proteins, adhesion molecules, receptor molecules, cytokines, oncogenes, growth factors, and interleukins .
  • Target genes or RNAs can be associated with any pathological condition such as inflammatory conditions, cardiovascular disorders, immune reactions, cancer, viral infections, bacterial infections and the like.
  • Oligomers of the present invention are suitable for both in vivo and ex vivo therapeutic applications.
  • Indications for ex vivo uses include treatment of cells such as bone marrow or peripheral blood in conditions such as leukemia or viral infection.
  • Genes that can serve as targets for cancer treatments include oncogenes, such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl or overexpressed sequences such as mdm2 , oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as bcr/abl, or RNAs encoded by such genes.
  • Viral gene sequences such as polymerase or reverse transcriptase genes of CMV, HSV-1, HSV-2, HTLV-1, HIV-1, HIV-2, HBV, HPV, VZV, influenza virus, rhinovirus and the like or RNAs encoded by these are also suitable targets.
  • Application of specifically binding oligomers can be used in conjunction with other therapeutic treatments.
  • oligomers of the invention include (1) modulation of inflammatory responses by modulating expression of genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selectin that play a role in mediating inflammation and (2) modulation of cellular proliferation in conditions such as arterial occlusion (restenosis) after angioplasty by modulating the expression of (a) growth or mitogenic factors such as non-muscle myosin, myc, fos, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors such as c-myb.
  • growth or mitogenic factors such as non-muscle myosin, myc, fos, PCNA, PDGF or FGF or their receptors
  • cell proliferation factors such as c-myb.
  • Other suitable extracellular proliferation factors such as TGF ⁇ , IL-6, ⁇ INF, protein kinase C may be targeted for treatment of psoriasis or other conditions.
  • EGF receptor, TGF ⁇ or MHC alleles may be
  • oligomers of the invention into cells can be enhanced by any suitable method including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by the use of cationic anionic and/or neutral lipid compositions or liposomes by methods described, for example, in International Publication Nos. WO 90/14074, WO 91/16024, WO 91/17424, and U.S. Pat. No. 4,897,355, the disclosure of which are incorporated by reference herein.
  • the oligomers can be introduced into cells by complexation with cationic lipids such as DOTMA (which may or may not form liposomes) , which complex is then contacted with the cells.
  • DOTMA cationic lipids
  • Suitable cationic lipids include but are not limited to N- (2 , 3-di (9- (Z) -octadecenyloxyl) ) -prop-l-yl-N,N,N-trimethylammonium (DOTMA) and its salts, l-0-oleyl-2-0-oleyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium and its salts and 1, 2-bis (oleyloxy) -3- (trimethylammonio) propane and its salts.
  • DOTMA 3-di (9- (Z) -octadecenyloxyl) ) -prop-l-yl-N,N,N-trimethylammonium
  • DOTMA l-0-oleyl-2-0-oleyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium and its salts
  • 1, 2-bis (oleyloxy) -3- (trimethylammonio) propane and its salts
  • lipopolyamine complexes using compounds such as lipospermine (Behr, J.-P., et al , Proc Natl Acad Sci (1989) 86:6982-6986; Loeffler, J. P., et al J Neurochem (1990) 54:1812-1815);
  • anionic, neutral or pH sensitive lipids using compounds including anionic phospholipids such as phosphatidyl glycerol , cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, K.-D., et al , Biochim Biophys ACTA (1992) 1103:185-197; Cheddar, G.
  • Delivery of the oligomers into cells can be via cotransfection with other nucleic acids such as (i) expressible DNA fragments encoding a protein(s) or a protein fragment or (ii) translatable RNAs that encode a protein (s) or a protein fragment.
  • nucleic acids such as (i) expressible DNA fragments encoding a protein(s) or a protein fragment or (ii) translatable RNAs that encode a protein (s) or a protein fragment.
  • oligomers can thus be incorporated into any suitable formulation that enhances delivery of the oligomers into cells.
  • suitable pharmaceutical formulations also include those commonly used in applications where compounds are delivered into cells or tissues by topical administration.
  • Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines or lipopolyamines can be used in topical preparations that contain the oligomers.
  • the modified nucleomonomers described herein may be used effectively to inhibit expression of a selected protein or proteins in a subject or in cells, wherein the proteins are encoded by DNA sequences and the proteins are translated from RNA sequences by introducing an oligomer of the invention into the cells; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereby expression of the protein or proteins is inhibited.
  • the method is suitable for modulating gene expression in both procaryotic and eucaryotic cells such as bacterial, parasite, yeast and mammalian cells. The following methods and experimental designs are provided to facilitate the practice of the present invention.
  • Duplex ODN-A has a d (A) 15 :d (T) 15 duplex target for the third strand and the other (ODN-B) contains several G:C base pairs.
  • the synthesis of the phosphoramidites has already been achieved for the dU analogues using the scheme shown in Figure 1.
  • the synthesis of ODNs in which the 3 ' -residue is modified requires a non-standard approach since the 3 ' -residue of the ODN is normally directly attached to the solid support.
  • the solid support that is used has an attached abasic site with a ribose sugar ring ( Figure 2) .
  • the ODN synthesis is performed as usual, which affords an ODN with an extra ribose sugar residue on the 3' -terminus.
  • the ribose sugar is removed during the deprotection step by treatment with 0.5 M NaOH in MeOH/H 2 0 (1:1) for 1 h at R.T as recommended by Glenn Research (Sterling, VA) . This approach gives excellent yields using the 5P ⁇ NH 2 -dU modified DNA.
  • duplex targets and ODNs 1 structure of duplex-A and third structure of duplex-B and third strand strand duplex-A 5 ' - duplex-B 5 ' -TGAGAAAAAGAGAGAAACCAA TGAGAAAAAAAAAAAAACCAA 3 ' -ACTCTTTTTCTCTCTCTTTGGTT
  • T M studies can be done as described in Example 1. See legend of Table 2.
  • An alternative approach using a more "physiological" buffer is also contemplated to be within the scope of the present invention.
  • This buffer consists of 25 mM sodium phosphate (pH 7.0) containing 70 mM KCl, 2 mM MgCl 2 , and 400 ⁇ M spermine.
  • the T M results are readily converted into binding constants, and thermodynamic properties.
  • the length of the incubation of the third strand with the duplex at 4 °C will be varied: 1, 3, 6, 9, 12 and 24 h.
  • the ⁇ A bsorbance at 260 and 280 nm as a function of temperature will be used to determine triplex concentration.
  • the T M values will also be determined as a function of pH (5.0, 5.5, 6.0, 6.5, 7.0 and 7.5) .
  • unmodified ODN with C residues require acidic pH to form an N3-protonated-dC: the protonated C binds to G via Hoogsteen base pairing.
  • the pKa of 5P ⁇ NH 2 -dC will be determined.
  • the protonation requirement for dC in the third strand in general limits triplex stability at physiological pH.
  • the 5P ⁇ NH 2 -dC modifications should stabilize triplex formation.
  • Experiments will also be conducted to determine whether the stabilization is due to protonation and/or the cationic appendage. This will be done by titrating the free 5P NH 2 -dC with HCl while monitoring the UV: the ⁇ for dC shifts 10 nm upon protonation (Singer, B. and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, N.Y.
  • 5-Methyl-dC has a pKa of 4.4
  • 5P ⁇ H-dU has a pKa of 3.5
  • the duplex substrates can be shortened from the 33mers described in Example I to 23mers with shorter ends, and the use of the 32 P-labeled ODN should permit resolution of the triplex band more clearly.
  • the duplex was end-labeled. End-labeling the purine strand of the duplex should confirm that the band being monitored corresponds to the triplex because it is possible that the labeled-ODN could be involved in a strand displacement and form a duplex with the purine rich top strand in ODN-A and -B.
  • the length of the incubation of the third strand with the duplex at 4°C can be varied: 1, 3, 6, 9, 12 and 24 h.
  • the ratio of triplex to ODN bands as a function of incubation time should provide rate data. Measuring the relative intensities of the triplex and ODN bands gives the triplex fraction (q) .
  • concentrations of the labeled ODN (0, 0.05, 0.10, 0.25, 0.5, 0.75 ⁇ M using 20 ⁇ M duplex), it should be possible to calculate a binding rate as follows:
  • the second order rate constant can be formulated as a pseudo-first order rate equation:
  • the duplex can be incubated for 24 h with different equivalents of the labeled third strand (unmodified and sidechain modified.
  • the same triplex fraction (q) can be calculated and presented as a function of ODN concentration to obtain binding constants.
  • the ODNs (a and q) in Table B below, are to serve as controls.
  • the T M ' s of the different potential triplexes can be determined and compared to those without a mismatch (duplex-A with ODN-a and duplex-B with ODN-q) . From the T M values, the ⁇ G between the mismatch vs the normal sequence can be calculated.
  • the duplex target is 5 ' -end labeled on the top strand using T4 kinase and [ 32 P]y-ATP.
  • Approximately 0.5 pmol of duplex is dissolved in the buffer described for the T M studies (see above) , and 20 pmol of the third strand (unmodified or modified with the different sidechains) is added.
  • the incubation is kept at room temperature overnight and then treated with dimethyl sulfate using standard Maxam-Gilbert G-lane conditions (Maxam and Gilbert, supra) .
  • the resulting N7- methylguanine lesions are converted into single-strand breaks using hot piperidine and the breaks sequenced on a 20% denaturing PAGE gel.
  • the Maxam-Gilbert G, G+A and C marker lanes are included for reference.
  • the presence of the triplex should block N7- methylguanine formation at the 4 G's that are within the central 15 base pair duplex target sequence, ...A 5 GAGAGAGA 3 ... (see Table A). This should result in a loss of cleavage bands that correspond to G's in the triplex region. No change in 7-methylguanine should be observed outside of the triplex forming region.
  • EXAMPLE I A. Effect of 5-substituted-2 ' -deoxypyrimidine modification of duplex stability.
  • T M 's of duplexes with 5- (3-aminopropyn-l-yl) -2 ' - deoxyuridine substitutions (5 ⁇ PNH 2 -dU) , 5- (3- aminopropyl) - 2 ' -deoxyuridine (5P-NH 2 -dU) and 5- (3- hydroxypropyl) -2 ' -deoxyuridine (5P-OH-dU) are shown in Table I along with the sequences studied Heystek et al . , supra) . Oligomers with one or more 5P ⁇ NH 2 -dU substitutions show a very significant increase in T M (Table 1).
  • the T M ' S °f tne oligomers should not be as sensitive as unmodified ODNs to ionic strength.
  • the relationship between salt concentration vs T M for the different ODNs is shown in Table 1. As anticipated, the ⁇ T M over the range of NaCl concentration (50 to 500 mM) is smaller for ODN-3 (7.8 °C) vs unmodified duplex ODN-1 (13.6 °C) .
  • ODN Duplex b 50 100 200 500
  • the neutral 5P H-dU sidechain stabilizes triplex formation, but the increase in triplex T M is 2.4 °C per residue (Froehler et al . , supra) .
  • the dC analogue destabilizes triplex formation by ⁇ 3.4 °C per residue (Froehler et al . , supra) . It appears that the propyne group reduces the pKa for protonation of the N3-C position to -3.5 vs . 4.4 for 5-methyl-dC (Froehler et al . , supra) .
  • the flexible sidechains 5P-NH 2 - dU sidechains adopt a conformation that places it toward the 3 '-residue and near the floor of the major groove ( Figure 3, Y) .
  • a similar orientation is predicted for the 5P-OH-dU residue.
  • the rigid 5P ⁇ NH 2 -dU sidechain points toward the 5' -residue (see Figure 3, X) and can make a salt bridge with the non-bridging phosphate oxygen (Heystek et al . , supra) .
  • the foregoing results demonstrate that the introduction of rigid 3-aminopropyn-l-yl sidechains at the 5-position of deoxyuridine results in ODNs with a marked increase in duplex (DNA-DNA and RNA-DNA) and triplex stability.
  • duplexes there is no decrease in base pairing fidelity. This should also be the case in triplex formation.
  • the 5P ⁇ NH 2 -dU (and presumably 5P ⁇ NH 2 -dC) modified oligomers thus comprise potential antigene molecules that have both in vi tro and in vivo to regulate gene expression and to detect the presence or absence of particular target sequences.

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Abstract

On a utilisé des résidus de 2'-deoxyuridine 5'-non substituée et modifiée auxquels on a ajouté sous forme d'appendices des chaînes latérales de 3-aminopropyle, 3-hydroxylpropyle et 3-aminopropyn-1-yle afin d'étudier les effets de charges localisées sur des oligomères d'ADN duplex. Les substitutions de 3-aminopropyle se sont avérées induire une incurvation d'environ 8° dans le duplex et on a établi que l'amine liée a créé un pont de sel avec le 5' phosphate. Cependant, la caractérisation de différents appendices indique que seule la chaîne latérale de 3-aminopropyn-1-yle peut créer un pont de sel.
PCT/US1999/019029 1998-08-22 1999-08-20 Nucleomonomeres modifies et leurs procedes d'utilisation Ceased WO2000011013A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROBINS ET AL: "Nucleic Acid Related Compounds. 39. Efficient Conversion of 5-Iodo to 5-Alkynyl and Derived 5-Substituted Uracil Bases and Nucleosides", J. ORG. CHEM., vol. 48, no. 11, January 1983 (1983-01-01), pages 1854 - 1862, XP002069924 *
SZABOLCS ET AL: "Unnatural Nucleosides and Nucleotides, III, Preparation of 2-(14)C and 4-(14)C Labelled 5-Alkyluracils and 5-Alkyl-2'-Deoxyuridines", JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, vol. 14, no. 5, January 1978 (1978-01-01), pages 713 - 726, XP002922335 *

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