WO2002059093A1 - Procedes et compositions de fixation de molecules d"acide nucleique - Google Patents

Procedes et compositions de fixation de molecules d"acide nucleique Download PDF

Info

Publication number
WO2002059093A1
WO2002059093A1 PCT/US2001/045165 US0145165W WO02059093A1 WO 2002059093 A1 WO2002059093 A1 WO 2002059093A1 US 0145165 W US0145165 W US 0145165W WO 02059093 A1 WO02059093 A1 WO 02059093A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
group
composition
acid binding
formula
Prior art date
Application number
PCT/US2001/045165
Other languages
English (en)
Inventor
Randal A. Goffe
Original Assignee
Genespan Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genespan Corporation filed Critical Genespan Corporation
Publication of WO2002059093A1 publication Critical patent/WO2002059093A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/74—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

  • This invention relates to methods of binding nucleic acid molecules, including methods for isolating nucleic acid molecules.
  • nucleic acids The ability to achieve successful affinity binding of nucleic acids would provide a major advancement in many areas of biomedical research, development and therapeutics.
  • Selective nucleic acid isolation and high quality purification is essential for DNA sequencing, PCR, nucleic acid transfections and gene therapy.
  • Affinity binding of nucleic acids is also useful for gene regulation in a specific and highly selective manner in a variety of cell culture and in vivo therapeutic applications.
  • Useful affinity binding methods must be capable of selectively binding nucleic acid molecules in the presence of other macromolecules, including proteins, that bind competitively to the affinity binding ligand based on physiochemical parameters such as charge and hydrophobicity.
  • a variety of strategies have been developed to address the challenge of nucleic acid purification.
  • the present invention provides methods for binding nucleic acid molecules to a nucleic acid binding composition in vitro.
  • the methods of this aspect of the invention comprise the step of contacting a nucleic acid binding composition with nucleic acid molecules under conditions that enable binding of the nucleic acid binding composition to the nucleic acid molecules, wherein the nucleic acid binding composition is defined by formula I, formula ⁇ , or formula HI, as defined herein.
  • a nucleic acid binding composition is contacted with nucleic acid molecules under low salt conditions.
  • the nucleic acid binding composition includes a W group, as defined herein, which yields a positive spectral shift (such as a spectral shift having a value of from 1 to 500, such as from 10 to 100) in the assay disclosed in Example 7 herein.
  • the present invention also provides methods for binding nucleic acid molecules in vivo to a nucleic acid binding composition.
  • the methods of this aspect of the invention comprise the step of introducing a nucleic acid binding composition into a living body, under conditions that enable binding of the nucleic acid molecules to the nucleic acid binding composition, wherein the nucleic acid binding composition is defined by formula I, formula ⁇ , or fo mula III, as defined herein.
  • the nucleic acid binding composition includes a W group, as defined herein, which yields a positive spectral shift (such as a spectral shift having a value of from 1 to 500, such as from 10 to 100) in the assay disclosed in Example 7 herein.
  • a positive spectral shift such as a spectral shift having a value of from 1 to 500, such as from 10 to 100
  • nucleic acid molecules such as DNA or RNA molecules.
  • the methods of the invention can be used to isolate nucleic acid molecules, such as nucleic acid molecules present in a cellular extract that contains other macromolecules, such as proteins.
  • nucleic acid binding compositions defined by formula I, formula II, or formula III, as defined herein can be attached to beads which are formed into a column through which a solution containing nucleic acid molecules is passed, so that the nucleic acid molecules bind to the beads from which they can be subsequently eluted.
  • the methods of the invention for binding nucleic acid molecules to nucleic acid binding compositions in vivo can be used, for example, to bind specific target mRNA molecules within a cell to inhibit expression of the bound mRNA molecules.
  • plasmid DNA used for transfection can be bound to such nucleic acid binding compositions to protect the DNA from nuclease digestion in living cells.
  • the present invention provides nucleic acid binding compositions defined by formula I, II or m, wherein substrate, group W, groups X, Y, L and Z are as described herein.
  • the W group yields a positive spectral shift (such as a spectral shift having a value of from 1 to 500, such as from 10 to 100) in the assay disclosed in Example 7 herein.
  • the nucleic acid binding compositions of the present invention are useful for binding nucleic acid molecules in vitro or in vivo. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGURE 1 A-C illustrates a reaction scheme for the synthesis of some nucleic acid binding compositions defined by formula I.
  • FIGURE 2 graphically illustrates the binding capacity for mammalian genomic
  • DNA of a representative nucleic acid binding composition of formula I was synthesized by reacting 3,5- dichloro-2,4,6-trifluoropyridine (DCTFP) with W group 4-(Dimethylamino)pyridine (DMAP), X group EDTA at pH 12.0, and substrate Cytopore.
  • DCTFP 3,5- dichloro-2,4,6-trifluoropyridine
  • DMAP W group 4-(Dimethylamino)pyridine
  • X group EDTA at pH 12.0
  • substrate Cytopore substrate Cytopore.
  • the equilibrium binding of mammalian genomic DNA was performed by incubating DNA with 50 ⁇ l of the nucleic acid binding composition for one hour.
  • the DNA binding profile of the representative nucleic acid binding composition was determined at different DNA offering levels by measuring the amount of unbound DNA remaining in the supernatant.
  • FIGURE 3 graphically illustrates the depletion of biologically active adenovirus particles by a representative nucleic acid binding composition of formula I which was synthesized from Cytopore substrate, DCTFP, W group DMAP and X group ⁇ -mercaptoethanol.
  • 10 9 adenovirus particles were loaded onto a 5mL column at 0.5mL minute. This one-step column resulted in a thousand-fold virus depletion in the flow through, with no viral particles detected in the wash or eluent samples.
  • the quantitation of live adenovirus was achieved by a transfection-based bioassay.
  • FIGURE 4 graphically illustrates the scalability of a plasmid DNA purification protocol that is described in Example 3.
  • the nucleic acid binding composition was synthesized as described in the legend to FIGURE 2 and is composed of a composition based upon formula I with W group DMAP, X group EDTA and substrate Cytopore. E.coli paste was used as the source for crude plasmid DNA and the nucleic acid binding composition was packed into columns of various sizes to verify the scalability of plasmid DNA purification.
  • FIGURE 5 shows the change in absorbance (A) across a spectrum of wavelengths for a DNA binding composition of formula I, wherein W is DMAP, in the presence or absence of plasmid DNA.
  • low salt when used in connection with binding nucleic acid molecules to a nucleic acid binding composition, means a phosphate or acetate salt in the concentration range of 0 to 100 mM.
  • molysaccharide means natural polymers composed of sugar or saccharide subunits that are key components of mucus, extra-cellular matrix and connective tissue.
  • chondroitin sulfate type-6 refers to a chondroitin sulfate molecule disclosed in U.S. Patent No. 6,146,847, incorporated herein by reference in its entirety.
  • Cytopore refers to a cellulose-based porous microparticle ( ⁇ 200 ⁇ m) manufactured by Asahi Chemicals Co. Ltd., Tokyo, Japan, and distributed by Amersham Biotech, 800 Centennial Ave. Piscataway, N.J. USA.
  • Sepharose CL-4B refers to an agarose polysaccharide microparticle (-200 ⁇ m) with 4% cross-linkage, available from Amersham Biotech, 800 Centennial Ave. Piscataway, N.J. USA.
  • Sepharose-6FF refers to an agarose polysaccharide microparticle
  • Sephacryl S-1000 refers to a copolymer microparticle (40 to 105 ⁇ m) composed of dextran and acrylamide, available from Amersham Biotech, 800 Centennial Ave. Piscataway, N.J. USA.
  • Sephacryl S-200 refers to a co-polymer microparticle (50 ⁇ m) composed of dextran and acrylamide, manufactured by Amersham Biotech, 800 Centennial Ave. Piscataway, N.J. USA.
  • the present invention provides methods for binding nucleic acid molecules to a nucleic acid binding composition in vitro.
  • the methods of this aspect of the invention comprise the step of contacting a nucleic acid binding composition with nucleic acid molecules under conditions that enable binding of the nucleic acid binding composition to the nucleic acid molecules, wherein the nucleic acid binding composition is defined by formula I, formula II or formula HI.
  • Sub is a macromolecule
  • H is a heteroatom selected from the group consisting of oxygen, sulfur and nitrogen;
  • W is a nitrogen containing moiety
  • X has the formula: -Y-L-Z, wherein Y is a nucleophilic functional group, Z is a functional group that modulates nucleic acid binding specificity and L is a linking group that is independently selected from the group consisting of alkyl, aryl and arylalkyl and can include at least one heteroatom. L typically contains from 1 to 1000 atoms, more typically from 1 to 500 atoms, such as 1 to 100, such as 10 to 100, or such as 10 to 50 atoms.
  • the methods of this aspect of the invention are useful for binding nucleic acid molecules in any situation where it is desired to bind nucleic acid molecules.
  • the methods of this aspect of the invention are useful for isolating DNA that can be introduced into prokaryotic or eukaryotic cells to inhibit, enhance, or otherwise modify gene expression within the cells.
  • the methods of this aspect of the invention can be used to isolate plasmid DNA that is then introduced into mammalian cells in vivo, wherein one or more proteins encoded by the plasmid DNA are expressed and confer a desirable phenotype on the cells.
  • Non-limiting, exemplary, uses for nucleic acid molecules isolated in accordance with this aspect of the invention include use in DNA sequencing, PCR, transfections and gene therapy.
  • the methods of this aspect of the invention utilize nucleic acid binding compositions composed of a substrate ("sub") covalently linked to an affinity ligand that binds nucleic acids.
  • the substrate may be any macromolecule suitable for covalent attachment to a nucleic acid binding ligand.
  • the substrate is chosen with regard to the requirements of the individual situation, such as the type of nucleic acid binding ligand to be coupled to the substrate.
  • the substrate can be made from any polymer which contains at least one nucleophilic group, such as a hydroxyl, amino or sulfhydryl bonded to a carbon atom, which is available for activation and coupling to a nucleic acid affinity ligand.
  • the substrate may be made from natural, semi-synthetic, or synthetic materials containing at least one nucleophilic group.
  • useful natural and semi-synthetic substrates are polysaccharides such as cellulose, agarose, dextran and cross-linked derivatives thereof.
  • useful, polysaccharide-based, substrates that are commercially readily available include: several microparticles supplied by Amersham Pharmacia Biotech such as Cytopore, Cytopore-underivatized, Sepharose CL-4B, Sepharose-6FF, Sephacryl S-1000, Sephacryl S-200 and soluble polymer chondroitin sulfate type-6 (Genespan Corporation, Bothell, WA).
  • Non-limiting examples of synthetic substrates include poly(ethylene glycol), poly(vinyl alcohol), poly(hydroxyethyl methacrylate) hydroxyethyl cellulose, polyacrylamide based polymers and nylon.
  • the substrates useful in the practice of this aspect of the invention may be water- soluble polymers.
  • the water-soluble forms can be used for coating structures.
  • Such coatings can be applied to porous structures (e.g., membranes), surfaces (e.g., the wells of micro titer plates) or particles, such as paramagnetic (magnetite) particles or spherical beads.
  • Coated beads can be used, for example, to form a column through which a solution containing nucleic acid molecules can be passed.
  • Some substrates useful in the practice of this aspect of the invention are structurally robust and can be formed into structures, such as high-surface area, porous, structures that are suitable for adsorption of nucleic acid molecules.
  • Exemplary, structurally robust substrates useful in the practice of this aspect of the invention include Cytopore microparticles (-200 ⁇ m diameter). Some substrates bind non-specifically to protein. For example, sepharose-based substrates such as Sepharose CL-4B, Sephacryl S-200 and S-1000 bind immunoglobulins. When utilizing these substrates, a mixture containing nucleic acid molecules and proteins is typically first purged of protein by any useful means, such as the method described by Goffe et al. in copending U.S. Patent Application No. 09/733,240, incorporated herein by reference, or by gel filtration chromatography.
  • any useful means such as the method described by Goffe et al. in copending U.S. Patent Application No. 09/733,240, incorporated herein by reference, or by gel filtration chromatography.
  • substrates bind little or no proteins. These substrates are especially useful in any situation where nucleic acid molecules are present in a mixture of macromolecules including proteins (e.g., a cell extract).
  • substrates that bind little or no protein include cellulose substrates, such as underivatized Cytopore, sulfonated mucopolysaccharide substrates, such as chondroitin sulfate and heparan sulfate, and carboxylated mucopolysaccharide substrates, such as hyaluronic acid.
  • chondroitin sulfate is one of the least efficient at interacting with proteins or polypeptides. This tendency for low protein binding, combined with the ability of the polyanionic chondroitin sulfate to interact with negatively charged nucleic acids, makes this mucopolysaccharide a useful substrate to attach to affinity ligands to bind nucleic acids. Due to the negatively charged sulfonic and carboxylic acid groups on chondroitin sulfate, it is an unexpected result that this substrate is useful to maintain plasmid DNA in solution with ethanol, and therefore effectively serves as a nucleic acid solubility enhancer.
  • a presently preferred chondroitin sulfate is chondroitin sulfate type 6 disclosed in U.S. Patent No. 6,146,847 to Goffe et al., which patent is incorporated herein by reference in its entirety.
  • the nitrogen containing moiety (W) serves to enhance the resonance stability of the nucleic acid binding composition, and effectively creates an electron sink due to the electron withdrawing effects of the halogens.
  • the greater the resonance stability the higher will be the rate of binding of nucleic acid molecules to the nucleic acid binding compositions, and the lower the rate of disassociation of bound nucleic acid molecules.
  • the relatively electron rich bases in nucleic acid molecules are able to donate lone pair electrons in the electron sink formed by a compound with formula I, H or HI and derivatives thereof.
  • the nitrogen moieties might participate in hydrogen bonding with nucleotide groups between base pairs in the manner defined by Watson and Crick.
  • (W) contain an increased number of electron mobilizing groups such as for example, CH 3 , S-CH 3 and OH, in order to balance the increased electron donating effects that occur due to the increased number of N atoms in pyrimidine and cyanuric based structures.
  • the nitrogen containing moiety (W) can be a primary, secondary, or tertiary amine, (including aliphatic or heterocyclic amino compounds).
  • heterocyclic amino compounds are preferred due to their relatively higher level of reactivity and greater efficiency in contributing to the resonance stabilization of the final affinity agent.
  • Useful heterocyclic nitrogen containing moieties include, but are not limited to, 5 and 6 membered rings and two or more fused ring molecules.
  • heterocyclic nitrogen containing substituted and unsubstituted moieties include: 4-(dimethylamino)pyridine (DMAP), triethylamine (TEA), pyrrole, pyrazole, imidazole, pyrazole, 4-azabenimidazole, l,2,4-triazolo[l,5-a] pyrimidine, lH-l,2,3-triazolo[4,5-b]pyridine, pyridine, imidazo-pyridine, azidoaniline, trizolo-pyridine, azabenzimidazole, guanidine HC1, 1-hydroxybenztriazole, benzimidazole, 2-methyl benzimidazole, 2-methyl thiobenzimidazole, and I-methyl imidazole.
  • DMAP dimethylamino)pyridine
  • TAA triethylamine
  • pyrrole pyrazole
  • imidazole imidazole
  • pyrazole 4-azabenimidazo
  • Example 7 herein sets forth a method for identifying useful (W) compounds.
  • the method measures the ultraviolet to visible spectral shift ( ⁇ nm) when candidate (W) compounds are used to form a nucleic acid binding composition of formula I, ⁇ , or HI, or to form a synthetic intermediate of a nucleic acid binding composition of formula I, II, or HI.
  • Useful (W) compounds produce a positive spectral shift (i.e., a shift towards the visible range).
  • ⁇ nm values of between +1 to +500 are desirable, with values beween +10 and 100 nm preferred.
  • the (W) group is defined by the following structure:
  • each of Xj is hydrogen or optionally substituted alkyl, aryl or aralkyl; and at least one Y ⁇ is hydrogen and the other is hydrogen or optionally substituted alkyl, aryl or aralkyl; and R ⁇ is hydrogen, optionally substituted alkyl, aryl or aralkyl, or -NR2R3, in which R 2 and R 3 are the same or different and are optionally substituted alkyl, aryl or aralkyl; and sub is a polymer.
  • Z" is a suitable counterion.
  • alkyl refers to straight- or branched-chain alkyl of one to about 20 carbon atoms, as well as aliphatic cyclic substituents (such as cyclopentyl and cyclohexyl); "aryl” refers to aromatic hydrocarbon (such as benzyl, naphthyl, anthracyl, etc.) substituents; and “aralkyl” refers to benzyl, alkylphenyl, alkylnaphthyl, etc. By “optionally substituted” herein is meant that the subject alkyl, aryl or aralkyl group may bear one or more substituents which are the same or different.
  • the (W) group can be selected from heterocyclic compounds known to mimic and/or bind to nucleic acids.
  • useful heterocyclic compounds include purines, pyrimidines, and imidazoles. This provides a means for rational design of nucleic acid binding specificity.
  • the capping compound (X) contains two functional groups (Y) and (Z) connected by a linker group (L).
  • the functional group (Y) participates in the capping reaction and is chosen to modulate the on-rate and off-rate of nucleic acid binding.
  • the functional group (Z) modulates nucleic acid binding and selectivity.
  • the nature of (Y) is an important determining factor for enhancing the resonance structure of the nucleic acid binding compositions by electron donating effects from lone pairs of electrons (e.g., the electron pair present on nitrogen), or destabiUzation of the resonance structure by electron withdrawing effects by elements such as sulfur.
  • Table 1 shows the nucleophilic reactivity series (NRS) for representative (Y) groups.
  • nucleic acid binding compositions of the invention that include mercaptoproprionic acid as a Y group bind RNA less efficiently than DNA.
  • nucleic acid binding compositions of the invention that include mercaptopropanol as a Y group bind RNA and DNA with approximately equal efficiency.
  • X is PolyA, so that RNA binding is dominant over DNA.
  • nucleic acid binding specificity can be systematically designed into the nucleic acid binding compositions of the invention. The rational design model to predictively obtain selective nucleic acid adsorption and desorption properties is further discussed in Example 7 herein.
  • Examples of compounds useful as the (Y) functional group include: cysteine, mercaptoethanol, mercaptoacetic acid, mercaptopropionic acid, mercaptosulfonic acid, glycine, guanidine hydrochloride, EDTA, taurine, aspartic acid, 1H- 1,2,3 Triazo[4,5b]pyridine, imidizole, azo compounds, including various dyes such as Fast Sulfone Black, hydroxyl ions in alkaline solution, and ethylene glycol.
  • the functional group (Z) may be any functional group that modulates nucleic acid binding specificity.
  • the (Z) group encompasses compounds that function by a series of complex affinity-type interactions between the nucleic acid and the nucleic acid binding composition.
  • the (Z) functional group has a relative non-covalent interaction strength for binding nucleic acid molecules as follows: RNA>Genomic>Open Circle Plasmid>Nicked Plasmid>Supercoiled Plasmid DNA.
  • the relative interaction strength of the (Z) functional group is distinguishable from an anion exchanger which has a higher relative interaction strength for plasmid and genomic DNA than for RNA.
  • Non-limiting examples of suitable (Z) functional groups useful for the practice of this invention include: ⁇ -mercaptoethanol, cysteine, cystine, homocysteine, EDTA, polyA, ethylenediamine, guanidine, AMP, Xylenol Orange, l-(2,3,-xylyl)piperazine, Xylene Cyanol, 5,10,15,20 tetra (4-pyridyl) 21H, 23H-porphine, 2-aminomethyl crown-5, 4'amino5'nitro benzo-15-crown-5, TEA, ethylamine, ethanolamine, NaOH, 3-mercapto-l- propanol, mercaptoacetic acid, 3-mercapto-l-propionic acid, 2-mercaptoethane sulfonic acid, lysine, proline, histidine, phenylalanine, arginine, tryptophan, glycine, ⁇ -alanine
  • the compounds of formulae I II or HI may be prepared, for example, by the synthetic route exemplified in FIGURE 1 A-C.
  • Compounds of formula I may be synthesized from 3,5-dichloro-2,4,6-trifluoropyridine (DCTFP)
  • compounds of formula H may be synthesized from monochloro-2,4,6-trifluoropyrimidine (MCTFP)
  • compounds of formula HI may be synthesized from cyanuric fluoric.
  • DCTFP is reacted with a nitrogen containing moiety (W) to form a reaction intermediate.
  • W nitrogen containing moiety
  • hydroxyl groups on the substrate react with the DCTFP intermediate.
  • the capping compound (X) reacts with the DCTFP intermediate resulting in the nucleophilic displacement of the fluoride (F-) leaving group from DCTFP to provide the nucleic acid binding compositions of formula I.
  • nucleic acid molecules are bound to nucleic acid binding compositions under low salt conditions, and in the absence of surfactant.
  • Low salt conditions are preferred for substrates such as Sepharose and Sephacryl that are known to bind proteins because high salt concentration is required for protein binding to these substrates.
  • the nucleic acid binding compositions useful in the practice of the methods of this aspect of the invention effectively bind nucleic acid molecules in the pH range of 3.5 to 9.5.
  • the methods of this aspect of the invention are useful to purify nucleic acid molecules such as plasmid DNA, that has a high level of functionality, when used to express protein in vivo or in vitro, due to the low level of contaminants that are co- purified, such as, for example, endotoxins, spermidine, mucopolysaccharides and putrescine. Endotoxins are negatively charged membrane components of gram negative bacteria, and are especially detrimental for most biological and all in vivo applications of plasmid DNA. There typically exists a correlation between a low level of RNA contamination and a high level of functionality for plasmid DNA. Goffe et al. (U.S. Patent Application No. 09/733,240) has shown a correlation between low mucosaccharide and DNA functionality.
  • nucleic acid purification methods that utilize an anion exchanger which utilizes chaotropic (high salt) conditions to force the nucleic acid onto the anion exchange substrate based upon the relative hydrophilicity of the substrate compared with the chaotropic solution.
  • high salt conditions protein contaminants such as endotoxins and mucopolysaccharides from bacteria can co-purify with nucleic acids, resulting in plasmid DNA with a low level of functionality.
  • an eluent is used to elute the nucleic acid that is bound to the nucleic acid binding composition (such as nucleic acid binding composition that has been formed into, or onto, spheres that are packed together to form a column through which a solution containing nucleic acid molecules is passed, thereby binding the nucleic acid molecules to the nucleic acid binding composition).
  • nucleic acid binding composition such as nucleic acid binding composition that has been formed into, or onto, spheres that are packed together to form a column through which a solution containing nucleic acid molecules is passed, thereby binding the nucleic acid molecules to the nucleic acid binding composition.
  • Some suitable eluents for use in this invention are effective under low salt and a range of pH from 3.5 to 9.5. These include, but are not limited to, amino/cationic compounds and diazo compounds (which are dyes).
  • compounds in each class may be combined to achieve optimal elution efficiency with a visual indication.
  • dyes of different colors may be used for different types of nucleic acids, such as plasmid DNA, RNA, animal genomic DNA and plant DNA.
  • a kit may be prepared that contains different colored dye eluents that are designated for each of the forms of nucleic acid of commercial interest.
  • the diazo compounds useful to elute bound nucleic acid molecules function by displacing nucleic acids and form a complex with the nucleic acid binding composition. This interaction allows for a visual indication of successful elution of nucleic acid. For example, a gel substrate would be colored upon successful elution, leaving a clear and colorless solution containing the eluted target molecule.
  • useful diazo compounds include: Congo Red, Trypan Blue, Fast Sulphon Black, Ponceau SS, Ponceau S, Biebrich Scarlet, Xylidine Ponceau 2R and Polar Yellow.
  • amino/cationic compounds useful in the methods of this aspect of the invention to elute bound nucleic acid molecules work by preferentially binding to the negative charges of the phosphate group of nucleic acids.
  • An amino/cationic compound is selected to obtain a stronger interaction with the target nucleic acid than that formed when the nucleic acid is bound to the nucleic acid binding composition.
  • useful eluents include: glycine, arginine, histidine, tryptophan, cysteine, ⁇ -alanine, ethylenediamine, ethylenediamine tetra acetic acid tetra sodium salt (EDTA) and guanidine HC1.
  • the nucleic acid binding compositions can be regenerated and sanitized. Regeneration can be achieved through the use of nuclease enzymes, detergents, guanidine hydrochloride, and/or washing with sodium hydroxide.
  • Useful nuclease enzymes include RNase and DNase.
  • a column containing a nucleic acid binding composition that includes the substrate Cytopore bound to ⁇ -mercaptoethanol may be effectively regenerated multiple times with the addition of sodium hydroxide.
  • sodium hydroxide is utilized at a concentration in the range of from 0.01 N to 5.0 N.
  • nucleases are used at a concentration in the range of from 2 ⁇ g/ml to 1000 ⁇ g/ml, such as 200 ⁇ g/ml.
  • the invention provides methods for binding nucleic acid molecules (such as DNA and RNA) in vivo.
  • the methods of this aspect of the invention include the step of introducing a nucleic acid binding composition into a living body under conditions that enable binding of the nucleic acid molecules to the nucleic acid binding composition.
  • Nucleic acid binding compositions useful in this aspect of the invention are those defined by structural formulae I, H, and IH.
  • the methods of this aspect of the invention can be used, for example, in applications in which targeted affinity binding to nucleic acids is desired in intact cells (such as prokaryotic and eukaryotic cells) to affect gene expression.
  • the methods of this aspect of the invention can be used to specifically bind RNA, such as mRNA, thereby killing target microorganisms, viruses or cancer cells.
  • Target mRNAs may be any mRNA in which gene expression modulation is desired, including pathogen- specific mRNAs, tissue-specific cellular mRNAs, and/or disease-specific mRNAs.
  • the targeted mRNA may be the mRNA of a gene which plays a critical role in the survival of the pathogen, or which is essential to the pathogen's life cycle.
  • Substrates (sub) useful for this aspect of the invention include natural cellulose and other low protein binding polysaccharides (e.g., chondroitin sulfate, heparan sulfate and hyaluronic acid). These molecules have the added advantage of being metabolized by normal physiological processes of the body and, at least in some cases, are actively transported into living cells (see, e.g., M. Ishihara, N.S. Fedarko and H.E. Conrad, "Transport of Heparan Sulfate into the Nuclei of Hepatocytes", J. Biol. Chem., Vol. 261(29), October 15, 1986 pp. 13575-80).
  • natural cellulose and other low protein binding polysaccharides e.g., chondroitin sulfate, heparan sulfate and hyaluronic acid.
  • binding of the nucleic acid binding composition is sequence specific.
  • pyrrole-imidazole polyamides can recognize and bind specific DNA sequences have been pursued by several groups world wide including Dervan et al. This can be achieved by selecting a capping compound (X) with component (Z) selected to have specific nucleic acid sequence binding characteristics.
  • component (Z) is a series of adenine-based nucleotides, that specifically binds RNA over DNA.
  • a nucleic acid binding composition can be delivered into a living body by any suitable means.
  • a nucleic acid binding composition can be introduced into a living body by application to a bodily membrane capable of absorbing the composition, for example the nasal, gastrointestinal and rectal membranes.
  • the nucleic acid complex may be combined with other suitable ingredients, such as carriers and/or adjuvants.
  • suitable ingredients include ointments, creams, gels, or suspensions, with or without purified collagen.
  • Methods of delivery of nucleic acid binding complex also include administration by oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IN) or subcutaneous injection), inhalation (such as via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual routes of administration, and can be formulated in dosage forms appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous (IN) or subcutaneous injection
  • inhalation such as via a fine powder formulation
  • transdermal nasal, vaginal, rectal, or sublingual routes of administration
  • the present invention provides nucleic acid binding compositions defined by formulae I, H or HI
  • Sub is a macromolecule
  • H is a heteroatom selected from the group consisting of oxygen, sulfur and nitrogen;
  • W is a nitrogen containing moiety
  • X has the formula -Y-L-Z, wherein Y is a nucleophilic functional group, Z is a functional group that modulates nucleic acid binding specificity, and L is a linking group.
  • substrate (sub), W and X groups are described more fully in the discussion of the methods for binding nucleic acid molecules to nucleic acid binding compositions disclosed herein.
  • Example 1 This example shows a general chemical procedure for synthesizing nucleic acid binding compositions of the present invention, and shows nucleic acid binding properties of several representative nucleic acid binding compositions of the invention.
  • the following protocol is adapted for a 1000 ml gel batch size. 1.
  • Gel washing a. Wash and filter gel three times with 1000 mLs deonized water (DI-H 2 O) b. Suspend gel for five minutes with 1000 mLs DI-H 2 O c. Filter d. Suspend gel in 500 mLs DI-H 2 O and stir e. Stir in 5000 mLs acetone over one hour: i. 0-8 minutes: 50 mLs acetone every 2 minutes ii.
  • DI-H 2 O deonized water
  • MBG- H2O iv. Wash and filter gel in 2000 mLs MBG- H2O v. Wash and filter gel in 2000 mLs Phosphate Buffered Saline (PBS) in MBG- H 2 O vi. Suspend gel in 2000 mLs PBS in MBG- H 2 O and store at 2°-8°C for twenty-four hours, vii. Filter viii. Wash and filter gel in 1000 mLs PBS in MBG- H 2 O ix. Wash and filter gel in 1000 mLs PBS in MBG- H 2 O
  • PBS Phosphate Buffered Saline
  • nucleic acid binding composition in the form of a gel
  • the gel was washed 6X with 500 ⁇ l Bind buffer (10 mM
  • Bind buffer was aspirated off and discarded.
  • 75 ⁇ g DNA was added to 500 ⁇ l of Bind buffer, mixed, added to the prepared gel and vortexed for 25 minutes. 50 ⁇ l samples of DNA/Bind buffer were then aliquoted into separate eppendorf tubes and an additional 50 ⁇ l of Bind buffer was added.
  • Assay for Binding Capacity The starting material was briefly centrifuged to settle the gel. The supernatant was removed and stored. 500 ⁇ l of wash 1 buffer (10 mM Na 2 HPO pH 8, also use as wash 4 buffer) was added, vortexed for 5 minutes, the supernatant was removed and stored. Then 500 ⁇ l of wash 2 buffer (0.1 M Guanidine- HC1 pH 8, also used as wash 3 buffer) was added, vortexed for 5 minutes, the supernatant was removed and stored. These wash steps were repeated with wash 3 and wash 4 buffers. The samples were then eluted with elution buffer (1.0 M Ethylenediamine pH 8).
  • RNA Equilibrium Binding To quantitate the amount of DNA eluted, 10 ⁇ l of each sample, including the starting sample was aliquoted into separate eppendorf tubes and digested with EcoRl restriction enzyme for a minimum of 3 hours. Restriction digests were then analyzed by 1% gel electrophoresis. DNA concentrations were quantitated based on ethidium bromide staining intensity as compared to DNA ladder standards. Assay for RNA Equilibrium Binding:
  • nucleic acid binding composition in the form of a gel
  • the gel was washed 3X with 1 ml Bind buffer (10 mM Na 2 HPO 4 , pH 5).
  • Bind buffer 10 mM Na 2 HPO 4 , pH 5
  • An RNA solution was prepared from E. coli rRNA (Roche Catalog # 206938) at 0.2 mg/ml in Bind buffer (this solution should be kept on ice or stored at -20°C).
  • RNA solution 500 ⁇ l of RNA solution was loaded onto the prepared gels, vortexed and incubated for 25 minutes, inverting tubes 5X every 1-2 minutes. After 25 minutes, the samples were pulse-centrifuged in a microfuge and the supernatant was carefully removed and stored in a tube labeled "bind.” The sample was then washed with 500 ⁇ l of wash 1 buffer (10 mM Na 2 HPO 4 , pH 8.0). The spin was repeated and supernatant was placed into 'bind" tube. The sample was washed with 1000 ⁇ l wash 2 buffer (0.1 M guanidine HC1, 10 mM sodium phosphate, pH 8.0). The spin was repeated and supernatant placed into the "bind" tube.
  • wash 1 buffer 10 mM Na 2 HPO 4 , pH 8.0
  • the total volume in the "bind" tube should be 2.5 ml.
  • the sample was eluted 5X with 500 ⁇ l elution buffer (1.0M Ethylenediamine, pH 8.0). These supernatants were placed in a tube labeled "elute”. Total eluted volume should be 2.5 ml.
  • elute buffer 1.0M Ethylenediamine, pH 8.0.
  • Total eluted volume should be 2.5 ml.
  • a set of standards were prepared by aliquoting 200 ⁇ l of the starting material into a fresh eppendorf tube, then preparing serial dilutions. The samples and standards were analyzed on a 1% agarose TAE gel to determine the percent RNA bound.
  • a column was attached to a peristaltic pump, PBS was washed through the lines, and a bottom frit was loaded onto the column.
  • One milliliter (by volume) of a nucleic acid binding composition of the invention was prepared and loaded onto the column and a top frit was loaded onto the gel.
  • the column was then washed with PBS, and the flow was reversed several times to remove any bubbles.
  • the column was then washed with 15 ml of 20% methanol, followed by 15 ml of PBS. 3 ml of polyclonal antibody in goat serum was mixed with 12 ml of PBS, and this 15 ml mixture was loaded onto the prepared column.
  • a flow rate was set at 0.5 ml/min.
  • the column was washed with 15 ml PBS, and then eluted with 15 ml 0.1 M NaOAc pH 3.0.
  • a 4.5 ml residual PBS volume was collected and discarded, and the remaining volume was recorded and collected in a 50 ml tube.
  • a spectrophotometer reading was taken of 1 ml of the eluate at OD 280.
  • Absorbance/1.35X Volume X dilution factor total mg polyclonal bound/1 ml of gel.
  • Cytopore is not an efficient substrate for affinity binding of immunoglobulins, whereas Sepharose-based substrates bind immunoglogulins more efficiently.
  • nucleic acid binding compositions efficiently bound nucleic acids, with a few specifically designed exceptions as follows:
  • the nucleic acid binding composition has a lower affinity for nucleic acid binding.
  • cysteine i.e., #10 in Table 2
  • cysteine which is technically also a mercaptocompound and equivalent to mercaptoproprionic acid, except for the presence of an additional NH 2 group in the (Z) group
  • NH 2 is this (NH 2 ) group that completely reverses the nucleic binding characteristics for affinity agent #10 relative to #4, particularly for RNA.
  • polyclonal antibody binding is completely insensitive to these subtle changes in the structure of the nucleic acid binding composition.
  • the binding capacity of a representative nucleic acid binding composition for mammalian genomic DNA was determined using affinity agent #2 from Table 2.
  • Affinity agent #2 was synthesized by reacting DCTFP with (W) group DMAP, (X) group EDTA, and substrate Cytopore.
  • the equilibrium binding of mammalian genomic DNA was performed by incubating DNA with 50 ⁇ l of affinity agent #2 for one hour.
  • the DNA binding profile of affinity agent #2 was determined at different DNA offering levels by measuring the amount of unbound DNA remaining in the supernatant. Using this method the equilibrium binding capacity of affinity agent #2 for mammalian genomic DNA was found to be 700 ug DNA/mL gel as shown in FIGURE 2.
  • (X) is poly A or some combination with adenine monophosphate (AMP), e.g., affinity agent #23, DNA binding is suppressed while RNA binding remains high.
  • AMP adenine monophosphate
  • (Z) group on the capping compound (X) See also affinity agents #35-39].
  • nitrogen groups from adenine can provide the (Y) function, and the (Z) group in this case specifically binds RNA, due to the higher relative affinity of poly A for RNA than for DNA.
  • capping compound (X) When EDTA is used as capping compound (X) at pH 12 (see #34, Table 2) and at pH 4 (see #22), both DNA and RNA bind strongly, but at pH 4, adsorption is not reversible. Likewise, even adenovirus can bind irreversibly at very high capacity with DMAP reacted with DCTFP and ⁇ -mercaptoethanol as capping agent (X) (see FIGURE 3). This suggests that these affinity agents are suitable for use as affinity agents to bind DNA, and, very surprisingly, adenovirus removal from recombinant and other protein containing fluids.
  • FIGURE 3 This is illustrated in FIGURE 3, where 10 9 adenovirus particles were loaded on to a 5 ml column of affinity agent #2 (from TABLE 2) at 0.5 ml/min where a thousand-fold depletion from the flow through was obtained. It is particularly surprising that adenovirus bound so well because this virus has surface proteins. As these affinity agents do not bind proteins well, the mechanism is unclear at this time. It would be obvious to also attempt to bind retroviruses and lentiviruses as well.
  • a EDTA is tetrasodium EDTA at pH 12 unless otherwise specified.
  • *CPB is the abbreviation for the underivatized Cytopore substrate. **Acetonitrile solvent, 4.5:1 molar ratio TEA:DCTFP, 80°C for 24 hrs.
  • the starting compound is the nitrogen-containing cyclic compound used as the starting compound in the synthesis of a nucleic acid binding composition of the invention (e.g., in accordance with the scheme shown in FIGURE 1).
  • This example demonstrates the utility of amino/cationic compounds and diazo dye compounds as eluents for eluting nucleic acid molecules bound to nucleic acid binding compositions.
  • results using amino/cationic compounds as eluents The amino groups on three of the compounds in Table 3 were quite effective eluents, which functioned by interacting with the negatively charged phosphate groups on the nucleic acid.
  • the effective eluents were: ethylenediamine, ethylenediamine tetra acetic acid tetra sodium salt and guanidine hydrochloride.
  • a list of diazo dye eleunts is set forth in Table 4. These compounds function by displacing nucleic acids and form a complex with the nucleic acid binding composition. This interaction is extremely valuable commercially as a visual indication of successful elution of nucleic acid, i.e., coloring the gel substrate while leaving a clear and colorless solution containing the eluted target molecule.
  • Compounds in Tables 3 and Table 4 can be combined (e.g., 1.0 M ethylenediamine and 1.0 mM Fast Sulphon Black), to achieve optimal elution efficiency with a visual indication.
  • different dyes i.e., colors
  • nucleic acids such as plasmid DNA, RNA, animal genomic DNA, and plant DNA.
  • This example sets forth a presently preferred method of isolating plasmid DNA using the methods and compounds of the present invention.
  • Table 5 describes the typical expected yield of plasmid DNA from various starting culture sizes.
  • Reagent #1 is an aqueous solution containing 50 mM Dextrose, 25 mM TRIS base, 10 mM EDTA, 50 units/mL ⁇ -amylase enzyme, pH 8.0.
  • Reagent #2 is prepared prior to use by adding 6.4 mL of 1.0 N NaOH to the Reagent #2 bottle and mixing to yield an aqueous Reagent #2 solution containing Sodium Dodecyl Sulfate (SDS) at 0.005 g/mL, 0.0025 mL Surfynol (S-485)/mL.
  • SDS Sodium Dodecyl Sulfate
  • Reagent #2 10 mL of Reagent #2 is then added to the suspended pellet, inverted gently 5X and incubated at room temperature for 5 min.
  • 10 mL of refrigerated Reagent #3 (neutralization buffer) is then added to the lysed cell suspension, inverted gently 5X and incubated on ice for 10 min.
  • Reagent #3 contains 3.1 M Potassium Acetate in water, titrated to pH 5.5 with Acetic acid (approximately 0.115 mIJmL). The mixture is then centrifuged, 10,000 x g at 4°C for 10 min.
  • the supernatant is transferred to a fresh 50 mL tube by filtering it through a lOO ⁇ m cell strainer. 0.7 volumes of 100% isopropyl alcohol (IP A) is added and mixed by inversion 5X. This mixture is then centrifuged, 5,000 x g at 4°C for 10 min. After centrifugation, the supernatant is removed using a pipet or by decanting off the liquid. The pellet is washed with 3 mL IPA and centrifuged, 5,000 x g at 4°C for 5 min. The IPA wash is then removed with a micropipette to ensure removal of all residual IPA.
  • IP A isopropyl alcohol
  • Reagent #4 Pre-Column Pellet Treatment 1).
  • Reagent #4 is 10 mM TRIS base in water, pH 8.0.
  • the suspension is transferred to a fresh tube and incubated at room temperature with gently swirling until the pellet is fully dissolved.
  • 3 mL of Reagent #5 (stored at -20°C) is added and mixed by inversion 5X.
  • Reagent #5 is 5 M Lithium Chloride in water.
  • the mixture is then centrifuged, 5,000 x g at 4°C for 10 min. After centrifugation, the supernatant is transferred to a fresh tube and the pellet is discarded. 6mL of IPA is added to the supernatant and mixed by inversion 5X.
  • Reagent #6 (Bind Buffer). The pellet may take several minutes to dissolve with gently swirling.
  • Reagent #6 is 10 mM Sodium phophate monobasic, in water, titrated with 1 N sodium hydroxide to pH 5.
  • a maxi column is prepared for use by first removing the top cap and then the lower cap in order to prevent pulling air up into the column.
  • the composition of the nucleic acid binding composition in the column is DCTFP with DMAP as the W group and EDTA (pH 12) as capping agent.
  • the size of the Maxi Column is 5 mL gel (nucleic acid binding composition) in a 10 mL column.
  • the maxi column is washed with 20 mL of molecular biology grade water which is DNase free.
  • the column is then equilibrated with 15 mL of Reagent #6 (Bind buffer).
  • Reagent #7 Reagent #7
  • Reagent #7 is composed of 2mg of RNase A per mL of 10 mM TRIS base in water, pH 8.0. After dilution in use the final concentration of RNase A is 200 microgram per mL. The sample is then immediately loaded onto the equilibrated maxi column and incubated on the column for 5 min.
  • Reagent #6 Bod buffer
  • Reagent #8 Cold wash Buffer 1
  • Reagent #9 Cold Wash Buffer 2
  • Reagent #8 is 10 mM sodium phosphate in water titrated to pH 8 with sodium hydroxide
  • Reagent #9 is 0.1 M Guanidine hydrochloride in 10 mM sodium phosphate in water, pH 8.
  • Reagent #10 Cold Elute Buffer
  • the plasmid is then eluted off the column by adding 12.5 mL of Reagent #10 and the flow through is collected.
  • the column is then incubated at room temp, for 20 min.
  • Another 10 mL of Reagent #10 is then placed onto the column and collected in the same tube to give a total of 22.5 mL of effluent.
  • Reagent #10 is a 1 M ethylenediamine/ethylenediamine hydrochloride solution in water, pH 8.
  • the effluent is mixed with 15.75 mL IPA, inverted 10X and centrifuged for
  • Example 4 This example demonstrates a procedure for column regeneration that is useful for regenerating a number of nucleic acid binding compositions useful in the practice of the methods of the invention. Maxi scale columns were used in a series of experiments designed to demonstrate the chemical stability of the nucleic acid binding compositions. Reusability confers considerable economic advantage in high volume usage and large scale (e.g., cGMP) situations. The following procedure for column regeneration was used with a number of nucleic acid binding compositions that contained Cytopore as the substrate. Procedure for column regeneration: After elution, the column was washed with
  • MBG molecular biology grade
  • This example illustrates the use of Sephacryl-based nucleic acid binding compositions for plasmid DNA purification with equivalent regeneration procedures as those previously described in Example 4.
  • Sephacryl is known to be a relatively high protein binding substrate
  • the procedure for plasmid DNA purification from Example 3 in combination with cleaning procedures allow it to be a very effective and economical alternative to other substrates.
  • Cleaning procedures include the use of RNase and/or DNase, Tween 20 detergent or guanidine-HCl, followed by a washing procedure as described in Tables 11, 12 and 13.
  • GFC gel filtration chromatography
  • GFC gel filtration column
  • Plasmid DNA began eluting in fraction 8, and RNA began eluting in fraction 11. Plasmid DNA was precipitated from fractions 8-10 (sample A) and fractions 11-12 (sample B) with 0.1 volumes of 3 M NaOAc and 0.7 volumes of IPA. The resulting DNA pellets were dissolved in 1 ml Bind buffer and each sample was run over a 5 ml column packed with a nucleic acid binding composition based on formula I, W group DMAP, X group EDTA at pH 12.0 and substrate Sephacryl.
  • results of the Gel Filtration Column integrated procedure demonstrate the utility of this method.
  • Table 14 contains data from fractions A and B eluted from the combined GFC and nucleic acid binding composition column procedure. The table shows that approximately 69 ⁇ g of DNA (by gel analysis method) was obtained in each fraction, to give a total of about 138 ⁇ g. A yield of 500 ⁇ g is typical with RNAse enzyme used to remove RNA. This procedure may be optimized by increasing the GFC column size and related parameters, in order to increase both DNA yield and purity.
  • Example 7 The rational design of nucleic acid binding compositions of the present invention is facilitated by a multi-level screening procedure described in this example.
  • Level 1 The rational design of nucleic acid binding compositions of the present invention is facilitated by a multi-level screening procedure described in this example.
  • Level 1 The rational design of nucleic acid binding compositions of the present invention is facilitated by a multi-level screening procedure described in this example.
  • Candidates for compound (W) are selected for reactions with the various base structures to obtain compounds represented by the formula I, ⁇ , or DI (prior to addition of (X) or (Sub) groups).
  • the general procedure is as follows:
  • Graphs are plotted by overlaying the individual spectra and the reaction mixture onto the same graphs and observing for spectral shifts.
  • Spectral shifts are quantified by drawing a tangent along the main peak (usually 300 to 500 nm) and estimating the x intercept as compared to that of the reactants.
  • the absorbance shift shown by ⁇ nm when DMAP and DCTFP (or substitute compounds) are reacted must have a positive value in order to be effective for nucleic acid binding. Furthermore, the larger the ⁇ nm value, the higher the probability for nucleic acid binding. Typically, a positive value of at least 10 nm is required for a (W) compound to be useful in the practice of the present invention. A shift of greater than +100 is desirable.
  • a (W) compound useful in the practice of the present invention possesses one or more "electron mobilizing groups". These groups include alkyl (e.g., CH 3 ), tertiary amines, hydroxyl, and thiomethyl groups. Undesirable functional groups attached to candidate (W) compounds that cause negative absorbance shifts under uncertain conditions include: halogen, nitro, sulfhydryl, and carboxylic acid moieties.
  • a second level of screening is accomplished by exposing the reaction product prepared using the level 1 protocol with nucleic acid molecules (such as DNA or RNA). Any complex formed is then recovered by precipitating the DNA and washing thoroughly (e.g., with methanol) to remove any unbound or "loosely" bound nucleic acid binding composition. UV-Nis analysis of both the washing residue and the recovered nucleic acid complex confers confidence in the determination.
  • nucleic acid binding cability of a particular composition for binding specific types of nucleic acid molecules can be rapidly ascertained before embarking on an extensive chemistry project involving the use of substrates.
  • FIGURE 5 shows the change in absorbance across a spectrum of wavelengths for a DNA binding composition of formula I, wherein W is DMAP, in the presence or absence of plasmid DNA.
  • W is DMAP
  • the bound DNA complex was precipitated with ethanol and washed thoroughly with methanol until no absorbance was seen in the washing supernatants.
  • the results show that nucleic acid binding occurs without the need for linking the synthetic reagent to a substrate.
  • Levels 3 and 4 The (X) functionality for formula I, ⁇ , and HI is the basis for nucleic acid binding specificity. Screening of these candidates is achieved prior to reaction to a substrate by modifying procedures employed in Levels 1 and 2 screening.
  • Both synthetic moieties e.g., polyamides composed of aminoacids, imidazole, pyrrole, hydroxypyrrole, and poly A
  • naturally occurring groups such as DNA or RNA binding nucleotide sequences

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L"invention porte sur des procédés de fixation de molécules d"acide nucléique à une composition de fixation d"acide nucléique consistant à mettre en contact une telle composition avec des molécules d"acide nucléique dans des conditions permettant ladite fixation, la composition étant définie par la formule (I), la formule (II) ou la formule (III) de la description. L"invention porte également sur les compositions de fixation d"acides nucléiques de formule (I), de formule (II) ou de formule (III).
PCT/US2001/045165 2000-10-19 2001-10-19 Procedes et compositions de fixation de molecules d"acide nucleique WO2002059093A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US24161500P 2000-10-19 2000-10-19
US60/241,615 2000-10-19
US26621201P 2001-02-02 2001-02-02
US60/266,212 2001-02-02
US27656201P 2001-03-14 2001-03-14
US60/276,562 2001-03-14
US28671901P 2001-04-26 2001-04-26
US60/286,719 2001-04-26

Publications (1)

Publication Number Publication Date
WO2002059093A1 true WO2002059093A1 (fr) 2002-08-01

Family

ID=27500047

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/045165 WO2002059093A1 (fr) 2000-10-19 2001-10-19 Procedes et compositions de fixation de molecules d"acide nucleique

Country Status (1)

Country Link
WO (1) WO2002059093A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007531794A (ja) * 2004-04-05 2007-11-08 アルニラム ファーマスーティカルズ インコーポレイテッド オリゴヌクレオチドの合成および精製に使用する方法および反応試薬

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981961A (en) * 1988-09-12 1991-01-01 Bioprobe International, Inc. Synthetic affinity ligand compositions and methods for purification and recovery of organic molecules
US5053499A (en) * 1989-02-27 1991-10-01 Sanyo-Kokusaku Pulp Co., Ltd. 2',3'-dideoxy purine nucleoside

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981961A (en) * 1988-09-12 1991-01-01 Bioprobe International, Inc. Synthetic affinity ligand compositions and methods for purification and recovery of organic molecules
US5053499A (en) * 1989-02-27 1991-10-01 Sanyo-Kokusaku Pulp Co., Ltd. 2',3'-dideoxy purine nucleoside

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007531794A (ja) * 2004-04-05 2007-11-08 アルニラム ファーマスーティカルズ インコーポレイテッド オリゴヌクレオチドの合成および精製に使用する方法および反応試薬
US8431693B2 (en) 2004-04-05 2013-04-30 Alnylam Pharmaceuticals, Inc. Process for desilylation of oligonucleotides

Similar Documents

Publication Publication Date Title
ES2629615T3 (es) Ácido L-nucleico modificado
RU2451022C2 (ru) Катионные олигонуклеотиды, автоматизированные способы их получения и их применение
US5698685A (en) Morpholino-subunit combinatorial library and method
ES2668619T3 (es) Compuestos que se dirigen a ARN y procedimientos para prepararlos y usarlos
WO2019149198A1 (fr) Banque de composés codés par adn et procédé de criblage de composés
Uhlenbeck et al. Interaction of R17 coat protein with its RNA binding site for translational repression
WO2009077173A2 (fr) Bibliothèques de produits chimiques codés par adn
JPH06508526A (ja) 配列−特異結合する、二重らせん核酸のためのポリマー
JPH06509063A (ja) 診断薬および分析方法における核酸類縁体の使用・用途
WO2003054232A2 (fr) Procedes de suppression des oligonucleotides bicatenaires contenant des erreurs de sequençage a l'aide de proteines de reconnaissance de mesappariements
AU2003214031A1 (en) An improved method for synthesising templated molecules
KR20180073584A (ko) 올리고뉴클레오타이드 접합 방법
US20220372196A1 (en) Biomagnetic microsphere and preparation method and use method therefor
EP1731615A1 (fr) Adn et arn de localisation cytoplasmique
JPH10257888A (ja) ポリエーテル核酸
CA2169535A1 (fr) Evolution systematique des ligands par enrichissement exponentiel; photoselection de ligands d'acide nucleique et utilisation de la methode selex
US20130231480A1 (en) Sequence specific double-stranded dna/rna binding compounds and uses thereof
ES2300379T3 (es) Procedimiento de deteccion selectiva de transfeccion de oligonucleotidos.
WO2002059093A1 (fr) Procedes et compositions de fixation de molecules d"acide nucleique
US11987904B2 (en) Oligonucleotides and methods for preparing
EP4359418A1 (fr) Dérivés d'hydrates de carbone et kits pour le marquage de surface cellulaire
JP2002523059A (ja) 基質置換での酵素活性スクリーン
CN105821046A (zh) 一种特异性的l-丝氨酸适配体及其用途
ES2310969B1 (es) Biotina-soporte poroso, metodos de obtencion y usos.
WO2021153717A1 (fr) Chromatographie d'affinité à base d'acide nucléique artificiel

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 69(1) EPC.(EPO LETTER FORM 1205A HAS BEEN SENT ON 07/08/2003)

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP