WO1998027425A1 - Purification a grande echelle d'oligonucleotides de longueur totale par extraction par affinite solide-liquide - Google Patents

Purification a grande echelle d'oligonucleotides de longueur totale par extraction par affinite solide-liquide Download PDF

Info

Publication number
WO1998027425A1
WO1998027425A1 PCT/US1997/023284 US9723284W WO9827425A1 WO 1998027425 A1 WO1998027425 A1 WO 1998027425A1 US 9723284 W US9723284 W US 9723284W WO 9827425 A1 WO9827425 A1 WO 9827425A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
target oligonucleotide
information
matrix
affinity unit
Prior art date
Application number
PCT/US1997/023284
Other languages
English (en)
Inventor
Danhua Chen
Githa Susan Srivatsa
Douglas L. Cole
Original Assignee
Isis Pharmaceuticals, Inc.
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 Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to AU62371/98A priority Critical patent/AU6237198A/en
Publication of WO1998027425A1 publication Critical patent/WO1998027425A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • This invention relates to methods for purifying a desired full length synthetic oligonucleotide (the target oligonucleotide) from a mixture containing undesired contaminants (e.g., deletion derivatives of the target oligonucleotide) using an immobilized affinity unit that selectively and reversibly binds the target oligonucleotide.
  • an immobilized affinity unit that selectively and reversibly binds the target oligonucleotide.
  • hybridization between the target oligonucleotide and the affinity unit which comprises a nucleobase sequence having reverse complementary to the nucleobase sequence of at least a central portion of the target oligonucleotide, results in the selective retention of the desired full length (n) target oligonucleotide.
  • Undesired derivatives e.g., all forms of (n-1), (n-2), etc.
  • treatments e.g., changes in pH, ionic strength, temperature, and the like
  • the desired full length (n) target oligonucleotide is thus rapidly, cost-effectively and efficaciously separated from undesired contaminants including the undesired derivatives (e.g., all forms of (n- 1), (n-2), etc.) of the target oligonucleotide that result from, e.g., incomplete oligomerization during synthesis or degradative processes.
  • Preferred matrices for practicing the methods of the invention are also herein provided.
  • a process of oligonucleotide synthesis generally comprises the steps of (1) blocking chemically reactive sites on the base portion of a first and second selected nucleoside with unreactive "blocking groups," (2) coupling the first selected base-blocked nucleoside monomer to an inorganic support via a 3' hydroxyl linkage from the pentose portion of the first nucleoside monomer, (3) "protecting" the 5' hydroxyl position of the pentose portion of the second selected base-blocked nucleoside monomer, for example, by chemically attaching a dimethoxytrityl (D Tr) group thereto, (4) attaching the second selected base- and 5
  • D Tr dimethoxytrityl
  • the oligonucleotide Upon completion of a desired number of such cycles, the oligonucleotide is "deblocked" (i.e., the blocking groups attached to the bases of the oligonucleotide are removed) and a desired biological activity is then realized.
  • Such methods of oligonucleotide synthesis include, for example, those commonly known as the "phosphite triester method," the “phosphotriester method” and the "H-phosphonate method.” Methods for the solution phase synthesis of oligonucleotides have also been described (see U.S. Patent No. 5,210,264 to Yau, assigned to the present applicants; Reese et al . , J. Chem . Soc . Perkin Trans .
  • the stepwise yield for each nucleoside addition is typically about 99%.
  • approximately 1% of the oligomers fail nucleoside monomer addition in each step.
  • Such failures of addition result from incomplete coupling, incomplete capping, incomplete detritylation, undesired retritylation through oxidation or by other mechanisms.
  • the resulting oligonucleotides are, e.g., one oligonucleotide (n-1), two oligonucleotides (n-2), etc.
  • High Pressure Liquid Chromatography HPLC
  • reverse-phase chromatography reverse-phase chromatography
  • ion-exchange chromatography are examples of commonly used traditional techniques for the purification of crude synthetic oligonucleotides (Warren et al . , Chapter 9 In : Methods in Molecular Biology, Vol . 26 : Protocols for Oligonucleotide Conjugates , Agrawal , S., Ed., 1994, Humana Press Inc., Totowa, NJ, pages 233-264).
  • the present invention provides new methods for the large-scale purification of oligonucleotide substances with superior selectivity and product yield, by solid-liquid affinity extraction using immobilized affinity units that preferentially bind the desired full length oligonucleotide.
  • the affinity unit comprises an immobilized nucleobase sequence having reverse complementarity to the desired target oligonucleotide over a central portion thereof, the essentially full-length (p, as defined herein) thereof, or the full-length (n) thereof.
  • the method of the invention allows for the purification of a desired full length synthetic oligonucleotide from a mixture of heterogeneous failed sequence oligonucleotides.
  • Reese (Tetrahedron, 1978, 34 , 3143) describes methods for synthesizing nucleoside building blocks for oligonucleotides and early attempts to achieve significant amounts of oligonucleotide synthesis using the phosphotriester method.
  • Caruthers ( Science, 1985, 230 , 281) describes the phosphite triester method of oligonucleotide synthesis and at least partially automated machines for carrying out this method.
  • Uhlmann et al . Chem . Reviews , 1990, 90 , 543) review the then-prevailing state of the art of synthesis of unmodified and modified oligonucleotides.
  • Yashima et al . J. Chroma tography, 1992, 603 , 111) describe the separation of nucleosides and nucleotide dimers via the use of affinity chromatography on silica gel columns comprising immobilized nucleic acid analogs.
  • oligonucleotides are also separable by the method of Yashima et al . , it is noted that the resolving power of the system decreases as the target oligonucleotide length increases. Temsamani et al . (Nucl . Acids Res .
  • the invention provides methods and matrices for the rapid, cost-effective and efficacious removal of undesired derivatives of a desired target oligonucleotide. More particularly, the invention provides methods and matrices for the removal of all forms of undesired deletion derivatives [i.e., (n-1), (n-2), etc.] of the desired full length (n) oligonucleotide, including those undesired derivatives having an internal or 3' deletion of one or more oligonucleotides. A desired level of large-scale purification of the desired oligonucleotide is achieved according to the methods and matrices of the invention.
  • the affinity unit comprises a nucleobase sequence having a sequence that is the reverse complement of the target oligonucleotide over a central portion thereof, the essentially full-length (p, as defined herein) thereof, or the full-length (n) thereof.
  • a mixture comprising the desired target oligonucleotide is contacted with a matrix of the invention comprising the immobilized affinity unit under stringent hybridization conditions, i.e., conditions under which the hybridization (binding) of the desired target oligonucleotide is preferentially achieved.
  • the undesired [e.g., (n-1), (n-2), etc.] derivatives of the target oligonucleotide hybridize poorly, or not at all, to the matrix of the invention under these conditions and are separated by relatively simple techniques (e.g., washing, centrifugation, etc.).
  • the hybridization reaction occurs under conditions wherein some undesired derivatives of the target oligonucleotide are initially bound but are subsequently removed by a first chemical or physical treatment (e.g., changes in pH, ionic strength, temperature, and the like) which result in a change in the environment of the mixture.
  • the bound target oligonucleotide is eluted from the affinity unit by application of a second chemical or physical treatment.
  • the affinity unit remains immobilized to the matrix of the invention and, in a preferred embodiment, is reused for purifying further batches of the desired target oligonucleotide .
  • Figure 1 shows the modification of a primary amine, attached to a support (indicated by the open circle) by a linker (indicated by (CH 2 ) n ), by 1, 4-phenylene diisthiocyanate to form a modified support having a phenylisothiocyanate group.
  • Figure 2 shows the reaction of the primary amine group of a probe (i.e., affinity unit and spacer) with the isothiocyanate group of the modified support to form a thiocarbamyl adduct, thereby covalently attaching the probe portion to the support via the linker.
  • Figure 3 shows an electropherogram of crude ISIS 2302.
  • Figure 4 shows an electropherogram of ISIS 2302 prepared by a method of the invention.
  • the present invention is directed to methods for purifying a desired, full length oligonucleotide by the rapid, cost-effective and efficacious removal of contaminants, including but not limited to undesired deletion (e.g., n-1, n-2, etc.) derivatives of the desired (target) oligonucleotide. This is accomplished via solid-liquid extraction of the oligonucleotide through selective hybridization to an affinity unit that specifically binds the desired oligonucleotide. Matrices comprising such affinity units, useful for practicing the methods of the invention, are also herein provided.
  • deletion derivatives of the desired target oligonucleotide are contaminants of particular concern, it will be appreciated by those skilled in the art that other contaminants can be removed by the method of the invention as well. For example, a significant portion of undesired salts can be removed using the method of the invention.
  • the target oligonucleotides that are purified by the methods and matrices of the invention include, in particular, those intended for uses requiring a relatively low concentration of undesired or uncharacterized contaminants.
  • the invention is drawn to the purification of target oligonucleotides, particularly antisense oligonucleotides, intended for therapeutic delivery to an animal, including a human.
  • Certain preferred embodiments of the present invention are drawn to the purification of target oligonucleotides designed to have therapeutic activity in an animal, such as a human; such target oligonucleotides may be formulated into a pharmaceutical composition.
  • the target oligonucleotide is designed to function in a manner that is prophylactic, palliative or curative with regard to (1) a disorder caused by a hyperproliferation of cells (e.g., cancer), a pathogen (e.g., malaria, AIDS), or from causes that appear to relate to neither pathogens nor hyperproliferative cells (e.g., Alzheimer's disease) or (2) the symptoms of such a disorder.
  • the invention is also drawn to the purification of target oligonucleotides that modulate the expression of a cellular protein, including cell surface proteins.
  • “designed to modulate” means designed to either effect an increase (stimulate) or a decrease (inhibit) in the expression of a gene.
  • modulation can be achieved by a variety of mechanisms known in the art, including but not limited to transcriptional arrest; effects on RNA processing (capping, polyadenylation and splicing) and transportation; enhancement of cellular degradation of the target nucleic acid; and translational arrest (Crooke, S.T., et al . , Exp. Opin . Ther. Patents, 1996, 6, 1) .
  • Such desired target oligonucleotides include, but are not limited to, those designed to modulate the expression of cellular surface proteins (Table 1) , and those designed to have therapeutic activity against disorders associated with cellular hyperproliferation (Table 2) or having no apparent pathological or hyperproliferative-related cause (Table 3) and diseases resulting from eukaryotic pathogens (Table 4) , retroviruses such as human immunodeficency virus (HIV; Table 5) or viral pathogens other than retroviruses (Table 6) .
  • TABLE 1 TARGET OLIGONUCLEOTIDES DESIGNED TO MODULATE CELL SURFACE PROTEINS AND AFFINITY UNITS THEREFOR
  • Target oligonucleotides that may be purified according to the methods and matrices of the invention include those consisting of naturally occurring nucleotides as well as those comprising one or more chemical modifications. Specific examples of some modified oligonucleotides that can be incorporated into the target oligonucleotide include those containing phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, cycloalkyl, heteroatomic or heterocyclic intersugar linkages.
  • such oligonucleotides include those having phosphorothioates intersugar linkages, those with heteroatomic intersugar linkages including CH 2 -NH-0-CH 2 , CH 2 - N(CH 3 )-0-CH 2 [known as a methylene (methylimino) or MMI backbone], CH 2 -0-N (CH 3 ) -CH 2 , CH 2 -N (CH 3 ) -N (CH 3 ) -CH, and 0-N(CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as 0-P-0-CH 2 ) , thoses with heterocyclic linkages including the morpholino sugar-backbone structures (Summerton and Weller, U.S.
  • Patent 5,034,506 or those with a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone wherein the nucleobases is bound directly or indirectly to an aza nitrogen atoms of the polyamide backbone (Nielsen et al . , Science, 1991, 254, 1497).
  • PNA peptide nucleic acid
  • modified oligonucleotides also include oligonucleotides containing one or more substituted sugar moieties [i.e., sugar moieties comprising one of the following at the 2' position: -F; -Cl; -Br; -OH; -SH; -SCH 3 ; -OCN; -OCH 2 OCH 3 , -0 (CH 2 ) n O (CH2 ) m CH 3 (i .
  • substituted sugar moieties i.e., sugar moieties comprising one of the following at the 2' position: -F; -Cl; -Br; -OH; -SH; -SCH 3 ; -OCN; -OCH 2 OCH 3 , -0 (CH 2 ) n O (CH2 ) m CH 3 (i .
  • alkoxyalkoxy , - 0(CH 2 ) n NH 2 or -0(CH 2 ) n CH 3
  • m is from 0 to about 6 and n is from 1 to about 10; C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; -CN; -CF 3 ; -OCF 3 ; 0-, S-, or N- alkyl or substituted alkyl; 0-, S-, or N-alkenyl; -S0CH 3 ; - S0 2 CH 3 ; -ON0 2 ; -N0 2 ; -N 3 ; -NH 2 ; heterocycloalkyl ; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide;
  • 2 -alkoxyalkoxy substituents such as 2 ' -O-methoxyethoxy [2 ' -0-CH 2 CH 2 OCH 3 , also known as 2 ' - 0- (2-methoxyethyl) ] (Martin et al . , Helv. Chim . Acta , 1995, 78, 486), 2'-methoxy (2'-
  • Additional modified oligonucleotides include those having similar modifications at other positions on the oligonucleotide (particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide), oligonucleotides having sugar mimetics such as cyclobutyls in place of the pentofuranosyl group or base modifications or substitutions (e.g., with a "universal" base such as inosine) .
  • a further modification of the target oligonucleotide involves chemically linking to the target oligonucleotide one or more lipophilic moieties which enhance the cellular uptake of the oligonucleotide.
  • lipophilic moieties include but are not limited to a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
  • a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Letts., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 15, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-0-hexadecyl-rac-glycero-3-H- phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.
  • a phospholipid e.g., di-hexade
  • Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides, are disclosed in U.S. Patents No. 5,138,045, No. 5,218,105 and No. 5,459,255.
  • the target oligonucleotide can also be an oligonucleotide which is a chimeric oligonucleotide including a "gapmer" or a "hemimer.”
  • Chimeric oligonucleotides are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one terminal region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the intracellular target nucleic acid.
  • a single terminal (either 5' or 3') region is so modified in the oligonucleotide structure.
  • the oligonucleotide is called a Agapmer ⁇ and the modified 5'- and 3 '-terminal regions are referred to as "wings"; an additional, typically central, region (typically referred to as the "gap” or “core”) of the oligonucleotide may serve as a substrate for cellular enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids.
  • the 5' and 3' wings can be modified in the same or different manner depending on what properties it is desired to achieve.
  • the length of a target oligonucleotide is defined as "n" nucleobases, wherein "n” is a positive whole number.
  • the length of target oligonucleotides that can be purified according to the methods and matrices of the invention is one having from 5 to about 60 nucleobases, preferably from 7 to about 50 nucleobases, more preferably from 8 to about 40 nucleobases, even more preferably from 9 to about 30 nucleobases and most preferably from 10 to about 25 nucleobases.
  • the method of synthesis of the target oligonucleotide does not typically effect the methods of the invention; in general, any method of synthesis, including methods for the solution phase synthesis of oligonucleotides (see U.S. Patent No. 5,210,264 to Yau, assigned to the present applicants; Reese et al . , J. Chem. Soc . Perkin Trans . , 1993, 1, 2291; and Wada et al . , Tetrahedron, 1993, 49, 2043) may be used.
  • the matrices of the invention comprise a plurality of a molecular composition that comprises several parts: (1) a support, (2) an optional linker to the support, (3) an optional spacer and (4) a unit having a high degree of specific affinity to the target oligonucleotide (affinity unit) .
  • affinity unit a unit having a high degree of specific affinity to the target oligonucleotide
  • the linker (2) and spacer (3) provide a bridge between the support (1) and the affinity unit (4) in such a way as to not significantly alter or reduce the binding capacity of the latter element .
  • the probe of the matrix of the invention comprises parts (4) and, optionally, (3) . The following paragraphs describe the matrices of the invention in more detail.
  • a suitable support has preferred characteristics of non-reactivity with compounds introduced during the various steps of oligonucleotide synthesis, accessibility to solvents utilized during such syntheses, and a tendency towards minimal barrier layer diffusion.
  • a barrier layer is created by an ordering of solvent molecules on the surface of a solid phase support. As this barrier layer is composed of ordered molecules, it is difficult to get consistent reagent diffusion across such a barrier to the molecules of interest which are attached to the support . It will be appreciated by those skilled in the art that the chemical composition of the affinity unit (4) , the optional linker (2) and/or the optional spacer (3) may influence the choice of the support (1) .
  • the support may be insoluble
  • solid or soluble.
  • graft polymers U.S. Patent No. 4,908,405 to Bayer and Rapp
  • polyacrylamide Fahy et al . , Nucl . Acids Res . , 1993, 21 , 1819
  • polyacrylmorpholide Polystyrene and derivatized polystyrene resins (Syvanen et al . , Nucl . Acids Res . , 1988, 16, 11327; U.S. Patent Nos.
  • the solid support is a crosslinked copolymer of N-vinylpyrrolidone, other N-vinyl - lactam monomers and an ethylenically unsaturated monomer having at least one amine or amine-displacable functionality as disclosed in U.S. Patent No. 5,391,667.
  • polystyrene or long chain alkyl CPG (controlled pore glass) beads are employed as the solid dupport .
  • the support is soluble and is composed of, for example, modified polethylene glycol (PEG) units (Bonora et al . , Nucleic Acids Res .
  • any chemical group or chain capable of forming a stable chemical linkage, or a stable association, between the support (1) and the affinity unit (4) , or between the support (1) and the optional spacer (3) may be employed.
  • a suitable linker has preferred characteristic of non-reactivity with compounds introduced during the various steps of oligonucleotide synthesis. It will be appreciated by those skilled in the art that the chemical composition of the support (1) and the affinity unit (4) and/or the optional spacer (3) may influence the choice of the linker.
  • linkers will comprise a primary amine group at either or both termini, as many chemical reactions are known in the art for linking primary amine groups to a variety of other chemical groups .
  • Suitable linkers include, but are not limited to, linkers having a terminal thiol group for introducing a disulfide linkages to the support (Day et al . , Biochem . J. , 1991, 278 , 735; Zuckermann et al . , Nucl . Acids Res .
  • linkers having a terminal bromoacetyl group for introducing a thiol-bromoacetyl linkage to the support (Fahy et al . , Nucl . Acids Res . , 1993, 21 , 1819); linkers having a terminal amino group which can be reacted with an activated 5' phosphate of an oligonucleotide (Takeda et al . , Tetrahedron Letts . , 1983, 24 , 245; Smith et al . , Nucl . Acids Res . , 1985, 13 , 2399; Zarytova et al . , Anal . Biochem .
  • an n-aminoalkyl chain is the linker.
  • an n-aminohexyl chain [i.e., NH 2 -(CH 2 ) 6 ] is the linker (2).
  • a suitable spacer has preferred characteristic of non-reactivity with compounds introduced during the various steps of oligonucleotide synthesis. It will be appreciated by those skilled in the art that the chemical composition of the support (1) and the affinity unit (4) and/or the optional linker (2) may influence the choice of the spacer.
  • suitable spacers include, but are not limited to, oligopeptides; oligonucleotides; alkyl chains; polyamines; polyethylene glycols; oligosaccharides ; and art-recognized equivalents of any of the preceding spacers.
  • the spacer is an alkyl chain, most preferably a C 1 -C 20 alkyl chain.
  • the spacer is an oligonucleotide chain, particularly an oligonucleotide chain that comprises one or more chemical modifications that render it resistant to chemical attack.
  • an oligodeoxyribonucleotide chain is particularly preferred.
  • oligo (dT) 5-30 acts as the spacer of the matrix of the invention.
  • This preferred spacer has the following advantages .
  • This spacer is composed of nucleotides and is thus closely related in chemical properties to a preferred affinity unit, i.e., an oligonucleotide.
  • This chemical relatedness provides the benefit of placing the affinity unit in a context that is likely to be appropriate for nucleic acid hybridization duplexing.
  • oligonucleotides e.g., oligo (dA), oligo (dG) or oligo (dC)
  • a preferred spacer is more chemically stable.
  • linker (2) and the optional spacer (3) can be combined into one unit.
  • linker and spacers need not comprise distinct chemical groups or chains.
  • an appropriate oligopeptide or oligonucleotide chain could function as a combined linker and spacer of the matrix of the invention.
  • suitable linker/spacers include, but are not limited, to the linker and spacers described above. Methods of determining an appropriate (i.e., providing the optimal degree and specificity of hybridization between the affinity unit and the target oligonucleotide) length of linker/spacers are known in the art (see, for example, Day et al . , Biochem. J.
  • the linker (2) or spacer (3) of the matrix of the invention would not include carbonate groups.
  • the carbonate moiety is relatively unstable to basic reagents used in some oligonucleotide syntheses and to contaminants (mainly bases) that may be found in solvents utilized in such oligonucleotide synthesis.
  • this portion of the matrix of the invention has the characteristic of binding specifically (or at least preferentially) yet reversibly to a portion of a target oligonucleotide, the purification of the target oligonucleotide being the object of the invention.
  • the portion of the target oligonucleotide that is specifically bound by the affinity unit is referred to as its "hybridizing portion" herein.
  • a preferred affinity unit is one that comprises a chemical composition having a nucleobase sequence that is the reverse complement of the hybridizing portion of the nucleobase sequence of the target oligonucleotide.
  • the hybridizing portion may be a central portion, a terminal portion, or the majority or even entirety of the nucleobase sequence of the target oligonucleotide.
  • the term "a central portion” is intended to encompass preferably from at least five, or from at least ten, contiguous nucleobases derived from the section of the target oligonucleotide' s sequence that is internal from the target oligonucleotide' s 3' and 5' "terminal portions.”
  • a terminal portion includes the most 3' or 5' nucleobase of a target oligonucleotide and comprises an additional number, r, of immediately contiguous nucleobases of the target oligonucleotide, wherein r is from 2 to about 10 nucleobases.
  • an affinity unit having a nucleobase sequence that is the reverse complement of a central portion of the nucleobase sequence of the target oligonucleotide will hybridize with high affinity to the target oligonucleotide, but not to, e.g., deletion derivatives lacking one or more nucleobases in the central portion.
  • the nucleobase sequence of the affinity unit is "full- length", i.e., the same length (n) of the target oligonucleotide, and which is the reverse complement of that of the target oligonucleotide.
  • the nucleobase sequence of the affinity oligonucleotide can be "essentially full-length", i.e., having a length, p, wherein p is a positive whole number ranging from 4 to n+ , wherein, in a duplex between the target oligonucleotide and the nucleobase sequence of the affinity oligonucleotide, neither the 5' overhang nor the 3' overhang of said target oligonucleotide is greater than two nucleobases, provided that, over length p, the nucleobase sequence of the affinity oligonucleotide is the reverse complement of the nucleotide sequence of the target oligonucleotide.
  • the nucleobase sequence of the affinity unit can be from 5 to 60 nucleobases in length, preferably from 10 to 40 nucleobases in length, more preferably from 11 to 30 nucleobases in length and most preferably from 12 to 25 nucleobases in length.
  • Affinity units of differing chemical compositions e.g., oligodeoxynucleotides, oligoribonucleotides and peptide nucleic acids
  • compositions that are relatively nuclease resistant might be preferred.
  • Such relatively nuclease resistant compositions include, for example, oligodeoxyribonucleotides and peptide nucleic acids.
  • RNA nucleases for which no "universal" inhibitor is known, all characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents such as EDTA (Jarrett, J " . Chroma togr.
  • oligodeoxyribonucleotides can thus be more simply prevented from nuclease degradation than oligoribonucleotides.
  • Peptide nucleic acids which are not degraded by either nucleases or proteases, exhibit particularly stringent specificities for their complementary oligonucleotides, and may thus provide the best separation from undesired derivative oligonucleotides in some instances .
  • the method of synthesis of the affinity unit does not typically effect the methods of the invention; in general, any method of synthesis of the particular sort of affinity unit, including methods for the solution phase synthesis of oligonucleotides (see U.S. Patent No.
  • the presence of deletion derivatives in the affinity unit may result in the undesired binding and retention of deletion derivatives of the target oligonucleotide, it may be necessary to purify the affinity unit in a manner that achieves significant purity thereof at the expense of a reduced yield of the affinity unit.
  • certain embodiments of the invention are more tolerant of impurities in the affinity unit than others. For example, purification of oligonucleotides via multiple rounds of affinity chromatography, wherein a different affinity unit is used during each round of purification (see Example 8) , exposes the target oligonucleotide to two or more "screens"
  • oligonucleotide of the affinity unit has a sequence that is the "reverse complement" of that of the nucleotide sequence, the following features are intended.
  • a nucleic acid duplex is formed of two antiparallel strands, i.e., strands that hybridize to each other in a "head-to- tail” fashion:
  • nucleobases in the interior of a nucleic acid duplex bind to specific partner nucleobases.
  • guanine (G) binds to cytosine (C)
  • adenine (A) binds to thymine (T) or uracil (U) .
  • Strand 2 will have a nucleotide sequence that is the reverse complement of Strand 1, i.e., Strand 2 will have, in "reverse” (3' to 5 ' ) order, the partner ("complement") nucleobases to those of Strand 1.
  • the sequence of the oligonucleotide of the affinity unit can have reverse complementarity to the target oligonucleotide through a variety of equivalents.
  • other naturally occurring nucleobase equivalents including 5-methylcytosine, 5-hydroxy ⁇ nethylcytosine (HMC) , glycosyl HMC, gentiobiosyl HMC (C equivalents) , and 5- hydroxymethyluracil (U equivalent) .
  • synthetic nucleobases which retain partner specificity are known in the art and include, for example, 7-deaza-guanine, which retains specificity for C.
  • reverse complementarity will not be altered by any chemical modification to a nucleobase in the nucleotide sequence of the affinity oligonucleotide which does not alter its specificity for the partner nucleobase in the target oligonucleotide.
  • reverse complementarity can be achieved by inserting a "universal" base partner, e.g., hypoxanthine (inosine, I, is the corresponding nucleotide) at the corresponding position in the affinity unit.
  • an affinity unit having an affinity oligonucleotide having the nucleotide sequence 5'-GGGICGCG has a sequence that is the reverse complement of the target oligonucleotide mixture [5 ' -CGCGACCC, 5'-CGCGGCCC, 5'-CGCGTCCC and 5'- CGCGCCCC] .
  • the duplex between the nucleobase sequence of the affinity unit and the hybridizing portion of the target oligonucleotide will result in the target oligonucleotide having either a "3' overhang” or a "5' overhang” or, in some instances, both types of overhangs.
  • a "3' overhang” consists of unpaired nucleotides on the 3' terminus of the target oligonucleotide, whereas a "5' overhang” consists of unpaired nucleotides on the 5' terminus of the target oligonucleotide.
  • the two types of overhangs may be diagramed as follows :
  • Affinity unit (length p) 3' ⁇ 5'
  • Affinity unit (length p) 3' ⁇ 5'
  • a preferred affinity unit will selectively bind and/or retain the target oligonucleotide with high affinity and specificity under conditions wherein derivatives of said target oligonucleotide having one or more mismatches with the nucleobase sequence of said affinity unit are not bound and/or retained.
  • the affinity unit can incorporate one or more chemical modifications for the purpose of enhancing specific interactions with the target oligonucleotide, as is described in more detail in Example 6. Such modifications may additionally or alternatively result in the affinity unit having increased resistance to degradative contaminants, e.g., exonucleases .
  • the target oligonucleotide may additionally or alternatively comprise such modifications, so long as reverse complementarity is maintained between the target oligonucleotide and that of the affinity unit over the above-defined “hybridizing" portion of the target oligonucleotide or lengths "n" (full- length) or “p” (essentially full-length) thereof.
  • the optional linker (2) , optional spacer (3) and affinity unit (4) can be combined into one unit.
  • the linker, spacer and affinity unit need not comprise distinct chemical groups or chains.
  • an appropriate oligonucleotide chain could function as the linker, spacer and affinity unit of the matrix of the invention.
  • the probe of the matrix of the invention comprising affinity unit (4) and, optionally, the spacer (3), can be combined into one unit.
  • the spacer and affinity unit need not comprise distinct chemical groups or chains.
  • an aminohexyl group is the linker (2) to the support (1) , as it is easily attached to the 5' end of a oligonucleotide by a solid phase synthesizer.
  • the probe includes an oligonucleotide which comprises a first nucleotide sequence, which functions as the spacer (3) , and a second nucleotide sequence, which serves as the affinity unit (4) .
  • the affinity unit (4) can be attached to the spacer (3) , linker (2) or support (1) at any position thereof so long as the potential for hybridization with the target oligonucleotide is not negatively effected. That is, the affinity unit can be attached to the spacer, linker or support at its 3' or 5 ' terminus, or through a position on its backbone or one or more of its sugar residues or nucleobases, so long as the portion of the affinity unit that hybridizes to the hybridizing portion of the target oligonucleotide remains accessible for binding.
  • the affinity unit (4) is synthesized directly on the support (i.e., in si tu) rather than being separately synthesized and subsequently attached to the support.
  • This embodiment is particularly useful when the components of the affinity unit, and the components linking it to the support, are stable under the various conditions of synthesis and subsequent chemical steps (deprotection, deblocking and the like) necessary to prepare the matrix for use in the method of the invention.
  • Examples of in si tu synthesis of oligonucleotides on both soluble and insoluble supports are known in the art (Bonora et al . , Nucl ei c Acids Res . , 1993, 21 , 1213; Bagno et al . , Chem . Biochem . Eng. Q . , 1994, 8 , 183; Matson et al . , Anal .
  • the method of the invention comprises at least 2 steps: (a) contacting a mixture comprising the target oligonucleotide and undesired deletion sequence oligonucleotides to the matrix of the invention under conditions such that a hybridization reaction preferentially occurs between the target oligonucleotide and the affinity unit, and (c) dissociating and recovering the target oligonucleotide from the matrix of the invention.
  • the method of the invention additionally comprises step (b) , removing unbound, undesired deletion sequence oligonucleotides or other undesirable contaminants from the matrix by, for example, washing the matrix while the target oligonucleotide is bound thereto.
  • Step (a) is a hybridization step in which a mixture of crude synthetic oligonucleotides, comprising the desired full length n-mer as well as undesired derivatives [i.e., (n-1), (n-2), etc.] is contacted to the matrix of the invention and allowed to hybridize to the affinity unit.
  • the degree of hybridization between the affinity unit and the full length (n) target oligonucleotide is dependent upon parameters such as the ionic strength of the buffer solution in which the hybridization occurs, temperature, base composition and length of the duplex formed between the target oligonucleotide and the affinity unit, concentration of the affinity unit, concentration of the target oligonucleotide, and the concentration (s) of duplex destabilizing agent (s) .
  • the method of the invention is designed to maximize the affinity of the affinity unit for the full length target oligonucleotide while achieving the least degree of affinity for undesired (deletion sequence) oligonucleotides.
  • SSPE buffer (lx-5x) ; 5x SSPE buffer is 0.75 M NaCl, 50 mM NaH 2 P0 4 , pH 7.4, and 5 mM EDTA; and
  • nucleases and duplex destabilizing agents from such buffers before their use in hybridization reactions. This can be achieved by, for example, autoclaving the buffers .
  • Temperature can be another important parameter for hybridization reactions.
  • the temperature of the hybridization reaction is adjusted so that only full length target oligonucleotide will quantitatively hybridize to the affinity unit.
  • the formation of duplexes between the affinity unit and undesired oligonucleotides (deletion sequences) will be thermodynamically disfavored.
  • optimum temperatures can be estimated for various chromatographic methods involving nucleic acids, some degree of "fine tuning" will be required in many instances. For example, Jarrett (J. Chromatogr . , 1993, 618, 315) opines that the equation given by Sambrook et al .
  • the methods of the invention optionally comprise the step (b) , removing undesired oligonucleotides and/or other undesired contaminants, which may be carried out, for example, by placing the hybridized matrix: target oligonucleotide complexes into a suitable washing buffer which has a composition that is similar, or even identical, to that of the hybridization buffer of step (a) but which is different in concentration.
  • the washing buffer can be from 0.4x to 2x, preferably from 0.5x to 1.6x, and most preferably from 0.6x to 1.2x the concentration of the hybridization buffer.
  • the washing buffer is from 1.2x to 6x SSPE buffer, preferably from 1.5x to 4.8x SSPE buffer, and most preferably from 1.8x to 3.6x SSPE buffer.
  • the temperature at which the hybridization reaction occurs is increased so that undesired oligonucleotides do not hybridize as well to the affinity unit.
  • the temperature for hybridization reactions should be between the melting temperature of the full length oligonucleotide duplex, T m n , and the highest melting temperature of deletion sequence oligonucleotides, T m d . That is, the temperature range at which the hybridization reaction is performed, T, is defined by the equation
  • step (b) The purpose of optional step (b) is to remove as many deletion sequence oligonucleotide molecules and other undesirable contaminating molecules as possible while maintaining the highest possible concentration of hybridized full length oligonucleotide bound to the affinity unit of the support. Accordingly, the specific conditions at which these steps are carried out may be adjusted by monitoring these parameters and others known to those skilled in the art.
  • Step (c) dissociation and recovery of the full length target oligonucleotide, is achieved, for example, by placing the matrix: target oligonucleotide complexes into distilled water.
  • the target oligonucleotide is thus easily and readily dissociated from the matrix of the invention and is recovered in the distilled water. Temperature and time are two parameters that can be adjusted to achieve optimal elution.
  • the full length oligonucleotide is dissociated and recovered (1) in distilled water, ensuring a low salt content in the final product and thus eliminating an expensive desalting step, and (2) at a temperature higher than that at which the hybridization step (a) occurred in order to facilitate the complete release of the target oligonucleotide.
  • This preferred embodiment eliminates the need for an expensive and time-consuming desalting step that is present in many other methods of oligonucleotide purification.
  • oligonucleotides in the flow-through during the recovery step (c) can be monitored by a variety of methods known in the art (see, for example, Jarrett, J. Chromatogr. , 1993, 618, 315) .
  • the methods and matrices of the invention are used to purify oligonucleotides prepared by a synthesis procedure that is "blockwise," i.e., one in which one or more coupling steps results in the incorporation of a "blockmer” of nucleobase units (e.g., a dinucleotide or trinucleotide) .
  • a "blockmer” refers to a chemically linked sequence of 2, 3, 4, 5, 6, 7 or 8 nucleobases that is incorporated into an oligonucleotide en toto .
  • Oligonucleotide synthesis procedures that utilize at least one step in which several nucleobases are incorporated in one step are known in the art (see, e.g., Kumar et al . , J. Org. Chem. , 1984, 49 , 4905; Bannwarth, Helv. Chimica Acta , 1985, 68 , 1907; Cosstick et al . , Biochemistry, 1985, 24 , 3630; Wolter et al . , Nucleosides & Nucleotides , 1986, 5, 65; Miura et al . , Chem. Phar . Bull . , 1987, 35, 833; Yau et al . , Tetrahedron Letts . , 1990, 31 , 1953) .
  • target oligonucleotides prepared by blockwise synthesis procedures is a preferred embodiment of the invention for the following reasons.
  • a synthesis procedure that couples, for example, dinucleotides rather than mononucleotides results in a final reaction mixture wherein the contaminating undesired products are, for the most part, (n-2), (n-4), (n-6) , etc. in length relative to the desired n-mer target oligonucleotide.
  • an [(n-2), (n-4), (n-6), etc.] mixture has the inherent advantage over final reaction mixtures comprising (n-1) derivatives, because some (n-1) derivatives can be particularly difficult to separate from the desired n-mer.
  • the method of purification of a desired target oligonucleotide of the invention which is effective in selectively removing single base (n-1) deletions from a mixture, is expected to be particularly effective when the method is applied to a "blockwise" final reaction mixture that has fewer undesired derivative oligonucleotides of the (n-1) type, albeit containing undesired derivative oligonucleotides having deletions greater than one nucleobase [i.e., (n-2), (n-3), (n-4), (n-5) or (n-6) bases] .
  • a desired chemical feature e.g., a stereospecific chemical linkage
  • blockwise procedures should produce final reaction mixtures that contain fewer non- nucleobase containing contaminants (e.g., solvents) as well, a feature that further enhances the use of such procedures with the methods and materials of the invention herein disclosed.
  • Example 1 Affinity Design
  • the matrix of the invention consists of several parts: a support (1), an optional linker (2) to the support, an optional spacer (3) and an affinity unit (4) designed to specifically hybridize to the target oligonucleotide.
  • the probe of the matrix of the invention comprises parts (4) and, optionally, (3) .
  • All four parts are used.
  • An aminohexyl group, a preferred linker (2) to the support (1) as it is easily attached to the 5' end of a oligonucleotide by a solid phase synthesizer, is used in Examples 1 to 3.
  • oligo (dT) 5 . 30 acts as the optional spacer (3); in Examples 1 to 3, (dT) 15 is used.
  • an oligomer complementary to the target oligonucleotide in sequence is a preferred affinity unit.
  • the affinity unit of this matrix will hybridize with ISIS 2302 to form a duplex of 20 base pairs (hybridization between bases is indicated by the "j" symbol) :
  • any (n-1), (n-2), etc. derivative of the desired oligonucleotide will have either one or more mismatches to the affinity unit or a shorter hybridizing sequence thereto.
  • the following representative deletion sequences serve as illustrations of this principle (deleted nucleotides relative to SEQ ID NO:l are indicated by an asterisk, "*") :
  • the hybridization structures with one or more one- or two-base “bulges” are unstable because of the steric hindrance (s) and thermodynamic consequence (s) of excluding a base from the duplex. Accordingly, although not wishing to be bound by any theory in particular, the hybridization structures that might preferentially form would be those having fewer matches but lacking "bulges . "
  • the (n-1) deletion sequence with the terminal base deleted is the hardest to separate from the full length oligonucleotide. In that case, perfect matches will be formed for the terminal (n-1) deletion sequence compared with n perfect matches for the full length oligonucleotides. All of the other sequences will have either fewer matches and/or at least one mismatch, and, due to the formation of energetically disfavored duplexes with the affinity unit, thus can be separated from the full length oligonucleotide based on differential affinity.
  • Example 2 Preparation of Support :Activated Linker Polystyrene, controlled pore glass (CPG) and polyethylene glycol (PEG) , with PEG being reversibly precipitable, are some preferred supports.
  • CPG controlled pore glass
  • PEG polyethylene glycol
  • a solid support a CPG bead with a terminal primary amine group (CPG, Inc., Lincoln Park, N.J.) was used as the support (1) and linker (2) .
  • the CPG bead has a mean pore diameter of 569 D and a volume of 1.44 ml/g.
  • the surface area is 55 m 2 /g, therefore, 10 mg of CPG beads has the same surface area as one 0.5 m x 0.5 m glass slide.
  • the primary amine of the linker was modified as follows. 1, 4-phenylene diisothiocyanate was dissolved in 100 ml pyrimine and 900 ml dimethyl formamide and reacted with 49.81 mg CPG at 37°C for 3 hours. The concentration of 1, 4-phenylene diisothiocyanate was in large excess to minimize the dimer formation.
  • the modified CPG was washed once with acetone, twice with methanol, spun down to remove the solvent, and allowed to dry.
  • the linker of the resultant modified beads comprises a terminal isothiocyanate group .
  • the probe comprising the affinity unit (4) and optional spacer (3) is, in this example, an oligonucleotide that comprises a first oligonucleotide sequence, dT n , where n is 15, that serves as the spacer, and a second oligonucleotide sequence, SEQ ID NO: 2, that is the reverse complement of the target oligonucleotide (ISIS 2302) and which serves as the affinity unit .
  • the probe further comprises a primary aminohexyl group, -NH 2 (CH 2 ) 6 -, which is attached to the spacer at the 5' position of the terminal thymine residue.
  • activated linker (Example 2) was added 0.5 ml of probe (145.9 nmol/ml) solution in Ix Tris buffer (pH 8.0) . The mixture was incubated at 37°C for 4 hours. Under these mildly alkaline conditions, the primary amine group of the probe reacted with the isothiocyanate group of the modified beads to form a thiocarbamyl adduct. The probe was thereby covalently attached to the surface of the solid support via the linker ( Figure 2) . Before and after immobilization, the concentration of the probe solution was measured by UV absorbtion at 260 nm.
  • step (a) hybridization was achieved by adding 1 ml of 10 fold diluted crude ISIS 2302 in 3x SSPE buffer solution to the immobilized CPG, followed by incubation at 37°C for 4 hours. By measuring the absorbance of the solution before and after the hybridization and after washing, it was determined that 46.9 nmol of ISIS 2302 had been hybridized to the probe on the support.
  • a preferred hybridization structure is represented as follows:
  • the ratio of target oligonucleotide to the probe was 0.9607, that is to say, the efficiency of hybridization is 96%, or close to 1:1.
  • the hybridized CPG may be washed once or twice with the extraction buffer.
  • the removal (step (b) ) of undesired oligonucleotides was accomplished as follows. The bound oligonucleotides were rinsed with 0.5 ml of 2x SSPE buffer. The rinse was performed 2x at 37°C.
  • hybridized ISIS 2302 was dissociated and recovered (step (c) ) by incubation with distilled water at 90°C for 30 minutes.
  • the affinity unit may be or comprise a peptide nucleic acid (PNA) .
  • PNA peptide nucleic acid
  • ammonium hydroxide can be added, at a concentration of from about 0.5% to about 10% (wt/vol), to a matrix to which target oligonucleotide is bound in order to effect dissociation of the target oligonucleotide from the matrix. Ammonium hydroxide can then be removed from the purified target oligonucleotide according to methods known in the art.
  • the purity of the purified full length oligonucleotide was measured by capillary gel electrophoresis (CGE) . Both crude and extracted ISIS 2302 solutions were analyzed by CGE. The resultant electropherograms ( Figure 3, crude ISIS 2302; Figure 4, extracted ISIS 2303) show that the purity of the extracted oligonucleotide solution is much higher than the original one. The earlier eluting (n-1), (n-2), etc. undesired derivative oligonucleotides are to the left of the large, narrow peak of desired target oligonucleotide. Integration of the areas under the peaks indicates that the crude ISIS 2302 preparation contains 13.5% undesired derivative oligonucleotides. In contrast, ISIS 2302 purified by the method of the invention contains only 2.8% undesired derivative oligonucleotides. Thus, the purity of the desired target oligonucleotide has been increased from 86.5% to 97.2%.
  • crude ISIS 2303, HPLC-purified ISIS 2303 and ISIS 2303 purified according to the method of the invention are electrophoretically separated under the same conditions.
  • one or more nucleotides of the affinity unit comprises at least one chemical modification which (i) lowers the affinity of the probe for one or more undesired oligonucleotides but does not adversely impact the probe's affinity for the desired oligonucleotide, (ii) raises the affinity of the probe for the target oligonucleotide but does not enhance the probe's affinity for the undesired oligonucleotides, or (iii) achieves both of goals (i) and (ii) .
  • modified oligonucleotides that can be incorporated into the probe include those containing phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • Patent 5,034,506 or a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al .
  • PNA peptide nucleic acid
  • modified oligonucleotides containing one or more substituted sugar moieties i.e., sugar moieties comprising one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 2 OCH 3 , OCH 2 0 (CH 2 ) n CH 3 , 0(CH 2 ) n NH 2 or 0(CH 2 ) n CH 3 where n is from 1 to about 10; C 1 to C 10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl ; Cl ; Br; CN; CF 3 ; 0CF 3 ; 0-, S-, or N- alkyl; O- , S-, or N-alkenyl; S0CH 3 ; S0 2 CH 3 ; 0N0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl ; hetero
  • oligonucleotides having sugar mimetics such as cyclobutyls in place of the pentofuranosyl group or base modifications or substitutions (e.g., with a "universal" base such as inosine) can be used in this embodiment.
  • the target oligonucleotide may also comprise one or more the above modifications.
  • a further modification of the target oligonucleotide involves chemically linking to the target oligonucleotide one or more lipophilic moieties which enhance the cellular uptake of the oligonucleotide.
  • lipophilic moieties include but are not limited to a cholesteryl moiety (Letsinger et al . , Proc . Natl . Acad. Sci . USA, 1989, 86, 6553), cholic acid (Manoharan et al . , Bioorg. Med. Chem . Let .
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al . , Ann . N. Y. ⁇ Acad . Sci . , 1992, 660 , 306; Manoharan et al . , Bioorg. Med . Chem . Let . , 1993, 3, 2765), a thiocholesterol (Oberhauser et al . , Nucl . Acids Res .
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di- O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al . , Tetrahedron Lett . , 1995, 36, 3651; Shea et al . , Nucl . Acids Res . , 1990, 18 , 3777), a polyamine or a polyethylene glycol chain (Manoharan et al .
  • the target oligonucleotide, an oligonucleotide present in the probe, or both, can also be oligonucleotides which are chimeric oligonucleotides including "gapmers" and "hemimers . " Chimeric oligonucleotides are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one terminal region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the intracellular target nucleic acid.
  • a single terminal (either 5' or 3') region is so modified in the oligonucleotide structure.
  • the oligonucleotide is called a "gapmer” and the modified 5'- and 3' -terminal regions are referred to as "wings"; an additional, typically central, region (typically referred to as the "gap” or “core”) of the oligonucleotide may serve as a substrate for cellular enzymes capable of cleaving RNA: DNA or RNA:RNA hybrids.
  • the 5' and 3' wings can be modified in the same or different manner depending on what properties it is desired to achieve.
  • a preferred modification in many chimeric oligonucleotides designed for antisense purposes is the inclusion of one or more residues modified at the 2' position.
  • An example is the 2'-0-methyl modification, which, when incorporated into synthetic oligonucleotides, results in increased duplex stability (nearly 0.8 kcal/mol per modification) between such oigonucleotides and RNA targets.
  • the 2'-0-methyl modification slightly destabilizes the duplex formed between the oligonucleotide and a DNA target (Freier, Chapter 5 In : Antisense Research and Applications, Crooke et al .
  • RNA molecules Modifications that enhance the affinity for, and/or duplex stability with, RNA molecules are preferred for applications wherein such oligonucleotides are intended for antisense purposes, wherein the oligonucleotide is designed to selectively bind to, and modulate the expression of, a particular RNA species.
  • affinity units comprising nucleobase sequences complementary to such modified portions of target oligonucleotides are designed to reflect the chemical modification (s) present in the target oligonucleotide.
  • RNA-like nucleotide it is meant that such an oligonucleotide, or such portion thereof, has an ability to form a stable duplex with an RNA target that exceeds its ability to form a stable duplex with a DNA target under comparable conditions.
  • RLO refers to "RNA- like oligonucleotide" herein.
  • RNA: RNA, RNA: RLO and RLO : RLO duplexes differ from DNA: DNA and DNA: RNA duplexes in several significant conformational respects (Lesnik et al . , Biochemistry, 1993, 32, 7832; Lesnik et al . , Biochemistry, 1995, 34 , 10807), and that these conformational differences contribute to the relative stabilities of structurally distinct duplex partners.
  • target antisense oligonucleotides comprising an RNA or RNA-like hybridizing portion are preferentially purified using a matrix having an affinity unit comprising a hybridizing portion with one or more the following modifications, which enhance the stability of duplexes formed with such modified affinity units and RNA or RLO molecules, and/or enhance the affinity of such modified affinity units for RNA or RLO molecules, and are thus preferred.
  • target ASO's comprising one or more of the following modifications in their hybridizing portion are preferably purified using affinity units having an RNA or RNA-like structure in their corresponding hybridizing portions.
  • preferred modifications include but are not limited to sugar modifications, backbone modifications, nucleobase modifications or combinations thereof .
  • Patent No. 5,610,289 formacetal/ketal type linkages, such as, for example, those disclosed in U.S. Patent No. 5,264,562; and backbones incorporating HNA (1, 5-anhydrohexitol) (Herdewijn, Liebigs Ann . , 1996, 1337; published PCT patent application WO 96/05213 ) .
  • a combination of 2 ' -fluoro-5-propynyl deoxyuridine is especially preferred.
  • Preferred purine modifications include 7- modified-7-deaza purines and 2-amino-adenosine . Certain combinations of the above modifications are also preferred in this embodiment of the invention.
  • nucleobases include, but are not limited to, 2'- fluoro-propynyl uridine, substituted for uridine or thymine, and 2'-0-methyl, 2-amino-adenosine in substitution for adenosine.
  • oligonucleotide-based affinity units particularly preferred are 2'-0-methyl MMI backbones, 2'-0- methyl amide 3 backbones, and 2' -fluoro, N3'->P5' phosphoramidite oligonucleotides.
  • oligonucleotides comprising certain of these modifications (e.g., amide 3 and amide 4) have the tendency to organize themselves, prior to hybridization, into conformations more favorable for duplex formation.
  • certain of these modifications favor the C3 ' endo pucker conformation which, it is believed, RNA:RNA and RNA: RLO duplexes adopt (Lesnik et al . , Biochemistry, 1993, 32 , 7832; Kawasaki et al . , J. Med. Chem. , 1993, 36, 831; Griffey et al .
  • modification (s) to be incorporated into the affinity unit is thus influenced by the chemical nature of the target oligonucleotide. Furthermore, modifications can be strategically placed within the affinity unit in order to maximize the separation of undesired oligonucleotides that are particularly refractory to separation from the desired full length oligonucleotide.
  • the hybridization structure of Example 4 is modified in the following manner: SEQ ID N0 : 1
  • SEQ ID NO: 2 where the underlined nucleotides in the affinity unit (SEQ ID NO: 2) are ribonucleotides rather than deoxyribonucleotides, and the target oligonucleotide (SEQ ID NO:l) comprises terminal 2 ' -O-methoxyethoxy modifications, as indicated by the asterisks.
  • the above hybridization structure has a significantly greater stability when bound to the desired target chimeric oligonucleotide than when bound to undesired (n-1) derivatives lacking a single terminal nucleotide, i.e.,
  • the desired hybridization structure is expected to have an increased stability of about 0.8 kcal/mol when compared with either of the duplexes which incorporate an undesired terminal (n-1) oligonucleotide.
  • the nucleobase sequence of the affinity unit may be "full-length", i.e., of the same length, n, as the target oligonucleotide
  • affinity units having "essentially full-length" nucleobase sequences may also be used.
  • the affinity unit has a nucleobase sequence that is of a length, p, that is less than the length, n, of the target oligonucleotide.
  • the nucleobase sequence of the affinity unit is nonetheless the reverse complement of the target oligonucleotide over length p.
  • p is a number ranging from n-4 to n, wherein, in a duplex between the target oligonucleotide and the nucleobase sequence of the affinity unit, neither the 5' overhang nor the 3' overhang of said target oligonucleotide is greater than two nucleotides .
  • SEQ ID NOS: 8 and 9 SEQ ID NO:l
  • oligonucleotides in the flow- through during the extraction step (b) or elution step (c) can be monitored by a variety of methods known in the art. See, for example, Jarrett, J. Chromatogr. , 1993, 618, 315.
  • the mismatched oligonucleotides potentially bound and/or retained from the matrix comprising the first affinity unit are removed:
  • the affinity units are complementary to hybridizing portions of the target oligonucleotide located at 5' and 3' termini
  • hybridizing portions of the target oligonucleotide should typically be distinct from one another so that different undesirable contaminating derivatives of the target oligonucleotide are removed by each matrix.
  • the first and second hybridizing portions can each be located at a 5 ' terminus, a 3' terminus, or within a central portion of the target oligonucleotide .
  • the temporal ordering of the two purification steps is not typically important; in the above scheme, for example, the "Second Affinity Unit” might just as well be used before the "First Affinity Unit” in most instances.
  • Temporal ordering of the two purification steps may be a consideration, however, when the matrices comprising the two affinity units are of different chemical composition and/or depending on the means used to dissociate and recover target oligonucleotide from the first affinity unit to which it is bound. For example, consider an instance wherein the eluent from affinity unit "A" is prepared in such a way so as to
  • affinity unit "B” include one or more components (e.g., salts, solvents, ions, etc.) that would chemically disrupt the matrix into which affinity unit "B” is incorporated.
  • components e.g., salts, solvents, ions, etc.
  • the number of affinity units and stepwise purifications need not be limited to two and that, in some instances, several such stepwise purifications, and appropriate affinity units and matrices, may be needed.
  • the two affinity units can be of the same or different chemical composition, depending on the chemical nature of the target oligonucleotide.
  • the affinity unit for the RNA- like 3 ' hybridizing portion would preferably incorporate one or more of the modifications detailed in Example 6, whereas no such preference would exist for an affinity unit designed to hybridize to the DNA-like 5' portion of such an oligonucleotide.
  • VEGF Vascular Endothelial Growth factor
  • VEGF Vascular Endothelial Growth factor
  • Vm Vascular Endothelial Growth factor
  • VEGF Vascular Endothelial Growth factor
  • AUTHORS Robinson, G.S., et al .
  • TITLE Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy (SEQ ID NO: M3 )
  • VEGF Vascular Endothelial Growth factor
  • VEGF Vascular Endothelial Growth factor
  • VEGF Vascular Endothelial Growth factor
  • VEGF Vascular Endothelial Growth factor
  • VEGF Endothelial Growth factor
  • VEGF Endothelial Growth factor

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Manufacturing & Machinery (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne des procédés et des matrices de purification d'un oligonucléotide cible voulu au moyen d'une unité d'affinité immobilisée se fixant sélectivement à l'oligonucléotide cible. L'unité d'affinité immobilisée comprend de préférence une séquence de nucléobase, laquelle comprend le complément inverse de l'oligonucléotide cible. Les procédés préférés de l'invention permettent d'obtenir un séparation rapide, peu coûteuse et efficace de la plupart des contaminants indésirables, notamment des dérivés [par exemple, (n-1), (n-2), etc.] de délétion indésirable de l'oligonucléotide de longueur totale voulue n résultant de l'oligomérisation incomplète ou de processus de dégradation, notamment les dérivés indésirables présentant des délétions internes ou en 3' d'un ou de plusieurs nucléotides.
PCT/US1997/023284 1996-12-19 1997-12-18 Purification a grande echelle d'oligonucleotides de longueur totale par extraction par affinite solide-liquide WO1998027425A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU62371/98A AU6237198A (en) 1996-12-19 1997-12-18 Large-scale purification of full length oligonucleotides by solid-liquid affinity extraction

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US76995196A 1996-12-19 1996-12-19
US08/769,951 1996-12-19

Publications (1)

Publication Number Publication Date
WO1998027425A1 true WO1998027425A1 (fr) 1998-06-25

Family

ID=25087017

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/023284 WO1998027425A1 (fr) 1996-12-19 1997-12-18 Purification a grande echelle d'oligonucleotides de longueur totale par extraction par affinite solide-liquide

Country Status (2)

Country Link
AU (1) AU6237198A (fr)
WO (1) WO1998027425A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19850186A1 (de) * 1998-10-30 2000-05-25 Roche Diagnostics Gmbh Neue Primer und Sonden zum Nachweis von HIV
EP2314691A3 (fr) * 2002-11-14 2012-01-18 Dharmacon, Inc. SIRNA fonctionnel et hyperfonctionnel
US8546349B2 (en) 2010-07-28 2013-10-01 Thermo Fisher Scientific Biosciences Inc. siRNA targeting VEGFA and methods for treatment in vivo
US8575329B2 (en) 2002-11-14 2013-11-05 Thermo Fisher Scientific Biosciences Inc. siRNA targeting kinase insert domain receptor (KDR)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J. CHROMATOG., 1990, Vol. 508, GOSS et al., "High Performance Affinity Chromatography of DNA", pages 279-287. *
J. CHROMATOG., 1992, Vol. 603, YASHIMA et al., "High-Performance Affinity Chromatography of Oligonucleotides on Nucleic Acid Analogue Immobilized Silica Gel Columns", pages 111-119. *
NATURE, 13 November 1986, Vol. 324, SAIKI et al., "Analysis of Enzymatically Amplified B-Globin and HLA-DQ DNA with Allele-Specific Oligonucleotide Probes", pages 163-166. *
NUCLEIC ACIDS RES., 1995, Vol. 23, No. 11, TEMSAMANI et al., "Sequence Identity of the N-1 Product of a Synthetic Oligonucleotide", pages 1841-1844. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19850186A1 (de) * 1998-10-30 2000-05-25 Roche Diagnostics Gmbh Neue Primer und Sonden zum Nachweis von HIV
EP2314691A3 (fr) * 2002-11-14 2012-01-18 Dharmacon, Inc. SIRNA fonctionnel et hyperfonctionnel
US8575329B2 (en) 2002-11-14 2013-11-05 Thermo Fisher Scientific Biosciences Inc. siRNA targeting kinase insert domain receptor (KDR)
US8546349B2 (en) 2010-07-28 2013-10-01 Thermo Fisher Scientific Biosciences Inc. siRNA targeting VEGFA and methods for treatment in vivo
US9150862B2 (en) 2010-07-28 2015-10-06 Thermo Fisher Scientific Inc. siRNA targeting VEGFA and methods for treatment in vivo

Also Published As

Publication number Publication date
AU6237198A (en) 1998-07-15

Similar Documents

Publication Publication Date Title
US6140491A (en) Nucleozymes
Giannaris et al. Oligoribonucleotides containing 2′, 5′-phosphodiester linkages exhibit binding selectivity for 3′, 5′-RNA over 3′, 5′-ssDNA
JP3482209B2 (ja) オリゴヌクレオチドｎ３’→ｐ５’ホスホルアミデート：合成および化合物；ハイブリダイゼーションおよびヌクレアーゼ耐性特性
EP1470144B1 (fr) Oligonucleotides comportant des segments alternatifs et utilisations associees
AU750469B2 (en) 2'-O-aminoethyloxyethyl-modified oligonucleotides
AU2003202376A1 (en) Oligonucleotides comprising alternating segments and uses thereof
CA2593583A1 (fr) Deprotection et purification d'oligonucleotides et leurs derives
US20220049240A1 (en) Purification methods for guanine-rich oligonucleotides
EP4065711A1 (fr) Procédés de synthèse de molécules d'arn
US20170275610A1 (en) Process for separation of oligonucleotide of interest from a mixture
CA2176259A1 (fr) Composes oligonucleosidiques chimeriques
WO1998027425A1 (fr) Purification a grande echelle d'oligonucleotides de longueur totale par extraction par affinite solide-liquide
Engels et al. Chemistry of oligonucleotides
WO1999011820A1 (fr) Compositions et procedes d'identification et de quantification d'oligonucleotides a sequence de deletion dans des preparations d'oligonucleotides de synthese
JP2003507037A (ja) プロパンジオールを含有するキメラｄｎａ／ｒｎａリボザイム
TW202323526A (zh) 分離富含鳥嘌呤的寡核苷酸的分子物質之方法
Noe et al. Chemistry of antisense oligonucleotides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

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

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
CFP Corrected version of a pamphlet front page

Free format text: ADD INID NUMBER (63) "RELATED BY CONTINUATION (CON) OR CONTINUATION-IN-PART (CIP) TO EARLIER APPLICATION" WHICH WAS INADVERTENTLY OMITTED FROM THE FRONT PAGE

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

122 Ep: pct application non-entry in european phase