Europaisches Patentamt
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`European Patent Office
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`Office europeen des brevets
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`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`29.08.2001 Bulletin 2001/35
`
`(21) Application number: 93902851.0
`
`(22) Date of filing: 23.12.1992
`
`(51) Int CI.7: CO7H 21/04
`
`(86) International application number:
`PCT/US92/11339
`
`(87) International publication number:
`WO 93/13121 (08.07.1993 Gazette 1993/16)
`
`(54) ANTISENSE OLIGONUCLEOTIDES
`
`ANTISENSE OLIGONUKLEOTIDE
`
`OLIGONUCLEOTIDES ANTISENSE
`
`(84) Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL
`PT SE
`
`(30) Priority: 24.12.1991 US 814961
`
`(43) Date of publication of application:
`12.10.1994 Bulletin 1994/41
`
`(60) Divisional application:
`00202252.3 / 1 044 987
`
`(73) Proprietor: ISIS PHARMACEUTICALS, INC.
`Carlsbad, CA 92008 (US)
`
`(72) Inventors:
`• COOK, Phillip, Dan
`Carlsbad, CA 92009 (US)
`• MONIA, Brett P.
`California 92008 (US)
`
`(74) Representative: Hallybone, Huw George
`CARPMAELS AND RANSFORD
`43 Bloomsbury Square
`London WC1A 2RA (GB)
`
`(56) References cited:
`WO-A-86/05518
`WO-A-91/12323
`WO-A-92/20703
`
`WO-A-86/05519
`WO-A-92/20702
`WO-A-93/12129
`
`• Annual Reports in Medicinal Chemistry, 23,
`Chapter 30, copyright 1988, MILLER et al.,
`"Oligonucleotide Inhibitors of Gene Expression
`in Living Cells: New Opportunities in Drug
`Design", pages 295-304, see the entire
`document.
`
`• Proceedings of the National Academy of
`Science, USA, Vol. 85, issued October 1988,
`AGRAWAL et al., "Oligodeoxynucleoside
`Phosphoramidates and Phosphorothioates as
`inhibitors of Human Immunodeficiency Virus",
`pages 7079-7083, see the Abstract.
`• The EMBO Journal, Vol. 10, No. 5, issued 1991,
`SAISON-BEHMOARAS et al., "Short modified
`anti-sense oligonucleotides directed against
`Ha-ras point mutation induce selective cleavage
`of the mRNA and inhibit T24 cells proliferation",
`pages 1111-1118, see the entire document.
`• Nucleic Acids Research, Vol. 19, No. 8, issued
`1991, DAGLE et al.
`• Tetrahedron Letters, Vol. 31, No. 6, issued 1990,
`PETERSEN et al., "Chemical Synthesis of Dimer
`Ribonucleotides Containing Internucleotidic
`Phosphoradithioate Linkages", pages 911-914,
`see pages 911-912.
`• Antisense Research and Development, No. 1,
`issued 1991, DAGLE et al., "Pathways of
`Degradation and Mechanism of Action of
`Antisense Oligonucleotides in Xenopus laevis
`Embryos", pages 11-20, see the Abstract.
`• Anti-Cancer Drug Design, No. 2, issued 1987,
`MILLER et al.: "A new approach to
`chemotherapy based on molecular biology and
`nucleic acid chemistry: Matagen (masking tape
`for gene expression)", pages 117-128, see the
`summary.
`• COHEN, "Oligodeoxynucleotides", published
`1989, by CRC Press, Inc, Boca Raton, (F1), pages
`1-255, note pages 16, 35, 36, 38, 55, 62, 66, 67,
`79-82, 85.
`
`Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
`notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
`a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
`99(1) European Patent Convention).
`
`Printed by Jouve, 75001 PARIS (FR)
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`(Cont. next page)
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`• The Journal of the American Chemical Society,
`Vol. 113, issued 1991, BRILL et al., "Synthesis of
`Deoxydinucleotide Phosphorodithioates",
`pages 3972-3980, see pages 3972-3973.
`• KAWASAKI et al., disclosed January 1991,
`"Synthesis and Biophysical Studies of
`2'-dR1B0-2'-F Modified Oligonucleotides",
`pages 1-9, see entire document.
`
`• Nucleic Acid Research, Vol. 18, No. 16, issued
`1990, DAGLE et al., "Targeted degradation of
`mRNA in Xenopus oocytes and embryos
`directed by modified oligonucleotides: Stufies
`of An2 and Cyclin in Embryogenesis", pages
`4751-4757, see 4751-4752.
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`Description
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`[0001] This invention is directed to the synthesis and use of oligonucleotides to elicit RNase H for strand cleavage
`in an opposing strand. Included in the invention are oligonucleotides wherein at least some of the nucleotides of the
`oligonucleotides are functionalized to be nuclease resistant, at least some of the nucleotides of the oligonucleotide
`include a substituent that potentiates hybridization of the oligonucleotide to a complementary strand, and at least some
`of the nucleotides of the oligonucleotide include 2'-deoxy-erythro-pentofuranosyl sugar moieties. The oligonucleotides
`are useful for therapeutics, diagnostics and as research reagents.
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`BACKGROUND OF THE INVENTION
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`[0002] It is well known that most of the bodily states in mammals including most disease states, are effected by
`proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to
`many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins
`in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been
`made to moderate the actual production of such proteins by interactions with messenger RNA (mRNA) or other intra-
`cellular RNA's that direct protein synthesis. It is generally the object of such therapeutic approaches to interfere with
`or otherwise modulate gene expression leading to undesired protein formation.
`[0003] Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-
`stranded RNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are
`disrupted. Hybridization is the sequence specific hydrogen bonding via Watson-Crick base pairs of the heterocyclic
`bases of oligonucleotides to RNA or DNA. Such base pairs are said to be complementary to one another.
`[0004] Naturally occurring events that provide for the disruption of the nucleic acid function, as discussed by Cohen
`in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fl (1989) are thought to
`be of two types. The first is hybridization arrest. This denotes the terminating event in which an oligonucleotide inhibitor
`binds to target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often
`ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (see, e.g., Miller, et al., Anti-Cancer Drug Design
`1987, 2, 117) and a-anomer oligonucleotides are the two most extensively studied antisense agents that are thought
`to disrupt nucleic acid function by hybridization arrest.
`[0005]
`In determining the extent of hybridization arrest of an oligonucleotide, the relative ability of an oligonucleotide
`to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hy-
`bridization complex. The melting temperature (Tm), a characteristic physical property of double helixes, denotes the
`temperature in degrees centigrade at which 50% helical (hybridized) versus coil (un hybridized) forms are present. Tm
`is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base
`stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Conse-
`quently a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding
`of the strands. Non-Watson-Crick base pairing, i.e. base mismatch, has a strong destabilizing effect on the Tm.
`[0006] The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the
`targeted RNA by intracellular RNase H. The mechanism of such RNase H cleavages requires that a 2'-deoxyribofuran-
`osyl oligonucleotide hybridize to a targeted RNA. The resulting DNA-RNA duplex activates the RNase H enzyme; the
`activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the RNA.
`Phosphorothioate oligonucleotides are one prominent example of antisense agents that operate by this type of termi-
`nating event. For a DNA oligonucleotide to be useful for activation of RNase H, the oligonucleotide must be reasonably
`stable to nucleases in order to survive in a cell for a time sufficient for the RNase H activation.
`[0007] Several recent publications of Walder, et al. further describe the interaction of RNase H and oligonucleotides.
`Of particular interest are: (1) Dagle, et al., Nucleic Acids Research 1990, 18, 4751; (2) Dagle, et al., Antisense Research
`And Development 1991, 1, 11; (3) Eder, et al., J. Biol. Chem. 1991, 266, 6472; and (4) Dagle, et al., Nucleic Acids
`Research 1991, 19, 1805. In these papers, Walder, et al. note that DNA oligonucleotides having both unmodified
`phosphodiester internucleoside linkages and modified, phosphorothioate internucleoside linkages are substrates for
`cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by the RNase H. However, the
`authors further note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also
`subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonu-
`cleotide available for RNase H activation.
`[0008] As described in references (1); (2), and (4), to stabilize their oligonucleotides against nuclease degradation
`while still providing for RNase H activation, Walder, et a/. constructed 2'-deoxy oligonucleotides having a short section
`of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phos-
`photriester linkages. While the phosphoamidate-containing oligonucleotides were stabilized against exonucleases, in
`reference (4) the authors noted that each phosphoramidate linkage resulted in a loss of 1.6°C in the measured Tm
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`value of the phosphoramidate containing oligonucleotides. Such decrease in the', value is indicative of an undesirable
`decrease in the hybridization between the oligonucleotide and its target strand.
`[0009] Kawasaki, et al. disclosed phosphorothioate oligonucleotides with 2'-deoxy-2'-fluoro nucleosides at the Con-
`ference on Nucleic Acid Therapeutics, Clearwater, FL in January, 1991. These oligonucleotides were shown to form
`thermodynamically stable duplexes when hybridised with RNA and to possess stability to nucleases present in heat-
`inactivated fetal calf serum, relative to unmodified oligonucleotides.
`[0010]
`International patent application W091/12323 provides oligonucleotides which activate RNase H. This is
`achieved by selective modification of the internucleoside linkages while incorporating regions of consecutive nucle-
`otides having unmodified internal linkages.
`[0011] Other authors have commented on the effect such a loss of hybridization between an antisense oligonucleotide
`and its targeted strand can have. Saison-Behmoaras, et al., EMBO Joumall 991, 10, 1111, observed that even through
`an oligonucleotide could be a substrate for RNase H, cleavage efficiency by RNase H was low because of weak hy-
`bridization to the mRNA. The authors also noted that the inclusion of an acridine substitution at the 3' end of the
`oligonucleotide protected the oligonucleotide from exonucleases.
`[0012] While it has been recognized that cleavage of a target RNA strand using an antisense oligonucleotide and
`RNase H would be useful, nuclease resistance of the oligonucleotide and fidelity of the hybridization are also of great
`importance. Heretofore, there have been no suggestion in the art of methods or materials that could both activate
`RNase H while concurrently maintaining or improving hybridization properties and providing nuclease resistance even
`though there has been a long felt need for such methods and materials. Accordingly, there remains a long-felt need
`for such methods and materials.
`
`OBJECTS OF THE INVENTION
`
`[0013]
`It is an object of this invention to provide oligonucleotides that both activate RNase H upon hybridization with
`a target strand and resist nuclease degradation.
`[0014]
`It is a further object to provide oligonucleotides that activate RNase H, inhibit nuclease degradation, and
`provide improved binding affinity between the oligonucleotide and the target strand.
`[0015] A still further object is to provide research and diagnostic methods and materials for assaying bodily states
`in animals, especially diseased states.
`[0016] Another object is to provide therapeutic and research methods and materials for the treatment of diseases
`through modulation of the activity of DNA and RNA.
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`[0017]
`In accordance with one embodiment of this invention there are provided oligonucleotides formed from a se-
`quence of nucleotide units. The oligonucleotides incorporate a least one nucleotide unitthat is functionalized to increase
`nuclease resistance of the oligonucleotides. Further, at least some of the nucleotide units of the oligonucleotides are
`functionalized with a substituent group to increase binding affinity of the oligonucleotides to target RNAs, and at least
`some of the nucleotide units have 2'-deoxy-erythro-pentofuranosyl sugar moieties.
`[0018]
`In preferred oligonucleotides of the invention, nucleotide units that are functionalized for increased binding
`affinity are functionalized to include a 2'-substituent group. In even more preferred embodiments, the 2'-substituent
`group is fluoro, C1-C9 alkoxy, C1-C9 aminoalkoxy including aminopropoxy, allyloxy, C1-C9-alkyl-imidazole and poly-
`ethylene glycol. Preferred alkoxy substituents include methoxy, ethoxy and propoxy. A preferred aminoalkoxy unit is
`aminopropoxy. A preferred alkyl-imidazole is 1-propy1-3-(imidazoy1).
`[0019]
`In certain preferred oligonucleotides of the invention having increased nuclease resistance, each nucleotide
`unit of the oligonucleotides is a phosphorothioate or phosphorodithioate nucleotide. In other preferred oligonucleotides,
`the 3' terminal nucleotide unit is functionalized with either or both of a 2' or a 3' substituent.
`[0020] The oligonucleotides include a plurality of nucleotide units bearing substituent groups that increase binding
`affinity of the oligonucleotide to a complementary strand of nucleic acid. In certain preferred embodiments, the nucle-
`otide units that bear such substituents can be divided into a first nucleotide unit sub-sequence and a second nucleotide
`unit sub-sequence, with 2'-deoxy-erythro-pentofuranosyl structures being positioned within the oligonucleotide be-
`tween the first nucleotide unit sub-sequence and the second nucleotide unit sub-sequence. It is preferred that all such
`intervening nucleotide units be 2'-deoxy-erythro-pentofuranosyl units.
`[0021]
`In further preferred oligonucleotides of the invention, nucleotide units bearing substituents that increase bind-
`ing affinity are located at one or both of the 3' or the 5' termini of the oligonucleotide. There can be from one to about
`eight nucleotide units that are substituted with substituent groups. Preferably, at least five sequential nucleotide units
`are 2'-deoxy-erythro-pentofuranosyl sugar moieties.
`[0022] Antisense methodology can be used for treating an organism having a disease characterized by the undesired
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`production of an protein. These methods include contacting the organism with an oligonucleotide having a sequence
`of nucleotides capable of specifically hybridizing to a complementary strand of nucleic acid where at least one of the
`nucleotides is functionalized to increase nuclease resistance of the oligonucleotide to nucleases, where a substituent
`group located thereon to increase binding affinity of the oligonucleotide to the complementary strand of nucleic acid
`and where a plurality of the nucleotides have 2'-deoxyerythroregions;-pentofuranosyl sugar moieties.
`[0023] Further in accordance with this invention there are provided compositions including a pharmaceutically effec-
`tive amount of an oligonucleotide having a sequence of nucleotides capable of specifically hybridizing to a comple-
`mentary strand of nucleic acid and where at least one of the nucleotides is functionalized to increase nuclease resist-
`ance of the oligonucleotide to nucleases and where a plurality of the nucleotides have a substituent group located
`thereon to increase binding affinity of the oligonucleotide to the complementary strand of nucleic acid and where a
`plurality of the nucleotides have 2'-deoxy-erythro-pentofuranosyl sugar moieties. The composition further include a
`pharmaceutically acceptable diluent or carrier.
`[0024] Further in accordance with this invention there are provided methods for in vitro modification of a sequence
`specific nucleic acid including contacting a test solution containing an RNase H enzyme and said nucleic acid with an
`oligonucleotide having a sequence of nucleotides capable of specifically hybridizing to a complementary strand of
`nucleic acid and where at least one of the nucleotides is functionalized to increase nuclease resistance of the oligo-
`nucleotide to nucleases and where a plurality of the nucleotides have a substituent group located thereon to increase
`binding affinity of the oligonucleotideto the complementary strand of nucleic acid and where a plurality of the nucleotides
`have 2'-deoxy-erythro-pentofuranosyl sugar moieties.
`[0025] There are also provided methods of concurrently enhancing hybridization and RNase H enzyme activation in
`an organism that includes contacting the organism with an oligonucleotide having a sequence of nucleotides capable
`of specifically hybridizing to a complementary strand of nucleic acid and where at least one of the nucleotides is func-
`tionalized to increase nuclease resistance of the oligonucleotide to nucleases and where a plurality of the nucleotides
`have a substituent group located thereon to increase binding affinity of the oligonucleotide to the complementary strand
`of nucleic acid and where a plurality of the nucleotides have 2'-deoxy-erythro-pentofuranosyl sugar moieties.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0026] This invention will be better understood when taken in conjunction with the drawings wherein:
`
`Figure 1 is a graph showing dose response activity of oligonucleotides of the invention and a reference compound;
`and
`Figure 2 is a bar chart showing dose response activity of oligonucleotides of the invention and reference com-
`pounds.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0027]
`In accordance with the objects of this invention, novel oligonucleotides that, at once, have increased nuclease
`resistance, increased binding affinity to complementary strands and that are substrates for RNase H are provided. The
`oligonucleotides of the invention are assembled from a plurality of nucleotide, nucleoside or nucleobase sub-units.
`Each oligonucleotide of the invention includes at least one nucleotide, nucleoside or nucleobase unit that is function-
`alized to increase the nuclease resistances of the oligonucleotide. Further, at least some of the nucleotide or nucleoside
`units bear a substituent group that increases the binding affinity of the oligonucleotide to a complementary strand of
`nucleic acid. Additionally at least some of the nucleotide units comprise a 2'-deoxy-erythro-pentofuranosyl group as
`their sugar moiety and which are consecutively located in said sequence.
`[0028]
`In conjunction with the above guidelines, each nucleotide unit of an oligonucleotides of the invention, alter-
`natively referred to as a subunit, can be a "natural" or a "synthetic" moiety. Thus, in the context of this invention, the
`term "oligonucleotide" in a first instance refers to a polynucleotide formed from a plurality of joined nucleotide units.
`The nucleotides units are joined together via native internucleoside, phosphodiester linkages. The nucleotide units are
`formed from naturally-occurring bases and pentofuranosyl sugars groups. The term "oligonucleotide" thus effectively
`includes naturally occurring species or synthetic species formed from naturally occurring nucleotide units.
`[0029] Oligonucleotides of the invention also can include modified subunits. The modifications can occur on the base
`portion of a nucleotide, on the sugar portion of a nucleotide or on the linkage joining one nucleotide to the next. In
`addition, nucleoside units can be joined via connecting groups that substitute for the inter-nucleoside phosphate link-
`ages. Macromolecules of the type have been identified as oligonucleosides. In such oligonucleosides the linkages )
`include an -O-CH2-CH2-O- linkage (i.e., an ethylene glycol linkage) as well as other novel linkages disclosed in the
`following United States patent applications: Serial Number 566,836, filed August 13, 1990, entitled Novel Nucleoside
`Analogs; Serial Number 703,619, filed May 21, 1991, entitled Backbone Modified Oligonucleotide Analogs; and Serial
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`Number 903,160, filed June 24, 1992, entitled Heteroatomic Oligonucleotide Linkage. Other modifications can be made
`to the sugar, to the base, or to the phosphate group of the nucleotide. Representative modifications are disclosed in
`the following United States patent applications: Serial Number 463,358, filed January 11, 1990, entitled Compositions
`And Methods For Detecting And Modulating RNA Activity; Serial Number 566,977, filed August 13: 1990, entitled Sugar
`Modified Oligonucleotides That Detect And Modulate Gene Expression; Serial Number 558,663, filed July 27, 1990,
`entitled Novel Polyamine Conjugated Oligonucleotides; Serial Number 558,806, filed July 27, 1991, entitled Nuclease
`Resistant Pyrimidine Modified Oligonucleotides That Detect And Modulate Gene Expression; and Serial Number PCT/
`US91/00243, filed January 11, 1991, entitled Compositions and Methods For Detecting And Modulating RNA Activity,
`all assigned to the assignee of this invention.
`[0030] Thus, the terms oligonucleotide is intended to include naturally occurring structures as well as non-naturally
`occurring or "modified" structures -- including modified sugar moieties, modified base moieties or modified sugar linking
`moieties -- that function similarly to natural bases, natural sugars and natural phosphodiester linkages. Thus, Oligo-
`nucleotides can have altered base moieties, altered sugar moieties or altered inter-sugar linkages. Exemplary among
`these are phosphorothioate, phosphorodithioate, methyl phosphonate, phosphotriester, phosphoramidate, phospho-
`roselenate and phosphorodiselenate inter-nucleoside linkages used in place of phosphodiester inter-nucleoside link-
`ages; deaza or aza purines and pyrimidines used in place of natural purine and pyrimidine bases; pyrimidine bases
`having substituent groups at the 5 or 6 position; purine bases having altered or replacement substituent groups at the
`2, 6 or 8 positions; or sugars having substituent groups at their 2' position, substitutions for one or more of the hydrogen
`atoms of the sugar, or carbocyclic or acyclic sugar analogs. They may also comprise other modifications consistent
`with the spirit of this invention. Such oligonucleotides are best described as being functionally interchangeable with
`natural oligonucleotides (or synthesized oligonucleotides along natural lines), but which have one or more differences
`from natural structure. All such oligonucleotides are comprehended by this invention so long as they function effectively
`to mimic the structure of a desired RNA or DNA strand.
`[0031]
`In one preferred embodiment of this invention, nuclease resistance is achieved by utilizing phosphorothioate
`internucleoside linkages. Contrary to the reports of Walder, et al. note above: I have found that in systems such as
`fetal calf serum containing a variety of a-exonucleases, modification of the internucleoside linkage from a phosphodi-
`ester linkage to a phosphorothioate linkage provides nuclease resistance.
`[0032] Brill, et al., J. Am. Chem. Soc. 1991, 113, 3972, recently reported that phosphorodithioate oligonucleotides
`also exhibit nuclease resistance. These authors also reported that phosphorodithioate oligonucleotide bind with corn-
`plementary deoxyoligonucleotides, stimulate RNase H and stimulate the binding of lac repressor and cro repressor.
`In view of these properties, phosphorodithioates linkages also may be useful to increase nuclease resistance of oligo-
`nucleotides of the invention.
`[0033] Nuclease resistance further can be achieved by locating a group at the 3' terminus of the oligonucleotide
`utilizing the methods of Saison-Behmoraras, et al., supra, wherein a dodecanol group is attached to the 3' terminus of
`the oligonucleotide. Other suitable groups for providing increased nuclease resistance may include steroid molecules
`and other lipids, reporter molecules, conjugates and non-aromatic lipophilic molecules including alicyclic hydrocarbons,
`saturated and unsaturated fatty acids, waxes, terpenes and polyalicyclic hydrocarbons including adamantane and
`buckminsterfullerenes. Particularly useful as steroid molecules for this purpose are the bile acids including cholic acid,
`deoxycholic acid and dehydrocholic acid. Other steroids include cortisone, digoxigenin, testosterone and cholesterol
`and even cationic steroids such as cortisone having a trimethylaminomethyl hydrazide group attached via a double
`bond at the 3 position of the cortisone ring. Particularly useful reporter molecules are biotin and fluorescein dyes. Such
`groups can be attached to the 2' hydroxyl group or 3' hydroxyl group of the 3' terminal nucleotide either directly or
`utilizing an appropriate connector in the manner described in United States Patent Application Serial Number 782,374,
`filed October 24, 1991 entitled Derivatized Oligonucleotides Having Improved Uptake and Other Properties, assigned
`to the assignee as this application, the entire contents of which are herein incorporated by reference.
`[0034] Attachment of functional groups at the 5' terminus of compounds of the invention also may contribute to
`nuclease resistance. Such groups include acridine groups (which also serves as an intercalator) or other groups that
`exhibit either beneficial pharmacokinetic or pharmacodynamic properties. Groups that exhibit pharmacodynamic prop-
`erties, in the context of this invention, include groups that improve oligonucleotide uptake, enhance oligonucleotide
`resistance to degradation, and/or strengthened sequence-specific hybridization with RNA. Groups that exhibit phar-
`macokinetic properties, in the context of this invention: include groups that improve oligonucleotide uptake: distribution,
`metabolism or excretion.
`[0035] Further nuclease resistance is expect to be conferred utilizing linkages such as the above identified -O-CH2-
`CH2-O-linkage and similar linkages of the above identified United State Patent Applications Serial Number 566,836,
`Serial Number 703,619, and Serial Number 903,160, since these types of linkages do not utilize natural phosphate
`ester-containing backbones that are the natural substrates for nucleases. When nuclease resistance is conferred upon
`an oligonucleotide of the invention by the use of a phosphorothioate or other nuclease resistant internucleotide linkages,
`such linkages will reside in each internucleotide sites. In other embodiments, less than all of the internucleotide linkages
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`will be modified to phosphorothioate or other nuclease resistant linkages.
`[0036]
`I have found that binding affinity of oligonucleotides of the invention can be increased by locating substituent
`groups on nucleotide subunits of the oligonucleotides of the invention. Preferred substituent groups are 2' substituent
`groups, (cid:9)
`substituent groups located at the 2' position of the sugar moiety of the nucleotide subunits of the oligonu-
`cleotides of the invention. Presently preferred substituent groups include but are not limited to 2'-fluoro, 2'-alkoxy, 2'-
`aminoalkoxy, 2'-allyloxy, 2'-imidazole-alkoxy and 2'-poly(ethylene oxide). Alkoxy and aminoalkoxy groups generally
`include lower alkyl groups, particularly C1-C9 alkyl. Poly(ethylene glycols) are of the structure (0-CH2-CH2)n-0-alkyl.
`Particularly preferred substituent groups are 2'-fluoro, 2'-methoxy, 2'-ethoxy, 2'-propoxy, 2'-aminopropoxy, 2'-imida-
`zolepropoxy, 2'-imidazolebutoxy, and 2'-allyloxy groups.
`[0037] Binding affinity also can be increased by the use of certain modified bases in the nucleotide units that make
`up the oligonucleotides of the invention. Such modified bases may include 6-azapyrimidines and N-2, N-6 and 0-6
`substituted purines including 2-aminopropyladenine. Other modified pyrimidine and purine base are expected to in-
`crease the binding affinity of oligonucleotides to a complementary strand of nucleic acid.
`[0038] The use of 2'-substituent groups increases the binding affinity of the substituted oligonucleotides of the in-
`vention. In a published study, Kawasaki and Cook, et al., Synthesis and Biophysical Studies of 2'-dRIBO-F Modified
`Oligonucleotides, Conference On Nucleic Acid Therapeutics, Clearwater, FL, January 13, 1991, the inventor has re-
`ported a binding affinity increase of 1.6°C per substituted nucleotide unit of the oligonucleotide. This is compared to
`an unsubstituted oligonucleotide for a 15 mer phosphodiester oligonucleotide having 2'-deoxy-2'-fluoro groups as a
`substituent group on five of the nucleotides of the oligonucleotide. When 11 of the nucleotides of the oligonucleotide
`bore such 2'-deoxy-2'-fluoro substituent groups, the binding affinity increased to 1.8°C per substituted nucleotide unit.
`[0039]
`In that same study, the 15 mer phosphodiester oligonucleotide was derivatized to the corresponding phos-
`phorothioate analog. When the 15 mer phosphodiester oligonucleotide was compared to its phosphorothioate analog,
`the phosphorothioate analog had a binding affinity of only about 66% of that of the 15 mer phosphodiester oligonucle-
`otide. Stated otherwise, binding affinity was lost in derivatizing the oligonucleotide to its phosphorothioate analog.
`However, when 2'-deoxy-2'-fluoro substituents were located at 11 of the nucleotides of the 15 mer phosphorothioate
`oligonucleotide, the binding affinity of the 2'-substituent groups more than overcame the decrease noted by derivatizing
`the 15 mer oligonucleotide to its phosphorothioate analog. In this compound, i.e., a 15 mer phosphorothioate oligonu-
`cleotide having 11 nucleotide substituted with 2'-fluoro groups, the binding affinity was increased to 2.5°C per substit-
`uent group. In this study no attempt was made to include an appropriate consecutive sequence of nucleotides have
`2'-deoxy-erythro-pentofuranosyl sugars that would elicit RNase H enzyme cleavage of a RNA target complementary
`to the oligonucleotide of the study.
`[0040]
`In order to elicit RNase H enzyme cleavage of a target RNA, an oligonucleotide of the invention must include
`a segment or sub-sequence therein that is a DNA type segment. Stated otherwise, at least some of the nucleotide
`subunits of the oligonucleotides of the invention must have 2'-deoxy-erythro-pentofuranosyl sugar moieties. I have
`found that a sub-sequence having more than three consecutive, linked 2'-deoxy-erythro-pentofuranosyl-containing
`nucleotide sub-units likely is necessary in order to elicit RNase H activity upon hybridization of an oligonucleotide of
`the invention with a target RNA. It is presently preferred to have a sub-sequence of 5 or more consecutive 2'-deoxy-
`erythro-pentofuranosyl containing nucleotide subunits in an oligonucleotide of the invention. Use of at least 7 consec-
`utive 2'-deoxy-erythro-pentofuranosyl-containing nucleotide subunits is particularly preferred.
`[0041] The mechanism of action o