CATIONIC OLIGONUCLEOTIDES, AND RELATED METHODS OF SYNTHESIS AND USE

Abstract: Novel oligonucleotides are provided having cationic internucleoside linkages. The cationic internucleoside linkage has structure (I), wherein W, X, Y, Z, R<1>, R<2> and R<3> are as defined herein, P* represents an asymetric phosphorus atom capable of existing in two distinct stereoisomeric configurations, and further wherein the internucleoside linkage is stereouniform. Certain of these oligonucleotides may have alternating anionic and cationic internucleoside linkages, which are not necessarily stereouniform. A method for synthesizing the compounds are provided as well, as are methods for using the compounds, e.g., as antisense molecules and in nucleic acid hybridization assays.

Description: CATIONIC OLIGONUCLEOTIDES. AND RELATED METHODS OF SYNTHESIS AND USE Technical Field This invention relates generally to nucleic acid chemistry. More particularly, the invention relates to oligonucleotides containing cationic intemucleoside linkages. In addition, the invention relates to methods and reagents for preparing such oligonucleotides. The invention has applications in antisense therapeutics and in nucleic acid hybridization assays for diagnostic or clinical monitoring purposes. Background Sequence-specific oligonucleotides containing modified intemucleoside phosphodiester linkages have utility as antisense molecules for therapeutic applications and nucleic acid hybridization probes for diagnostic or therapeutic efficacy-monitoring applications. Successful antisense molecules and nucleic acid hybridization probes must bind specifically to the single-stranded or double-stranded target nucleic acid sequence of interest under physiological conditions. Such molecules and probes must also be effectively taken up by intact cells and must be resistant to nuclease degradation. The phosphodiester backbone has been modified in an attempt to satisfy these criteria. For example, the phosphodiester backbone has been replaced by phosphonate (Miller et al. (1980) J . Biol . Chem . 255:9659-9665) . phosphotriester (Pless et al. (1977) Biochemistry 16:1239- 1250) or phosphorothioate backbones (Stec et al. (1984) J. Am . Chem . Soc . 106:6077-6079) . One approach to oligonucleotide backbone modification has been to remove the negative charge of the intemucleoside phosphodiester ("PDE") linkage to produce neutral backbones such as, for example, methyl phosphonates (Vyazovkina et al. (1994) Nucleic Acids Res . £2:2404-2409), phosphoramidates (Jager et al. (1988) Biochemistry 21_:7237-7246) or peptide nucleic acids (Egholm et al. (1992) J . Am . Chem . Soc . 114:1895-1897) . An alternative approach to modifying the oligonucleotide backbone has been to replace anionic PDE groups with cationic groups. Cationic substituents have been attached to the intemucleoside phosphorus atom via phosphoramidate linkages (Letsinger et al. (1988) J . Am . Chem . Soc . 110:4470-4471) . Cationic groups can also be introduced into oligonucleotides via phosphonate derivatives in which the anionic oxygen is replaced by a cationic group via a phosphorus-carbon bond. Thus, the preparation of oligonucleotides in which the backbone consists of alternating phosphodiester and stereoiso erically pure (2-aminoethyl) -phosphonate linkages, and oligonucleotide containing backbones consisting of (aminomethyl) -phosphonates have been respectively reported in Fathi et al. (1994) Nucleic Acids Res . 2:5416-5424 and Fathi et al. (1994) Bioconjugate Chem . 5 :47-57. Patil et al. (1994) Biorg . Medicinal Chem . Lett . 4 . :2663-2666 reported the synthesis of oligothymidylate analogs containing stereoisomers of phosphorothioates using stereoisomerically pure modified dinucleosides to synthesize decathymidylates with alternating stereoregulated anionic phosphorothioate and anionic PDE linkages. Peyrottes et al. (1994) Nucleosides & Nucleotides JL3 . :2135-2149 prepared oligomers with methoxyphosphoramidate intemucleoside linkages using preformed dimers. Furthermore, when compared with oligo(dT), the oligomer with the methoxyphosphoramidate intemucleoside linkages showed reduced thermal stability when hybridized to poly(dA) or poly(A) . There is a need in the art for oligonucleotides having cationic intemucleoside linkages that have greater affinity target nucleic acids than oligonucleotides having exclusively standard phosphodiester intemucleoside linkages. Such oligonucleotides can serve as antisense therapeutic agents and as probes in nucleic acid hybridization assays. Summary of the Invention Accordingly, it is an object of the invention to provide an oligonucleotide having cationic intemucleoside linkages useful in antisense therapeutics and in nucleic acid hybridization assays for diagnostic or clinical monitoring purposes. It is another aspect of the invention to provide a method of synthesizing oligonucleotides containing cationic intemucleoside linkages. It is yet another object of the invention to provide nucleic acid hybridization assays using oligonucleotide probes containing cationic intemucleoside linkages. In one embodiment of the invention, an oligonucleotide is provided having a cationic intemucleoside linkage having the structure (I) wherein: W is selected from the group consisting of O, S and Se; X and Y are independently selected from the group consisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consisting of H and Ci-Cg alkyl, and NR 6 where R 6 is H or C-^C g alkyl; Z is selected from the group consisting of 0, S, C- L -Cg alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C^C g alkyl, with the proviso that when , X and Y are O, Z is O, S or NR 7 ; R 1 is selected from the group consisting of C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the group consisting of H, ^-C 8 alkyl, C j -C 8 alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or R 2 and R 3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and wherein P* represents an asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage is stereouniform. In another embodiment of the invention, an oligonucleotide is provided having alternating cationic and anionic intemucleoside linkages wherein the cationic inte ucleoside linkages have the structure (II) wherein , X, Y, Z, R 1 , R 2 and R 3 are as defined above, with the proviso that when W, X and Y are O, Z is 0 or S, and wherein P is a phosphorus atom that may or may not be capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage may or may not be stereouniform. In still another embodiment of the invention, an oligonucleotide is provided having at least one cationic intemucleoside linkage having the structure (II) wherein W, X, Y, Z, R 1 , R 2 and R 3 are as defined above, with the proviso that when , X and Y are O, Z is O or S. In a further embodiment of the invention, a method is provided for making an oligonucleotide containing at least one cationic intemucleoside linkage having the structure (I) wherein: is selected from the group consisting of O, S and Se; X and Y are independently selected from the group consisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consisting of H and C-L-Cg alkyl, and NR 6 where R 6 is H or C^C g alkyl; Z is selected from the group consisting of 0, S, C-^Cg alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C 1 -C 6 alkyl, with the proviso that when W, X and Y are O, Z is O, S or NR 7 ; R 1 is selected from the group consisting of C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the group consisting of H, C j -C 8 alkyl, C j^ -C 8 alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or R 2 and R 3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and wherein P* represents an asymmetric phosphorus atom capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage is stereouniform, said method comprising: (a) synthesizing a point racemic mixture of protected cationic nucleotide dimers comprising the cationic intemucleoside linkage and a 3'-0-TBDMS protecting group; (b) optionally resolving the stereoiso ers in the mixture; (c) deprotecting the cationic nucleotide dimer isolated in step (b) ; (d) converting the deprotected cationic nucleotide dimer provided in step (c) into the corresponding 3'-0-CH 2 CH 2 CN phosphoramidite derivative by reaction with Cl-P(N(iPr) 2 ) -O-BCE; and (e) coupling the 3'-0-CH 2 CH 2 CN phosphoramidite derivative to an unprotected hydroxy1- containing terminal unit of an oligonucleotide chain. In yet another embodiment of the invention, a nucleic acid hybridization assay is provided comprising: (a) providing a labeled oligonucleotide probe containing at least one cationic intemucleoside linkage; (b) hybridizing the probe to a single- stranded analyte nucleic acid to produce a labeled duplex; and (c) detecting the labeled duplexes. Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. Detailed Description of the Invention Definitions and nomenclature: Before the present invention is disclosed and described in detail, it is to be understood that this invention is not limited to specific assay formats, materials or reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an oligonucleotide containing "a cationic intemucleoside linkage" includes polynucleotides containing two or more cationic intemucleoside linkages and the like. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: As used herein, the terms "poiynucleotide" and "oligonucleotide" shall be generic to polydeoxyribonu- cleotides (containing 2'-deoxy-D-ribose) , to polyribonu- cleotides (containing D-ribose) , to any other type of poiynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs) ) and polymorpholino (commer¬ cially available from the Anti-Virals, Inc., Corvallis, Oregon, as Neugene™ polymers) , and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the term "poiynucleotide" and "oligonu¬ cleotide," and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single- stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with negatively charged linkages (e.g. , phosphorothioates, phosphorodi- thioates, etc.), those containing 2'-0- internucleotide linkages of 3'-oxy or 3'-deoxy ribose moieties, those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxida¬ tive metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the poiynucleotide or oligonucleotide. It will be appreciated that, as used herein, the terms "nucleoside" and "nucleotide" will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" include those moieties which contain not only conventional ribose and deoxyribose sugars, but also other sugars as well. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like. The designation "3 M as used in a structural representation of an intemucleoside linkage refers to a bond to the 3' carbon of the ribose moiety of the nucleoside situated 5' to the linkage. The designation "5 ' " as used in a structural representation of an intemucleoside linkage refers to a bond to the 5' carbon of the ribose moiety of the nucleoside situated 3' to the linkage. However, as indicated above, the invention is not intended to be limited to oligonucleotides including ribose sugars as part of the backbone. Accordingly, one of ordinary skill in the art would know that the novel oligonucleotides containing cationic intemucleoside linkages as depicted herein need not be limited to traditional 3' and 5' intemucleoside bonds. The term "cationic" as used herein refers to a chemical moiety that carries a positive charge at pH less than about 9, preferably less than about 8. More preferably, when in an aqueous solution near neutrality, i.e., in the range of about pH 4 to pH 8, preferably about pH 7, most preferably about pH 7.3, a cationic moiety will be protonated to carry a positive charge. Thus, a "cationic intemucleoside linkage" is such a linkage that includes a substituent chemical moiety that is positively charged under the above-described conditions. A "cationic oligonucleotide" is used herein to indicate an oligonucleotide having one or more cationic intemucleoside linkages. The term "alkyl" or "lower alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl ("iPr") , n-butyl, isobutyl, t- butyl and the like. The term "alkylene" or "lower alkylene" as used herein refers to a bifunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 6 carbon atoms, and includes, for example, methylene (-CH 2 -) , ethylene (-CH 2 -CH 2 -) , propylene (-CH 2 -CH 2 -CH 2 -) , 2-methylpropylene [-CH 2 - CH(CH 3 )-CH 2 -] , hexylene [-(CH 2 ) 6 -] and the like. The term "alkenylene" or "lower alkenylene" as used herein refers to a bifunctional branched or unbranched hydrocarbon chain containing 2 to 6 carbon atoms and at least one double bond. The term "alkynylene" or "lower alkynylene" as used herein refers to a bifunctional branched or unbranched hydrocarbon chain containing 2 to 6 carbon atoms and at least one triple bond. The term "aryl" as used herein refers to an aromatic species containing 1 to 5 aromatic rings, either unsubstituted or substituted with 1 or more substituents typically selected from the group consisting of halogen and C-^C g alkyl. The term "arylene" refers to a difunctional aromatic moiety; "monocyclic arylene" refers to a phenylene group. These groups may be substituted with up to four ring substituents as outlined above. The term "heterocycle" is used in its conventional meaning to include subεtituted or unsubstituted aromatic and nonaromatic cyclic molecules containing heteroatoms such as O, N, S, P and halogens. Examples of such heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, morpholine, quinoline, indole, pyrimidine, piperazine, pipecoline, imidazole, benzimidazole, purine and the like. These groups may also be substituted as outlined above. By "purified" or "homogeneous" is meant, when referring to a polypeptide or nucleotide sequence that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type or stereoisomeric configuration. The term "purified" or "homogeneous" as used herein preferably means at least about 90% by weight, more preferably at least about 95% by weight, and most preferably at least about 98% by weight, of biological macromolecules of the same type present. The term "stereoisomer" is used in its conventional sense to refer to a chemical compound having at least one asymmetric atom such that the compound can exist in two or more forms that have the same number and kind of atoms and the same atomic arrangement, but differ in their spatial relationship. In the case of an asymmetric intemucleoside phosphorus atom, there are two possible stereoisomeric configurations. By the term "stereoisomerically pure" or "stereochemically pure" is meant that one stereoisomer is present in the substantial absence of other stereoisomer. When referring to a molecule containing at least one asymmetric intemucleoside phosphorus atom, the terms mean that one stereoisomeric configuration of the asymmetric intemucleoside phosphorus atom is present in the substantial absence of the other stereoisomeric configuration. The term "stereochemically pure" as used herein preferably means that at least about 70% by weight, more preferably at least about 80% by weight, and most preferably at least about 90% by weight, of a dimer or oligonucleotide of a particular stereoisomeric configuration is present to the exclusion of the other stereoisomeric configuration. "Resolution" of stereoisomers indicates a means by which the stereoisomers may be separated from each other to yield stereochemically pure isomers. A "point racemic mixture" or "point race ate" is defined herein to be a mixture of stereoisomers in which both stereoisomers at a particular asymmetric phosphorus atom are present. Such a "point racemic mixture" will typically, although not necessarily, contain on the order of 40% to 60% of one stereoisomer and, correspondingly, 60% to 40% of the other stereoisomer, although, generally, the two stereoisomers will be present in approximately equal quantities. For example, in a dimer having an asymmetric intemucleoside phosphorus, or in an oligonucleotide having one such asymmetric phosphorus, a point racemic mixture will have generally contain approximately equal amounts of each stereoisomer. The term "stereouniform" when referring to a particular cationic intemucleoside linkage having an asymmetric phosphorus atom such that the intemucleoside linkage is capable of existing in one of two distinct stereoisomeric configurations intends that a substantial portion of such molecules containing the intemucleoside linkage have the linkage present in a distinct stereoisomeric configuration. Preferably, a "substantial portion" intends that greater than 70%, more preferably greater than 80% and most preferably greater than 90% of such intemucleoside linkages are present in a stereoisomeric configuration. Stereoisomers may be resolved by any of a variety of methods known in the art. For example, some racemic mixtures crystallize in such a manner that molecules of like stereoisomeric configuration assemble into visibly asymmetric crystals. Such crystals may be physically separated to yield stereochemically pure stereoisomers. A second method that is well known in the art involves a chemical procedure by which a racemic mixture is allowed to react with a second, standard asymmetric molecule, e.g., if the racemate is an acid an optically active amine such as quinine, brucine or strychnine may be used to resolve the mixture. This method creates two stereoisomers that may be separated by standard physical means, e.g., distillation, crystallization, chromatography and the like. In the case of oligonucleotides containing substituents attached to the intemucleoside phosphorus, the isomers determined by chirality at the intemucleoside phosphorus are actually diastereomers, since the sugar components of the oligonucleotide backbone are chiral; diastereomers can be separated by conventional methods such as distillation, crystallization, chromatography or the like. Stereoisomers which are diastereomers may be separated using thin layer chromatography ("TLC") or column chromatography using an achiral medium. TLC resolution of the stereoisomers may be done on an appropriate TLC plate containing a εolid phase, e.g., silica, which optionally contain chiral reagents, or the like and which are effective to resolve stereoiεomerε. The stereoisomers separated by TLC may be characterized by their differential solubility in the TLC substrate and a solvent used to develop the plate. Typically, the differential solubility is expressed by determining the migration of the compound on the plate in a particular solvent system relative to the migration of the solvent system used to develop the plate. Thus, the location of the stereoisomers on the plate after development thereof is expressed as the distance moved from the spot where the compound is applied relative to the location of the solvent front; this ratio is typically referred to as the R f of the stereoisomer. Column chro atographic resolution of the stereoisomers may also be done using a solid phase matrix, e.g., silica gel, which optionally contains chiral reagents using techniques and reagents that are well known in the art. The stereoisomer having the higher R f , i.e., higher mobility on a TLC plate in a particular solvent system, or eluting first from the column is designated herein the "first-eluting" isomer while the stereoisomer having the lower R f or eluting second from the column is designated herein the "second-eluting" isomer. The absolute stereochemistry at phosphorus atoms can be determined by the 2D-NMR method of Loschner et al. (1990) Nucleic Acids Res . 18 . :5083-5088 as described by Fathi et al. (1994), Nucleic Acids Res . , supra . The absolute stereochemical configuration may also be determined by conventional methods well known in the art such as by optical rotatory dispersion or circular dichroism measurements. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase "optionally substituted alkylene" means that an alkylene moiety may or may not be substituted and that the description includes both unsubstituted alkylene and alkylene where there is substitution. The Novel Oligonucleotides The novel oligonucleotides of this invention contain cationic intemucleoside linkages having the structure (I) with W, X, Y, Z, R 1 , R 2 and R 3 as defined above for structure (I) . In the structure, P* represents an asymmetric phosphorus atom present in, for example, a phosphotriester, a phosphoramidate, a phosphothioester or an alkylaminophosphonate linkage. The oligonucleotide may contain any combination of anionic and/or cationic intemucleoside linkages. In an oligonucleotide, the cationic intemucleoside linkages may be any combination of phosphotriester, phosphoramidate, phosphothioester, alkylaminophosphonate linkages. As a result of the presence of an asymmetric phosphorus atom, the cationic intemucleoside linkages are capable of existing in one of two stereoisomeric configurations. The oligonucleotide may be prepared as described and exemplified below such that only one of the two stereoisomers is present at any predetermined intemucleoside linkage, i.e., such that a predetermined intemucleoside linkage in an oligonucleotide is stereouniform. As noted above, W may be O, S and Se. In a particularly preferred embodiment, wherein P* is an asymmetric phosphorus atom and each such linkage is stereouniform, W is S. X and Y may be independently 0, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consisting of H and C^-C 8 alkyl, and NR 6 where R 6 is H or c l ~ c 6 alkyl. Preferably, X and Y are independently O, S CH 2 or NH, more preferably X and Y are both O. Z can be O, S, ^-C 8 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C 1 -C 6 alkyl. When P* is an asymmetric phosphorus atom and each cationic intemucleoside linkage is stereouniform Z is O, S or NR 7 when W, X and Y are O. In a preferred stereouniform cationic intemucleoside linkage, Z is O or NH. In other preferred embodiments of the invention, the cationic intemucleoside linkages are not necessarily stereouniform. In these embodiments, when W, X and Y are O, Z is O or S. In one set of these embodiments, the oligonucleotide contains alternating cationic and anionic intemucleoside linkages. R 1 is selected from the group consisting of lower alkylene, lower alkenylene, lower alkynylene, monocyclic arylene and a bond. Thus, for example, when R 1 is a bond, Z is linked directly to NR 2 R 3 . R 1 is selected to provide an spacer between the intemucleoside phosphorus and the cationic center and may be chosen to optimize the affinity of the oligonucleotide containing cationic intemucleoside linkages for hybridizing with nucleic acids. Particularly preferred groups useful as R 1 include (CH 2 ) 2 or (CH 2 ) 3 . R 2 and R 3 may be any combination of H, lower alkyl, lower alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl. Alternatively, or R 2 and R 3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle. It is preferred that, when P* is an asymmetric phosphorus atom and each such linkage is stereouniform, R 2 and R 3 are H, CH 3 or lower alkyl terminally substituted with 1 NH 2 group. More preferably, R 2 and R 3 are CH 2 NH 2 or CH 2 CH 2 NH 2 . Preferred heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, morpholine, quinoline, indole, pyrimidine, piperazine, pipecoline, imidazole, benzimidazole and purine, which may be unsubstituted or substituted with halogen or C-^C g alkyl. Preferred heterocycles include imidazole, morpholine, pyrrolidine, piperazine pipecoline, methylpiperazine. Generally, W, X, Y, Z, R 1 , R 2 and R 3 are chosen to provide an oligonucleotide with cationic oligonucleoside linkages that are chemically insensitive to hydrolysis under physiological condition, that provide an oligonucleotide that is resistant to nucleases and/or that form stable duplexes with complementary oligonucleotides. In one particularly preferred embodiment, the cationic intemucleoside linkage has the structure (III) (III) wherein P* represents an asymmetric phosphorus atom, such that the linkage exists in stereoisomeric configuration that corresponds to the configuration of the first-eluted stereoisomer when a point racemic mixture of a nucleotide dimer containing the intemucleoside linkage is resolved using silica gel column chromatography. The oligonucleotide disclosed and claimed herein may have any proportion of anionic PDE linkages replaced by cationic intemucleoside linkages. Thus, the oligonucleotide may contain as few as about one of the anionic PDE linkage replaced by a cationic intemucleoside linkage and may have as many as 100% of the anionic PDE linkages replaced with cationic linkages. The proportion of negative and positive charges can be varied to yield an oligonucleotide having a desired net charge; the oligonucleotide may have an overall positive, neutral or negative charge depending on the number of cationic intemucleoside linkages incorporated therein. The oligonucleotide may be prepared to contain alternating anionic and cationic intemucleoside linkages. As described hereinbelow in Example 4, oligonucleotides containing alternating anionic and cationic intemucleoside linkages form more stable duplex with DNA or RNA than the anionic counterpart. The anionic and cationic intemucleoside linkages may be randomly distributed throughout the oligonucleotide or may be present in the oligonucleotide in blocks of anionic linkages and cationic linkages. In one preferred embodiment of the invention, the oligonucleotide has a stereouniform cationic intemucleoside linkage between selected nucleosides in the oligonucleotide. Oligonucleotides having cationic intemucleoside linkages may be prepared using conventional oligonucleotide synthetic techniques. For example, an oligonucleotide containing a cationic intemucleoside linkage having a predetermined stereoisomeric configuration at that linkage can be prepared by synthesizing dimer blocks having the desired phosphotriester, phosphoramidate or alkylaminophosphonate linkage, resolving the stereoisomers of the dimer block and incorporating the resolved stereoisomer of the dimer block into an oligonucleotide using solid phase oligonucleotide synthetic techniques. Either the first- or second-eluting stereoisomer may be incorporated into an oligonucleotide. For example, the cationic thymidylate dimer having the two stereoisomeric structures (IVa) and (IVb) where T represents a thymidylate nucleoside, can be prepared by reacting 5'-HO-T-θ-t-butyldimethyldisilyl ("TBDMS") with dimethoxytrityl ("DMT") -T-0-P(OCH 3 ) -N(iPr) 2 as described in Example 1 and depicted in Scheme 1. Scheme 1 DMT-T-O-P(OCH 3 )-N(CHCH 3 ) 2 ) (V) was reacted with 5'-HO-T-0-TBDMS (VI) in tetrazole. After the reaction was essentially complete, the reaction mixture was concentrated, diluted with CH 2 C1 2 and extracted with NaHC0 3 and 80%-saturated NaCI. The organic phase was dried, filtered and evaporated to yield the crude phosphite-triester intermediate (VII) . The intermediate was reacted with 3-diaminopropylamine in the presence of iodine. The product of the final reaction is a point racemic mixture of crude DMT-T-O-P(O) -(NH-(CH 2 ) -N(CH 3 ) 2 ) - O-T-O-TBDMS (IVa and IVb) . In addition, the point racemate may be used to prepare an oligonucleotide having a cationic intemucleoside linkage where the configuration of the linkage does not need be stereospecific. Oligomers having cationic phosphoramidate intemucleoside linkages using the two-step method described in Example 6. The reaction involves oxidation of a pre-synthesized dimer having a methyl phosphite- triester linkage with dithiodipyridine to give an activated S-Pyr-phosphorothiodiester. The newly formed stereoisomers may be resolved and the S-Pyr group thereafter displaced with reaction amine such as 3- dimethylpropylamine to yield a dimer having the desired phosphoramidate diester linkage in the desired stereochemical configuration. The two-step method described above and in Example 6 may be used for solid phase synthesis of cationic phosphoramidate-linked oligomers. During chain elongation, the oligomer is not exposed to amine solutions and at the conclusion of the reaction all S-Pyr- phosphbrothiodiester linkages are converted to phosphoramidate linkages. This method of modifying intemucleoside linkages during solid phase synthesis does not result in stereospecificity at the phosphoramidate linkages. Other methods by which oligonucleotides containing cationic intemucleoside linkages will be apparent to those skilled in the art. Oligonucleotides having cationic intemucleoside linkages find utility as probes for nucleic acids as a result of the increased stability of duplexes formed therewith. Cationic and zwitterionic oligonucleotides offer the possibility of using salt and/or pH to further control duplex formation involving nucleic acid targets. Typically, hybridization of oligonucleotides with RNA targets can be difficult because of extensive secondary and tertiary structures in the RNA; however, at low salt concentrations these structures are much less stable. Hybridization of RNA targets with cationic oligonucleotides which may be controlled by salt concentration, i.e., which form stable duplexes at low salt, allows stable duplex formation. For example, oligonucleotides having increased duplex stability will find utility in nucleic acid hybridization assays commonly used in genetic research, biomedical research and clinical diagnostics. In a basic nucleic acid hybridization assay, single- stranded analyte nucleic acid is hybridized to a labeled single-stranded nucleic acid probe and resulting labeled duplexes are detected. Hybridization steps in such assays are performed under appropriate stringency conditions. Stringency can be controlled by altering a parameter which is a thermodynamic variable. Such variables are well known in the art, and include formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent content, and temperature. Preferred stringency controls are pH and salt concentration. The stringency will be varied depending on the length and nature of the analyte sequence. Variations of this basic scheme have been developed to enhance accuracy, facilitate the separation of the duplexes to be detected from extraneous materials, and/or amplify the signal that is detected. One such assay is described in detail in commonly assigned U.S. Patent No. 4,868,105 to Urdea et al., the disclosure of which is incorporated herein by reference. In addition, the ability to control duplex formation by varying salt concentration and/or pH makes oligonucleotides having cationic intemucleoside linkages particular useful in nucleic acid hybridization assays for decreasing background noise due to nonspecific hybridization as described in commonly assigned U.S. Patent Application Serial No. 08/298,073 to Collins et al., the disclosure of which is incorporated herein by reference. Another application in which the construction of hybridizing oligonucleotides containing cationic intemucleoside linkages finds utility is in the design of antisense compounds. Antisense compounds, as explained, for example, in Ching et al. (1989) Proc . Natl . Acad . Sci . U .S .A . ]3j5: 10006-10010, Broder et al. (1990) Ann. Int . Med . 111:604-618, Loreau et al. (1990) FEBS Letters 274:53-56. and PCT Publication Nos. W091/11535, WO91/09865, WO91/04753, WO90/13641, WO91/13080 and, WO 91/06629, are oligonucleotides that bind to and disable or prevent the production of the mRNA responsible for generating a particular protein. Conventional antisense molecules are generally capable of reacting with a variety of oligonucleotide species. A triplex structure, e.g., an antisense molecule hybridized to a double-stranded oligonucleotide target, can also provide an antisense function. The oligonucleotides of the invention have properties that make them more desirable than the naturally linked oligonucleotides as antisense molecules. Oligonucleotides having cationic intemucleoside linkages are more resistant to nucleases which hydrolyze naturally occurring oligonucleotide; therefore, oligonucleotides having cationic inte ucleoside linkages have a longer half-life in cells. Such oligonucleotides can interact with complementary oligonucleotides in the cells with greater stability than naturally occurring counterparts. The formation of this substantially stable complex within the cell by an oligonucleotide having a cationic intemucleoside linkage allows the selective inhibition of gene expression. Experimental The practice of the present invention will employ, unless otherwise indicated, conventional techniques of synthetic organic chemistry, biochemistry, molecular biology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual. Second Edition (1989) ; Oligonucleotide Synthesis (M.J. Gait, ed. , 1984) ; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, eds., 1984); and the series, Methods in Enzymology (Academic Press, Inc.). All patents, patent applications, and publications mentioned herein, both supra and infra , are hereby incorporated by reference. It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the description above as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Temperature is always given in degrees C and, unless otherwise indicated, pressure is at or near atmospheric. NMR Spectroseopy. In the following Examples, NMR spectra were recorded on a Varian 300 MHz instrument. 31 P spectra were run at 121 MHz with reference to trimethylphosphite set at 140 ppm. High Performance Liquid Chromatography. Reverse phase high performance liquid chromatography ("RP- HPLC") analysis and purification were performed on a Supelco LC-18 column 5 micron (25 cm x 4.6 mm) eluted with a 0-50% gradient over 25 minutes of CH 3 CN into 5% CH 3 CN in 0.1 M triethylammonium acetate buffer, pH 7.5, flow rate 1 mL/min. EXAMPLE 1 Synthesis of Cationic Dimer DMT-T-O-PO ( H- (CH 2 ) 3 -N (CH 3 ) 2 ) -0-T-O-TBDMS 5'-HO-T-O-TBDMS (VI) (3.7 grams; 10.4 mmole) and tetrazole (20 mmole) were coevaporated with dry acetonitrile (2 x 100 mL) and the residue was dissolved in dry acetonitrile (50 mL) . DMT-T-O-P(OCH 3 ) - (iPr) 2 (V) (10 mmole) was dissolved in dry acetonitrile (50 mL) in a separate round bottom flask and then slowly added to the 5'-HO-T-0-TBDMS/tetrazole solution over a five minute period of time. Thin layer chromatography analysis on silica plates developed twice in 5% methanol/CH 2 Cl 2 , where the DMT-positive product migrated slightly faster than 5'- HO-T-O-TBDMS, indicated that the reaction appeared to be complete in less than 5 minutes. The reaction mixture was gently concentrated to a small volume, diluted with 400 mL CH 2 C1 2 and the organic phase extracted with 5% aqueous NaHC0 3 (400 mL) and 80%-saturated aqueous NaCI (400 mL) . The organic phase was dried over εolid NaS0 4 , filtered and evaporated to dryness. The residue was coevaporated with toluene (100 mL) and acetonitrile (2 x 200 L) to give 10 gramε of a crude phoεphite-triester intermediate (VII) . The crude intermediate, which was used directly without further purification, was dissolved in acetonitrile (200 mL) . Undiluted 3-diaminopropylamine (10 mL; 16 mmole) waε added directly to the solution followed by the drop-wise addition of a solution of iodine (2.53 grams; 10 mmole) in acetonitrile (100 L) with rapid stirring. The violet color of iodine faded immediately on addition and a slightly yellow color persisted after addition concluded. The reaction mixture was left at 4°C for 18 hours. The reaction mixture was then gently concentrated to a small volume and diluted with 400 mL CH 2 C1 2 . The organic phase of this preparation was extracted with 15% sodium bisulfite (300 mL) , 5% aqueous NaHC0 3 (2 x 400 mL) and 80% saturated aqueous NaCI (2 x 400 mL) . The organic phase was dried over solid NaS0 4 , filtered and evaporated to dryness. The residue waε coevaporated with toluene (100 mL) and acetonitrile (2 x 200 mL) to give 12 gramε of crude DMT-T-0-PO(NH-(CH 2 ) 3 - N(CH 3 ) 2 )-0-T-0-TBDMS ((IVa) and (IVb)). The product waε fractionated by εilica gel chromatography ("600 mL" Merck silica gel 60 poured in a solvent system of 2% triethylamine ("TEA") /CH 2 C1 2 ) using a gradient of methanol (0-6%) in 2% TEA/CH 2 C1 2 taking 100 mL fractions. Fractions 16-25 were pooled and concentrated. The fractionation waε repeated by silica gel chromatography as described above with a drawn-out gradient of methanol (2% (8 fractions) , 4% (16 fractionε) , 5% (16 fractions) and 6% (16 fractions) methanol) in 2% TEA/CH 2 C1 2 taking 50 mL fractions. Three pools were isolated: #1, fractions 19-22; #2, fractions 23-32; and #3, fractionε 33-40. Each pool was concentrated and coevaporated with toluene and acetonitrile to yield three pools with the following characteriεticε: Pool Weight Amount NMR TLC 2 (gm) (mmole) (purity) 1 1 1.17 0.95 8.85 ppm faster (100%) 100% 2 4.17 3.5 9.05/8.85 faster/slower (60:40) 60:40 3 0.82 0.7 9.1/8.9 faster/slower (95:5) 95:5 1 31 P NMR experiments were performed with a approximately 0.05 M solution of the respective dimer pool in acetonitrile using d 5 -DMS0 as external standard; Varian 300. 2 TLC: Merck silica 60F plates on alumina; pre- developed in 8% MeOH/2% TEA/CH 2 C1 2 followed by applying about 0.05 M sample and developing in the same εolvent system; the first-eluting fraction had an R f of 0.62 while the second-eluting had an R f of 0.40. Pool #2 from the initial purification of DMT- T-0-PO(NH-(CH 2 ) 3 -N(CH 3 ) 2 )-0-T-0-TBDMS was further fractionated by silica gel chromatography ("400 mL" Merck silica gel 60 poured in a solvent syεtem of 2% TEA/CH 2 C1 2 ) with a gradient of methanol (2% (16 50-mL fractions) , 3% (8 x 50 mL, 10 x 25 mL) , 4% (16 x 50 mL) and 5% (6 x 50 mL) ) in 2% TEA/CH 2 C1 2 . Three pools were isolated as follows. Pool #1, fractions 24-34: first-eluting isomer, 1.62 grams; Pool #2, fractionε 23-32: mixture of isomers, 0.73 gram; and Pool #3, fractions 33-40: second-eluting iεomer, 1.2 gramε. Removal of the TBDMS group waε achieved with tetrabutylammonium fluoride ("TBAF") . Silica purified DMT-T-0-P 0 (NH-(CH 2 ) 3 -N(CH 3 ) 2 )-0-T-0-TBDMS, Pool #1 from the initial purification (1.14 grams; 0.95 mmole), in 19 L acetonitrile was treated with 5 equivalents of TBAF (5 L of 1 M of TBAF in THF) for 2.5 hours at which time the reaction was more than virtually complete. The reaction mixture was diluted with 250 mL methylene chloride and extracted with 5% aqueous NaHC0 3 (250 mL) and 80%-saturated aqueous NaCI (250 mL) . The organic phase was dried over solid NaS0 4 , filtered and evaporated to dryness. The residue was coevaporated with toluene (100 mL) and acetonitrile (2 x 200 mL) . Crude DMT-T-0-P0(NH- (CH 2 )-N(CH 2 ) 2 )-0-T-OH from Pool #1 was purified by silica gel chromatography ("250 mL" Merck silica gel 60 poured in a solvent system of 2% TEA/CH 2 C1 2 ) using a gradient of methanol (0-18%) in 2% TEA/CH 2 C1 2 (0% (4 fractions) , 3% (4 fractions) , 6% (8 fractions) , 9% (8 fractions) , 12% (8 fractions), 15% (8 fractions), 18% (8 fractions)) in 2% TEA/CH 2 C1 2 taking 50 mL fractions. Fractions 32-40 were pooled. Concentration of the pooled fractions 32-40 yielded pure DMT-T-0-PO(NH-(CH 2 ) 3 -N(CH 3 ) 2 )-O-T-OH (first- eluting isomer; 0.61 gram; 0.57 mmole). 31 P MNR in CH 2 C1 2 with dg-DMSO as external standard: 8.3 ppm (100%); 31 P MNR in CH 3 CN with d 6 -DMSO as external standard: 8.87 ppm (100%) . Similarly, silica-purified DMT-T-0-PO(NH- (CH 2 ) 3 -N(CH 3 ) 2 )-0-T-0-TBDMS, Pool #3 from initial purification (0.8 gram; 0.7 mmole), was treated with 5 equivalents of TBAF. Workup and purification, aε deεcribed above for DMT-T-0-PO(NH-(CH 2 ) 3 -N(CH 3 ) 2 ) -0-T-O- TBDMS, Pool #1, yielded pure DMT-T-0-P0(NH-(CH 2 ) 3 - N(CH 3 ) 2 )-0-T-OH (εlower isomer; 0.57 gram; 0.53 mmole). 31 P NMR (CH 2 C1 2 with d g -DMSO as external standard) : 8.57 ppm (98%) . EXAMPLE 2 Synthesis of DMT-T-0-PO (NH- (CH 2 ) 3 ~N (CH 3 ) 2 ) -0-T-O-P (N (iPr) 2 ) -O-BCE DMT-T-0-P0(NH-(CH 2 ) 3 -N(CH 3 ) 2 ) -0-T-OH (first- eluting isomer; 0.61 gram; 0.57 mmole) in CH 2 C1 2 containing DIPEA (5.7 mmole) was reacted with Cl- P(N(iPr) 2 ) -O-BCE (1.14 mmole; two-fold excess) , wherein BCE is 3-cyanoethyl. The progresε of the reaction was monitored by TLC in 8% methanol in 2% TEA/CH 2 C1 2 ) . The reaction mixture was left at 4°C for 18 hours. The reaction mixture was diluted with 250 mL methylene chloride and extracted with 5% aqueous NaHC0 3 (250 mL) and 80% saturated aqueous NaCI (250 mL) . The organic phase was dried over solid NaS0 4 , filtered and evaporated to drynesε. The reεidue waε coevaporated with toluene (50 mL) and acetonitrile (2 x 100 mL) to give 0.6 gramε (0.47 mmole) . 31 P NMR (CH 3 CN with d 6 -DMSO as external standard): 148.5. 148.3. 8.87 ppm (integration: 1/1 phosphoramidite to phosphoramidate) . DMT-T-O-PO(NH-(CH 2 ) 3 ~N(CH 3 ) 2 ) -O-T-OH (second- eluting isomer; 0.57 gram; 0.53 mmole) was converted to the BCE phosphoramidite using the same procedure to give 0.6 grams product. 31 P NMR (CH 3 CN with d g -DMSO as external standard): 148.5. 148.3. 9.07 ppm (integration: 1/1 phosphoramidite to phosphoramidate) . The stereoiεomers of DMT-T-0-PO(NH-(CH 2 ) 3 - N(CH 3 ) 2 )-0-T-0-TBDMS were readily separated by TLC and column chromatography on silica, but after removal of the 3 ' -0-TBDMS group it was not possible to resolve them by TLC in any solvent syεtem. NMR data for DMT-T-0-PO(NH-(CH 2 ) 3 -N(CH 3 ) 2 ) -O- T-0-P(N(iPr) 2 ) -(O-BCE) . (a) firεt-eluting isomer. 1 H NMR (CDC1 3 ) δ (ppm) : 9.0 (s, IH, NH, exchangeable by D) ; 7.48 (ε, IH, H6) ; 7.45 (ε, IH, H6) ; 7.45-6.8 (m, 13H, trityl); 6.16 (t, IH, HI'); 6.15 (t, IH, HI'); 5.05 (m, IH, H3' ) ; 4.16 (m, IH, H3'); 4.1 (m, IH, H4' ) ; 4.05-3.9 (m, 2H, H5', 5'); 3.85 (m, IH, H4' ) ; 3.75 (ε, 6H, OCH3); 3.28-3.21 (m, 2H, H5') ; 2.9-2.7 (m, 4H, NCH2); 2.46 (m, 2H, H2' ) ; 2.3 (ε, 6H) ; 2.06 (m, 2H, H2' ) ; 1.74 (s, 3H, CH3) ; 1.7-1.5 ( , 2H, CH2) ; 1.43 (s, 3H, CH3); 1.0 (s, 9H) ; 0.0 (s, 6H) ; 31 P NMR (CDC1 3 ) : 8.75 ppm. (b) second-eluting isomer: 31 P NMR (CDC1 3 ) : 9.1 ppm. NMR data for DMT-T-0-PO(NH-(CH 2 ) 3 -N(CH 3 ) 2 ) -O- T-3'-O-P(N(iPr) 2 )-O-BCE. (a) First-eluting isomer: 31 P NMR 148.5 ppm, 147.9 ppm, 8.75 ppm (integration ratio of phosphoramidite/phosphoramidate = 1:1). (b) Second-eluting: 148.45 ppm, 147.96 ppm, 9.1 ppm (integration ratio of phosphoramidite/phosphoramidate - 1:1). EXAMPLE 3 Synthesis of 5'-O-DMT-Uf2'-O-(CH 3 n -3 -O-P ( 0 ) (NH- (CH 2 ) 3 -N(CH 3 ) 2 ) - 5'-0-U(2'-0-(CH 3 ) )-3'-OR 5'-O-DMT-U(2'-0-CH 3 )-3'-O-P(O) (NH-(CH 2 ) 3 - N(CH 3 ) 2 )-5'-0-U(2'-0-CH 3 )-3'-OR, wherein R is TBDMS, H or P(0-CH 2 CH 2 CN)-N(iPr) 2 was synthesized as follows. 5'-O-DMT-U(2'-0-CH 3 )-3'-O-P(0-CH 3 ) -N(iPr) 2 (MW 721) 7.9 mmole, was prepared from commercially available 5'-0-DMT-U(2'-0-CH 3 ) -3'-OH, purchased from ChemGenes (Waltham, MA). 5'-0-U(2'-0-CH 3 ) -3'-0-TBDMS waε prepared from 5'-0-DMT-U(2'-0-CH 3 ) -3'-OH purchaεed from Monomer Scienceε (Huntεville, AL) . The fully protected derivative waε prepared by firεt coupling 5'-O-DMT-U(2'-0- CH 3 )-3'-0-P(0-CH 3 )-N(iPr) 2 (MW 7210) (7.9 mmole) and 5'-0- U(2'-0-CH 3 ) -3'-0-TBDMS (8 mmoles) in the presence of tetrazole (16 mmoles) to give the phosphitetrieεter intermediate. The phosphitetriester intermediate (VII) , used directly after aqueous workup essentially as described in Example 1, was mixed with a two-fold excesε of 3-dimethylaminopropylamine and reacted with 80 mL of 0.1 M εolution of iodine in CH 3 CN. After aqueouε workup aε described in Example 1, this reaction yielded 8.54 grams of crude product. 31 P-NMR (in CH 3 CN with dg-DMSO as external reference) : 8.9 and 9.5 ppm. Purification by silica gel chromatography ("700 mL" Merck silica gel 60) performed as described in Examples 1 and 2 yielded two fraction pools: the first- eluting pools (fraction noε. 32-44, 1.69 grams, 31 P-NMR 8.9 ppm (100%)) and fraction nos. 47-60, SLOWER, 1.62 grams, 31 P-NMR 8.9 ppm (> 5%), 9.5 ppm (80%), 10.2 + 10.7 ppm (total 18%) (unknown identity) . EXAMPLE 4 Preparation of Cationic Oligomers with Alternating Stereo-defined Phosphoramidate Linkages Oligonucleotides having alternating anionic and stereo-uniform cationic phosphoramidate linkages were prepared by incorporating cationic stereoisomerically pure Tp(+)T dimer phosphoramidites, wherein Tp(+)T indicates a cationic dimethylaminopropylamido substituent on the intemucleoside phoεphorus atom and Tp(-)T indicates a conventional phosphodiester intemucleoside linkage. The following oligomers were synthesized using the methods described in Examples 1-3: (Tp(+)Tp) 7 T (using the first-eluting isomer) ; (Tp(+)Tp) 7 T (using the second-eluting isomer); and (Tp(+)Tp) 7 T (point racemate, i.e., a random mixture of first- and second-eluting isomerε at any one poεition) . Oligomer syntheseε uεing cationic dimerε were performed on a Millipore Expedite DNA synthesizer using a 10 minutes coupling step. DMT-Tp(+)T BCE (first-eluting iεomer or εecond-eluting isomer, or a point racemate (approximately equal amounts of first- and second-eluting isomerε) ) was dissolved in dry acetonitrile to a concentration of 0.1 M. The oligomer sequence synthesized was DMT-(Tp(+)Tp) 7 T by seven cycles of dimer addition; the final DMT was not removed. The crude oligomer was cleaved from the support with aqueous NH 4 OH (1 hour/20°C) . The supernatant was concentrated and the residue was redissolved in 400 μL water. Analysis of the crude oligomer product by high performance liquid chromatography ("HPLC") done as described in the Experimental section in all cases showed one major peak eluting around 20 minutes. The full-length DMT product was purified by reverse phase-HPLC ("RP-HPLC") by injecting 100 μL (about 43 A 260 units) . The peak eluting at around 20 minutes was collected (about 1 mL) and evaporated to dryness. The residue was redissolved in 200 μL 80% aqueous acetic acid for 1 hour to remove the 5'-terminal DMT group. The acid solution was removed by evaporation and the residue redisεolved in 100 μL 80% aqueouε acetic acid. The solution was allowed to stand for 1 hr at room temperature and was then evaporated to dryness. The residue was dissolved in about 0.5 mL water. The aqueous solution was washed twice with about 0.5 mL ethyl acetate and then lyophilized. Fine particular matter was removed by centrifugation. The detritylated oligomer was finally purified by RP-HPLC using the same procedure as described above. EXAMPLE 5 Preparation of Dimer Blocks with Isomer Reεolution The methodε described in this Example can be used to εyntheεize cationic oligomers from monomer units on a solid support using one monomer type. This method was exemplified using nucleoside methyl phoεphoramiditeε. A. The phoεphorothioate dimer Tp(S)T waε treated with 2-4-fold exceεs MeS0 2 -Cl in pyridine. The starting material was completely consumed to yield two new 31 P NMR signals at 67/68 ppm. When the reaction mixture was quenched with dimethylaminopropylamine ("DMAPA") the 67/68 ppm signals were immediately replaced with a new pair at 72/73 ppm; no signals corresponding to Nu-O-P- (0) (NH-R) -O-Nu at 8.8/9.4 ppm were observed. B. MeS0 2 -Cl (20μL; 2-fold excess) was added to 5'-0-DMT-T-0-P(S)0-0-5'-T-3'-0-TBDMS in pyridine (0.6 mL of a 0.2 M εolution) at 20°C. The reaction was complete in minutes as judged by 31 P NMR with two new signals at 67/6 and 67/0 ppm. DMAPA was added (4-fold excess over the phosphorothioate and the reaction was allowed to proceed. After lesε than 5 minutes 31 P NMR showed that the two signalε at 67.6 and 67.0 ppm had completely disappeared and were replaced by two new signals at 73.5 and 72.9 ppm. The reaction mixture was diluted with ethyl acetate, extracted with NaHC0 3 and NaCI. TLC of the crude product showed two DMT/εugar poεitive spots which migrated slightly faster than 5'-0- DMT-T-O-P(O) (NH-(CH 2 ) 3 -N(CH 3 ) 2 )-0-5'-T-3'-0-TBDMS in 2% TEA/8% MeOH/90% CH 2 C1 2 . Thus, the reaction leads exclusively to O-activation without any S-activation. EXAMPLE 6 Preparation of Cationic Dimers Having a Cationic Intemucleoside Phosphoramidate Linkage This example describes a two-step synthesis of cationic phoεphoramidate linked oligomerε. The reaction involves oxidation of a methyl phosphite-trieεter linkage with dithiodipyridine to give an activated S-Pyr- phosphorothiodiester followed by displacement with amine to yield the desired phosphoramidate diester linkage. This scheme is attractive for solid phase synthesiε of cationic phosphoramidate-linked oligomers. During chain elongation, the oligomer is not exposed to amine εolutionε and at the conclusion of the reaction all S-Pyr- phosphorothiodiester linkages are converted to phoεphoramidate linkageε. Triethylamine (0.1 mL) waε added to a solution of DMT-0 5 '-T-0 3' -P(0-(CH 3 ) )-0 5' -0 3 ' -TBDMS in CH 3 CN (0.3 mL; 0.2 M) . A five-fold excess of dithiopyridine in CH 3 CN (0.3 mL; 1.0 M) was added and the reaction was allowed to proceed at 20°C. The progress of the reaction was followed by NMR. The initial two signals at 140 ppm corresponding to the two stereoisomers of the starting material, disappeared after 3 hours and were replaced with two new signals at 20.3/20.6 ppm corresponding to the two stereoisomers of DMT-0 5' -T-0 3 '-PO(S-Pyr)-0 5' -T-0 3' -TBDMS. 0.2 mL of 3-dimethylpropylamine was added directly to the NMR tube. After about 1 hour, the 20.3/20.6 ppm signals had been replaced by two new signals at 9.1/8.8 ppm corresponding to the two stereoisomers of DMT-O 5 ' -T-0 3' -PO(NH-(CH 2 ) 3 -N(CH 3 ) 2 )-O 5 '-T-O 3' -TBDMS. Thin layer chromatography analysis confirmed that only nucleotidic material comigrated with a genuine sample of the compound prepared by the method described in Example 1. EXAMPLE 7 Effect of Cationic Phosphoramidate Linkage on Oligonucleotide Hybridization Properties The effect of cationic intemucleoside linkages on hybridization propertieε of oligonucleotides was assessed using oligonucleotides having alternating anionic and cationic phosphoramidate linkages, the cationic linkages being stereoisomerically pure. Thus, the dimer block used to synthesize the cationic oligonucleotide was a stereoisomerically pure Tp(+)T dimer phosphoramidate prepared as described in Example 1. The point racemic [Tp(+)Tp(-) ] 7 T, wherein p(+) and p(-) are as defined above, was synthesized as described in Letsinger et al. (1988) J " . Am. Chem . Soc . 110:4470-4471. Oligonucleotide analogε having the structure [Tp(+)Tp(-) ] 7 T were syntheεized on a εolid support using either the pure stereoisomer of 5'-0-DMT-Tp(+)T-3'- P(N(iPr) 2 ) -0-CH 2 CH 2 CN that eluted first or second from a silica gel column, or the point racemic mixture of the firεt- and εecond-eluting iεomerε, uεing standard phosphoramidite chemistry. The product of each εyntheεiε waε purified by RP-HPLC in the DMT form followed by detritylation with 80% aqueouε acetic acid. The final product waε lyophilized for storage. The hybridization properties of the cationic homothymidine oligonucleotides containing alternating positive and negative linkages were assessed against poly (dA) and poly(A) target sequences. Thermal melt analyseε were performed on a Perkin Elmer Lambda 2 UV/Viε spectrophotometer in 15 mM phosphate buffer, pH 7.3. The concentration of cationic oligonucleotide was approximately 5 μM. The changes in absorbance were measured at 260, 280 and 330 n with a temperature ramping of l°C/minute. The T m is the temperature corresponding to the midpoint of the region of maximum slope in a plot of the A 260 versus temperature. The oligonucleotide sequenceε and the melting temperatureε for duplex formation are εhown in Tables 1 and 2. Tp (+) T indicates a cationic dimethylaminopropylamido εubstituent on the intemucleoside phosphorus atom and Tp ( -) T indicates a conventional phosphodiester intemucleoside linkage . Tp(+)T indicates a cationic dimethylaminopropylamido substituent on the intemucleoside phosphorus atom and Tp(-)T indicates a conventional phosphodiester intemucleoside linkage. The hybridization data in Tables 1 and 2 indicate that cationic oligonucleotides with stereoisomeric phosphoramidate linkages that correspond to the fast-eluting stereoisomer form very stable duplexes with DNA targets. In addition, these data shown that the duplex stability for the cationic oligonucleotides tested (first-eluting or point racemate) were esεentially independent of εalt concentration. This hybridization behavior is quite different from that of naturally occurring all-anionic oligonucleotides which form duplexes with their DNA target that are highly dependent upon the salt concentration. Generally, cationic oligonucleotides with stereoisomeric phosphoramidate linkages that correspond to the εecond-eluting stereoisomer do not form stable duplexes with DNA targets. For example, in this experiment the T m for the normal all-anionic oligonucleotide-poly(dA) duplex decreased from 53°C to 35°C to 25°C when the salt concentration was lowered from 1.0 M to 0.1 M to no added salt. In contrast, the cationic oligonucleotide prepared from the first-eluting Tp(+)T isomer had the highest T m valueε which were independent of the salt concentration. Even in the absence of salt, under which conditions a dramatic loεε in duplex εtability for the natural oligomer was observed, the T m value obtained with the cationic oligonucleotide prepared from the first-eluting Tp(+)T iεomer was 56°C. The cationic oligonucleotide prepared from the second-eluting Tp(+)τ isomer did not form a stable duplex with poly(dA) at no salt and 0.1 M NaCI. Even at 1.0 M NaCI the second-eluting iεomer showed lower T m values than that prepared from the first-eluting isomer (-17°C) . The cationic oligonucleotide prepared from a point racemic mixture of Tp(+)T formed duplexes with intermediate stability at no and low salt concentrations; however, the T m values for the point racemate were salt- independent and when no added salt was present, the T m values obtained with the point racemate was greater than the all anionic oligonucleotide. As reflected by the higher T m values, duplex stability with a poly(A) target was higher for the first- eluting cationic oligomer than for the all-anionic oligomer. The second-eluting iεomer did not form stable duplexes with poly(A) while the point racemate showed intermediate duplex stability. EXAMPLE 8 Effect of Cationic Phosphotriester Linkage on Oligonucleotide Hybridization Properties Three thymidine oligonucleotides were prepared having: (1) all εtandard phoεphodieεter intemucleoside linkages (5'-TTTTTTTTTTTTTTT-3' ) ; (2) alternating anionic εtandard PDE linkageε and cationic phoεphoramidate internucleoεide linkageε, wherein the cationic N-substituent group is dimethylaminopropyl (designated [Tp(N+)Tp(-) ] 7 T) (Letsinger et al. (1988) J . Am . Chem . Soc . 110:4471-4472) ; and (3) alternating standard PDE linkages and cationic phosphotriester intemucleoside linkages, wherein the cationic substituent group is dimethylaminopropyl (designated "[Tp(0+)Tp(-) ] 7 T") (synthesized on a solid support using standard phosphoramidite chemistry) . The hybridization properties of the cationic homothymidine oligonucleotides containing alternating positive and negative linkages were assessed against poly (dA) as described in Example 7. The results are shown in Table 3. The data in Table 3 show that the [Tp(0+)Tp(-) ] 7 T oligomer has a T m that is independent of salt concentration and is 10°C (0.1 M NaCI) and 21°C (no NaCI) higher than the all anionic thymidine 15-mer. In addition, the [Tp(0+)Tp(-) ] 7 T oligomer forms a more stable duplex with poly(dA) than the [Tp(N+)Tp(-) ] 7 T oligomer. EXAMPLE 9 Hybridization of Thymidine and 2'-O-Methyluridine Zwitterionic Derivatives to a Duplex Target The hybridization of zwitterionic thymidine and 2'-0-methyluridine derivatives to a duplex target d(T 15 C 4 A 15 ) was studied to show the utility of such oligonucleotides as probes for double-stranded DNA. In the absence of other oligomers, d(T 15 C 4 A 15 ) forms a self- complementary structure with a dT.dA stem of high stability (T m 62°C in 0.1 M NaCI; T m 78°C in 1 M NaCI) . All stereouniform zwitterionic oligomers were prepared using the procedures described in Examples 1 through 6 above or as described in Horn et al. (1996) Tetrahedron Lett . 12:743-745 and Chaturvedi et al. (1996) Nucleic Acids Res . 24 . :2318-2323. As above, the stereoisomer having the higher R f is designated herein the "first- eluting" isomer while the stereoisomer having the lower R f is designated herein the "second-eluting" isomer. The affinities of the oligothymidylate derivatives for the duplex target d(T 15 C 4 A 15 ) depend strongly on the charge and stereochemistry of the probe and the ionic εtrength of the εolution (Table 4) . One of the zwitterionic iεomers, d(T+T-) 7 T (first-eluting) , formed a relatively stable triple-stranded complex with the target even in 0.1 M NaCI. The melting curve for an equimolar mixture of d(T+T-) 7 T (firεt-eluting) and d(T 15 C 4 A 15 ) εhowed a tranεition (T m 24°C) for dissociation of the zwitterionic strand from the duplex εegment, as well as a transition (T ro 68°C) for denaturation of d(T 15 C 4 A 15 ); and a plot of A 260 versus titrant for titration of d(T+T-) 7 T (first-eluting) with d(T 15 C 4 A 15 ) at 0°C displayed a break at equimolar concentrations of the two oligomers (data not shown) , in accord for a complex with the dT.dA.dT motif. Under the εame conditionε, neither dT 15 nor the zwitterionic iεomeric oligomer d(T+T- ) 7 T (εecond-eluting) interacted εignificantly with d(T 15 C 4 A 15 ). In a high εalt solution (1.0 M NaCI) dT 15 did bind to d(T 15 C 4 A 15 ) (T m 30°C) . The stability of the complex formed by d(T+T-) 7 T (first-eluting) also increased with an increase in the salt concentration (T m 32°C in 1.0 M NaCI) ; however, the rise in T m was lesε than in the case of the all anionic probe. Oligomer d(T+T-) 7 T (second- eluting) did not bind εignificantly to d(T 15 C 4 A 15 ) even in 1 M NaCI. Since it haε been εhown that replacement of thymidine by 2'-O-methyluridine in a phoεphodiester oligonucleotide probe enhances stability of triple- stranded complexes formed with the probe (Escude et al. (1992) CR . Acad . Sci . Paris III , 315:521-525) . The stereoisomeric oligomers (U'+U'-) 7 dT (first-eluting) and (U'+U'-) 7 dT (second-eluting) were prepared to test the effect of this εubεtitution on binding of zwitterionic oligonucleotides to double-stranded targets. (U'+U'-) 7 dT (firεt-eluting) bound to d(T 15 C 4 A 15 ) more effectively (T m 35°C, 0.1 M NaCI) than the thymidine analogue, d(T+T-) 7 T (first-eluting) , (T m 24°C, 0.1 M NaCI) . Neither the isomeric oligomer, (U'+U'-) 7 dT (second-eluting), nor the corresponding phosphodiester control, U' 14 dT, interacted significantly with d(T 15 C 4 A 15 ) under these conditions. In contrast to the resultε for the triple-stranded complexes, the 2'-0-methyluridine derivative, (U'+U'-) 7 dT (first- eluting) , was found to bind lesε effectively than the thymidine analogue, d(T+T-) 7 T (first-eluting) to an equivalent of poly(dA) . The T m values for formation of the double-stranded complex in 0 M NaCI, 0.1 M NaCI, and 1.0 M NaCI solutions were, respectively, 35°C, 36°C, and 41°C for (U'+U'-) 7 dT (firεt-eluting) ; and 58°C, 58°C, and 58°C for d(T+T-) 7 T (first-eluting) (see Horn et al. Tetrahedron Lett . , supra) . Data for the mixed dT,dC oligomers d(T+T-) 2 (T+C-) 5 T (first-eluting) and d(T+T-) 2 (T+C-) 5 T) (second-eluting) are presented in Table 5. The target d[A 5 (GA) 5 T 4 (TC) 5 T 5 ] , self-associates under the conditions employed (T m 68°C at pH 7.0 and T m 67°C at pH 6.0; 0.1 M NaCI) . The dT,dC pentadecamers bound to d[A 5 (GA) 5 T 4 (TC) 5 T 5 ] with higher affinity than the dT or dU' pentadecamers bound to d(T 15 C 4 A 15 ), and, as expected for triple-stranded structures containing dC (Lipsett, M.N., J. Biol . Chem . 239:1256-1260 (1964)), the affinity was greater at pH 6 than at pH 7. The thermal stability of the triple-stranded complex formed by one of the zwitterionic probes, d(T+T-) 2 (T+C-) 5 Tb (first-eluting), and target d[A 5 (GA) 5 T 4 (TC) 5 T 5 ] in a low salt εolution (0.1 M NaCI) at pH 7. The T m value was 23°C higher than that for the complex formed by the corresponding all- phosphodiester probe. That the high affinity of the zwitterionic oligonucleotideε depends on sequence as well as ionic charge and stereochemistry at phosphorus was demonstrated by experimentε in which d(T+T-) 7 T (first- eluting) was paired with a mismatched target, d[A 5 (GA) 5 T 4 (TC) 5 T 5 ] , and d(T-T-) 2 (T+C-) 5 Tb (first-eluting) was similarly paired with d(T 15 C 4 A 15 ) . Neither of these systems afforded stable-triple stranded complexes (Tables 4 and 5) . a Also <0 °C in 1 . 0 M NaCI , Three pairε of stereoisomeric zwitterionic 15mers were prepared for thiε εtudy. A firεt pair waε derived from thymidine, a second pair from 2'-0- methyluridine and a third pair from thymidine and deoxycytidine. In each case, all phosphoramidate linkages in a given oligomer had the same configuration. Thermal denaturation experimentε εhowed that participation of these alternating cationic-anionic oligonucleotides in formation of triple-stranded complexes is highly dependent on chirality at the modified phosphate linkages. In each case one of the stereoisomeric probes, designated as the first-eluting isomer, binds more effectively to a complementary double-stranded DNA target than the corresponding all phosphodiester probe does. In 0.1 M aqueous NaCI at pH 7, the enhancement in T m is of the order of 20°C for the dT and the dT,dC zwitterionic 15mers (d(T+T-) 7 T (first-eluting) and d(T+T-) 2 (T+C-) 5 T (firεt-eluting) ) and ~35°C for the 2'-O-methyluridine derivative ((U'+U'-) 7 dT (firεt-eluting) ) . The enhancement in T m in 1 M NaCI solutions is less, but still significant. It is noteworthy that the affinity of d(T+T- ) 2 (T+C-) 5 T (first-eluting) is relatively high at pH 7, even though the probe contains several dC units. Strong binding of thiε unεymmetrical, mixed-baεe probe to the double-εtranded target iε conεiεtent with a structure in which the third strand is oriented parallel to the purine strand, as in other triple-stranded complexeε derived from two pyrimidine and one purine strand. In contrast to the results with the first- eluting stereoiεomeric oligomers, the phosphoramidate oligomers with the opposite configuration at phosphorus (second-eluting) exhibited very low affinity for the double-stranded targets. Since the absolute configuration of the isomerε haε not yet been definitively aεεigned, and structural information on the geometry of the triple strand complex is limited, speculation on the reasonε for these differences is premature. It is interesting, however, that the configuration at the phosphoramidate links that favorε binding of an oligonucleotide probe to double-stranded DNA is the εame that favorε binding to a single-stranded target to give a double-stranded complex (Horn et al. Tetrahedron Lett . , supra) . Gryaznov et al. have shown that oligodeoxy¬ ribonucleotide N3'-P5' phosphoramidate derivatives bind to DNA duplexes to give unusually εtable pyr.pur.pyr and pur.pur.pyr complexes (Gryaznov et al. (1994) J . Am . Chem . Soc . 116:3143-3144 and Gryaznov et al. (1995) Proc . Natl . Acad . Sci . USA, 9_2:5798-580) . Nielsen et al. have found that λ peptide nucleic acids' (PNAs) interact with appropriate DNA sequences by strand invasion to give PNA.pur.PNA triple-stranded segments (Nielsen et al. (1991) Science 254:1497-1500; Cherny et al. (1993) Proc . Natl . Acad . Sci . USA 9.0:1667-1670; and Nielsen et al. (1993) Nucleic Acids Res . 21:197-200). The stereo-uniform cationic phosphoramidate derivatives compriεe another family of oligonucleotide analogueε that bind εtrongly to double stranded DNA targets. Since these three families differ from one another in physical and chemical properties and, no doubt, in behavior in biological systems as well, they offer the chemist rich opportunities for tailoring DNA binding agents for specific applications. EXAMPLE 10 Horn et al. Tetrahedron Lett, supra previously noted an unusual feature in the melting curve obtained at 260 nm for a 1/1 dT/dA mixture of d(T+T-) 7 T (second-eluting) and poly(dA) in 1 M NaCI solution. The melting curve showed two transitions, differing in T m by ~20°C. The first transition appears to stem from a reversible disproportionation of two d(T+T-) 7 T (second- eluting) .poly(dA) duplex segments to give a triplex with the dA.dA.dT motif pluε a strand of d(T+T-) 7 T (second- eluting) , and that the second transition represents reversible dissociation of the triplex to give free poly(dA) and d(T+T-) 7 T (second-eluting). Several lines of evidence support this concluεion. (i) Two transitions appear in the melting curve measured at 280 nm as well aε the one at 260 nm (see Chaturvedi et al. Nucleic Acids Res, supra) . The higher temperature transition in the 280 nm curve is hypochromic. Characteristically, tranεitionε for dissociation of dT.dA duplexes and dT.dA.dT triplexes are hyperchromic at these wavelengths. This result points to dissociation of a complex with a novel structure. (ii) Melting curves obtained in low salt solutions (0-0.1 M NaCI) showed a single transition (- T m 22°C) that was independent of the salt concentration. As the salt concentration was increased incrementally, a second transition appeared at progresεively higher temperatureε. The salt dependence for the second transition is consiεtent with expectations for dissociation of a triple-stranded complex containing a zwitterionic and two anionic strandε. (iii) Asεociation curves obtained by cooling solutionε of the oligomers slowly from 80°C to 0°C demonstrated that the transitionε are fully reverεible. As a further test, a ldT/ldA mixture of d(T+T-) 7 T (second- eluting) and poly(dA) in 1 M NaCI waε allowed to cool from 80°C to 25°C and held at the lower temperature for two hourε to permit a εlow equilibration to take place. No change in abεorbance occurred during the two hours, indicating that the syεtem was stable at this temperature. When the temperature was then further reduced through the range for the lower transition to 18°C, the absorbance at 260 nm dropped rapidly and equilibrium was reached within two minutes. (iv) A single transition (T m 42°C; 1 M NaCI) was observed on heating a mixture of d(T+T-) 7 T (second- eluting) and poly(dA) at a ldT/2dA nucleotide ratio from 0°C to 80°C, demonstrating that the triple-εtranded complex waε stable at 0°C in absence of excess d(T+T-) 7 T (second-eluting) . (v) In agreement with these results, titration of d(T+T-) 7 T (second-eluting) in 1 M NaCI (pH 7.0) with poly(dA) consumed twice as much poly(dA) at 30°C as at 0°C, and the breaks corresponded to formation of a ldT/2dA complex at 30°C and a ldT/ldA complex at 0°C. As controlε, dT 15 waε alεo titrated with poly(dA) under the εame conditionε. Theεe experiments showed breaks corresponding to 2dT/ldA, ldT/ldA and ldT/ldA, as expected, for titrations at 0°C in 1.0 M NaCI, 30°C in 1.0 M NaCI and 0°C in 0.1 M NaCI, respectively. Also, titration of d(T+T-) 7 T (second-eluting) at 0°C in 0.1 M NaCI gave a ldT/ldA ratio. (vi) A single tranεition (T m 26°C) waε found on heating a mixture of d(T+T-) 7 T (second-eluting) with either one or two equivalents of d(CCA 15 CC) in 1.0 M NaCI. This result suggeεted that appearance of the εecond transition in the case of poly(dA) might be related to the fact poly(dA) could fold to a hairpin structure that would stabilize a dA.dA.dT triple-stranded complex. We therefore examined hybridization of d(T+T-) 7 T (second- eluting) with a shorter, well defined oligomer, d(A 15 C 4 A 15 ) , which also could fold to a hairpin structure. This target indeed simulated poly(dA) in behavior, although the complexes formed were somewhat less stable (T m - 17°C for the disproportionation reaction and -32°C for dissociation of the triplex in 1.0 M NaCI; see Chaturvedi et al. Nucleic Acid Res . , supra) . A comparison with the heating curve for d(A 15 C 4 A 15 ) alone showed that these breaks indeed reflect interaction of d(T+T-) 7 T (second-eluting) with this target oligonucleotide. Aε in the case with poly(dA) , the transitions were reversible. Formation of two distinct complexes from d(T+T-) 7 T (second-eluting) and poly(dA) is further supported by CD spectral data (see Chaturvedi et al. Nucleic Acids Res . , supra) . The CD spectrum for a mixture of d(T+T-) 7 T (second-eluting) and poly(dA) containing equivalent concentrations of dT and dA units in 1 M NaCI at 5°C is very similar to the CD spectrum for the same combination of d(T+T-) 7 T (second-eluting) and poly(dA) in 0.1 M NaCI and for the spectra of poly(dT) .poly(dA) in 0.1 M and in 1 M NaCI. The two peaks and the trough observed in the 255-300 nm region are diεtinctive (Wellε et al. (1970) J . Mol . Biol . 5 :465-497) and serve as a signature for the poly(dT) .poly(dA) duplex. The similarity in the spectra for all these systems indicates that the base stacking is much the same in all caseε. These data therefore point to a conventional duplex structure for the complex derived from d(T+T-) 7 T (second-eluting) and poly(dA) in 1 M NaCI at 5°C. A very different spectrum was obtained when the solution of d(T+T-) 7 T (second- eluting) and poly(dA) was warmed to 27°C. The trough at 247 nm deepened, the peak at 259 nm fell, the trough at 268 nm disappeared, and the peak at 282 nm increased. When the solution was then cooled, the spectrum obtained at 27°C readily reverted to the spectrum obtained at 0°C. These changes correlate well with the UV absorbance changes observed on heating and cooling the solution. Furthermore, the spectrum obtained at 27°C is quite different from the spectrum expected for a mixture of free d(T+T-) 7 T (second-eluting)and poly(dA). We attribute the spectrum obtained at 27°C to the presence of a dA.dA.dT triple stranded complex derived from poly(dA) and d(T+T- ) 7 T (second-eluting). This assignment leads to the prediction that the CD spectrum of a 2dA/ldT ratio of poly(dA) and d(T+T-) 7 T (second-eluting) in 1 M NaCI at 27°C would be similar to the spectrum obtained for a IdA/dT ratio of poly(dA) and d(T+T-) 7 T (second-eluting) at 27°C and that it would remain unchanged on cooling to 5°C. These reεults were indeed obεerved. The CD spectrum for a ldT/ldA mixture of oligomer d(T+T-) 7 T (first-eluting) and poly(dA) in 1 M NaCI at 27°C is very εimilar to the εpectrum of the poly(dT) .poly(dA) duplex. In contraεt to the caεe with d(T+T-) 7 T (second-eluting), no evidence for formation of a dA.dA.dT triplex was found. The CD spectrum was virtually unchanged on cooling to 5°C and was not altered significantly by addition of a second equivalent of poly(dA) . These results buttresε data from the melting curveε indicating that d(T+T-) 7 T (firεt-eluting) formε a duplex, but not a dA.dA.dT triplex, with poly(dA) . A εurprising set of equilibria was found for systems containing the second-eluting d(T+T-) 7 T isomer and poly(dA) or d(A 15 C 4 A 15 ) in 1 M NaCI solution. For a syεtem containing the second-eluting d(T+T-) 7 T isomer and poly(dA) in a ldT/ldA ratio, at 0°C the components form a double-stranded complex; at 30°C they exist as a dA.dA.dT triple-stranded complex and an equivalent of free d(T+T-) 7 T (second-eluting) ; and at 50°C the complex is fully dissociated. The transitionε between the states are relatively sharp (T m 22°C and T m 42°C) and equilibrium is established rapidly both on heating and cooling the solution. The system involving d(A 15 C 4 A 15 ) fits the same scheme, but with a dC strand serving as the linker to facilitate alignment of two dA 15 segments. The importance of the configuration of the phosphoramidate linkages is shown by the fact that oligomer d(T+T-) 7 T (first-eluting), in contrast to d(T+T-) 7 T (second-eluting), did not form triple stranded complexes of this type. Two featureε in these equilibria are unusual, though not without some related precedents: (i) formation of a stable triple-stranded complex containing exclusively dA.dA.dT triads; and (ii) formation of a triple-εtranded complex by a thermally induced disproportionation of a double-stranded complex. Although a number of examples of pur.pur.pyr triplexes have been reported (see Lipsett (1964) J . Biol . Chem . ,23_9_: 1256-1260; Anna et al. (1989) Science 241:456-459; Beal et al. (1992) Nucleic Acids Res . 2Q_:2773-2776; and Pilch et al. (1991) Biochemistry 3_0:6081-6087 for representative cases), the stable complexes containing dA.dA.dT triads that have been described have also contained dG units in one of the purine strands. Pilch et al. have noted that "...the presence of guanine residueε appearε to be crucial for stabilization of short pur.pur.pyr triplexeε" (Pilch et al. Biochemistry , supra) . For the ribonucleotide family, however, Broitman et al. (1987) Proc . Natl . Acad . Sci . USA J34 . :5120-5124 have described a triple-stranded complex, poly(A) .poly(A) .poly(U) , which can form when the degree of polymerization of poly(A) falls in the range of -28-150, and Lauceri et al. (1996) Angew . Chem . Int . Ed . Engl . 15:215-216 have shown that cationic porphyrins in low concentration induce formation of poly(A) .poly(A) .poly(U) triple-stranded complexes from high molecular weight poly(A) (> 400 bp) . With respect to thermally induced disproportions, examples affording pyr.pur.pyr complexes have been reported for both ribonucleotide, [poly(A) .poly(U) ] (Miles et al. (1964) Biochem . Biophys . Res . Comm . JL4.:129-136; Stevens et al. (1964) Biopolymers 2:293-314; and Broitman et al. Proc . Natl . Acad . Sci . USA, supra , and deoxyribonucleotide polymers, [poly(dTdC) .poly(dGdA) ] (Lee et al. (1979) Nucleic Acids Res . j5:3073-3091; Lee et al. (1984) Nucleic Acids Res . 11:6603-6613; and Hampel et al. (1991) Biochemistry 10:4455-4459) . It appears that the unusual stability of the dA.dA.dT complexes containing oligonucleotide d(T+T-) 7 T (second-eluting) , and the disproportionation reaction leading to their formation, must reflect the presence of the stereouniform side chainε in the probe. The propertieε of zwitterionic oligomerε with thiε chirality may prove useful in designing novel self-aεεembly systemε baεed on oligonucleotide hybridization.

Claims: WE CLAIM : 1. An oligonucleotide having at least one cationic intemucleoside linkage having the structure (I) wherein: W is εelected from the group consisting of O, S and Se; X and Y are independently selected from the group consisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consiεting of H and C 1 -C 6 alkyl, and NR 6 where R 6 is H or C^C g alkyl; Z is selected from the group consisting of O, S, ^-C 8 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C-^C g alkyl, with the proviεo that when W, X and Y are O, Z iε O, S or NR 7 ; R 1 is selected from the group consisting of C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the group consiεting of H, C j -C 8 alkyl, C-^C g alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or R 2 and R 3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and wherein P* repreεentε an aεymmetric phoεphoruε atom capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage is stereouniform. 2. The oligonucleotide of claim 1, wherein the stereoiεomeric configuration of the cationic internucleoεide linkage correεpondε to the configuration of the firεt-eluted εtereoiεomer when a point racemic mixture of a nucleotide dimer containing the intemucleoside linkage is resolved using silica gel column chromatography. 3. The oligonucleotide of claim 1, wherein the stereoisomeric configuration of the cationic intemucleoside linkage correspondε to the configuration of the second-eluted stereoisomer when a point racemic mixture of a nucleotide dimer containing the intemucleoside linkage is resolved using silica gel column chromatography. The oligonucleotide of claim 2, wherein Z is NR' 5. The oligonucleotide of claim 4, wherein R' is H, 6. The oligonucleotide of claim 4, wherein X and Y are O. 7. The oligonucleotide of claim 4, wherein R 1 is ^-C 8 alkylene. 8. The oligonucleotide of claim 7, wherein R 1 is (CH 2 ) 3 . 9. The oligonucleotide of claim 4, wherein R 2 and R 3 are C-^C g alkyl. 10. The oligonucleotide of claim 9, wherein R 2 and R 3 are CH 3 . 11. The oligonucleotide of claim 4, wherein W is S. 12. The oligonucleotide of claim 1, wherein Z is NR 5 . 13. The oligonucleotide of claim 12, wherein R= is H. 14. The oligonucleotide of claim 12, wherein R 1 is C j -C 8 alkylene. 15. The oligonucleotide of claim 14, wherein R ** - is (CH 2 ) 2 , 16. The oligonucleotide of claim 12, wherein R 2 and R 3 are C-^C g alkyl substituted with 1 to 4 NH 2 groups. 17. The oligonucleotide of claim 16, wherein R 2 and R 3 are -CH 2 CH 2 NH 2 . 18. An oligonucleotide having at least one cationic intemucleoside linkage having the structure (I) wherein: W is S; X and Y are 0; Z is NH; R 1 is (CH 2 ) 3 ; R 2 and R 3 are CH 3 ; and wherein P* representε an aεymmetric phoεphoruε atom, such that the linkage existε in stereoisomeric configuration that correspondε to the configuration of the firεt-eluted stereoisomer when a 5 point racemic mixture of a nucleotide dimer containing the intemucleoside linkage is resolved using silica gel column chromatography. 19. An oligonucleotide having alternating 10 cationic and anionic intemucleoside linkageε wherein the cationic internucleoεide linkageε have the εtructure (II) wherein: 20 W iε εelected from the group conεiεting of 0, S and Se; X and Y are independently εelected from the group conεisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consisting of H and 25 C-^C g alkyl, and NR 6 where R 6 is H or ^-C 8 alkyl; Z is εelected from the group consisting of 0, S, C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 iε H or C-^C g alkyl, with the proviεo that when W, X and Y are 0, Z is O or S; _ R 1 is selected from the group consisting of C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the group consisting of H, C^-C 8 alkyl, C- j^ -C 8 alkyl _ 5 substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or wherein R 2 and R 3 are linked to form a five- or six- membered alkyl or aryl ring or N-, O- or S-containing heterocycle; and wherein P iε a phoεphoruε atom that may or may not be capable of existing in two distinct stereoisomeric configurations and further wherein the linkage may or may not be stereouniform. 20. The oligonucleotide of claim 19, wherein Z is NR 5 . 21. The oligonucleotide of claim 20, wherein R b is H. 22. The oligonucleotide of claim 20, wherein X and Y are O. 23. The oligonucleotide of claim 20, wherein R 1 is C 1 -C 6 alkylene. 24. The oligonucleotide of claim 23, wherein R 1 is (CH 2 ) 3 . 25. The oligonucleotide of claim 20, wherein R 2 and R 3 are C 1 -C 6 alkyl. 26. The oligonucleotide of claim 25, wherein R 2 and R 3 are CH 3 . 27. The oligonucleotide of claim 20, wherein W is S. 28. The oligonucleotide of claim 20, wherein R 1 is C^C g alkylene. 29. The oligonucleotide of claim 28, wherein R 1 is (CH 2 ) 2 . 30. The oligonucleotide of claim 20, wherein 5 R 2 and R 3 are C-^C g alkyl substituted with 1 to 4 NH 2 groupε. 31. The oligonucleotide of claim 30, wherein R 2 and R 3 are -CH 2 CH 2 NH 2 . 10 32. An oligonucleotide having at least one cationic intemucleoside linkage having the structure (II) 20 wherein: W is selected from the group conεiεting of 0, S and Se; X and Y are independently εelected from the group conεiεting of O, S, C(R 4 )R 5 where R 4 and R 5 are 25 independently εelected from the group conεiεting of H and c l _c 6 alkyl, and NR 6 where R 6 iε H or C-^Cg alkyl; Z is selected from the group consisting of 0, S, C-^C g alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C- j ^C g alkyl, with the proviso that _ 0 when W, X and Y are O, Z iε O or S; R 1 is selected from the group consiεting of C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the _ 5 group consisting of H, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or wherein R 2 and R 3 are linked to form a five- or εix- membered alkyl or aryl ring or N-, O- or S-containing heterocycle; and wherein P is a phosphorus atom that may or may not be capable of existing in two distinct stereoisomeric configurations, and further wherein the linkage may or may not be stereouniform. 33. A method for making an oligonucleotide containing at least one cationic intemucleoside linkage having the structure (I) wherein: W is selected from the group consisting of O, S and Se; X and Y are independently selected from the group consisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently εelected from the group consisting of H and C 1 -C 6 alkyl, and NR 6 where R 6 is H or C 1 -C 6 alkyl; Z is selected from the group consiεting of O, S, C- j^ -C 8 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C^C g alkyl, with the proviso that when W, X and Y are 0, Z is O, S or NR 7 ; R 1 is selected from the group consisting of j -C 8 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the group conεiεting of H, C^-C 8 alkyl, C-^Cg alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or R 2 and R 3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, o- or S-containing heterocycle; and wherein P* represents an asymmetric phosphorus atom capable of existing in two distinct εtereoiεomeric configurationε, and further wherein the linkage is stereouniform, said method comprising: (a) syntheεizing a point racemic mixture of protected cationic nucleotide dimerε comprising the cationic intemucleoside linkage and a 3'-0-TBDMS protecting group; (b) optionally resolving the stereoiεomerε in the mixture; (c) deprotecting the cationic nucleotide dimer iεolated in εtep (b) ; (d) converting the deprotected cationic nucleotide dimer provided in εtep (c) into the correεponding 3'-0-CH 2 CH 2 CN phosphoramidite derivative by reaction with Cl-P(N(iPr) 2 ) -O-BCE; and (e) coupling the 3'-0-CH 2 CH 2 CN phosphoramidite derivative to an unprotected hydroxyl- containing terminal unit of an oligonucleotide chain. 34. A nucleic acid hybridization assay comprising: (a) providing a labeled oligonucleotide probe containing at least one cationic intemucleoside linkage; (b) hybridizing the probe to a εingle- stranded analyte nucleic acid to produce a labeled duplex; and (c) detecting the labeled duplexes. 35. The nucleic acid hybridization assay of claim 34, wherein the cationic internucleotide linkage has the εtructure (I) wherein: W is selected from the group consisting of O, S and Se; X and Y are independently selected from the group consiεting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently εelected from the group conεiεting of H and C^Cg alkyl, and NR 6 where R 6 iε H or C 1 -C 6 alkyl; Z iε εelected from the group consisting of O, S, C 1 -C 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 iε H or C^C g alkyl, with the proviεo that when W, X and Y are O, Z iε 0, S or NR 7 ; R 1 iε εelected from the group consisting of c l ~ c 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; R 2 and R 3 are independently selected from the group consiεting of H, C-^C g alkyl, C 1 -C 6 alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or R 2 and R 3 may be linked to form a five- or six-membered alkyl or aryl ring or an N-, O- or S-containing heterocycle; and wherein P* representε an aεymmetric phoεphoruε atom capable of exiεting in two diεtinct εtereoisomeric configurations, and further wherein the linkage is stereouniform. 36. The nucleic acid hybridization asεay of claim 34, wherein the oligonucleotide probe haε alternating cationic and anionic intemucleoside linkages wherein the cationic intemucleoside linkages have the structure (II) wherein: W is selected from the group consisting of O, S and Se; X and Y are independently selected from the group consisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consisting of H and C^Cg alkyl, and NR 6 where R 6 is H or C 1 -C 6 alkyl; Z is selected from the group consisting of 0, S, ^-C 8 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 is H or C 1 -C 6 alkyl, with the proviso that when W, X and Y are O, Z is O or S; R 1 is selected from the group consisting of i~ c 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; and R 2 and R 3 are independently selected from the group consisting of H, C-^C g alkyl, C α -C 6 alkyl subεtituted with 1 to 4 NH 2 groups, and monocyclic aryl, or wherein R 2 and R 3 are linked to form a five- or six- membered alkyl or aryl ring or N-, O- or S-containing heterocycle. 37. The nucleic acid hybridization asεay of claim 34, wherein the cationic intemucleoside linkage has the structure (II) wherein: W is selected from the group consisting of O, S and Se; X and Y are independently selected from the group consisting of O, S, C(R 4 )R 5 where R 4 and R 5 are independently selected from the group consiεting of H and C 1 -C 6 alkyl, and NR 6 where R 6 iε H or C 1 -C 6 alkyl; Z iε εelected from the group conεiεting of O, S, ^-C 8 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene and NR 7 where R 7 iε H or ^-C alkyl, with the proviso that when W, X and Y are O, Z iε 0 or S; R 1 is selected from the group consisting of c l ~ c 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, monocyclic arylene and a bond; and R 2 and R 3 are independently selected from the group consiεting of H, C-^C g alkyl, C 1 -C 6 alkyl substituted with 1 to 4 NH 2 groups, and monocyclic aryl, or wherein R 2 and R 3 are linked to form a five- or six- membered alkyl or aryl ring or N-, O- or S-containing heterocycle.

Inventors: BALASUBRAMANIAM TANJORE NARAYA, HORN THOMAS, LETSINGER ROBERT L, BALASUBRAMANIAM, TANJORE, NARAYANASWAMY, HORN, THOMAS, LETSINGER, ROBERT, L.
Applicants: CHIRON CORP, UNIV NORTHWESTERN, CHIRON CORPORATION, NORTHWESTERN UNIVERSITY
Application year: 1996
Main classification: C07J21
IPC codes: C07H21, C12N15, C12Q1