Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-02-12
2002-04-30
Fredman, Jeffrey (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024310, C536S024320, C536S024500, C536S028520, C536S028530, C435S006120
Reexamination Certificate
active
06380368
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to novel nucleomonomer and oligomer analogs, and to oligonucleotide-based therapy and diagnosis by binding of the oligonucleotide analogs to single or double-stranded nucleic acid target sequences. More specifically, the invention concerns oligomers containing certain 5-substituted pyrimidine base residues and intermediates in their synthesis.
BACKGROUND ART
Sequence-specific binding of oligonucleotides both to single-stranded RNA and DNA and to duplex DNA has been demonstrated. The appropriate sequence recognition for binding to single-stranded targets is well known: the A-T and G-C pairing characteristic of duplex formation has been established as the basis for DNA replication and transcription.
More recently, oligonucleotides have been shown to bind in a sequence-specific manner to duplex DNA to form triplexes. Single-stranded nucleic acid, primarily RNA, is the target molecule for oligonucleotides that are used to inhibit gene expression by an “antisense” mechanism (Uhlmann, E., et al,
Chem Reviews
(1990) 90:543-584; van der Krol, A. R., et al,
Biotechniques
(1988) 6:958-976). Antisense oligonucleotides are postulated to exert an effect on target gene expression by hybridizing with a complementary RNA sequence. In this model, the hybrid RNA-oligonucleotide duplex interferes with one or more aspects of RNA metabolism including processing, translation and metabolic turnover. Chemically modified oligonucleotides have been used to enhance their nuclease stability.
Duplex DNA can be specifically recognized by oligomers based on a recognizable nucleomonomer sequence. Two major recognition motifs have been recognized. In an earlier description of a “CT” motif, protonated cytosine residues recognize G-C basepairs while thymine residues recognize A-T basepairs in the duplex. These recognition rules are outlined by Maher III, L. J., et al.,
Science
(1989) 245:725-730; Moser, H. E., et al.,
Science
(1987) 238:645-650. More recently, an additional motif, termed “GT” recognition, has been described (Beal, P. A., et al,
Science
(1992) 25:1360-1363; Cooney, M., et al.,
Science
(1988) 241:456-459; Hogan, M. E., et al., EP Publication 375408). In the G-T motif, A-T pairs are recognized by adenine or thymine residues and G-C pairs by guanine residues.
In both of these binding motifs, the recognition sequence of the oligomer must align with the complementary sequence of the purine chain of the duplex; thus, recognition, for example, of an A-T pair by a thymine, depends on the location of the adenyl residues along the purine chain of the duplex. An exception to the foregoing is the recent report by Griffin, L. C., et al.,
Science
(1989) 245:967-971, that limited numbers of guanine residues can be provided within pyrimidine-rich oligomers and specifically recognize thymine-adenine base pairs; this permits the inclusion of at least a limited number of pyrimidine residues in the homopurine target.
The two motifs exhibit opposite binding orientations with regard to homopurine target chains in the duplex. In the CT motif, the targeting oligonucleotide is oriented parallel to the target purine-rich sequence; in the GT motif, the oligonucleotide is oriented antiparallel (Beal, P. A., et al.,
Science
(1990) 25:1360-1363).
The efficiency of binding by C residues in CT motif oligomers is reduced as the pH of hybridization is increased. The protonated tautomer of C (C
+
) is the binding competent species in Hoogsteen binding, but is present at only low levels at physiological pH. This is consonant with the pK
a
of cytosine which is 4.25. Base analogs such as 5-methylcytosine, pK
a
4.35, (Lee, J. S. et al.,
Nucleic Acids Res
(1984) 12:6603-6614), 8-oxo-N
6
-methyladenine (Krawczyk, S. H. et al,
Proc Natl Acad Sci
(1992) 89:3761-3764; International Application No. PCT/US91/08811), pseudoisocytidine (Ono, A., et al,
J Org Chem
(1992) 57:3225-3230; International Application No. PCT/US90/03275) or carbocyclic cytidine (Froehler, B. C., et al,
J Am Chem Soc
(1992) 114:8320-8322; U.S. patent application Ser. No. 07/864,873 incorporated herein by reference) have been utilized to obtain binding of CT motif oligomers over an extended pH range.
Sequence-specific targeting of both single-stranded and duplex target sequences has applications in diagnosis, analysis, and therapy. Under some circumstances wherein such binding is to be effected, it is advantageous to stabilize the resulting duplex or triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one approach to prolong stabilization. Sequence-specific recognition of single-stranded DNA accompanied by covalent crosslinking has been reported by several groups. For example, Vlassov, V. V., et al.,
Nucleic Acids Res
(1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleomonomers complementary to target sequences. A report of similar work by the same group is that by Knorre, D. G., et al.,
Biochimie
(1985) 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleomonomer which was capable of activating cleavage (
J Am Chem Soc
(1987) 109:1241-1243). Meyer, R. B., et al.,
J Am Chem Soc
(1989) 111:8517-8519, effect covalent crosslinking to a target nucleomonomer using an alkylating agent complementary to the single-stranded target nucleomonomer sequence. Photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al.,
Biochemistry
(1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Horne, et al.,
J Am Chem Soc
(1990) 112:2435-2437.
Use of N
4
,N
4
-ethanocytosine as an alkylating agent to crosslink to single-stranded and double-stranded oligomers has also been described (Webb and Matteucci,
J Am Chem Soc
(1986) 108:2764-2765;
Nucleic Acids Res
(1986) 14:7661-7674; Shaw, J. P., et al,
J Am Chem Soc
(1991) 13:7765-7766). These papers also describe the synthesis of oligonucleotides containing the derivatized cytosine. Matteucci and Webb, in a later article in
Tetrahedron Letters
(1987) 28:2469-2472, describe the synthesis of oligomers containing N
6
,N
6
-ethanoadenine and the crosslinking properties of this residue in the context of an oligonucleotide binding to a single-stranded DNA.
In a recent paper, Praseuth, D., et al.,
Proc Natl Acad Sci
(
USA
) (1988) 85:1349-1353, described sequence-specific binding of an octathymidylate conjugated to a photoactivatable crosslinking agent to both single-stranded and double-stranded DNA.
In addition, Vlassov, V. V. et al.,
Gene
(1988) 313-322 and Fedorova, O. S. et al.,
FEBS
(1988) 228:273-276, describe targeting duplex DNA with an alkylating agent linked through a 5′-phosphate of an oligonucleotide.
In effecting binding to obtain a triplex, to provide for instances wherein purine residues are concentrated on one chain of the target and then on the opposite chain, oligomers of inverted polarity can be provided. By “inverted polarity” is meant that the oligomer contains tandem sequences which have opposite polarity, i. e., one having polarity 5′→3′ followed by another with polarity 3′→5′, or vice versa. This implies that these sequences are joined by linkages which can be thought of as effectively a 3′-3′ internucleoside junction (however the linkage is accomplished), or effectively a 5′-5′ internucleoside junction. Such oligomers have been suggested as by-products of reactions to obtain cyclic oligonucleotides by Capobionco, M. L., et al.,
Nucleic Acids Res
(1990) 18:2661-2669. Compositions of “parallel-stranded DNA” designed to form hairpins secured with AT linkages using either a 3′-3′ inversion or a 5′-5′ inversion have been synthesized by van de Sande, J. H., et al.,
Science
(1988) 241:551-557. In addition, triple helix formation using oligomers which conta
Froehler Brian
Gutierrez Arnold J.
Jones Robert J.
Mattencci Mark
Pudlo Jeff
Fredman Jeffrey
ISIS Pharmaceuticals Inc.
Woodcock Wahsburn Kurtz Mackiewicz & Norris LLP
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