Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Patent
1993-09-22
1997-11-04
Marschel, Ardin H.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
435 691, 4351723, 436501, 536 231, 536 241, 536 243, 536 2431, 536 2432, 536 2433, A61K 4800
Patent
active
056839855
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to novel oligodeoxynucleotides and novel oligonucleotides, and the use thereof to modulate the production of selected messenger or other cellular RNAs.
BACKGROUND OF THE INVENTION
The rate of transcription of many genes depends on the interaction of control proteins (e.g., transcription factors, repressors, and the like) with specific short DNA sequences, generally located close to the promoter. The CREB protein (also referred to as CRE-BP), for example, binds tightly to double-stranded DNA containing the sequence 5'-TGACGTCA-3'.
Double stranded DNA containing such a target sequence can be introduced into the system as a decoy, diverting control proteins from their endogenous DNA target. By diverting the control proteins from their endogenous target, the regulatory effects of such proteins can be altered.
Double stranded DNAs containing such a target sequence are typically prepared by first synthesizing the two complementary oligonucleotide strands, and then hybridizing them together. Introduction of such double stranded DNAs into whole cells, as will be required for many therapeutic applications, will be useful only if the construct is reasonably stable under physiological conditions under which cells remain viable. For example, if the sequence length of the double stranded DNA is insufficient, the two strands will tend to dissociate. In addition, relatively short DNA sequences will be particularly prone to nuclease digestion by enzymes in the growth medium.
Therefore, it would be desirable to develop DNA double-helixes which contain target sequences which bind to control proteins, and which are stable to physiological conditions which would otherwise degrade (and inactivate) such DNA.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, we have developed improved DNA structures which contain target sequences which bind to control proteins. The structures of the present invention are stable with respect to strand separation and to enzyme-mediated degradation, and are effective as decoys for control proteins, thereby enabling one to modulate the transcriptional control normally exerted by such control proteins.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic diagram of a hairpin DNA of the present invention.
FIG. 2 is a schematic diagram of a dumbbell DNA of the present invention.
FIGS. 3A-3F are schematic diagrams of several modified forms of the dumbbell DNA of the present invention.
FIG. 4 is a schematic diagram of covalently bound DNA of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a composition comprising an oligonucleotide comprising, reading from the 5'-end of said oligonucleotide: residues, or analogs thereof; wherein said first segment, when hybridized with its complement, forms at least one transcription control recognition sequence of at least 6 nucleotides, nucleotide residues, or analogs thereof, sufficient to allow the formation of a first loop structure between said first segment and the third segment, and nucleotide residues, or analogs thereof, wherein said third segment is substantially the complement to said first segment.
In accordance with another embodiment of the present invention, there is provided a composition comprising a double-stranded DNA fragment, wherein said DNA fragment contains at least one transcription control recognition sequence of at least 6 nucleotide base pairs, and wherein one strand of said DNA fragment is attached to the other strand by means of at least one linker that is covalently bound to each of the strands of said DNA.
In accordance with yet another embodiment of the present invention, there is provided a method to modulate the transcription of products which are subject to regulation by transcription control recognition sequences, said method comprising administering a therapeutically effective amount of at least one of the above-described compositions to a subject. In accordance with
REFERENCES:
patent: 4582789 (1986-04-01), Sheldon, III et al.
Bielinska et al., "Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides" Science 250:997-1000 (1990).
Clusel et al., "Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbell oligonucleotides" Nucleic Acids Research 21(15) 3405-3411 (1993).
Erie et al., "A Dumbbell-Shaped, Double-Hairpin Structure of DNA: A Theromodynamic Investigation" Biochemistry 26(22):7150-7159 (1987).
Marky et al., "Loop formation in polynucleotide chains. I. Theory of hairpin loop closure" Chemical Abstracts Abstract No. 48885t, vol. 98(7), p. 265, col. 1 (1983).
Sriprakash et al., "Hairpin extension: a general method for the improvement of sensitivity of oligonucleotide probes" Chemical Abstracts Abstract No. 18772y, vol. 111(3), p. 176, col. 2 (1989).
van de Sande et al., "Parallel Stranded DNA" Science 241:551-557 (1988).
Chu et al. (1989) Nucleic Acids Research, vol. 17, No. 12, pp. 4783-4798.
Chu Barbara Chen Fei
Orgel Leslie
Marschel Ardin H.
Reiter Stephen E.
The Salk Institute for Biological Studies
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