Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2002-01-22
2004-12-14
Epps-Ford, Janet L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S026130, C536S027600
Reexamination Certificate
active
06831072
ABSTRACT:
TECHNICAL FIELD
The invention relates to novel oligomer analogs and to oligonucleotide-based diagnostics by binding of the oligomers to single and double-stratided nucleic acids target sequences. More specifically, the invention concerns oligomers containing 8-aminopurine base residues and their triple-helix stabilization properties.
BACKGROUND OF THE INVENTION
It has been shown that oligonucleotides could bind to homopurine-homopyrimidine sequences of double stranded DNA by forming triple helices. The formation of nucleic acid triple helices offers the possibility of designing sequence-specific DNA binding molecules, which may have important uses as diagnostic tools as well as therapeutic treatments. For example, triple helices are used for the extraction and purification of specific nucleotide sequences, control of gene expression, mapping genomic DNA, detection of mutations in homopurine DNA sequences, site-directed mutagenesis, triplex-mediated inhibition of viral DNA integration, nonenzymatic ligation of double-helical DNA and quantitation of polymerase chain reactions (for reviews see references 1-3).
One of the problems for the development of applications based on triple helix formation is the low stability of triple helices especially at neutral pH. Another problem associated with the use of triplex forming oligonucleotides (TFO) is the presence of interruptions in the homopurine-homopyrimidine tracks. In order to overcome this problem a lot of effort has been put into the design and preparation of modified oligonucleotides in order to enhance triple helix stability. See references 39 and 40. One of the most successful modifications is to replace natural bases with some modified bases such as 5-methylcytidine, 5-bromouracil, 5-aminouracil, N
4
-spermnine-5-methylcytidine, or 5-methyl-2,6(1H,3H)-pyrimidinedione.
The most studied type of triple helix formation is the so-called purine: pyrimidine: pyrimidine motif. (FIG.
1
). In this motif, the purine: pyrimidine strands correspond to the target double-stranded DNA sequence (known as the Watson-Crick purine and pyrimidine strands) and the Hoogsteen strand is a pyrimidine strand used for the specific recognition of the double-stranded DNA. See U.S. Pat. Nos. 5,422,251 and 5,693,471. For these reasons, most of the base analogues studied for triple helix stabilization are modified pyrimidines located at the Hoogsteen strand, though there are some recent disclosures of purine analogs. For example, see U.S. Pat. Nos. 5,739,308; 5,645,985; and 5,594,121.
In order to obtain a triplex, in some occasions, purine residues are concentrated on one chain and are linked to a pyrimidine chain of inverted polarity. By “inverted polarity” is meant that the oligomer contains tandem sequences which have opposite polarity, i.e. one segment or region of sequences 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 as a
3′-3
′ or a 5′-5′ internucleotide junction. Such oligomers named “parallel-stranded DNA” have been synthesized See References 34, 42 and 43.
Recent results have shown that the introduction of an amino group at position 8 of adenine increases the stability of triple helix due to the combined effect of the gain in one Hoogsteen purine-pyrimidine H-bond, (see references 4-6) and the ability of the amino group to be integrated into the “spine of hydration” located in the minor-Major groove of the triplex structure (FIG.
2
). (See references 4-7). A similar behavior has been observed with 8-amino-2-deoxyguanosine and 8-amino-2′-deoxyinosine (FIG.
2
). (See reference 11). The preparation and the characterization of the binding properties of oligonucleotides containing 8-aminopurines has been described, but these oligonucleotides can not be directly used for the specific recognition of double-stranded DNA sequences because the modified bases are purines that are in the target sequence and not in the Hoogsteen strand used for the specific recognition of double-stranded DNA.
Synthetic oligonucleotides probes have been proven very useful in the detection of cloned DNA sequences. When a partial protein sequence is available, a mixture of oligonucleotides presenting all possible DNA sequences can be successfully used as a probe or as PCR or sequencing primers for screening of cloned DNA (or amplification of DNA. See reference 44. The mixed probe approach may have two principal drawbacks when the complexity of the mixture is very high. First, the oligonucleotide probes must, for reasons of practicality, be synthesized together on the same support. Thus, the products of the synthesis can never be adequately characterized. Second, since the exact coding sequence is not known, it is difficult to set appropriate stringent conditions for the hybridisation and subsequent washings. A universal baseone that could base pair equally well with any of the four natural basesould resolve these two difficulties. A number of compounds have been tested as possible universal bases, with being 2′-deoxyinosine one of the most successfully used. See references 45-50.
Other molecular biological techniques, such as selective restriction of nucleic acids and target detection, can be improved. Though current techniques are adequate, there are inherent problems in the amplification steps. The PCR system will amplify any DNA added to the mixture, regardless of whether it contains the correct target sequence. If DNA fragments are selected based only on the size of the fragment that is created by the restriction enzymes, then the target sequence may be missed entirely. Additionally, if the restriction enzymes become contaminated, there is no assurance that the correct sequences are being restricted and that the target sequence is being selected. Obtaining and maintaining purified nucleases is often problematic in laboratory settings and is even more of a problem in automated systems.
Thus, methods and compositions are needed that are capable of specifically selecting target nucleic acid sequences that do not require amplification or that can be used with amplification techniques but provide for more target specific amplification. Additionally, what is needed are compositions and methods that require less use of enzymes.
Though several techniques are currently available for modification of nucleic acid structures, what is needed are compositions and methods for binding to specific target regions of DNA or RNA sequences. Specifically what is needed are triplex structures that are stabile at neutral pHs and modified oligonucletides that can bind to specific sequences in a target nucleic acid to form triple helix structures. Modified oligonucleotides that can be used for synthesis of oligonucleotides in the 5′ to 3′ direction, reverse of the normal 3′ to 5′ synthesis direction are also needed.
What is also needed are spacer arms that can link oligonucleotides in 5′ to 5′ orientation or 3′ to 3′ orientation. Particularly needed are simple and economic methods for the synthesis of such spacer arms and such paired oligonucleotide structures.
Compositions and methods for incorporation of modified nucleic acid bases are also needed. What is particularly needed are methods and compositions comprising a base labeled with active compounds, such as intercalating agents, photoreactive agents and cleavage agents that are attached to the base through a linker arm.
SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions of nucleic acid structures that are used for detection of specific nucleic acid sequences. One of the embodiments of the present invention comprises compositions and methods for the preparation of oligonucleotides carrying modified nucleic acids, such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine, that are connected 3′ to 3′ or 5′ to 5′ (head-to-head or tail-to-tail) to a Hoogste
Eritja Ramón
García Ramón Güimil
Buchanan & Ingersoll PC
Cygene, Inc.
Epps-Ford Janet L.
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