Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-05
2001-10-16
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S026600
Reexamination Certificate
active
06303312
ABSTRACT:
INTRODUCTION
BACKGROUND
With the explosion of techniques for the synthesis, analysis and manipulation of nucleic acids, numerous new opportunities have arisen in diagnostics and therapeutics. In research there is substantial interest in being able to identify DNA sequences, which may be associated with specific organisms, alleles, mutations, and the like, to understand particular genetic processes, to identify diseases, for forensic medicine, etc. Also, for many purposes, one may wish to modulate the activity of a particular gene, so as to identify the function of a particular gene, the effect of changes in its cellular concentration on the function of the cell, or other cellular characteristic. In therapeutics, one may wish to inhibit the proliferation of cells, such as bacterial, fungal and chlamydia cells, which may act as pathogens, of viruses, of mammalian cells, where proliferation results in adverse effects on the host, or other situation. In vivo, one may provide for reversible or irreversible knock out, so that information can be developed on the development of a fetus, or the effect on the organism of reduced levels of one or more genetic products.
In a number of seminal papers, Peter Dervan's group has shown that oligomers of nitrogen heterocycles can be used to bind to dsDNA. It has been shown that there is specificity in that G/C is complemented by N-methyl imidazole (Im)/ N-methyl pyrrole (Py), C/G is complemented by Py/Im, A/T and T/A are redundantly complemented by Py/Py. In effect, N-methyl imidazole tends to be associated with guanosine, while N-methyl pyrrole is associated with cytosine, adenine, and thymidine. By providing for two chains of the heterocycles, as 1 or 2 molecules, a 2:1 complex with dsDNA is formed, with the two chains of the oligomer antiparallel, where G/C pairs have Im/Py in juxtaposition, C/G pairs have Py/Im, and T/A pairs have Py/Py in juxtaposition. The heterocycle oligomers are joined by amide carbamyl) groups, where the NH may participate in hydrogen bonding with nitrogen unpaired electrons, particularly of adenine. While the complexes were of substantial interest, the binding affinities for the most part were less than about 10
6
M
−1
. Furthermore, the discrimination between a target DNA sequence, and one involving a mismatch was frequently not better than about two-fold. Therefore, for many purposes, the complexes had limited utility.
Improvements in affinity were shown for a cyclic dimer, where the two oligomers were joined at their ends by &ggr;-aminobutyric acid, where the affinity was shown to be enhanced to about 10
9
M
−1
. However, the difference in affinity between the target sequence and single-base mismatch sequences were less than three-fold difference for three different single-base mismatch sequences. This would severely limit the applications for the compound in the presence of a large amount of naturally occurring dsDNA.
Also, for many applications, one wishes to be able to use the sequences with viable cells. There was no showing that these oligomers would be capable of being transported across a cellular membrane to the nucleus and, upon successful transport to the nucleus, they could bind to the chromosomal DNA, where the chromosomal DNA is present as nucleosomes.
Relevant Literature
Wade et al., J.AM.CHEM.SOC., 1992, 114, 8783-8794; Mrkish et al., PROC.NATL.ACAD.SCI. USA, 1992, 89, 7856-7590; Mrkish and Dervan, J.AM.CHEM.SOC., 1993, 115, 2572-2576; Wade et al., Biochemistry, 1993, 32, 11385-11389; Mrkish and Dervan, J.AM.CHEM.SOC., 1993, 115, 9892-9899; Dwyer et al., J.AM.CHEM.SOC., 1993, 115, 9900-9906; Mrkish and Dervan, J.AM.CHEM.SOC., 1994, 116, 3663-3664; Mrkish et al, J.AM.CHEM.SOC., 1994, 116, 7983-7988; Mrkish and Dervan J.AM.CHEM.SOC., 1995, 117, 3325-3332; Cho et al., PROC.NATL.ACAD.SCI. USA, 1995, 92, 10389-10392; Geierstanger, Nature Structural Biology, 1996, 3, 321-324; Parks et al., J.AM.CHEM.SOC., 1996, 118, 6147-6152; Parks et al., J.AM.CHEM.SOC., 1996, 118, 6153-6159; Baird and Dervan, J.AM.CHEM.SOC., 1996, 118, 6141-6146; Swalley et al., J.AM.CHEM.SOC., 1996, 118, 8198-8206; Trauger et al., J.AM.CHEM.SOC., 1996, 118, 6160-6166; Szewczyk et al., J.AM.CHEM.SOC., 1996, 118, 6778-6779; Trauger et al., Chemistry & Biology, 1996, 3, 369-377; Trauger et al., Nature, 1996, 382, 559-561; Kelly et al., PROC.NATL.ACAD.SCI. USA, 1996, 93, 6981-6985; Szewczyk et al., ANGEW.CHEM.INT.ED.ENGL., 1996, 35, 1487-1489; Pilch et al., PROC.NATL.ACAD.SCI. USA, 1996, 93, 8306-8311; White et al., Biochemistry, 1996, 35, 12532-12537.
SUMMARY OF THE INVENTION
Methods and compositions are provided for selectively producing a complex intracellularly at low concentrations, between dsDNA and an oligomer of organic cyclic groups, wherein at least 60% of the cyclic groups are heterocyclics, and at least 60% of the heterocycles have at least one nitrogen annular member. The heterocycles form complementary pairs, where at least two of the nucleotide pairs are preferentially paired with a specific pair of heterocycles. There are at least three complementary pairs of organic cyclic groups in the complex, either as a result of a hairpin turn in a single oligomer, or the complementation between organic cyclic groups of two oligomeric molecules. Usually, a small aliphatic amino acid will be interspersed in or divide what would otherwise be a chain of six or more consecutive organic cyclic groups. To further enhance binding, a terminus may have at least one aliphatic amino acid of from two to six carbon atoms and/or an alkyl chain having a polar group proximal to the linkage of the alkyl chain. By appropriate selection of the target sequence, the complementary pairs, unpaired organic cyclic groups, the aliphatic amino acids, and the polar-substituted alkyl chain, complexes may be formed with high affinity, low dissociation constants, and significant disparities in affinity between the target sequence and single-base mismatches. Modifications of the oligomers are used to provide for specific properties and are permitted at sites which do not significantly interfere with the oligomers positioning in the minor groove. The compositions are found to be able to enter viable cells and inhibit transcription of genes comprising the target sequence, cleave at particular sites, become covalently bonded at specific sites, direct selected molecules to a target site, as well as perform other activities of interest.
The oligomers are combined with cells in culture, in vivo and ex vivo under conditions where the oligomers are transported across the cell membrane to the nucleus, where the oligomers bind to the dsDNA with high affinity. Binding of the oligomers with inhibition of transcription can be used in research, modification of phenotype for change in characteristics of a host, for example, in animal husbandry and breeding, in prophylactic and therapeutic treatment, and the like.
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Brenowitz et al.
Dervan Peter B.
Gottesfeld Joel M.
California Institute of Technology
Houtteman Scott W.
Morrison & Foerster / LLP
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