Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-04
2001-12-04
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C536S022100, C536S025300
Reexamination Certificate
active
06326479
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is related to the field of synthetic polymers. More specifically, this invention relates to methods, kits and compositions suitable for modulating the solubility of synthetic polymers and, in particular, peptide nucleic acid (PNA) oligomers.
2. Description of the Related Art
Peptides and nucleic acids are naturally occurring compositions which are increasingly utilized in research, diagnostic and therapeutic applications. Though naturally occurring peptides and nucleic acids are generally soluble in aqueous solutions, the solubility of individual compositions of differing sequence can vary substantially, with certain compositions exhibiting little or no solubility in aqueous solution. Additionally, the introduction of products and methods for the synthetic production of peptides and nucleic acids has made available sequence variations which are not known to, or may in fact not, exist in nature. The absence of certain biopolymer sequences in nature may at least partially be due to the limited solubility of the composition.
The limited solubility of certain peptide and nucleic acid oligomers can prohibit what would otherwise be a useful research, diagnostic or therapeutic application for that polymer. Therefore, methods and compositions suitable for improving the solubility of peptides and nucleic acids in aqueous solutions may prove essential to the enablement of new technology which utilizes peptides and nucleic acids which otherwise have little intrinsic water solubility. However, compositions which modulate the solubility of synthetic polymers should preferably be simple and achiral since the effectiveness of complex macromolecules such as nucleic acids and peptides in research, diagnostic or therapeutic applications can be adversely affected by the size, complexity or chirality of attached ligands.
Peptide nucleic acids (PNAs) are non-naturally occurring polyamides (also properly characterized as pseudopeptides) which can hybridize to nucleic acids (DNA and RNA) with sequence specificity. (See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,571 and Egholm et al.,
Nature
365: 566-568 (1993)). PNAs are candidates for investigation as alternatives/substitutes to nucleic acid probes in probe-based hybridization assays because they exhibit several desirable properties. In preferred embodiments, PNAs are achiral polymers which hybridize to nucleic acids to form hybrids which are more thermodynamically stable than a corresponding nucleic acid
ucleic acid complex (See: Egholm et al.,
Nature
365: 566-568 (1993)). Being non-naturally occurring molecules, they are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids. Therefore, PNAs should be stable in biological samples, as well as, have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength under conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (See: Egholm et. al.,
Nature
, p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (See: Tomac et al.,
J. Am. Chem. Soc
. 118: 5544-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (See: Egholm et al.,
Nature
, p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay appears to be somewhat sequence dependent (See: Nielsen et al.
Anti
-
Cancer Drug Design
8: 53-65 (1993) and Weiler et al.,
Nucl. Acids Res
. 25: 2792-2799 (1997)). As an additional advantage, PNAs hybridize to nucleic acid in both a parallel and antiparallel orientation, though the antiparallel orientation is preferred (See: Egholm et al.,
Nature
, p. 566).
PNAs are synthesized by adaptation of standard peptide synthesis procedures in a format which is now commercially available. (For a general review of the preparation of PNA monomers and oligomers please see: Dueholm et al.,
New J. Chem
. 21: 19-31 (1997) or Hyrup et. al.,
Bioorganic
&
Med. Chem
. 4: 5-23 (1996)). Labeled and unlabeled PNA oligomers can be purchased (See: PerSeptive Biosystems Promotional Literature: BioConcepts, Publication No. NL612, Practical PNA,
Review
and Practical PNA, Vol 1, Iss. 2) or prepared using the commercially available products.
Limited aqueous solubility and a tendency toward self-aggregation has been a long established and well documented restriction on applications of PNA (See for example: Lee, Morse & Olsvik,
Nucleic Acid Amplification Technologies: Application to Disease Diagnositics
, Chapter 3 by Ørum et al., BioTechniques Book Div. of Eaton Publishing (1997) pp. 29-48, at p. 40, lns. 14-26; Corey, D. R.,
TIBTECH
, 15: 224-229, June (1997) at p. 225, col. 1, ln. 37 to col. 2, ln. 2; p. 226, col. 2, lns. 24-30 and p. 229, col. 1, lns. 14-33; Lesnik et al.,
Nucleosides
&
Nucleotides
, 16: 177-51779 (1997) at p. 1775, lns 1-5; Peyman et al.,
Angew. Chem. Int. Ed. Engl
., 35: 2636-2638 (1996) at p. 2636, col. 1, lns. 13-24; van der Laan et al.,
Tetrahedron Letters
, 37: 7857-7860 (1996) at p. 7857, lns. 1-10; Bergman et al.,
Tetrahedron Letters
, 36: 6823-6826 (1995) and Egholm et al.,
J. Am. Chem. Soc
., 114: 1895-1897 (1992). The solubility properties of PNA oligomers in aqueous solution is known to be very sequence dependent. Purine-rich PNA oligomers are known to be particularly difficult to purify and/or characterize at least partially due to their limited solubility. Similarly, the solubility of PNA tends to decrease as the polymer length increases thereby resulting in a preference for shorter PNAs. Self-aggregation is another property which tends to limit the utility of PNA oligomers. Because certain PNA oligomers cannot be adequately purified or characterized, there are presently a large number of potentially useful PNA sequence variations which are unavailable for evaluation in research, diagnostic or therapeutic applications.
By way of example, the product literature of a commercial vendor of custom PNA oligomers states “For most applications an oligomer of 12-15 is optimal. Longer PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterize” (See: Guidelines For Sequence Design of PNA Oligomers: PerSeptive Biosystems, Inc. Promotional Literature; 1997-1998 Synthesis Products Catalog, col. 2, lns. 6-11, p. 45). Additionally, this document sets forth several rules for the design of a PNA oligomer which will avoid these limitations. Under the heading “Specific Design Rules” (col. 3, lns. 1-18), the text reads “Length: We will not synthesize any sequences with more than 18 bases, not including linkers, amino acids and labels. Purine Content: Purine rich PNA oligomers tend to aggregate and have low solubility. To avoid that follow these specific guidelines: 1. Of any stretch of 10 bases in the sequence do not have more than 6 purines 2. NO more than 4-5 purines in a row, specifically no more than 3 G's in a row”. The vendor suggests that one consider analyzing the other strand if it is otherwise impossible to comply with the limitations set forth in guidelines 1 and 2.
A number of modifications have been made to peptide nucleic acids (PNAs) in order to improve their aqueous solubility or minimize polymer self-aggregation. A commonly used modification of PNA which was first used by the inventors involves the incorporation of one or more positively charged terminal lysine residues (See: Egholm et al.,
J. Am. Chem. Soc
., 114: 1895-1897 (1992) at p. 1896, col. 1, ln. 23 to col. 2, ln. 2). The inventors of PNA, as well as others, have also advocated the preparation of PNAs having backbone modifications which comprise one or more alkyl amine groups (See: U.S. Pat. No. 5,719,262 and Lesnik et al.,
Nucleosides
Coull James M.
Gildea Brian D.
Boston Probes, Inc.
Gildea Brian D.
Riley Jezia
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