Peptide nucleic acid precursors and methods of preparing same

Details Peptide nucleic acid precursors and methods of preparing same Peptide nucleic acid precursors and methods of preparing same

2000-01-05

2002-08-13

Shah, Mukund J. (Department: 1624)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S333000, C536S022100, C536S026700, C536S026800, C536S025300, C544S264000, C544S265000, C544S296000, C544S310000

Reexamination Certificate

active

06433134

ABSTRACT:

BACKGROUND OF THE INVENTION
Reagents that selectively bind to DNA or RNA are of significant interest in molecular biology and medicinal chemistry as they may be developed into gene-targeted drugs for diagnostic and therapeutic applications and may be used as tools for sequence-specific modification of DNA. To date, research directed at identifying such reagents has focused primarily upon development of various oligonucleotides and their close analogs having modified backbones, such as, phosphorothioate or methyl phosphonate backbones instead of the natural phosphodiester backbone. These reagents, however, have been found to have serious shortcomings, especially with respect to stability against biological degradation, solubility, cellular uptake properties and ease of synthesis. For these reasons, alternative concepts for oligonucleotide mimics have been attracting interest.
Peptide nucleic acids (PNAs) are a recently developed class of oligonucleotide mimics wherein the entire deoxyribose phosphate backbone has been replaced by a chemically different, structurally homomorphous backbone composed of (2-aminoethyl)glycine units. Despite this dramatic change in chemical makeup, PNAs recognize complementary DNA and RNA by Watson-Crick base pairing. Furthermore, PNAs have been shown to have numerous advantages over DNA and RNA oligomers. For example, PNAs lack 3′ to 5′ polarity and thus can bind in either a parallel or an antiparallel orientation to DNA or RNA (Egholm, M. et al., Nature 365:566, 1993). It has been demonstrated that PNAs can bind double-stranded DNA by invading the DNA duplex and displacing one strand to form a stable D-loop structure (Peffer et al.,
Proc. Natl. Acad. Sci. USA
90:10648, 1993). A further advantage of PNAs is that they are less susceptible to enzymatic degradation (Demidov et al.
Biochem. Pharmacol
. 48:1310, 1994) and bind RNA with higher affinity than analogous DNA oligomers (Norton et al.
Nature Biotechnology
14:615, 1996). Quite advantageously, selective hybridization of PNA to DNA is less tolerant of base pair mismatches than DNA-DNA hybridization. For example, a single base mismatch within a 16 bp PNA-DNA duplex can reduce the T
m
by up to 15° C., compared to 10° C. in the case of a 16 bp DNA-DNA duplex (Egholm, M. et al.
Nature
365:566, 1993). Finally, in at least one example, a PNA molecule has been shown capable of mimicing a transcription factor and acting as a promoter, thus demonstrating the potential use of PNAs as gene-specific activating agents (Mollegaard et al.
Proc Natl Acad Sci USA
91:3892, 1994).
The success of an oligonucleotide analog as an antisense drug requires that the oligonucleotide be taken up by cells in large enough quantities to reach its target at a concentration sufficient to cause the desired effect. Until recently PNAs have shown low phospholipid membrane permeability (Wittung et al.
FEBS Letters
365:27, 1995) and have been reported to be taken up by cells very poorly (Hanvey et al. Science 258:1481, 1992; Nielsen et al.
Bioconiugate Chem
. 5:3, 1994; Bonham et al.
Nucleic Acid Res
. 23:1197, 1995), initially suggesting their potential use as anti-gene and anti-sense agents would be quite limited.
Strategies to improve the cellular uptake of PNAs by conjugating the PNA sequence to a carrier molecule have met with some limited success (Basu et al.
Bioconiugate Chem
. 8:481, 1997). Conjugation of PNA molecules to receptor ligand molecules has increased cellular uptake of the PNA; however, the ability of these receptor ligand-conjugated PNA oligomers to influence biological activity once inside the target cells remains unproven. Further, using such a conjugation strategy permits the PNA oligomers to enter only those cells expressing the particular targeted receptor. Thus, an appropriate ligand molecule would have to be designed for each cell type of interest.
However, recently it has been discovered that unconjugated (aka “naked”) PNA oligomers administered extracellularly can both cross cell membranes (Gray, G. D.
Biochem. Pharmacol
. 53:1465, 1997) and elicit a sequence-specific biological response in living cells (Richelson, E.
FEBS Letters
421:280, 1998). Thus, PNAs possess the following characteristics suggesting they are well suited as therapeutic and diagnostic candidates: cell permeability in vivo; higher specificity and stronger binding to its complementary DNA or RNA than oligonucleotides or their analogs; resistance to enzymes like nucleases and proteases thereby showing long biological half-life; chemical stability over a wide pH range; no action as a primer; and an ability to act as a gene promoter.
Improvements in genomic research have increased the rate of generation of information on the identity, structure and function of a number of human genes, thereby producing a diverse group of novel molecular targets for therapeutic and diagnostic applications. However, gene sequencing and characterization is still a slow and often arduous process, as evidenced by the fact that, to date, only a fraction of the entire human genome has been sequenced. The same advantageous binding and chemical stability properties that make PNAs useful as therapeutics and diagnostics also suggest such compounds will be useful in determining the sequence, structure and/or function of DNA and RNA.
In addition to completely characterizing a gene, the tasks of unraveling the details of the interactions of the gene with its DNA binding proteins and determining the mechanisms whereby such proteins mediate gene expression, replication and transduction of the gene require a great deal of time and effort. Further, understanding the genetic malfunctions of dysfunctional genes that cause the many complex genetic disorders found in man still requires extensive research. Thus, here too, PNAs can be useful.
While PNAs appear to be particularly well-suited for use as diagnostics, therapeutics and/or research tools, identification of appropriate PNAS for a specific purpose can be difficult, time consuming and expensive. For example, identifying which region of a gene should be targeted in order to provide a desired effect, such as blocking transcription thereof, or which region, if any, may be activated to promote transcription thereof, generally requires sequencing most, if not all, of the gene and then testing various PNA fragments complementary thereto.
Recently, combinatorial libraries of random-sequence oligonucleotides, polypeptides and/or synthetic oligomers have been employed to facilitate the isolation and identification of compounds capable of producing a desired biological effect or useful as diagnostics. Compounds so identified may mimic or block natural ligands, may interfere with the natural interactions of the target molecule or may simply be useful as tools for designing and developing other molecules with more desirable properties.
Combinatorial libraries useful in this general application may be formed by a variety of solution-phase or solid-phase methods in which mixtures of different subunits are added in a stepwise manner to growing oligomers, until a desired oligomer size is reached. Alternatively, the library may be formed by solid-phase synthetic methods in which beads containing different sequence oligomers that form the library are alternately mixed and separated with one of a selected number of subunits being added to each group of separated beads at each step. An advantage of this method is that each bead contains only one oligomer species, allowing the beads themselves to be used for oligomer screening (Furka, et al.,
Int. J. Pept. Protein Res
. 37:487-493 (1991); Sebestyen, et al.,
Bioorg. Med. Chem. Letter
3:413-418 (1993).)
Still another approach that has been proposed involves the synthesis of a combinatorial library on spatially segregated arrays (see, Fodor, et al.,
Science
, 251:767-773, 1991). This approach has generally been limited in the number of different library sequences that can be generated.
Because the chance of finding useful ligands increases with the size of the c

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