Peptide nucleic acid monomers and oligomers

Details Peptide nucleic acid monomers and oligomers Peptide nucleic acid monomers and oligomers

1998-05-22

2003-10-14

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C435S006120, C536S023100, C536S024300, C536S024310, C536S024320, C514S045000, C514S049000, C530S332000, C530S323000

Reexamination Certificate

active

06632919

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to oligomeric compounds and their constituent monomers, especially peptide nucleic acid (PNA) oligomers and monomers. The peptide nucleic acid oligomers are useful for forming triple helix (triplex) structures with nucleic acids with increased binding specificity. In one aspect of the present invention novel PNA oligomers have increased specificity for thymidine and deoxyuridine in triplex structures.
BACKGROUND OF THE INVENTION
Peptide nucleic acids are useful surrogates for oligonucleotides in binding to both DNA and RNA. See Egholm et al.,
Nature
, 1993, 365, 566-568 and references cited therein).
PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as evidence by their higher melting temperatures (Tm). This high thermal stability has been attributed to the neutrality of the PNA backbone, which does not encounter the charge repulsion present in DNA or RNA duplexes. The neutral backbone of the PNA also renders the Tms of PNA/DNA(RNA) duplexes practically independent of salt concentration. Thus the PNA/DNA duplex offers a further advantage over DNA/DNA duplex interactions which are highly dependent on ionic strength. Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)
2
/DNA(RNA) triplexes of high thermal stability (see, e.g., Nielsen, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 9677).
In addition to increased affinity, PNA has also been shown to bind to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex there is seen an 8 to 20° C. drop in the Tm. This magnitude of a drop in Tm is not seen with the corresponding DNA/DNA duplex with a mismatch present. See Egholm, M., et al.,
Nature
1993 365 p. 566.
The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti-parallel when the DNA or RNA strand in a 5′ to 3′ orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5′ end of the DNA or RNA and amino end of the PNA is directed towards the 3′ end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are in reverse orientation with respect to the 5′-3′ direction of the DNA or RNA.
Because of their properties, PNAs are known to be useful in several different applications. In particular, PNAs have been used to form duplexes and triplexes with complementary RNA or DNA (see e.g., Knudsen et al.,
Nucleic Acids Res
., 1996, 24, 494-500; and Nielsen et al.,
J. Am. Chem. Soc
., 1996, 118, 2287-2288). Additionally, several review articles have recently been published in this area. See e.g., Hyrup et al.,
Bioorganic
&
Med. Chem
., 1996, 4, 5-23; Nielsen, “Peptide nucleic acid (PNA): A lead for gene therapeutic drugs,” in Trainor (Ed.),
Perspectives Drug Disc. Des
., 1996, 4, 76-84.
Since PNAs have stronger binding and greater specificity than oligonucleotides, they are of great utility as probes in cloning, blotting procedures, and in applications such as fluorescence in situ hybridization (FISH). Homopyrimidine PNAs are used for strand displacement in homopurine targets. The local triplex inhibits gene transcription. Additionally, the restriction sites that overlap with or are adjacent to the D-loop will not be cleaved by restriction enzymes. The binding of PNAs to specific restriction sites within a DNA fragment can inhibit cleavage at those sites. Such inhibition is useful in cloning and subcloning procedures. Labeled PNAs are also used to directly map DNA molecules by hybridizing PNA molecules having a fluorescent or other type of detectable label to complementary sequences in duplex DNA using strand invasion.
PNAs also have been used to detect point mutations in PCR-based assays (PCR clamping). In PCR clamping, PNA is used to detect point mutations in a PCR-based assay, e.g. the distinction between a common wild type allele and a mutant allele, in a segment of DNA under investigation. Typically, a PNA oligomer complementary to the wild type sequence is synthesized and included in the PCR reaction mixture with two DNA primers, one of which is complementary to the mutant sequence. The wild type PNA oligomer and the DNA primer compete for hybridization to the target. Hybridization of the DNA primer and subsequent amplification will only occur if the target is a mutant allele. With this method, the presence and exact identity of a mutant can be determined.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics. For many uses, the oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express activity.
PCT/EP/01219 describes novel peptide nucleic acid (PNA) compounds which bind complementary DNA and RNA more tightly than the corresponding DNA. It is desirable to append to these compounds groups which modulate or otherwise influence their activity or their membrane or cellular transport. One method for increasing such transport is by the attachment of a pendant lipophilic group.
The synthesis of peptide nucleic acids via preformed monomers has been described in International patent applications WO 92/20702 and WO 92/20703, the contents of each of which are incorporated herein by reference in their entirety. Recent advances have also been reported on the synthesis, structure, biological properties, and uses of PNAs. See for example WO 93/12129 and U.S. Pat. No. 5,539,083 to Cook et al., Egholm et al.,
Nature
, 1993, 365, 566-568, Nielsen et al.,
Science
, 1991, 254, 1497-1500; and Egholm et al.,
J. Am. Chem. Soc
., 1992, 114, 1895-1897. Peptide nucleic acids also have been demonstrated to effect strand displacement of double stranded DNA (see Patel, D. J.,
Nature
, 1993, 365, 490-492). The contents of each of the foregoing patents and publications are incorporated herein by reference in their entirety.
Triple helix formation by oligonucleotides has been an area of intense investigation since sequence-specific cleavage of double-stranded deoxyribonucleic acid (DNA) was demonstrated by Moser et al.,
Science
, 1987, 238, 645-650. Triplex-forming oligonucleotides are believed to be of potential use in gene therapy, diagnostic probing, and other biomedical applications. See e.g., Uhlmann et al.,
Chemical Reviews
, 1990, 90, 543-584.
Pyrimidine oligonucleotides have been shown to form triple helix structures through binding to homopurine targets in double-stranded DNA. In these structures the new pyrimidine strand is oriented parallel to the purine Watson-Crick strand in the major groove of the DNA, and binds through sequence-specific Hoogsteen hydrogen bonds. The sequence-specificity is derived from thymine recognizing adenine (T:A-T) and protonated cytosine recognizing guanine (C
+
:G-C). See Best et al.,
J. Am. Chem. Soc
., 1995, 117, 1187-1193). In a less well-studied triplex motif, purine-rich oligonucleotides bind to purine targets of double-stranded DNA. The orientation of the third strand in this motif is anti-parallel to the purine Watson-Crick strand, and the specificity is derived from guanine recognizing guanine (G:G-C) and thymine or adenine recognizing adenine (A:A-T or T:A-T). See Greenberg et al.,
J. Am. Chem. Soc
., 1995, 117, 5016-5022.
Homopyrimidine PNAs form highly stable PNA:DNA-PNA complexes with complementary oligonucleotides. The formation of triple helix structures involving two PNA strands and one nucleotide strand has been previously reported in U.S. patent application Ser. No. 08/088,661, filed Jul. 2, 1993, entitled Double-Stranded Peptide Nucleic Acids, the contents of which are incorpora

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