Higher order structure and binding of peptide nucleic acids

Abstract

Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest, transcription initiation, and site specific cleavage of nucleic acids.

Description

[0001] This patent application is related to the patent application Ser. No. ______ entitled Double-Stranded Peptide Nucleic Acids, filed herewith bearing attorney docket number ISIS-1108. This patent application also is a continuation-in-part of patent application Ser. No. 08 / 054,363, filed Apr. 26, 1993, which is a continuation-in-part of application PCT EP92 / 01219, filed May 19, 1992 and published Nov. 26, 1992 as WO 92 / 20702. The entire contents of each of the foregoing patent applications are incorporated herein by reference.[0002] This invention is directed to compounds that form triple-stranded structures with single-stranded and double-stranded nucleic acids. It is further directed to the use of such compounds to cause strand displacement in double-stranded nucleic acids. The invention further is directed to processes for modifying double-stranded nucleic acid utilizing such strand displacement. Such processes for modifying double-stranded nucleic acids include cleavage of t...

AI Technical Summary

Benefits of technology

[0023] We have further surprisingly found that the PNA compounds are able to form triple helices with dsDNA. We have found that PNA compounds are able to form triple helices with RNA and ssDNA. The resulting triplexes, e.g., (PNA).sub.2 / DNA or (PNA).sub.2 / RNA, surprisingly have very high thermal stability. It has been found that the PNA binds with a DNA or RNA in either orientation, i.e., the antiparallel orientation where the amino-terminal of the PNA faces the 3' end of the nucleic acid or the parallel orientation where the amino-terminal of the PNA faces the 5' end of the nucleic acid. PNAs are able to form triple helices wherein a first PNA strand binds with RNA or ssDNA and a second PNA strand binds with the resulting double helix or with the first PNA strand.

[0127] In some cases it may be of interest to attach ligands at either terminus (Q, I) to modulate the binding characteristics of the PNAS. Representative ligands include DNA intercalators, which improve dsDNA binding or basic groups, such as lysine or polylysine, which strengthen the binding of the PNA due to electrostatic interaction. To decrease electrostatic repulsion charged groups such as carboxyl and sulfo groups could be used. The design of the synthons further allows such other moieties to be located on non-terminal positions. Oligonucleotides and / or oligonucleoside can be covalently bound to terminal positions Q or I to form chimeras containing PNA portions and oligonucleotide and / or oligonucleoside portions. Nucleosides and / or nucleotides (mono, di or tri-phosphates) also can be attached to the terminal positions.

[0131] Triple helix formation, wherein an oligonucleotide is triplexed to a dsDNA, is believed to be the only means known in the art for sequence-specific recognition of dsDNA. However, triple helix formation is largely limited to recognition of homopurine-homopyrimidine sequences. Triplexing with strand displacement using PNAS of this invention is superior to oligonucleotide-dsDNA triple helix recognition in that it may allow for recognition of any sequence by use of the four natural bases.

[0137] As an illustrative example of use as an artificial restriction enzyme, the targets are cloned within a pUC19 polylinker and the plasmids are linearized with the Cfr10I restriction enzyme. In the linear DNA, the resulting polylinker region is in the middle (1.33 kb from one end and 1.36 kb from the other). A pT10 plasmid carrying a known sequence is then inserted in the unique BamHl site in the polylinker. This is complexed with a complementary PNA compound and subjected to treatment with S.sub.1 nuclease. Upon nuclease treatment, a significant fraction of DNA is cut, as shown by gel electrophoresis of the resulting fragments. Cross reactivity experiments show that the targeting is sequence-specific; only corresponding PNAs mediated cutting of the targets. The yield of digested molecules is high and one can reach the quantitative digestion with increasing exposure to nuclease.

[0138] The results of such treatment demonstrate a novel method for sequence-selective cleavage of double-stranded DNA. If two closely positioned homo-pyrimidine stretches can be identified (as for example 7-10 bases, preferably on opposite strands), this can be done by synthesizing pairs of PNAs complementary to two parts of this DNA sequence and separated by several base pairs. If this strand displacement binding mode is extended to include PNAs recognizing DNA sequences containing thymine and cytosine, this strategy allows targeting and specific cleavage of any desired sequence of 10-20 base pairs. In effecting such cleavage, the high stability of PNA-DNA complexes consisting of two PNA strands (one "Watson-Crick" and one "Hoogsteen") and one DNA strand must be considered, as must the sequence of the "Hoogsteen-like" strand.

[0145] In our studies we have shown that homo-pyrimidine PNA binds strongly to ssDNA, dsDNA or RNA with 2:1 stoichiometry (triplexing) and effects stable strand displacement complexes with dsDNA. Further, purine-pyrimidine PNA binds to Watson-Crick complementary oligonucleotides, either RNA or DNA, with 1:1 stoichiometry. Utilizing these findings, one embodiment of this invention is directed to use of PNA compounds as effective inhibitors of the elongation activity of RNA and DNA polymerases. As contrasted to oligonucleotides, this allows use of PNA compounds to target genes not only at promoter or regulatory regions of the genome but to other regions as well. Furthermore, only decamer targets are required for efficient binding of the PNA to dsDNA. Consequently, it is easier to identify suitable PNA targets as compared to oligonucleotide triple helix targets.

Problems solved by technology

However, there are a number of drawbacks associated with oligonucleotide triple helix formation.

For example, triple helix formation generally has only been obtained using homopurine sequences and requires unphysiologically high ionic strength and low pH.

Whether used as antisense reagents or a triplexing structures, unmodified oligonucleotides are unpractical because they have short in vivo half-lives.

However, restriction enzymes have been identified and isolated only for a small subset of all possible sequences within these constraints.

Images