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Higher order structure and binding of peptide nucleic acids

a peptide nucleic acid, higher order technology, applied in the direction of peptides, peptide/protein ingredients, organic chemistry, etc., can solve the problems of unpractical unmodified oligonucleotides, unphysiologically high ionic strength and low ph, and generally the formation of triple helixes, so as to facilitate the identification of suitable pna targets, stable strand displacement complexes, and certain effects on transcription

Inactive Publication Date: 2005-03-03
IONIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

PNAs provide high thermal stability and specificity in binding to DNA and RNA, enabling efficient sequence-specific cleavage and modulation of gene expression, overcoming the limitations of existing oligonucleotides by enhancing stability and membrane penetration.

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.

Method used

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  • Higher order structure and binding of peptide nucleic acids

Examples

Experimental program
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Effect test

example 2

[0158] Site-Specific S.sub.1 Nuclease Digestion of the pT10 Plasmid Linearized with Cfr10I Restriction Enzyme and Complexed with PNA (a Double-Stranded Target)

[0159] PNA H-T.sub.10-LysNH.sub.2 (SEQ ID NO:2) was synthesized as described in Example 1. A pT10 plasmid was prepared from the pUC19 plasmid by inserting the dA.sub.10 / dT10 (SEQ ID NO:3) sequence into the BamH1 site of the polylinker as per the method of Egholm, M et al., J.A.C.S. 1992 114:1895-1897. The pT10 plasmid was linearized with Cfr10I restriction enzyme in the unique site. To form complex with the PNA, about 0.1 .mu.g of the linearized plasmid was incubated with 2 o.u. / ml of PNA in 3 .mu.l of the TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 7.4) at 37.degree. C. To perform the S.sub.1 nuclease reaction, 10 .mu.l of Na-Acetate buffer (33 mM NaAc; 50 mM NaCl; 10 mM ZnSO.sub.4; 0.5% of glycerol, pH 4.6) and 145 units of S.sub.1 nuclease (Sigma) were added and incubated for various time periods at room temperature. The react...

example 3

[0165] Site-Specific S.sub.1 Nuclease Digestion of the pT9C Plasmid Linearized by CfrlOl Restriction Enzyme and Complexed with PNA H-T.sub.5CT.sub.4-LysNH.sub.2

[0166] This example illustrates the use of one molecule of PNA hybridized to a target DNA to define a restriction site.

[0167] PNA 1, 2 and 3 were synthesized as Example 1 and the results are shown in FIG. 2. A pT9C plasmid carried the insert:

3 5'-A.sub.5GA.sub.4GTCGACA.sub.5GA.sub.4-3' (SEQ ID NO: 30) 3'-T.sub.5CT.sub.4CAGCTGT.sub.5CT.sub.4-5',

[0168] cloned in the Sal1 site of the pUC19 polylinker. The pT9C-5 plasmid carried the single insert A.sub.5GA.sub.4 / CT.sub.5 cloned in the same site. The PNA-DNA complexes were prepared as described in Example 2 with the only difference being the duration of the incubation was 2 hours. Digestion by 30 units of the S.sub.1 nuclease was performed in the same buffer as described in Example 2 with two exceptions, lanes 2 and 4. In lane 2, 15 units of the enzyme were used, whereas in lane 4...

example 4

[0170] Site Specific S.sub.1 Nuclease Cleavage of the Plasmids pT9C-5, pT9CT9C and pT9CA9GKS (Linearized with Sca1) Targeted by PNA T.sub.4CT.sub.5-LysNH.sub.2

[0171] This example illustrates the use of two molecules of PNA bound to opposite strands of a DNA target. A further plasmid pT9CA9GKS was produced by cloning the insert GTCGACA.sub.5GA.sub.4GTCGACT.sub.4CT.sub.5-GTCGAC (SEQ ID NO:32) into pUC19 at the Sal I site and linearizing with Sca I restriction enzyme. The linearized plasmid has two hybridization sites for PNA H-T.sub.4CT.sub.5-Lys NH.sub.2 (SEQ ID NO:8) on opposite strands spaced by six base pairs. The protocol of Example 3 was use except the samples were treated using 1 U / .mu.l of S.sub.1 and 15 min incubation at 37.degree. C. The results are shown in FIG. 3 Lanes 1-4: pT9C-5; lanes 5-8: pT9CT9C; lanes 9-12: pT9CA9GKS. Lanes 1, 5 & 9: no PNA; lanes 2, 6 & 10: 50 .mu.M; lanes 3, 7, 11: 500 uM; lanes 4, 8, 12: 5 mM.

EXAMPLE 5

[0172] Use of PNA / Nuclease to Cut Selectively ...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07H21/00C07K14/00C12N15/113C12Q1/68
CPCA61K38/00B82Y5/00C07H21/00C07K14/003C12N15/113C12N2310/15C12Q1/6813C12N2310/3181C12Q2525/107C12Q2537/119
Inventor ECKER, DAVID J.BUCHARDT, OLEEGHOLM, MICHAELNIELSEN, PETER E.BERG, ROLF H.MOLLEGAARD, NIELS E.
Owner IONIS PHARMA INC
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