Berg, Rolf Henrik

Buchardt, Ole

Ecker, David J.

Engholm, Michael

Møllegaard, Niels E.

Nielsen, Peter E.

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

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Downloaded from orbit.dtu.dk on: Aug 12, 2016Cleaving Double-Stranded DNA with Peptide Nucleic AcidsEcker, David J.; Buchardt, Ole; Engholm, Michael; Nielsen, Peter E.; Berg, Rolf Henrik; Møllegaard, NielsE.Publication date:1997Link to publicationCitation (APA):Ecker, D. J., Buchardt, O., Engholm, M., Nielsen, P. E., Berg, R. H., & Møllegaard, N. E. (1997). Patent No.US5641625.United States Patent [191 Ecker et al. [54] CLEAVING DOUBLE-STRANDED DNA WITH PEPTIDE NUCLEIC ACIDS [75] Inventors: David J. Ecker, Leucadia. Calif.; Ole Buchardt, Vaerlose, Denmark; Michael Egholm, Fredriksberg, Denmark; Peter E. Nielsen, .Hjortevanget 509, DK 2980 Koddedal, Denmark; Rolf H. Berg, Rungsted Kyst, Denmark; Niels E. Mollegaard, Virum, Denmark [73] Assignees: ISIS Pharmaceuticals, Inc., Carlsbad, Calif.; Peter E. Nielsen, Koddedal, Denmark [21] Appl. No.: 88,658 [22] Filed: Jul. 2, 1993 Related US. Application Data [63] Continuation-impart of Ser. No. 54,363, ?led as PCI‘IEP92/ 01219, May 19, 1992, published as WO92/20702, Pat. No. 5,539,082. [51] Int. (:1.6 ..................................................... .. C12Q 1/68 [52] us. Cl. ............................................... .. 435/6; 536/243 [58] Field of Search ............................... .. 435/6, 4, 91.42, 435191.51; 536/243, 24.5 [56] References Cited U.S. PATENT DOCUIVIENTS 5,134,066 7/1992 Rogersetal. .......................... .. 435/91 FOREIGN PATENT DOCUMENTS WO92/20703 of 0000 WlPO . OTHER PUBLICATIONS Demidov et al., Nuc. Acids Res., 21(l9):2103—2107, May 11, 1993. Egholm, J. Chem. Soc., Chem. Commun, pp. 800-801, May 1993. McQndy et al., “Deoxyoligonucleotides with Inverted Polarity: Synthesis and Use in Triple-Helix Formation,” Nucleosides and Nucleotides, 10:287-290, 1991. Nielsen et al., “Sequence-Selective Recognition of DNA by Strand Displacement with a ThymineqSubstituted Polya mide,” Science, 254:1497-1500, 1991. Foehler et al., “Oligodeoxynucleotides Containing C—5 Pro pyne Analogs of 2’—Deoxyuridine and 2’-Deoxycytidine,” Tetrahedron Letters, 33:5307-5310, 1992. Sagi et al., “Base-Modi?ed Oligodeoxynucleotides. I. Effect of 5-Alkyl, 5-(1-Alkenyl) and 5-(1-Alkynyl) Substitution of teh Pyrirnidines on Duplex Stability and Hydrophobic ity,” Tetrahedron Letters, 34:2191-2194, 1993. 5,641,625 Jun. 24, 1997 US005641625A [11] Patent Number: [45] Date of Patent: Egholrn et al., “Peptide Nucleic Acids (PNA). Oligonucle otide Analogues with and Achiral Peptide backbone,” J. Am. Chem. Soc., 114:1895-1897, 1992. Spalholtz et al., “Bovive papillomavirus Transcriptional Regulation: Localization of the E2-Responsive Elements of the Long Control Region,” J. ViroL, 61:2128-2137, 1987. Dubochet et al., “A New Preparation Method for Dark-Field Electron Microscopy of Biomacromolecules,” J. Ultra struct. Res., 35:147-167, 1971. Vickers et al., “Inhibition of HIV-LTR gene expression by oligonucleotides targeted to the TAR element,” Nucleic Acids Research, 193359-3368, 1991. Stenberg et al., “Promoter-Speci?c trans Activation and Repression by Human Cytomegalovirus Immediate-Early Proteins Invovles Common and Unique Protein Domains,” J. ViroL, 64:1556-1565, 1990. Hahn et al., “Molecular cloning and characterization of the HTLV-III virus associated with AIDS,” Nature, 312:166-169, 1984. Depto et al., “Regulated Expression of the Human Cytome galovirus pp65 Gene: Octarner Sequence in the Promoter Is Required for Activation by Viral Gene Products,” J. ViroL, 1989, 63:1232-1238, 1989. Tibauyenda et al., “The effect of single base-pair mis matches on the duplex stability of d(T-A-T -T-A—A—T—A-T-C-A-A-G-T—T—G) . d(C—A-A-C-T —T-G—A—T—A—T—T—A—A—T—A),” Eur. J. Biochem, 139:19-27, 1984. Vasseur et al., “Oligonucleosides: Synthesis of a Novel Methylhydroxylarnine-Linked Nucleoside Dimer and Its Incorporation into Antisense Sequences,” J. Am. Chem. Soc., 114:4006-4007, 1992. Lal et al., “Dipheuylphosphoryl Azide A Novel Reagant for the Stereospeci?c Synthesis of Azides from Alcohols,” Tetrahedron Letters, 23:1977-1980, 1977. Doel et al., Tetrahedron, 30:2755-2759 1974. Primary Examiner—Scott W. Houtteman Attomey, Agent, or Fimz-Woodcock Washburn Kurtz Mackiewicz & Norris [57] 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 modu late protein activity through, for example, transcription arrest, transcription initiation, and site speci?c cleavage of nucleic acids. 18 Claims, 40 Drawing Sheets U.S. Patent waiimméiarmim Jun. 24, 1997 Sheet 1 of 40 5,641,625 w M 5m US. Patent Jun. 24, 1997 Sheet 3 of 40 5,641,625 ‘ 2% 5E US. Patent Jun. 24, 1997 Sheet 4 0f 40 5,641,625 my 1% apsawgg US. Patent Jun. 24, 1997 Sheet 5 of 40 5,641,625 US. Patent Jun. 24, 1997 Sheet 6 of 40 5,641,625 120 100 80 40 60 Time (min) 20 FIG.6 5-9-80 Q5 Dissociation Temperature FIG.7 US. Patent Jun. 24, 1997 Sheet 8 of 40 5,641,625 TTCTTCTTTT. / PUHCBTC PUHCGTC ______ CTBCHG ______ PUT-FUCHS 1_n-r I—H-T +E-H- C CTTTT -_— PUHHHH _ TFFFF FIG.9 U S. Patent Jun. 24, 1997 Sheet 9 of 40 5,641,625 alwwwmwwmww U.S. Patent Jun. 24, 1997 Sheet 10 of 40 5,641,625 US. Patent Jun. 24, 1997 Sheet 11 0f 40 5,641,625 / “w Amgnsak US. Patent Jun. 24, 1997 Sheet 12 of 40 5,641,625 FIG“ 83B US. Patent ~ Jun. 24, 1997 Sheet 13 of 40 5,641,625 Z5142 PIQE25x42 Paul71 6 5 4 I o I I i I Ill-7J1’ I I I I mMJDOMJOE 0mmZDZwIP0 IO 20 30 4o 50 so 70 so 90160 LENGTH , °/o US. Patent Jun. 24, 1997 Sheet 14 0f 40 5,641,625 is 425%? iiiszzasmim iifaiaziamqaiza? US. Patent Jun. 24, 1997 Sheet 15 0f 40 5,641,625 U.S. Patent Jun. 24, 1997 Sheet 16 of 40 5,641,625 Km? US. Patent Jun. 24, 1997 Sheet 17 of 40 5,641,625 @wBi U S. Patent Jun. 24, 1997 Sheet 18 of 40 5,641,625 a g, 22 $ US. Patent Jun. 24, 1997 Sheet 19 of 40 5,641,625 393%? 3 

Cleaving Double-Stranded DNA with Peptide Nucleic Acids

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Cleaving Double-Stranded DNA with Peptide Nucleic Acids

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