Tenascin-C nucleic acid ligands

• US 6,596,491 B2
• Filed: 05/14/2001
• Issued: 07/22/2003
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Warren, Stephen, Parma, David, Gold, Larry, Hicke, Brian
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/854,662
• Publication Number:
  US 20030104377A1
• Time in Patent Office: 799 Days
• Field of Search: 435/6, 435/91, 435/2, 530/350, 530/387.1, 536/22.1, 536/25.4, 536/24.2
• US Class Current: 435/5
• CPC Class Codes: A61K 2123/00 Preparations for testing in vivo
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/317 with an inverted bond, e.g. a cap structure
• CPC Class Codes: C12N 2310/3183 Diol linkers, e.g. glycols or propanediols
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/3517 : Marker; Tag
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/53 : partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 : RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 : involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2541/101 : Selex
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 2333/16 : HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin G01N2333/665, corticotropin G01N2333/695
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/9726 : Tissue plasminogen activator
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 2333/976 : Trypsin; Chymotrypsin
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Methods are described for the identification and preparation of nucleic acid ligands to tenascin-C. Included in the invention are specific RNA ligands to tenascin-C identified by the SELEX method. Further included in the invention are methods for detecting the presence of a disease condition in a biological tissue in which tenascin-C is expressed.

Claims

1. A complex for use in in vivo diagnostics comprising a tenascin-C nucleic acid ligand and a marker.

2. The complex of claim 1 wherein said tenascin-C nucleic acid ligand is identified by the method comprising:

a) contacting a candidate mixture of nucleic acids with tenascin-C, wherein nucleic acids having an increased affinity to tenascin-C relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

c) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to tenascin-C, whereby a nucleic acid ligand of tenascin-C may be identified.

3. The complex of claim 2 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

4. The complex of claim 3 wherein said single stranded nucleic acids are ribonucleic acids.

5. The complex of claim 4 wherein said candidate mixture of nucleic acids comprises 2′

-F (2′

-fluoro) modified ribonucleic acids.

6. The complex of claim 1 wherein said marker is selected from the group consisting of radionuclides, fluorophores, magnetic compounds, and biotin.

7. The complex of claim 6 wherein said radionuclide is selected from the group consisting of technetium-99m (Tc-99m), Re-188, Cu-64, Cu-67, F-18, ¹²⁵I, ¹³¹I, ³²P, ¹⁸⁶Re.

8. The complex of claim 7 wherein said marker is technetium-99m.

9. The complex of claim 8 further comprising a linker.

10. The complex of claim 9, wherein said linker has the structure

11. The complex of claim 10, wherein said complex is

12. A method for the preparation of a complex comprised of a tenascin-C nucleic acid ligand and a marker, said method comprising:

a) contacting a candidate mixture of nucleic acids with tenascin-C, wherein nucleic acids having an increased affinity to tenascin-C relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

c) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to tenascin-C to yield a tenascin-C nucleic acid ligand; and

d) covalently linking said tenascin-C nucleic acid ligand with a marker.

13. The method of claim 12 wherein said marker is selected from the group consisting of radionuclides, fluorophores, magnetic compounds, and biotin.

14. The method of claim 13 wherein said radionuclide is selected from the group consisting of Tc-99m, Re-188, Cu-64, Cu-67, F-18, ¹²⁵I, ¹³¹I, ³²P, ¹⁸⁶Re.

15. The method of claim 14 wherein said marker is Tc-99m.

16. The method of claim 15 further comprising a linker.

17. The complex of claim 16, wherein said linker has the structure

18. The complex of claim 17, wherein said complex is