Nucleic acid ligands of tissue target

• US 5,688,935 A
• Filed: 05/03/1995
• Issued: 11/18/1997
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc., Schering Ag (Merck & Co., Inc.)
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.), Schering Ag (Merck & Co., Inc.)
• Inventors: Schneider, Dan J., Gold, Larry, Speck, Ulrich, Stephens, Andrew
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/433,126
• Time in Patent Office: 930 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 935/77, 935/78
• US Class Current: 536/23.1
• 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: B01D 15/3804 Affinity chromatography
• 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: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C07K 7/22 Tachykinins, e.g. Eledoisins , Substance P; Related peptides
• 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 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2740/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/70 : involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/703 : Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 : incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2541/101 : Selex
• 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 2333/4724 : Lectins
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/575 : Hormones
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/7056 : Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• 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 33/53 : Immunoassay; Biospecific binding assay; Materials therefor
• 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

This invention discloses high-affinity oligonucleotide ligands to complex tissue targets, specifically nucleic acid ligands having the ability to bind to complex tissue targets, and the methods for obtaining such ligands. Tissue targets comprise cells, subcellular components, aggregates or cells, collections of cells, and higher ordered structures. Specifically, nucleic acid ligands to peripheral blood mononuclear cells (PBMC), fibrin clots, and carotid arteries are described.

Claims

1. A nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries identified according to a method comprising:

a) preparing a candidate mixture of nucleic acids having a mixture of nucleic acid sequences;

b) contacting said candidate mixture of nucleic acids with said target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

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

d) amplifying the increased affinity nucleic acids

e) repeating steps a) through d) as required to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said target, whereby a nucleic acid ligand of said target may be identified.

2. A purified and isolated non-naturally occurring nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries.

3. The purified and isolated nucleic acid ligand of claim 2 wherein said nucleic acid ligand is a non-naturally occurring nucleic acid ligand having a specific binding affinity for said target, such target being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target.

4. The nucleic acid ligand of claim 2 which is a deoxyribonucleic acid ligand.

5. The nucleic acid ligand of claim 2 which is a ribonucleic acid ligand.

6. The nucleic acid ligand of claim 2 wherein said target is a peripheral blood mononuclear cell.

7. The nucleic acid ligand to a peripheral blood mononuclear cell of claim 6 wherein said ligand is a DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 2, SEQ ID NOS:

7-39.

8. The nucleic acid ligand of claim 2 wherein said target is a fibrin clot.

9. The nucleic acid ligand to a fibrin clot of claim 8 wherein said ligand is a RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 5, SEQ ID NOS:

43-130.

10. The nucleic acid ligand of claim 2 wherein said target is a carotid artery.

11. The nucleic acid ligand to a carotid artery of claim 10 wherein said ligand is a RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 8, SEQ ID NOS:

141-237.

12. A nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids having a mixture of nucleic acid sequences;

(b) contacting the candidate mixture with a first target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries, wherein nucleic acids having an increased affinity to the first target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

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

(d) contacting the increased affinity nucleic acids with a second target, wherein nucleic acids with affinity to the second target are removed;

(e) amplifying the nucleic acids with increased affinity to said first target

(f) repeating steps (a) through (e) as required to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said target; and

(g) identifying said nucleic acid ligand of said first target.

13. A nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids having a mixture of nucleic acid sequences;

(b) contacting the candidate mixture with a first target, wherein nucleic acids with affinity to the first target are removed;

(c) contacting the candidate mixture from (b) with a second target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries, wherein nucleic acids having an increased affinity to the second target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

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

(e) amplifying the nucleic acids with increased affinity to said second target and

(f) repeating steps (a) through (e) as required to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said target; and

(g) identifying said nucleic acid ligand of said second target.