Systematic evolution of ligands by exponential enrichment: tissue selex

• US 5,789,157 A
• Filed: 05/03/1995
• Issued: 08/04/1998
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Chen, Hang, Gold, Larry, Stephens, Andrew, Morris, Kevin N., Jensen, Kirk B.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/434,425
• Time in Patent Office: 1,189 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1, 536/25.4, 935/77, 935/78
• US Class Current: 435/6.11
• 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: 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: 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 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• 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 33/558 : using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/57407 : Specifically defined cancers
• CPC Class Codes: G01N 33/57426 : leukemia
• 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
• CPC Class Codes: G01N 33/80 : involving blood groups or blood types or red blood cells white blood cells G01N33/56972

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 red blood cells ghosts, glioblastomas, and lymphomas are described.

Claims

1. A method for identifying nucleic acid ligands to a protein component of a biological tissue comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said biological tissue, wherein nucleic acids having an increased affinity to the biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with an increased affinity and an increased specificity for binding to said protein, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

2. The method of claim 1 wherein said nucleic acids of the candidate mixture are comprised of single-stranded nucleic acids.

3. The method of claim 2 wherein said single-stranded nucleic acids are ribonucleic acids.

4. The method of claim 2 wherein said single-stranded nucleic acids are deoxyribonucleic acids.

5. The method of claim 1 wherein said biological tissue is selected from the group consisting of red blood cell ghosts, glioblastoma, and lymphoma.

6. A method for identifying nucleic acid ligands to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids having an increased affinity to the first biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

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

(e) amplifying the remaining nucleic acids with specific affinity to said protein components to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein components, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

7. A method for identifying nucleic acid ligands to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids with affinity to the first biological tissue relative to the candidate mixture are removed from the candidate mixture;

(c) contacting the remaining candidate mixture from (b) with a second biological tissue, wherein nucleic acids having an increased affinity to the second biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity for a protein component of said second biological tissue;

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

(e) amplifying the nucleic acids with specific affinity to said protein component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein components, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

8. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said biological tissue, wherein nucleic acids having an increased affinity to the biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with an increased affinity and an increased specificity for binding to said protein component; and

e) repeating steps b)-d) as necessary, whereby a nucleic acid ligand to protein component of a biological tissue is identified.

9. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids having an increased affinity to the first biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

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

(e) amplifying the remaining nucleic acids with specific affinity to said protein component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein component; and

(f) repeating steps (b)-(e) as necessary, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.

10. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids with affinity to the first biological tissue relative to the candidate mixture are removed from the candidate mixture;

(c) contacting the remaining candidate mixture from (b) with a second biological tissue, wherein nucleic acids having an increased affinity to the second biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity for protein component of said second biological tissue;

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

(e) amplifying the nucleic acids with specific affinity to said protein component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein component; and

(f) repeating steps (b)-(e) as necessary, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.

11. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said biological tissue, wherein nucleic acids having an increased affinity to the biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with an increased affinity and an increased specificity for binding to said protein component;

e) cloning said increased affinity nucleic acids;

f) determining the nucleic acid sequences of said cloned nucleic acids; and

g) determining the specificity of said cloned nucleic acids for binding said protein component of a biological tissue, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.