Methods of producing nucleic acid ligands

• US 6,344,318 B1
• Filed: 10/02/1998
• Issued: 02/05/2002
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Tuerk, Craig, Gold, Larry
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US09/165,616
• Time in Patent Office: 1,222 Days
• Field of Search: 455/6, 455/91.2, 536/24.3, 536/25.4, 935/77.78
• US Class Current: 435/5
• 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 47/547 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: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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: 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 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 : incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/301 : Hairpin oligonucleotides
• 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 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/575 : Hormones
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/918 : Carboxylic ester hydrolases (3.1.1)
• 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 33/531 : 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 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

The present invention includes methods for the identification and production of improved nucleic acid ligands based on the SELEX process. Also included are nucleic acid ligands to the HIV-RT protein identified according to the methods described therein.

Claims

1. A method for preparing a modified nucleic acid ligand having a specific affinity for a given target comprising:

a) identifying a nucleic acid ligand to said target, wherein said nucleic acid ligand has specific affinity for said target;

b) determining the structure of said nucleic acid ligand; and

c) modifying said nucleic acid ligand, wherein said modifying comprises replacing one or more nucleotides with a modified monomer to produce said modified nucleic acid ligand, wherein said modified nucleic acid ligand mimics the structure of said nucleic acid ligand and wherein said modified nucleic acid ligand has a specific affinity to said target.

5. A modified nucleic acid ligand having specific affinity for a target, wherein said modified nucleic acid ligand is identified by the method of claim 1.

2. A method for preparing a modified nucleic acid ligand, wherein said modified nucleic acid ligand is a ligand to a given target, comprising:

a) contacting a candidate mixture of nucleic acids with said target, 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;

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

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids;

d) repeating steps a)-c), as necessary, to identify a nucleic acid ligand of a given target;

e) determining the structure of said nucleic acid ligand; and

f) modifying said nucleic acid ligand, wherein said modifying comprises replacing one or more nucleotides with a modified monomer to produce a modified nucleic acid ligand, wherein said modified nucleic acid ligand mimics the structure of said nucleic acid ligand and wherein said modified nucleic acid ligand has a specific affinity to said target.

6. A modified nucleic acid ligand having specific affinity for a target, wherein said modified nucleic acid ligand is identified by the method of claim 2.

3. A method for preparing a small molecule ligand having a specific affinity for a given target, comprising:

a) identifying a nucleic acid ligand to a given target, wherein said nucleic acid ligand has a specific affinity to said target;

b) determining the structure of said nucleic acid ligand; and

c) modifying said nucleic acid ligand, wherein said modifying comprises replacing each nucleotide of said nucleic acid ligand with a modified monomer to produce the small molecule ligand, wherein said small molecule ligand mimics the structure of said nucleic acid ligand and wherein said small molecule ligand has a specific affinity for said target.

7. A small molecule ligand having specific affinity for a target, wherein said small molecule ligand is identified by the method of claim 3.

4. A method for preparing a small molecule ligand having a specific affinity for a given target, comprising:

a) contacting a candidate mixture of nucleic acids with said target, 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;

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

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids;

d) repeating steps a)-c), as necessary, to identify a nucleic acid ligand of a given target;

e) determining the three-dimensional structure of said nucleic acid ligand; and

f) modifying said nucleic acid ligand, wherein said modifying comprises replacing each nucleotide of said nucleic acid ligand with a modified monomer to produce said small molecule ligand, wherein said small molecule ligand mimics the structure of said nucleic acid ligand and wherein said small molecule ligand has a specific affinity for said target.

8. A small molecule ligand having specific affinity for a target, wherein said small molecule ligand is identified by the method of claim 4.