High-affinity oligonucleotide ligands to the tachykinin substance P

• US 5,648,214 A
• Filed: 09/09/1994
• Issued: 07/15/1997
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: University of Colorado System
• Inventors: Gold, Larry, Wecker, Matthew, Nieuwlandt, Dan T.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/303,362
• Time in Patent Office: 1,040 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• 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 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: 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: 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 2333/96433 : 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 33/76 : 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 Substance P (SP). Included in the invention are specific RNA ligands to SP identified by the SELEX method.

Claims

1. A method of identifying nucleic acid ligands to Substance P (SP), comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with SP, wherein nucleic acids having an increased affinity to SP 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 to yield a mixture of nucleic acids enriched for nucleic acids with said increased affinity to SP, whereby nucleic acid ligands of SP may be identified.

2. The method of claim 1 further comprising:

e) repeating steps b), c) and d).

3. The method of claim 1 further comprising:

e) contacting, between steps (c) and (d), the increased affinity nucleic acids with one or more non-substance P molecules, wherein nucleic acids with an affinity to the non-Substance P molecules(s) is are removed.

4. The method of claim 1 further comprising:

e) preparing a second candidate mixture of nucleic acids by mutagenizing the mixture of increased affinity nucleic acids; and

f) repeating steps (b), (c) and (d), whereby nucleic acid ligands of SP may be identified.

5. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single-stranded nucleic acids.

6. The method of claim 5 wherein said single-stranded nucleic acids are ribonucleic acids.

7. The method of claim 5 wherein said single-stranded nucleic acids are deoxyribonucleic acids.

8. A method for identifying nucleic acid ligands from a candidate mixture of nucleic acids, said nucleic acid ligands being a ligand of a given target molecule comprising:

a) contacting said candidate mixture with the target molecule, wherein nucleic acids having increased affinity to the target molecule 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) preparing a second candidate mixture of nucleic acids by mutagenizing the ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the given target molecule may be identified.

9. The method of claim 8 further comprising:

e) repeating steps (a), (b), and (c).