Systematic evolution of ligands by exponential enrichment: blended SELEX

• US 5,683,867 A
• Filed: 04/28/1994
• Issued: 11/04/1997
• 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: Gold, Larry, Jayasena, Sumedha D., Smith, Drew, Kirschenheuter, Gary P., Biesecker, Greg
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/234,997
• Time in Patent Office: 1,286 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/25.4, 935/77, 935/78
• US Class Current: 435/6.11
• 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: 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: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G01N 2333/163 Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• 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 2333/96455 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

A method is described for generating blended nucleic acid ligands containing non-nucleic acid functional units. Specifically, a SELEX identified RNA ligand to the integrin gpIIbIIIa is conjugated to the peptide Gly-Arg-Gly-Asp-Thr-Pro (SEQ ID NO:1). This blended RNA ligand inhibits the biological activity of gpIIbIIIa with high specificity. Also described is a single-stranded DNA ligand to elastase coupled to N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl (SEQ ID NO:2) ketone. This elastase blended nucleic acid ligand inhibits the biological activity of elastase.

Claims

1. A method for identifying blended nucleic acid ligands of a target compound from a blended candidate mixture comprised of blended nucleic acids wherein each blended nucleic acid comprises a nucleic acid sequence region and a functional unit, wherein said functional unit is a chemical species not naturally associated with nucleic acids and wherein said functional unit imparts at least one of the following functions to the blended nucleic acid ligand:

enhanced binding affinity, enhanced membrane partitioning, enhanced membrane permeability, enhanced stability, diagnostic reporting, covalent bond formation, inhibition of biological activity, said method comprising;

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

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

c) amplifying the increased affinity blended nucleic acids to yield a ligand-enriched mixture of blended nucleic acids, whereby blended nucleic acid ligands of the target compound are identified.

2. The method of claim 1 wherein said nucleic acid sequence region in the blended nucleic acid is composed of a fixed region and a randomized region.

3. The method of claim 1 wherein said functional unit is attached to an oligonucleotide hybridized to said nucleic acid sequence fixed region.

4. A blended nucleic acid ligand identified according to the method of claim 1.

6. A blended nucleic acid ligand prepared according to the method of claim 3.

8. The method of claim 1 wherein said functional unit is a chemical species not naturally associated with nucleic acids and is selected from the group consisting of proteins, peptides, amino acids, aliphatic groups, photoreactive groups, chemically-reactive groups, active site directed compounds, catalytic groups, chloromethylketones, lipids, biotin, and fluorescent compounds.

10. The blended nucleic acid ligand of claim 4 wherein said functional unit is a chemical species not naturally associated with nucleic acids and is selected from the group consisting of proteins, peptides, amino acids, aliphatic groups, photoreactive groups, chemically-reactive groups, active site directed compounds, catalytic groups, chloromethylketones, lipids, biotin, and fluorescent compounds.

12. The method of claim 1 wherein said nucleic acid sequence region is selected from the group consisting of single-stranded DNA or single-stranded RNA.

14. The blended nucleic acid ligand of claim 4 wherein said nucleic acid sequence region is a ribonucleotide and said functional unit is an Arg-Gly-Asp peptide, wherein said blended nucleic acid ligand binds to gpIIbIIIa.

15. The blended nucleic acid ligand of claim 4 wherein said nucleic acid sequence region is a deoxyribonucleotide and said functional unit is N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (SEQ ID NO:

2), wherein said blended nucleic acid ligand binds to elastase.

16. The blended nucleic acid ligand of claim 6 wherein said at least one functional unit is valyl phosphonate.

5. A method for preparing blended nucleic acid ligands of a target compound comprising:

a) identifying a nucleic acid ligand of a target compound from a candidate mixture comprised of nucleic acids each having a region of randomized sequence by a method comprising;

i) contacting the candidate mixture with the 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;

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

iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target compound are identified; and

b) attaching a functional unit, wherein said functional unit is a chemical species not naturally associated with nucleic acids, and wherein said functional unit imparts at least one of the following functions to the nucleic acid ligand;

enhanced binding affinity, enhanced membrane partitioning, enhanced membrane permeability, enhanced stability, diagnostic reporting, covalent bond formation, inhibition of biological activity, to said nucleic acid ligand to yield a blended nucleic acid ligand of the target compound.

7. A blended nucleic acid ligand prepared according to the method of claim 5.

9. The method of claim 5 wherein said functional unit is a chemical species not naturally associated with nucleic acids and is selected from the group consisting of proteins, peptides, amino acids, aliphatic groups, photoreactive groups, chemically-reactive groups, active site directed compounds, catalytic groups, chloromethylketones, lipids, biotin, and fluorescent compounds.

11. The blended nucleic acid ligand of claim 7 wherein said functional unit is a chemical species not naturally associated with nucleic acids and is selected from the group consisting of proteins, peptides, amino acids, aliphatic groups, photoreactive groups, chemically-reactive groups, active site directed compounds, catalytic groups, chloromethylketones, lipids, biotin, and fluorescent compounds.

13. The method of claim 5 wherein said nucleic acid sequence region is selected from the group consisting of single-stranded DNA or single-stranded RNA.