Systematic evolution of ligands by exponential enrichment: blended SELEX

• US 7,176,295 B2
• Filed: 10/02/2002
• Issued: 02/13/2007
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Gold, Larry, Biesecker, Gregory, Smith, Drew, Kirschenheuter, Gary P., Jayasena, Sumeda
• Primary Examiner(s): Schultz; James
• Assistant Examiner(s): Vivlemore; Tracy
• Application Number:
  US10/263,456
• Publication Number:
  US 20030077646A1
• Time in Patent Office: 1,595 Days
• Field of Search: 536/23.1, 536/25.4, 435/6, 435/91.1, 435/91.5
• US Class Current: 536/23.1
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 5/1008 the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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 ketone (SEQ ID NO:2). This elastase blended nucleic acid ligand inhibits the biological activity of elastase.

Claims

1. A blended nucleic acid ligand of a target compound prepared by a method comprising the steps of:

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 may be identified; and

b) attaching at least one functional unit to said nucleic acid ligand to yield a blended nucleic acid ligand of the target compound, wherein the functional unit is selected from the group consisting of proteins, peptides, photoreactive groups, chemically-reactive groups, active site directed compounds, lipids, biotin, and fluorescent compounds.

2. The blended nucleic acid ligand of claim 1 wherein said at least one functional unit is attached to an oligonucleotide capable of hybridizing to said nucleic acid ligand, and wherein step b) is accomplished by hybridizing said oligonucleotide to said nucleic acid ligand.

3. A diagnostic reagent comprising a blended nucleic acid ligand of a target compound and a reporter molecule, wherein said diagnostic reagent is prepared by a method comprising the steps of:

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;

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 a nucleic acid ligand of the target compound may be identified;

b) attaching at least one functional unit to said nucleic acid ligand to yield a blended nucleic acid ligand of the target compound, wherein the functional unit is selected from the group consisting of proteins, peptides, photoreactive groups, chemically-reactive groups, active site directed compounds, lipids, biotin, and fluorescent compounds; and

c) attaching at least one reporter molecule to said blended nucleic acid ligand, whereby a diagnostic reagent is prepared.

4. The diagnostic reagent of claim 3 wherein said at least one functional unit is attached to an oligonucleotide capable of hybridizing to said nucleic acid ligand, and wherein step b) is accomplished by hybridizing said oligonucleotide to said nucleic acid ligand.