Systematic evolution of ligands by exponential enrichment: tissue selex

• US 6,114,120 A
• Filed: 10/28/1997
• Issued: 09/05/2000
• Est. Priority Date: 05/03/1995
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Morris, Kevin N., Jensen, Kirk B, Chen, Hang, Gold, Larry, Stephens, Andrew
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/945,909
• Time in Patent Office: 1,043 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1, 536/25.4, 536/24.3, 935/77.78
• 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: C12N 15/1048 SELEX
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: G01N 2333/96433 Serine endopeptidases (3.4.21)

Abstract

A new class of nucleic acid compounds, referred to as nucleic acid ligands, have been shown to exist that have a specific binding affinity for three dimensional molecular targets, including cell surface macromolecules. The nucleic acid ligands are identified by the method of the invention referred to as the Systematic Evolution of Ligands by EXponential enrichment (SELEX), wherein a candidate mixture of nucleic acids are iteratively enriched and the high affinity nucleic acids are amplified for further partitioning. The high affinity nucleic acid ligands are useful in capturing target cells.

Claims

1. A nucleic acid ligand to a cell target macromolecule identified according to the method comprising:

a) preparing a candidate mixture of nucleic acid sequences;

b) contacting said candidate mixture of nucleic acids with said cell, wherein nucleic acids having an increased affinity to the cell macromolecule 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) optionally amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said cell,

whereby said nucleic acid ligand to said macromolecule may be identified.

2. A purified and isolated non-naturally occuring nucleic acid ligand to a cell macromolecule.

3. The purified nucleic acid ligand of claim 2 which is a non-naturally occurring nucleic acid ligand having a specific binding affinity for the cell target macromolecule, said macromolecule being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.

4. The nucleic acid ligand of claim 2 which is a deoxyribonucleic acid ligand.

5. The nucleic acid ligand of claim 2 which is a ribonucleic acid ligand.

6. A method of purifying macromolecule component of a cell comprising:

a) identifying a nucleic acid ligand to said macromolecule according to the method comprising;

i) preparing a candidate mixture of nucleic acid sequences;

ii) contacting said candidate mixture of nucleic acids with said cell, wherein nucleic acids having an increased affinity to the cell macromolecule relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

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

iv) optionally amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said cell

whereby said nucleic acid ligand to said macromolecule may be identified;

b) separating said macromolecule component of said cell away from the remainder of said cell on the basis of affinity between said cell macromolecule and said nucleic acid ligand; and

c) purifying said macromolecule.

7. The method of claim 6 wherein said macromolecule is selected from the group consisting of a protein, lipid and carbohydrate.

8. A purified macromolecule identified according to the method of claim 6.

9. The purified macromolecule of claim 8 which is selected from the group consisting of a protein, lipid and carbohydrate.

10. The purified macromolecule of claim 9 which is a tumor associated antigen.