Systematic evolution of ligands by exponential enrichment: chemi-SELEX

• US 5,998,142 A
• Filed: 08/21/1997
• Issued: 12/07/1999
• Est. Priority Date: 09/08/1993
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Jensen, Kirk, Gold, Larry, Eaton, Bruce, Smith, Drew, Wecker, Matthew
• Primary Examiner(s): Ztonmer, Stephanie
• Application Number:
  US08/894,578
• Time in Patent Office: 838 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/25.4, 536/24.3, 935/77, 935/78
• US Class Current: 435/6.11
• 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: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2541/101 Selex

Abstract

This application provides methods for identifying nucleic acid ligands capable of covalently interacting with targets of interest. The nucleic acids can be associated with various functional units. The method also allows for the identification of nucleic acids that have facilitating activities as measured by their ability to facilitate formation of a covalent bond between the nucleic acid, including its associated functional unit, and its target.

Claims

1. Nucleic acid ligands that bind covalently with a protein, wherein said nucleic acid ligands comprise a chemically reactive functional unit, wherein said nucleic acid ligands are produced by the method comprising the steps of:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture with said protein, wherein nucleic acids which bind covalently with said protein may be partitioned from the remainder of the candidate mixture;

c) partitioning the nucleic acids that bind covalently with the protein from the remainder of the candidate mixture; and

d) amplifying the nucleic acids that bind covalently with the protein, whereby nucleic acid ligands that bind covalently with the protein may be produced.

2. The nucleic acid ligands of claim 1 which are selected from the sequences listed in Table VI (SEQ ID NOS:

108-224).

3. A facilitating nucleic acid, wherein said nucleic acid comprises a chemically reactive functional unit, identified according to the method comprising the steps of:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture with a protein, wherein nucleic acids having a facilitating activity, as indicated by a covalent bond being formed between said protein and said nucleic acid, relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the nucleic acids having a facilitating activity from the remainder of the candidate mixture; and

d) amplifying the nucleic acids having a facilitating activity, whereby a facilitating nucleic acid may be identified.

4. A method for partitioning nucleic acids from a nucleic acid candidate mixture, wherein said nucleic acid ligands comprise a chemically reactive functional unit, said method comprising:

a) preparing a nucleic acid candidate mixture;

b) contacting the nucleic acid candidate mixture with a protein under conditions wherein nucleic acids form a covalent bond with said protein; and

c) partitioning away the remainder of the nucleic acid candidate mixture which did not form a covalent bond with the protein, leaving only nucleic acids which have formed a covalent bond with the protein.

5. A purified and isolated non-naturally occurring nucleic acid ligand which binds covalently with a protein, wherein said nucleic acid ligand comprises a chemically reactive functional unit.