Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex

• US 6,291,184 B1
• Filed: 12/13/1999
• Issued: 09/18/2001
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Somalogic Incorporated
• Original Assignee: Somalogic Incorporated
• Inventors: Gold, Larry, Jensen, Kirk, Atkinson, Brent, Willis, Michael, Koch, Tad, Ringquist, Steven
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/459,553
• Time in Patent Office: 645 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3, 536/35.4, 530/350
• 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: C07K 14/001 by chemical synthesis
• 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 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: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 four
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin G01N2333/665, corticotropin G01N2333/695
• 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 for identifying nucleic acid ligands to target molecules using the SELEX procedure wherein the candidate nucleic acids contain photoreactive groups and nucleic acid ligands identified thereby are claimed. The complexes of increased affinity nucleic acids and target molecules formed in the procedure are crosslinked by irradiation to facilitate separation from unbound nucleic acids. In other methods partitioning of high and low affinity nucleic acids is facilitated by primer extension steps as shown in the figure in which chain termination nucleotides, digestion resistant nucleotides or nucleotides that allow retention of the cDNA product on an affinity matrix are differentially incorporated into the cDNA products of either the high or low affinity nucleic acids and the cDNA products are treated accordingly to amplification, enzymatic or chemical digestion or by contact with an affinity matrix.

Claims

1. A method for detecting the presence of a target molecule that is associated with a disease state in a biological substance which may contain said target molecule comprising:

a) exposing a biological substance which may contain said target molecule that is associated with a disease state to a nucleic acid ligand identified according to the method comprising;

i) identifying a nucleic acid ligand that photocrosslinks to a target molecule from a candidate mixture of nucleic acids, wherein each member of said candidate mixture contains a photoreactive group, said method comprising;

1) contacting said candidate mixture of nucleic acids with a first biological substance which contains a target molecule that is associated with said disease state, wherein nucleic acids having an increased affinity to a molecule of said first biological substance relative to the candidate mixture form nucleic acid-molecule complexes with said molecule;

2) irradiating said complexes, wherein said nucleic acid and molecule photocrosslink;

3) partitioning the photocrosslinked nucleic acid-molecule complexes from the remainder of the candidate mixture; and

4) identifying nucleic acid ligands that photocrosslink to said target molecule;

ii) contacting a second biological substance which does not contain said target molecule that is associated with said disease state with said nucleic acid ligands identified in step

     4), wherein the nucleic acids with affinity to a molecule that is not associated with the disease state in the second biological substance is removed; and

iii) amplifying the remaining nucleic acids with specific affinity to said target molecule that is associated with a disease state to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said target molecule that is associated with said disease state, whereby a nucleic acid ligand to a target molecule that is associated with a disease state in a biological substance is identified;

b) irradiating said biological substance containing said nucleic acid ligand; and

c) detecting whether a nucleic acid ligand-molecule complex has been formed, whereby said target molecule that is associated with said disease state is detected.

2. A method for detecting the presence of a target molecule that is associated with a disease state in a biological substance which may contain said molecule comprising:

a) exposing a biological substance which may contain said target molecule that is associated with a disease state to a nucleic acid ligand identified according to a method comprising;

i) identifying a nucleic acid ligand that photocrosslinks to a target molecule from a candidate mixture of nucleic acids, said method comprising;

1) contacting said candidate mixture of nucleic acids with a first biological substance which contains a target molecule that is associated with said disease state, wherein nucleic acids having an increased affinity to a molecule of said first biological substance relative to the candidate mixture form nucleic acid-molecule complexes with said molecule;

2) partitioning the complexed increased affinity nucleic acids from the remainder of the candidate mixture;

3) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids,

4) incorporating photoreactive groups into said amplified increased affinity nucleic acids;

5) irradiating said increased affinity nucleic acids, wherein said nucleic acid-molecule complexes photocrosslink;

6) partitioning the photocrosslinked nucleic acid-molecule complexes from the remainder of the candidate mixture; and

7) identifying nucleic acid ligands that photocrosslink to the molecule;

ii) contacting a second biological substance which does not contain said target molecule that is associated with said disease with said nucleic acid ligands identified in step vii), wherein the nucleic acids with affinity to a molecule that is not associated with said disease is removed; and

iii) amplifying the remaining nucleic acids with specific affinity to said target molecule that is associated with said disease state to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said target molecule that is associated with said disease state, whereby nucleic acid ligands to a target molecule that is associated with a disease state in a biological substance is identified;

b) irradiating said biological substance containing said nucleic acid ligand;

c) detecting whether a nucleic acid ligand-molecule complex has been formed, whereby said target molecule that is associated with said disease state is detected.