Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands

• US 6,482,594 B2
• Filed: 06/14/2001
• Issued: 11/19/2002
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Somalogic Incorporated
• Original Assignee: Somalogic Incorporated
• Inventors: Jensen, Kirk, Atkinson, Brent, Ringquist, Steven, Gold, Larry, Willis, Michael, Koch, Tad
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US09/882,246
• Publication Number:
  US 20020106652A1
• Time in Patent Office: 523 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.33, 536/25.4, 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

Methods to obtain nucleic acid ligands that photocrosslink to target molecules associated with a disease state are provided. The methods presented are variations on the photoSELEX methods for obtaining nucleic acid ligands. In one method, a candidate mixture of photocrosslinkable nucleic acids is contacted with a biological substance obtained from a source associated with a disease state suspected of containing a target molecule to form nucleic acid-target molecule complexes, the complexes are irradiated to form crosslinked complexes, the photocrosslinked complexes are partitioned from the remainder of the candidate mixture; and the nucleic acid ligands that photocrosslink to molecule are retained. These nucleic acids are then contacted with a second biological substance of the same type as the first, but obtained from a source not associated with a disease state. This removes nucleic acids with affinity to molecules that are not associated with the disease state. In another method, photocrosslinkable nucleic acids are not incorporated in the initial candidate mixture, but rather are incorporated in an enriched candidate mixture obtained by contacting a biological substance associated with a disease state containing a target molecule to form nucleic acid-target molecule complexes, partitioning the complexes away from the candidate mixture.

Claims

1. A nucleic acid ligand that photocrosslinks to a target molecule that is associated with a disease state, said target molecule is a protein, wherein said nucleic acid ligand is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for said target molecule, wherein said target molecule is not a nucleic acid binding molecule, and wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, identified by the process of:

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

i) contacting said candidate mixture of nucleic acids with a first biological substance obtained from a source associated with said disease state which is suspected of containing the target molecule that is associated with said disease state, wherein nucleic acids having an increased affinity to molecules of said first biological substance relative to the candidate mixture form nucleic acid-target molecule complexes with the target molecule;

ii) irradiating said complexes, wherein said nucleic acids and target molecules photocrosslink;

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

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

b) contacting a second biological substance of the same type as the first biological substance, and obtained from a source not associated with said disease state and which does not contain said target molecule that is associated with said disease state with said nucleic acid ligands identified in step iv), wherein the nucleic acids with affinity to the molecules in the second biological substance that are not associated with said disease state are removed; and

c) amplifying the remaining nucleic acids 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 that photocrosslinks to a target molecule that is associated with a disease state in a biological substance is identified.

2. The nucleic acid ligand of claim 1, wherein the nucleic acid ligand is comprised of one or more of the photoreactive groups selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2′

-deoxyadenine, 5-iodo-2′

-deoxyuracil, 5-iodo-2′

-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

5. The method of claim 1, wherein said first biological substance is serum from a patient with a disease, and said second biological substance is serum from a patient not having said disease.

6. The method of claim 1, wherein said first biological substance is an abnormal cell, and said second biological substance is a normal cell of the same type.

3. A nucleic acid ligand that photocrosslinks to a target molecule that is associated with a disease state, wherein said target molecule is protein, wherein said nucleic acid ligand is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for said target molecule, wherein said target molecule is not a nucleic acid binding molecule, and wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, identified by the process of:

a) identifying a nucleic acid ligand that photocrosslinks to a target molecule that is associated with the disease state from a candidate mixture of nucleic acids, said method comprising;

i) contacting said candidate mixture of nucleic acids with a first biological substance obtained from a source associated with said disease state which is suspected of containing the target molecule that is associated with said disease state, wherein nucleic acids having an increased affinity to molecules of said first biological substance relative to the candidate mixture form nucleic acid-target molecule complexes with the target molecule;

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

iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids;

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

v) contacting said increased affinity nucleic acids with a biological substance obtained from a source associated with said disease state which is suspected of containing the target molecule that is associated with said disease state;

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

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

viii) identifying nucleic acid ligands that photocrosslink to the target molecule;

b) contacting a second biological substance of the same type as the first biological substance, and obtained from a source not associated with said disease state and which does not contain said target molecule that is associated with said disease state with said nucleic acid ligand identified in step vii), wherein the nucleic acids with affinity to molecules not associated with said disease state are removed; and

;

c) amplifying the remaining nucleic acids 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 that photocrosslinks to a target molecule that is associated with a disease state in a biological substance is identified.

4. The nucleic acid ligand of claim 3,wherein the nucleic acid ligand is comprised of one or more of the photoreactive groups selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2′

-deoxyadenine, 5-iodo-2′

-deoxyuracil, 5-iodo-2′

-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

7. The method of claim 3, wherein said first biological substance is serum from a patient with a disease, and said second biological substance is serum from a patient not having said disease.

8. The method of claim 3, wherein said first biological substance is an abnormal cell, and said second biological substance is a normal cell of the same type.