Use of nucleic acid ligands in flow cytometry

• US 5,853,984 A
• Filed: 06/07/1995
• Issued: 12/29/1998
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Davis, Ken, Jayasena, Sumedha
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/479,729
• Time in Patent Office: 1,301 Days
• Field of Search: 435/6, 436/501, 536/26.6, 536/23.1
• US Class Current: 435/6.11
• 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 1/68 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 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 15/14 : Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals

Abstract

This invention discloses the use of high-affinity oligonucleotide ligands in flow cytometry diagnostic applications. Specifically, DNA ligands having one or more fluorophore molecules attached are disclosed which are useful in flow cytometry.

Claims

1. A method for detecting the presence of a target compound in a substance which may contain said target compound, wherein said target compound or said substance is particulate, comprising:

a) identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of said target compound, by the method comprising;

i) contacting the candidate mixture with said target compound, wherein nucleic acids having an increased affinity to said target compound 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;

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

iv) identifying said nucleic acid ligand;

b) attaching said nucleic acid ligand with a fluorophore to form a fluorophore-labelled nucleic acid ligand;

c) mixing said fluorophore-labelled nucleic acid ligand with a substance which may contain said target compound under conditions suitable for binding of the fluorophore-labelled nucleic acid ligand to said target compound; and

d) analyzing said mixture by flow cytometry to detect whether said fluorophore-labelled nucleic acid ligand bound to said target compound in said substance, whereby the presence of a target compound in a substance can be detected.

2. The method of claim 1 wherein said fluorophore of said fluorophore labelled nucleic acid ligand is selected from the group consisting of fluorescein, rhodamine, Cy5 reactive dye, Cy3 reactive dye, allophycocyanin, peridinine chlorophyll-a protein (PerCP), phycoerythrin, and green fluorescein protein (GFP).

3. The method of claim 1 wherein said substance is a biological fluid.

4. The method of claim 1 wherein said substance comprises cells.

5. The method of claim 3 wherein said substance is selected from the group consisting of blood, plasma, serum, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, and macerated tissue.

6. The method of claim 5 wherein said substance is selected from the group consisting of blood, plasma, serum and macerated tissue.

7. The method of claim 1 wherein said target compound is on the surface of a cell.

8. The method of claim 1 wherein said target compound is intracellular.

9. The method of claim 8 wherein said target compound is cytoplasmic.

10. The method of claim 1 wherein unbound fluorophore-labelled nucleic acid ligand is removed from said mixture between steps c) and d).

11. A method for detecting the presence of a soluble target compound in a substance which may contain said target compound by flow cytometry comprising:

a) immobilizing on a particulate solid support a capture molecule capable of binding to said target compound;

b) exposing a substance which may contain said target compound to said capture molecule;

c) adding to said capture molecule;

target compound complex a fluorophore-labelled detector molecule capable of binding to said target compound; and

d) detecting said capture molecule;

target compound;

detector molecule complex by flow cytometry, wherein said capture molecule, detector molecule or both are a nucleic acid ligand to said target compound, wherein said nucleic acid ligand is identified by the method comprising;

i) preparing a candidate mixture of nucleic acids;

ii) contacting the candidate mixture with said target compound, wherein nucleic acids having an increased affinity to said target compound 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,

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

v) identifying said nucleic acid ligand.

12. The method of claim 11 wherein said particulate solid support is a bead.

13. The method of claim 11 wherein steps b) and c) are done simultaneously.

14. A method for detecting the presence of a non-nucleic acid target compound in a substance which may contain said non-nucleic acid target compound, wherein said non-nucleic acid target compound or said substance is particulate, comprising mixing said substance with a fluorophore labelled nucleic acid ligand which is capable of binding to said non-nucleic acid target compound and analyzing said mixture by flow cytometry to detect whether said fluorophore labelled nucleic acid ligand bound to said non-nucleic acid target compound in said substance, whereby the presence of a non-nucleic acid target compound in a substance can be detected, wherein said nucleic acid ligand is identified by the method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said target compound, wherein nucleic acids having an increased affinity to said target compound 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;

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

e) identifying said nucleic acid ligand.