Spectroscopically detectable nucleic acid ligands

• US 5,641,629 A
• Filed: 01/20/1995
• Issued: 06/24/1997
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Douglas P. Malinowski, Glenn P. Vonk, James B. Pitner, Larry Gold
• Inventors: Malinowski, Douglas P., Vonk, Glenn P., Pitner, James B., Gold, Larry
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/376,329
• Time in Patent Office: 886 Days
• Field of Search: 435/6, 435/91.2, 435/7.1, 435/7.8, 435/7.93, 935/77, 935/78
• US Class Current: 435/6.19
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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 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 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• 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

The present invention relates to methods of using spectroscopically detectable labeled receptor molecules to determine the presence or absence of a target compound in a sample. In one embodiment, spectroscopically detectable labeled nucleic acid ligands are used to determine the presence or absence of biological targets of interest in biological samples.

Claims

1. A method for determining the presence of a target compound in a sample, said target compound selected from the group consisting of proteins, peptides, cell surface markers, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, co-factors, inhibitors, drugs, dyes, nutrients, growth factors, amino acids, ATP, theophylline, whole cells and viral particles comprising:

(a) measuring at least one of the fluorescence polarization, anisotropy values or rotational correlation times of a spectroscopically detectable labeled nucleic acid ligand to said target compound,

(b) adding to the sample the spectroscopically detectable labeled nucleic acid ligand whereby the nucleic acid ligand binds to the target compound; and

(c) determining whether there is a difference between at least one of the fluorescence polarization, anisotropy values or rotational correlation times of (i) the mixture of spectroscopically detectable nucleic acid ligand bound to the target compound and sample in (b), and (ii) the spectroscopically detectable nucleic acid ligand alone in (a) whereby an increase in at least one of the fluorescence polarization, anisotropy values or rotational correlation times in the mixture relative to the nucleic acid ligand alone is indicative of the presence of the target compound in the sample.

2. The method of claim 1 wherein the spectroscopically detectable nucleic acid ligand is labeled with fluorescein.

3. The method of claim 2 wherein the fluorescein label is attached to the nucleic acid ligand at its 3'"'"' end.

4. The method of claim 2 wherein the fluorescein label is attached to the nucleic acid ligand at its 5'"'"' end.

5. The method of claim 4 wherein the nucleic acid ligand binds thrombin.

6. The method of claim 5 wherein the spectroscopically detectable labeled nucleic acid ligand has the formula of Compound 1.

7. The method of claim 5 wherein the spectroscopically detectable labeled nucleic acid ligand has the formula of Compound 2.

8. The method of claim 2 wherein the fluorescein label is attached at an internal position on the nucleic acid ligand.

9. The method of claim 8 wherein the nucleic acid ligand binds thrombin.

10. The method of claim 9 wherein the spectroscopically detectable nucleic acid ligand has the formula of Compound 6.

11. The method of claim 1 wherein the nucleic acid ligand is labeled with thiazole orange.

12. The method of claim 11 wherein the thiazole orange label is attached to the nucleic acid ligand at its 3'"'"' end.

13. The method of claim 11 wherein the thiazole orange label is attached at an internal position on the nucleic acid ligand.

14. The method of claim 11 wherein the thiazole orange label is attached to the nucleic acid ligand at its 5'"'"' end.

15. The method of claim 5 wherein the spectroscopically detectable labeled nucleic acid ligand has the formula of Compound 3.

16. The method of claim 4 wherein the nucleic acid ligand binds elastase.

17. The method of claim 16 wherein the spectroscopically detectable labeled nucleic acid ligand has the formula of Compound 5.

18. The method of claim 1 wherein the spectroscopically detectable labeled nucleic acid ligand is a bi-directional nucleic acid ligand.

19. The method of claim 1 wherein the determination of step (c) is made by comparison of fluorescence polarization values of (i) and (ii).

20. The method of claim 1 wherein the determination of step (c) is made by comparison of anisotropy values of (i) and (ii).

21. The method of claim 1 wherein the determination of step (c) is made by comparison of rotational correlation times of (i) and (ii).

22. The method of claim 1 wherein the sample is a biological sample.

23. The method of claim 22 wherein the sample is blood.

24. The method of claim 1 wherein the target compound is thrombin.

25. The method of claim 1 wherein the target compound is elastase.

26. The method of claim 1 wherein the target compound is a cell surface marker.

27. The method of claim 1 wherein the target compound is a growth factor.

28. The method of claim 27 wherein the target compound is basic fibroblast growth factor.

29. The method of claim 27 wherein the target compound is vascular endothelial growth factor.

30. The method of claim 1 wherein the target compound is a growth factor receptor.

31. The method of claim 1 wherein the target compound is human chorionic gonadotropin.

32. The method of claim 1 wherein the target compound is a whole cell.

33. The method of claim 32 wherein the whole cell is a bacterial cell.

34. The method of claim 1 wherein the target compound is a viral particle.

35. A method of monitoring the binding of a target compound to its receptor in a sample using a spectroscopically detectable nucleic acid ligand to the receptor in a competition-based assay, comprising:

(a) measuring the spectroscopic emissions of the spectroscopically detectable nucleic acid ligand;

(b) mixing the spectroscopically detectable nucleic acid ligand with the sample, whereby the spectroscopically detectable nucleic acid ligand binds to the receptor in competition with the target compound;

(c) measuring the spectroscopic emissions of the complex of the spectroscopically detectable nucleic acid ligand bound to the receptor of step (b);

(d) determining the degree of binding of the spectroscopically detectable nucleic acid ligand to the receptor from the spectroscopic emissions measured in step (c); and

(e) calculating the degree of binding of target compound to its receptor from the determination of step (d).

36. The method of claim 35 wherein the target compound is a growth factor. b1 In this embodiment, the method comprises the steps of adding to the sample a spectroscopically detectable labeled nucleic acid ligand that binds to the receptor;

measuring the spectroscopic emissions of the complex of receptor and spectroscopically detectable nucleic acid ligand;

determining from the spectroscopic emissions of the complex the degree of binding of the spectroscopically detectable labeled nucleic acid ligand to the receptor; and

calculating from the degree of binding of the spectroscopically detectable labeled nucleic acid ligand to the receptor, the degree of binding of target compound to the receptor.