Target detection method using spectroscopically detectable nucleic acid ligands

• US 5,650,275 A
• Filed: 07/18/1994
• Issued: 07/22/1997
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Douglas P. Malinowski, Glenn P. Vonk, Larry Gold, Pitner J. Bruce
• Inventors: Malinowski, Douglas P., Pitner, J. Bruce, Gold, Larry, Vonk, Glenn P.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/276,271
• Time in Patent Office: 1,100 Days
• Field of Search: 435/6, 935/77, 935/78
• US Class Current: 435/6.19
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 2333/974 Thrombin
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The 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 detecting 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, whole cells and viral particles using a spectroscopically detectable labeled nucleic acid ligand comprising:

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

(b) mixing the sample with the spectroscopically detectable labeled nucleic acid ligand whereby the spectroscopically detectable labeled nucleic acid ligand complexes with the target compound; and

(c) determining whether there is a difference between (i) spectroscopic emissions of the spectroscopically detectable labeled nucleic acid ligand as determined in step (a) and (ii) spectroscopic emissions of the complex of spectroscopically detectable labeled nucleic acid ligand and target compound, whereby an increase in the spectroscopic emission of the complex of step (b) relative to the nucleic acid ligand of step (a) is indicative of the presence of the target compound in the sample.

2. The method of claim 1 wherein the 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 target compound is the protein 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 target compound is the protein 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 14 wherein the spectroscopically detectable labeled nucleic acid ligand has the formula of Compound 3.

16. The method of claim 4 wherein the target compound is the protein 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 bi-directional.

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

20. The method of claim 1 wherein the determination of step (c) is made by comparison of fluorescence anisotropy values of the spectroscopic emissions 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 the spectroscopic emissions 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 the protein thrombin.

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

31. The method of claim 1 wherein the target compound is the hormone 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 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 complexes to the receptor in competition with the target compound;

(c) measuring the spectroscopic emissions of the complex of the spectroscopically detectable nucleic acid ligand and 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 complexing of target compound to its receptor from the determination of the degree of binding in step (d).

36. The method of claim 35 wherein the target compound is a growth factor.