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Details for Patent: 5,641,629

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Details for Patent: 5,641,629

Title: Spectroscopically detectable nucleic acid ligands
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.
Inventor(s): Pitner; James B. (Durham, NC), Malinowski; Douglas P. (Hillborough, NC), Vonk; Glenn P. (Fuquay-Varina, NC), Gold; Larry (Boulder, CO)
Assignee:
Filing Date:Jan 20, 1995
Application Number:08/376,329
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.
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