Nucleotide-Based Probes and Methods for the Detection and Quantification of Macromolecules and Other Analytes

• US 20130288923A1
• Filed: 11/21/2011
• Published: 10/31/2013
• Est. Priority Date: 11/24/2010
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ricci, Francesco, Plaxco, Kevin W., Valle-Blisle, Alexis, White, Ryan, Bonham, Andrew J.
• Granted Patent: US 9,828,628 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

Provided are unimolecular oligonucleotide probes for detecting a target in a sample. The probes use target binding-induced structural changes to detect the presence of the target in the sample. Also provided are methods of using the probes to detect a target in a sample.

Claims

1. A system for detecting one or more targets in a sample, the system comprising:

a unimolecular oligonucleotide probe configured to produce a detectable change in signal when contacted by the one or more targets, the probe comprising;

a first target binding moiety and a second target binding moiety;

a first hybridization sequence and a second hybridization sequence, wherein the first hybridization sequence and the second hybridization sequence are configured to form a duplex in the absence of the target binding to both the first target binding moiety and the second target binding moiety, such that the first target binding moiety is positioned adjacent the second target binding moiety; and

a first signaling moiety and a second signaling moiety configured such that the position of the first signaling moiety is changed relative to the second signaling moiety upon binding of the one or more targets to both the first target binding moiety and the second target binding moiety, and

wherein in the presence of binding of the one or more targets to both the first target binding moiety and the second target binding moiety, formation of the duplex is inhibited such that the probe is configured to position the first signaling moiety relative to the second signaling moiety such that the probe produces a detectable change in signal.

2. The system of claim 1, wherein the probe, comprises a stem-loop structure in the absence of the one or more targets binding to the first target binding moiety and the second target binding moiety.

3. The system of claim 1, wherein the first target binding moiety and the second target binding moiety are bound directly to the probe.

4. The system of claim 1, wherein the first target binding moiety and the second target binding moiety are bound indirectly to the probe.

5. The system of claim 4, wherein at least one of the first target binding moiety and the second target binding moiety is bound to the probe through a linker moiety.

6. The system of claim 4, wherein the probe further comprises a third hybridization sequence and a fourth hybridization sequence, and wherein the first target binding moiety is bound to a fifth hybridization sequence complementary to the third hybridization sequence and the second target binding moiety is bound to a sixth hybridization sequence complementary to the fourth hybridization sequence.

7. The system of claim 6, wherein the third hybridization sequence and the fourth hybridization sequence are substantially the same, the fifth hybridization sequence and the sixth hybridization sequence are substantially the same, and wherein the probe comprises a frame inversion between the third hybridization sequence and the fourth hybridization sequence.

8. The system of claim 7, wherein the frame inversion is a 3′

to 3′

or a 5′

to 5′

frame inversion.

9. The system of claim 1, wherein the first target binding moiety and the second target binding moiety comprise antigens, and wherein the target comprises an antibody specific for the antigens.

10. The system of claim 1, wherein the first target binding moiety and the second target binding moiety comprise polypeptides that specifically bind to a macromolecule, and wherein the target comprises the macromolecule.

11. The system of claim 1, wherein the first target binding moiety and the second target binding moiety comprise aptamers that specifically bind to a macromolecule, and wherein the target comprises the macromolecule.

12. The system of claim 1, wherein the first target binding moiety and the second target binding moiety comprise DNA or RNA sequences that specifically bind to a macromolecule, and wherein the target comprises the macromolecule.

13. The system of claim 1, wherein the target has a concentration ranging from 1 pM to 100 nM.

14. The system of claim 1, wherein the first signaling moiety comprises a fluorophore and the second signaling moiety comprises a quencher.

15. The system of claim 1, wherein the first signaling moiety comprises a first fluorophore and the second signaling moiety comprises a second fluorophore.

16. The system of claim 1, wherein the first signaling moiety comprises a nanoparticle and the second signaling moiety comprises a quencher.

17. The system of claim 1, wherein the first signaling moiety comprises a first nanoparticle and the second signaling moiety comprises a second nanoparticle.

18. The system of claim 1, wherein the first signaling moiety comprises an electrochemical reporter and the second signaling moiety comprises an electrode.

19. The system of claim 18, wherein the probe is immobilized on a surface of the electrode.

20. The system of claim 19, wherein the system comprises an array of probes.

21. The system of claim 1, wherein the first signaling moiety comprises a macromolecule having a catalytic activity and the second signaling moiety comprises an inhibitor or an activator of the catalytic activity.

22. A method of detecting a target in a sample, the method comprising:

contacting a unimolecular oligonucleotide probe of claim 1 with the sample, whereby the target selectively binds to both the first target binding sequence and the second target binding sequence to form a target-probe hybrid; and

detecting the presence or absence of the target-probe hybrid.

23. The method of claim 22, wherein the sample comprises a complex sample.

24. The method of claim 23, wherein the sample comprises whole blood.

25. A method of detecting a second target in a sample, the method comprising:

contacting a unimolecular oligonucleotide probe of claim 1 with the sample, whereby the target selectively binds to both the first target binding sequence and the second target binding sequence to form a target-probe hybrid;

contacting the target-probe hybrid with a second target, whereby the second target selectively binds the target and inhibits formation of the target-probe hybrid; and

detecting the presence or absence of the target-probe hybrid.

26. A system for detecting a DNA binding protein in a sample, the system comprising:

a unimolecular oligonucleotide probe configured to produce a detectable change in signal when contacted with the DNA binding protein, the probe comprising;

a first recognition sequence and a second recognition sequence, wherein the first and second recognition sequences are configured to form a recognition duplex specifically bound by the DNA binding protein in the sample;

a first hybridization sequence and a second hybridization sequence, wherein the first and second hybridization sequences are configured to form a second duplex in the absence of binding of the DNA binding protein to the recognition duplex;

a third hybridization sequence and a fourth hybridization sequence, wherein the third and fourth hybridization sequences are configured to form a third duplex in the absence of binding of the DNA binding protein to the recognition duplex; and

a first signaling moiety and a second signaling moiety configured such that the position of the first signaling moiety is changed relative to the second signaling moiety upon binding of the DNA binding protein to the recognition duplex, and

wherein in the presence of binding of the DNA binding protein to the recognition duplex, formation of the second and third duplexes is inhibited such that the probe is configured to position the first signaling moiety relative to the second signaling moiety such that the probe produces a detectable change in signal.

27. The system of claim 26, wherein at least a portion of the first recognition sequence is positioned between the first and second hybridization sequences and at least a portion of the second recognition sequence is positioned between the third and fourth hybridization sequences.

28. The system of claim 26, further comprising a fifth hybridization sequence and a sixth hybridization sequence, wherein fifth and sixth hybridization sequences are configured to form a fourth duplex in the absence of binding of the DNA binding protein to the recognition duplex.

29. The system of claim 27, wherein the probe is configured to be in an equilibrium between formation of the second and third duplexes and formation of the recognition duplex.

30. The system of claim 29, wherein in the absence of binding of the DNA binding protein to the recognition duplex, the equilibrium is shifted towards the formation of the second and third duplexes.

31. The system of claim 29, wherein in the presence of binding of the DNA binding protein to the recognition duplex, a DNA binding protein-probe hybrid is formed and the equilibrium is shifted towards the formation of the recognition duplex.

32. The system of claim 31, wherein in the presence of a single-stranded DNA sequence configured to stabilize the DNA binding protein-probe hybrid, the equilibrium is shifted towards the formation of the recognition duplex.

33. The system of claim 26, wherein the DNA binding protein has a concentration ranging from 1 nM to 1 μ

M.

34. The system of claim 26, wherein the first signaling moiety comprises a fluorophore and the second signaling moiety comprises a quencher.

35. The system of claim 26, wherein the first signaling moiety comprises a first fluorophore and the second signaling moiety comprises a second fluorophore.

36. The system of claim 26, wherein the first signaling moiety comprises a nanoparticle and the second signaling moiety comprises a quencher.

37. The system of claim 26, wherein the first signaling moiety comprises a first nanoparticle and the second signaling moiety comprises a second nanoparticle.

38. The system of claim 26, wherein the first signaling moiety comprises an electrochemical reporter and the second signaling moiety comprises an electrode.

39. The system of claim 38, wherein the probe is immobilized on a surface of the electrode.

40. The system of claim 39, wherein the system comprises an array of probes.

41. The system of claim 26, wherein the first signaling moiety comprises a macromolecule having a catalytic activity and the second signaling moiety comprises an inhibitor or an activator of the catalytic activity.

42. A method of detecting a DNA binding protein in a sample, the method comprising:

contacting a unimolecular oligonucleotide probe of claim 26 with the sample, whereby the DNA binding protein selectively binds to the recognition duplex to form a DNA binding protein-probe hybrid; and

detecting the presence or absence of the DNA binding protein-probe hybrid.

43. The method of claim 42, wherein the detecting comprises quantifying the concentration of the DNA binding protein-probe hybrid by comparing the signal from the sample to:

(1) a saturating concentration of a competitive DNA binding sequence;

(2) a saturating concentration of a transcription factor;

or

(3) a saturating concentration of a single-stranded DNA configured to stabilize the DNA binding protein-probe hybrid.

44. The method of claim 42, wherein the sample comprises a complex sample.

45. The method of claim 44, wherein the sample comprises whole blood.

46. The method of claim 44, wherein the sample comprises a crude nuclear extract.