Nucleotide-based probes and methods for the detection and quantification of macromolecules and other analytes

• US 9,828,628 B2
• Filed: 11/21/2011
• Issued: 11/28/2017
• 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: Vall- Blisle, Alexis, Ricci, Francesco, White, Ryan, Bonham, Andrew J., Plaxco, Kevin W.
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US13/988,487
• Publication Number:
  US 20130288923A1
• Time in Patent Office: 2,199 Days
• Field of Search: None
• 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) a first oligonucleotide configured to produce a detectable change in signal when bound by a target in a sample, wherein the first oligonucleotide comprises;

a first hybridization sequences;

a second hybridization sequence complementary to the first hybridization sequence such that a duplex is formed in the absence of the target;

a third hybridization sequence;

a fourth hybridization sequence;

a first signaling moiety attached to the first oligonucleotide, wherein the first signaling moiety comprises a fluorophore, a quencher, a nanoparticle, an electrochemical reporter, or an electrode; and

a second signaling moiety attached to the first oligonucleotide, wherein the second signaling moiety comprises a fluorophore, a quencher, a nanoparticle, an electrochemical reporter, or an electrode;

(b) a second oligonucleotide attached to a first target binding moiety and comprising a fifth hybridization sequence complementary to the third hybridization sequence, wherein the first target binding moiety is configured to specifically bind the target in the sample and comprises a protein, a polypeptide, a carbohydrate, a nucleic acid, a lipid, or a small molecule; and

(c) a third oligonucleotide attached to a second target binding moiety and comprising a sixth hybridization sequence complementary to the fourth hybridization sequence such that the second target binding moiety is positioned adjacent to the first target binding moiety in the absence of the target, wherein the second target binding moiety is configured to specifically bind the target in the sample and comprises a protein, a polypeptide, a carbohydrate, a nucleic acid, a lipid, or a small molecule,

wherein in the presence of binding of the target to both the first target binding moiety and the second target binding moiety, formation of the duplex is inhibited such that the position of the first signaling moiety is changed relative to the second signaling moiety such that the first oligonucleotide produces a detectable change in signal.

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

3. The system of claim 1, 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 first oligonucleotide comprises a frame inversion between the third hybridization sequence and the fourth hybridization sequence.

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

to 3′

or a 5′

to 5′

frame inversion.

5. 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.

6. 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.

7. 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.

8. 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.

9. The system of claim 1, wherein the sample comprises the system and a target and the target is present in a concentration ranging from 1 pM to 100 nM.

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

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

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

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

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

15. The system of claim 14, wherein the first oligonucleotide is immobilized on a surface of the electrode.

16. The system of claim 15, wherein the system comprises an array of first oligonucleotides.

17. 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.

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

contacting the system 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 hybrid; and

detecting the presence or absence of the hybrid.

19. The method of claim 18, wherein the sample comprises a complex sample.

20. The method of claim 19, wherein the sample comprises whole blood.

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

contacting the system 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 hybrid;

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

detecting the presence or absence of the hybrid.

22. The system of claim 1, wherein the system is configured to detect the target at a concentration ranging from 1 pM to 100 nM in the sample.

23. The system of claim 1, further comprising a detector configured to detect the detectable change in signal.