IP Library Granted Patent US 10,982,289
Granted Patent B2
US 10,982,289 · App. 14/848,976 · Granted Apr 20, 2021

Hybridization probes and uses thereof

Inventors: Paul Blainey (Cambridge, MA); Jacob de Riba Borrajo (Petaluma, CA)
Assignees: The Broad Institute Inc.; Massachusetts Institute of Technology
C12Q1/689C07H21/00C12Q1/6816G01N21/6428G01N21/78C12Q2600/158G01N2021/6439G01N2201/062Y02A50/30
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Quick Facts
Patent No.
US 10,982,289
App. No.
14/848,976
Granted
Apr 20, 2021
Kind
B2
Abstract

The present invention relates to novel hybridization probes useful for rapid hybridization to realize a practicable and affordable pathogen diagnostic based on RNA signature detection, technology and hardware. In particular, the present invention relates to a nucleic acid structure which may comprise nucleic acid tiles, wherein each nucleic acid tile comprises nucleic acid oligomers, wherein each nucleic acid oligomer hybridizes to each other thereby forming a nucleic acid tile.

Claims (42)

1. A nucleic acid structure, comprising:

(a) fifteen or more three-way junction DNA monomers assembled together, wherein each three-way junction DNA monomer comprises three or more hybridizing DNA strands and at least two overhangs:

wherein the two overhangs are overhangs A and B or overhangs A′ and B′,

wherein A and A′ are complementary overhangs and B and B′ are complementary overhangs;

wherein at least one three-way junction DNA monomer is detectably labeled; and

wherein the fifteen or more three-way junction DNA monomers are synthesized in a single step or by successive addition of three-way junction DNA monomers hybridizing at overhangs A and A′ or B and B′ thereby forming the nucleic acid structure comprising three-way junction DNA monomers;

or

(b) fifteen or more four-way junction DNA monomers assembled together, wherein each four-way junction DNA monomer comprises at least two overhangs,

wherein the two overhangs are overhangs A and B or overhangs A′ and B′, wherein A and A′ are complementary overhangs and B and B′ are complementary overhangs,

wherein at least one four-way junction DNA monomer is labeled with a label, and

wherein the fifteen or more four-way junction DNA monomers are synthesized in a single step or by successive addition of four-way junction DNA monomers hybridizing at overhangs A and A′ or B and B′ thereby forming the nucleic acid structure comprising four-way junction DNA monomers.

2. The nucleic acid structure of claim 1 ,

wherein each three-way or four-way-junction DNA monomer is labeled with a single label, wherein each nucleic acid structure is distinguishable based upon a varying intensity of the label or

wherein the at least one three-way junction DNA monomer or the at least one four-way junction DNA monomer is labeled with one, two, three, four, five, six, seven, or eight labels wherein each nucleic acid structure is distinguishable based upon a varying intensity of the collection of labels or

wherein the at least one nucleic acid structure is labeled with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 labels and/or combinations of different types of labels.

3. The nucleic acid structure of claim 1 , wherein the label is a dye molecule, a fluorescent dye molecule, a quantum dot, a metal cluster, or a phosphorescent label.

4. The nucleic acid structure of claim 3 , wherein the fluorescent dye molecule is a terbium chelate derivative.

5. The nucleic acid structure of claim 4 , wherein the terbium chelate derivative is Cy3.

6. The nucleic acid structure of claim 3 , wherein the fluorescent dye molecule is fluorescein or rhodamine.

7. The nucleic acid structure of claim 1 , wherein the label is an organic label.

8. The nucleic acid structure of claim 1 wherein Y 0 is a set of all three-way or four-way junction DNA monomers which are capable of being ligated to Y 1 , wherein Y 1 is Y N when N=1, and Y N is the set of all DNA monomers which can be ligated to Y N−1 or to both Y N−1 and Y N+1 (at different ligation sites), wherein N is an integer greater than 0.

9. The nucleic acid structure of claim 8 ,

wherein Y 1 ligates to Y 0 or

wherein N=2 and Y 2 ligates to Y 1 or

wherein Y 1 comprises Y 1a and Y 1b , wherein Y 1a and Y 1b are both capable of being ligated to Y 2 or

wherein the nucleic acid structure comprises four three-way or four-way junction DNA monomers wherein each three-way or four-way junction DNA monomer is Y 0 , Y 1a , Y 1b , Y 2 or any combination thereof or

wherein the nucleic acid structure comprises seven three-way or four-way junction DNA monomers.

10. The nucleic acid structure of claim 1 ,

wherein the nucleic acid structure is a nucleic acid dendrimer or

wherein the nucleic acid structure is a DNA structure.

11. A probe set comprising an optical probe comprising the nucleic acid structure of claim 1 , wherein the optical probe is conjugated to a hybridization probe, wherein the hybridization probe targets a nucleic acid.

12. A method of detecting a target nucleic acid sequence comprising adding the probe set of claim 11 , to a sample comprising the target nucleic acid sequence and identifying the target nucleic acid sequence bound to the probe set, wherein the identifying comprises detection of at least one label.

13. The method of claim 12 ,

wherein the detection of a label comprises measuring the intensity of the at least one label or

further comprising photobleaching the at least one label or

further comprising analyzing time-dependent intensity variations of the at least one label or

further comprising measuring a duration of emission from the at least one label.

14. The method of claim 12 , wherein the target nucleic acid sequence is a stress response gene.

15. The method of claim 14 , further comprising exposing an organism comprising the stress response gene to an antibiotic and determining if the stress response gene is expressed.

16. The method of claim 15 , wherein the organism is a microbe.

17. The method of claim 14 , wherein the absence of a stress response gene indicates an antibiotic resistant organism.

18. The method of claim 12 , wherein the target nucleic acid originated in a human cell.

Assignments (3)
CONFIRMATORY LICENSE Recorded Jul 27, 2017
From: BROAD INSTITUTE, INC.
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 043350/0388 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 12, 2016
From: BLAINEY, PAUL
To: THE BROAD INSTITUTE INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 040708/0605 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 12, 2016
From: BORRAJO, JACOB DE RIBA
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 040708/0781 →
Continuity (3)
Continuation In Part PCTUS2014030415 · Mar 17, 2014
Provisional Application 61786931 · Mar 15, 2013
Related Publication 20160083783A1 · Mar 24, 2016