Methods for enhancing nucleic acid hybridization

• US 8,916,345 B2
• Filed: 03/28/2011
• Issued: 12/23/2014
• Est. Priority Date: 03/26/2010
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Inventors: Rose, Scott, Behlke, Mark A., Owczarzy, Richard, Walder, Joseph A., Thomas, Derek M., Marvin, Michael R.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/073,866
• Publication Number:
  US 20110236898A1
• Time in Patent Office: 1,366 Days
• Field of Search: 435/6.2, 435/91.1, 435/6.1, 536/23.1, 536/26.6
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

A composition comprising an oligonucleotide having the structure 5′-Y₁-L₁-X-L₂-Y₂-3′. Y₁ comprises a sequence of one or more DNA or RNA nucleotides, including a first nucleotide N₁ having a 3′ phosphate covalently linked to L₁. Y₂ comprises a sequence of one or more DNA or RNA nucleotides, including a second nucleotide N₂ having a 5′ phosphate covalently linked to L₂. L₁ and L₂ each independently are a direct bond or a C₁-C₇ alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group. X is

R₁ is a hydrogen or a C₁-C₈ alkyl. M is a label or ligand comprising a fused polycyclic aromatic moiety.

Claims

1. A method of detecting a target oligonucleotide in a sample, comprising:

contacting the sample with a composition comprising a first oligonucleotide having the structure 5′

-Y₁-L₁-X-L₂-Y₂-3′

, wherein

Y₁ comprises a sequence of DNA or RNA nucleotides, including a first nucleotide N₁ having a 3′

phosphate covalently linked to L₁;

Y₂ comprises a sequence of DNA or RNA nucleotides, including a second nucleotide N₂ having a 5′

phosphate covalently linked to L₂;

L₁ and L₂ each independently are a direct bond or a C₁-C₇ alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group;

X is

2. The method of claim 1, wherein fluorescence is reduced by fluorescence resonance energy transfer when the first oligonucleotide is not hybridized to the target oligonucleotide.

3. The method of claim 1, wherein fluorescence is reduced by ground state quenching when the first oligonucleotide is not hybridized to the target oligonucleotide.

4. The method of claim 1, wherein fluorescence is reduced by both fluorescence resonance energy transfer and ground state quenching when the first oligonucleotide is not hybridized to the target oligonucleotide.

5. The method of claim 1, wherein the increase in fluorescence arises from cleavage of the target oligonucleotide.

6. The method of claim 1, wherein the oligonucleotide forms a random-coil conformation when the oligonucleotide is unhybridized, such that the fluorescence of the fluorophore is reduced.

7. The method of claim 1, wherein the oligonucleotide comprises a self-complimentary sequence and wherein the quencher and the fluorophore are attached to the oligonucleotide such that the fluorescence of the fluorophore is quenched by the quencher when the nucleic acid polymer undergoes intramolecular base pairing.

8. The method of claim 1, wherein the method is used in a PCR reaction wherein synthesis of PCR product results in an increase in fluorescence.

9. The method of claim 1, wherein the target nucleotide is the second nucleotide.

10. The method of claim 1, wherein R₉ is

11. The method of claim 1, wherein the fluorophore is 6-carboxyfluorescein (FAM), 2′

7′

-dimethoxy-4′

5′

-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;

N′

-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX);

1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2′

-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarin dye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-1-carboxy-pentyl)-3′

-ethyl-5,5′

-dimethyloxacarbocyanine (CyA);

1H,5H,11H,15H-Xantheno[2,3,4-ij;

5,6,7-i′

j′

]diquinolizin-18-ium, 9-[2(or

     4)-[[[6[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or

     2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or Texas Red), a BODIPY™

dye, benzoxaazole, stilbene or pyrene.

12. The method of claim 1, wherein the oligonucleotide is labeled with a second quencher.

13. The method of claim 12, wherein the second quencher is dabcyl, Eclipse®

quencher, BHQ1, BHQ2 and BHQ3, Iowa Black®

FQ, Iowa Black®

RQ-n1 or Iowa Black®

RQ-n2.

14. The method of claim 13, wherein the second quencher is Iowa Black®

FQ, Iowa Black®

RQ-n1 or Iowa Black®

RQ-n2.

15. A method of detecting a target oligonucleotide in a sample, comprising:

contacting the sample with a composition comprising a first oligonucleotide having the structure 5′

-Y₁-L₁-X-L₂-Y₂-3′

, wherein

Y₁ comprises a sequence of DNA or RNA nucleotides, including a first nucleotide N₁ having a 3′

phosphate covalently linked to L₁;

Y₂ comprises a sequence of DNA or RNA nucleotides, including a second nucleotide N₂ having a 5′

phosphate covalently linked to L₂;

L₁ and L₂ each independently are a direct bond or a C₁-C₇ alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group;

X is

16. The method of claim 15, wherein fluorescence is reduced by fluorescence resonance energy transfer when the first oligonucleotide is not hybridized to the target oligonucleotide.

17. The method of claim 15, wherein fluorescence is reduced by ground state quenching when the first oligonucleotide is not hybridized to the target oligonucleotide.

18. The method of claim 15, wherein fluorescence is reduced by both fluorescence resonance energy transfer and ground state quenching when the first oligonucleotide is not hybridized to the target oligonucleotide.

19. The method of claim 15, wherein the increase in fluorescence arises from cleavage of the target oligonucleotide.

20. The method of claim 15, wherein the oligonucleotide forms a random-coil conformation when the oligonucleotide is unhybridized, such that the fluorescence of the fluorophore is reduced.

21. The method of claim 15, wherein the oligonucleotide comprises a self-complimentary sequence and wherein the quencher and the fluorophore are attached to the oligonucleotide such that the fluorescence of the fluorophore is quenched by the quencher when the nucleic acid polymer undergoes intramolecular base pairing.

22. The method of claim 15, wherein the method is used in a PCR reaction wherein synthesis of PCR product results in an increase in fluorescence.

23. The method of claim 15, wherein the target nucleotide is the second nucleotide.

24. The method of claim 15, wherein the fluorophore is 6-carboxyfluorescein (FAM), 2′

7′

-dimethoxy-4′

5′

-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;

N′

-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX);

1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl -6-naphthalene sulfonate, 5-(2′

-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarin dye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-1-carboxy-pentyl)-3′

-ethyl-5,5′

-dimethyloxacarbocyanine (CyA);

1H,5H,11H,15H-Xantheno[2,3,4-ij;

5,6,7-i′

j′

]diquinolizin-18-ium, 9-[2(or

     4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or

     2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or Texas Red), a BODIPY™

dye, benzoxaazole, stilbene or pyrene.

25. The method of claim 15, wherein the oligonucleotide is labeled with a second quencher.

26. The method of claim 25, wherein the second quencher is dabcyl, Eclipse®

quencher, BHQ1, BHQ2 and BHQ3, Iowa Black®

FQ, Iowa Black®

RQ-n1 or Iowa Black®

RQ-n2.

27. The method of claim 26, wherein the second quencher is Iowa Black®

FQ, Iowa Black®

RQ-n1 or Iowa Black®

RQ-n2.