METHODS FOR ENHANCING NUCLEIC ACID HYBRIDIZATION

• US 20110236898A1
• Filed: 03/28/2011
• Published: 09/29/2011
• Est. Priority Date: 03/26/2010
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Owczarzy, Richard, Thomas, Derek M., Walder, Joseph A., Marvin, Michael R., Rose, Scott, Behlke, Mark A.
• Granted Patent: US 8,916,345 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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 composition comprising:

an oligonucleotide having the structure 5′

-Y₁-L₁-X-L₂-Y₂-3′

, wherein

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

2. The composition of claim 1, wherein the oligonucleotide comprises 15-35 nucleotides.

3. The composition of claim 1, wherein L₁ and L₂ each is a C₁-C₇ alkyl.

4. The composition of claim 3 wherein L₁ and L₂ each are C₂ alkyls.

5. The composition of claim 1, wherein the 3′

phosphate that is covalently linked to L₁, and the 5′

phosphate that is covalently linked to L₂, each independently are a phosphodiester, a phosphothioate, a phosphodithioate, a methyl phosphonate, a phosphoramidate, a phosphoramidite or a phosphotriester.

6. The composition of claim 1, wherein:

M is

7. The composition of claim 6, wherein the one of R₂-R₆ comprising the fused polycyclic aromatic moiety is

8. The composition of claim 6, wherein the electron withdrawing group is selected from the group consisting of —

NO₂, —

SO₃⁻

, —

SO₂⁻

, —

CN, —

NCS, a ketone, an alkoxyl, an ether, a carboxylic acid and a sulfonyl.

9. The composition of claim 6, wherein the electron donating group is selected from the group consisting of an alkoxyl, a heteroalkoxyl, an alkyl, a cycloalkyl, a heteroalkyl, an amino, an alkylamino, or an arylamino.

10. The composition of claim 7, wherein:

P is

11. The composition of claim 10, wherein:

R₉ is

12. The composition of claim 7, wherein:

P is and

13. The composition of claim 1, wherein M is -L₄-P, wherein L₄ is an alkyl, an alkynyl, an alkenyl, a heteroalkyl, a substituted alkyl, or an alkoxyl group, and P is a fused polycyclic aromatic moiety.

14. The composition of claim 13, wherein L₄ is —

CH2—

O—

CH2—

CH2—

NH—

.

15. The composition of claim 13, wherein:

P is

16. The composition of claim 15, wherein:

R₉ is

17. The composition of claim 1, wherein the oligonucleotide is labeled with a fluorophore.

18. The composition of claim 17, 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, benzoxanzole, stilbene or pyrene.

19. The composition of claim 17, wherein the fluorophore is attached to the 5′

phosphate at the end of the oligonucleotide.

20. The composition of claim 17, wherein the oligonucleotide is labeled with a second quencher.

21. The composition of claim 20, wherein the second quencher is dabcyl, Eclipse®

quencher, BHQ1, BHQ2 and BHQ3, Iowa Black®

FQ, Iowa Black®

RQ-n1 or Iowa Black®

RQ-n2.

22. The composition of claim 21, wherein the second quencher is Iowa Black®

FQ, Iowa Black®

RQ-n1 or Iowa Black®

RQ-n2.

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

contacting the sample with the composition of claim 1, wherein the oligonucleotide is adapted to hybridize to the target nucleic acid, the oligonucleotide is labeled with a fluorophore, and fluorescence of the fluorophore is reduced by fluorescence resonance energy transfer to the quencher or by ground state quenching by the quencher when the oligonucleotide is not hybridized to the second oligonucleotide; and

detecting an increase in fluorescence indicating the presence of the second oligonucleotide in the sample.

24. The method of claim 23, wherein fluorescence is reduced by fluorescence resonance energy transfer when the first oligonucleotide is not hybridized to the second oligonucleotide.

25. The method of claim 23, wherein fluorescence is reduced by ground state quenching when the first oligonucleotide is not hybridized to the second oligonucleotide.

26. The method of claim 23, wherein fluorescence is reduced by both fluorescence resonance energy transfer and ground state quenching when the first oligonucleotide is not hybridized to the second oligonucleotide.

27. The method of claim 23, wherein the increase in fluorescence arises from cleavage of the labeled oligonucleotide.

28. The method of claim 23, wherein the oligonucleotide forms a random-coil conformation when the oligonucleotide is unhybridized, such that the fluorescence of the fluorophore is reduced.

29. The method of claim 23, 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.

30. The method of claim 23, wherein the method is used in a PCR reaction wherein synthesis of PCR product results in an increase in fluorescence.