Methods for enhancing nucleic acid hybridization

• US 9,234,233 B2
• Filed: 12/20/2014
• Issued: 01/12/2016
• Est. Priority Date: 03/26/2010
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (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:
  US14/578,399
• Publication Number:
  US 20150141635A1
• Time in Patent Office: 388 Days
• Field of Search: 536/23.1, 536/26.6, 435/6.1
• US Class Current: 1/1
• 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 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 composition of claim 1, wherein L₁ and L₂ each is a C₁-C₇ alkyl.

3. The composition of claim 2, wherein L₁ and L₂ are C₂ alkyls.

4. 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.

5. The composition of claim 1, wherein:

R₉ is

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

7. The composition of claim 6, 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.

8. The composition of claim 6, wherein the fluorophore is attached to the 5′

phosphate at the end of the oligonucleotide.

9. The composition of claim 6, wherein the first oligonucleotide is labeled with a second quencher.