Covalently linked oligonucleotide minor groove binder conjugates

• US 6,486,308 B2
• Filed: 12/18/2000
• Issued: 11/26/2002
• Est. Priority Date: 04/03/1995
• Status: Expired due to Term

• Current Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Meyer, Rich B. Jr., Lukhtanov, Eugeny A., Kutyavin, Igor V., Gamper, Howard B.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/739,928
• Publication Number:
  US 20020052482A1
• Time in Patent Office: 708 Days
• Field of Search: 514/44, 514/419, 536/23.1, 536/25.3, 536/25.32, 546/270.1, 546/271.1, 548/311.1, 548/312.4
• US Class Current: 536/23.1
• CPC Class Codes: C07D 207/34 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 403/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

Minor groove binding molecules are covalently bound to oligonucleotides which in their base sequence are complementary to a target sequence of single stranded or double stranded DNA, RNA or hybrids thereof. The covalently bound oligonucleotide minor groove binder conjugates strogly bind to the target sequence of the complementary strand.

Claims

1. An oligonucleotide-minor groove binder-reporter group combination comprising an oligonucleotide having a plurality of nucleotide units, a 3′

-end and a 5′

-end, a minor groove binder moiety attached to at least one of said nucleotide units through a linking group which covalently binds the minor groove binder moiety to the oligonucleotide, and a reporter group;

wherein the minor groove binder moiety is a radical of a molecule having a molecular weight of approximately 150 to approximately 2000 Daltons that binds in a non-intercalating manner into the minor groove of double stranded DNA, RNA or hybrids thereof with an association constant greater than approximately 10³ M<sup>−

1</sup>.

2. An oligonucleotide-minor groove binder-reporter group combination of claim 1 wherein the minor groove binding moiety is attached to the 5′

-end of the oligonucleotide.

3. An oligonucleotide-minor groove binder-reporter group combination of claim 1 wherein the minor groove binding moiety is attached to the 3′

-end of the oligonucleotide.

4. An oligonucleotide-minor groove binder-reporter group combination of claim 1 wherein the minor groove binder moiety is attached to a nucleotide unit which is neither at the 3′

nor at the 5′

end of the oligonucleotide.

5. An oligonucleotide-minor groove binder-reporter group combination of claim 1 wherein the minor groove binder moiety is attached to the heterocyclic base portion of a nucleotide unit.

6. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein the reporter group is covalently attached to the minor groove binder moiety.

7. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said reporter group is detectable by color, ultraviolet spectrum, or a physical or chemical characteristic.

8. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said reporter group is a diazobenzene moiety.

9. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said reporter group is attached to said minor groove binder moiety via a—

NH(CH₂)_mCOO(CH₂)_mS(CH₂)_m—

bridge wherein each subscript m is an integer between 1 and 10.

10. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said minor groove binder moiety is represented by the formula:

wherein

the subscript m is an integer of from 2 to 5; and

R is a selected from the group consisting of tBOC and CONH₂.

11. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said oligonucleotide is an oligodeoxyribonucleotide.

12. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said oligonucleotide is an oligodeoxyribonucleotide having nucleotide units interconnected by phosphodiester linkages, phosphorothioate linkages, methylphosphonate linkages or combinations thereof.

13. An oligonucleotide-minor groove binder-reporter group combination of claim 1, wherein said oligonucleotide comprises bases selected from the group consisting of uracil, guanine, adenine, cytosine, thymine, hypoxanthine, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-N⁴ ethenocytosine, 4-aminopyrazolo[3,4-d]pyrimidine and 6-amino-4-hydroxy-[3,4-d]pyrimidine.