Non-aggregating non-quenching oligomer comprising nucelotide analogs, method of synthesis and use thereof

• US 6,972,328 B2
• Filed: 11/04/2003
• Issued: 12/06/2005
• Est. Priority Date: 11/23/1999
• Status: Expired due to Term

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Gall, Alexander A., Dempcy, Robert O., Vermeulen, Nicolaas M. J., Kutyavin, Igor V.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/702,007
• Publication Number:
  US 20040116689A1
• Time in Patent Office: 763 Days
• Field of Search: 536/22.1, 536/23.1, 536/24.1, 536/24.3, 536/24.33
• US Class Current: 536/22.1
• CPC Class Codes: A61K 31/519 ortho- or peri-condensed with heterocyclic rings
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• 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: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C07K 5/06026 the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
• CPC Class Codes: C07K 5/06139 with the first amino acid being heterocyclic
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid

Abstract

The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.

Claims

1. An oligonucleotide probe comprising a 5′

-end, a 3′

-end, one or more detectable fluorescent labels, and a qenching agent which qenches the fluorescence emission of the one or more fluorescent labels wherein the probe comprises at least 4 consecutive guanine residues in which at least one of the four consecutive guanine residues has been replaced by a PPG residue, the probe exhibiting reduced quenching of the one or more fluorescent labels due to said replacement of residues.

2. An oligonucleotide probe of claim 1, further comprising an attached minor groove binder.

3. An oligonucleotide probe of claim 1, wherein the label is a fluorescein.

4. An oligonucleotide probe of claim 1, wherein the label is a cyanine.

5. An oligonucleotide probe of claim 1, wherein the label is a rhodamine.

6. An oligonucleotide probe of claim 2, wherein the minor groove binder is located at the oligonucleotide 5′

end.

7. An oligonucleotide probe of claim 2, wherein the minor groove binder is located at the oligonucleotide 3′

end.

8. An oligonucleotide probe of claim 1, wherein the label is located at the oligonucleotide 5′

end.

9. An oligonucleotide probe of claim 1, wherein the label is located at the oligonucleotide 3′

end.

10. An oligonucleotide probe of claim 2, wherein the minor groove binder is selected from the group consisting of a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).

11. An oligonucleotide probe of claim 2, further comprising multiple fluorescent labels.

12. An oligonucleotide probe of claim 11, wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.

13. An oligomeric probe of claim 1, comprising from 4 to 9 consecutive guanine residues in which at least one of the guanine residues has been replaced by a PPG residue.

14. An oligomeric probe of claim 1, comprising at least 4 consecutive guanine residues in which all of the at least four consecutive guanine residues have been replaced by PPG residues.

15. An oligomeric probe of claim 14 comprising from 4 to 9 consecutive guanine residues in which all of the guanine residues have been replaced by PPG residues.

16. An oligonucleotide probe of claim 1, wherein at least one guanine radical that is directly adjacent to a fluorescent label has been replaced by a PPG residue.

17. An oligonucleotide probe of claim 16, comprising from 4 to 9 consecutive guanine radicals directly adjacent to a fluorescent label in which at least one of the guanine residues has been replaced by a PPG residue.

18. An oligomeric probe of claim 17 comprising from 4 to 9 consecutive guanine residues in which all of the guanine residues have been replaced by PPG residues.