Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

• US 7,794,945 B2
• Filed: 07/14/2008
• Issued: 09/14/2010
• Est. Priority Date: 04/03/1995
• Status: Expired due to Fees

• Current Assignee: ELITechGroup B.V. (ELITech Group SAS)
• Original Assignee: Elitech Holding BV (ELITech Group SAS)
• Inventors: Meyer, Rich B. Jr., Lukhtanov, Eugeny A., Kutyavin, Igor V., Hedgpeth, Joel, Belousov, Evgeniy S., Afonina, Irina A.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US12/172,999
• Publication Number:
  US 20090048427A1
• Time in Patent Office: 792 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• 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

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI₃ group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

Claims

1. An oligonucleotide probe comprising a 5′

-end, a 3′

-end and one or more detectable labels, wherein the probe is a MGB-oligonucleotide conjugate, wherein the MGB is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 10³ M<sup>−

1</sup>.

2. The probe according to claim 1, wherein said MGB is a radical of a compound selected from the group consisting of netropsin, distamycin and lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, diarylamidines, pentamidine, stilbamidine, berenil, CC-1065, pyrroloindole, indole polypeptides, Hoechst 33258, and 4′

-6-diamino-2-phenylindole.

3. The probe according to claim 1, wherein MGB is crescent shaped.

4. The probe according to claim 1, wherein MGB is selected from the group consisting of a dimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₂), a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃), tetramer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₄) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).

5. The probe according to claim 1, wherein said MGB is an analog of a compound selected from the group consisting of netropsin, distamycin and lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, diarylamidines, pentamidine, stilbamidine, berenil, CC-1065, pyrroloindole, indole polypeptides, Hoechst 33258, and 4′

-6-diamino-2-phenylindole.