Fluorescent quenching detection reagents and methods

• US 6,699,975 B2
• Filed: 03/07/2002
• Issued: 03/02/2004
• Est. Priority Date: 12/08/1999
• Status: Expired due to Term

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Gall, Alexander A., Lukhtanov, Eugeny Alexander, Reed, Michael W., Dempcy, Robert O.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/093,769
• Publication Number:
  US 20020155484A1
• Time in Patent Office: 726 Days
• Field of Search: 435/6, 536/23.1, 536/25.3, 536/26.6, 534/558
• US Class Current: 534/558
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 245/08 with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/572 Five-membered rings
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• 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: C09B 29/0813 substituted by OH, O-C(=X)-R, O-C(=X)-X-R, O-R (X being O,S,NR; R being hydrocarbonyl)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Oligonucleotide-fluorophore-quencher conjugates wherein the fluorophore moiety has emission wavelengths in the range of about 300 to about 800 nm, and or where the quencher includes a substituted 4-(phenyldiazenyl)phenylamine structure provide improved signal to noise ratios and other advantageous characteristics in hybridization and related assays. The oligonucleotide-fluorophore-quencher conjugates can be synthesized by utilizing novel phosphoramidite reagents that incorporate the quencher moiety based on the substituted 4-(phenyidiazenyl)phenylamine structure, and or novel phosphoramidite reagents that incorporate a fluorophore moiety based on the substituted coumarin, substituted 7-hydroxy-3H-phenoxazin-3-one, or substituted 5,10-dihydro-10-[phenyl]pyrido[2,3-d;6,5-d′]dipyrimidine-2,4,6,8-(1H,3H,7H,9H,10H)-tetrone structure. Oligonucleotide-fluorophore-quencher-minor groove binder conjugates including a pyrrolo[4,5-e]indolin-7-yl}carbonyl) pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indoline-7-carboxylate (DPI3) moiety as the minor groove binder and the substituted 4-(phenyldiazenyl)phenylamine moiety as the quencher, were synthesized and have substantially improved hybridization and signal to noise ratio properties.

Claims

1. A phosphoramidite-containing compound comprising a quencher moiety with an absorption spectrum in the region of about 400 to 800 nm based on a 4-(4-nitrophenylazo)phenylamine or a 4-(nitrophenylazo)naphthylamine.

2. A compound of claim 1 based on a 4-(4-nitrophenylazo)phenylamine structure.

3. A compound of claim 1 based on a 4-(4-nitrophenylazo)naphthylamine structure.

4. A compound of claim 1 wherein the quencher moiety is described by the structure

wherein R₁, R₂, R₃ and R₄ are independently —

H, halogen, —

O(CH₂)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_nCH₃]₂ where n′

=0 to 5 or —

CN;

R₅=—

H or —

(CH₂)_n—

CH₃ where n″

=0 to 5;

R₆=—

(CH₂)_n* where n*=1 to 5 and q=1 to 20.

5. A compound covalently linked to a solid support comprising a quencher moiety with an absorption spectrum in the region of about 400 to 800 nm based on a 4-(4-nitrophenylazo)phenylamine or a 4-(nitrophenylazo)naphthylamine structure.

6. A compound of claim 5 based on a 4-(4-nitrophenylazo)phenylamine structure.

7. A compound of claim 5 based on a 4-(4-nitrophenylazo)naphthylamine structure.

8. A compound of claim 5 wherein the quencher moiety is described by the structure

wherein R₁, R₂, R₃ and R₄ are independently —

H, halogen, —

O(CH₂)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_n′

CH₃]₂ where n′

=0 to 5 or —

CN;

R₅=—

H or —

(CH₂)_n—

CH₃ where n″

=0 to 5;

R₆=—

(CH₂)_n* where n*=1 to 5 and q=1 to 20.