Nitro-substituted non-fluorescent asymmetric cyanine dye compounds
View Patent ↗The invention provides an asymmetric cyanine dye compound having the structure including substituted forms thereof, wherein, at least one of R 1 and R 2 is linking group, X is O, S, or Se, and n ranges from 0 to 2. The invention further provides reporter-quencher dye pairs comprising the asymmetric cyanine dyes, dye-labelled polynucleotides incorporating the asymmetric cyanine dyes, and hybridization detection methods utilizing the dye-labelled polynucleotides.
1. A labelled oligonucleotide comprising:
an oligonucleotide; and
a reporter-quencher energy-transfer dye pair comprising a reporter dye and quencher dye wherein the quencher dye is an asymmetric cyanine dye compound having the structure:
and including any associated counter ions, wherein:
n ranges from 0 to 2;
X is O, S or Se;
R 1 is selected from the group consisting of methyl and a linking group, or when taken together with the proximate carbon of the methine bridge forms a ring structure having from 5 to 7 members;
R 2 is a linking group;
R 3 is hydrogen, or when taken together with R 4 forms a fused aromatic bridge substituted with one or more nitro groups;
R 4 is hydrogen, or when taken together with either R 3 or R 5 forms a fused aromatic bridge substituted with one or more nitro groups;
R 5 is nitro, or when taken together with either R 4 or R 6 forms a fused aromatic bridge substituted with one or more nitro groups; and
R 6 is hydrogen, or when taken together with either R 5 forms a fused aromatic bridge substituted with one or more nitro groups.
2. The labelled oligonucleotide of claim 1 in which the reporter dye is selected from the group consisting of xanthene, coumarin, naphthylamine, cyanine and bodipy dyes.
3. The labelled oligonucleotide of claim 2 in which the reporter dye is a xanthene dye.
4. The labelled oligonucleotide of claim 3 in which the xanthene dye is a fluorescein dye or a rhodamine dye.
5. The labelled oligonucleotide of claim 1 in which the reporter dye is 5- or 6-carboxy-fluorescein (FAM) and the quencher dye is of the formula:
6. The labelled oligonucleotide of claim 1 in which the reporter dye is selected from the group consisting of 5- or 6-carboxy-fluorescein (FAM), 2′,4′, 1,4-tetrachlorofluorescein (TET) and 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED) and the quencher dye is of the formula:
7. The labelled oligonucleotide of claim 1 in which the quencher dye is of the formula:
8. The labelled oligonucleotide of claim 1 in which the quencher dye is of the formula:
9. The labelled oligonucleotide of claim 1 in which the quencher dye is of the formula:
10. The labelled oligonucleotide of claim 1 in which the quencher dye is of the formula:
11. The labelled oligonucleotide of claim 1 in which the quencher dye is of the formula:
12. The labelled oligonucleotide of claim 1 in which the quencher dye is of the formula:
13. The labelled oligonucleotide of claim 1 wherein the location of the reporter dye and the quencher dye is such that when the labelled oligonucleotide is hybridized to a target nucleic acid sequence the reporter dye is not effectively quenched by the quencher dye, and when the labelled oligonucleotide is not hybridized to a target nucleic acid sequence the reporter dye is effectively quenched by the quencher dye.
14. The labelled oligonucleotide of claim 1 wherein one of the reporter and quencher dyes is attached at a 3′ end of the oligonucleotide and the other is attached at a 5′-end of the oligonucleotide.
15. The labelled oligonucleotide of claim 1 wherein the oligonucleotide sequence is designed such that a hairpin forms at the ends.
16. The labelled oligonucleotide of claim wherein the oligonucleotide is cut by 5'—3'nuclease activity of a polymerase whereby the reporter dye and quencher dye are separated.