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.
