Dark quenchers for donor-acceptor energy transfer
The present invention provides a family of dark quenchers, termed Black Hole Quenchers (“BHQs”), that are efficient quenchers of excited state energy but which are themselves substantially non-fluorescent. Also provided are methods of using the BHQs, probes incorporating the BHQs and methods of using the probes.
1. A quencher of excited state energy having a structure comprising a first radical, a second radical and a third radical, wherein said first radical, said second radical and said third radical are each independently selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and combinations thereof, wherein said first radical is covalently attached to said second radical through an azo bond and said second radical is covalently attached to said third radical through an azo bond,
wherein said quencher bears a reactive functional group located on a chain attached to an aryl nucleus,
wherein said reactive functional group is a member selected from a phosphoramidite and an N-hydroxysuccinimide ester.
2. The quencher of excited state energy according to claim 1 , wherein at least one of said first radical, said second radical and said third radical is aryl substituted with an alkoxy moiety.
3. The quencher of excited state energy according to claim 2 , in which said first radical attached to said second radical through said azo bond forms a moiety having the formula:
4. The quencher of excited state energy according to claim 3 , having the formula:
in which
R 3 is a member selected from substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl; and
Y′ is said reactive functional group located on a chain attached to R 3 .
5. The quencher of excited state energy according to claim 4 , wherein R 3 is substituted aryl.
6. The quencher of excited state energy according to claim 4 , wherein said chain comprises the moiety:
wherein DMT is 4,4′-dimethoxytrityl.