Duplex stabilizing fluorescence quenchers for nucleic acid probes
Diaryl-azo derivatives are efficient fluorescence quenchers as well as nucleic acid duplex-stabilizing agents and are useful in oligonucleotide conjugates and probes. The oligonucleotide-quencher conjugates may be used in detection methods for nucleic acid targets.
1. A quencher reagent for oligonucleotide labeling having the formula:
wherein Ar 1 and Ar 2 are aromatic or hetero-aromatic moieties;
X is hydroxyl, a leaving group, a linker with a functional group or a protected functional group, or a linker connected to a synthesis solid support; and
n is from 1 to 3.
2. The quencher reagent of claim 1 , wherein one or both of Ar 1 and Ar 2 are substituted with a reactive group, a linker with a functional group or a protected functional group, or a linker connected to a synthesis solid support.
3. The quencher reagent of claim 1 , having the formula:
wherein Ar 2 is an aromatic or hetero-aromatic ring with an exocyclic amino group.
4. The quencher reagent of claim 3 , wherein the exocyclic amino group is substituted with one or two of an alkyl, a linker with a functional group or protected functional group, or a linker connected to a synthesis solid support.
5. The quencher reagent of claim 3 , having the formula:
wherein X is hydroxyl, —OPFP, or a leaving group.
6. A method for preparing an oligonucleotide conjugate labeled with a quencher, comprising:
reacting an oligonucleotide with the quencher reagent of claim 1 to produce an oligonucleotide conjugate labeled with a quencher.
7. A method for preparing an oligonucleotide conjugate labeled with a quencher, comprising:
reacting an oligonucleotide with the quencher reagent of claim 3 to produce an oligonucleotide conjugate labeled with a quencher.
8. A method for preparing an oligonucleotide conjugate labeled with a quencher, comprising:
reacting an oligonucleotide with the quencher reagent of claim 5 to produce an oligonucleotide conjugate labeled with a quencher.
9. A quencher reagent for oligonucleotide labeling having the formula:
wherein Ar 1 and Ar 2 are aromatic or hetero-aromatic moieties;
Y is NH, N-alkyl, N(C═O)O-alkyl, N—P, O, S, or Se, wherein P is a protecting group;
R 1 is H or an alkyl connecting with R 2 to form a five-member ring;
R 2 , R 3 , R 4 , and R 5 are H, alkyl, aryl, heteroaryl, O-alkyl, O-aryl, NHR 6 , NHC(═O)R 6 , F, Cl, or Br, wherein R 6 is H, alkyl, aryl, heteroaryl, O-alkyl, O-aryl, F, Cl, or Br;
X is hydroxyl, a leaving group, a linker with a functional group or a protected functional group, or a linker connected to a synthesis solid support; and
n is from 1 to 3.
10. The quencher reagent of claim 9 , wherein one or both of Ar 1 and Ar 2 are substituted with a reactive group, a linker with a functional group or a protected functional group, or a linker connected to a synthesis solid support.