MULTI-ACCEPTOR MOLECULAR PROBES AND APPLICATIONS THEREOF

• US 20100248385A1
• Filed: 06/17/2005
• Published: 09/30/2010
• Est. Priority Date: 06/17/2004
• Status: Active Grant

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Yang, Chaoyong, Tan, Weihong
• Granted Patent: US 8,652,851 B2
• Time in Patent Office: Days
• US Class Current: 436/172
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An oligonucleotide-based molecular probe includes at least one pin loop, the pin loop including a loop sequence complementary to a target sequence. A first stem sequence is attached to one end of the pin loop, the first stem having at least one fluorescent label attached thereto. A second stem sequence is attached to the other end of the pin loop. The second stem has a plurality of quencher molecules attached thereto.

Claims

1. A molecular probe, comprising:

at least one pin loop, said pin loop including a loop sequence complementary to a target sequence;

a first stem sequence attached to one end of said pin loop, said first stem having at least one fluorescent label attached thereto, and

a second stem sequence attached to the other end of said pin loop, said second stem having a plurality of quencher molecules attached thereto.

2. The probe of claim 1, wherein said plurality of quencher molecules are attached to a single base comprising said second stem sequence.

3. The probe of claim 2, wherein a dendrimer phosphoramidite is used to link said single base to said plurality of quencher molecules.

4. The probe of claim 3, wherein said dendrimer phosphoramidite is at least one selected from the group consisting of Tris-2,2,2-[3-(4,4′

-dimethoxytrityloxy) propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 1,3-bis-[5-(4,4′

-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

5. The probe of claim 1, wherein said plurality of quencher molecules are attached to a plurality of bases comprising said second stem sequence.

6. The probe of claim 1, wherein said quencher molecule and fluorescent label possess spectrum overlap.

7. A molecular probe comprising:

an aptamer having both a 5′

-end and a 3′

-end;

at least one fluorescent molecule attached to one of the 5′

-end and the 3′

-end; and

a plurality of quencher molecules attached to the other of the 5′

-end and the 3′

-end.

8. The molecular probe of claim 7, wherein the at least one fluorescent molecule is attached to the 5′

-end and the plurality of quencher molecules are attached to the 3′

-end.

9. The molecular probe of claim 7, wherein the at least one fluorescent molecule is quenched by the plurality of quencher molecules.

10. The molecular probe of claim 7, wherein a dendrimer phosphoramidite links said plurality of quencher molecules to the aptamer.

11. The molecular probe of claim 10, wherein said dendrimer phosphoramidite is at least one selected from the group consisting of Tris-2,2,2-[3-(4,4′

-dimethoxytrityloxy) propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 1,3-bis-[5-(4,4′

-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

12. The molecular probe of claim 7, wherein said plurality of quencher molecules and fluorescent label possess spectrum overlap.

13. A detection system comprising:

at least one excitation light source providing excitation radiation; and

at least one oligonucleotide-based molecular probe comprising at least one pin loop, said pin loop comprising a loop sequence complementary to a target sequence, a first stem sequence attached to one end of said pin loop, said first stem having at least one fluorescent label attached thereto, and a second stem sequence attached to the other end of said pin loop, said second stem having a plurality of quencher molecules attached thereto.

14. A method for forming molecular beacons each having a plurality of quenchers, the method comprising the steps of:

providing at least one fluorophore labeled nucleotide monomer, a plurality of quencher labeled nucleotide monomers, and a plurality of bases; and

coupling a programmed sequence of said at least one fluorophore labeled nucleotide monomer, said plurality of bases, and said plurality of quencher labeled nucleotide monomers.

15. A method for forming molecular beacons each having a plurality of quenchers, the method comprising the steps of:

coupling a programmed sequence of bases to form a sequence;

coupling dendrimeric phosphoramidite to said sequence, wherein said dendrimeric phosphoramidite generates multiple reactive OH groups; and

coupling a plurality of quencher molecules to said sequence at said reactive OH groups.

16. A method for forming molecular beacons each having a plurality of quenchers, the method comprising the steps of:

coupling a plurality of quencher molecules to at least one base to form a multiple quencher moiety; and

attaching said multiple quencher moiety to an oligonucleotide comprising at least one pin loop having a first end and a second end, said pin loop including a loop sequence complementary to a target sequence, a first stem sequence attached to the first end of said pin loop, said first stem having at least one fluorescent label attached thereto, and a second stem sequence attached to the second end of said pin loop, said second stem having a plurality of quencher molecules attached thereto.

17. A method of detecting the presence of at least one target molecule in a sample, the method comprising the steps of:

providing an aptamer comprising a binding region that binds to the target molecule and both a 5′

-end and a 3′

-end, a fluorophore attached to one of the 5′

-end and the 3′

-end, and at least one quencher attached to the other of the 5′

-end and the 3′

-end;

providing the sample;

contacting the aptamer with the sample and allowing binding of the aptamer to said at least one target molecule,

wherein binding of the at least one target molecule to the aptamer results in either an increase or decrease of fluorescence intensity compared to a baseline fluorescence of unbound aptamer; and

measuring the resultant fluorescence intensity.

22. The method of claim 17, wherein binding of the aptamer to the at least one target molecule results in a decrease of fluorescence intensity as compared to a baseline fluorescence of unbound aptamer.

23. The method of claim 17, wherein binding of the at least one target molecule to the aptamer results in an increase of fluorescence intensity as compared to a baseline fluorescence of unbound aptamer.

24. The method of claim 17, wherein the fluorophore is attached to the 5′

-end and the at least one quencher is attached to the 3′

-end and wherein the at least one target molecule is selected from the group consisting of protein, organic molecule, and nucleic acid molecule.

18-21. -21. (canceled)