Multi-acceptor molecular probes and applications thereof

• US 8,652,851 B2
• Filed: 06/17/2005
• Issued: 02/18/2014
• Est. Priority Date: 06/17/2004
• Status: Expired due to Fees

• 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: Tan, Weihong, Yang, Chaoyong
• Primary Examiner(s): Xu, Robert
• Application Number:
  US11/570,491
• Publication Number:
  US 20100248385A1
• Time in Patent Office: 3,168 Days
• Field of Search: None
• 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 having two ends and including a loop sequence complementary to a target sequence;

a first stem sequence attached to the 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 to a single terminal base of the second stem sequence, wherein a dendrimer phosphoramidite is used to link said single base to said plurality of quencher molecules, wherein said quencher molecules and fluorescent label(s) possess spectrum overlap.

2. The probe of claim 1, 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.

8. The probe of claim 1, wherein the molecular probe is an aptamer.

3. A detection system comprising:

at least one excitation light source providing excitation radiation; and

at least one molecular probe, comprising;

at least one pin loop, said pin loop having two ends and including a loop sequence complementary to a target sequence;

a first stem sequence attached to the 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 to a single terminal base of the second stem sequence, wherein a dendrimer phosphoramidite is used to link said single base to said plurality of quencher molecules wherein said quencher molecules and fluorescent label(s) possess spectrum overlap.

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

providing at least one molecular probe, comprising;

at least one pin loop, said pin loop having two ends and including a loop sequence complementary to a target sequence;

a first stem sequence attached to the 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 to a single terminal base of the second stem sequence, wherein a dendrimer phosphoramidite is used to link said single base to said plurality of quencher molecules, wherein said quencher molecules and fluorescent label(s) possess spectrum overlap;

providing the sample;

contacting the at least one molecular probe with the sample and allowing binding of the molecular probe(s) to said at least one target molecule, wherein binding of the at least one target molecule to the at least one molecular probe results in either an increase or decrease of fluorescence intensity compared to a baseline fluorescence of unbound molecular probe(s); and

measuring the resultant fluorescence intensity.

5. The method of claim 4, wherein binding of the at least one molecular probe to the at least one target molecule results in a decrease of fluorescence intensity as compared to a baseline fluorescence of unbound molecular probe(s).

6. The method of claim 4, wherein binding of the at least one target molecule to the at least one molecular probe results in an increase of fluorescence intensity as compared to a baseline fluorescence of unbound molecular probe(s).

7. The method of claim 4, wherein the at least one target molecule is selected from the group consisting of protein, organic molecule, and nucleic acid molecule.