Homogeneous detection of a target through nucleic acid ligand-ligand beacon interaction

• US 5,989,823 A
• Filed: 09/18/1998
• Issued: 11/23/1999
• Est. Priority Date: 09/18/1998
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Jayasena, Sumedha
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/157,206
• Time in Patent Office: 431 Days
• Field of Search: 435/6, 435/810, 435/7.1, 536/24.3, 536/23.1
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement

Abstract

The presence of a target in a test mixture or the concentration of such target can be determined by a method relying on fluorescence emission measurement. Such method utilizes a nucleic acid ligand to the target and a ligand beacon comprising: i) a nucleic acid sequence complimentary to at least a portion of said nucleic acid ligand, ii) a fluorescent group, and iii) a fluorescence-modifying group. The emission profile of the ligand beacon is altered based on the presence and/or concentration change of the target in the test mixture.

Claims

1. A method for identifying the presence of a target molecule in a test mixture that may contain such target molecule comprising:

a) introducing a nucleic acid ligand to said target and a ligand beacon to said test mixture;

wherein said ligand beacon comprises i) a nucleic acid sequence complementary to at least a portion of said nucleic acid ligand, ii) a fluorescent group, and iii) a fluorescence-modifying group; and

wherein the emission profile of said fluorescent group is different when said target molecule is present in the test mixture from when said target molecule is not present; and

b) measuring the fluorescence emission of said ligand beacon, whereby the presence of said target molecule is determined.

2. The method of claim 1 wherein said fluorescence-modifying group is a quenching group.

3. The method of claim 2 wherein said quenching group is (4-dimethylamino-phenylazo)benzoic acid (DABCYL).

4. The method of claim 1 wherein said fluorescent group is selected from the group consisting of fluorescein, tetramethylrhodamine, 5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), Texas Red, BODIPY, Lucifer yellow, and coumarin.

5. A method for measuring the concentration of a target molecule in a test mixture comprising:

a) introducing a nucleic acid ligand to said target and a ligand beacon to said test mixture;

wherein said ligand beacon comprises;

i) a nucleic acid sequence complementary to at least a portion of said nucleic acid ligand, ii) a fluorescent group, and iii) a fluorescence-modifying group; and

wherein the emission profile of said fluorescent group can be correlated to the concentration of said target molecule in the test mixture; and

b) measuring the fluorescence emission of said ligand beacon whereby the concentration of said target molecule is determined.

6. The method of claim 5 wherein said fluorescence-modifying group is a quenching group.

7. The method of claim 6 wherein said quenching group is (4-dimethylamino-phenylazo)benzoic acid (DABCYL).

8. The method of claim 5 wherein said fluorescent group is selected from the group consisting of fluorescein, tetramethylrhodamine, 5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), Texas Red, BODIPY, Lucifer yellow, and coumarin.

9. A method for determining the concentration of a target molecule in a test mixture, comprising:

a) mixing a nucleic acid ligand to said target; and

a ligand beacon comprising a nucleic acid capable of hybridizing to said nucleic acid ligand, wherein said nucleic acid is labeled at discrete nucleotide positions with at least one fluorescent group and at least one fluorescence-modifying group, wherein said ligand beacon adopts a primary configuration having a primary emission profile in the absence of said nucleic acid ligand, and a secondary configuration having a secondary emission profile when hybridized to said nucleic acid ligand, and wherein the secondary emission profile is different from the primary emission profile as a result of a movement of said fluorescence-modifying groups relative to said fluorescent groups upon adoption of said secondary configuration;

b) making a first measurement of fluorescence emission of said ligand beacon in said primary configuration;

c) contacting said test mixture with said nucleic acid ligand and said ligand beacon; and

d) making a second measurement of fluorescence emission of said ligand beacon;

whereby the difference between said first and said second measurements is used to determine the concentration of said target molecule in said test mixture.

10. The method of claim 9 wherein said ligand beacon hybridizes to said nucleic acid ligand only in the absence of said target.

11. The method of claim 9 wherein said ligand beacon hybridizes to said nuclei only in the presence of said target.

12. The method of claim 9 wherein said fluorescence-modifying group is a quenching group.

13. The method of claim 12 wherein said quenching group is (4-dimethylamino-phenylazo)benzoic acid (DABCYL).

14. The method of claim 9 wherein said fluorescent group is selected from the group consisting of fluorescein, tetramethylrhodamine, 5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), Texas Red, BODIPY, Lucifer yellow, and coumarin.

15. The method of claim 9 wherein said primary configuration is a stem-loop configuration.

16. The method of claim 15 wherein said fluorescent group and said fluorescence-modifying group are located at nucleotide positions on opposite strands of the stem of said stem-loop.

17. The method of claim 16 wherein said fluorescent group and said fluorescence-modifying group arc located at the terminal nucleotides of said stem-loop.

18. The method of claim 15 wherein the loop of said stem-loop comprises a sequence at least partially complementary to said nucleic acid ligand.

19. The method of claim 9 wherein said ligand beacon in said primary configuration comprises:

(1) a loop comprising a single-stranded nucleic acid with a sequence complementary to at least part of said nucleic acid ligand;

(2) a stem comprising a two-stranded duplex nucleic acid, the 5'"'"' terminus of one strand connected to the 3'"'"' terminus of the other strand by said loop, wherein said stem unwinds upon hybridization of said loop to said nucleic acid ligand to adopt said secondary configuration;

wherein said fluorescent group is covalently attached to a first nucleotide on one strand of the stem, wherein said fluorescence-modifying group is a quenching group covalently attached to a second nucleotide on the other strand of the stem, said fluorescent group being sufficiently close to said quenching group in said primary configuration such that the fluorescence emission of said fluorescent group is at least partially quenched by said fluorescence-modifying group, and wherein said fluorescence-group is sufficiently distant from said fluorescent group in said second configuration that the fluorescence emission is quenched to a lesser degree than in said primary configuration.

20. The method of claim 9 wherein said ligand beacon is capable of forming a G-quartet.

21. The method of claim 9 wherein said nucleic acid ligand is attached to a solid support.

22. A kit for detecting for the presence or measuring the concentration of a target molecule in a test mixture, comprising;

a) a nucleic acid ligand to said target molecule; and

b) ligand beacon comprising i) a nucleic acid sequence complementary to at least a portion of said nucleic acid ligand, (ii) a fluorescent group, and iii) a fluorescence modifying group.