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

• US 6,261,783 B1
• Filed: 08/11/2000
• Issued: 07/17/2001
• Est. Priority Date: 12/15/1997
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Gold, Larry, Jayasena, Sumedha
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/581,326
• Time in Patent Office: 340 Days
• Field of Search: 435/6, 435/810, 436/94
• 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
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for detecting a target molecule in a test mixture suspected of containing said target molecule is described. The nucleic acid ligand capable of binding to the target molecule has a first sequence A and a second sequence B, which are partially complementary sequences that form an imperfect intramolecular duplex, which unwinds upon the binding of the target to the nucleic acid ligand. Sequences A and B are able to participate in extramolecular hybridization reactions only when the duplex is unwound. Three different cascade nucleic acids contain a first sequence and a second sequence, which are partially complementary sequences. At least one sequence is exactly complementary to A or B. The second sequence may be complementary to A or B, or may be a third sequence C, or its complement. The test mixture suspected of containing the target molecule is contacted with the nucleic acid ligand, causing the duplex of the nucleic acid ligand to unwind such that sequences A and B become available for extramolecular hybridization. This mixture is contacted with the first, second, and third cascade nucleic acids so that the unpaired A and B sequences triggers a cascade of intermolecular hybridization involving the cascade nucleic acids in which intermolecular hybridization takes place between A and its complement, B and its complement, and between C its complement, leading to the formation of a multimolecular hybridization complex. The presence of the multimolecular hybridization complex is then detected.

Claims

1. A method for detecting a target molecule in a test mixture suspected of containing said target molecule, the method comprising:

A) i) providing a nucleic acid ligand capable of binding to said target molecule, said nucleic acid ligand comprising a first sequence A and a second sequence B wherein A and B are partially complementary sequences that form an imperfect intramolecular duplex, wherein said duplex unwinds upon the binding of said target to said nucleic acid ligand, and wherein said sequences A and B are able to participate in extramolecular hybridization reactions only when said duplex is unwound;

ii) providing a first cascade nucleic acid comprising a first sequence A′ and

a second sequence C′

, wherein A′ and

C′

are partially complementary sequences that form an imperfect intramolecular duplex, wherein A′

is exactly complementary to sequence A and wherein the duplex of said first cascade nucleic acid unwinds upon the hybridization of A′

to an unpaired A sequence;

iii) providing a second cascade nucleic acid, said second cascade nucleic acid comprising a first sequence B′ and

a second sequence C wherein B′ and

C are partially complementary sequences that form an imperfect intramolecular duplex, wherein B′

is exactly complementary to sequence B and C is exactly complementary to C′

, and wherein the duplex of said second cascade nucleic acid unwinds upon the hybridization of B′

to an unpaired B sequence or upon the hybridization of C′

to an unpaired C sequence;

iv) providing a third cascade nucleic acid, said third cascade nucleic acid comprising a first sequence A and a second sequence B that form an imperfect intramolecular duplex and wherein the duplex of said third cascade nucleic acid unwinds upon the hybridization of A to A′

or upon the hybridization of B to B′

;

B) contacting said test mixture suspected of containing said target molecule with said nucleic acid ligand, whereby said duplex of said nucleic acid ligand unwinds in the presence of said target whereby sequences A and B become available for extramolecular hybridization;

C) contacting said test mixture and said nucleic acid ligand with said first, second, and third cascade nucleic acids, wherein the presence of unpaired A and B sequences on said nucleic acid ligand triggers a cascade of intermolecular hybridization involving said cascade nucleic acids in which intermolecular hybridization takes place between A and A′

, between B and B′ and

between C and C′

, leading to the formation of a multimolecular hybridization complex;

D) detecting the presence of said multimolecular hybridization complex.

2. The method of claim 1 wherein at least one of said cascade nucleic acids is labeled at discrete nucleotide positions with at least one fluorescent group and at least one fluorescence-modifying group, wherein the fluorescence emission profile of each said labeled cascade nucleic acid changes measurably upon unwinding of said intramolecular duplex, wherein said change results from movement of said fluorescence groups relative to said fluorescence-modifying groups, and wherein step D is accomplished by monitoring the fluorescence emission profile of said labeled cascade nucleic acid in said target mixture.

3. The method of claim 2 wherein each said fluorescence-modifying group is a quenching group, and wherein each said fluorescent group and each said quenching group are located on opposite strands of said intramolecular duplex such that fluorescence emission from said fluorescent group is quenched by said quenching group, wherein said fluorescent group and said quenching group become spatially separated upon the unwinding of said stem such that fluorescence emission from said fluorescence group is no longer quenched by said quenching group.