Patent ID: 6261783
Filing Date: 2001-07-17
Classification: C07H,C12Q,Y10T

Abstract:
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.