Molecular biosensors capable of signal amplification

A method for determining the presence of a target molecule in a sample, the method comprising:

• a) combining a molecular biosensor with a target molecule, the molecular biosensor consisting of a plurality of constructs, the constructs consisting:
• R1—R2—R3;
• R4—R5—R6;
• and
• at least one R7—R8—R9;
• wherein:
• R1 is an epitope-binding agent that binds to a first epitope on a target molecule;
• R2 is a flexible linker attaching R1 to R3;
• R3 and R6 are a first pair of nucleotide sequences that are complementary to two distinct regions on R8;
• R5 is a flexible linker attaching R4 to R6;
• R4 is an epitope-binding agent that binds to a second epitope on a target molecule;
• R7 is a signaling molecule;
• R8 is an oligonucleotide construct comprising a first region that is complementary to R3 and a second region that is complementary to R6, such that in the absence of said target molecule R7 and R8 do not produce detectable signal, and in the presence of said target molecule R1 and R4 bind to the said target molecule and R3 and R6 bind to R8, creating a double stranded restriction endonuclease site that is cleaved by a restriction endonuclease that recognizes the double stranded restriction endonuclease site, wherein upon cleavage of the double stranded restriction endonuclease site created by R3, R6 and R8, R7 dissociates, producing detectable signal;
• and
• R9 is a solid support;
• b) adding a restriction endonuclease that recognizes the double-stranded restriction endonuclease recognition site formed by R3, R6 and R8;
• c) measuring the release of the R7 signaling molecule from the R9 solid support, wherein an increase in signal indicates the presence of a target molecule.

A molecular biosensor consisting of a restriction enzyme and a plurality of constructs, the constructs consisting of:

• R1—R2—R3;
• R4—R5—R6;
• and
• at least one R7—R8;
• (II)
• wherein:
• R1 is an epitope-binding agent that binds to a first epitope on a target molecule;
• R2 is a flexible linker attaching R1 to R3;
• R3 and R6 are a first pair of nucleotide sequences that are complementary to two distinct regions on R8;
• R5 is a flexible linker attaching R4 to R6;
• R4 is an epitope-binding agent that binds to a second epitope on a target molecule;
• R7 is a signaling molecule;
• and
• R8 is an oligonucleotide construct comprising a first region that is complementary to R3 and a second region that is complementary to R6, such that in the absence of said target molecule R7 and R8 do not produce detectable signal, and in the presence of said target molecule R1 and R4 bind to the said target molecule and R3 and R6 bind to R8, creating a double stranded restriction endonuclease site that is cleaved by a restriction endonuclease that recognizes the double stranded restriction endonuclease site, wherein upon cleavage of the double stranded restriction endonuclease site created by R3, R6 and R8, R7 dissociates, producing detectable signal.

A method for determining the presence of a target molecule in a sample, the method comprising:

• a) combining a molecular biosensor with a target molecule, the molecular biosensor consisting of a plurality of constructs, the constructs consisting of:
• R1—R2—R3;
• R4—R5—R6;
• and
• at least one R7—R8;
• (II)
• wherein:
• R1 is an epitope-binding agent that binds to a first epitope on a target molecule;
• R2 is a flexible linker attaching R1 to R3;
• R3 and R6 are a first pair of nucleotide sequences that are complementary to two distinct regions on R8;
• R5 is a flexible linker attaching R4 to R6;
• R4 is an epitope-binding agent that binds to a second epitope on a target molecule;
• R7 is a signaling molecule;
• and
• R8 is an oligonucleotide construct comprising a first region that is complementary to R3 and a second region that is complementary to R6, such that in the absence of said target molecule R7 and R8 do not produce detectable signal, and in the presence of said target molecule R1 and R4 bind to the said target molecule and R3 and R6 bind to R8, creating a double stranded restriction endonuclease site that is cleaved by a restriction endonuclease that recognizes the double stranded restriction endonuclease site, wherein upon cleavage of the double stranded restriction endonuclease site created by R3, R6 and R8, R7 dissociates, producing detectable signal
• b) contacting the molecular biosensor with a restriction endonuclease that recognizes the double-stranded restriction endonuclease recognition site formed by R3, R6 and R8;
• c) measuring the release of the R7 signaling molecule from R8.