# US 9,040,287 B2 · Molecular biosensors capable of signal amplification

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- Number: US 9,040,287 B2
- Document ID: US9040287B2
- Status: Granted
- Kind: B2
- Application number: 13/578,718
- Filed: 2011-02-11
- Published: 2013-02-07
- Granted: 2015-05-26
- Earliest claimed: 2010-02-12
- Assignees: Mediomics, LLC; Saint Louis University
- Applicants: Yie-Hwa Chang; Ling Tian; Tomasz Heyduk
- Inventors: Yie-Hwa Chang; Ling Tian; Tomasz Heyduk
- Web page: https://patentgrep.com/patent/US9040287B2

## CPC

- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6823: Release of bound markers
- G01N 33/54326: {Magnetic particles}
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention provides molecular biosensors capable of signal amplification, and methods of using the molecular biosensors to detect the presence of a target molecule.

## Claims

### Claim 1

A 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.

### Claim 2 (depends on 1)

The molecular biosensor of claim 1, wherein the free energy for association of R3 and R8, and R6 and R8 are from about −5.5 kcal/mole to about −8.0 kcal/mole at a temperature from about 21° C. to about 40° C., and a salt concentration from about 1 mM to about 100 mM.

### Claim 3 (depends on 1)

The molecular biosensor of claim 1, wherein R3 and R6 are independently from about 2 to about 20 nucleotides in length.

### Claim 4 (depends on 1)

The molecular biosensor of claim 1, further comprising a plurality of R7—R8—R9.

### Claim 5

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.

### Claim 6

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.

### Claim 7 (depends on 6)

The molecular biosensor of claim 6, wherein the free energy for association of R3 and R8, and R6 and R8 are from about −5.5 kcal/mole to about −8.0 kcal/mole at a temperature from about 21° C. to about 40° C., and a salt concentration from about 1 mM to about 100 mM.

### Claim 8 (depends on 6)

The molecular biosensor of claim 6, wherein R3 and R6 are independently from about 2 to about 20 nucleotides in length.

### Claim 9 (depends on 6)

The molecular biosensor of claim 6, further comprising a plurality of R7—R8.

### Claim 10

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.