Molecular biosensors capable of signal amplification

• US 9,040,287 B2
• Filed: 02/11/2011
• Issued: 05/26/2015
• Est. Priority Date: 02/12/2010
• Status: Active Grant

• Current Assignee: Mediomics, LLC, Saint Louis University
• Original Assignee: Mediomics, LLC, Saint Louis University
• Inventors: Chang, Yie-Hwa, Tian, Ling, Heyduk, Tomasz
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US13/578,718
• Publication Number:
  US 20130034846A1
• Time in Patent Office: 1,565 Days
• Field of Search: 435/4, 435/6.1, 435/7.2, 435/183, 435/287.2, 536/24.3, 422/430
• US Class Current: 435/287.2
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

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

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.

2. 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.

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

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

R⁸—

R⁹.

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.

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.

7. 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.

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

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

R⁸.

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.