Three-component biosensors for detecting macromolecules and other analytes
The invention generally provides three-component molecular biosensors. The molecular biosensors are useful in several methods including in the identification and quantification of target molecules.
1. A three-component molecular biosensor, the molecular biosensor comprising two epitope binding agent constructs and a signal construct, which together have the formula:
R 36 —R 37 —R 38 ;
R 39 —R 40 —R 41 ;
R 44 —R 43 —R 42 —R 45 —R 46
wherein:
R 36 is an epitope binding agent that binds to a first epitope on a target molecule;
R 37 is a flexible linker attaching R 36 to R 38 ;
R 38 and R 41 are a pair of nucleotide sequences that are not complementary to each other, but are complementary to two distinct regions in R 42 , such that when R 38 and R 41 bind to R 42 in the presence of said target molecule, a restriction endonuclease site recognized by a restriction endonuclease that cleaves double stranded DNA is created;
R 39 is an epitope binding agent that binds to a second epitope on the target molecule;
R 40 is a flexible linker attaching R 39 to R 41 ;
R 42 is a single-stranded nucleotide sequence comprising a first region that is complementary to R 38 , and a second region that is complementary to R 41 , such that when R 38 and R 41 bind to R 42 in the presence of said target molecule, a restriction endonuclease site recognized by a restriction endonuclease that cleaves double stranded DNA is created;
R 43 is a flexible linker attaching R 42 to R 44 ;
R 44 is a first nucleotide sequence that is complementary to R 48 , and is attached to a first detection means;
R 45 is a flexible linker attaching R 42 to R 46 ; and
R 46 is a second nucleotide sequence that is complementary to R 44 , and is attached to a second detection means, such that in the absence of said target molecule R 44 and R 46 associate and a detectable signal is produced, and in the presence of said target molecule, R 36 and R 39 bind to the said target molecule and R 38 and R 41 bind to R 42 , 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 R 42 , R 44 and R 46 dissociate, reducing the detectable signal.
2. The three-component molecular biosensor of claim 1 , wherein the restriction endonuclease site of R 42 overlaps a first region that is complementary to R 38 and overlaps a second region that is complementary to R 41 , such that the endonuclease cleavage site is between the first region complementary to R 38 and the second region complementary to R 41 .
3. The three-component molecular biosensor of claim 1 , wherein the target molecule is selected from the group consisting of an analyte, a prion, a protein, a polypeptide, a nucleic acid, a lipid, a carbohydrate, a macromolecular complex, a fungus, and a microbial organism.
4. The three-component molecular biosensor of claim 1 , wherein R 36 and R 39 are independently selected from the group consisting of an aptamer, an antibody, an antibody fragment, a double-stranded DNA sequence, modified nucleic acids, nucleic acid mimics, a ligand, a ligand fragment, a receptor, a receptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator, an allosteric molecule, and an ion.
5. The three-component molecular biosensor of claim 1 , wherein R 37 , R 40 , R 43 , and R 45 are from about 50 to about 250 angstroms in length and are independently selected from the group consisting of a heterobifunctional chemical linker, a homobifunctional chemical linker, polyethylene glycol, and nucleic acid.
6. The three-component molecular biosensor of claim 1 , wherein R 38 and R 41 are from about 2 to about 20 nucleotides in length.
7. The three-component molecular biosensor of claim 1 , wherein R 38 has a free energy for association with the sequence of R 42 complementary to R 38 from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. at and at a salt concentration from about 1 mM to about 100 mM and R 41 has a free energy for association with the sequence of R 42 complementary to R 41 from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. at and at a salt concentration from about 1 mM to about 100 mM.
8. The three-component molecular biosensor of claim 1 , wherein R 44 and R 46 have a free energy for association from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. at and at a salt concentration from about 1 mM to about 100 mM.
9. The three-component molecular biosensor of claim 1 , wherein the first and second detection means are independently selected from the group consisting of fluorescence resonance electron transfer (FRET), lanthamide resonance electron transfer (LRET), fluorescence cross-correlation spectroscopy, flourescence quenching, fluorescence polarization, flow cytometry, scintillation proximity, luminescence resonance energy transfer, direct quenching, ground-state complex formation, chemiluminescence energy transfer, bioluminescence resonance energy transfer, excimer formation, colorimetric substrates detection, phosphorescence, electro-chemical changes, and redox potential changes.
10. A method for detecting a target molecule in a sample, the method comprising: (a) contacting a biosensor comprising two epitope binding agent constructs and a signal construct with a sample comprising the said target molecule, under conditions such that:
i. R 36 and R 39 bind to the said target molecule,
ii. R 38 associates with the first region of R 42 that is complementary to R 38 ,
iii. R 41 associates with the second region of R 42 that is complementary to R 41 , and
iv. R 44 associates with R 46 , such that the first and second detection means produce a detectable signal;
wherein the two epitope binding agent constructs and the signal construct together have the formula:
R 36 —R 37 —R 38 ;
R 39 —R 40 —R 41 ;
R 44 —R 43 —R 42 —R 45 —R 46
wherein:
R 36 is an epitope binding agent that binds to a first epitope on a target molecule;
R 37 is a flexible linker attaching R 36 to R 38 ;
R 38 and R 41 are a pair of nucleotide sequences that are not complementary to each other, but are complementary to two distinct regions in R 42 , such that when R 38 and R 41 bind to R 42 in the presence of said target molecule, a restriction endonuclease site recognized by a restriction endonuclease that cleaves double stranded DNA is created;
R 39 is an epitope binding agent that binds to a second epitope on the target molecule;
R 40 is a flexible linker attaching R 39 to R 41 ;
R 42 is a single-stranded nucleotide sequence comprising a first region that is complementary to R 38 , and a second region that is complementary to R 41 , such that when R 38 and R 41 bind to R 42 in the presence of said target molecule, a restriction endonuclease site recognized by a restriction endonuclease that cleaves double stranded DNA is created;
R 43 is a flexible linker attaching R 42 to R 44 ;
R 44 is a first nucleotide sequence that is complementary to R 48 , and is attached to a first detection means;
R 45 is a flexible linker attaching R 42 to R 46 ; and
R 46 is a second nucleotide sequence that is complementary to R 44 , and is attached to a second detection means, such that in the absence of said target molecule R 44 and R 46 associate and a detectable signal is produced, and in the presence of said target molecule, R 36 and R 39 bind to the said target molecule and R 38 and R 41 bind to R 42 , 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 R 42 , R 44 and R 46 dissociate, reducing the detectable signal;
(b) contacting the biosensor with a restriction endonuclease that recognizes the restriction endonuclease site of R 42 , and
(c) detecting the signal change when the restriction endonuclease cleaves R 42 such that R 44 disassociates from R 46 and the first and second detection means no longer produce a detectable signal.
11. The method of claim 10 , wherein the restriction endonuclease site of R 42 overlaps a first region that is complementary to R 38 and overlaps a second region that is complementary to R 41 , such that the endonuclease cleavage site is between the first region complementary to R 38 and the second region complementary to R 41 .
12. The method of claim 10 , wherein the target molecule is selected from the group consisting of an analyte, a prion, a protein, a polypeptide, a nucleic acid, a lipid, a carbohydrate, a macromolecular complex, a fungus, and a microbial organism.
13. The method of claim 10 , wherein R 36 and R 39 are independently selected from the group consisting of an aptamer, an antibody, an antibody fragment, a double-stranded DNA sequence, modified nucleic acids, nucleic acid mimics, a ligand, a ligand fragment, a receptor, a receptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator, an allosteric molecule, and an ion.
14. The method of claim 10 , wherein R 37 , R 40 , R 43 , and R 45 are from about 50 to about 250 angstroms in length and are independently selected from the group consisting of a heterobifunctional chemical linker, a homobifunctional chemical linker, polyethylene glycol, and nucleic acid.
15. The method of claim 10 , wherein R 38 and R 41 are from about 2 to about 20 nucleotides in length.
16. The method of claim 10 , wherein R 38 has a free energy for association with the sequence of R 42 complementary to R 38 from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. at and at a salt concentration from about 1 mM to about 100 mM and R 41 has a free energy for association with the sequence of R 42 complementary to R 41 from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. at and at a salt concentration from about 1 mM to about 100 mM.
17. The method of claim 10 , wherein R 44 and R 46 have a free energy for association from about 5.5 kcal/mole to about 8.0 kcal/mole at a temperature from about 21° C. to about 40° C. at and at a salt concentration from about 1 mM to about 100 mM.
18. The method of claim 10 , wherein the first and second detection means are independently selected from the group consisting of fluorescence resonance electron transfer (FRET), lanthamide resonance electron transfer (LRET), fluorescence cross-correlation spectroscopy, flourescence quenching, fluorescence polarization, flow cytometry, scintillation proximity, luminescence resonance energy transfer, direct quenching, ground-state complex formation, chemiluminescence energy transfer, bioluminescence resonance energy transfer, excimer formation, colorimetric substrates detection, phosphorescence, electro-chemical changes, and redox potential changes.