Linked peptides fluorogenic biosensors
Biosensors, compositions comprising biosensors, methods of producing biosensors, and methods of using biosensors are disclosed. The biosensors comprise a fluorogen-activating peptide and a blocking peptide. The fluorogen-activating peptide and blocking peptide are covalently linked through a peptide linker. The blocking peptide associates with the fluorogen-activating peptide thereby blocking an active domain of the fluorogen-activating peptide when the linker is in an unmodified state. The peptide linker may contain an amino acid sequence that is specifically recognized as a modification substrate by a cognate enzyme. The fluorogen-activating peptide and the blocking peptide at least partially disassociate when the linker is modified by an enzyme, thereby allowing the fluorogen-activating peptide to bind a cognate fluorogen and modulate a fluorescence signal.
1. A biosensor comprising:
a fluorogen-activating peptide having an active domain; and
a blocking peptide linked to the fluorogen-activating peptide through a peptide linker comprising an amino acid sequence that is specifically recognized by furin;
wherein one of the fluorogen-activating peptide and the blocking peptide comprises a variable heavy chain domain of an antibody and the other peptide comprises a variable light chain domain of a different antibody;
wherein the blocking peptide associates with the fluorogen-activating peptide thereby blocking the active domain of the fluorogen-activating peptide when the peptide linker is intact, and
wherein the fluorogen-activating peptide and the blocking peptide at least partially disassociate when the peptide linker is cleaved, thereby allowing the fluorogen-activating peptide to interact with a cognate fluorogen and modulate a fluorescence signal.
2. The biosensor of claim 1 , wherein the fluorogen-activating peptide comprises a variable heavy chain domain of an antibody and the blocking peptide comprises a variable light chain domain of a different antibody.
3. The biosensor of claim 1 , wherein the fluorogen-activating peptide comprises a variable light chain domain of an antibody and the blocking peptide comprises a variable heavy chain domain of a different antibody.
4. The biosensor of claim 1 , wherein the fluorogen-activating peptide specifically binds to a cognate fluorogen selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
5. The biosensor of claim 1 , wherein the peptide linker comprises an amino acid sequence comprising the sequence Arg-Xaa-(Arg/Lys)-Arg.
6. The biosensor of claim 1 , wherein the peptide linker comprises an amino acid sequence comprising SEQ ID NO:3.
7. The biosensor of claim 1 , wherein the peptide linker comprises an amino acid sequence comprising SEQ ID NO:5.
8. A composition comprising:
a biosensor according to claim 1 ; and
a fluorogen.
9. The composition of claim 8 , wherein the fluorogen is selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
10. A method for analyzing furin enzyme activity comprising:
contacting a medium comprising an analyte enzyme with the composition according to claim 1 ; and
detecting a fluorescence signal produced by an interaction between the fluorogen-activating peptide and the fluorogen.
11. The method of claim 10 , wherein the fluorogen is selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
12. A biosensor comprising:
a fluorogen-activating peptide comprising a variable domain of an antibody; and
a blocking peptide comprising a variable domain of an antibody;
wherein one of the fluorogen-activating peptide and the blocking peptide comprises a variable heavy chain domain of an antibody and the other peptide comprises a variable light chain domain of a different antibody;
wherein the blocking peptide is linked to the fluorogen-activating peptide through a peptide linker comprising an amino acid sequence that is specifically recognized as a cleavage substrate by furin;
wherein the blocking peptide associates with the fluorogen-activating peptide thereby blocking an active domain of the fluorogen-activating peptide when the peptide linker is intact; and
wherein the fluorogen-activating peptide and the blocking peptide disassociate when the peptide linker is cleaved, thereby allowing the fluorogen-activating peptide to interact with a cognate fluorogen and modulate a fluorescence signal.
13. A composition comprising:
a biosensor according to claim 12 ; and
a fluorogen.
14. The composition of claim 13 , wherein the fluorogen is selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
15. A method for analyzing furin enzyme activity comprising:
contacting a medium comprising an analyte enzyme with the composition according to claim 13 ; and
detecting a fluorescence signal produced by an interaction between the fluorogen-activating peptide and the fluorogen.
16. The method of claim 15 , wherein the fluorogen is selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
17. A method for analyzing furin enzyme activity comprising:
contacting a reaction medium comprising an analyte enzyme with a composition comprising:
a fluorogen; and
a biosensor comprising:
a fluorogen-activating peptide; and
a blocking peptide linked to the fluorogen-activating peptide through a peptide linker comprising an amino acid sequence that is specifically recognized as a cleavage substrate by furin, wherein one of the fluorogen-activating peptide and the blocking peptide comprises a variable heavy chain domain of an antibody and the other of the fluorogen-activating peptide and the blocking peptide comprises a variable light chain domain of a different antibody;
wherein the blocking peptide associates with the fluorogen-activating peptide thereby blocking an active domain of the fluorogen-activating peptide when the peptide linker is intact, and wherein the fluorogen-activating peptide and the blocking peptide at least partially disassociate when the peptide linker is cleaved, thereby allowing the fluorogen-activating peptide to bind a cognate fluorogen and modulate a fluorescence signal; and
detecting a fluorescence signal produced by an interaction between the fluorogen-activating peptide and the fluorogen.
18. A biosensor comprising:
a fluorogen-activating peptide having an active domain comprised of a variable heavy chain domain; and
a blocking peptide comprising a variable light chain domain, the blocking peptide linked to the fluorogen-activating peptide through a peptide linker comprising an amino acid sequence that is specifically recognized by furin;
wherein the blocking peptide associates with the fluorogen-activating peptide thereby blocking an active domain of the fluorogen-activating peptide when the peptide linker is unmodified, and
wherein the fluorogen-activating peptide and the blocking peptide at least partially disassociate when the peptide linker is cleaved by furin, thereby allowing the fluorogen-activating peptide to interact with a cognate fluorogen and modulate a fluorescence signal.