Linked peptide 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 and positioned to block the active domain of the fluorogen-activating peptide;
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;
wherein the blocking peptide is linked to the fluorogen-activating peptide through a cleavable peptide linker comprising an amino acid sequence recognized by a protease enzyme, wherein the blocking peptide blocks 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; and
wherein the fluorogen-activating peptide that at least partially disassociates when the peptide linker is cleaved is able to bind to the cognate fluorogen thereby modulating a fluorescence signal of the cognate fluorogen,
wherein:
the variable heavy chain domain comprises the amino acid sequence of SEQ ID NO: 33, and the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 21; or
the variable heavy chain domain comprises the amino acid sequence of SEQ ID NO: 23, and the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 25.
2. The biosensor of claim 1 , wherein the fluorogen-activating peptide specifically binds to the cognate fluorogen selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
3. The biosensor of claim 1 , wherein the peptide linker comprises an amino acid sequence that is recognized by a serine protease, a threonine protease, a cysteine protease, an aspartic acid protease, a matrix metalloproteinase, or a glutamic acid protease.
4. The biosensor of claim 1 , wherein:
the peptide linker comprises Glu Asn Leu Tyr Phe Gln Gly (SEQ ID NO:7) and is recognized by TEV protease;
the peptide linker comprises Leu Glu Val Leu Phe Gln Gly Pro (SEQ ID NO:9) and is recognized by a HRV-3C protease;
the peptide linker comprises Tyr Val Ala Asp (SEQ ID NO:11) and is recognized by a caspase 1 protease;
the peptide linker comprises Asp Glu Val Asp (SEQ ID NO:13) and is recognized by a caspase 3 protease; or
the peptide linker comprises Val Met Arg Leu Val Val (SEQ ID NO:15) and is recognized by MMP25 protease.
5. The biosensor of claim 1 , wherein the variable heavy chain domain consists of the amino acid sequence of SEQ ID NO: 33, and the variable light chain domain consists of the amino acid sequence of SEQ ID NO: 21.
6. The biosensor of claim 1 , wherein the variable heavy chain domain consists of the amino acid sequence of SEQ ID NO: 23, and the variable light chain domain consists of the amino acid sequence of SEQ ID NO: 25.
7. A composition comprising: a biosensor according to claim 1 and a fluorogen.
8. The composition of claim 7 , wherein the fluorogen is selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.
9. A method for analyzing protease enzyme activity comprising:
contacting a medium comprising an analyte protease enzyme with the composition according to claim 7 ; and
detecting a fluorescence signal produced by an interaction between the fluorogen activating peptide and the fluorogen.
10. The method of claim 9 , wherein the fluorogen is selected from the group consisting of thiazole orange, malachite green, dimethyl indole red, and derivatives thereof.