Bimolecular protease-based biosensor

Patent metadata

Field	Value
Publication number	US-9772328-B2
Application number	US-201415021494-A
Country	US
Kind code	B2
Filing date	Sep 12, 2014
Priority date	Sep 12, 2013
Publication date	Sep 26, 2017
Grant date	Sep 26, 2017

Abstract

A biosensor comprises first and second molecular components and is capable of displaying protease activity in response to a binding event mediated by first and second binding partners of the biosensor. The first and second binding partners may bind each other directly or may both bind a target molecule. At least the first molecular component comprises an autoinhibited protease, whereby the binding event switches the protease frora an autoinhibited inactive state to a protease active state. The second molecular component may activate the protease of the first molecular component by binding a cross-binder which releases the autoinhibitor or by cleaving a linker which releases the autoinhibitor. The first and second molecular components may both have autoinhibited proteases which reciprocally activate each other.

First claim

The invention claimed is:

1. A biosensor that comprises first and second molecular components wherein: the first molecular component comprises: a first binding partner, a protease amino acid sequence and an inhibitor of the protease activity of said protease; and the second molecular component comprises: a second binding partner and a subcomponent capable of facilitating: (i) at least partial release of inhibition of the protease of the first molecular component by the inhibitor to switch the protease of the first molecular component from a protease inactive to a protease active state; or (ii) at least partial inhibition of the protease of the first molecular component by the inhibitor to switch the protease of the first molecular component from a protease active to a protease inactive state; upon a binding interaction between the first and second binding partners.

2. The biosensor of claim 1 , wherein the first binding partner and the second binding partner are capable of directly binding, coupling, interacting or forming a complex to thereby co-localize the first molecular component and the second molecular component.

3. The biosensor of claim 1 , wherein the first binding partner and the second binding partner are capable of binding, coupling, interacting or forming a complex with a target molecule to thereby co-localize the first molecular component and the second molecular component.

4. The biosensor of claim 3 , wherein the first binding partner and the second binding partner are capable of binding, interacting or forming a complex with the same target molecule to thereby co-localize the first molecular component and the second molecular component.

5. The biosensor of claim 3 , wherein the first binding partner and the second binding partner are capable of co-operatively binding the target molecule.

6. The biosensor of claim 1 , wherein the subcomponent of the second molecular component comprises an amino acid sequence of a protein or fragment thereof.

7. The biosensor of claim 6 , wherein the protein or fragment thereof is not a protease or protease fragment.

8. The biosensor of claim 7 , wherein the first molecular component comprises a cross-binder that is capable of binding the protein or protein fragment that is not a protease.

9. The biosensor of claim 8 , wherein the cross-binder is coupled, fused, connected or contiguous with the inhibitor of the protease activity of the first molecular component.

10. The biosensor of claim 9 , wherein upon a binding interaction between the first binding partner, the second binding partner and optionally, a target molecule, the cross-binder is capable of binding the protein of the second molecular component to thereby at least partly release inhibition of the protease of the first molecular component by the inhibitor to thereby switch the protease of the first molecular component from a protease inactive to a protease active state.

11. The biosensor of claim 7 , wherein the protein is, or comprises calmodulin or a fragment thereof.

12. The biosensor of claim 11 , wherein the cross-binder is a peptide capable of binding of interacting with calmodulin.

13. The biosensor of claim 7 , wherein the protein is, or comprises, an affinity clamp; wherein the affinity clamp comprises a recognition domain and wherein the recognition domain is capable of binding one or more target molecules.

14. The biosensor of claim 13 , wherein the cross-binder is a peptide capable of binding or interacting with the affinity clamp.

15. The biosensor of claim 13 , wherein the affinity clamp further comprises an enhancer domain.

16. The biosensor of claim 15 , wherein the recognition domain is a PDZ domain.

17. The biosensor of claim 1 , wherein the first binding partner and/or the second binding partner is or comprises an antibody or antibody fragment.

18. The biosensor of claim 1 , wherein the first binding partner and/or the second binding partner is or comprises one or a plurality of epitopes.

19. The biosensor of claim 1 , wherein the subcomponent of the second molecular component comprises an amino acid sequence of another protease or protease fragment.

20. The biosensor of claim 19 , wherein the first molecular component further comprises at least one protease cleavage site cleavable by said another protease of the second molecular component to at least partly release inhibition of the protease of the first molecular component by the inhibitor and thereby switch the first molecular component of the biosensor from a protease inactive to a protease active state.

21. The biosensor of claim 20 , wherein the second molecular component does not comprise an inhibitor of said another protease.

22. The biosensor of claim 21 , wherein the second molecular component further comprises at least one protease cleavage site cleavable by the protease of the first molecular component and an inhibitor of said another protease, whereby cleavage of the protease cleavage site of the second molecular component by the protease of the first molecular component at least partly releases inhibition of said another protease by the inhibitor to thereby switch the second molecular component of the biosensor from a protease inactive to a protease active state.

23. The biosensor of claim 1 , wherein the protease of the first molecular component and/or the second molecular component is a cysteine protease, a serine protease, an aspartate protease, a metalloprotease, a threonine protease, or a glutamic acid protease.

24. The biosensor of claim 23 , wherein the protease is derived or obtainable from a virus.

25. The biosensor of claim 24 , wherein the virus is a Potyvirus or a Flavivirus.

26. The biosensor of claim 25 , wherein the Potyvirus is SMV, TEV or TVMV.

27. The biosensor of claim 26 , wherein the Flaivivirus is HCV.

28. The biosensor of claim 27 , wherein the protease is an NIa protease.

29. The biosensor of claim 1 , wherein the inhibitor of the protease of the first molecular component and/or the second molecular component is a peptide.

30. The biosensor of claim 29 , wherein the inhibitor is an autoinhibitory peptide.

31. The biosensor of claim 30 , wherein the autoinhibitory peptide is encoded by a Potyvirus genome or a Flavivirus genome.

32. The biosensor of claim 1 , wherein: (a) the first molecular component comprises an amino acid sequence set forth in any one of SEQ ID Nos: 1-10; and/or (b) the second molecular component comprises an amino acid sequence set forth in any one of SEQ ID Nos: 1-10.

33. A composition or kit comprising the biosensor of claim 1 and a substrate.

34. The composition or kit of claim 33 , wherein the substrate comprises an amino acid sequence cleavable by the protease of the biosensor.

35. The composition or kit of claim 33 , further comprising an amplifier molecule.

36. The composition or kit of claim 35 , wherein the amplifier molecule comprises: (i) an amino acid sequence of a protease that is different to the protease(s) of the biosensor; (ii) an inhibitor of the protease of (i); and (iii) a linker amino acid sequence which comprises a cleavage site for the protease of the biosensor.

37. The composition or kit of claim 35 , wherein the amplifier molecule com

Assignees

• Univ Queensland

Inventors

• Stein Viktor
• Alexandrov Kirill

Classifications

• G01N2333/08

  RNA viruses · CPC title

• G01N2333/185

  Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue · CPC title

• G01N33/542 Primary

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C07K19/00 Primary

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title

• C07K2319/50

  containing protease site · CPC title