Claims
- 1. A protease biosensor that detects lethal factor from Bacillus anthracis comprising
a) a peptide comprising an amino acid sequence of the general formula I:X-Pro-Y-Z-W wherein
X is between 5 and 13 amino acids, wherein at least one amino acid is selected from the group consisting of Arg, Lys, or His, and wherein none are Asp or Glu; Y is selected from the group consisting of hydrophobic amino acids or Gly; Z is selected from the group consisting of uncharged amino acids; and W is between 2 and 10 amino acids of any type; b) a fluorescent donor molecule attached to the peptide that is capable of participating in fluorescence resonance energy transfer; and c) an acceptor molecule attached to the peptide that has an absorption spectrum overlapping the emission spectrum of the donor molecule, wherein the fluorescence donor molecule and the acceptor molecule are attached to amino acid residues that are on opposite sides of the cleavage site.
- 2. The protease biosensor of claim 1 wherein the acceptor molecule is fluorescent.
- 3. The protease biosensor of claim 2 wherein Y is selected from the group consisting of Ile, Tyr, Val, Leu, Ala, Phe, and Gly.
- 4. The protease biosensor of claim 3 wherein Z is selected from the group consisting of Ile, Tyr, Val, Leu, Ala, Phe, Gly, Gln, Asn, Ser, Thr, Trp, Pro, Met, and Ile.
- 5. The protease biosensor of claim 4 wherein the peptide portion of the protease biosensor is between 10 and 18 amino acids in length.
- 6. The protease biosensor of claim 4 wherein the peptide portion of the protease biosensor is between 10 and 15 amino acids in length.
- 7. The protease biosensor of claim 5 wherein the fluorescent donor molecule and the acceptor molecule are attached to opposite terminal residues of the peptide.
- 8. The protease biosensor of claim 5 wherein a linker is used to attach one or both of the fluorescent donor molecule and the acceptor molecule to the peptide.
- 9. The protease biosensor of claim 8 wherein the linker is selected from the group consisting of isothiocyanate, succinimide ester, maleimide, iodoacetamide, straight or branched-chain carbon linkers, heterocyclic carbon linkers, and peptide linkers.
- 10. The protease biosensor of claim 5, wherein the fluorescent donor molecule comprises a compound selected from the group consisting of fluorescein and ALEXA® 488.
- 11. The protease biosensor of claim 5 wherein the acceptor molecule comprises a compound selected from the group consisting of rhodamine, eosin, erythrosin, QSY-7, ALEXA® 546, BODIPY®-TMR, Cy3, and ALEXA® 532.
- 12. The protease biosensor of claim 5 wherein the fluorescent donor molecule comprises fluorescein.
- 13. The protease biosensor of claim 12 wherein the acceptor molecule comprises ALEXA® 546.
- 14. The protease biosensor of claim 5, wherein the fluorescent donor molecule comprises ALEXA® 532.
- 15. The protease biosensor of claim 14 wherein the acceptor molecule comprises a compound selected from the group consisting of ALEXA® 546 or rhodamine.
- 16. The protease biosensor of claim 5, wherein the fluorescent donor molecule comprises ALEXA® 350.
- 17. The protease biosensor of claim 16 wherein the acceptor molecule comprises ALEXA® 430.
- 18. The protease biosensor of claim 5, wherein the fluorescent donor molecule comprises ALEXA® 430.
- 19. The protease biosensor of claim 18 wherein the acceptor molecule comprises a compound selected from the group consisting of ALEXA® 532, eosin, rhodamine, or Cy3.
- 20. The protease biosensor of claim 1 wherein the peptide comprises an amino acid sequence of the general formula II:
- 21. The protease biosensor of claim 20 wherein the acceptor molecule is fluorescent.
- 22. The protease biosensor of claim 20 wherein at least two of R1, R2, R3, R4, and R5 are selected from the group consisting of Arg, Lys, and His.
- 23. The protease biosensor of claim 20 wherein at least three of R1, R2, R3, R4, and R5 are selected from the group consisting of Arg, Lys, and His.
- 24. The protease biosensor of claim 20 wherein R1, R2, and R3 are selected from the group consisting of Arg, Lys, and His.
- 25. The protease biosensor of claim 21 wherein R6 is selected from the group consisting of Ile, Tyr, Val, Leu, Ala, Phe, and Gly.
- 26. The protease biosensor of claim 25 wherein R7 is selected from the group consisting of Ile, Tyr, Val, Leu, Ala, Phe, Gly, Gln, Asn, Ser, Thr, Trp, Pro, Met, and Ile.
- 27. The protease biosensor of claim 26 wherein the peptide is between 10 and 18 amino acids in length.
- 28. The protease biosensor of claim 26 wherein the peptide is between 10 and 15 amino acids in length.
- 29. The protease biosensor of claim 26 wherein R9 is selected from the group consisting of Cys, Lys, and N-methylaminooxy amino acid; wherein if R9 is Lys, then none of R1, R2, R3, R4, and R5 are Lys.
- 30. The protease biosensor of claim 29 wherein R9 is Cys.
- 31. The protease biosensor of claim 30 wherein a thiol-specific fluorophore is attached to the Cys at the R9 position.
- 32. The protease biosensor of claim 31 wherein the thiol-specific fluorophore is selected from the group consisting of ALEXA-FLUOR® 546 maleimide, BODIPY® 530/550 iodoacetamide, eosin-5-maleimide, and QSY-7 maleimide.
- 33. The protease biosensor of claim 32 wherein the thiol-specific fluorophore is ALEXA-FLUOR® 546 maleimide
- 34. The protease biosensor of claim 26 wherein the acceptor molecule comprises fluorescein.
- 35. The protease biosensor of claim 26 wherein the fluorescent donor molecule and the acceptor molecule are attached to opposite terminal residues of the peptide.
- 36. The protease biosensor of claim 26 wherein a linker is used to attach one or both of the fluorescent donor molecule and the acceptor molecule to the peptide.
- 37. The protease biosensor of claim 36 wherein the linker is selected from the group consisting of isothiocyanate, succinimide ester, maleimide, iodoacetamide, straight or branched-chain carbon linkers, heterocyclic carbon linkers, and peptide linkers.
- 38. The protease biosensor of claim 26, wherein the fluorescent donor molecule comprises a compound selected from the group consisting of fluorescein and ALEXA® 488.
- 39. The protease biosensor of claim 26 wherein the acceptor molecule comprises a compound selected from the group consisting of rhodamine, eosin, erythrosin, QSY-7, ALEXA® 546, BODIPY®-TMR, Cy3, and ALEXA® 532.
- 40. The protease biosensor of claim 34 wherein the acceptor molecule comprises ALEXA® 546.
- 41. The protease biosensor of claim 26, wherein the fluorescent donor molecule comprises ALEXA® 532.
- 42. The protease biosensor of claim 41 wherein the acceptor molecule comprises a compound selected from the group consisting of ALEXA® 546 or rhodamine.
- 43. The protease biosensor of claim 26, wherein the fluorescent donor molecule comprises ALEXA® 350.
- 44. The protease biosensor of claim 43 wherein the acceptor molecule comprises ALEXA® 430.
- 45. The protease biosensor of claim 26, wherein the fluorescent donor molecule comprises ALEXA® 430.
- 46. The protease biosensor of claim 45 wherein the acceptor molecule comprises a compound selected from the group consisting of ALEXA® 532, eosin, rhodamine, or Cy3.
- 47. The protease biosensor of claim 1 wherein the peptide comprises the general formula III:
- 48. The protease biosensor of claim 47 wherein the acceptor molecule is fluorescent.
- 49. The protease biosensor of claim 47 wherein at least two of R1, R2, and R3 are selected from the group consisting of Arg, Lys, and His.
- 50. The protease biosensor of claim 47 wherein f R1, R2, and R3 are selected from the group consisting of Arg, Lys, and His.
- 51. The protease biosensor of claim 47 wherein the peptide is between 10 and 18 amino acids in length.
- 52. The protease biosensor of claim 47 wherein the peptide is between 10 and 15 amino acids in length.
- 53. The protease biosensor of claim 47 wherein the peptide comprises a sequence selected from the group consisting of:
a) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro (SEQ ID NO:2); b) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:3); c) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:4); d) Met-Pro-Lys-Lys-Lys-Pro-His-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:5); e) Met-Pro-Lys-Lys-Lys-Pro-His-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:6); f) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Tyr-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:7); g) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Pro-Leu-Asn-Pro-Cys (SEQ ID NO:8); h) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:9); i) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:10); j) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Val-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:11); k) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Phe-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:12); l) Met-Pro-Lys-Lys-Lys-Pro-Arg-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:13); m) Arg-Arg-Lys-Pro-Val-Leu-Pro-Ala-Leu-Thr-Ile (SEQ ID NO:14); and n) Ser-Gln-Gln-Arg-Asn-Pro-Gly-Leu-Ile-Pro-Lys (SEQ ID NO:15).
- 54. The protease biosensor of claim 47 wherein the peptide comprises a sequence selected from the group consisting of:
a) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro (SEQ ID NO:2); b) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:3); c) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:9); d) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:10); e) Arg-Arg-Lys-Pro-Val-Leu-Pro-Ala-Leu-Thr-Ile (SEQ ID NO:14); and f) Ser-Gln-Gln-Arg-Asn-Pro-Gly-Leu-Ile-Pro-Lys (SEQ ID NO:15).
- 55. The protease biosensor of claim 26, wherein the peptide consists essentially of a sequence of general formula II.
- 56. The protease biosensor of claim 47, wherein the peptide consists essentially of a sequence of general formula III.
- 57. The protease biosensor of claim 47 wherein the peptide consists essentially of a sequence selected from the group consisting of:
a) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro (SEQ ID NO:2); b) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:3); c) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:4); d) Met-Pro-Lys-Lys-Lys-Pro-His-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:5); e) Met-Pro-Lys-Lys-Lys-Pro-His-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:6); f) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Tyr-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:7); g) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Pro-Leu-Asn-Pro-Cys (SEQ ID NO:8); h) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:9); i) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:10); j) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Val-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:11); k) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Phe-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:12); l) Met-Pro-Lys-Lys-Lys-Pro-Arg-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:13); m) Arg-Arg-Lys-Pro-Val-Leu-Pro-Ala-Leu-Thr-Ile (SEQ ID NO:14); and n) Ser-Gln-Gln-Arg-Asn-Pro-Gly-Leu-Ile-Pro-Lys (SEQ ID NO:15).
- 58. The protease biosensor of claim 47 wherein the peptide consists essentially of a sequence selected from the group consisting of:
a) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro (SEQ ID NO:2); b) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:3); c) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:9); d) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:10); e) Arg-Arg-Lys-Pro-Val-Leu-Pro-Ala-Leu-Thr-Ile (SEQ ID NO:14); and f) Ser-Gln-Gln-Arg-Asn-Pro-Gly-Leu-Ile-Pro-Lys (SEQ ID NO:15), on linkers, and peptide linkers
- 59. The protease biosensor of claim 20 wherein R9 is Lys, and wherein an amine-reactive fluorescent donor molecule or acceptor molecule is attached to the R9 position.
- 60. The protease biosensor of claim 59 wherein the fluorescent donor molecule or acceptor molecule comprises a fluorophore derivatized with an amine-reactive compound selected from the group consisting of isothiocyanate or succinimide esters.
- 61. The protease biosensor of claim 47 wherein R11 is Lys, and wherein an amine-reactive fluorescent donor molecule or acceptor molecule is attached to the R11 position.
- 62. The protease biosensor of claim 61 wherein the fluorescent donor molecule or acceptor molecule comprises a fluorophore derivatized with an amine-reactive compound selected from the group consisting of isothiocyanate or succinimide esters.
- 63. The protease biosensor of claim 20 wherein R9 is Cys and wherein one of the fluorescent donor molecule or the acceptor molecule are thiol specific and bind to the Cys residue at the R9 position.
- 64. The protease biosensor of claim 63 wherein the thiol-specific fluorescent molecule comprises ALEXA® 546.65. The protease biosensor of claim 47 wherein R11 is Cys and wherein one of the fluorescent donor molecule or the acceptor molecule are thiol specific and bind to the Cys residue at the R11 position.
- 66. The protease biosensor of claim 65 wherein the thiol-specific fluorescent molecule comprises ALEXA® 546.
- 67. The protease biosensor of claim 47, wherein the fluorescent donor molecule comprises a compound selected from the group consisting of fluorescein and ALEXA® 488.
- 68. The protease biosensor of claim 67 wherein the acceptor molecule comprises a compound selected from the group consisting of rhodamine, eosin, erythrosin, QSY-7, ALEXA® 546, BODIPY®-TMR, Cy3, and ALEXA® 532.
- 69. The protease biosensor of claim 47 wherein the fluorescent donor molecule comprises fluorescein.
- 70. The protease biosensor of claim 69 wherein the acceptor molecule comprises ALEXA® 546.
- 71. The protease biosensor of claim 47, wherein the fluorescent donor molecule comprises ALEXA® 532.
- 72. The protease biosensor of claim 71 wherein the acceptor molecule comprises a compound selected from the group consisting of ALEXA® 546 or rhodamine.
- 73. The protease biosensor of claim 47, wherein the fluorescent donor molecule comprises ALEXA® 350.
- 74. The protease biosensor of claim 73 wherein the acceptor molecule comprises ALEXA® 430.
- 75. The protease biosensor of claim 47, wherein the fluorescent donor molecule comprises ALEXA® 430.
- 76. The protease biosensor of claim 75 wherein the acceptor molecule comprises a compound selected from the group consisting of ALEXA® 532, eosin, rhodamine, or Cy3.
- 77. A method for detecting the presence of Bacillus anthracis in a test sample, comprising
a) providing the protease biosensor of claim 1;b) contacting the protease biosensor with the test sample; c) measuring fluorescence resonance energy transfer from the protease biosensor, wherein an increase in intensity of emission spectra of the donor molecule indicates the presence of lethal factor from Bacillus anthracis in the test sample.
- 78. A cell-based method for detecting the presence of Bacillus anthracis in a test sample, comprising
a) providing the protease biosensor of claim 1;b) loading the protease biosensor into cells to be analyzed; c) contacting the cells with the test sample; c) measuring cell-based fluorescence resonance energy transfer from the protease biosensor, wherein an increase in intensity of emission spectra of the donor molecule indicates the presence of lethal factor from Bacillus anthracis in the test sample.
CROSS REFERENCE
[0001] This application claims priority to U.S. patent application Ser. No. 60/182,011 filed Feb. 11, 2000 and is related to U.S. patent application Ser. No. 09/430,656 filed Oct. 29, 1999.
U.S. GOVERNMENT RIGHTS
[0002] This invention was made in part with support from the U.S. Government under Contract No. N00014-98-C-0326, awarded by the U.S. Office of Naval Research, an organization of the U.S. Department of Defense. The U.S. Government may have certain nonexclusive rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60182011 |
Feb 2000 |
US |