Bimolecular optical probes

• US 7,619,059 B2
• Filed: 09/09/2004
• Issued: 11/17/2009
• Est. Priority Date: 07/29/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Somberg, Richard, Kupcho, Kevin R., Kleman-Leyer, Karen M., Lasky, David A., Klink, Tony A., Beebe, Jane A., Werner, Elizabeth A., Vogel, Kurt
• Primary Examiner(s): Tsang; Cecilia
• Assistant Examiner(s): Bradley; Christina
• Application Number:
  US10/937,042
• Publication Number:
  US 20050170442A1
• Time in Patent Office: 1,895 Days
• Field of Search: None
• US Class Current: 530/327
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2440/14 phosphorylation

Abstract

Compositions, methods, and kits for detecting and monitoring post-translational modification activities, including kinase or phosphatase activities, are described. The compositions typically include a peptide, a first detectable moiety, a first binding member, and a protease cleavage site. Modification of a composition by a post-translational modification enzyme, such as a kinase or phosphatase, alters the proteolytic sensitivity of the peptide, resulting in a change of a detectable property of the composition when it is associated noncovalently with a probe composition that includes a second binding member and a second detectable moiety. Panel assays for determining substrates or modulators of enzymatic activities are also described.

Claims

1. A composition of matter comprising:

a) a peptide wherein the peptide is selected from the group consisting of;

ALRRFSLGEK (SEQ ID NO;

     47),

ALRRFS*LGEK (SEQ ID NO;

     97),

RGGLFSTTPGGTK (SEQ ID NO;

     50),

RGGLFS*TTPGGTK (SEQ ID NO;

     100),

KLNRVFSVAC (SEQ ID NO;

     58);

KLNRVFS*VAC (SEQ ID NO;

     108),

RPRTFSSLAEGK (SEQ ID NO;

     63),

RPRTFSS*LAEGK (SEQ ID NO;

     113),

AKHPFSPKKAK (SEQ ID NO;

     69),

AKHPFS*PKKAK (SEQ ID NO;

     119),

DDEFTAYVATRK (SEQ ID NO;

     75),

DDEFT*AYVATRK (SEQ ID NO;

     125),

TGIFTEYVATRK (SEQ ID NO;

     76),

TGIFT*EYVATRK (SEQ ID NO;

     126),

TGVFTQHVATRK (SEQ ID NO;

     77),

TGVFT*QHVATRK (SEQ ID NO;

     127),

QRIFSIANSIVK (SEQ ID NO;

     80),

QRIFS*IANSIVK (SEQ ID NO;

     130),

RIDSIAFSIVGK (SEQ ID NO;

     83),

RIDSIAFS*IVGK (SEQ ID NO;

     133),

EEDAFSIIGK (SEQ ID NO;

     90),

EEDAFS*IIGK (SEQ ID NO;

     140),

REDEFSQNEEK (SEQ ID NO;

     91),

REDEFS*QNEEK (SEQ ID NO;

     141),

IIKIFSPDVEK (SEQ ID NO;

     70),

IIKIFS*PDVEK (SEQ ID NO;

     120),

EGDYSQVLEK (SEQ ID NO;

     22), and

EGDYS*QVLEK (SEQ ID NO;

     144),

wherein S* represents a phosphorylated serine and T* represents a phosphorylated threonine;

b) a first detectable moiety; and

c) a second detectable moiety.

2. The composition of claim 1, wherein the composition exhibits an optical property, a magnetic property, or a radioactive property.

3. The composition of claim 2, wherein the composition exhibits an optical property selected from the group consisting of a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, an anisotropy, a polarization of emitted light, a resonance energy transfer, and a quenching of emission at a wavelength.

4. The composition of claim 2, wherein the optical property is selected from the group consisting of a fluorescence excitation spectrum, a fluorescence emission spectrum, a fluorescence excitation wavelength maximum, a fluorescence emission wavelength maximum, a ratio of fluorescence excitation amplitudes at two wavelengths, a ratio of fluorescence emission amplitudes at two wavelengths, a fluorescence excited state lifetime, a fluorescence anisotropy, and a quenching of fluorescence emission at a wavelength.

5. The composition of claim 2, wherein the composition exhibits a fluorescence excitation maximum in the range from 250 to 750 nm and/or a fluorescence emission maximum in the range from 450 to 800 nm.

6. The composition of claim 5, wherein the first detectable moiety is selected from the group consisting of 5-FAM, 6-FAM, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

7. The composition of claim 1, wherein the first detectable moiety is 5-FAM.

8. The composition of claim 1, wherein the first detectable moiety is covalently linked to the peptide via a linker.

9. The composition of claim 8, wherein the linker is selected from the group consisting of GABA, an amino acid, a small peptide, diaminopentanyl, and aminohexanoyl.

10. An article of manufacture comprising:

a) packaging material; and

b) a composition of matter according to claim 1 associated with the packaging material.

11. The article of claim 10, further comprising a protein kinase or a protein phosphatase.

12. The article of claim 10, further comprising a protease.

13. The article of claim 10, further comprising ATP.

14. The composition of claim 1, wherein the first detectable moiety or the second detectable moiety is a luminescent metal complex.

15. The composition of claim 14, wherein the luminescent metal complex is a lanthanide metal complex.

16. The composition of claim 15, wherein the lanthanide metal complex comprises an organic antenna moiety, a metal liganding moiety, a lanthanide metal ion, and an optional linker.

17. The composition of claim 16, wherein the lanthanide metal ion is selected from the group consisting of Sm(III), Ru(III), Eu(III), Gd(III), Tb(III), and Dy(III).

18. The composition of claim 17, wherein the lanthanide metal ion is Eu(III) or Tb(III).

19. The composition of claim 18, wherein the lanthanide metal ion is Tb(III).

20. The composition of claim 16, wherein the organic antenna moiety is selected from the group consisting of rhodamine 560, fluorescein 575, fluorescein 590, 2-quinolone, 4-quinolone, 4-trifluoromethylcoumarin (TFC), 7-diethyl-amino-coumarin-3-carbohydrazide, 7-amino-4-methyl-2-coumarin, 7-amino-4-methyl-2-coumarin, 7-amino-4-trifluoromethyl-2-coumarin, and aminomethyltrimethylpsoralen.

21. The composition of claim 16, wherein the metal liganding moiety is selected from the group consisting of EDTA, DTPA, TTHA ,DOTA, NTA, HDTA, DTPP, EDTP, HDTP, NTP, DOTP, DO3A, DOTAGA, and NOTA.

22. The composition of claim 5, wherein the second detectable moiety is selected from the group consisting of 5-FAM, 6-FAM, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

23. The composition of claim 1, wherein the first detectable moiety and the second detectable moiety form a FRET pair.

24. The composition of claim 1, wherein the first detectable moiety and the second detectable moiety form a TR-RET pair.