Functional illumination in living cells

Patent metadata

Field	Value
Publication number	US-10598667-B2
Application number	US-201414780484-A
Country	US
Kind code	B2
Filing date	Mar 26, 2014
Priority date	Mar 26, 2013
Publication date	Mar 24, 2020
Grant date	Mar 24, 2020

Abstract

The present invention provides kits and methods for detecting peptides that change of the fluorescence of dyes upon binding to the dye. In addition, the invention provides methods for identifying said peptides.

First claim

What is claimed is:

1. A method of detecting a peptide, comprising:
contacting the peptide with an organic dye and a chelating metal ion, such that the peptide specifically binds to the dye changing the fluorescence properties of the dye, wherein the peptide is 12 to 100 amino acids in length and is conformationally constrained by the chelating metal ion, and wherein the organic dye is selected from the group consisting of triarylmethane dyes, benzylidene imidazolinone dyes, and variants thereof; and
detecting the change in fluorescence properties of the dye, thereby detecting the peptide, wherein the peptide comprises the formula:

(IIb)

(SEQ ID NO: 186)

DX 2 X 3 X 4 X 5 GX 7 X 8 X 9 X 10 X 11 E,

wherein:
each of X 2 , X 3 , X 4 , and X 5 is an amino acid independently selected from the group consisting of D, A, Y, and W, wherein at least one of X 2 , X 3 , X 4 , and X 5 is D and at least one of X 2 , X 3 , X 4 , and X 5 is W; and
each of X 7 , X 8 , X 9 , X 10 , and X 11 is an amino acid independently selected from the group consisting of V, L, I, M, F, N, E, Q, H, K, R, D, G, A, S, T, Y, W, C and P.

2. The method of claim 1 , wherein the peptide is 12 to 50 amino acids in length.

3. The method of claim 1 , wherein the peptide is a linear peptide.

4. The method of claim 1 , wherein the peptide is a peptide cyclized by a disulfide bond.

5. The method of claim 1 , wherein the chelating metal ion is Ca 2+ or Zn 2+ .

6. The method of claim 1 , wherein the peptide has at least one post-translational modification.

7. The method of claim 1 , wherein the peptide is further fused to a protein.

8. The method of claim 7 , wherein the protein comprises a targeting moiety.

9. The method of claim 8 , wherein the targeting moiety comprises an antibody, antibody fragment, peptide aptamer or variant thereof.

10. The method of claim 8 , wherein the targeting moiety comprises a viral protein.

11. The method of claim 8 , wherein the targeting moiety comprises an ion channel or a membrane receptor.

12. The method of claim 1 , wherein the peptide is selected from the group consisting of:

(SEQ ID NO: 204)

DAYWDGTGHIYE,

(SEQ ID NO: 31)

DWWDWGNHGYTE,

and

(SEQ ID NO: 23)

DWWWDGFERLEE.

Assignees

• Univ California

Inventors

• Lam Kit S
• Tian Lin
• Ahadi Sara
• Liu Ruiwu

Classifications

• G01N33/6803 Primary

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C07K2319/90

  containing a motif for post-translational modification · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title