Genetically encoded fluorescent sensors for detecting intracellular signalling through diacylglycerol pathways
Described herein are novel fluorescent sensors for Diacyl Glycerol (DAG) and hosphatidylinositol 4,5-bisphosphate (PIP2) that are based on circularly permuted fluorescent proteins. These sensors use less visible spectrum than FRET-based sensors, produce robust changes in fluorescence, and can be combined with one another, or with other sensors, in a multiplex assay on standard fluorescent plate readers or live cell imaging systems.
1. A diacylglycerol (DAG) sensor fusion protein comprising
a. a protein kinase C (PKC) protein comprising a DAG binding domain and a fusion region, and
b. a circularly permuted fluorescent protein,
wherein the fusion region is located between the pseudo substrate domain and the DAG binding domain, or within the DAG binding domain;
wherein the fluorescent protein is fused with the PKC protein at a fusion site present within the fusion region;
and wherein the fluorescence of the DAG sensor fusion protein changes upon binding to DAG.
2. The DAG sensor fusion protein of claim 1 , wherein the PKC protein is selected from the group consisting of PKC-δ (delta), PKC-ε (epsilon), PKC-θ (theta), PKC-η (eta), PKC-α (alpha), PKC-β1 (beta 1), PKC-β11 (beta 11), PKC-γ (gamma), and PKC-ξ (zeta).
3. The DAG sensor fusion protein of claim 1 , wherein the PKC protein is selected from the group consisting of PKC-δ (delta), PKC-ε (epsilon), PKC-θ (theta) and PKC-η (eta).
4. The DAG sensor fusion protein of claim 1 , wherein the fusion region comprises additions or deletions of amino acids.
5. The DAG sensor fusion protein of claim 1 , wherein the fusion region further comprises linker sequences.
6. The DAG sensor fusion protein of claim 1 , comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
7. The DAG sensor fusion protein of claim 1 , comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:30.
8. A multiplex system for detecting one or more analytes comprising the DAG sensor fusion protein of claim 1 , and one or more additional fluorescent sensor that specifically detects an analyte other than DAG, wherein the one or more additional fluorescent sensor comprises a) a fluorescent sensor fusion protein comprising a fluorescent protein, or b) a fluorescent dye.
9. The multiplex system of claim 8 , wherein the DAG sensor fusion protein comprises a fluorescent protein that is fluorescent in one region of the spectrum and the additional fluorescent sensor is fluorescent in another region of the spectrum.
10. The multiplex system of claim 8 , wherein the additional fluorescent sensor is
a. a phosphatidylinositol biphosphate (PIP2) sensor, wherein the fluorescence of the PIP2 sensor changes upon binding to PIP2, or
b. a Calcium sensor, wherein the fluorescence of the Calcium sensor changes upon binding to Calcium, or
c. both.
11. A nucleic acid molecule encoding the DAG sensor fusion protein of claim 1 .
12. A cell comprising the nucleic acid molecule of claim 11 .
13. The cell of claim 12 , wherein the nucleic acid molecule encoding the DAG sensor fusion protein is located in the genome of the cell.
14. The cell of claim 12 , further comprising one or more additional nucleic acid molecules that encode one or more additional fluorescent sensor proteins that specifically detect an analyte other than DAG.