Probe and method for detecting membrane-associated molecules in living cells

The method of detecting active or inactive form of a target membrane-associated molecule in cells, said method comprising the following steps:

• providing a protein-based probe;
• contacting the probe with a reference sample comprising cells that contain the target membrane-associated molecule either in active form or in inactive form;
• contacting the probe with a test sample comprising cells in which the active or inactive form of the target membrane-associated molecule is to be detected;
• observing the cells in the reference sample and in the test sample by means of polarization microscopy;
• quantifying the fluorescence polarization exhibited by the probes in each of the reference sample and test sample;
• and
• determining whether the fluorescence polarization exhibited by the probes differs between the two samples;
• wherein when the fluorescence polarization exhibited by the probes differ, it is determined that the target membrane-associated molecule is present in the cells of the test sample in a different form than in the reference sample;
• and when the fluorescence polarization exhibited by the probes do not differ, it is determined that the target membrane-associated molecule is present in the cells of the test sample in the same form as in the reference sample;
• wherein the protein-based probe comprises:
• a) an anchoring moiety comprising at least one lipidated peptide and/or at least one transmembrane α-helical peptide,
• b) a peptide linker moiety having a length of at least 5 amino acids, wherein at least 50% of the amino acids forming the peptide linker are selected from glycine, serine, and threonine,
• c) a fluorescent moiety, and
• d) an affinity binding moiety capable of binding the target membrane-associated molecule,
• wherein the moieties are arranged in the order a-b-c-d or d-c-b-a in the direction from the N-terminus to the C-terminus;
• and
• wherein the step of providing a protein-based probe is performed by introducing into the cells a DNA that encodes the protein-based probe, and by causing the cells to produce the protein-based probe.

A method of detecting activation or inactivation of a target membrane-associated molecule in cells, said method comprising the following steps:

• providing a protein-based probe;
• contacting the probe with a test sample comprising cells in which the active or inactive form of the target membrane-associated molecule is to be detected;
• observing the cells in the test sample by means of polarization microscopy in at least two time points;
• quantifying the fluorescence polarization exhibited by the probe in each time point;
• determining whether the fluorescence polarization exhibited by the probe differs between the time points;
• wherein when the fluorescence polarization exhibited by the probes differ between the time points, it is determined that the target membrane-associated molecule in the cells of the test sample has undergone activation or inactivation;
• and when the fluorescence polarization exhibited by the probes do not differ, it is determined that the target membrane-associated molecule in the cells of the test sample has not undergone activation or inactivation;
• and
• wherein the protein-based probe comprises:
• a) an anchoring moiety comprising at least one lipidated peptide and/or at least one transmembrane a-helical peptide,
• b) a peptide linker moiety having a length of at least 5 amino acids, wherein at least 50% of the amino acids forming the peptide linker are selected from glycine, serine, and threonine,
• c) a fluorescent moiety, and
• d) an affinity binding moiety capable of binding the target membrane-associated molecule,
• wherein the moieties are arranged in the order a-b-c-d or d-c-b-a in the direction from the N-terminus to the C-terminus;
• and
• wherein the step of providing a protein-based probe is performed by introducing into the cells a DNA that encodes the protein-based probe, and by causing the cells to produce the protein-based probe.