US-12618828-B2 - Probe and method for detecting membrane-associated molecules in living cells

US12618828B2US 12618828 B2US12618828 B2US 12618828B2US-12618828-B2

Abstract

A protein-based probe for detecting the presence of one of two distinct states of a target membrane-associated molecule by means of polarization microscopy is disclosed. The probe contains an anchoring moiety consisting of at least one lipidated peptide and/or at least one transmembrane α-helical peptide, a peptide linker moiety having the length of at least 5 amino acids, wherein at least 50% of the amino acids forming the linker are selected from glycine, serine, and threonine, a fluorescent moiety, and an affinity binding moiety capable of binding the target membrane-associated molecule. 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. Methods of detecting presence or absence of the target molecule, detecting activated or inactive forms of the target molecule, and detecting the activation of the target molecule are also described.

Inventors

Josef Lazar
Alexey BONDAR

Assignees

Josef Lazar

Dates

Publication Date: 20260505
Application Date: 20221121
Priority Date: 20211122

Claims (20)

1 . A method of detecting a presence or an absence 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 do not contain the target membrane-associated molecule; contacting the probe with a test sample comprising cells in which the presence or absence 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; 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; and when the fluorescence polarization exhibited by the probes do not differ, it is determined that the target membrane-associated molecule is absent from the cells of the test sample; 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 of invention, and by causing the cells to produce the protein-based probe.
2 . The method according to claim 1 , wherein the moieties are arranged in the order d-c-b-a in the direction from the N-terminus to the C-terminus.
3 . The method according to claim 1 , wherein the peptide linker moiety contains between 5 and 30 amino acids, of which at least 50% are serine and/or glycine and/or threonine, and the remaining amino acids are lysine and/or arginine and/or glutamate.
4 . The method according to claim 3 , wherein at least 70% of the amino acids in the peptide linker moiety are serine and/or glycine and/or threonine.
5 . The method according to claim 1 , wherein the fluorescent moiety is a fluorescent protein, or a protein configured to become fluorescent when covalently or non-covalently bound to a fluorescent or non-fluorescent ligand, wherein the fluorescent moiety is selected from the group consisting of fluorogenic protein tag based on haloalkane dehalogenase, a fluorogenic protein tag based on alkylguanine-DNA alkyltransferase, bacteriophytochrome-based near-infrared fluorescent protein 2.0(IFP2.0), allophycocyanin-derived small ultra-red fluorescent protein (smURFP), fatty acid binding protein UnaG, photoactive yellow protein and circularly permuted versions thereof.
6 . The method according to claim 1 , wherein the polarization microscopy is selected from excitation polarization resolved fluorescence microscopy and fluorescence polarization resolved fluorescence microscopy.
7 . 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.
8 . The method according to claim 7 , wherein the polarization microscopy is selected from excitation polarization resolved fluorescence microscopy and fluorescence polarization resolved fluorescence microscopy.
9 . 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.
10 . The method according to claim 9 , wherein the polarization microscopy is selected from excitation polarization resolved fluorescence microscopy and fluorescence polarization resolved fluorescence microscopy.
11 . The method according to claim 3 , wherein at least 85% of the amino acids in the peptide linker moiety are serine and/or glycine and/or threonine.
12 . The method according to claim 7 , wherein the moieties are arranged in the order d-c-b-a in the direction from the N-terminus to the C-terminus.
13 . The method according to claim 7 , wherein the peptide linker moiety contains between 5 and 30 amino acids, of which at least 50% are serine and/or glycine and/or threonine, and the remaining amino acids are lysine and/or arginine and/or glutamate.
14 . The method according to claim 13 , wherein at least 70% of the amino acids in the peptide linker moiety are serine and/or glycine and/or threonine.
15 . The method according to claim 13 , wherein at least 85 % of the amino acids in the peptide linker moiety are serine and/or glycine and/or threonine.
16 . The method according to claim 7 , wherein the fluorescent moiety is a fluorescent protein, or a protein configured to become fluorescent when covalently or non-covalently bound to a fluorescent or non-fluorescent ligand, wherein the fluorescent moiety is selected from the group consisting of a fluorogenic protein tag based on haloalkane dehalogenase, a fluorogenic protein tag based on alkylguanine-DNA alkyltransferase, bacteriophytochrome-based near-infrared fluorescent protein 2.0 (IFP2.0), allophycocyanin-derived small ultra-red fluorescent protein (smURFP), fatty acid binding protein UnaG, photoactive yellow protein and circularly permuted versions thereof.
17 . The method according to claim 9 , wherein the moieties are arranged in the order d-c-b-a in the direction from the N-terminus to the C-terminus.
18 . The method according to claim 9 , wherein the peptide linker moiety contains between 5 and 30 amino acids, of which at least 50% are serine and/or glycine and/or threonine, and the remaining amino acids are lysine and/or arginine and/or glutamate.
19 . The method according to claim 18 , wherein at least 70% of the amino acids in the peptide linker moiety are serine and/or glycine and/or threonine.
20 . The method according to claim 18 , wherein at least 85% of the amino acids in the peptide linker moiety are serine and/or glycine and/or threonine.

Description

SEQUENCE LISTING This application contains a sequence listing which has been submitted electronically in ASCII format in a text file and is hereby incorporated by reference in its entirety. This ASCII text file, created on Nov. 11, 2022, is named P1939US00-SeqList.xml and is 332 kilobytes in size. FIELD OF THE INVENTION The invention described herein concerns detection and visualization of molecular processes taking place in living cells. BACKGROUND ART In order to survive and to carry out their functions within living organisms, cells receive, process, and transmit molecular signals. Many such signals involve molecules associated with, or embedded within the cell membrane (herein referred to as membrane-associated molecules). Changes in concentration of such molecules, in their chemical modifications, in their conformation, and in their mode and extent of interactions with other molecules carry information in biological sy stems. Molecular events that involve membrane-associated molecules can be observed through the use of optically detectable molecular probes, using suitable techniques of optical microscopy. In order to observe molecular events in living cells, the target molecules are often modified to bear optically detectable labels. These labels allow specificity in observing processes pertaining to the target molecules. However, since the presence of molecular labels can affect the processes to be observed, it is of high scientific and practical interest to develop means to observe the presence or absence of a target molecule, or its particular molecular state, without modifying the target molecule by introducing an optical label. To detect by means of optical microscopy, with specificity, a target membrane membrane-associated molecule that bears no optical label, genetically encoded optically active probes that bind specifically to the target molecule have been used. Binding of such probes to the target molecule is then detected. Four distinct means of detecting an interaction between a non-labeled target molecule and an optically active molecular probe could be identified: 1) through observations of changes in cellular localization of the probe, 2) through observations of the diffusion rate of the probe, 3) through observations of changes in resonant energy transfer within the probe, and 4) through observations of changes in molecular orientation of the probe. However, all of these approaches bear significant disadvantages. Quantification of cellular localization is problematic due to optical overlap between the distinct cellular compartments. Quantification of rates of diffusion (achievable through fluorescence correlation spectroscopy or single molecule tracking) is experimentally challenging and time consuming. Probes relying on bioluminescence resonant energy transfer generate only low amounts of light, which generally requires integration of signal from many cells, over periods of time of seconds to minutes, precluding imaging of dynamic molecular processes. Probes relying on fluorescence resonant energy transfer generally do not allow multiplexing. Despite their potential advantages, probes relying on changes in molecular orientation for detection of processes involving membrane-associated molecules have been used only in a small number of cases (Benninger R K & al., Biophysical Journal. 2009 Jan. 21; 96(2):L13-5; Kress A & al., Biophysical Journal. 2011 Jul. 20; 101(2):468-76; Lazar J. & al., Nature Methods. 2011 August; 8(8):684-90. Bondar A. Lazar J., Journal of Biological Chemistry. 2014 Jan. 1; 289(3):1271-81; Han Z & al., PloS One. 2014 Nov. 24; 9(11):e113873; Bondar A, Lazar J., Journal of Biological Chemistry. 2017 Jul. 1; 292(23):9690-8; Bondar A & al., Communications Biology. 2021 Feb. 12; 4(1):1-2). This is due to the non-rigid character of the cell membrane, only partially restricting the orientation of the potential molecular probes, and therefore severely limiting their ability to function as probes. DISCLOSURE OF THE INVENTION The present invention aims to overcome the limitations of existing technologies and to enable the use of polarization microscopy-based methods using genetically encoded probes for observing and/or detecting membrane-associated processes and/or membrane-associated molecules (preferably membrane-associated proteins), by implementing a modular molecular probe design which maximizes differences in optical properties detectable by polarization microscopy, said differences conveying information on two distinct states of the target molecule (preferably target protein), such as absence and presence of the target molecule. The molecular probes of the invention convert the two distinct states of the target membrane-associated molecule, such as presence or absence of the target molecule, into changes of molecular orientation of a fluorescent moiety of the molecular probe. These changes in molecular orientation of the fluorescent moiety are then observed by means of