Artificial transmembrane proteins for detecting intracellular or intravesicular biomolecular interactions

Abstract

This invention provides an artificial transmembrane protein for use in biomolecular detection devices for detecting intracellular or intravesicular biomolecular interactions. [Solution] Disclosed herein is an artificial transmembrane protein comprising an extracellular or extravesicle-binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicle-receptor structure having an intracellular or intravesicle-receptor structure, wherein the receptor structure is configured to interact with the intracellular or intravesicle-component of a biomolecular interaction to be detected, and the extracellular or extravesicle-binding structure is configured to bind to membrane recognition elements positioned along a plurality of predetermined lines of a biomolecular detection device.

Description

[Technical Field]

[0001] The present invention is particularly in the field of artificial transmembrane proteins for detecting intracellular or intravesicular biomolecular interactions. The present invention further relates to biomolecular detection devices for analyzing cells, vesicles, or cellular components or vesicular components, including artificial transmembrane proteins, and to methods for detecting intracellular or intravesicular biomolecular interactions in cells, cellular components, or vesicles or vesicular components. [Background technology]

[0002] Detection devices are used, for example, as biosensors in a variety of applications. One specific application is the detection or monitoring of binding affinity or process. For example, various assays can be performed using such biosensors to detect the binding of a target sample to a binding site. Typically, many such assays are performed on the biosensor in spots arranged in a two-dimensional microarray on the surface of the biosensor. The use of microarrays provides a means for the simultaneous detection of the binding affinity or process of different target samples in highly efficient screening. To detect the affinity of a target sample to a specific binding site, or the affinity of a target molecule to a specific capture molecule, numerous capture molecules are immobilized on the outer surface of the biosensor in individual spots (e.g., by inkjet spotting or photolithography). Each spot forms an individual measurement zone for a given type of capture molecule. The binding of the target molecule to a specific type of capture molecule is detected and used to provide information about the binding affinity of the target molecule with respect to that specific capture molecule.

[0003] Known techniques for detecting the binding affinity of a target sample utilize fluorescent labeling. Fluorescent labeling can emit fluorescence upon excitation. The emitted fluorescence has a characteristic emission spectrum that identifies the fluorescent label of the present invention at a specific spot. The identified fluorescent labeling indicates that the labeled target molecule has bound to a specific type of binding site present at that spot.

[0004] The sensor for detecting labeled target molecules is described in the paper "Zeptosens' protein microarrays: A novel high performance microarray platform for low abundance". This is described in "protein analysis," Proteomics 2002, 2, pp. 383-393, Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany. The sensor described in this paper includes a plane waveguide placed on a substrate. The plane waveguide has an outer surface on which multiple binding sites can be attached. Furthermore, the plane waveguide has multiple unbinding lines for coupling a beam of coherent light in such a manner that the beam of coherent light propagates along the plane waveguide. The coherent light propagates through the plane waveguide under total internal reflection, and the evanescent field of the coherent light propagates along the outer surface of the plane waveguide. The depth of penetration of the evanescent field into the medium with the lower refractive index at the outer surface of the plane waveguide is approximately a fraction of the wavelength of the coherent light propagating through the plane waveguide. The evanescent field excites the fluorescent label of a labeled target sample bound to the binding sites located on the surface of the plane waveguide. Because the penetration depth of the evanescent field into the optically thinner medium at the outer surface of the plane waveguide is very small, only labeled samples bound to fixed coupling sites on the outer surface of the plane waveguide are excited. The fluorescence emitted by these labels is then detected using a CCD camera.

[0005] While detecting binding affinity using fluorescent labeling is theoretically possible, this technique has the drawback that the detected signal is produced by the fluorescent labeling rather than the binding partner itself. Furthermore, labeling the target sample requires additional preparation steps. Moreover, labeled target samples are relatively expensive. Another drawback is distortion of the results caused by steric hindrance of the fluorescent labeling on the target sample, which can inhibit the binding of the target sample to the capture molecule. A further drawback is distortion of the results due to fading or quenching effects of the labeling. In addition, fluorescent labeling can significantly affect the chemical, biological, pharmacological, and physical properties of the compound of interest. Thus, measurements that rely solely on fluorescent labeling can be distorted by the presence of such labeling. Furthermore, fluorescence spectroscopy requires labeling any compound or cellular component of interest. Thus, while it is possible to observe interactions with a specific labeled compound, further interactions cannot be observed. [Overview of the Initiative]

[0006] A crucial requirement for analyzing biological samples is the distinction between specific and nonspecific binding of a compound or structural part of interest to its binding site. Known strategies to address this problem, such as surface plasmon resonance (SPR) or Mach-Zehnder interference spectroscopy, heavily rely on reference measurements and are only suitable for measurements under static conditions. Thus, such techniques are typically unsuitable for measurements in complex environments, such as detecting biomolecular interactions within living cells. SPR measures the change in refractive index upon receptor-ligand binding near the sensor surface. However, this technique has the drawback of being susceptible to changes in refractive index near the sensor surface. Therefore, nonspecific binding remains a significant problem. In particular, refractometer sensors cannot distinguish between molecules that actually bind to the target of interest and the sole presence of other compounds, nor can they provide additional effects that affect the refractive index.

[0007] G protein-coupled receptors (GPCRs) have been developed as specific and excellent targets for drug candidates due to their involvement in the onset and progression of many diseases, including pain, asthma, inflammation, obesity, and cancer. Therefore, a detailed analysis of these receptors in living cells is highly desirable. Whole-cell assays to determine GPCR activity are used to analyze different intracellular second messengers (cAMP, Ca). 2+ These assays traditionally rely on the detection of fluorescently tagged proteins, relocalization of the fluorescently tagged protein (arrestin recruitment to the receptor or receptor internalization), or expression of the reporter gene under the control of the GPCR-activated signaling cascade. However, as noted above, fluorescent labeling requires significant biomolecular modifications, such as protein overexpression or the introduction of fluorescent labels, which can alter cell physiology or drug pharmacology and are therefore not always feasible or desirable. Furthermore, GPCRs typically modulate more than one effector, which can lead to cross-sensitivity in these assays.

[0008] Label-free cell assays, such as resonant waveguide grid biosensors, do not require any molecular labeling (see Paulsen et al., Photonics Nanostruct. Fundam. Appl. 2017, 26, 69). Typically, such refractometer sensors monitor the refractive index on the sensor chip by a propagating evanescent wave that defines the sensing volume by its penetration depth. The redistribution of cellular contents within this sensing volume results in an overall change in refractive index, producing a highly valuable overall picture of dynamic mass redistribution (DMR). In exchange, DMR is inherently cross-sensitive, meaning that different GPCR-mediated signaling pathways cannot be spatiotemporally deconvoluted by the sensor.

[0009] As an alternative to whole-cell analysis that does not use light labeling, such as SPR, resonant waveguide grids (RW) are being used. Based on G), dynamic mass redistribution, symmetric waveguide sensors, and quantitative phase imaging were used to investigate morphological changes and phenotypic cellular responses for drug discovery. However, it should be noted that these methods can only provide information on cytoplasmic mass, absolute concentration, and volume changes, but cannot monitor intracellular or intravesicular processes.

[0010] Therefore, the overall objective of the present invention is to improve the state of the art regarding the detection of intracellular or intravesicular interactions, thereby preferably completely or partially avoiding the drawbacks of the prior art.

[0011] In a preferred embodiment, an artificial transmembrane protein is provided that is configured to interact with specific intracellular or intravesicular components and can be specifically manipulated to enable monitoring of direct or indirect interactions with the intravesicular or intracellular components.

[0012] In a more preferred embodiment, an artificial transmembrane protein is provided that enables single-cell measurement.

[0013] The overall objective is generally achieved by the subject matter of the independent claims. Further advantages and exemplary embodiments are derived from the following description and drawings.

[0014] According to a first aspect of the present invention, the overall objective is achieved by an artificial transmembrane protein for use in a biomolecular detection device for detecting intracellular or intravesicular biomolecular interactions. The artificial transmembrane protein comprises an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicular domain including an intracellular or intravesicular receptor structure. The receptor structure is configured to interact with the intracellular or intravesicular component of the biomolecular interaction to be detected, and the extracellular or extravesicular binding structure is configured to bind to membrane recognition elements positioned along a plurality of predetermined lines of the biomolecular detection device. The interaction between the receptor structure and the intracellular or intravesicular component of the biomolecular interaction to be detected is understood to refer to an interaction in the biomolecular sense. Thus, such an interaction may be, for example, the binding of a compound to the receptor. The intracellular or intravesicular receptor structure can be specifically designed, for example, by genetic engineering to interact with a given intracellular or intravesicular component of interest. Therefore, the artificial transmembrane protein is a biomimetic membrane receptor designed by available biotechnological procedures for a particular purpose.

[0015] Preferably, a biomolecular detection device that can be used in combination with an artificial transmembrane protein according to any embodiment described herein for detecting a biomolecular interaction of interest includes an evanescent illuminator, which includes a photo-coupled unit configured to generate an evanescent field from coherent light having a specified wavelength on a first surface of the evanescent illuminator. The first surface of the evanescent illuminator includes a template nanopattern containing a plurality of predetermined lines of coherent arrangements on which membrane recognition elements are positioned for a cell, vesicle, or cellular component or vesicular component of an artificial transmembrane protein, preferably a laterally diffusive artificial transmembrane protein-binding structure. A laterally diffusive transmembrane protein is a transmembrane protein that can diffuse within the membrane of a cell or vesicle. The membrane recognition elements are configured to bind to a laterally diffusive artificial transmembrane protein-binding structure to form a transmembrane nanopattern within a cell, vesicle, or cellular component or vesicular component based on the template nanopattern of the evanescent illuminator, by locally immobilizing the artificial membrane protein such that the light of the evanescent field is scattered by the cell, vesicle, or cellular component or vesicular component bound to the membrane recognition element. As anyone skilled in the art will understand, an evanescent illuminator is a light source This element is capable of generating an evanescent field from coherent light. The predetermined lines on the evanescent illuminator are arranged such that light scattered by cells, vesicles, or cellular or vesicular components bound to the membrane recognition element constructively interferes at predetermined detection sites having differences in optical path length that are integer multiples of a predetermined wavelength of the coherent light. As those skilled in the art will understand, optical path length refers to the product of the geometric length of the path through which light travels in a given system and the refractive index of the medium in which it propagates. Extracellular or extravesicular binding structures of artificial transmembrane proteins are configured to bind to membrane recognition elements arranged along multiple predetermined lines of the biomolecular detection device, and the template nanopatterns of the biomolecular detection device are introduced into living cells by selectively binding to transmembrane proteins diffusing laterally within the cell, thus generating transmembrane nanopatterns within the cell itself. Typically, multiple membrane recognition elements on multiple different predetermined lines bind to the same cell; that is, more than one membrane recognition element binds to or is configured to bind to a single cell, vesicle, or cellular or vesicular component.

[0016] Artificial transmembrane proteins provide a detection window that is specific to the detection of a biomolecule of interest, due to the fact that extracellular or extravesicle-binding structures are configured to bind to membrane recognition elements positioned along multiple predetermined lines of a biomolecule detection device, and that receptor structures can be specifically designed by genetic engineering to interact with, for example, any given intracellular or intravesicle component of interest. The template nanopattern of the biomolecule detection device allows for the introduction or regeneration of the template nanopattern of the biomolecule detection device into the living cell membrane by selectively binding to artificial transmembrane proteins that diffuse laterally within the cell. As a result, any biomolecule interaction, including interactions of artificial transmembrane proteins, and even intercellular processes, can be selectively detected. Upon binding of the membrane recognition elements to the binding structures of the artificial transmembrane proteins, evanescent light is scattered and constructively interferes at the designated detection site. The constructive interference of scattered light is associated with all bound transmembrane proteins and results in a quadratic scaling of the intensity measured with respect to the number of transmembrane proteins. Importantly, random scattering of background molecules that do not bind to the molecular recognition elements interferes both constructively and destructively with equal probability. As a result, cross-sensitivity is significantly reduced. Surprisingly, cross-sensitivity is rarely observed due to cell scattering, particularly membrane scattering, even when living cells and vesicles contain surfaces that are highly uneven on a nanometer scale. Although measurements in living cells can be easily hindered by signals that neutralize light scattered by the bound membrane recognition element, no significant loss of sensitivity was observed, only a small distortion of the concentrated diffusion signal.

[0017] Typically, coherent light has a predetermined wavelength, particularly at a single wavelength and preferably is monochromatic. Usually, visible light or near infrared light can be used. A part of the evanescent field is coherently scattered by scattering centers composed of cells, vesicles or biomolecules derived from cell components or vesicle components bound to membrane recognition elements arranged on different predetermined lines. The scattered electric field at any position can be determined by computer calculation of the intensity by adding the contributions from each individual scattering center and then squaring the resulting phase vector. At a predetermined detection position, the predetermined line is arranged such that the optical path lengths of the light scattered by different scattering centers differ by an integer multiple of the wavelength of the light, so the maximum value of the scattering intensity is located at the predetermined detection position. The requirement of constructive interference is satisfied by any scattered light that adds a detectable signal at the detection position. The intensity pattern at the predetermined detection position preferentially forms a diffraction-limited Airy disk, although it is not limited to this. Essentially, any shape accessible by Fourier optics is possible. For any shape, the signal can be optimally recovered using a matched filter.

[0018] In a medium with refractive index n0 under plane polarization, the scattering of an isolated membrane recognition element - transmembrane complex with refractive index n R is

Number

Number

[0019] Thus, depending on the experimental design, the intensity change of the scattered light provides access to the molecular mass of the interaction partner, the total binding mass or number of receptors involved in the biomolecular interaction involving the artificial membrane-penetrating protein. For example, when a specific messenger compound binds to the artificial membrane-penetrating protein, the corresponding mass increase can be calculated. For example, an intracellular or intravesicular receptor construct can be configured to interact with a first component A of a biomolecular interaction having a known molecular mass. When component A binds, a change in the diffraction intensity is observed. This intensity change is used to computer-calculate the number of receptors that interact with component A. The mass of the complex can be calculated. If the second component B binds to component A, or also to the receptor structure, a further change in intensity is observed. Then, when components A and / or B are released from the receptor structure, the mass of the corresponding complex decreases again, and the signal intensity changes. Assuming that B binds to the same amount of the binding site as A, the molecular mass of the complex and one molecule of B can be calculated at any given time of measurement, thereby enabling real-time monitoring of biomolecular interactions over time. Due to the difference in mass, information can be obtained about compounds that directly or indirectly bind to or are released from the artificial transmembrane protein.

[0020] In some embodiments, the artificial transmembrane protein further comprises a linker domain configured to facilitate interaction between an intracellular or intravesicular receptor structure and the intracellular or intravesicular component of the biomolecular interaction to be detected, wherein the linker domain is positioned between the intracellular or intravesicular receptor structure and the hydrophobic transmembrane domain. Preferably, the linker domain does not contain bulky hydrophobic residues such as tryptophan that may interfere with protein folding. Thus, the linker domain may consist only of amino acids such as glycine, serine, alanine, glutamine, proline, and phenylalanine or glycine. Smaller amino acids such as glycine can provide greater flexibility of the linker domain. Polar residues such as glutamine increase the solubility of the linker in water. The linker domain may generally have a length of 4 to 25 residues, preferably 4 to 20 residues.

[0021] In further embodiments, the extracellular or extravesicle-binding structure is configured to establish covalent bonds with membrane recognition elements positioned along a plurality of predetermined lines of a biomolecule detection device. For example, the extracellular or extravesicle-binding structure may include a chemical moiety that enables the generation of covalent bonds, such as an electrophile, nucleophile, dienophile, diene, 1,3-dipole, or dipolarophile.

[0022] In certain embodiments, the extracellular or extracellular vesicle-binding construct comprises a nucleophile, preferably a thiol or thiolate.

[0023] In further embodiments, the extracellular or extracellular vesicle-binding construct comprises or consists of a SNAP-tag or a CLIP-tag. The SNAP-tag is an 182-residue polypeptide that accepts O 6 -benzylguanine derivatives (19.4 kDa, Crivat et al., Trends in Biotechnology 30(1):8-16, doi:10.1016 / j.tibtech.2011.08.002; Juillerat et al., Chemistry and Biology 10(4):313-317, doi:10.1016 / S1074-5521(03)00068-1; Molliwtz et al., Biochemistry 51(5):986-994, doi:10.1021 / bi2016537). The CLIP-tag is related to the SNAP-tag and accepts O 6 -benzylcytosine derivatives as substrates instead of O 2 -benzylguanine (Gautier et al., Chemistry and Biology 15(2):128-136, doi:10.1016 / j.chembiol.2008.01.007).

[0024] In some embodiments, the artificial membrane protein is a single-pass transmembrane protein.

[0025] In further embodiments, the artificial transmembrane protein is (a) of type I, wherein the intracellular or intravesicular domain is disposed adjacent to the C-terminus and the extracellular or extracellular vesicle domain is disposed adjacent to the N-terminus; or (b) of type II, wherein the intracellular or intravesicular domain is disposed adjacent to the N-terminus and the extracellular or extracellular vesicle domain is disposed adjacent to the C-terminus thereof.

[0026] In type I artificial transmembrane proteins, the N-terminus is configured to face the extracellular space, while the C-terminus remains in the cytosol. In contrast, in type II artificial transmembrane proteins, the N-terminus is configured to remain in the cytosol, while the C-terminus faces the extracellular space.

[0027] In more specific embodiments of type I, the domain order from N-terminus to C-terminus may preferably be an extracellular or extravesicle-binding structure having a SNAP or CLIP tag, followed by a hydrophobic transmembrane domain, then optionally a linker domain, followed by an intracellular or intravesicle-binding domain having an intracellular or intravesicle-receptor structure. Preferably, positively charged amino acids are positioned before the linker domain. Optionally, the extracellular or extravesicle-binding structure may preferably include a cleavable signal peptide positioned before the SNAP or CLIP tag, i.e., closest to the N-terminus.

[0028] In more specific embodiments of type II, the domain order from N-terminus to C-terminus may be an intracellular or intravesicular domain having an intracellular or intravesicular receptor structure, followed optionally by a linker domain, followed by a hydrophobic transmembrane domain, followed preferably by an extracellular or extravesicular binding structure having a SNAP or CLIP tag. Optionally, the intracellular or intravesicular domain may contain a cleavable signal peptide, which may preferably be positioned prior to the receptor structure.

[0029] In some embodiments, the artificial transmembrane protein is type I or type II, and the intracellular or intravesicular domain contains a greater amount of positively charged amino acid residues than the extracellular or extravesicular domain. In particular, the positively charged amino acids can be selected from lysine, arginine, or histidine, preferably lysine. These amino acids may be present in 3 to 4 times greater amounts than the rest of the artificial transmembrane protein, either intracellular or intravesicular. The greater amount of positively charged amino acids allows for more efficient orientation of the artificial transmembrane helix within the membrane.

[0030] In further embodiments, the intracellular or intravesicular receptor structure is configured to interact with the β-subunit of a protein kinase in the cAMP pathway or with a receptor tyrosine kinase (RTK). When the intracellular or intravesicular receptor structure is configured to interact with an RTK, the receptor structure may include the Grb2 protein.

[0031] In some embodiments, the intracellular or intravesicular receptor structure may include a fluorescent protein such as eYFP.

[0032] In some embodiments, the intracellular or intravesicular receptor structure is a designed receptor, an artificial binder, or another functional molecule such as an antibody, antibody fragment, nanobody, or affimer.

[0033] In further embodiments, the artificial transmembrane protein includes a cleavable signal peptide adjacent to the N-terminus of the artificial transmembrane protein for interaction with the protein transport system and for controlling the translocation of the artificial transmembrane protein.

[0034] Typically, a cleavable signal peptide consists of 18 to 26 amino acids and may form a positively charged N-terminal n-region, a central hydrophobic h-region, and a polar C-terminal c-region. Preferably, the cleavage site is contained within the c-region and is configured to be recognized by a signal peptidase.

[0035] In some embodiments, the extracellular or extravesicle-binding structure is the membrane of the biomolecular detection device. The system includes an affinity tag that interacts with the recognition element, preferably configured to interact selectively. For example, the affinity tag may be HA (human influenza hemagglutinin), FLAG, or 6His affinity tag. The use of affinity tags is beneficial because it can modify protein conformation through molecular interactions, which can restrict or prevent an antibody acting as a membrane recognition element from recognizing extracellular or extravesicle-bound structures of an artificial transmembrane protein. This can be avoided by affinity tags in extracellular or extravesicle-bound structures.

[0036] In some embodiments, the artificial transmembrane protein includes a protein configured to interact with other intracellular elements, such as intracellular or intravesicular fluorescent proteins or biomolecular tags, for example, for intracellular saltase-mediated immobilization of the protein. Preferably, the protein is configured to interact specifically with other intracellular elements.

[0037] In some embodiments, the transmembrane protein is unlabeled, particularly unfluorescently labeled. Preferably, the transmembrane protein does not contain an artificially labeled moiety that can be excited upon irradiation. The unfluorescently labeled transmembrane protein used herein is a transmembrane protein that does not contain small fluorescent molecules, i.e., molecules with a molecular weight of less than 900 Da.

[0038] In a further aspect, the present invention relates to cells, vesicles, or cellular components or vesicle components comprising an artificial transmembrane protein according to any embodiment described herein or a nucleic acid sequence encoding an artificial transmembrane protein according to any embodiment described herein. For use in biomolecule detection devices, the nucleic acid sequence can be introduced into cells, preferably in vitro, and expressed therein. In a further aspect, the present invention relates to recombinant nucleic acid molecules comprising at least one nucleic acid sequence encoding an artificial transmembrane protein according to any embodiment described herein.

[0039] In a further embodiment, the present invention relates to a vector, preferably a plasmid vector, comprising a recombinant nucleic acid molecule as described in any embodiment herein.

[0040] In a further embodiment, the present invention relates to the use of a vector according to any embodiment described herein for expressing an artificial transmembrane protein in vitro, comprising the steps of preparing cells, introducing a vector according to any embodiment described herein into cells, and expressing an artificial transmembrane protein.

[0041] In some embodiments, use further includes the step of cleaving a signal peptide from an artificial transmembrane protein.

[0042] In a further embodiment, the present invention relates to a biomolecular detection device for analyzing cells, vesicles, or cellular components or vesicular components containing artificial transmembrane proteins according to any embodiment described herein, wherein the biomolecular detection device includes an evanescent illuminator comprising a photo-coupling unit configured to generate an evanescent field from coherent light having a specified wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nanopattern containing a plurality of predetermined linear coherent arrangements on which membrane recognition elements are arranged for binding structures of artificial transmembrane proteins in cells, vesicles, or cellular components or vesicular components, and the membrane recognition elements are binding structures of artificial transmembrane proteins to form a transmembrane nanopattern within cells, vesicles, or cellular components or vesicular components based on the template nanopattern of the evanescent illuminator, such that the light of the evanescent field is scattered by cells, vesicles, or cellular components or vesicular components bound to the membrane recognition elements. The present invention relates to a biomolecular detection device configured to bind to a membrane recognition element, wherein a predetermined line is positioned to constructively interfere at a predetermined detection site having a difference in optical path length where the light scattered by cells, vesicles, or cellular or vesicular components bound to the membrane recognition element is an integer multiple of a predetermined wavelength of coherent light.

[0043] The template nanopattern selectively binds to transmembrane proteins diffusing laterally within the cell, thus transferring the template nanopattern of the biomolecular detection device to living cells by generating the transmembrane nanopattern within the cell itself. Typically, multiple different lines of membrane recognition elements bind to the same cell; i.e., more than one membrane recognition element binds to or is configured to bind to a single cell, vesicle, or cellular component or vesicle component. As a result, any biomolecular interaction, including transmembrane protein interactions, as well as intercellular or intracellular processes, can be selectively detected. Upon binding of the membrane recognition element to the transmembrane protein binding structure, evanescent light is scattered, constructively interfering at the designated detection site. The constructive interference of scattered light is associated with all bound transmembrane proteins, resulting in a quadratic scaling of the intensity measured with respect to the number of transmembrane proteins. Importantly, random scattering of background molecules that do not bind to the molecular recognition element interferes both constructively and destructively with equal probability. As a result, cross-sensitivity is efficiently suppressed. Surprisingly, cross-sensitivity is rarely observed due to cell scattering, particularly membrane scattering, even when living cells and vesicles contain highly uneven surfaces on a nanometer scale. While measurements of living cells can be easily hindered by signals that neutralize light scattered by the bound membrane recognition elements, no significant loss of sensitivity was observed. This is surprising because cells, vesicles, or cellular or vesicle components are significantly larger than the distances between predetermined lines in a nanopattern. Thus, generally, in the embodiments disclosed herein, membrane recognition elements on multiple different predetermined lines bind to the same cell. Typically, the distance between two directly adjacent predetermined lines is 1 / 2 to 1 / 100th, preferably 1 / 30 to 1 / 100th, the size of a single cell.

[0044] In some embodiments, the evanescent illuminator includes or is an evanescent carrier having a planar waveguide arranged on the surface of the carrier, and an optical coupler as an optical coupling unit for coupling coherent light of a specified wavelength in the waveguide so that coherent light propagates through the planar waveguide having an evanescent field of coherent light propagating along a first surface of the planar waveguide including a template nanopattern.

[0045] In some embodiments, the evanescent illuminator is a total internal reflection system configured to provide a beam of coherent light at a predetermined wavelength and angle onto a first surface of the evanescent illuminator by an optical coupling unit, in particular a prism or any other suitable optical element.

[0046] In some embodiments, a plurality of predetermined lines include curves having a curved portion configured such that light from the evanescent field scattered by cells, vesicles or vesicle components or cellular components, or their biomolecules, bound to the membrane recognition element interferes at a specified detection site.

[0047] Preferably, the overall shape of the template nanopattern may be round, particularly circular.

[0048] In a preferred embodiment, the curves are arranged such that the distance between adjacent lines decreases in the direction of light propagation in order to concentrate light scattered by cells, vesicles, or cellular or vesicular components to a predetermined detection site. Alternatively, a plurality of predetermined lines may include straight lines arranged at a predetermined angle to the propagation of light coupled to an evanescent illuminator. Additional couplers may be used to concentrate diffracted light to a predetermined detection site.

[0049] In a further embodiment, a plurality of predetermined lines are x j , y j Their positions in coordinates are given by:

number

number

[0050] The selected integer A0 assigns negative x values ​​to the center of the line with negative j values, and positive x values ​​to the center of the line with positive j values. In other words, the integer A0 defines the origin of the x,y coordinate frame used for the position of the line on the outer surface of the evanescent illuminator; the selected A0 value places the detection position at x=0, y=0, z=-f.

[0051] In some embodiments, at least one cell, vesicle, or cellular component or vesicle component binds to a membrane recognition element via an artificial transmembrane protein-binding structure.

[0052] In another embodiment, the present invention relates to a kit of parts comprising an artificial transmembrane protein according to any embodiment described herein, or a cell according to any embodiment described herein, or a recombinant nucleic acid molecule according to any embodiment described herein, or a vector according to any embodiment described herein, and a biomolecule detection device according to any embodiment described herein.

[0053] In some embodiments, the kit of parts further includes the protein of interest configured to interact with intracellular or intravesicular biomolecules, particularly gold nanoparticles, of which the protein of interest is a high-mass moiety, in particular. The use of such high-mass moieties is beneficial because the higher molecular weight has a beneficial effect on the intensity of the resulting signal, thus enabling even single-cell measurements. As those skilled in the art will understand, high-mass moieties typically have a significantly larger molecular weight than artificial transmembrane proteins. For example, the high-mass moiety may be a protein or an overexpressed protein aggregate. Preferably, the mass of the high-mass moiety may be at least 150 kD.

[0054] In another aspect, the present invention provides a label-free method for detecting intracellular or intravesicular biomolecular interactions in cells, cellular components, or vesicles or vesicular components using a biomolecular detection device according to any embodiment described herein, - A step of preparing cells, cellular components, or vesicles or vesicle components or vesicle components containing an artificial transmembrane protein according to any embodiment described herein, if necessary; - Steps of applying cells, cellular components, or vesicles or vesicular components to the membrane recognition element of a biomolecular detection device, or preparing a biomolecular detection device in which at least one cell, vesicle, or cellular component or vesicular component is bound to the membrane recognition element via an artificial transmembrane protein binding structure; - A step of generating a beam of coherent light at a predetermined beam generation position for multiple predetermined lines that are incident on a membrane recognition element containing a bound transmembrane protein, such that the beam of coherent light has a predetermined wavelength, and the diffracted portion of the incident coherent light beam constructively interferes at a predetermined detection site with multiple predetermined lines having path length differences that are integer multiples of the predetermined wavelength of the coherent light, thereby providing a signal at the predetermined detection site that represents the membrane recognition element to which an artificial transmembrane protein of a cell, vesicle, or cellular component or vesicle component is bound; - A step to measure a signal representative of a membrane recognition element to which an artificial transmembrane protein of a cell, vesicle, or cellular component or vesicle component is bound. Regarding methods including

[0055] Label-free methods for detecting intracellular or intravesicular biomolecular interactions in cells, cellular components, or vesicles or vesicular components are methods that do not rely on the interaction between light from a light source, particularly coherent light, and a label. Such labels are artificial label portions that can be excited upon irradiation, such as fluorescent labels. Commonly used fluorescent labels are small fluorescent molecules, i.e., fluorescent molecules with a molecular weight lower than 900 Da.

[0056] In some embodiments, the method further includes the step of providing an artificial transmembrane protein receptor structure, or a substance that interacts with intracellular or intravesicular components of the biomolecular interaction to be detected, such as a drug, drug candidate, small molecule, or antibody.

[0057] In certain embodiments, cells or cellular components containing an artificial transmembrane protein are provided by transfecting cells in vitro with a vector according to any embodiment described herein, expressing the artificial transmembrane protein in the cells, and, if necessary, removing a portion of the cell membrane to provide the cellular component.

[0058] In some embodiments, the present invention is described by the following clauses:

[0059] Clause 1: An artificial transmembrane protein for use in a biomolecular detection device for detecting intracellular or intravesicular biomolecular interactions, wherein the artificial transmembrane protein comprises an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicular domain having an intracellular or intravesicular receptor structure, the receptor structure being configured to interact with the intracellular or intravesicular component of the biomolecular interaction to be detected, and the extracellular or extravesicular binding structure being configured to bind to membrane recognition elements positioned along a plurality of predetermined lines of the biomolecular detection device.

[0060] Clause 2: The artificial transmembrane protein according to Clause 1, further comprising a linker domain configured to facilitate interaction between an intracellular or intravesicular receptor structure and an intracellular or intravesicular component of a biomolecular interaction to be detected, wherein the linker domain is positioned between the intracellular or intravesicular receptor structure and a hydrophobic transmembrane domain.

[0061] Clause 3: The extracellular or extravesicle-binding structure is configured to establish covalent bonds with membrane recognition elements positioned along multiple predetermined lines of the biomolecular detection device. , artificial transmembrane proteins as described in Clause 1 or 2.

[0062] Clause 4: An artificial transmembrane protein according to any of the preceding clauses, wherein the extracellular or extravesicle-binding structure comprises a nucleophile, preferably a thiol or thiolate.

[0063] Clause 5: An artificial transmembrane protein according to either of the preceding clauses, wherein the extracellular or extravesicle-binding structure is a SNAP tag or a CLIP tag.

[0064] Clause 6: Artificial transmembrane proteins, (a) Type I, in which the intracellular or intravesicular domain is located adjacent to the C-terminus and the extracellular or extravesicular domain is located adjacent to the N-terminus; or (b) An artificial transmembrane protein according to any of the preceding clauses, which is of type II, having an intracellular or intravesicular domain located adjacent to the N-terminus and an extracellular or extravesicular domain located adjacent to the C-terminus.

[0065] Clause 7: The artificial transmembrane protein according to Clause 7, wherein the artificial transmembrane protein is of type II, and the intracellular or intravesicular domain contains a greater amount of positively charged amino acid residues than the extracellular or extravesicular domain.

[0066] Clause 8: An artificial transmembrane protein according to any of the preceding clauses, wherein the intracellular or intravesicular receptor structure is a designed receptor or other functional molecule such as an antibody, antibody fragment, nanobody, or affimer.

[0067] Clause 9: An artificial transmembrane protein according to any of the preceding clauses, further comprising a cleavable signal peptide adjacent to the N-terminus of the artificial transmembrane protein for interaction with a protein transport system and for controlling the migration of the artificial transmembrane protein.

[0068] Clause 10: An artificial transmembrane protein according to any of the preceding clauses, comprising an affinity tag whose extracellular or extravesicle-binding structure is configured to interact with a membrane recognition element.

[0069] Clause 11: An artificial transmembrane protein according to any of the preceding clauses, wherein the transmembrane protein is unlabeled, in particular, unfluorescently labeled.

[0070] Clause 12: A cell, vesicle, or cellular component or vesicle component comprising a nucleic acid sequence encoding an artificial transmembrane protein as described in any of Clauses 1 to 11 or an artificial transmembrane protein as described in any of Clauses 1 to 10.

[0071] Clause 13: A recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding an artificial transmembrane protein as described in any of Clauses 1 to 10.

[0072] Clause 14: A vector, preferably a plasmid vector, comprising the recombinant nucleic acid molecule described in Clause 12.

[0073] Clause 15: Use of the vector described in Clause 13 for in vitro expression of an artificial transmembrane protein, comprising the steps of preparing cells, introducing the vector described in Clause 13 into cells, and expressing the artificial transmembrane protein.

[0074] Clause 16: Use as described in Clause 14, further comprising the step of cleaving a signal peptide from an artificial transmembrane protein.

[0075] Clause 17: A biomolecular detection device for analyzing cells, vesicles, or cellular components or vesicular components containing artificial transmembrane proteins as described in any of Clauses 1 to 10, wherein the biomolecular detection device includes an evanescent illuminator comprising a photo-coupling unit configured to generate an evanescent field from coherent light having a specified wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nanopattern containing a plurality of predetermined lines of coherent arrangements on which membrane recognition elements are located for binding structures of artificial transmembrane proteins of cells, vesicles, or cellular components or vesicular components, and the membrane A biomolecular detection device in which a recognition element is configured to bind to an artificial transmembrane protein-binding structure to form a transmembrane nanopattern within a cell, vesicle, or cellular component or vesicular component based on a template nanopattern of an evanescent illuminator such that light from the evanescent field is scattered by a cell, vesicle, or cellular component or vesicular component bound to the membrane recognition element, and predetermined lines are arranged to constructively interfere at predetermined detection sites having differences in optical path length where the light scattered by the cell, vesicle, or cellular component or vesicular component bound to the membrane recognition element is an integer multiple of a predetermined wavelength of coherent light.

[0076] Clause 18: The biomolecule detection device according to Clause 17, wherein at least one cell, vesicle, or cellular component or vesicle component binds to a membrane recognition element via an artificial transmembrane protein-binding structure.

[0077] Clause 19:a. An artificial transmembrane protein as described in any of Clauses 1 to 11, or Cells as described in Clause 12, or Recombinant nucleic acid molecules as described in Clause 13, or The vectors described in Clause 14, and b. Biomolecular detection devices as described in Clause 16 or 17 A kit of parts including parts.

[0078] Clause 20: The kit according to Clause 19, further comprising the protein of interest configured to interact with intracellular or intravesicular biomolecules, particularly gold nanoparticles, of which the protein of interest comprises a high-mass portion.

[0079] Clause 21: A label-free method for detecting intracellular or intravesicular biomolecular interactions in cells, cellular components, or vesicles or vesicular components using the biomolecular detection device described in Clause 16 or 17, - A step of preparing cells, cellular components, or vesicles or vesicle components containing an artificial transmembrane protein as described in any of clauses 1 to 11; - A step of applying cells, cellular components, vesicles, or vesicle components to the membrane recognition element of a biomolecular detection device; - A step of generating a beam of coherent light at a predetermined beam generation position for multiple predetermined lines that are incident on a membrane recognition element containing a bound transmembrane protein, such that the beam of coherent light has a predetermined wavelength, and the diffracted portion of the incident coherent light beam constructively interferes at a predetermined detection site with multiple predetermined lines having path length differences that are integer multiples of the predetermined wavelength of the coherent light, thereby providing a signal at the predetermined detection site that represents the membrane recognition element to which an artificial transmembrane protein of a cell, vesicle, or cellular component or vesicle component is bound; - A step to measure a signal representative of a membrane recognition element to which an artificial transmembrane protein of a cell, vesicle, or cellular component or vesicle component is bound. A method that includes this.

[0080] Clause 22: Receptor structures of artificial transmembrane proteins, or biomolecules to be detected. The method according to clause 21, further comprising the step of providing a substance that interacts with intracellular or intravesicular components of an interaction.

[0081] Clause 23: The method according to Clause 21 or 22, wherein cells or cellular components containing an artificial transmembrane protein are provided by the steps of transfecting cells with the vector described in Clause 14 in vitro, expressing the artificial transmembrane protein in the cells, and optionally removing a portion of the cell membrane to provide the cellular component. [Brief explanation of the drawing]

[0082] [Figure 1] A schematic diagram of a biomolecular detection device according to an embodiment of the present invention. [Figure 2] This is a cross-sectional view of a biomolecule detection device according to another embodiment of the present invention. [Figure 3a] This figure shows an artificial transmembrane protein according to an embodiment of the present invention for detecting intracellular interactions of the cAMP pathway. [Figure 3b] This figure shows the expected signals from control experiments using wild-type cells and G protein knockout cells during cAMP pathway detection. [Figure 3c] This figure shows the signals obtained from control experiments using wild-type cells and G protein knockout cells during the detection of the cAMP pathway. [Figure 4a] This figure shows two different artificial transmembrane proteins according to another embodiment of the present invention for detecting intracellular interactions of the ERK pathway. [Figure 4b]This figure shows the signals associated with artificial transmembrane proteins obtained during detection of the ERK pathway when the ERK pathway is activated by growth factors. [Figure 4c] This figure shows the signals associated with artificial transmembrane proteins obtained during ERK pathway detection when downstream kinases of the ERK pathway are inhibited. [Figure 4d] The figure shows that the signal remained within baseline range for approximately 20 minutes after peptide injection, after which an increase in response was observed from cells with artificial transmembrane proteins containing Grb2 as the receptor structure, while the signal from cells with artificial transmembrane proteins containing eYFP as the receptor structure remained constant at baseline. [Figure 5] Figures 5a and 5b are schematic diagrams of a biomolecular detection device according to another embodiment of the present invention. [Figure 6] Figures 6a and 6b are schematic diagrams of a biomolecule detection device according to another embodiment of the present invention. [Modes for carrying out the invention]

[0083] Figure 1 shows a biomolecule detection device (1) according to an embodiment of the present invention. The biomolecule detection device (1) includes a carrier (2) having a surface on which a plane waveguide (3) is arranged. The detection device further includes an optical coupler (4) for coupling coherent light (L) of a specified wavelength into the plane waveguide (3) such that coherent light propagates through the plane waveguide having an evanescent field of coherent light propagating along a first surface of the plane waveguide (3). The first surface of the plane waveguide is the surface facing outward as viewed from the carrier (2), i.e., the surface shown in Figure 1. In addition to the optical coupler (4), the first surface of the waveguide (3) includes a template nanopattern (5) containing a plurality of predetermined lines (not shown in Figure 1) on which film recognition elements are arranged. Generally, a light source (6) is used to provide an incident beam of coherent light (L) toward the optical coupler (4). The optical coupler (4) couples the coherent light (L) in the plane waveguide (3) as the coherent light (L) propagates toward the template nanopattern (5) in the direction of the arrow shown in Figure 1. When the membrane recognition element binds to a cell, vesicle, or cellular component or vesicular component via an artificial transmembrane protein according to any embodiment described herein, the light is scattered and due to predetermined lines arranged such that the light scattered by the cell, vesicle, or cellular component or vesicular component bound to the membrane recognition element constructively interferes at a predetermined detection site (7). Specific embodiments shown In this configuration, the predetermined line is a curved section configured such that light from the evanescent field scattered by cells, vesicles, or cellular or vesicle components bound to the membrane recognition element interferes at a predetermined detection site (7). The distance between each membrane recognition element of the template nanopattern (5) and the detection site (7) is referred to as the optical path length.

[0084] Figure 2 shows a cross-sectional view of a biomolecule detection device (1) passing through a template nanopattern (5) aligned with the propagation direction of light (L) through a planar waveguide (3). The device (1) contains a carrier (2) on which the waveguide (3) is located on the surface. A living cell (8) is located at the top of the first surface of the planar waveguide (3). Furthermore, the template nanopattern (5) includes protrusions (51) and grooves (52). The protrusions (51) are regions where membrane recognition elements are located. The grooves (52) are regions that do not contain membrane recognition elements. Thus, the binding structures of the transmembrane proteins of the cell (8) can bind to the nanopattern (5) only at the corresponding protrusions. In general, the artificial transmembrane proteins described herein can diffuse laterally within the cell membrane or vesicle membrane. Thus, artificial transmembrane proteins diffuse laterally across the membrane until they approach membrane recognition elements, during which time covalent bonds may be formed to establish nanopatterns within cells, vesicles, or cellular or vesicular components. It should be noted that the widths of cells, nanopatterns, waveguides, and carriers do not provide an indicator of their actual width or width ratio.

[0085] Figure 3a shows a schematic diagram of the cyclic AMP (cAMP) pathway monitored using an artificial transmembrane protein according to an embodiment of the present invention. The cAMP pathway is a G protein-coupled receptor-induced signaling cascade that plays a fundamental role in cellular responses. In resting cells, G proteins bind to the intracellular site of GPCRs. Upon activation of GPCRs by an external ligand, the α-subunit of the G protein detaches from the GPCR complex and activates adenylate cyclase (AC), a membrane-bound enzyme with an intracellular active site. AC then converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), one of the most common second messengers in cells. Furthermore, AC plays a role in signal amplification because the attack by the external ligand leads to the synthesis of thousands of second messenger molecules within seconds. Increased cAMP concentration stimulates cAMP-dependent protein kinases (PKAs). PKA is a tetramer composed of two regulatory subunits (type I and type II), each bound by two cAMP molecules and two catalytic subunits (α and β). Binding of the cAMP molecules results in phosphorylation of the two catalytic subunits, leading to their dissociation from the two regulatory dimers. These phosphorylated subunits activate various targets, which may be other signaling proteins as well as effector proteins. The final step of the cAMP pathway was chosen as a proof-of-concept for the operating principle of the artificial transmembrane protein according to the present invention. To explore the dissociation of the catalytic β subunit upon GPCR induction, an artificial transmembrane protein was constructed having an intracellular or intravesicular domain with an intracellular or intravesicular receptor structure presenting the PKA type II-beta regulatory subunit on its cytosolic region (R2). As seen in Figure 3b, a decrease in signal intensity should be expected, which is associated with a decrease in the complex mass upon dissociation of the catalytic β subunit (dashed line). At the same time, in the absence of the G protein, activation of AC activity cannot be achieved, and therefore, dissociation of the catalytic β subunit cannot be obtained. Consequently, no change in signaling should be detected in G protein knockout cells.Figure 3c shows experimentally obtained signals for both HER293 cells (wild-type) and G protein knockout cells. The knockout cells show no change in signal intensity, while the wild-type cells show a decrease in signal intensity upon dissociation of the catalytic β subunit.

[0086] Figure 4a shows a schematic diagram of the ERK signaling pathway as monitored by two different artificial transmembrane proteins. The ERK pathway is one of the most common types of enzyme-bound surface receptors. One such receptor is an external ligand-binding tyrosine kinase receptor (RTK), which initiates the process. Generally, an inactive RTK receptor consists of two one-pass monomers that are activated and dimerized once the ligand binds to the extracellular domain. Dimerization involves trans-autophosphorylation on specific tyrosine residues (one monomer phosphorylating the other, and vice versa). The phosphorylated tyrosine increases the kinase activity of the RTK and acts as a binding site for certain intracellular proteins. These proteins can recognize the phosphorylated tyrosine and the conformation of the RTK around it, thus the binding event occurs. Proteins that associate with the RTK then often associate with the adapter protein Grb2 via its SH2 domain. Grb2 also has two other SH3 domains for interactions with other proteins. One of the SH3 domains often interacts with Sos, a protein that facilitates the exchange of GDP bound to Ras by GTP. When Sos activates Ras, a monomeric GTPase with immobilized GDP (when inactive) replaces GDP with GTP, and Ras then further activates Sos in a positive feedback loop. Ras activates Raf, which then, via Mek, activates an extracellular signal-regulated kinase commonly known as the ERK module, using a cascade mechanism. ERK proteins are found in either an activated or inactivated state, corresponding to their phosphorylated and unphosphorylated forms, respectively. Phosphorylated ERK translocates to the cell nucleus and acts on gene-regulating proteins. The ERK module is often terminated by ERK itself, which inactivates Raf via negative feedback. To monitor the initial cascade effects of growth factors along the ERK pathway, a first artificial transmembrane protein is provided with the Grb2 protein as a receptor structure. Furthermore, downstream effects were monitored by a second artificial transmembrane protein containing an artificial binder as a receptor structure for the phosphorylated ERK component.Figure 4b shows the expected signals for the first artificial transmembrane protein (dashed line) and the second artificial transmembrane protein (solid line) upon activation of the ERK cascade by growth factor stimulation. Figure 4c shows the expected signals for the first artificial transmembrane protein (Grb2, dashed line) and the second artificial transmembrane protein (artificial binder, solid line) upon inhibition of the downstream kinase, which should result in a decrease in the signal obtained only from the second artificial transmembrane protein.

[0087] First, we tested whether the SH3-binding domain of the Grb2 protein as a receptor structure for the first artificial transmembrane protein (SPF) was functional. In this event, cells containing an SPF with Grb2 as the receptor structure and cells containing an SPF with eYFP (enhanced yellow fluorescent protein) were treated with a protein that specifically targets Grb2. As seen in Figure 4d, the signal remained within baseline range for approximately 20 minutes after peptide injection. Subsequently, an increase in response was observed from cells containing an SPF with Grb2 as the receptor structure, while the signal for cells containing an SPF with eYFP as the receptor structure remained constant at baseline. The lag between injection and the change in response is likely due to the time required for the peptide to diffuse across the cell membrane and bind to the Grb2 SH3 domain. Thus, the method enables real-time monitoring of intracellular biomolecular interactions within living cells. Furthermore, these results indicate that peptides targeting Grb2 specifically bind to Grb2 receptor structures within living cells, but do not bind to non-specific receptor structures such as eYFP.

[0088] Figures 5a and 5b show a biomolecule detection device (1') according to an embodiment of the present invention. The device (1') includes an evanescent illuminator (2') having a photo-coupling unit (4') configured to generate an evanescent field (9') from coherent light having a specified wavelength from a light source (6') on a first surface of the evanescent illuminator (2'). The first surface of the evanescent illuminator (2') is the binding structure of transmembrane proteins (81) of cells (8'). The nanopattern (5') includes a template nanopattern containing a coherent arrangement of multiple predetermined lines along membrane recognition elements for the body. As seen, by establishing chemical bonds between the membrane recognition elements and laterally diffusible transmembrane proteins (81), the nanopattern (5') of the evanescent illuminator (2') is transported to the cell as a transmembrane nanopattern. In the embodiment in Figure 5a, the light source (6') and the detection unit (7') are physically separated components, but in the embodiment shown in Figure 5b, they are integral parts of the evanescent illuminator (2').

[0089] Figures 6a and 6b show alternative embodiments of the biomolecule detection device (1'') according to the present invention. In these particular embodiments, the biomolecule detection device (1) includes an evanescent illuminator, which is a total internal reflection system configured to provide a beam of coherent light from a light source (6'') at a predetermined wavelength and angle onto a first surface of the evanescent illuminator via an optical coupling unit (4''). In these embodiments, the optical coupling unit is a prism. In the embodiment shown in Figure 6a, the evanescent illuminator includes a refractive index matching medium (11'') such as a refractive index matching oil, DMSO, glycerol, a water mixture, or a hydrogel, and a carrier slide (12'') containing a template nanopattern (5''). Alternatively, as shown in Figure 6b, the evanescent illuminator may not include the refractive index matching medium (11'') and the carrier slide (12''). In this case, the nanopattern (5'') is provided directly onto the optical coupling unit (4''), i.e., the first surface of the prism.

[0090] material and method DNA plasmids encoding different artificial transmembrane proteins were used by Thermo Fisher Scientific's Invitrogen GeneArt Gene The synthesis was purchased from a synthesis service. All synthetic genes were assembled from synthetic oligonucleotides and / or PCR products and inserted into the pcDNA3.1(+) vector backbone. Plasmid DNA was purified from transformed bacteria, its concentration was determined by UV spectroscopy, and the construct was validated by manufacturer sequencing. Sequence identity within the insertion site was 100%. Plasmids were delivered in TE buffer at a concentration of approximately 1 mg / ml and stored in working aliquots at -80°C.

[0091] The three signal peptides tested are identified in Table 3.1, but their further amino acid sequences can be found in Table 1. [Table 1]

[0092] Table 2 shows the structural characteristics of plasmid vectors encoding some of the artificial transmembrane proteins that were tested. [Table 2]

[0093] Cell culture and transfection HEK293 wild-type and G protein knockout cells were cultured in a cell incubator with 5% CO2 at 37°C in complete medium (DMEM medium containing 10% fetal bovine serum). To generate cells expressing artificial transmembrane proteins, the cells were transfected using Lipofectamine 3000 transfection reagent according to the manufacturer's protocol.

[0094] To establish stable cell lines, transiently transfected cells were grown in complete medium supplemented with 1 mg / ml of G418 for approximately 20 days. Subsequently, neomycin-resistant cells were stained with SNAP-Surface 649 dye and selected by flow cytometry.

[0095] For fluorescence imaging, cells were seeded at a 50% concentration density on 24-well glass-bottom plates and transfected after 24 hours as previously described. The transfection medium was replaced with complete medium after 12 hours. Cells were imaged 12, 24, 36, and 48 hours after transfection using an Olympus FluoView FV3000 confocal laser scanning microscope. Prior to imaging, cells were incubated with SNAP-Surface 649 dye for 30 minutes and then washed three times with warm PBS. During imaging, cells were maintained at 37°C with 5% CO2. eYFP and SNAP-Surface 649 channels were simultaneously acquired using a 20x objective lens with a 514 nm excitation / 527 nm emission wavelength for the green channel and a 651 nm excitation / 667 nm emission wavelength for the red channel.

[0096] Biomolecular detection devices Thin-film optical waveguides from Zeptosens were processed using a standard procedure (Gatterdam et al., Nature Nanotechnology, 12(11):1089-1095, September 2017) and coated with grafted PAA-g-PEG polymer. The polymer's amine groups were protected by photosensitive PhSNPPOC groups to allow for further processing. Subsequently, molograms were patterned on the optical waveguides using a previously described reactive immersion lithography process. Briefly, the polymer-coated waveguide tips were mounted on custom holders that allowed for the alignment of phase masks. After placing the phase masks on the holders, the tips were subjected to 2000 mJ / cm². 2The photosensitive groups were cleaved and removed from the protrusions of the nanopattern by illumination at a wavelength of 405 nm with a dose of [percentage missing]. The activated amine moieties were incubated with either a BG-GLA-NHS or BC-GLA-NHS substrate to bind to either a SNAP tag or a CLIP tag, respectively. Subsequently, full-field illumination under UV light was performed to remove any remaining sensitive groups from the grooves and surrounding areas. The photoactive group was removed. The resulting amine group was functionalized with the GRGDSPGSC peptide.

[0097] measurement Cells were seeded onto a planar waveguide to 100% density and allowed to adhere in complete culture medium for 2-3 hours while the planar waveguide was kept inside a cell incubator. The medium was then replaced with HEPES-buffered complete medium or HEPES-buffered HBSS adjusted to pH 7.4. Measurements were performed on an F3000 ZeptoReader maintained at 35°C with 5% CO2. Images were acquired every 15 seconds using a 635 nm laser with exposure times ranging from 0.1 s to 1 s. After establishing a 10-minute baseline (30 images), pharmacological manipulations were performed on the chip.

[0098] amino acid sequence [Table 3-1] [Table 3-2]

Claims

1. An artificial transmembrane protein for use in a biomolecular detection device for detecting intracellular or intravesicular biomolecular interactions, wherein the artificial transmembrane protein comprises an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicular domain having an intracellular or intravesicular receptor structure, the receptor structure being configured to interact with the intracellular or intravesicular component of the biomolecular interaction to be detected, and the extracellular or extravesicular binding structure being configured to bind to membrane recognition elements positioned along a plurality of predetermined lines of the biomolecular detection device.

2. The artificial transmembrane protein according to claim 1, further comprising a linker domain configured to facilitate interaction between an intracellular or intravesicular receptor structure and an intracellular or intravesicular component of a biomolecular interaction to be detected, wherein the linker domain is positioned between the intracellular or intravesicular receptor structure and a hydrophobic transmembrane domain.

3. The artificial transmembrane protein according to claim 1 or 2, wherein an extracellular or extravesicle-binding structure is configured to establish a covalent bond with membrane recognition elements positioned along a plurality of predetermined lines of a biomolecular detection device.

4. An artificial transmembrane protein according to any one of claims 1 to 3, wherein the extracellular or extravesicle-binding structure contains a nucleophile, preferably a thiol or thiolate.

5. An artificial transmembrane protein according to any one of claims 1 to 4, wherein the extracellular or extravesicle-binding structure is a SNAP tag or a CLIP tag.

6. Artificial transmembrane proteins, (a) Type I, in which the intracellular or intravesicular domain is located adjacent to the C-terminus and the extracellular or extravesicular domain is located adjacent to the N-terminus; or (b) Type II, in which the intracellular or intravesicular domain is located adjacent to the N-terminus and the extracellular or extravesicular domain is located adjacent to the C-terminus. The artificial transmembrane protein according to any one of claims 1 to 5.

7. The artificial transmembrane protein according to claim 6, wherein the intracellular or intravesicular domain contains a greater amount of positively charged amino acid residues than the extracellular or extravesicular domain.

8. The artificial transmembrane protein according to any one of claims 1 to 7, wherein the intracellular or intravesicular receptor structure is a designed receptor or antibody, antibody fragment, nanobody or other functional molecule such as an affimer.

9. An artificial transmembrane protein according to any one of claims 1 to 8, further comprising a cleavable signal peptide adjacent to the N-terminus of the artificial transmembrane protein for interaction with protein transport systems and for controlling the migration of the artificial transmembrane protein.

10. An artificial transmembrane protein according to any one of claims 1 to 9, comprising an affinity tag in which an extracellular or extravesicle-binding structure is configured to interact with a membrane recognition element.

11. An artificial transmembrane protein according to any one of claims 1 to 10, wherein the transmembrane protein is unlabeled, particularly unfluorescently labeled.

12. The artificial transmembrane protein according to any one of claims 1 to 11 or the artificial transmembrane protein according to claim 1 to 11 A cell, vesicle, or cellular component or vesicle component containing a nucleic acid sequence encoding an artificial transmembrane protein as described in any one of the items.

13. A recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding an artificial transmembrane protein according to any one of claims 1 to 11.

14. A vector, preferably a plasmid vector, comprising the recombinant nucleic acid molecule described in claim 13.

15. The use of the vector according to claim 14 for in vitro expression of an artificial transmembrane protein, comprising the steps of preparing cells, introducing the vector according to claim 14 into cells, and expressing the artificial transmembrane protein.

16. A biomolecular detection device (1) for analyzing cells, vesicles, or cellular components or vesicle components containing an artificial transmembrane protein according to any one of claims 1 to 11, wherein the biomolecular detection device includes an evanescent illuminator comprising a photo-coupling unit configured to generate an evanescent field from coherent light (L) having a specified wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nanopattern (5) containing a plurality of predetermined lines of coherent arrangements on which membrane recognition elements are arranged for binding structures of the artificial transmembrane protein of the cell, vesicle, or cellular component or vesicle component (8), and the membrane recognition elements A biomolecular detection device wherein a ment is configured to bind to an artificial transmembrane protein-binding structure to form a transmembrane nanopattern within cells, vesicles, or cellular components or vesicle components (8) based on a template nanopattern (5) of an evanescent illuminator such that light from the evanescent field is scattered by cells, vesicles, or cellular components or vesicle components (8) bound to a membrane recognition element, and predetermined lines are arranged to constructively interfere at predetermined detection sites (7) having differences in optical path length where the light scattered by cells, vesicles, or cellular components or vesicle components (8) bound to a membrane recognition element is an integer multiple of a predetermined wavelength of coherent light (L).

17. a. An artificial transmembrane protein according to any one of claims 1 to 11, or a cell according to claim 12, or a recombinant nucleic acid molecule according to claim 13, or a vector according to claim 14; and b. The biomolecular detection device according to claim 16; and, if necessary, c. The target protein is configured to interact with intracellular or vesicular biomolecules, particularly those containing gold nanoparticles, in a high-mass region. A kit of parts, including...

18. A label-free method for detecting intracellular or intravesicular biomolecular interactions in cells, cellular components, or vesicles or vesicular components using the biomolecular detection device described in claim 15, - A step of preparing cells, cellular components, or vesicles or vesicle components containing the artificial transmembrane protein described in any one of claims 1 to 10; - A step of applying cells, cellular components, or vesicles or vesicle components to the membrane recognition element of a biomolecular detection device; - A coherent light beam having a specified wavelength, and the diffracted portion of the incident coherent light beam constructively interfering at a specified detection site with multiple predetermined lines having path length differences that are integer multiples of the specified wavelength of the coherent light, thereby providing a signal at the specified detection site to which artificial transmembrane proteins of cells, vesicles, or cellular or vesicle components are bound to a membrane recognition element, wherein a coherent light beam is generated at a specified beam generation position for multiple predetermined lines incident on a membrane recognition element containing the bound transmembrane protein. Step; - A step to measure a signal representative of a membrane recognition element to which an artificial transmembrane protein of a cell, vesicle, or cellular component or vesicle component is bound. A method that includes this.

Images