Method for fabricating composite arrays

Abstract

The present invention relates to a high-throughput process for the simultaneous and targeted mixing of a molecule with a number of other molecules. The obtained molecule-molecular complex can then be captured on a surface, thereby creating a microarray. Subsequently, the molecule-molecular complex can be characterized and measured (e.g., for reactions with other molecules) using the microarray.

Description

Background Technology

[0001] In biology, complexes can be composed of various components. They are typically combinations of at least two molecules that interact with each other in a non-covalent manner. Molecular complexes typically have functions different from those of individual molecules. Typical examples include protein-protein complexes, RNA-protein complexes, or DNA-protein complexes. Examples of such complexes include ribosomes or nucleosomes. For instance, MHC / HLA molecules form complexes with various peptides. Typically, 8 to 11 peptides are incorporated, thereby stabilizing the complex. The complex is presented on the cell, and binding with T-cell receptors can occur.

[0002] The analysis or testing of various complexes can be associated with a great many questions. Therefore, microarrays containing such complexes are receiving attention.

[0003] A microarray is an integration of many different small dots (spots) containing molecules on a solid substrate. The fabrication of microarrays is basically divided into four different fabrication types:

[0004] 1. Spot-type microarray

[0005] a. Microarray spotter [1]

[0006] 2. In-situ synthesis microarray

[0007] a. Spot composite; inkjet printing [2]

[0008] b. Photolithography using a photomask [3]

[0009] c. Photolithography by micromirror [4]

[0010] 3. Synthesis using DNA polymerase

[0011] A relatively recent method for fabricating DNA microarrays consists of synthesizing DNA on a surface using a polymerase based on a DNA template (WO2009034181A2_stellacci, WO2010100265A1_roth). In this method, a primer is provided on a solid surface (a starting point for synthesis for the DNA polymerase). Subsequently, a mixture consisting of individual synthesis components, DNA polymerase, and a template is applied to the surface. Synthesis proceeds in a massively parallel manner, reaching up to thousands of spots. The reaction spaces of each of these spots are physically separated from one another to ensure independent synthesis reactions. This can be achieved by various means, ranging from spatial separation through microcavities to the restriction of diffusion.

[0012] 4. Synthesis using in vitro translation mixtures

[0013] DNA microarrays can be converted into protein microarrays using a cell-free expression mixture, by first translating expressible DNA into RNA and then translating the RNA into protein. This principle has already been demonstrated in a number of diverse applications and models, but at the core of it is always cell-free expression of protein. Only the technical implementation and protein capture on the surface differ [5, 16]. In all described applications, the goal is to produce protein microarrays with the purest possible monoclonal protein spots.

[0014] The fundamental difference between the fabrication methods is that the molecule is produced in advance in the first method mentioned, Method 1, while it is produced during the fabrication of the microarray in the other methods.

[0015] There also exist approaches and methods aimed at replicating existing microarrays. Examples are as follows:

[0016] 5. Amplification of DNA microarrays by hybridization [6-10].

[0017] 6. Amplification of DNA microarrays through hybridization and extension by DNA polymerase [11-13].

[0018] 7. Amplification by a master cavity chip and subsequent PCR [14, 15].

[0019] The goal of all the methods described above for fabricating microarrays is to generate a spot of a target molecule that is monoclonal as much as possible. The target molecule does not interact with any other molecules. In all known synthetic methods, the synthesis of the target molecule requires the interaction of various molecules (synthetic building blocks, DNA, RNA, proteins). In most cases, these molecules are no longer present in the final microarray. If they are present, they are considered merely appendages and will no longer interact with the target molecule in any appropriate manner. In other words, these methods are highly suitable for producing microarrays with the purest possible target molecule.

[0020] However, in reality, there are often cases where a specific molecule must first be activated by another molecule or form a so-called complex with another molecule to reach an activated state on its own. Microarrays containing molecules activated in this way cannot be fabricated with current state-of-the-art technology, or can only be fabricated using complex methods.

[0021] In biology and related industries, pipetting robots are often used (micro to macro) to deliver molecules into so-called reaction chambers. Typically, microplates with 6, 12, 24, 48, 96, 384, 1536, or 3456 reaction chambers (wells) are used. This is essential, especially when the number of samples to be analyzed is very large. Here, it is common and modern technology to also mix molecules in these reaction chambers to realize multiple biological tests, such as ELISA, activity tests, and enzyme tests. In this way, molecular complexes can also be generated and measured through a high-throughput process.

[0022] However, it is common practice to measure individual reactions separately. It is possible to generate molecular complexes in a reaction chamber and then print them onto a surface using traditional microarray fabrication methods. This type of fabrication is time-consuming and costly. Additionally, it has been suggested that particularly complex molecules, such as receptors or enzymes, are damaged due to the long delivery process, becoming partially or completely inactive or exhibiting unnatural behavior. Generally, attempts are made to add more complex molecules as late as possible, or preferably even to rinse them out with solution from an already completed array. Therefore, when measuring antigen-antibody interactions, antigen arrays (because they are less complex) are much more prevalent than antibody arrays (because they are more complex).

[0023] Literature US8105845B2 is prior art describing a method for fabricating and measuring an array of complexes. The method is relatively complex and uses a channel system. A surface is coated with molecules through six channels. This configuration is then rotated 90 degrees, and a second coating is applied through the same channels, resulting in the formation of molecular complexes. Subsequently, an analyte passes through the channels, and the interaction between the analyte and the complexes on the surface can be measured. Using this configuration, potentially 36 molecular complexes on the surface can be measured.

[0024] Publications US 8211382 B2 and US 9682396 B2 are included in the prior art and describe a so-called flow printing method. In this method, a print head is pressed onto a surface to create many small, closed microfluidic channels. Subsequently, molecules are injected through these channels to specifically contact the surface. In this system, the number of channels in the print head is also limited.

[0025] A method for fabricating a microarray according to the prior art involves simultaneously transferring molecules from a cavity chip having many small reaction chambers to a predetermined surface. Such a method is disclosed, for example, in WO 2010100265 A1. Here, molecules are supplied to a carrier system (e.g., a cavity chip) and amplified in a reaction chamber. The molecules or formed derivatives are subsequently captured on a capture surface. In this regard, the formation of complexes is neither described nor considered. Additionally, an essential component of the method is an amplification step.

[0026] WO 2013174942 A1 also describes, as prior art, a method in which another molecule is produced from a template molecule within a carrier system (e.g., a cavity chip) so that the product can then be captured on a capture surface. Its purpose is to fabricate a microarray as pure as possible consisting of pure spots of monoclonals. A specific mixture of two types of molecules intended to form a complex was not considered.

[0027] WO 2013 045700 A1 is also included in the prior art and describes a method in which another molecule can be generated from exactly one template molecule present in a cavity. To this end, an amplification mixture is filled. Subsequently, the generated product is captured on a capture surface. The method is intended to produce a microarray as pure as possible consisting of pure spots of monoclonal. In the described method, it is essential to amplify the molecule, and no specific mixture of molecules is provided. Therefore, it is not possible to generate a microarray of molecular complexes by this method.

[0028] WO 2013186359 A1 is included in the prior art and describes a method for analyzing molecular properties or reaction conditions, wherein an array having monoclonal molecular spots is preferentially fabricated. In this process, product molecules are produced and delivered. Complex formation is not included in the intended reaction spectrum.

[0029] DE 102018122546 B3 is also prior art. The above disclosure describes possible uses of an MHC complex array, in particular, for which stabilized MHC is used. Measurements are performed by BLI (biological interferometric measurement). However, the fabrication of the array was not disclosed.

[0030] As such, prior art has not yet provided a method to fabricate microarrays having molecular complexes in a simple and cost-effective manner.

[0031] Accordingly, the object of the present invention was to provide a method for fabricating molecular complex arrays that can overcome the disadvantages of the prior art and provide various arrays for analysis in a simple, inexpensive, and rapid manner. This object is resolved by the independent claim. Particularly advantageous embodiments may be identified in the dependent claim.

[0032] In a first preferred embodiment, the present invention relates to a method for fabricating a molecular complex microarray in situ, comprising the following steps:

[0033] A step of providing a first surface comprising a plurality of independent active regions,

[0034] A step of introducing a first molecule into a plurality of active regions,

[0035] A step of adding a second molecule to each active region where a first molecule is present,

[0036] A step of closing the active region to the second surface,

[0037] The stage of complex formation between molecules,

[0038] A step of immobilizing the formed composite on a capture surface.

[0039] A method for fabricating a molecular complex microarray in situ, comprising the following steps, is particularly preferred:

[0040] a) providing a first surface comprising a plurality of independent active regions,

[0041] b) a step of introducing a first molecule into a plurality of active regions,

[0042] c) a step of fixing the provided molecule to the surface,

[0043] d) a step of adding a second molecule to each active region where the first molecule is present,

[0044] e) A step of closing the active region to the second surface,

[0045] f) Complex formation stage between molecules,

[0046] g) a step of immobilizing the formed composite on a capture surface, preferably on a surface from e).

[0047] Accordingly, in the method according to the present invention, the formed molecular complexes can be simultaneously transferred to a capture surface and then to a final surface without the need to remove them individually from the reaction chamber (by microfluidization or through a carrier medium). This represents a significant simplification compared to the method of the prior art and results in very accurate results as well as savings in time and cost.

[0048] Therefore, an essential aspect of the present invention is that the molecules to form a composite or to be tested for their composite-forming properties are not premixed. That is, the composite is not spotted onto an array, but rather the composite formation occurs only on the surface. This has the advantage of not requiring the creation of a premix, which would consume complex and relatively large amounts of materials as well as resources. In particular, due to multiple possible combinations, the method of the prior art quickly reaches its limits. If multiple different composites are to be contained on an array, a large amount of premixing would have to be performed, but this is not necessary in the method according to the present invention. In contrast, the method according to the present invention is significantly faster and consumes less material, resources, and human time.

[0049] In a complex, two or more molecules typically engage in non-covalent interactions. In the context of the present invention, it is desirable that the resulting complex fulfills tasks and / or functions that individual molecules alone could not perform.

[0050] Different first molecules may be used on a surface. If more than one type of first molecule is used on a surface, they may exist individually in individual active regions, so that only one type of molecule is provided in each active region. However, it is also possible to provide multiple types of first molecules within a single active region. In addition, it is also possible to introduce different types of first molecules in sequence.

[0051] If more than one type of first molecule is introduced into the active region, it is possible to use more than one type of first molecule in the complex formed. The method according to the present invention preferably does not include an amplification step and / or the first molecule does not undergo derivatization. Therefore, there is no need to provide a reaction mixture.

[0052] With the method according to the present invention, it is possible to significantly facilitate and accelerate the fabrication of composite microarrays.

[0053] It is preferable that the capture surface be a second surface. This enables the complex to be attached during complex formation. The method is particularly suitable when the same second molecule is used for the entire array.

[0054] However, the second surface itself may also be, for example, a microarray containing the second molecule.

[0055] The active region is preferably a cavity and / or spot. It is important that the active regions on the first surface are independent of each other and that the molecules cannot be mixed.

[0056] The surface can be made of various materials, for example, glass or PDMS.

[0057] Preferably, the first surface, the second surface and / or the capture surface have dimensions of 5 mm - 75 mm x 3 mm - 25 mm, more preferably 10 mm - 25 mm x 10 mm - 25 mm, and most preferably 15 mm x 15 mm.

[0058] The number of active areas per surface is preferably 50 to 20,000, particularly preferably 300 to 10,000.

[0059] The active regions can have completely different sizes. They are preferably circular regions, but other shapes are also possible. The diameter of individual active regions is preferably 50 μm to 1000 μm, particularly preferably 100 μm to 700 μm, and very particularly preferably 15 μm to 500 μm. The distance between active regions can also vary. The distance is preferably 10 μm to 200 μm, particularly preferably 20 μm to 100 μm, and most preferably 50 μm.

[0060] If the active region is a cavity, it preferably has a volume of 500 pl to 100 nl, particularly preferably 350 pl to 30 nl, and most preferably 500 pl to 5 nl.

[0061] The depth of the cavity is preferably 5 μm to 100 μm, more preferably 10 μm to 50 μm, and most preferably 30 μm.

[0062] Specific embodiments have, for example, the following dimensions:

[0063] 4,104 (54x76) active regions on a 16 mm x 10 mm area, with an active region diameter of 150 μm and a distance of 50 μm between active regions. If these are cavities, they have a depth of 30 μm and a volume of 530 pl.

[0064] 1,188 (27x44) active regions on a 16 mm x 10 mm area, with an active region diameter of 300 μm and a distance of 50 μm between active regions. If these are cavities, they have a depth of 30 μm and a volume of 2.12 nl.

[0065] Or 476 (28x17) active regions on an area of ​​16 mm x 10 mm, with an active region diameter of 500 μm and a distance of 50 μm between active regions. If these are cavities, they have a depth of 30 μm and a volume of 5.8 nl.

[0066] The present invention is by no means limited to these embodiments. In principle, all possible dimensions, number, shape, and arrangement of the surface and active areas can be considered. It is also possible to use a conventional chip, for example, a chip having 1,188 cavities.

[0067] Additionally, it is preferable that the introduced first molecule be immobilized on the surface in step c) via an immobilization tag, by adsorption, by ionic interactions, by van der Waals forces, and / or by drying.

[0068] When immobilization tags are used, they do not necessarily have to be covalently bonded to the surface. For example, bonding through intermolecular interactions is also possible.

[0069] Therefore, it may be desirable for the first molecule to include an immobilized tag.

[0070] It is desirable that the surface having the molecule remains durable for a long time after this step, which is a decisive advantage of the method of the present invention. Durability also depends on the molecule used. It is particularly desirable that the surface produced in this way can be stored for any period of time. Thus, depending on the molecule, weeks or months can easily pass between step c) and step d). It is most appropriate to dry the surface and store it in a region below room temperature, preferably below 10°C, and particularly preferably at 4°C.

[0071] Complexes are often composed of stable and unstable complex partners. Accordingly, the present invention is particularly advantageous because it is possible to introduce a stable complex partner (e.g., a peptide) as a first molecule and store it over a long period in this manner. Subsequently, a less stable complex partner (e.g., an MHC) is added as a second molecule immediately before a planned analysis or test.

[0072] The complex may also be used as a first or second molecule. However, they subsequently form a new complex with the first or second molecule, which is then captured on the surface as a complex in the context of the present invention. Therefore, the important point is not merely to bind the complex to the surface, but to specifically cause the complex to form and capture them.

[0073] A second molecule is added to the first molecule, or

[0074] A second molecule is present on a second surface, and contact between an active region containing a first molecule and a second molecule is established through liquid crosslinking.

[0075] It is desirable.

[0076] The second molecule can be added in various ways. It is important to minimize air retention in the active region between the two surfaces, as air can make molecule capture on the capture surface more difficult. Additionally, cross-contamination must be prevented as much as possible, and the active regions must remain isolated. This is particularly important when working with different first molecules on a single surface.

[0077] It is preferable that the second molecule includes an immobilization tag. The same immobilization tag as that for the first molecule is possible here.

[0078] It is possible to apply a second molecule to the surface as a large droplet. This procedure has the advantage that individual active regions can be filled almost without air. However, depending on the level of charge, molecules may flow out when the second surface is applied; therefore, this method is not suitable for all applications or must be implemented with particular precision.

[0079] Another method is the filling of small droplets. This can be performed, for example, using a printer. In this case, a second molecule is applied to the active area as a small droplet. If a cavity is used as the active area, it may be advantageous to select a droplet volume larger than the cavity volume to exclude as many air bubbles as possible. However, if the active area is overfilled, it can lead to cross-contamination because molecules can penetrate into neighboring active areas.

[0080] However, it has been proven particularly desirable to use small droplets, the volume of which is smaller than the volume of the active area. Excess air can be removed, for example, after applying a second surface, preferably by applying excess pressure. This procedure has the advantage of preventing the presence of air bubbles and cross-contamination. Therefore, with current measuring equipment, this method has provided the best results.

[0081] If second molecules are present on the second surface, they may exist in independent active regions or as a plane. In particular, if they are applied to the second surface as a plane, it is preferable to use only one type of second molecule per array. If the second surface is a microarray or a cavity array, different second molecules may also be used, in which case the different second molecules are spatially separated by active regions, preferably spots or cavities.

[0082] Thus, the active region on the first surface comes into contact with the second molecule. This can be done simultaneously or per active region. After or during charging, the active region is sealed with a capture surface, which can specifically capture the subsequently generated molecular complex.

[0083] Advantageously, complex formation occurs in a closed active region. These may be, for example, a closed cavity. A liquid crosslink formed between two surfaces may also involve a closed active region in view of the present invention.

[0084] It is desirable that the formation of the complex be made possible by the release of the first molecule. This can be accomplished in various ways depending on the type of immobilization, for example, by the release of the immobilized tag, by rehydration, or by the dissolution of intermolecular interactions. A person skilled in the art can select a suitable method without the need for new invention. Depending on the tag and the bonding, various methods for the release of the immobilized tag may be considered. Thus, release can be achieved through light of various wavelengths, for example, UV light, chemical cutting, enzymatic cutting, electric fields, magnetic fields, or also electrochemical cutting.

[0085] It is further preferable that the introduction of the first molecule into the active region of the first surface be achieved by one of the following methods:

[0086] a. Spotting of a liquid containing the first molecule,

[0087] b. Synthesis of the first molecule,

[0088] c. Application of particles containing the first molecule, and / or

[0089] d. Establishment of contact between the active region of the first surface and a DNA microarray containing a spot of DNA encoding the first molecule.

[0090] The first molecule is preferably selected from the group comprising proteins, peptides, DNA, RNA, small molecules, cells, preferably CRISPR-associated proteins and their mutants, gRNA, proteins of major histocompatibility complex classes and their mutants, proteins of antibody classes, T lymphocytes, and B lymphocytes. Thus, in this regard, cells may also be referred to as molecules. When a cell is used as the first molecule, the complex partner typically represents a surface protein or other molecular structure on the cell surface, which are typically referred to in biology as receptors, interactors, markers, or differentiation complexes (CDs). Lipids, phospholipids, sugar residues, or other surface structures may also serve as complex partners. It is particularly desirable that the molecule used as the first molecule be sufficiently stable to be immobilized and stored on a surface. Thus, proteins, peptides, DNA, RNA, and small molecules are particularly preferred first molecules.

[0091] The second molecule is preferably selected from the group comprising proteins, peptides, DNA, RNA, small molecules, cells, preferably CRISPR-associated proteins and their mutants, gRNA, proteins of the major histocompatibility complex class and their mutants, proteins of the antibody class, T-lymphocytes, and B-lymphocytes. Accordingly, in this regard, a cell may also be referred to as a molecule. When a cell is used as the second molecule, the complex partner typically represents a surface protein or other structure on the cell surface.

[0092] It is desirable for a protein-protein or protein-peptide complex to be formed. It is also desirable for a complex in which one complex partner is located on the cell surface. For example, this may apply when a cell is used as the first or second molecule.

[0093] A desirable protein-peptide complex is, for example, an MHC-peptide complex. An antibody-antigen complex is also possible.

[0094] The formation of an RNA-protein complex is also desirable. For example, gRNA and Cas9 may be used as the first or second molecule, respectively. This produces an RNA-protein complex that has the function of specifically cleaving and / or binding DNA. gRNA provides specificity, and Cas9 provides the enzymatic activity of the cleavage process.

[0095] DNA-protein complexes are also desirable.

[0096] Preferably, the capture surface comprises a capture molecule selected from the group comprising proteins, peptides, DNA, RNA, small molecules, preferably silanes, sugars, and protein immobilization tags.

[0097] It is possible for the capture molecule to specifically capture the first molecule, the second molecule, and / or the formed complex. For example, the formed complex may not occur in the individual molecules and may have a tertiary structure that is specifically recognized by the capture molecule, e.g., an antibody.

[0098] In another preferred embodiment, the present invention relates to a described method in which a molecular complex microarray is analyzed, measured, and / or characterized. This may involve, for example, measuring interactions or examining the function of the complex. The analysis of interactions may relate to the complex formation itself or to the resulting interactions with one or more other molecules.

[0099] An important application of the method according to the present invention is MHC or HLA screening. The presentation of peptides on the cell surface by MHC / HLA molecules is a critical component in immune responses against infections and also against cancer cells. Adaptive cell therapy provides a novel and effective method for the direct and personalized treatment of diseases. For example, a patient's T-cells are genetically modified with specific T-cell receptors (TCRs) capable of specifically recognizing a particular cancer, thereby triggering an immune response targeting the patient's tumor. Another method to achieve the same result is to deliver "TCR-bispecific" molecules to the patient, designed to establish contact between abnormal cell types and T-cells. In both of these therapeutic approaches, it must be ensured that the administered novel TCRs do not interact with healthy cells to trigger an autoimmune response.

[0100] By the method described above, it is possible to construct an MHC or HLA assay specifically designed to screen thousands of different MHC or HLA peptide combinations. These MHC or HLA peptide combinations are key to distinguishing the body's native cells from foreign or abnormal cells. They also serve as binding sites for TCR molecules. Before TCR-based therapy, TCRs must be screened in a high-throughput manner to ensure that they bind only to specific HLA-peptide combinations present on cancer cells and not to those found on healthy cells

[17] . Screening can be used, for example, to examine the efficacy and specificity of TCR candidates.

[0101] To perform this screening, thousands of different peptides are specifically and individually mixed with the same MHC or HLA molecule. Typically, the peptide is the first molecule to be introduced. However, it is also possible for the MHC / HLA to be the first molecule and the peptide to be added as the second molecule. This leads to the formation of complexes between the MHC / HLA and the peptide. The individual complexes formed are then immobilized on a capture surface to create a microarray. Subsequently, the microarray comes into contact with the TCR molecules to be analyzed. These may be dissolved as analytes or present on the cell or parts of the cell. Finally, the interactions between the TCR and the HLA-peptide complex can be analyzed.

[0102] For the method according to the present invention, HLA or MHC does not need to be specifically stabilized. Screening can be performed using native, modified, mutated, or stabilized MHC / HLA molecules. This is also possible, particularly because the present invention enables spatially separated pre-storage of stable and long-term storable complex partners. A less stable partner can be added as a second molecule immediately before use of the array, so that the entire complex is formed immediately without showing signs of degradation due to storage.

[0103] It is additionally preferable that MHC / HLA screening be performed using T cells or parts thereof instead of TCRs, wherein these T cells have corresponding TCRs on their surfaces.

[0104] In a further embodiment, complex formation is initially prevented because the first or second molecule exists as a complex with a temporary molecule. Preferably, the MHC is already connected to the temporary peptide. This temporary peptide is bound to a peptide-binding pit or pocket of the MHC, so that the MHC cannot accommodate another peptide. A signal is used to break this binding, and the MHC is ready to form a complex with the desired peptide.

[0105] Preferably, it is possible to use an MHC comprising a UV-cleavable peptide acting as a placeholder. This peptide is subsequently replaced by a target peptide in the method of the present invention. To this end, when both molecules (MHC and the target peptide) are provided, a UV light source is used to illuminate the chip. The UV light cleaves the placeholder, and the corresponding site becomes available for the target peptide to form a complex with the MHC. In this case, complex formation is activated by an additional signal, in this case, a UV signal. This embodiment is particularly suitable when using an unstabilized MHC.

[0106] In another embodiment, an improperly folded MHC is used. Folding occurs only in the presence of a peptide that binds to the peptide-binding pit / pocket.

[0107] All embodiments of the present invention are suitable for use with both MHC class I and MHC class II.

[0108] Another possible application of the present invention is research in the field of gene therapy, for example. Cas proteins (e.g., Cas9) provide the possibility of highly precise genome editing, which plays a particularly important role in the field of gene therapy. In the case of Cas9, the protein is programmed by two specific RNA molecules (tracrRNA and crRNA). This programming provides specificity that causes Cas9 to bind to specific gene loci. In this process, tracrRNA and crRNA can also be fused to form so-called guide or gRNA. An advantage is that Cas9 only needs to be linked to a single molecule to provide corresponding specificity. Particularly in the field of personalized gene therapy, there may be a need to test many different gRNA molecules to investigate specificity and non-target activity for corresponding gene loci. The goal is to minimize the side effects of gene therapy for each patient

[18] .

[0109] When many different gRNAs are combined with corresponding Cas proteins, this is referred to as multiplexed CRISPR application. A very wide range of applications has already been described in prior art. Gene editing and transcriptional regulation are always distinguished. In the former case, targeted cleavage (single or double strand breakage) is common, and in the latter case, Cas proteins bind to corresponding locus to affect gene regulation

[18] .

[0110] With the novel method according to the present invention, it is possible to construct a microarray containing many different gRNA-Cas protein complexes. With such an array, binding to specific DNA regions can be investigated on the one hand (e.g., non-targeted analysis). On the other hand, individual active regions can also be combined with cells to specifically modify or regulate genes in a high-throughput manner. For example, an array is also possible in which multiple Cas mutants are combined with the same gRNA to generate / screen improved Cas mutants or proteins having modified PAM (protospacer adjacent motif) sequence recognition.

[0111] All methods known in the prior art in the field of CRISPR are based on all-in libraries in tubes for pull-down approaches or in cells for cell-based readouts. However, CRISPR microarrays have not been described in the prior art.

[0112] Accordingly, the present invention provides for the first time a simple method for fabricating a microarray of a complex that does not require an amplification reaction and can also use a more unstable complex partner. Brief explanation of the drawing

[0113] In the following, the present invention will be described with reference to drawings and examples, but is not limited thereto. Fig. 1The present invention presents a preferred embodiment of the invention. In the presented drawings, a first surface having an independent cavity as an active region is used. A to E show how the first molecule may be present or introduced. This may be accomplished by spotting a liquid containing a pure molecule (A), by spotting a liquid containing a molecule having a specific immobilization tag (B), by synthesizing a molecule having a specific immobilization tag (C), by spotting / applying a particle (bead) on which a molecule having a specific immobilization tag is immobilized (D), or by closing the cavity with a DNA microarray (spotted, synthesized...) containing a spot of DNA encoding the first complex partner (E). If immobilization has not yet occurred (C and D), the first molecule is applied to the surface of the cavity in the next step and immobilized thereon. This can be achieved by drying the existing liquid (F), by specific immobilization via an immobilization tag and subsequent washing or drying of the chip (G), and by the expression of the DNA molecule and subsequent specific immobilization via an immobilization tag and subsequent washing or drying of the chip (H). In (I), a second molecule is filled into the cavity. Complex formation occurs within a closed cavity by rehydration of the molecule from step 1 (J) or by specific splitting of the immobilization tag of the first molecule from step 1 (K). A microarray is formed by capturing the generated complex on a capture surface and washing the surface, which can be further measured and characterized (L + M). The capture surface may be a second surface from step I or another surface. Fig. 2 This presents an additional preferred embodiment of the method according to the present invention. In this example, one array is fabricated by the synthesis or spotting of multiple different first molecules, which are peptides (A). Another array is fabricated by the spotting of multiple second molecules (in this case, MHC complexes) (B). Subsequently, the two arrays are brought into close contact in such a manner that liquid crosslinks are formed between the individual arrays. It is important that the individual liquid crosslinks do not touch each other so that the active regions remain independent (C). The molecules of the first array (A) are rehydrated or specifically split from the surface, for example, by light. Subsequently, two molecules from each array are mixed together through this contact, and an MHC-peptide complex is formed (D). Subsequently, the MHC-peptide complex can be captured. The result is a microarray of MHC-peptide complexes (E). Fig. 3 FIG. 3 illustrates the application of the method according to the present invention in combination with MHC screening. To perform this screening, thousands of different peptides are specifically and individually mixed with the same MHC molecule (A). This leads to the formation of complexes between the MHC and the peptides. For a better illustration, the figure presents this process in a simplified form rather than in a closed active region. Subsequently, the individual complexes are immobilized on a surface to form a microarray (B). The microarray then comes into contact with the TCR molecule to be analyzed (C). Finally, the interaction between the TCR and the MHC-peptide complex can be analyzed (D). Fig. 4 FIG. 4 illustrates a preferred embodiment of the method according to the present invention. In this case, a first molecule, which is a peptide, is spotted onto a chip, e.g., a PDMS chip (A). In step B, the immobilization of the peptide can be verified by drying. In this case, storage at 4°C for an extended period is possible (C). In step D, a second molecule, which is an MHC complex, is added. In step E, the cavity of the first surface is closed by the capture surface to create a closed active region, where an MHC-peptide complex is formed. These are captured by a capture molecule on the capture surface. In step F, a T cell receptor is added and its binding characteristics are analyzed. Fig. 5 FIG. 5 presents a different embodiment of the method according to the present invention. In step A, a first molecule is introduced into an active region (in this case, a cavity). This is done in the form of a droplet. In step B, fixation can be confirmed, in this case, achieved by drying. In the corresponding example, the surface loaded in this manner can be stored for a long time, preferably at 4°C (C). The second line presents various methods for applying a second molecule. In the examples, MHC is used as the second molecule. 1 suggests that the second molecule can be applied by a large droplet, so that multiple active regions are filled simultaneously. In this example, the cavity is overfilled to prevent air pockets. In 2, MHC is applied to individual active regions in a more targeted manner by a smaller droplet. Here, too, the cavity is overfilled in this example. In 3, MHC is applied to individual active regions in a more targeted manner by a smaller droplet, where the volume of the droplet is smaller than the volume of the cavity. Complex formation occurs in the active region. Subsequently, a capture surface is applied in all three examples. The last line describes how the complex binds to the capture surface, and in this case, the binding characteristics with the T cell receptor are examined. Fig. 6 FIG. 6 presents various results of the method according to FIG. 5, where FIG. 6.3.2 presents excellent results when the method according to the present invention is performed correctly. FIG. 6.2.2 also presents evaluable results, but there was cross-contamination with neighboring cavities. Nevertheless, the interaction with T-cell receptors is already measurable here. FIG. 6.3.1 and FIG. 6.3.2 present desirable results when the method according to the present invention is performed appropriately. Here, a clean cavity that prevents cross-contamination can be identified. The interaction with T-cell receptors can be well measured. Various experiments were performed using MHC as a secondary molecule. For this purpose, different MHCs were used, and peptide-MHC (pMHC) complex arrays were prepared using the method of the present invention. Subsequently, the arrays were rinsed with T cell receptors, and binding with pMHC was indicated. The examples presented below are intended to illustrate the invention and are not intended to limit the scope of the application. In particular, both MHC class 1 and MHC class 2 molecules are suitable. Assays using soluble T-cell receptor analytes are examples of the scope of application. It is also possible to have the array come into contact with a T cell or a part thereof and determine its interaction. Of course, completely different assays are also possible, in which case the array comes into contact with each different component or assay partner. Specific details for implementing the invention

[0114] To explain in detail:

[0115] Example 1

[0116] Perform the experiment using stabilized MHC that does not contain peptides in the peptide-binding pocket (source: Tetramershop).

[0117] Provides streptavidin-coated glass slides and cavity chips.

[0118] Streptavidin-coated glass slides are used for the immobilization of biotin-tagged ligands.

[0119] The cavity chip (BioCopy cavity chip) includes a small cavity used as a reagent container for pMHC complex formation.

[0120] The peptides used for the pMHC complex are printed onto a prepared cavity chip. These can now be stored until further use.

[0121] In the next step, MHC molecules are printed onto a prepared peptide chip. Subsequently, binding of the peptide to the binding pocket of the MHC follows. The complex formed in this manner is captured on a streptavidin-coated surface to achieve microarray formation.

[0122] After the incubation phase, the glass slide-chip sandwich can be separated, and the pMHC microarray is now ready for immediate use.

[0123] Arrays fabricated in this manner are tested by rinsing with T cell receptors. Binding of pMHC spots is indicated, which provides excellent results.

[0124] Example 2

[0125] Experiments are performed using unstabilized MHC containing UV-cleavable or UV-sensitive peptides (sources: e.g., Sanquin, Biolegend).

[0126] Provides streptavidin-coated glass slides and cavity chips.

[0127] Streptavidin-coated glass slides are used for the immobilization of biotin-tagged ligands.

[0128] A cavity chip (biokopi cavity chip) includes a small cavity used as a reagent container for pMHC complex formation.

[0129] The peptides used for the pMHC complex are printed onto a prepared cavity chip. These can now be stored until further use.

[0130] In the next step, MHC molecules are printed onto a prepared peptide chip. To exchange UV-cleavable peptides located on the unstabilized MHC, the chip is illuminated using a UV light source. UV cleavage results in the exchange of the cleaved peptide with the provided (printed) peptide.

[0131] After peptide exchange, the complex formed in this way is captured on a streptavidin-coated surface to achieve microarray formation.

[0132] After the incubation phase, the glass slide-chip sandwich can be separated, and the pMHC microarray is now ready for immediate use.

[0133] When an array prepared in this way is rinsed with a T cell receptor, it can be indicated for binding with pMHC, which provides excellent results.

[0134] Example 3

[0135] Perform experiments using unstabilized HLA that needs to be folded (source: e.g., Immunodex). Unloaded MHC does not fold correctly. Folding occurs only in the presence of peptides.

[0136] Provides streptavidin-coated glass slides and cavity chips.

[0137] Streptavidin-coated glass slides are used for the immobilization of biotin-tagged ligands.

[0138] A cavity chip (biokopi cavity chip) includes a small cavity used as a reagent container for pMHC complex formation.

[0139] The peptides used for the pMHC complex are printed onto a prepared cavity chip. These can now be stored until further use.

[0140] In the next step, MHC molecules are printed onto a prepared peptide chip. Folding then occurs, and peptides bind to pockets of MHC molecules to form pMHC complexes.

[0141] The formed complex is captured on a streptavidin-coated surface to achieve microarray formation.

[0142] After the incubation phase, the glass slide-chip sandwich can be separated, and the pMHC microarray is now ready for immediate use.

[0143] Arrays prepared in this manner are also tested by rinsing with T cell receptors. Binded pMHC spots can be displayed, which presents excellent results.

[0144]

[0145]

Claims

Claim 1 A method for fabricating a molecular complex microarray in situ comprising the following steps: a) providing a first surface comprising a plurality of independent active regions; b) introducing a first molecule into the plurality of active regions; c) immobilizing the provided molecule on the surface; d) adding a second molecule to each active region where the first molecule is present; e) closing the active regions with the second surface; f) forming a complex between the molecules; g) immobilizing the formed complex on a capture surface. Claim 2 A method in which the capture surface is a second surface in paragraph 1. Claim 3 A method according to claim 1 or 2, wherein the active area is a cavity or a spot. Claim 4 A method according to claim 1 or 2, wherein the first molecule introduced is fixed to a surface in step c) by adsorption, by ionic interaction, by van der Waals force, by specific chemical reaction and / or by drying. Claim 5 In paragraph 1 or 2, A second molecule is added to the first molecule, or A method in which a second molecule is present on a second surface, and contact between an active region containing a first molecule and a second molecule is established through liquid crosslinking. Claim 6 A method according to claim 1 or 2, wherein the formation of the complex is made possible by the release of the first molecule. Claim 7 A method according to claim 1 or 2 in which the formation of a complex is initially prevented because the first or second molecule exists as a complex with a temporary molecule. Claim 8 A method according to claim 1 or 2 in which the complex formation is activated by a signal. Claim 9 In paragraph 8, the method in which the signal is a UV light signal. Claim 10 In paragraph 8, a method in which the signal separates the formation of a complex with a temporary molecule. Claim 11 A method according to claim 1 or 2, wherein the introduction of the first molecule into the active region of the first surface is achieved by one of the following methods: a. spotting of a liquid containing the first molecule, b. synthesis of the first molecule, c. application of a particle containing the first molecule, and / or d. establishment of contact between the active region of the first surface and a DNA microarray containing a spot of DNA encoding the first molecule. Claim 12 A method according to claim 1 or 2, wherein the first and / or second molecules comprise an immobilized tag. Claim 13 A method according to claim 1 or 2, wherein the first and / or second molecule is selected from the group comprising proteins, peptides, DNA, RNA, small molecules, cells, CRISPR-associated proteins and their mutants, gRNA, proteins of major histocompatibility complex classes and their mutants, proteins of antibody classes, T-lymphocytes, and B-lymphocytes. Claim 14 The method of claim 1 or 2, wherein the capture surface comprises a capture molecule selected from the group comprising: proteins, peptides, DNA, RNA, small molecules, silanes, sugars, and protein immobilization tags. Claim 15 A method according to claim 1 or 2 in which a molecular complex microarray is analyzed, measured and / or characterized. Claim 16 A method according to claim 15, wherein a molecular complex microarray comes into contact with a T cell receptor, a T cell, or a part thereof, and the interaction between the molecular complex and the T cell receptor, the T cell, or a part thereof is analyzed.

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