Parallel drug screening method using id-microparticles bound to target

Abstract

The present invention relates to a parallel drug screening method using ID-microparticles bound to a target. The present invention enables multiple target screening for a specific disease and thus has an advantage whereby costs and time may be saved as much as the number of target types compared to the existing DEL method. In addition, whereas the existing DEL method has a limitation in that a target is fixed to a surface of a solid support, thereby resulting in structural change and a specific direction being hidden, a target encapsulated in a hydrogel or bound to the hydrogel in a random direction may overcome such limitation.

Description

Parallel drug screening method using target-coupled ID-microparticles

[0001] A parallel drug screening method using target-coupled ID-microparticles is disclosed.

[0002] In the early stages of new drug development, identifying candidates that react with the target among numerous small molecule compounds is a crucial step that can increase the success rate of new drug development, but it requires significant time and cost. Traditional screening technologies such as high-throughput screening (HTS), virtual screening, phenotypic screening (VSPS), and fragment-based lead discovery (FBLD) have been developed to identify effective compounds. Recently, innovative methods such as DNA-Encoded Libraries (DELs), antibody-drug conjugates (ADCs), CRISPR-Cas9, and CAR-T have been utilized to screen candidates more quickly and at lower cost.

[0003] Among these, High-throughput screening (HTS) is currently the most widely used technology in companies to identify effective compounds. HTS is frequently used to evaluate thousands to millions of small molecule compounds against target proteins. However, effectively utilizing HTS in new drug development requires individual synthesis and testing of thousands to millions of compounds, which necessitates significant costs and systems for utilization and management. Furthermore, even with a library of one million compounds—the largest library currently available using this technology—the number is still insufficient to identify high-quality ligands for target proteins. While computational approaches like VSPS have been proposed as an alternative, their ability to reliably predict ligand binding has limitations. Therefore, DNA-Encoded Library (DEL) technology has been proposed as a method for generating a significantly larger library of compounds. Comparing the characteristics of conventional and DNA-encoded libraries, DEL has been reported to be more economical and efficient than conventional methods in terms of size, construction time, cost, screening time, and storage methods.

[0004] DEL was first introduced by Brenner & Lerner in 1992.11 S. Brenner and RA Lerner. Encoded combinatorial chemistry. Proc. Natl. Acad. Scie. USA, 1992, 89, 5381-5383. Unlike HTS, which physically separates each compound into 384 or 1536 wells to find hit ligands, this paper conceived the idea that if unique information could be encoded into each compound using DNA, screening would be possible in a single tube without physically dividing the space. The paper presented the core ideas that form the basis of DEL technology, such as a DEL synthesis method using the split and pool technique, an affinity-mediated selection method, and a method of reading the information of the combined compounds through PCR sequencing.

[0005] Referring to Figure 1, DEL technology can be broadly divided into two steps: DEL synthesis and DEL screening. First, DEL synthesis involves placing oligonucleotides containing a chemical linker portion into a 96-well plate, inserting different building blocks into each well, combining them, and extending corresponding short DNA barcodes (~6 nt) to the ends of the oligonucleotides. These are combined into one (pool), then dispensed back into the wells (split), and the process of combining building blocks and DNA barcodes is repeated three times. In theory, 96 x 96 x 96 = 884,736 libraries can be produced (see Figure 2). DEL synthesis utilizing the above split and pool method can synthesize a large amount of libraries in a short period of time. However, since the process of combining building blocks is mainly organic solvent-based, there is a problem that needs to be solved in that it does not conflict with the process of extending DNA barcodes in an aqueous solution using enzymes. The DEL screening process involves immobilizing purified target proteins on a solid support (beads, resin, etc.), reacting them with DEL in a single tube, leaving only compounds bound to the target protein, washing away unreacted compounds (affinity selection), and then amplifying and sequencing the DNA barcodes of the bound compounds to confirm their structures. While it offers the advantage of significantly reducing time and cost compared to existing HTS methods, the fact that most DEL screening technologies to date use affinity selection methods using immobilized proteins remains a challenge. Membrane proteins, live cells, and protein structures can lose their biological properties and be damaged during the protein purification and immobilization process, so there is a growing need for technological advancements that enable screening without purification or immobilization steps, if possible.

[0006] To overcome the limitations of existing DEL screening techniques, active research has recently been focused on technologies that allow proteins to be reacted in solution rather than immobilized on a solid support. In 2010, Professor Liu's research team at Harvard University presented IDPCR (Interaction-Dependent PCR), a solution-based screening method.

[0007] Referring to Figure 3, a key element of IDPCR technology is attaching a unique DNA tag to the target protein, allowing multiple proteins to be distinguished from each other within a single tube.

[0008] When a DNA-linked ligand binds to a DNA-linked protein, it induces hybridization of a short (6 nt) complementary DNA sequence. This hybridized region is extended to form a double-stranded DNA. When PCR is performed, only the complex of the ligand and protein bound to it contains both primer binding sequences, so it is selectively amplified. The structure of the bound compound can be confirmed through NGS. Screening using IDPCR showed that DNA encoding all five known protein-ligand pairs was amplified out of 67,599 possible sequences. This technology is evaluated to have pioneered the development of solution-based DEL screening technology by enabling DEL screening without immobilizing the protein on a solid support. However, there is a limitation in that purified proteins must be used because DNA tags must be attached to the target protein.

[0009] Accordingly, in 2014, the same research team proposed IDUP (Interaction determination using unpurified proteins) technology that enables screening in cell lysates to demonstrate that the above method can be applied even to complex biological samples. Referring to Figure 4, the technology is a method of encoding a target protein using DNA-linked antibodies in cell lysates, rather than directly modifying DNA tags on the surface of purified proteins. It was suggested that protein labeling is possible using not only antibodies but also self-labeling protein tags such as SNAP-tag, CLIP-tag, and HaloTag. Although screening results were shown using cell lysate, which is an actual biological sample, it is difficult to screen target proteins for which monoclonal antibodies do not exist, and it can be predicted that the cost of synthesizing DEL using antibodies will be considerable. The method of using self-labeling protein tags rather than antibodies also has a limited number of applicable proteins.

[0010] In addition, in 2015, the Danish pharmaceutical company Vipergen proposed BTE (Binder trap enrichment) technology as a method for in-solution DEL screening. Referring to Figure 5, BTE uses water-in-oil technology to trap binders that have bound to water droplets dispersed in oil. This method is characterized by low noise because amplification occurs within the prepared droplets during PCR in solution. However, similar to the limitations of the IDPCR technology previously proposed by the Liu group, since this method distinguishes by modifying DNA tags in the target protein, it is still an issue in that complex biological samples cannot be analyzed and purified proteins must be used.

[0011] In addition, a photo-crosslinking-based method was proposed by the Li group at Peking University in 2014. Referring to Figure 6, compared to previously proposed solution-based screening technologies such as IDPCR, IDUP, and BTE, this technology is characterized by its applicability to biological samples such as cell lysates because it does not directly modify the target protein. In the photo-crosslinking-based technology, a DNA-encoded small molecule compound hybridizes with a short 8-nt DNA strand (PC-DNA) containing a 5'-azidophenyl group at a specific temperature. After binding to the target, when light is irradiated, a DNA hairpin structure is formed, and the DNA-endcoded small molecule compound bound to the target forms a stable bond, and those that are not bound are degraded by exonuclease activity, so that only the bound structures can be amplified by PCR and sequenced.

[0012] In addition, in 2016, the Krusemark group at Purdue University proposed a crosslinking-based screening technology using reactive groups. Referring to Figure 7, the previously proposed photo-crosslinking-based technology of the Li group suggested that DEL is in the form of ssDNA, but there were limitations in its application because most DELs are synthesized in the form of dsDNA, which has a more stable structure. Therefore, the Krusemark group used a method of attaching ssDNA to the end of DEL in the form of dsDNA and covalently bonding the target protein and ligand using the reactive group to stabilize it. After crosslinking, the protein is denatured, and the complex is captured with an affinity tag such as biotin, washed, and then subjected to qPCR or DNA sequencing. In addition, in 2018, the Krylov group at York University proposed NECEEM (Nonequilibrium capillary electrophoresis of equilibrium mixtures) as a method for distinguishing DEL bound to a target in solution. Referring to Figure 8, the concept is that DEL can be separated due to the different electrophoretic mobilities of protein-bound and unbound structures. However, this method has only been tested in model systems, and its effectiveness on large-scale DEL has not yet been verified.

[0013] In the case of the HTS method, it is based on a 1:1 combination of the target and the compound, so it has the disadvantage of requiring a long screening time and being very expensive. In the case of the DEL method, it is based on a 1:n combination of the target and the compound, so it still has the disadvantage of requiring high costs.

[0014] Accordingly, the inventors of the present invention propose a method for innovatively reducing screening time based on the M:N combination of the target and the compound.

[0015] Accordingly, the present invention aims to provide a parallel drug screening method using ID-microparticles combined with a target.

[0016] Accordingly, the present invention comprises the steps of (1) reacting a microparticle library including a target molecule and ID information capable of distinguishing the type of the target molecule with a compound library combined with a DNA barcode capable of distinguishing the type of compound;

[0017] (2) When the target molecule and a specific compound are combined, a step of separating microparticles containing the combined target molecule and the specific compound; and

[0018] (3) A parallel drug screening method is provided, including a step of confirming the ID information of the microparticles separated in the above step (2) and the DNA barcode of the compound.

[0019] According to one embodiment of the present invention, the target molecule may be encapsulated in microparticles.

[0020] According to one embodiment of the present invention, the target molecule may form a chemical bond with the microparticle. The chemical bond may be a hydrogen bond or a covalent bond.

[0021] According to one embodiment of the present invention, the ID information may be formed using at least one of a QR code, an image of a microparticle, a superparamagnetic alignment axis pattern of a microparticle, and a barcode.

[0022] According to one embodiment of the present invention, the step of confirming in step (3) may be to align microparticles containing the compound separated in step (2) and the target molecule bound to the compound on a support, and then confirm the ID information of the microparticles and the DNA barcode information of the compound.

[0023] According to one embodiment of the present invention, the DNA barcode information of the compound may be confirmed in situ so that the positional information of the microparticle and the compound can be maintained.

[0024] According to one embodiment of the present invention, after step (2), a step of adding location information to the DNA barcode information of the compound may be further included.

[0025] According to one embodiment of the present invention, the step of adding the location information may be adding a nucleic acid molecule including the location information to the DNA barcode of the compound.

[0026] According to one embodiment of the present invention, a method for adding a nucleic acid molecule including the location information to a DNA barcode of the compound is provided.

[0027] The 3' terminal portion of the DNA barcode of the compound may include a nucleic acid sequence complementary to the 3' terminal nucleic acid sequence of the nucleic acid molecule including the positional information, and the 3' terminal of the DNA barcode of the compound and the 3' terminal of the nucleic acid molecule may be hybridized, and the sequence may be extended by DNA polymerase.

[0028] According to one embodiment of the present invention, a method for adding a nucleic acid molecule including the location information to a DNA barcode of the compound is provided.

[0029] The method may be such that the end of the DNA barcode of the above compound is extended by splint ligation to the end of a nucleic acid molecule containing the positional information.

[0030] According to one embodiment of the present invention, the support may be a hydrogel, and the support may be a hydrogel in which nucleic acid molecules containing positional information are clustered. In addition, the support may further include parallel partitions separating the hydrogels in which nucleic acid molecules containing positional information are clustered.

[0031] According to one embodiment of the present invention, the hydrogel comprises a DNA barcode S3;

[0032] H2 comprising a nucleic acid sequence S2 whose complementary sequence at the 3' terminal portion can act as a primer;

[0033] and a nucleic acid sequence H1 comprising a nucleic acid sequence S1 capable of acting as a primer at the 5' terminal region;

[0034] A hydrogel having at least one of the nucleic acid sequence S4 and its complementary nucleic acid sequence S4` fixed thereon,

[0035] The above nucleic acid sequence S4 or its complementary nucleic acid sequence S4' may be a hydrogel characterized in that nucleic acid sequences having the same DNA barcode S3 or its complementary DNA barcode S3' are clustered together and positioned at a predetermined distance from each other.

[0036] According to one embodiment of the present invention, the hydrogel comprises a DNA barcode S3;

[0037] H2 comprising a nucleic acid sequence S2 whose complementary sequence at the 3' terminal portion can act as a primer;

[0038] and a nucleic acid sequence H1 comprising a nucleic acid sequence S1 capable of acting as a primer at the 5' terminal region;

[0039] A hydrogel having at least one of the nucleic acid sequence S4 and its complementary nucleic acid sequence S4` fixed thereon,

[0040] The above nucleic acid sequence S4 or its complementary nucleic acid sequence S4' may be a hydrogel characterized in that nucleic acid sequences having the same DNA barcode S3 or its complementary DNA barcode S3' are clustered together and positioned spaced apart from each other in the microwells formed by the parallel partitions.

[0041] The present invention relates to a parallel drug screening method using ID-microparticles combined with targets. The present invention enables screening of multiple targets for a specific disease, and thus has the advantage of saving cost and time equivalent to the number of target types compared to the existing DEL method.

[0042] In addition, compared to the existing DEL method, which had limitations in that the target was fixed to the surface of a solid support, causing structural changes and a specific direction to be hidden, a target encapsulated in a hydrogel or bound to a hydrogel in a random direction can overcome the above limitations.

[0043] Figures 1 and 2 are drawings for explaining DEL technology.

[0044] Figure 3 is a drawing for explaining DPCR (Interaction-Dependent PCR) technology.

[0045] Figure 4 is a diagram for explaining IDUP (Interaction determination using unpurified proteins) technology.

[0046] Figure 5 is a drawing for explaining BTE (Binder trap enrichment) technology.

[0047] Figure 6 is a diagram for explaining photo-crosslinking-based methods.

[0048] Figure 7 is a diagram illustrating a cross-linking-based screening technique using a reactor group.

[0049] Figure 8 is a diagram for explaining NECEEM (Nonequilibrium capillary electrophoresis of equilibrium mixtures).

[0050] FIG. 9 and FIG. 10 are drawings for explaining the types of ID particles of the present invention.

[0051] Figure 11 is a drawing for explaining 'stop-flow maskless lithography' for QR-code generation.

[0052] Figures 12 to 15 are drawings for explaining ID-particles using superparamagnetic patterns.

[0053] Figure 16 is a drawing for explaining a technique for binding an antibody to the surface of a poly-(ethylene glycol) diacrylate (PEG-DA) hydrogel particle.

[0054] Figures 17 and 18 are drawings for explaining a technique for encapsulating a target within a hydrogel.

[0055] Figure 19 is a diagram showing examples of microparticles, including a. QR-codes, b. hydrogel microparticles that can be distinguished by an alignment axis, including superparamagnetic nanoparticles.

[0056] Figure 20 is a drawing showing a method of applying a QR code to microparticles.

[0057] Figure 21 is a drawing showing a QR code aligned on a slide glass.

[0058] Figure 22 is a drawing showing a process of generating n2 barcodes by combining microfluidic channels in a hydrogel with a parallel partition structure, introducing n types of X barcodes and n types of Y barcodes in the X and Y axes, and ligating them.

[0059] Figure 23 is a drawing showing a state in which a QR code is placed on a hydrogel assay.

[0060] Figure 24 is a diagram illustrating a spatial transcriptome analysis technique.

[0061] Figure 25 relates to a method of using a DNA barcode as ID information and combining it with a hydrogel having a parallel bulkhead structure.

[0062] FIG. 26 relates to a method of using a hydrogel including an ID-particle containing ID information and a DNA barcode based on location information.

[0063] Figures 27 to 30 relate to a method for replicating a DNA barcode based on location information within a hydrogel.

[0064] FIG. 31 and FIG. 32 relate to a method of applying the ID particle of the present invention into a hydrogel containing DNA position information.

[0065] Figure 33 is a diagram showing an example of analyzing a DEL sequence bound to a compound.

[0066] Figure 34 is a diagram showing an embodiment of the screening method of the present invention.

[0067] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below, along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention.

[0068] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components. The same reference numerals refer to the same components throughout the specification, and "and / or" includes each and every combination of one or more of the mentioned components.

[0069] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0070] In the present invention, the nucleic acid sequence can be used interchangeably with a nucleic acid molecule, a polynucleotide sequence, etc.

[0071] Hereinafter, the present invention will be described in detail with reference to the attached drawings.

[0072] Figures 9 and 10 are drawings for explaining the types of ID particles of the present invention. First, the term "ID particle" of the present invention may refer to a microparticle that holds ID information. The term "microparticle" of the present invention may be a hydrogel microparticle using various materials such as poly-AMPS, poly-SS, poly-DADMAC, poly-AA, poly-PEG, etc., but is not limited thereto.

[0073] The term "library" in the present invention means a cluster or collection of specific molecules, and a microparticle library means a cluster or collection of microparticles having various target molecules and ID information.

[0074] In the present invention, the term "hydrogel" generally refers to a material having a three-dimensional hydrophilic polymer network structure capable of containing a large amount of water. Hydrogels can absorb at least 20% of their total weight of water, and those that absorb more than 95% of water are called highly absorbent hydrogels. Hydrogels are composed of homopolymers or copolymers and form a structurally stable three-dimensional network structure with little fluidity due to external stress. This structure is formed by various factors such as covalent bonds, hydrogen bonds, van der Waals bonds, or physical cohesion. After swelling in an aqueous solution, they remain thermodynamically stable and possess mechanical and physicochemical properties that correspond to intermediate states between liquid and solid. Furthermore, the swelling degree of a hydrogel can be controlled by the chemical structure and hydrophilicity of the polymer, as well as the degree of crosslinking between polymer chains. Therefore, hydrogels with various shapes and properties can be manufactured depending on the components and manufacturing method.

[0075] The hydrogel of the present invention may include a polyacrylamide-based hydrogel such as a polyacrylamide hydrogel, a polyvinyl alcohol / polyacrylamide hydrogel, a gelatin-based hydrogel, an agarose-based hydrogel, etc. The polyacrylamide-based hydrogel has a relatively high density of a polymer network constituting the material, and exhibits excellent mechanical properties of the acrylamide monomer itself constituting the polymer. Gelatin-based hydrogels with little shape deformation may be gelatin hydrogel, polyvinyl alcohol (PVA) / gelatin hydrogel, sericin / gelatin hydrogel, polyvinyl alcohol / sericin / gelatin hydrogel, fibrinogen / gelatin hydrogel, hyaluronan / gelatin hydrogel, alginate / fibrinogel / gelatin hydrogel, etc. The agarose-based hydrogel may be agarose hydrogel, sodium alginate / agarose hydrogel, polyacrylamide / agarose hydrogel, collagen / agarose hydrogel, collagen / alginate / agarose hydrogel, etc.

[0076] The term "ID information" of the present invention refers to information contained within microparticles that can distinguish each target. Specifically, it may be, but is not limited to, 1) a QR code, 2) a superparamagnetic alignment axis pattern of a particle, 3) an image of a particle, or 4) a barcode.

[0077] The term "target molecule" of the present invention may be, but is not limited to, a protein associated with a specific disease.

[0078] Fig. 11 is a drawing for explaining 'stop-flow maskless lithography' for generating a QR code, and a QR code can be generated using the above method on microparticles.

[0079] Figures 12 to 15 are drawings for explaining ID particles using a superparamagnetic pattern. Referring to registered patent No. 10-2036434, ID information can be included in microparticles by using a superparamagnetic alignment axis pattern on the microparticles.

[0080] In the present invention, the target molecule and the microparticle may be 1) encapsulated within the microparticle, or 2) bonded, and the bond may be a covalent bond or a hydrogen bond.

[0081] Referring to Figure 16, a technique for binding antibodies to the surface of poly-(ethylene glycol) diacrylate (PEG-DA) hydrogel particles can be used to bind target and ID particles. More specifically, since the direction in which antibodies bind to the hydrogel particle surface is random, a sufficiently large number of antibodies can bind in an appropriate direction with their binding sites exposed. By binding antibodies of this small size, antigen specificity can be confirmed.

[0082] Referring to FIGS. 17 and 18, a target can be positioned within a porous hydrogel. More specifically, the microparticles can be hydrogels, and when encapsulated within the hydrogel pores or fixed to the linear polymer backbone of the hydrogel surface, proteins can be introduced in any direction, thereby ensuring that all surfaces of a type of protein introduced into the hydrogel are evenly exposed, thereby maximizing the interaction efficiency with the compound. A method for encapsulating a target within the hydrogel pores can be implemented in the form of microparticles through stop-flow lithography photopolymerization.

[0083] Fig. 19 is a diagram showing an embodiment of a drug screening method according to the present invention. Referring to Fig. 19, the parallel target screening (whole target screening) technology proposed in the present invention relates to a method of encapsulating or binding a target to a microparticle having an ID that can distinguish each target, preferably a hydrogel particle, and mixing and reacting the target with compounds of a compound library containing a DNA barcode in a reaction chamber in one pot. Each ID particle of the present invention can bind to one type of target, but each ID particle can bind to multiple or more types of targets.

[0084] Additionally, in the present invention, the compound library may refer to a library of compounds coupled with a DNA barcode, and it is preferable to match one type of compound with one DNA barcode. For example, compound A may be linked to the "AGCGT" sequence, and compound B may be linked to the "ACGAT" sequence, and the two compounds may be distinguished from each other by the DNA barcode. The connection between the DNA barcode and the compound may be via a linker, and the linker may be a photodegradable linker, but is not limited thereto, and any known linker may be used.

[0085] The term "DNA barcode" of the present invention may be an oligonucleotide having a base sequence of 3 to 22 nt in length for distinguishing the location of the originating cell or tissue of a transcript (mRNA) in the gene expression analysis results obtained after pooling multiple cells. In addition, the "DNA barcode" may be a method for detecting a biological target substance, such as a magnetic particle for separation having a probe complementary to the target substance attached to the surface, and DNA having a predetermined arrangement as an identification code. The method for generating a DNA barcode containing the location information of the present invention is described in detail in Republic of Korea Patent Application No. 10-2021-0128780, and may be generated using other known methods.

[0086] Figure 20 illustrates a method for identifying the type of target by identifying the ID information within the ID particle. That is, if one type of ID particle and one type of target are matched, the target can be identified by checking the QR code, image, superparamagnetic alignment axis pattern, barcode, etc.

[0087] Figure 21 is a diagram showing the process after the one-pot essay.

[0088] Referring to FIG. 21, the "one-pot assay" of the present invention refers to a method of reacting a target combined with an ID particle and a compound library combined with a specific barcode at once.

[0089] After the above one-pot assay is completed, the unbound compound library can be washed, and the ID particles can be positioned or aligned on a slide glass. In one embodiment, a QR code, each uniquely bound to a target, created using stop-flow lithography can be constructed at a sub-micron scale.

[0090] Referring to Fig. 21, it shows that the width and length are each 100 μm. If a QR code of this size is spread out as a single layer so that it self-aligns on a slide glass, 250 x 750 = 187,500 can be loaded simultaneously, as shown on the left side of Fig. 21. This is a sufficient number to analyze all existing targets at once, and therefore the screening method proposed in the present invention is called parallel target screening or "whole target screening." As described above, it is possible to know which target is located at which position from the QR-code image loaded on the slide. At this time, when there is a compound library (a compound bound to a DNA barcode; DNA encoded library) that hits the bound target as shown in the enlarged picture of the hydrogel polymer backbone of the QR-code, this is amplified by RCA (rolling circle amplification) and then the sequence is analyzed by performing fluorescent in-situ sequencing, thereby finding out which compound can act as an effective substance on which target at which position. Additionally, as shown on the right side of Figure 21, DNA can be recovered from a compound in which a hit has occurred and its sequence can be revealed by performing NGS (next generation sequencing) to identify the compound.

[0091] Figures 22 and 23 relate to a hydrogel of parallel bulkheads having positional information.

[0092] Referring to FIGS. 22 and 23, in the case of the existing 10x Visium method, the DNA barcode is fixed to the slide glass, so it cannot freely interact with the mRNA, and the capture efficiency is only a few %. In order to overcome this limitation, by encoding the barcode in combination with a hydrogel having a parallel partition structure, not only a DNA barcode array is constructed at a low cost, but also the DNA barcode is distributed in a 3-D hydrogel rather than a flat surface, so that a large amount can be loaded (see Korean Application No. 10-2021-0128780). Therefore, by combining a microfluidic channel with the hydrogel having the parallel partition structure and introducing n types of X barcodes and n types of Y barcodes in the X and Y axes, respectively, and ligating them, n2 barcodes can be generated.

[0093] Figures 24 to 26 are enlarged views of the inside of the parallel bulkhead after the X, Y axis barcodes are introduced, and the tissue is placed on the parallel bulkhead hydrogel barcode array for analysis. Using the above technology, it is possible to know which gene is expressed in which cell at which location within the tissue and to what extent. In the present invention, by applying this technology, it is possible to analyze which DNA barcode is located at which location in the QR code array described above. In particular, the QR code can be configured to self-align with the parallel bulkhead hydrogel barcode array, so that, for example, one QR code can be located in a 10x10 array as shown on the right side of the figure above. The parallel bulkhead hydrogel DNA barcode can include a poly-T tail to capture the poly-A tail of mRNA for spatial transcriptome analysis. When configuring the DEL, if the last part has poly-A, the two pieces of information can be combined by hybridizing with the parallel bulkhead hydrogel DNA barcode. This combination does not necessarily have to be poly-T and poly-A, but only if it consists of complementary sequences that can hybridize to a sufficient length on both sides. Through this, the X, Y-axis-specific barcode information can be combined with the DNA barcode of the compound that hit the target bound to each QR-code, and even if all the DNA is diluted and mixed and the sequence is analyzed by NGS, the DNA barcode and X, Y positions are read at the same time, so the information may not be mixed. If NGS is performed directly without the above process, when the DNA barcode bound to the compound that created a hit on the target is eluted, there is a limitation in that it is not possible to know which QR-code the DNA came from, that is, the position information is lost, and it is not possible to know which target the compound that was created hit.

[0094] Accordingly, in the present invention, the positional information of each DNA barcode can be maintained by applying spatial transcriptomics technology. According to one embodiment of the present invention, a hydrogel having positional information can be used. The hydrogel of the present invention is a hydrogel having at least one of a DNA barcode S3; a nucleic acid sequence H2 including a nucleic acid sequence S2 whose complementary sequence at the 3' end can act as a primer; and a nucleic acid sequence H1 including a nucleic acid sequence S1 which can act as a primer at the 5' end; and a nucleic acid sequence S4 including a nucleic acid sequence S4' complementary thereto, wherein the nucleic acid sequence S4 or the nucleic acid sequence S4' complementary thereto may be a hydrogel characterized in that nucleic acid sequences having the same DNA barcode S3 or the DNA barcode S3' complementary thereto are clustered together and positioned at a predetermined distance apart from each other, and may be positioned at a distance apart from each other in microwells forming parallel partitions.

[0095] FIGS. 27 to 32 relate to a method for replicating a DNA barcode based on positional information within a hydrogel, and as a method for clustering nucleic acid molecules having positional information within the hydrogel, the nucleic acid molecules having the positional information can be used to identify the reacted ID particles and compounds after a one-pot reaction using the hydrogel in which the nucleic acid molecules having the positional information are clustered within the hydrogel. That is, after positioning a support on which an identified compound and an ID-microparticle bound to the compound are positioned on the hydrogel in which the nucleic acid molecules having the positional information are clustered, the ID of the ID-microparticle is read, and then the DNA barcode of the compound is read, thereby identifying the target and the compound.

[0096] Therefore, the N:M reaction screening method of the target and compound of the present invention may require a step of aligning the specific compound and the id-particle after the reaction or binding with the id-particle to a support.

[0097] In addition, after the above alignment, it may be necessary to confirm 1) ID information of the ID-particle, 2) barcode information of the compound, and 3) location information on the support of the compound and ID-particle.

[0098] (1) According to one embodiment of the present invention, when the ID information of the ID-particle is in the form of a QR-code, after the one-pot reaction, the compound reacted on the support and the ID-particle are aligned, and then the QR-code is checked to confirm the location and type of the target. In addition, by using in-situ sequencing, the barcode of the compound can be checked to confirm which compound binds to which target. However, since the efficiency of the in-situ sequencing is not good, another method may be required.

[0099] (2) Therefore, according to one embodiment of the present invention, when using the QR code, after aligning the compound and ID particle reacted on the support, the QR code is checked to confirm the location and type of the target, and the location information is added to the barcode of the compound to confirm the location and type of the compound. Therefore, by matching the location of the target and the location of the compound, it is possible to confirm which target is combined with which compound. The method of assigning location information to the barcode information has been described above.

[0100] (3) In addition, according to one embodiment of the present invention, when the ID information of the ID-particle is a superparamagnetic alignment axis pattern, after the one-pot reaction, the compound reacted on the support and the ID-particle are aligned, and then a magnetic field is applied to confirm the superparamagnetic alignment axis pattern, thereby confirming the location and type of the target. In addition, by using in-situ sequencing, the barcode of the compound can be confirmed, thereby confirming which compound is bound to which target.

[0101] (4) According to one embodiment of the present invention, when using the superparamagnetic alignment axis pattern, after aligning the compound and ID-particle reacted on the support, a magnetic field is applied, and the superparamagnetic alignment axis pattern is confirmed to confirm the position and type of the target, and by adding position information to the barcode of the compound, the position and type of the compound can be confirmed. Therefore, by matching the position of the target and the position of the compound, it is possible to confirm which target is bound to which compound.

[0102] (5) In addition, according to one embodiment of the present invention, in the case of a barcode of an ID particle, after a one-pot reaction, the compound reacted on the support and the ID particle are aligned, and then the image is checked to confirm the location and type of the target. In addition, by using in-situ sequencing, the barcode of the compound can be confirmed to confirm which compound binds to which target.

[0103] (6) According to one embodiment of the present invention, when the ID information of the ID particle is a barcode, after aligning the compound and the ID particle reacted on the support, the image is checked to confirm the location and type of the target, and by adding the location information to the barcode of the compound, the location and type of the compound can be confirmed. Therefore, by matching the location of the target and the location of the compound, it is possible to confirm which target is bound to which compound.

Claims

1. (1) A step of reacting a microparticle library including a target molecule and ID information capable of distinguishing the type of the target molecule with a compound library combined with a DNA barcode capable of distinguishing the type of compound; (2) When the target molecule and a specific compound are combined, a step of separating microparticles containing the combined target molecule and the specific compound; and (3) A parallel drug screening method comprising a step of confirming the ID information of the microparticles separated in step (2) and the DNA barcode of the compound.

2. A method according to claim 1, wherein the target molecule is encapsulated in microparticles.

3. A method according to claim 1, wherein the target molecule forms a chemical bond with a microparticle.

4. A method according to claim 3, wherein the chemical bond is a hydrogen bond or a covalent bond.

5. A method according to claim 1, wherein the ID information uses at least one of a QR code, an image of a microparticle, a superparamagnetic alignment axis pattern of a microparticle, and a barcode.

6. In the first paragraph, the step of verifying in step (3) is a method in which the microparticles containing the compound separated in step (2) and the target molecule bound to the compound are aligned on a support, and then the ID information of the microparticles and the DNA barcode information of the compound are verified.

7. In the 6th paragraph, the DNA barcode information of the compound is a method for confirming the DNA barcode information of the compound in situ so that the location information of the microparticle and the compound can be maintained.

8. A method according to claim 1, further comprising a step of adding location information to the DNA barcode information of the compound after step (2).

9. A method according to claim 8, wherein the step of adding the location information is to add a nucleic acid molecule including the location information to the DNA barcode of the compound.

10. In the 9th paragraph, the method for adding a nucleic acid molecule including the position information to the DNA barcode of the compound is a method in which the 3' terminal portion of the DNA barcode of the compound includes a nucleic acid sequence complementary to the 3' terminal nucleic acid sequence of the nucleic acid molecule including the position information, and the 3' terminal of the DNA barcode of the compound and the 3' terminal of the nucleic acid molecule hybridize, so that the sequence is extended by a DNA polymerase.

11. In the 9th paragraph, the method of adding a nucleic acid molecule including the location information to the DNA barcode of the compound is as follows: A method wherein the end of the DNA barcode of the above compound is extended by splint ligation to the end of a nucleic acid molecule containing the positional information.

12. A method according to claim 6, wherein the support is a hydrogel.

13. A method according to claim 12, wherein the support is a hydrogel in which nucleic acid molecules containing positional information are clustered.

14. A method according to claim 13, wherein the support further comprises parallel partitions separating hydrogels in which nucleic acid molecules containing positional information are clustered.

15. In the 13th paragraph, the hydrogel comprises DNA barcode S3; H2 comprising a nucleic acid sequence S2 whose complementary sequence at the 3' terminal portion can act as a primer; and a nucleic acid sequence H1 comprising a nucleic acid sequence S1 capable of acting as a primer at the 5' terminal region; A hydrogel having at least one of the nucleic acid sequence S4 and its complementary nucleic acid sequence S4` fixed thereon, A method, wherein the nucleic acid sequence S4 or its complementary nucleic acid sequence S4' is a hydrogel characterized in that the nucleic acid sequences having the same DNA barcode S3 or its complementary DNA barcode S3' are clustered together and positioned at a predetermined distance from each other.

16. In the 14th paragraph, the hydrogel comprises DNA barcode S3; H2 comprising a nucleic acid sequence S2 whose complementary sequence at the 3' terminal portion can act as a primer; and a nucleic acid sequence H1 comprising a nucleic acid sequence S1 capable of acting as a primer at the 5' terminal region; A hydrogel having at least one of the nucleic acid sequence S4 and its complementary nucleic acid sequence S4` fixed thereon, A method, wherein the nucleic acid sequence S4 or its complementary nucleic acid sequence S4' is a hydrogel characterized in that the nucleic acid sequences having the same DNA barcode S3 or its complementary DNA barcode S3' are clustered together and positioned spaced apart from each other in the microwells formed by the parallel partition walls.

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