Hydrogel in which DNA barcode is replicated at high resolution and high density

Abstract

The present invention relates to a hydrogel in which a DNA barcode is replicated at high resolution and high density. The hydrogel of the present invention provides, first, a method by means of which gene expression levels can be easily, at high resolution, and further at a relatively low cost, analyzed while maintaining positional information within tissue of cells. Second, high capture efficiency at high resolution can be achieved at low cost for analysis of mRNA or the like. Third, since the hydrogel is formed in a three-dimensional structure, a barcode is generated at high density, thereby enabling achievement of various effects such as achieving high capture efficiency.

Description

Hydrogels with DNA barcodes replicated at high resolution and density

[0001] The present invention relates to a hydrogel in which a DNA barcode is replicated at high resolution and high density.

[0002] Analyzing mRNA or protein expression at the single-cell level is of great significance not only in the study of life phenomena but also in clinical applications such as cancer diagnosis.

[0003] In particular, this will play a major role in the implementation of personalized medicine and precision medicine by enabling accurate diagnosis based on the level of gene expression of each cell, moving beyond the old method of subjectively diagnosing tissue biopsy slides stained with H&E through a microscope.

[0004] To date, methods for analyzing mRNA expressed in single cells, such as Drop-seq and 10x Visium, have been commercialized and are rapidly developing, mainly in laboratories at leading universities around the world.

[0005] Referring to Figure 1, Drop-seq isolates cells from tissue at the single-cell level and flows them through a microfluidic chip, forming water-in-oil droplets in a 1:1 ratio with microbeads containing specific DNA barcodes. Within each droplet, mRNA binds to poly-T oligos, which are reverse-transcribed to create cDNA. Next-generation sequencing (NGS) analysis then analyzes mRNA from each cell, enabling the identification of differences between cells. However, because this method separates cells in advance, it has limitations in that it cannot determine the source of each cell.

[0006] Visium, commercialized by 10X Genomics, utilizes spatial transcriptomics to simultaneously analyze cell location information and gene expression within tissues.

[0007] Referring to Figures 2 and 3, each of the four squares on the slide glass is equipped with a unique DNA barcode corresponding to approximately 5,000 XY coordinates. Each DNA barcode contains a poly-T tail oligo, which attaches to mRNA diffused from the permeabilized tissue placed on the squares of the slide glass to generate cDNA. Then, gene expression in each part can be analyzed through NGS analysis. This has the advantage of being able to analyze tissue images and gene expression in each part together. Therefore, in the case of 10x Visium, there is the advantage of being able to analyze tissue images and gene expression in each part together.

[0008] Figure 4 is a diagram showing the conventional HDST (High density spatial transcriptomics) and Slide-seq. The difference from 10x Visium is that 10x Visium directly immobilizes oligos on the slide glass, whereas HDST and Slide-seq attach oligos to microbeads and then position them in the grooves of the slide glass (HDST) or place them as a monolayer on the slide glass (Slide-seq). The size of the microbeads in both methods is 2 μm and 10 μm, respectively, which is smaller than the 55 μm of 10x Visium, which is an advantage.

[0009] However, all three of the above methods have the disadvantage of having a very low capture efficiency of around 2%, as the mRNA must escape from the tissue and hybridize with the DNA of the substrate located below. The reasons for this low capture efficiency include that the efficiency of diffusion from the cell itself may be low, that the oligos fixed to the slide glass or microbeads may all have negative charges in a dense state, which may cause mutual repulsion, and that the number of oligos fixed to the surface of one spot is insufficient.

[0010] The Drop-seq, 10x Visium, HDST, and Slide-seq methods described above all label a single cell by combining the poly-A tail of mRNA with a barcode containing a poly-T tail to generate cDNA. Therefore, because as many barcodes as cells to be analyzed must be prepared in advance, a large number of different barcodes must be prepared, which has the disadvantage of requiring a large number of different barcodes. On the other hand, if DNA barcodes can be created through combination, the number of barcodes required can be significantly reduced.

[0011] In this regard, Cao and Junyue et al.'s paper, "Comprehensive single-cell transcriptional profiling of a multicellular organism (Science 357.6352 (2017): 661-667.)", proposes a combination of such barcodes. For convenience of explanation, the method proposed in the paper by Cao and Junyue et al. is named the "RT-P5 / P5 technique." Figures 5 and 6 are diagrams for explaining the existing RT-P5 / P7 technique.

[0012] Referring to Figure 5, cells are first separated into single cells (or nuclei) from tissue, and then placed into 96 wells using a FACS (Fluorescence activated cell sorter), with fewer than several dozen cells per well. Then, an oligo with a poly-T tail and a distinct DNA barcode is added to each well to generate cDNA. Then, these are mixed again and placed into 96 wells using FACS. During this process, for example, if there were 20 cells in a well located in row 1, column 1 of the first 96 wells, it is desirable that when these are mixed and placed into the second 96 wells, each of the 20 cells should be placed in a different well. Next, a process of attaching P5 and P7 for NGS analysis is performed, with each well assigned a different P5 and P7 index. The completed library through this process is as shown in Figure 6. Referring to Figure 6, the blue cDNA in the middle contains information on the mRNA to be originally analyzed, the green RT barcode on the left is an index for each well of the first 96 wells, and the blue i5 and i7 on both sides are indices corresponding to different wells of the second 96 wells.

[0013] That is, by assigning the index twice, with 96 RT barcodes and 96 P5 / P7 barcodes, a total of 192 barcodes are prepared, which has the advantage of enabling 96 x 96 = 9216 unique combinatorial indexing. However, the RT-P5 / P7 technique has the disadvantage of losing the location information of the cell within the tissue, making it undesirable.

[0014] To address this, application number 10-2020-0170938 proposes a technique for combinatorial indexing of specific cells at specific locations in a tissue using a linter. Liu et al., 2020, Cell 183, 1665-1681, proposes another method for combinatorial indexing.

[0015] Referring to Figure 7, a microfluidic chip with 50 channels is placed on a slide with tissue, and different DNA barcode oligos (referred to as RT-X) containing poly-T tails are flowed. The oligos penetrate into the cells and bind to the poly-A tail of the mRNA. Then, the microfluidic chip is removed, the channels are positioned orthogonally again, and a solution containing 50 different barcodes (referred to as RT-Y) is flowed. The location where the channels of the first chip and the channels of the second chip are orthogonal is where both the RT-X and RT-Y barcodes penetrate, and when the solution of the second microfluidic chip is flowed, a splint and a ligase are added together so that the two barcodes can be split-ligated. Through this, RT-X and RT-Y are connected, and the XY position information of the tissue can be obtained. The advantage of this method is that barcode oligos can be continuously supplied through the channels, so that a sufficient amount of barcode oligos can be introduced into the cells, thereby increasing the capture efficiency of mRNA analysis. However, the process of preparing a microfluidic chip as in B of the above-described FIG. 7 and manually inserting a barcode into each inlet without error has the disadvantages of being time-consuming and having low reliability.

[0016] A method that combines the advantages of the above methods was published by Srivatsan et al., Science 373, 111-117 (2021) (hereinafter referred to as the Gel-oligo method).

[0017] Referring to Fig. 8, a 100 μm agarose gel is coated on a slide glass, and a unique barcode oligo is positioned on the surface by spotting DNA oligos. Unlike the Drop-seq, 10x Visium, HDST, Slide-seq, RT-P5 / P7, and RT-RT methods described so far, the barcode oligo has a poly-A tail like mRNA, rather than an RT barcode with a poly-T tail. The circles expressed in different colors in the lower part of ii of Fig. 8 indicate the spotted oligo area, and the overlapping ovals represent the nuclei of each cell. When the agarose gel with the DNA oligos spotted on it is overlapped with the tissue slide, the DNA barcode oligo with the poly-A tail of the agarose gel enters the nucleus. After that, the tissue is separated into single cells, and each nucleus is distinguished and indexed in the same way as the RT-P5 / P7 method, and then NGS analysis is performed. As explained above, the RT-P5 / P7 method cannot provide information on the location of cells, but the Gel-oligo method contains a barcode oligo with a poly-A tail that includes location information for each nucleus (just like mRNA), so when NGS analysis is performed, when genetic information is classified for each cell (or nucleus), the barcode information corresponding to the location of the cell is read along with the mRNA, which has the advantage of being able to identify which location the cell came from. However, in the above method, since the DNA is placed on an agarose gel and positioned using a spotter, there is a possibility that the barcodes may be mixed up due to diffusion. Therefore, there is a limitation that the spatial resolution is not high enough with a spot size of 70 um and an interval of 220 um.

[0018] To solve the above problem, the inventor of the present invention discloses a parallel-barrier hydrogel DNA barcode device and a production process thereof (hereinafter referred to as the parallel-barrier gel method).

[0019] Referring to Fig. 9, by combining the advantages of the RT-RT method described above and the Gel-oligo method, the spatial resolution can be improved by preventing lateral diffusion by confining the hydrogel in the microwell, and in order to generate a barcode containing spatial information, there is no need to directly use inkjet printing, etc., but as in the RT-RT method, barcode x is flowed through the microchannel in the x-axis and bound to the hydrogel, and then barcode y is flowed through the microchannel in the y-axis and applied to the hydrogel, and then ligated to generate an xy barcode. This makes it possible to produce barcodes containing spatial information at a lower cost and more easily, and to increase the mRNA capture efficiency by ensuring that a sufficient amount of barcodes can be present in a three-dimensional structure on the hydrogel rather than on the glass surface. However, this method also has a problem in that the spatial resolution is limited by the microchannel spacing, and can only achieve a resolution at the level of 10x Visium.

[0020] A technology that can achieve high-resolution spatial transcriptomics is the Seq-Scope method announced by the University of Michigan (Cell 184, 3559-3572, June 24, 2021).

[0021] Referring to Figure 10, the Seq-Scope method is based on the Illumina NGS instrument. After applying a DNA barcode with a random address (including an adapter that can bind to P5 and P7 of the flow cell) to the Illumina NGS flow cell, each barcode is amplified through bridge PCR (bridge amplification), and NGS is performed to read the sequence of the barcode at each XY position. Then, the flow cell is disassembled, the tissue is placed on top of the barcode cluster, and the subsequent process captures and analyzes mRNA in a manner similar to 10x Visium. The advantage of this is that the barcodes can be positioned densely as high as the spatial resolution of the Illumina NGS instrument. This enables submicron-level resolution, which is higher than the single-cell level, and allows for the analysis of mRNA distribution patterns according to location even within a single cell. However, the disadvantages of this method are that the capture efficiency is low due to the small amount of barcodes in one bridge PCR-PCR barcode cluster, one Illumina NGS flow cell is consumed for one analysis, and NGS must be performed in advance to know the XY positions of the barcodes, so the analysis cost is very high.

[0022] A similar method was announced by BGI using a DNA nanoball patterned array (Cell 185(10):1777-1792.e21, 2022) (hereinafter referred to as the DNA nanoball method). This method has a resolution similar to that of Seq-scope, but suffers from the same drawbacks.

[0023] Accordingly, the present inventors provide a hydrogel, a method for manufacturing the same, and a method for using the same, which can analyze the level of gene expression more easily, with high resolution, and at low cost while maintaining the location information of cells within tissues.

[0024] Accordingly, the present invention relates to a DNA barcode S3;

[0025] A hydrogel having at least one of a nucleic acid sequence H2 including a nucleic acid sequence S2 capable of acting as a primer, a nucleic acid sequence H1 including a nucleic acid sequence S1 capable of acting as a primer, a nucleic acid sequence S4 including a nucleic acid sequence S1, and a complementary nucleic acid sequence S4' thereof fixed thereto, wherein the nucleic acid sequence H2 is located at the 3' terminal portion of S4, and the nucleic acid sequence H1 is located at the 5' terminal portion of S4.

[0026] The above nucleic acid sequence S4 or its complementary nucleic acid sequence S4' provides 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.

[0027] According to one embodiment of the present invention, the nucleic acid sequence S4 or the nucleic acid sequence S4' is a nucleic acid

[0028] A nucleic acid sequence S1; a nucleic acid sequence S2; and a DNA barcode S3; which is cloned by PCR from a substrate comprising a nucleic acid sequence S5 or a nucleic acid sequence S5' complementary thereto;

[0029] The above PCR may use a nucleic acid sequence H1 fixed to a hydrogel and a part of a nucleic acid sequence H2' complementary to the nucleic acid sequence H2 as primers, wherein the 3' end of the nucleic acid sequence H1 may include a nucleic acid sequence S1, and the 3' end of the nucleic acid sequence H2' may include a nucleic acid sequence S2' complementary to S2.

[0030] According to one embodiment of the present invention, the DNA barcode S3 is located between the nucleic acid sequence H1 and the nucleic acid sequence H2, and includes an oligo T sequence in the 3' direction of the DNA barcode S3, and a restriction enzyme site exists in the 3' direction of the oligo T, so that when the restriction enzyme is processed, the oligo T may be located at the 3' end.

[0031] According to one embodiment of the present invention, the 5' end of the nucleic acid sequence S4 or S4' may be linked to a linker and fixed to the hydrogel. The linker may be a linker that can be degraded by light.

[0032] According to one embodiment of the present invention, the DNA barcode S3 may include location information.

[0033] According to one embodiment of the present invention, the PCR may be a bridge PCR.

[0034] According to one embodiment of the present invention, H1 may further include a UMI, but may be positioned in the 5' direction of S1, and H2 may further include a UMI, but may be positioned in the 3' direction of S2.

[0035] In another aspect, the present invention provides (1) a hydrogel having a nucleic acid sequence H1 including a nucleic acid sequence S1 capable of acting as a primer; and a nucleic acid sequence H2` including a nucleic acid sequence S2` capable of acting as a primer fixed thereon; and

[0036] (2) A substrate comprising at least one of a nucleic acid sequence S5 including a DNA barcode S3; a nucleic acid sequence S2 having a complementary sequence S2' at the 3' end that can act as a primer; and a nucleic acid sequence S1 having a complementary sequence S1 at the 5' end that can act as a primer; and a nucleic acid sequence S5' complementary thereto;

[0037] A kit for replicating a DNA barcode including a nucleic acid sequence S1 and S2' into a hydrogel is provided, wherein the nucleic acid sequence S5 or the nucleic acid sequence S5' of the substrate is used as a primer to replicate the nucleic acid sequence S1 and S2' into a hydrogel.

[0038] According to one embodiment of the present invention, the DNA barcode S3 may include location information.

[0039] According to one embodiment of the present invention, the substrate may be a flow cell.

[0040] According to one embodiment of the present invention, the nucleic acid sequence S5 or the nucleic acid sequence S5' may be positioned spaced apart from each other by a predetermined distance while forming a cluster with nucleic acid sequences having the same DNA barcode S3 within the substrate.

[0041] In another aspect, the present invention comprises the steps of applying a hydrogel fixed with a nucleic acid sequence H1 including a nucleic acid sequence S1 capable of acting as a primer and a nucleic acid sequence H2` including a nucleic acid sequence S2` capable of acting as a primer to a substrate including a DNA barcode S3; a nucleic acid sequence S2 having a nucleic acid sequence complementary to a 3'-terminal portion thereof capable of acting as a primer; and a nucleic acid sequence S5 including a nucleic acid sequence S1 capable of acting as a primer at a 5'-terminal portion and a nucleic acid sequence S5` complementary thereto; and

[0042] (2) A step of performing PCR on the hydrogel and substrate to immobilize the nucleic acid sequence S4 or S4` including the S5 or its complementary nucleic acid sequence S5` to the hydrogel;

[0043] A method for replicating a DNA barcode in a hydrogel comprising:

[0044] According to one embodiment of the present invention, the DNA barcode S3 may include location information.

[0045] According to one embodiment of the present invention, the nucleic acid sequence S4 or S4' may be replicated in the hydrogel while forming the same cluster and being spaced apart from each other by a predetermined distance.

[0046] According to one embodiment of the present invention, the PCR may be a bridge PCR.

[0047] According to one embodiment of the present invention, the H1 may further include a UMI, but may be positioned in the 5' direction of S1, and the H2 may further include a UMI, but may be positioned in the 3' direction of S2.

[0048] According to one embodiment of the present invention, the S4 or the complementary sequence thereof, S4', may be connected to the hydrogel by a linker.

[0049] According to one embodiment of the present invention, the linker may be decomposed by light.

[0050] In another aspect of the present invention, (1) a step of plating a separated tissue on the hydrogel of claim 3 treated with a restriction enzyme;

[0051] (2) A method for analyzing a transcriptome of a tissue, comprising the step of isolating a nucleic acid sequence S6 or its complementary nucleic acid sequence S6` from a hydrogel and moving the nucleic acid sequence S6 or S6` into the tissue; wherein the nucleic acid sequence S6 or S6` is obtained by cleaving the nucleic acid sequence S4 or S4` at a restriction enzyme site by a restriction enzyme and includes an oligo T at the 3` end of the nucleic acid sequence S6 or S6`.

[0052] According to one embodiment of the present invention, after step (2), the method may further include a step of producing cDNA from the separated tissue using reverse transcriptase, and then obtaining cDNA containing location information.

[0053] According to one embodiment of the present invention, the nucleic acid sequence S6 or S6' may be connected to a hydrogel with a photodegradable linker, and when irradiated with light, the S6 or S6' may be separated from the hydrogel and move into the tissue.

[0054] According to one embodiment of the present invention, the step of separating the nucleic acid sequence S6 or S6` from the hydrogel and moving the nucleic acid sequence into the tissue may further include the step of performing electrophoresis by applying an electric field in a direction perpendicular to the hydrogel and the tissue surface.

[0055] In another aspect of the present invention, (1) a step of plating the separated tissue on the hydrogel of claim 3 treated with a restriction enzyme; and

[0056] (2) A method for analyzing a transcriptome of a tissue, comprising the step of moving mRNA of the tissue into a hydrogel; wherein the hydrogel comprises a nucleic acid sequence S6 or a complementary sequence S6', wherein the nucleic acid sequence S6 or S6' is obtained by cleaving the nucleic acid sequence S4 or S4' at a restriction enzyme site by a restriction enzyme, and comprises an oligo T at the 3' end of the nucleic acid sequence S6 or S6'.

[0057] According to one embodiment of the present invention, after step (2), the method may further include a step of producing cDNA using reverse transcriptase within the hydrogel and then obtaining cDNA containing location information.

[0058] According to one embodiment of the present invention, the nucleic acid sequence S6 or S6' of the hydrogel may be connected to the hydrogel with a photodegradable linker, and when irradiated with light, the S6 or S6' may be separated from the hydrogel, thereby obtaining cDNA.

[0059] According to one embodiment of the present invention, the step of moving mRNA within the tissue to a hydrogel may be a method for analyzing a transcriptome of a tissue, further comprising the step of performing electrophoresis by applying an electric field in a direction perpendicular to the hydrogel and the tissue surface.

[0060] According to the present invention having the above-described configuration, the following effects are realized. First, it provides a method for easily analyzing the level of gene expression with high resolution and at a relatively low cost while maintaining the location information of cells within the tissue. Second, it provides the advantage of achieving high capture efficiency at a low cost with high resolution in the analysis of mRNA, etc. Third, because it is made in the three-dimensional structure of the hydrogel, various effects are realized, such as high capture efficiency achieved by generating high-density barcodes.

[0061] Figure 1 is a diagram for explaining the conventional Drop-seq method.

[0062] Figures 2 and 3 are drawings illustrating Visium commercialized by 10X genomics.

[0063] Figure 4 is a diagram showing conventional HDST (High density spatial transcriptomics) and Slide-seq.

[0064] Figures 5 and 6 are drawings for explaining the existing RT-P5 / P7 technique.

[0065] Figure 7 is a drawing of a technique for combinatorial indexing of specific cells at specific locations in an organization using a conventional printer.

[0066] Figure 8 is a drawing of a conventional Gel-oligo method.

[0067] Figure 9 is a drawing for explaining the so-called parallel-barrier hydrogel DNA barcode device and its production process conducted by the inventor of the present invention.

[0068] Figure 10 is a diagram of the Seq-Scope method.

[0069] Figure 11 is a diagram illustrating the process of creating a cluster in an NGS flow cell.

[0070] Figure 12 is a drawing illustrating the process of introducing a DNA barcode into a hydrogel.

[0071] Figure 13 is a drawing for explaining the process of replicating a barcode in a hemispherical shape on a hydrogel through bridge PCR.

[0072] Figure 14 is a drawing for explaining the process of separating a hydrogel.

[0073] Figures 15 and 16 are drawings for explaining the process of flipping the hydrogel over and placing tissue on it.

[0074] Figure 17 is a drawing for explaining the process of using the hydrogel of the present invention for electrophoresis.

[0075] Figure 18 is a diagram showing the structure of a nucleic acid sequence included in the hydrogel of the present invention.

[0076] Figure 19 is a diagram showing a hydrogel structure required to cluster nucleic acid sequences having the same DNA barcode of the present invention.

[0077] Figure 20 is a diagram showing the structure of a nucleic acid sequence contained within the hydrogel of the present invention.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In the present invention, the nucleic acid sequence can be used interchangeably with a nucleic acid molecule, a polynucleotide sequence, etc.

[0082] Hereinafter, the present invention will be described in detail with reference to the attached drawings.

[0083] Fig. 11 relates to a process of clustering nucleic acid molecules containing specific location information on a substrate.

[0084] First, it relates to a process for clustering nucleic acid sequences having DNA barcodes containing the same positional information by performing PCR on a substrate having a DNA barcode containing specific positional information and a nucleic acid molecule containing an adapter sequence and a primer sequence or a complementary sequence thereof fixed within the adapter sequence.

[0085] A nucleic acid amplification method is described that allows amplification products to be immobilized on a solid support to form an array comprising clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. The product of the solid-phase amplification reaction is a so-called "bridged" structure formed by the annealing of pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at their 5' ends, preferably through covalent attachments. Cluster amplification methods are examples of methods for producing immobilized amplicons using immobilized nucleic acid templates. Other suitable methods can also be used to produce immobilized amplicons from immobilized DNA fragments produced according to the methods provided herein. For example, one or more clusters or colonies can be formed through solid-phase PCR, whether or not one or both primers of each pair of amplification primers are immobilized. Furthermore, the cluster amplification method described above can enable clustering of nucleic acid molecules with identical DNA barcodes. DNA barcodes can include location information within a tissue, but they can be indexed using a variety of methods. It is self-evident that these indexing methods can be implemented using methods disclosed in various existing literature.

[0086] The substrate of the present invention may be an NGS flow cell, and in the case of the third nucleic acid sequence included in the substrate, it includes an adapter sequence. The adapter sequence may be positioned as a first adapter sequence and a second adapter sequence at the 5' end and the 3' end, respectively, and a part or the entire sequence of the first adapter sequence and the second adapter sequence may be manufactured to be identical. In addition, in the present invention, a DNA barcode containing positional information may be included between the adapter sequences, and a DNA barcode containing positional information may be inserted within the adapter sequence.

[0087] 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 in the cell or tissue of origin of a transcript (mRNA) in the gene expression analysis results obtained after pooling multiple cells.

[0088] Additionally, a "DNA barcode" may be a DNA having a predetermined arrangement as an identification code and a magnetic particle for separation, with a probe complementary to the target substance attached to the surface, for detection of a biological target substance. A method for generating a DNA barcode containing location information of the present invention is described in detail in Republic of Korea Patent Application No. 10-2021-0128780, and can be generated using other known methods.

[0089] The "adapter sequence" of the present invention may include a sequencing primer binding site, an amplification primer binding site, and an index. For example, the adapter may include a P5 sequence, a P7 sequence, or a complementary sequence thereof. The P5 sequence may be a sequence defined by 5`-AATGATACGGCGACCACCGA-3`, and the P7 sequence may be 5`-CAAGCAGAAGACGGCATACGA-3`, but the P5 and P7 sequences are merely examples of the present invention and the present invention is not limited thereto.

[0090] In one embodiment of the present invention, the adapter sequence may further comprise a spacer polynucleotide, which may be 1 to 20, for example, 1 to 15, or 1 to 10 nucleotides in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the spacer comprises 10 nucleotides. The spacer is a polyT spacer, for example, a 10T spacer. The spacer nucleotide may be included at the 5' end of the polynucleotide, which may be attached to a suitable support via a linkage together with the 5' end of the polynucleotide.

[0091] Attachment can be achieved via a sulfur-containing nucleophile, a phosphorothioate group present at the 5' end of the polynucleotide. In some embodiments, the polynucleotide can comprise a polyT spacer and a 5' phosphorothioate group.

[0092] The term "index" of the present invention may be useful for identifying a source of a nucleic acid molecule. In some embodiments, the adapter may be modified to prevent the formation of concatemers, for example, by adding a blocking group at one or both ends that prevents extension of the adapter. Examples of 3' blocking groups include 3'-spacer C3, a dideoxynucleotide, and attachment to a substrate. Examples of 5' blocking groups include a dephosphorylated 5' nucleotide and attachment to a substrate. The adapter comprises a nucleic acid, such as a single-stranded nucleic acid. The adapter may comprise a short nucleic acid having a length less than, more than, equal to, or between any two of the above sizes, of about 5 nucleotides, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides.

[0093] Additionally, the present invention may utilize components useful for processing genetic material within a substrate, including lysozyme, proteinase K, random hexamers, polymerases (e.g., Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposases (e.g., Tn5), primers (e.g., P5 and P7 adapter sequences), ligases, catalytic enzymes, deoxynucleotide triphosphates, buffers, or divalent cations.

[0094] Figures 12 and 13 are drawings for explaining the process of introducing a DNA barcode having location information into a hydrogel. First, a monomer solution can be applied to the hydrogel and directly synthesized. When polymerizing the hydrogel, a monomer, a crosslinker can be included, and acrydite, a photocleavable linker, a primer sequence (e.g., P5, P7; in the present invention, DNA including primer sequence 1 or primer sequence 2 can be loaded together and polymerized to fix the primer sequence to the hydrogel structure. At this time, the primer sequence and the linker can be combined, and the linker can be combined with acrydite, and the acrydite can be fixed to the hydrogel.

[0095] After this, a substrate, such as the surface of a flow cell, can be exposed and brought into contact with the hydrogel. After bringing the substrate into contact with the hydrogel, a monomer solution and a DNA polymerase required for a PCR reaction can be applied to replicate the DNA barcode on the substrate into the hydrogel.

[0096] As illustrated in this figure, the barcode clusters in the flow cell are depicted as being replicated across the entire hydrogel layer, but this is merely an example and is not limiting. When performing bridge PCR in a hydrogel, the diameter of the clusters gradually increases with the number of PCR cycles, and adjacent clusters may overlap. Therefore, the number of PCR cycles can be appropriately controlled, and the resulting clusters can be formed into a hemispherical shape from the hydrogel surface.

[0097] Furthermore, since the spatial resolution of the barcode clusters on the flow cell produced by the present invention is at the sub-micron level, the diameter of the three-dimensional barcode clusters formed in the hydrogel can also be maintained at the same level. Accordingly, the barcode clusters can be replicated only within a 1 μm region of the entire hydrogel thickness that is in contact with the flow cell.

[0098] When polymerizing the hydrogel, if it is made too thin, it may tear during the separation process, and its mechanical strength may be weak, making it prone to stretching and deformation. Therefore, it can be made thicker than 1 μm. Making the hydrogel too thick also wastes the high cost of DNA containing photocleavable linkers. Therefore, by polymerizing a thin hydrogel, approximately 1 μm thick, with a monomer solution containing a photocleavable linker, and then adding and polymerizing a solution containing only the monomer and cross-linker, the cost increase factor when making a thick hydrogel can be suppressed.

[0099] In the present invention, the term "hydrogel" generally refers to a material with 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.

[0100] 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.

[0101] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free 3' terminal hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

[0102] The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modifications between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0103] The term "UMI" in the present invention may be used herein to refer to the sequence information of a polynucleotide and the physical polynucleotide itself. UMIs are similar to barcodes, which are commonly used to distinguish reads from one sample from reads from another, but instead, UMIs are used to distinguish nucleic acid template fragments from one another when many fragments from an individual sample are sequenced together. UMIs may be single- or double-stranded, and may be 5 or more bases, 6 or more bases, 7 or more bases, 8 or more bases, or more. In certain specific examples, UMIs may be 5 to 8 bases, 5 to 10 bases, 5 to 15 bases, 5 to 25 bases, 8 to 10 bases, 8 to 12 bases, 8 to 15 bases, or 8 to 25 bases in length, etc. Additionally, in certain embodiments, a UMI is 30 bases or less in length, 25 bases or less, 20 bases or less, or 15 bases or less in length.

[0104] Figures 14 to 17 illustrate a method of utilizing the hydrogel in which the DNA barcode has been replicated. As shown in Figure 14, the hydrogel can be removed from the flow cell and inverted to expose the three-dimensional barcode cluster. Then, tissue can be placed on the hydrogel to perform spatial transcriptomics analysis similar to 10x Visium, Seq-Scope. At this time, the tissue's mRNA can diffuse into the hydrogel to produce cDNA (Figure 15), or the barcode can be photodegraded by applying UV light, thereby diffusing into the tissue to produce cDNA (Figure 16). In addition, by applying an electric field (Fig. 17) by applying electrodes to both ends of the hydrogel and the tissue, it is possible to improve the electrophoretic migration speed of mRNA or barcode, and thus reduce lateral diffusion, enabling high-resolution analysis. Fig. 18 illustrates the structure S4 of the nucleic acid sequence included in the hydrogel of the present invention, and Fig. 19 illustrates the structure of the initial hydrogel used to cluster nucleic acid sequences having the same positional information in the hydrogel of the present invention. Fig. 20 is a diagram illustrating S6 or S6`, which is a preferred nucleic acid structure, when performing tissue transcriptome analysis using the hydrogel of the present invention.

[0105] More specifically, the DNA barcode S3 may be a barcode that holds location information inside the hydrogel. That is, in the present invention, groups with the same sequence of S3 may be clustered together. As shown in Fig. 19, by linking H1 or H2' to the hydrogel, a portion of H1, S1, and a portion of H2', S2', may act as primers, and a structure as shown in Fig. 18 may be synthesized through bridge PCR. H1 may include a UMI sequence for later analysis of the tissue transcriptome, and is preferably located 5' to S1 and 3' to S2.

[0106] More specifically, in order to replicate the positional information of the substrate into a hydrogel, the nucleic acid of the substrate needs at least an S1 sequence at the 5' end, and an S2 and S3 sequence at the 3' end, or a complementary sequence thereto, and within the substrate, nucleic acid sequences having the same S3 can be clustered together and positioned apart from each other by a predetermined distance. In the present invention, the above sequence may be referred to as S5 or S5'.

[0107] Referring to FIG. 20, S4 or S4' of the present invention may include oligo T in the 3' direction of S3 or S3', and a restriction enzyme site is located in the 3' direction of the oligo T, so that when the restriction enzyme is treated, oligo T is exposed at the 3' end, so that mRNA having an oligo A sequence can be captured for later analysis of the tissue transcriptome.

Claims

1. DNA Barcode S3; A hydrogel having at least one of a nucleic acid sequence H2 including a nucleic acid sequence S2 capable of acting as a primer, a nucleic acid sequence H1 including a nucleic acid sequence S1 capable of acting as a primer, a nucleic acid sequence S4 including a nucleic acid sequence S1, and a complementary nucleic acid sequence S4' thereof fixed thereto, wherein the nucleic acid sequence H2 is located at the 3' terminal portion of S4, and the nucleic acid sequence H1 is located at the 5' terminal portion of S4. A hydrogel characterized in that the nucleic acid sequence S4 or its complementary nucleic acid sequence S4' is clustered with nucleic acid sequences having the same DNA barcode S3 or its complementary DNA barcode S3' and are spaced apart from each other by a predetermined distance.

2. In the first paragraph, the nucleic acid sequence S4 or the nucleic acid sequence S4' is cloned through PCR from a substrate including the nucleic acid sequence S1; the nucleic acid sequence S2; and the nucleic acid sequence S5 including the DNA barcode S3 or the nucleic acid sequence S5' complementary thereto. The above PCR uses a nucleic acid sequence H1 fixed to the hydrogel and a part of the nucleic acid sequence H2` complementary to the nucleic acid sequence H2 as primers, wherein the 3` end of the nucleic acid sequence H1 includes the nucleic acid sequence S1, and the 3` end of the nucleic acid sequence H2` includes the nucleic acid sequence S2` complementary to S2.

3. In the hydrogel of claim 1, the DNA barcode S3 is located between the nucleic acid sequence H1 and the nucleic acid sequence H2, and includes an oligo T sequence in the 3' direction of the DNA barcode S3, and a restriction enzyme site exists in the 3' direction of the oligo T, so that when the restriction enzyme is processed, the oligo T is located at the 3' end.

4. A hydrogel according to claim 1, wherein the 5' end of the nucleic acid sequence S4 or S4' is linked to a linker and fixed to the hydrogel.

5. A hydrogel according to claim 4, wherein the linker is a linker that can be decomposed by light.

6. In the first paragraph, the hydrogel, wherein the DNA barcode S3 contains location information.

7. A hydrogel according to claim 2, wherein the PCR is a bridge PCR.

8. A hydrogel according to claim 1, wherein H1 further includes UMI and is positioned in the 5' direction of S1, and H2 further includes UMI and is positioned in the 3' direction of S2. 9.(1) A hydrogel having a nucleic acid sequence H1 including a nucleic acid sequence S1 capable of acting as a primer; and a nucleic acid sequence H2` including a nucleic acid sequence S2` capable of acting as a primer; fixed thereto; and (2) A substrate comprising at least one of a nucleic acid sequence S5 including a DNA barcode S3; a nucleic acid sequence S2 having a complementary sequence S2' at the 3' end that can act as a primer; and a nucleic acid sequence S1 having a complementary sequence S1 at the 5' end that can act as a primer; and a nucleic acid sequence S5' complementary thereto; A kit for replicating a DNA barcode including nucleic acid sequences S1 and S2' into a hydrogel, wherein the nucleic acid sequences S1 and S2' are used as primers to replicate the nucleic acid sequence S5 or the nucleic acid sequence S5' of the substrate into the hydrogel.

10. A kit according to claim 9, wherein the DNA barcode S3 contains location information.

11. A kit according to claim 9, wherein the substrate is a flow cell.

12. A kit according to claim 9, characterized in that the nucleic acid sequence S5 or the nucleic acid sequence S5' are clustered together with nucleic acid sequences having the same DNA barcode S3 within the substrate and are spaced apart from each other by a predetermined distance. 13.(1) A step of applying a hydrogel fixed with a nucleic acid sequence H1 including a nucleic acid sequence S1 that can act as a primer and a nucleic acid sequence H2` including a nucleic acid sequence S2` that can act as a primer to a substrate including at least one of a DNA barcode S3; a nucleic acid sequence S2 that can act as a primer with a nucleic acid sequence S2` complementary to the 3'-terminal portion; and a nucleic acid sequence S5 including a nucleic acid sequence S1 that can act as a primer at the 5'-terminal portion; and a complementary nucleic acid sequence S5`; and (2) A step of performing PCR on the hydrogel and substrate to immobilize the nucleic acid sequence S4 or S4` including the S5 or its complementary nucleic acid sequence S5` to the hydrogel; A method for replicating a DNA barcode into a hydrogel comprising:

14. A method according to claim 13, wherein the DNA barcode S3 includes location information.

15. A method according to claim 13, wherein the nucleic acid sequence S4 or S4' is replicated in the same cluster within the hydrogel and spaced apart from each other by a predetermined distance.

16. A method according to claim 13, wherein the PCR is a bridge PCR.

17. A method according to claim 13, wherein H1 further includes a UMI, but is positioned in the 5' direction of S1, and H2 further includes a UMI, but is positioned in the 3' direction of S2.

18. A method according to claim 13, wherein S4 or a sequence complementary thereto, S4`, is connected to the hydrogel by a linker.

19. A method according to claim 18, wherein the linker is decomposed by light. 20.(1) A step of plating the separated tissue on the hydrogel of paragraph 3 treated with a restriction enzyme; (2) A method for analyzing a transcriptome of a tissue, comprising the step of isolating a nucleic acid sequence S6 or its complementary nucleic acid sequence S6` from a hydrogel and moving the nucleic acid sequence S6 or S6` into the tissue; wherein the nucleic acid sequence S6 or S6` is obtained by cleaving the nucleic acid sequence S4 or S4` at a restriction enzyme site by a restriction enzyme and contains an oligo T at the 3` end of the nucleic acid sequence S6 or S6`.

21. A method according to claim 20, further comprising, after step (2), a step of producing cDNA from the separated tissue using reverse transcriptase, and then obtaining cDNA containing location information.

22. A method according to claim 20, wherein the nucleic acid sequence S6 or S6' is connected to a hydrogel and a photodegradable linker, and when irradiated with light, the S6 or S6' is separated from the hydrogel and moves into the tissue.

23. In paragraph 20, A method for analyzing a transcriptome of a tissue, wherein the step of separating the nucleic acid sequence S6 or S6` from the hydrogel and moving the nucleic acid sequence into the tissue further includes the step of performing electrophoresis by applying an electric field perpendicular to the hydrogel and the tissue surface. 24.(1) A step of plating the separated tissue on the hydrogel of paragraph 3 treated with a restriction enzyme; and (2) A method for analyzing a transcriptome of a tissue, comprising the step of moving mRNA of the tissue into a hydrogel; wherein the hydrogel comprises a nucleic acid sequence S6 or a complementary sequence S6`, wherein the nucleic acid sequence S6 or S6` is obtained by cleaving the nucleic acid sequence S4 or S4` at a restriction enzyme site by a restriction enzyme, and comprises an oligo T at the 3` end of the nucleic acid sequence S6 or S6`.

25. A method according to claim 24, further comprising, after step (2), a step of producing cDNA using reverse transcriptase within a hydrogel and then obtaining cDNA containing location information.

26. A method according to claim 25, wherein the nucleic acid sequence S6 or S6' of the hydrogel is connected to the hydrogel by a photodegradable linker, and when irradiated with light, the S6 or S6' is separated from the hydrogel, thereby obtaining cDNA.

27. In paragraph 24, The step of moving mRNA within the above tissue to the hydrogel is: A method for analyzing the transcriptome of a tissue, further comprising the step of performing electrophoresis by applying an electric field perpendicular to the hydrogel and the tissue surface.

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