Method of spatial sequencing of genes from tissue using padlocks with gaps on substrate

Abstract

To provide a method for obtaining sequencing and spatial information of genes on a subcellular level on tissue.SOLUTION: The present invention provides a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand, the method comprising the steps of a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker, b. providing a sample comprising at least one m-RNA strand on the surface, c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker, d. removing sample form surface, e. forming a padlock-shaped structure that is ligated to create a single strand circular template, f. performing rolling circle amplification to form rolonies, g. obtaining the sequence information of the rolonies, and h. linking the spatial information of the sample with the sequence information of the rolonies.SELECTED DRAWING: None

Description

Technical Field

[0001] Background The present invention relates to a method for obtaining sequence information and spatial information of genes at the intracellular level of tissues.

[0002] After retaining the spatial information, it has been difficult to detect all expressed genes at the intracellular level of tissues by sequencing for potential mutations of those genes. In particular, it has been difficult to achieve intracellular resolution. The method described in the present application enables a theoretical resolution reaching several tens of nanometers and enables the association of the spatial information of tissues with the sequence information of expressed genes.

[0003] Such techniques were developed by Spatial Transcriptomics and 10X Genomics. This technique preserves spatial information by tagging tissue RNA molecules with pre-spotted spatial identifiers on an array. The resulting library is then sequenced using, for example, Illumina's standard in vitro NGS sequencing method. The spotting process ensures that the positions of the spatial identifiers on the array are known before the sequencing process. After sequencing the spatial identifiers, the relevant RNA sequences can be assigned to their tissue locations. The resolution of this technique is one of its main limitations, as it depends on the shape and size of the spots on the array and is currently only applicable at the multicellular level. Spatial Transcriptomics holds several patents, but molecules are labeled with barcodes and sequenced off-substrate. Furthermore, library preparation is required for all barcoded molecules to be collected. The method presented herein sequences directly on the substrate, rather than removing the molecules from the substrate. The references are U.S. Patent Application Publication No. 2015 / 0344942, “Methods and Product for Optimizing Localized or Spatal Detection of Gene Expression in a Tissue,” and International Publication No. 2016 / 162309, “Spatially Distinguished, Multiplex Nucleic Acid Analysis of Biological Specimens.”

[0004] Another method for combining genetic and spatial information is disclosed in International Publication No. 2012 / 140224, Fredrik Salmen et al., in: Nature protocols 2018, “Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue”, and Sanja Vickovic et al., “High-density spatial transcriptomics arrays for in situ tissue profiling”, Nature methods, September 9, 2019.

[0005] overview The method described is used to detect tissue mRNA with high spatial resolution of 300–500 nm without requiring spatial identifiers, using a simplified workflow compared to other competing solutions currently on the market.

[0006] Therefore, an object of the present invention is a method for obtaining the spatial position and sequence information of a target sequence in a sample containing at least one mRNA strand, a. A process to provide a surface having a plurality of spacer units capable of binding to at least one mRNA chain and at least one reference marker. b. A step of supplying a sample containing at least one mRNA strand on a surface, wherein at least one mRNA strand of the sample binds to at least one spacer unit to generate at least one single-stranded oligomer. c. A step of taking a first image of the surface in order to obtain spatial information of the sample based on a reference marker. d. Steps to remove the sample from the surface e. A process to form a padlock-type structure by hybridizing at least one oligonucleotide containing 50 to 1000 nucleic acids having 5' and 3' ends with a complementary portion of a single-stranded oligomer, and then ligating this to generate a single-stranded circular template. f. The process of forming Rollonie by amplifying a single-stranded circular template into multiple DNA concatemers using a polymerase capable of rolling circle amplification. Steps to obtain sequence information for g. Loroney h. A process of relating the spatial information of the sample with the sequence information of the Roronie. This method includes [something].

[0007] In the first embodiment of the present invention shown in Figure 1, the spacer unit is selected from the group consisting of oligonucleotides containing at least five, preferably 5 to 50, single thymine molecules (referred to as "poly-T"), and the single-strand oligomer bond to at least one spacer unit is reverse transcribed onto the c-DNA strand, and the mRNA strand is removed by denaturation.

[0008] In the second embodiment of the present invention shown in Figure 3, the spacer unit is selected from the group consisting of an antibody, a Fab fragment of an antibody, a single-stranded Fv (scFv) fragment, a bivalent single-stranded antibody or diabody, or an aptamer. Preferably, a recombinant human antibody (eIF4E) or a monoclonal antibody (anti-7-methylguanosine (m7G) mAb) is used to target the cap end of the mRNA at the 5' end. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows a general workflow of the first embodiment of the present invention. [Figure 2] This figure shows the spatial arrangement of spacer units, reference markers, and tissue on the surface of the present invention. [Figure 3] This figure shows a general workflow according to a second embodiment of the present invention.

[0010] Detailed explanation The method of the present invention is carried out in several steps, which are described in detail below.

[0011] Process (a) First, a functionalized surface, such as an activated carboxylate group or succinimidyl ester, is conjugated onto a solid, flat, two-dimensional substrate. Spacer groups are then attached to this activated surface. In a variation of the poly-T group, this can be done using an amine-modified oligonucleotide, and in another variation, using an amino-conjugated antibody. The solid substrate also includes at least one, preferably two, independent reference marks.

[0012] Subsequently, mRNA molecules are loaded onto the surface with a buffer. The mRNA molecules interact with the functionalized surface and become randomly spatially distributed across the entire surface. The spatial or surface density of mRNA molecules can be controlled by the loading concentration.

[0013] It is preferable that spacer units, and therefore mRNA molecules, are randomly distributed on the substrate. The distribution density can be controlled by concentration, temperature, pH, and surface functionalization. Preferably, the sample is impregnated after being supplied to the surface.

[0014] Process (b) The tissue sample is brought into contact with a solid substrate on which all single molecules are arranged. Next, a 5-50 nucleotide polyT tail is hybridized with mRNA expressed in the tissue.

[0015] Process (c) Optionally, the tissue can be stained and visualized with, for example, DAPI, hematoxylin, or eosin. Images of reference marks are also captured, and their XY positions are recorded. Each sequence of individual molecules is recorded, and their spatial position relative to the reference marks is saved as an XY distance.

[0016] After supplying the sample to the surface, the sample can be stained to obtain its spatial position relative to a reference marker. Optionally, the sample can be stained to determine its morphology.

[0017] Process (d) Next, the tissue is removed from the substrate enzymatically or chemically, for example, using proteinase K. Thereafter, single molecules having mRNA can be further reverse transcribed into cDNA.

[0018] As a next step, the solid substrate to which single molecules are bound is denatured so that the double-stranded DNA molecule forms single-stranded oligonucleotides.

[0019] Optionally, the spatial position of the sample and the spatial position of the sequenced colony are overlaid based on the position of the reference marker.

[0020] Process (e) Padlock probes targeting various genes having gaps are added. If the gene is expressed, the padlock probe will bind to another mRNA. Thereafter, the gap is also reverse transcribed, and the padlock with the filled gap is ligated to form a circular template. Next, rolling circle amplification is performed on the padlock.

[0021] In a modified example, at least one oligonucleotide is hybridized with a complementary portion of at least one single-stranded oligomer to create a padlock unit having a gap between the 5' end and the 3' end of the oligonucleotide, and the gap of the padlock unit is filled with a nucleic acid complementary to the target sequence, and they are ligated to generate a single-stranded circular template, thereby implementing the method of the present invention.

[0022] In another modified example, at least one oligonucleotide is hybridized with a complementary portion of at least one single-stranded oligomer, and the 5' end and the 3' end of the oligonucleotide are ligated to generate a single-stranded circular template, thereby implementing the method of the present invention. In this case, the complementary portion of at least one single-stranded oligomer defines the target sequence.

[0023] Preferably, the oligonucleotide contains at least one primer sequence for a polymerase capable of rolling circle amplification. Alternatively, the oligonucleotide may have at least one primer sequence for a polymerase capable of rolling circle amplification by ligation of the primer oligonucleotide.

[0024] In another variation, the oligonucleotide has or contains at least two different primer sequences, which allows for sequential sequencing and effectively avoids optical crowding when multiple adjacent Roronys are fluorescent.

[0025] The location of each loronee on the substrate can be correlated with the original location of the tissue by taking one or more second images. Figure 2(A) shows how to take the first image with the tissue and reference marker. Figure 2(B) shows how to take the second image with the loronee and reference marker during sequencing.

[0026] Process (f) Finally, after adding sequencing primers, the gap-filled padlock is sequenced to determine each base. Subsequently, the position of each Roronie on the substrate can be correlated with its original position in the tissue. This means that the location of genes in the tissue can be determined, and whether or not there are mutations in the genes can be confirmed by sequencing.

[0027] Process (g) Preferably, sequencing information is obtained by sequencing by synthesis. Sequencing by synthesis is carried out by subsequently hybridizing a fluorescently labeled nucleotide with Rorony. In this case, the hybridized fluorescently labeled nucleotide provides a detectable fluorescent signal.

[0028] An exemplary workflow based on a modified example using polyT tails, as shown in Figure 1: • Identical oligonucleotides (spacer units) consisting of long-chain polyT tails are loaded onto a functionalized solid surface (e.g., a standard coverslip 25 × 75 × 1 mm) (Figure 1, A). • Single polyT molecules are randomly immobilized on the functionalized surface. A top view of the surface is shown in Figure 2. • The density of single molecules on the surface is controlled by concentration. • Single molecules can have a minimum distance of approximately 50-500 nm from each other. The tissue sample is brought into contact with a solid substrate on which all single molecules are arranged. Next, a 30-50 nucleotide polyT tail is hybridized with the mRNA expressed in the tissue. (Figure 1, B) • The tissue is stained (DAPI / hematoxylin, eosin) and visualized. The solid substrate also contains two independent reference marks. Images of the reference marks are also taken and their XY positions are recorded. Each sequence of individual single molecules is recorded and their spatial position relative to the reference marks is stored as an XY distance. The optional step involves performing periodic staining with different fluorescent dye-conjugated antibodies to obtain microscopic data, further characterizing the tissue sample, and identifying several different proteins expressed in the tissue. This provides more insight into the tissue and allows for later comparison of protein information with gene expression and mutant results. Next, the tissue is removed from the substrate. Then, a single molecule containing mRNA is reverse transcribed into cDNA. (Figure 1, C) • In the next step, the solid substrate to which the single molecules are bound is denatured so that the double-stranded DNA molecules form single-stranded oligonucleotides. (Figure 1, D) • A padlock probe with a gap is attached, targeting various genes. If the gene is expressed, the padlock probe will bind to another mRNA molecule. (Figure 1, E) Subsequently, the gap is also reverse-transferred, and the padlock with the gap filled is ligated to form an annular template. Next, rolling circle amplification is performed on the padlock. (Figure 1, E) Finally, after adding sequencing primers, the gap-filled padlock is sequenced to determine each base. Later, the position of each Roronie on the substrate can be correlated with its original position in the tissue. This means that the location of genes in the tissue can be determined, and the presence or absence of gene mutations can be confirmed by sequencing. (Figure 1, F)

[0029] An exemplary workflow based on a modified example using an antibody that captures the cap ends of mRNA, as shown in Figure 3: • Load the same antibody or aptamer onto a functionalized solid surface (e.g., a standard coverslip 25 × 75 × 1 mm) (Figure 3, A). • Antibodies or aptamers are randomly immobilized on the functionalized surface. A top view of the surface is shown in Figure 2. • The density of antibodies or aptamers on the surface is controlled by concentration. Antibodies or aptamers may have a minimum distance of approximately 50-500 nm from each other. • The tissue sample is brought into contact with a solid substrate on which all antibodies or aptamers are placed, and after penetration treatment, 7-methyl G is recognized by the antibodies or aptamers linked to the solid surface. • Stain the tissue (with DAPI / hematoxylin or eosin) and visualize it. The solid substrate also contains two independent reference marks. Images of the reference marks are also taken and their XY positions are recorded. Each sequence of individual single molecules is recorded and their spatial position relative to the reference marks is stored as an XY distance. The optional step involves performing periodic staining with different fluorescent dye-conjugated antibodies to obtain microscopic data, further characterizing the tissue sample, and identifying several different proteins expressed in the tissue. This provides more insight into the tissue and allows for later comparison of protein information with gene expression and mutant results. Next, remove the tissue from the substrate. (Figure 3, B) • A padlock probe with a gap is attached, targeting various genes. If the gene is expressed, the padlock probe will bind to another mRNA molecule. (Figure 3, C) Subsequently, the gap is also reverse-transferred, and the padlock with the gap filled is ligated to form a ring-shaped template. Next, rolling circle amplification is performed on the padlock. (Figure 3, D) Finally, after adding sequencing primers, the gap-filled padlocks are sequenced to determine each base. Later, the position of each Roronie on the substrate can be correlated with its original position in the tissue. This means that the location of genes in the tissue can be determined, and the presence or absence of gene mutations can be confirmed by sequencing. (Figure 3, E)

Claims

1. A method for obtaining spatial position and sequence information of a target sequence in a sample containing at least one m-RNA strand, comprising: a. providing a surface having a plurality of spacer units capable of binding to at least one m-RNA strand and at least one reference marker; b. supplying a sample containing at least one m-RNA strand to the surface, wherein at least one m-RNA strand of the sample binds to at least one spacer unit to generate at least one single-stranded oligomer; c. photographing a first image of the surface to obtain spatial information of the sample based on the reference marker; d. removing the sample from the surface; e. forming a padlock-type structure by hybridizing at least one oligonucleotide containing 50 to 1000 nucleic acids having 5'- and 3'-ends to a complementary portion of the single-stranded oligomer, and ligating them to generate a single-stranded circular template; f. forming rolling circles by amplifying the single-stranded circular template into a plurality of DNA concatemers with a polymerase capable of rolling circle amplification; g. obtaining sequence information of the rolling circles; h. associating the spatial information of the sample with the sequence information of the rolling circles. A method comprising the above steps.

2. The method according to claim 1, wherein the spacer unit is selected from the group consisting of an antibody, a Fab fragment of an antibody, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody or a diabody, or an aptamer.

3. The method according to claim 1, wherein the spacer unit is selected from the group consisting of oligonucleotides containing at least five thymine (poly-T) single molecules, the binding of the single-stranded oligomer to the at least one spacer unit is reverse transcribed into a c-DNA strand, and the m-RNA strand is removed by denaturation.

4. The method according to any one of claims 1 to 3, wherein the at least one oligonucleotide is hybridized to a complementary portion of the at least one single-stranded oligomer to create a padlock unit having a gap between the 5'-end and the 3'-end of the oligonucleotide, the gap of the padlock unit is filled with a nucleic acid complementary to the target sequence, and they are ligated to generate the single-stranded circular template.

5. Hybridizing the at least one oligonucleotide with a complementary portion of the at least one single-stranded oligomer, ligating the 5'-end and 3'-end of the oligonucleotide to generate the single-stranded circular template, and wherein the complementary portion of the at least one single-stranded oligomer defines a target sequence, the method according to any one of claims 1 to 3.

6. The method according to any one of claims 1 to 3, wherein the spacer units are randomly distributed on the substrate.

7. The method according to any one of claims 1 to 3, wherein the sample is subjected to a penetration treatment after being supplied to the surface.

8. The method according to any one of claims 1 to 3, wherein in step (d), the sample is enzymatically or chemically removed from the surface.

9. The method according to any one of claims 1 to 3, wherein the oligonucleotide contains at least one primer sequence for the polymerase capable of rolling circle amplification.

10. The method according to any one of claims 1 to 3, wherein the oligonucleotide comprises at least one primer sequence for the polymerase capable of rolling circle amplification by ligation of primer oligonucleotides.

11. The method according to claim 1, wherein the spatial position of the sample and the spatial position of the sequenced colony are superimposed based on the position of the reference marker.

12. The method according to any one of claims 1 to 3, wherein after supplying the sample to the surface, the sample is stained to obtain a spatial position based on the reference marker.

13. The method according to any one of claims 1 to 3, wherein sequencing information is obtained by the sequencing by synthesis method.

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