Spatial transcriptome detection in tissues using Rorony tagging

Abstract

This invention provides a method for obtaining sequence and spatial information of at least one RNA strand on a sample immobilized on a surface. [Solution] The present invention relates to the spatial detection of transcripts (mRNA) in tissue. The described method makes it possible to significantly increase the density of the detected transcripts.

Description

Technical Field

[0001] The present invention relates to a method for obtaining sequence information and spatial information of at least one RNA strand on a sample immobilized on a surface.

[0002] Background Art Some methods for spatial (comprehensive) transcriptome detection are known, as disclosed, for example, in US Patent Application Publication No. 20140066318, US Patent No. 10030261, US Patent No. 10774374, or US Patent Application Publication No. 20210292748.

[0003] All existing methods for spatial transcriptome detection use a glass surface, an array, a bead array, or a substrate, on which oligonucleotides are immobilized, and when a prepared tissue section is brought into contact with the glass surface, the array, or the substrate, they capture the mRNA expressed in the tissue section using those oligonucleotides, based on the concept.

[0004] Known methods have certain limitations in resolution and the length of genes to be sequenced. Figure 1 shows an overview of known methods in terms of their capabilities (source: Seferbekova, "Spatial biology of cancer evolution", Nature Sept. 2022).

[0005] One current approach is to directly target intracellular mRNA transcripts with padlock probes and then perform rolling circle amplification to generate nanoballs ("rolonies"). The size of these rolonies is typically 600 - 800 nm in diameter. Only one mRNA transcript can be detected per rolony. Therefore, in one cell, a total of approximately 50 - 100 mRNA transcripts can be detected. The method described herein makes it possible to significantly increase the number of spatially detectable mRNA transcripts per cell to a total of 2500 - 5600.

[0006] We found that by preparing a cyclic oligomer containing a specific unit described later that has a sequence complementary to the actual required sequence, amplifying this cyclic oligomer using RCA (rolling circle amplification), and thereby obtaining a concatemer loronie with the "correct" sequence, we can capture mRNA and molecularly characterize it.

[0007] Purpose of the invention The object of the present invention is a method for obtaining sequence information and spatial information of at least one RNA strand on a sample immobilized on a surface, a. A step of preparing a cyclic oligonucleotide having 50 to 1000 nucleotides, comprising a complementary cleavable unit having 5 to 50 nucleotides, a poly-A unit having 5 to 350 adenosine nucleotides, a complementary unique molecular identifier unit having 5 to 50 nucleotides, and a complementary sequencing primer unit having 5 to 50 nucleotides. b. A process to obtain roloney by amplifying a cyclic oligonucleotide using a polymerase capable of rolling circle amplification into multiple concatemers, each containing a cleavable unit, a poly-T unit, a unique molecular identifier (UMI) unit, and a sequencing primer unit. c. A step of providing loroney to a sample, thereby hybridizing loroney to at least one RNA strand via the poly T unit of the concatemer, d. A step of generating a linear oligonucleotide hybridized to at least one RNA strand by enzymatically cleaving loroni in the cleavable unit of the concatemer. A step of forming at least one cDNA strand by extending a hybridized linear oligonucleotide using e. mRNA as a template. f. A step of performing sequencing-by-synthesis of hybridized linear oligonucleotides in the 3' direction, starting from the sequencing primer unit, to obtain sequence information of the unique molecular identifier unit and simultaneously detect the spatial position of the hybridized linear oligonucleotides. g. The process of extracting cDNA from the sample. A step of obtaining sequence information of at least one RNA strand by sequencing h.cDNA. i. A step of matching the sequence information and spatial information of the unique molecular identifier unit obtained in step f) with the sequence information of at least one RNA strand obtained in step h). The method included [something].

[0008] Since the cyclic oligonucleotide is amplified by rolling circle amplification in step b), the resulting concatemer contains the complementary sequence of the starting material. In the following description, this will be referred to as the “final” sequence of the cleavable unit, unique molecular identifier, and sequencing primer unit, and accordingly, the respective “starting” sequences will be referred to as the “complementary” sequences, i.e., the “complementary cleavable unit,” “complementary unique molecular identifier,” and “complementary sequencing primer unit.” [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows various spatial resolution approaches developed for mapping DNA, RNA, and proteins. Typically, higher spatial resolution indicates smaller molecular amounts. Source: Zaira Seferbekova, "Spatial biology of cancer evolution," Nature Review Genetics, Sept 2022. [Figure 2] Figure 2 is a schematic diagram of steps a) to d) of the method of the present invention. [Figure 3] Figure 3 is a schematic diagram of step e) of the method of the present invention. [Figure 4] Figure 4 is a schematic diagram of a part of step f) of the method of the present invention. [Figure 5] Figure 5 shows the sequencing-by-synthesis portion of step f) of the method of the present invention. [Figure 6] Figure 6 shows an image of the experimental results obtained by the method of the present invention. [Figure 7] Figure 7 shows an image of the experimental results obtained using the method of the present invention.

[0010] Modes for carrying out the invention One current approach involves directly targeting intracellular mRNA transcripts with a padlock probe, followed by rolling circle amplification to generate nanoballs ("Rolonies"). These Rolonies are typically 600–800 nm in diameter. Only one mRNA transcript can be detected per Rolony. Therefore, a total of approximately 50–100 mRNA transcripts can be detected within a single cell. The method described herein allows for a significant increase in the number of spatially detectable mRNA transcripts per cell, reaching a total of 2500–5600.

[0011] The present invention relates to a method for obtaining sequence and spatial information of at least one RNA strand on a tissue sample immobilized on a surface, comprising the step of preparing a cyclic oligonucleotide containing a cleavable unit, a poly(A) unit, a unique molecular identifier (UMI) unit, and a sequencing primer unit.

[0012] Preferably, the cleavable unit includes a recognition site for a restriction enzyme selected from the group consisting of EcoRI, EcoRV, BamHI, HindIII, NotI, and PSstI, such as the recognition sequence 5'-GAATTC-3'.

[0013] This oligonucleotide is amplified by rolling circle amplification (RCA) to form concatemers called loronees, and these concatemers are directly hybridized to RNA strands on a tissue sample via poly-T units. Next, these loronees are enzymatically cleaved in cleavable units to generate linear oligonucleotides, and these are extended using RNA as a template to form cDNA.

[0014] The enzymatic cleavage of loronie in the cleavable unit in step d) can be performed by hybridizing oligonucleotides to the complementary unit of the cleavable unit and then cleaving them using restriction enzymes.

[0015] Sequencing-by-synthesis is performed directly on tissue samples (in situ sequencing) to obtain sequence information from UMI strands. Further steps include extracting cDNA, resequencing the cDNA and UMI, and matching the cDNA sequence with the UMI sequenced directly in the tissue. This leads to the detection of the transcript and the determination of its precise location in the tissue.

[0016] The surface is preferably a transparent surface, and sequencing bisynthesis of the hybridized linear oligonucleotide in step f) is preferably carried out by detecting fluorescence emission through the transparent surface.

[0017] Furthermore, the enzymatic cleavage of loronie in the cleavable unit in step d) can be carried out by hybridizing oligonucleotides to the units of the cleavable unit and then cleaving them using restriction enzymes.

[0018] Sequencing-by-synthesis is a method for determining the sequence of single-stranded DNA, and the present invention includes sequencing of cDNA reverse-transcribed from mRNA.

[0019] Using polymerase and nucleotides, the bases (A, C, G, T) of single-stranded DNA are replicated. Each nucleotide is modified and contains a blocking group and a fluorescent dye.

[0020] Each base (A, C, G, T) has its own unique fluorescent dye and can therefore be uniquely identified. The modified nucleotides are incorporated for each sequencing cycle. The blocking group prevents two or more nucleotides from being incorporated in one cycle.

[0021] By extending the single-stranded DNA in multiple cycles, the unknown bases are determined. Each cycle includes an extension step, an imaging step to determine the fluorescent color of each incorporated base, and a cleavage step to remove the blocking group.

[0022] In one variant of this method, after the cDNA strand is taken out of the sample, prior to sequencing, the cDNA strand is amplified by RCA.

[0023] Preferably, the cDNA strand is amplified by RCA by circularizing the cDNA with a ligase enzyme (such as T4) and using the circularized cDNA as a template for RCA.

[0024] The blocking group prevents two bases from being incorporated in one cycle.

[0025] Sequencing-by-synthesis is usually performed in a microfluidic channel where polymerase, nucleotides, and other chemical components are provided. Often, the temperature of the microfluidic channel is also controlled, and a higher temperature is beneficial for nucleotide incorporation (e.g., 65 °C).

[0026] The single-stranded DNA can be immobilized on a glass substrate where sequencing can be performed. Often, the single-stranded DNA is amplified before sequencing, and the target strand is replicated hundreds of times (bridge amplification, rolling circle amplification, etc.).

[0027] The spatial location of the unique molecular identifier unit in step f) can be achieved / performed by imaging the immobilized tissue sample in each cycle of the sequencing-by-synthesis process.

[0028] This makes it possible to detect much stronger fluorescence signals and distinguish them more clearly from background signals.

[0029] As described above, sequencing-by-synthesis is typically performed on single-stranded DNA immobilized on a glass substrate. Preferably, the sample is immobilized on a transparent surface by APTES, APTEM, silane, polymer, glycol, or thiol coating.

[0030] As a variation, for example, loroney may be crosslinked to the sample using a crosslinking agent such as bisPEG via UTP.

[0031] The method described herein involves directly applying sequencing-by-synthesis to single-stranded DNA immobilized on cells or tissue surfaces.

[0032] In this particular case, sequencing is used to decipher the unique molecular identifier portion of an oligomer that is part of a lorony. This lorony was generated by RCA.

[0033] This unique molecular identifier allows for the unique identification of Roronie and provides the spatial coordinates (x and y) of the location from which its sequence originates within the tissue.

[0034] The spatial position of the unique molecular identifier unit in step f) can be determined by imaging the immobilized tissue sample in each cycle of the sequencing-bi-synthesis process.

[0035] The method of the present invention may include one or more of the steps outlined in the following schematic workflow: 1. Begin with linear oligomers. Each oligomer is made up of synthetic oligonucleotides with the following sections: a. Poly(A) homopolymer oligosaccharide 10-300 bp b. Sequencing primer oligo 5-50 bp c. Cleavable oligo sequences 5-50 bp d. Unique Molecular Identifier (UMI) Oligonucleotides 5-50 bp e. Additional nucleotides required to form a padlock 2. Each individual oligomer (hereinafter referred to as padlock) possessing a UMI is cyclically formed, creating a closed ring. This is still a single-stranded oligo. Subsequently, the cyclic oligo is duplicated using a special polymerase, ultimately yielding 300-500 copies of the original oligo sequence. These copies are also called concatemers. A single tube contains millions of loroneys generated in parallel. Each of these loroneys will have its own unique molecular identifier. 3. Next, the generated loroney is brought into contact with the prepared tissue sample. The tissue sample can be a thin section of tumor tissue biopsy, typically 5-20 μm thick. The tissue itself has undergone multiple sample preparation steps. 4. Hybridization of each lorony to the poly-A tail of each expressed mRNA, and cross-linking of lorony to the tissue. Furthermore, the poly-T portion of lorony generated from poly-A on the padlock is hybridized to the mRNA tail. 5. Each loronie is enzymatically cleaved into each concatemer (cleavable sequence) to produce a 3' end that functions as a primer for a reverse transcription reaction that takes place directly on the tissue. Each concatemer has a UMI, and the expressed mRNA is attached to and captured by each concatemer. 6. cDNA synthesis of all captured mRNA strands containing cleavable sequences fragmented into loroney and possessing UMI. 7. After pre-staining with DAPI, image the entire tissue. Add sequencing primers to each lorony, then sequence the unique molecular identifier (UMI) for all lorony. Record the x and y coordinates of each lorony. 8. Extract DNA from the tissue slide. This contains a reverse-transcribed cDNA strand with a specific UMI. Further downstream library preparation. Rollonie reconstruction and subsequent ex situ sequencing of each concatemer again. Sequence the UMI again, and also sequence a portion of the target region of the captured mRNA. The x,y coordinates of the tissue location from which the mRNA originates can then be matched with the UMI determined in step 7.

[0036] Examples Experiments were conducted to verify the approach described in this application. These experiments were simplified, and the oligomers used contained only a very short unique molecular identifier and a sequencing primer region. The target tissue type in this application is formalin-fixed paraffin-embedded (FFPE) tissue. Therefore, the described process begins with FFPE tissue from a tissue block. In this particular example, it is an FFPE tonsil block.

[0037] Prepare the tissue, section it thinly with a microtome, and place it on a clear glass slide. The tissue block embedded in paraffin wax is trimmed with a microtome to remove excess paraffin and expose the tissue surface. This process, known as "surface preparation," creates a smooth, flat surface. To facilitate sectioning and improve section quality, the tissue block is often cooled, and in this embodiment, frozen at -20°C. The block is secured in the microtome chuck and held in a stable position to prevent movement during sectioning. Each section is set to the desired thickness, typically 4–10 micrometers (μm), in the microtome.

[0038] The microtome mechanism gradually advances the block towards a sharp steel or disposable blade. As the block advances, the blade bites into the tissue, creating thin, ribbon-like sections. These ribbons are then floated in a warm water bath. This helps flatten the tissue sections by gently loosening the paraffin. Individual sections are then removed from the water bath using a glass slide, typically with the tissue side facing up. The glass slides are then dried and stored at 4°C until use.

[0039] Further prepare the transparent glass slide on which the tissue is placed. The glass slides containing the tissue sections are typically placed in xylene or a similar solvent to dissolve and remove the paraffin (wax). The tissue is then rehydrated with a stepwise series of ethanol (e.g., 95%, 90%, 70%, 50%). Finally, the slides are rinsed with water to remove all residue.

[0040] Another crucial step in tissue preparation is to break the crosslinks formed during fixation. The tissue is heated in a buffer solution at a controlled pH. This can be done using a commercial pressure cooker or industrial steamer (water temperature between 95°C and 99°C).

[0041] The tissue may be treated with an RNase inhibitor to stabilize its RNA.

[0042] Roronie generation on tissue for mRNA detection In this experiment, four different mRNAs were targeted and detected directly in tissue. Therefore, the oligomers used contain a very short unique molecular identifier and sequencing primer region, as well as oligos in the arms of the padlock probe, designed to bind to the target mRNA on the target gene. When the probe encounters its mRNA target, both ends hybridize in a "lock and key" manner.

[0043] After hybridization, the two arms are ligated using a DNA ligase enzyme to form a circular probe. Following ligation, the circular probe serves as a template for rolling circle amplification (RCA). This prepares the tissue for the sequencing-by-synthesis step after the addition of sequencing primers to each rollony.

[0044] Whole tissue scan to obtain the morphology and location of each cell. Before performing sequencing-by-synthesis, the entire tissue is imaged using a fluorescence microscope.

[0045] Cells or tissues can be stained with DAPI (4',6-diamidino-2-phenylindole) to visualize the cell nucleus, or with an antibody marker to stain the cell membrane.

[0046] Figure 7 shows a fluorescence microscope image of an entire 3 × 7 mm tonsil tissue thin section with the nuclei stained with DAPI. Cells or tissues can be stained with DAPI (4',6-diamidino-2-phenylindole) to visualize the cell nuclei, or with an antibody marker to stain the cell membrane.

[0047] After imaging the entire tissue, sequencing bisynthesis can be performed to determine the sequence of single-stranded DNA.

[0048] Sequencing-by-synthesis is performed to identify the spatial location of mRNA within the tissue and UMI. The sequencing-by-synthesis process uses fluorescently labeled nucleotides. DNA polymerase sequentially incorporates each labeled nucleotide into the DNA strand based on a template, and a specific fluorescent signal is recorded after each cycle.

[0049] Figure 6 shows sequencing-by-synthesis on tissue for multiple cycles, illustrating Roronie's fluorescence with different bases A, C, G, or T.

[0050] As shown in the photograph, stained cells are directly visualized in each fluorescence channel. Applying sequencing-by-synthesis chemistry in each cycle reveals that Rorony's cells fluoresce in different channels.

[0051] Image software algorithms overlay images of the G, T, A, and C channels in each cycle to determine the sequence of each Roronie (e.g., "GCTAGCTT..."). Roronie fluoresces in either the G, T, A, or C channel, depending on the cycle. Along with the sequence, the spatial coordinates are recorded in x and y. In this way, each unique molecular identifier is identified, and its location in the tissue is determined.

[0052] This is an example of how to determine UMI codes for spatial transcriptome detection in an organization using Rollonie tagging.

[0053] In this case, the sequence is equal to the UMI of this particular Rorony, and together with the tissue image, its spatial location can be determined.

Claims

1. A method for obtaining sequence information and spatial information of at least one RNA strand on a sample immobilized on a surface, a. A step of preparing a cyclic oligonucleotide having 50 to 1000 nucleotides, comprising a complementary cleavable unit having 5 to 50 nucleotides, a poly-A unit having 5 to 350 adenosine nucleotides, a complementary unique molecular identifier unit having 5 to 50 nucleotides, and a complementary sequencing primer unit having 5 to 50 nucleotides. b. A step of amplifying the cyclic oligonucleotide into a plurality of concatemers, each comprising a cleavable unit, a poly-T unit, a unique molecular identifier (UMI) unit, and a sequencing primer unit, using a polymerase capable of rolling circle amplification, thereby obtaining roloney. c. A step of providing the lorony to the sample, thereby hybridizing the lorony to at least one RNA strand via the poly T unit of the concatemer. d. A step of generating a linear oligonucleotide hybridized to at least one RNA strand by enzymatically cleaving the loroney in the cleavable unit of the concatemer. e. A step of forming at least one cDNA strand by extending the hybridized linear oligonucleotide using the mRNA as a template. f. A step of obtaining sequence information of the unique molecular identifier unit and simultaneously detecting the spatial position of the hybridized linear oligonucleotide by performing sequencing bisynthesis of the hybridized linear oligonucleotide in the 3' direction starting from the sequencing primer unit. g. A step of extracting the cDNA from the sample, h. A step of obtaining sequence information of at least one RNA strand by sequencing the cDNA. i. A step of matching the sequence information and spatial information of the unique molecular identifier unit obtained in step f) with the sequence information of the at least one RNA strand obtained in step h). A method that includes this.

2. The method according to claim 1, characterized in that the surface is transparent, and the sequencing bisynthesis in step f) of the hybridized linear oligonucleotide is performed by detecting fluorescence emission through the transparent surface.

3. The method according to claim 1 or claim 2, characterized in that the cleavable unit includes a recognition site for a restriction enzyme selected from the group consisting of EcoRI, EcoRV, BamHI, HindIII, NotI, and PSstI.

4. The method according to any one of claims 1 to 3, characterized in that the severable unit includes the recognition sequence 5'-GAATTC-3'.

5. The method according to any one of claims 1 to 4, characterized in that the enzymatic cleavage of the loroney in the cleavable unit in step d) is carried out by hybridizing oligonucleotides to the cleavable unit and then cleaving them using restriction enzymes.

6. The method according to any one of claims 1 to 5, characterized in that the sequencing bisynthesis of the unique molecular identifier unit in step f) is performed by adding a polymerase capable of incorporating fluorescently labeled nucleotides and unlabeled nucleotides, and the cycle is repeated to determine the sequence of the unique molecular identifier unit.

7. The method according to any one of claims 1 to 6, characterized in that the spatial position of the unique molecular identifier unit in step f) is determined by imaging the immobilized tissue sample in each cycle of the sequencing-by-synthesis process.

8. The method according to any one of claims 1 to 7, characterized in that the lorony is crosslinked with the sample.

9. The method according to any one of claims 1 to 8, characterized in that the sample is stained before the cDNA strand is extracted from the tissue.

10. The method according to any one of claims 1 to 9, characterized in that, after the cDNA strand is removed from the sample, the cDNA strand is amplified by rolling circle amplification prior to sequencing.

11. The method according to claim 10, characterized in that the cDNA strand is amplified by rolling circle amplification by circularizing the cDNA with a ligase enzyme and using the circularized cDNA as a template for RCA.

12. The method according to any one of claims 1 to 11, characterized in that the sequencing of the cDNA in step h) is performed by a sequencing-by-synthesis process.

13. The method according to any one of claims 1 to 12, characterized in that the sample is immobilized on the transparent surface by an APTES, APTEM, silane, polymer, glycol, or thiol coating.

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