WO2023123065A1

WO2023123065A1 - Sequencing-based biological tissue imaging system, imaging method thereof and use thereof

Info

Publication number: WO2023123065A1
Application number: PCT/CN2021/142544
Authority: WO
Inventors: 徐臻; 吴天准; 舒伟良; 黄玉斌
Original assignee: 深圳先进技术研究院
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-07-06

Abstract

A sequencing-based biological tissue imaging system, an imaging method thereof and the use thereof, belonging to the technical field of synthetic biology. The imaging method is based on photoetching technology or combined temperature control and light control technology, a nucleic acid labeling sequence and an antibody carrying the nucleic acid labeling sequence are connected to biological macromolecules at preset spatial sites, and spatial coding is carried out on each coordinate point of a tissue sample, so that all coordinate points in the space of the tissue sample are endowed with specific nucleic acid labels. At least one of coding reaction components is caged or protected by one or more photosensitive components, and the nucleic acid labels are two-dimensional or three-dimensional. The imaging system using the imaging method may achieve high spatial resolution, and achieve the resolution of single cells and subcellular structures. In combination with proteome-based tissue structure analysis, the transcriptome expression condition of a specific structure in a tissue can be accurately tracked, and accurate positioning of different types of mRNAs in micron-sized subcellular structures can be realized.

Description

A sequencing-based biological tissue imaging system and its imaging method and application

technical field

The invention belongs to the technical field of synthetic biology, and in particular relates to a sequencing-based biological tissue imaging system and its imaging method and application.

Background technique

Accurately obtaining multi-omics spatio-temporal information of cellular substructures (such as the processes of neurons, the myelin sheath structure of oligodendrocytes, etc.) is a major problem in omics technology. Among the conventional techniques, in situ hybridization and immunohistochemistry are traditional molecular imaging techniques that rely on microscopes, but their throughput is low and they are not suitable for omics research. For example, high-throughput in situ hybridization technology (such as MERFISH) based on multiple rounds of combined encoding of nucleic acid tag sequences has achieved high-resolution transcriptome capture based on single-molecule imaging technology, which can resolve mRNA identities in cell substructures. Among many related technologies, MERFISH has been commercialized (VizgenMERFISH). However, this technology relies on known gene sequences, not de novo sequencing, and requires the synthesis of a large number of gene-specific probes, which has high synthesis costs and low parallel throughput. At the same time, the compatibility with proteomics technology is poor, and it is difficult to display cell substructure information in situ.

The principle of another high-resolution transcriptome capture technology is in situ sequencing, based on gene-specific probes, using probes as primers, and rolling circle replication to synthesize a large number of gene-specific tag sequences, and then using ligation-sequencing and The imaging method performs in situ sequencing of gene-specific tags to determine the spatial distribution of related mRNA molecules. Related products include CORTANA from 10x Genomics. FISSEQ, which is also based on the principle of in situ sequencing, uses random primers and rolling circle replication to amplify mRNA sequences, and then uses ligation-sequencing and imaging methods to determine the spatial distribution of related mRNA molecules and achieve high-resolution transcriptome analysis. However, although this technology is de novo sequencing, its sensitivity is poor, and it also has the problems of low parallel throughput and difficulty in displaying cell substructure information in situ. In addition, if the spatial encoding technology based on nucleic acid tag sequences can break through the micron scale, it can also reconstruct the location and identity information of individual biological macromolecules based on sequencing information, forming high-resolution images similar to in situ hybridization and immunohistochemical images. Flux biomacromolecule distribution images. The commercial product based on spatial coding technology is 10x Genomics Visium. However, its resolution is 100 μm, which cannot meet the micron and submicron scale requirements of cell substructure, and the transcriptome capture is extremely limited. The DBiT-seq technology based on microfluidic technology also has resolution problems, and has strict requirements for preventing cross-flow and blocking.

In the prior art, the patent with the international publication number WO 2020/076976 A1 (the applicant is READCOOR company incubated by MIT, which has been acquired by 10x Genomics) discloses: (1) using a synthetic transparent three-dimensional matrix to embed cells or Cell derivatives (such as mitochondria, exosomes, etc.), in which many spatially indexed nucleic acid molecules with a certain spatial position relationship are embedded in the three-dimensional matrix, (2) determine the spatial position of each element of the nucleic acid molecule assembly in the three-dimensional matrix, (3 ) Extracting the aforementioned nucleic acid molecule set from the three-dimensional matrix, (4) Determining the sequence of each element in the aforementioned nucleic acid set. The process of realizing (2) includes using the probe molecules in the three-dimensional matrix to determine the spatial position relationship of the index nucleic acid molecules. The patent first uses probes on an optical imaging platform to determine the spatial distribution of index nucleic acid molecules in a three-dimensional matrix. Since nucleic acid index fragments are connected to biological macromolecules, in vitro high-throughput deep sequencing can be used, based on index nucleic acid sequence-spatial coordinates Mapping relationship to determine the spatial relationship between biomacromolecules. The coupling between the three-dimensional matrix and the index nucleic acid molecules is carried out through artificially controllable reactions such as light-controlled reactions and heat-controlled reactions. However, for READCOOR’s spatial encoding patent, it is first necessary to use probes for in situ sequencing on an optical imaging platform to determine the spatial distribution of index nucleic acid molecules in a three-dimensional matrix and generate index nucleic acid sequence-spatial coordinate mapping relationships. Determination of spatial coordinates of biomacromolecules by in vitro sequencing. Its process is very cumbersome and complicated. The purpose of using light control technology is to anchor the index nucleic acid molecules to the three-dimensional matrix of embedded cells uniformly and controllably, so that a certain spatial position relationship is formed between the index fragments: since the three-dimensional matrix is a solid phase, the index Nucleic acid molecules are in liquid phase, and the two are directly mixed, so it is difficult to achieve uniform anchoring of indexing nucleic acid molecules in the matrix; it is necessary to pre-mix with indexing nucleic acid molecules before the matrix is solidified, and then use artificial stimulation such as light or heat to start indexing nucleic acid molecules Anchoring to the substrate. The advantage of using light control is that the anchoring response can be initiated at specific coordinates. However, due to the mixing of the matrix and the index fragments, it is impossible to anchor the index fragments of a specific sequence at specific spatial coordinates, and it is necessary to determine the index fragment-spatial coordinate mapping relationship through subsequent in situ sequencing.

Contents of the invention

In view of the above-mentioned problems in the prior art, the purpose of the present invention is to design and provide a biological tissue imaging system based on sequencing and its imaging method and application. Based on photolithography technology, the present invention uses photo-uncaging or temperature control in combination with photo-uncaging nucleic acid fragment synthesis reactions to accurately connect nucleic acid marker sequences and antibodies carrying nucleic acid marker sequences to biological macromolecules at predetermined spatial locations ( Nucleic acid, protein, etc.), use different fragment combination sequences to encode the coordinates of each point of biological tissue in high-density space (resolution <1 micron), and analyze the labeled biological macromolecules on the micron and sub-micron scales by sequencing Identity and its spatial location to reconstruct high-throughput molecular images of tissue specimens.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for imaging biological tissues based on sequencing, comprising the following steps:

(1) Take a tissue sample, and use a fixative to stabilize the biological macromolecules in the tissue sample;

(2) performing initial index marking on the omics information in the tissue specimen of the stabilized biomacromolecule obtained in the above step (1);

(3) fully filling the coding reaction system into the tissue sample obtained through the above step (2), wherein at least one component in the coding reaction system contains one or more photosensitive components caged or protected;

(4) Use a specific shape mask to cover the tissue sample processed in the above step (3), and irradiate part of the spatial coordinate X of the covered tissue sample with light of one or more wavelengths to start the coding reaction, and the light All the omics information at the coordinate X of the irradiation space is marked with the index fragment XN ⁰ of the known sequence, thoroughly cleaned, and added to the next encoding reaction system, wherein at least one component in the next encoding reaction system contains one or more photosensitive ingredients;

(5) switch the position of the mask plate, change the spatial site of one or more wavelengths of light irradiation, and mark the index segment YN ⁰ of known sequence for all the omics information at the spatial coordinate Y of the light irradiation;

Optionally, (6) overlapping the tissue samples obtained in step (5) according to the equal proportion of the mask position, and marking ZN ⁰ for all omics information;

(7) Steps (4)-(5) are repeated until all coordinate points in the space of the tissue sample are marked with specific nucleic acid markers.

In the imaging method, the stabilization in the step (1) includes immobilizing biomacromolecules in situ and/or cross-linking different biomacromolecules, and the method of immobilizing biomacromolecules in situ includes using alcohols to immobilize Methods for crosslinking different biomacromolecules include the use of aldehyde fixatives.

In the imaging method, the method of initially indexing the omics information in step (2) includes: reverse transcribing the mRNA into cDNA and linking it to the index fragment, and using an antibody carrying the index fragment to mark the protein, the The index fragment is a nucleic acid fragment, and the 5' end and/or internal site of the index fragment has a photosensitive component, and the sequence from the 5' end to the 3' end of the index fragment is: a fixed linker sequence, a variable coding sequence, and a variable linker sequence.

In the imaging method, the photosensitive components in the steps (3) and (4) include photocutting groups/photosensitive switch groups/photochromogenic groups, photosensitive metal ion chelating agents, photosensitive nucleic acid binding molecules and photosensitive At least one of the protonogenic reagents, preferably photocleavable groups including 1-(4,5-Dimethoxy-2-Nitrophenyl)ethyl (DMNPE), [7‐(diethylamino)coumarin‐4‐yl]methyl (DEACM) , one or more of 7-diethylamino-4-hydroxymethyl-thiocoumarin (Thio-DEACM), 7‐methoxycoumarin‐4‐yl]methyl (MCM), etc., the excitation wavelengths of the four are 365nm, 420nm, 490nm, respectively and 325nm, respectively used to cage different nucleic acid fragments for monochrome or multicolor lithography, preferably photocleavage group cage control ATP for monochrome lithography (such as DMNP-ATP), photosensitive switch groups (such as azobenzonzenyl, arylazopyrazole and its derivatives, etc.) to control the activation or depolymerization of the template-connection complex, photochromogenic groups (such as Diarylethene and its derivatives, etc.) control the melting temperature of the template-connection complex, and photosensitive chelators (such as DMNP-EDTA) controls the amount of Mg ²⁺ ions to perform monochrome photolithography, and the photoinduced nucleic acid binding molecules (including azobenzene derivatives, etc.) control the activation or depolymerization of the template-linkage complex.

In the imaging method, the encoding reaction in the step (4) includes one of an enzymatic reaction and a DNA fragment synthesis reaction, preferably the enzymatic reaction includes a DNA ligase reaction, a DNA terminal transferase reaction, and the DNA fragment synthesis reaction includes DNA chemical synthesis reaction.

In the imaging method, when the coding reaction in the step (4) is an enzymatic reaction, the coding reaction system in the step (3) includes one of a substrate carrying a photosensitive component, a catalyst carrying a photosensitive component or A variety of substrates preferably carrying photosensitive components and catalysts carrying photosensitive components include nucleoside triphosphates or activated derivatives thereof with photosensitive components, nucleoside triphosphates and derivatives thereof activated by light-controlled protons, 3' end Or the nucleic acid fragment whose 5' end or interior is protected by the photosensitive component, the nucleic acid fragment whose melting temperature is regulated by the photosensitive component, and the multivalent metal ion caged by the photosensitive component.

In the imaging method described above, when the coding reaction in the step (4) is a DNA ligase reaction, the components of the coding reaction system in the step (3) include a marker fragment, an index fragment, and a template sequence, preferably the template sequence starts from the 5' The end to the 3' end sequentially include a sequence fragment complementary to the variable joint sequence of the index fragment, and a sequence fragment complementary to the fixed variable joint sequence of the index fragment.

In the imaging method described above, when the coding reaction in the step (4) is a DNA terminal transferase reaction, the coding reaction system in the step (3) includes a polychromatic photosensitive component caged with four kinds of nucleoside triphosphates or activated Derivatives for four-color lithography, or monochrome photosensitive components cage four nucleoside triphosphates or Mo ²⁺ for monochrome lithography;

In the imaging method described above, when the coding reaction in the step (4) is a DNA chemical synthesis reaction, the coding reaction system in the step (3) includes polychromatic photosensitive components caged with four kinds of nucleoside triphosphates or their activated derivatives four-color lithography, or monochrome photosensitive components cage four nucleoside triphosphates or their activated derivatives for monochrome lithography (including phosphate and hydroxyl), or monochrome photosensitive components locally generate protons to initiate synthesis reactions .

In the imaging method, the next encoding reaction system in the step (4) includes an index fragment different from the previous circular spatial encoding, a new template adapted to the index fragment and a complementary fragment used to prevent the previous spatial encoding from being polluted, The 3' end of the index fragment has a photosensitive component;

In the imaging method, when the next encoding reaction system is a DNA ligase reaction system, preferably the next encoding reaction system includes two templates, an index segment and DNA ligase that are different from the previous cycle space encoding, and the two templates include: One template is a template complementary to the 3' end sequence and the 5' end sequence of the index fragment, and the second template is a template complementary to the 3' end sequence of the biomacromolecule straight chain fragment and the 5' end sequence of the index fragment.

In the imaging method, the annealing temperature of the template sequence-index fragment complex in the encoding reaction in step (4) is fixed or variable.

In the imaging method, when the annealing temperature is variable in the step (4), the annealing temperature is 25°C<Tm(B)<Tm(A)<Tm(C)<Tm(E)<Tm( D) <70°C, A is the complementary strand of the 5'-end sequence of the previous round of coding products, B is the 3'-end protection fragment of the index fragment, C is the 3'-end complementary strand of the index fragment to be connected, D is the index fragment to be connected, E For a template sequence, the difference in the annealing temperature is above 5°C.

A biological tissue imaging system based on sequencing, the biological tissue imaging system includes a microfluidic module, a photolithography machine module and a temperature control module.

The application of the sequencing-based biological tissue imaging system to track the expression of transcriptomes in tissue structures at the resolution of single cells and subcellular structures, and to locate different types of mRNAs in micron-scale subcellular structures.

The described sequencing-based biological tissue imaging system tracks the expression of transcriptomes in tissue structures at the resolution of single cells and subcellular structures, locates different types of mRNA in micron-scale subcellular structures, and can be used in cell classification and subcellular structures. and applications in sub-1 micron resolution for multi-omics.

The 5' end of the index fragment carries a photosensitive component, and its pairing method meets the following conditions:

①25℃<Tm(B)<Tm(A)<Tm(C)<Tm(E)<Tm(D)<70℃

②The difference between Tm(A) and Tm(B) enables the existence of the following temperature range: B is completely dissociated into single chains, while A can maintain a double chain state. Similarly, the difference between Tm(C) and Tm(A), Tm(E) and Tm(C), Tm(D) and Tm(E), and so on. Generally speaking, when designing the sequence, keep the annealing temperature difference above 5°C to achieve the above goals.

In the iterative lithography mode, if there are n kinds of photosensitive components used for light-controlled release, each of which can be decomposed by light of different wavelengths, the first round of reaction divides the sample space into n+1 parts, and the second round In response, all the subspaces in n+1 parts are divided into n+1 parts again, and so on, until the set minimum photolithography range (for example, 1 micron) is reached.

The sequencing-based biological tissue imaging technology of the present invention includes using high-resolution (less than 1 micron) maskless lithography technology to label biological macromolecules (including mRNA and protein) with identity tag sequences in tissues with different spatial coordinates DNA sequence, by sequencing the coordinate labels and identity, restores the identity and position information of biological macromolecules, and reconstructs it into a biological tissue image (two-dimensional or three-dimensional). The tag sequence information coupled with a specific protein molecule can be used as a localization mark of the cell substructure in the tissue, while the tag sequence information coupled with the mRNA is superimposed on the spatial information of the specific protein molecule in the cell substructure.

The present invention utilizes photolithography technology to realize multi-omics high-resolution (below 1 micron) pre-deterministic spatial encoding. Coordinate-oriented links to specific index fragments. The spatial coordinates of tissue sample multi-omics are encoded entirely by the ordered combination of pre-designed light spatio-temporal patterns and indexed nucleic acid fragments of known sequences. If different lithographic resolutions are set for adjacent slices of the same sample (such as below 1 micron and above 10 microns), tissue imaging at different scales can be realized, which can be used to study large-scale cell classification and small-scale subcellular structure respectively. .

The present invention utilizes a set of mask plates, through time sequence combination of different mask plates switching, combined with light-controlled nucleic acid fragment synthesis reaction, and based on fragment sequence, realizes spatial coding of multi-omics limited coordinates of biological tissue. The invention utilizes the maskless photolithography technology to realize the mask plate with any image pattern. Through the time series combination of different mask switching, combined with the light-controlled nucleic acid fragment synthesis reaction, based on the fragment sequence, high-resolution spatial encoding of arbitrary coordinates of biological tissue multi-omics is realized. The time series of the mask plate is formed, and the time required for spatial encoding and the number of reactions are shortened by combining encoding.

In the present invention, complex time series of mask plates are formed through repeated iterations of the same photolithography pattern, and multi-omics space coding is performed through combined coding, thereby simplifying the design of photolithography patterns.

The present invention utilizes the multi-band maskless photolithography technology to form a more complex time sequence of mask plates, and further shortens the time required for spatial encoding and the number of reactions by combining encoding. Correlative optical pathways enabling multi-omic spatial encoding based on multi-band maskless lithography. Use nucleic acid labeling fragments with a quantity within two digits to carry out light-controlled biomacromolecular nucleic acid labeling reactions.

The present invention uses multicolor photosensitive components to cage different marker segments or different nucleoside triphosphates, uses different excitation lights and maskless photolithography to combine complex spatio-temporal patterns, and further shortens the time required for spatial encoding.

The invention integrates microfluidic control and photolithography systems, and automatically completes multiple cycles of injection-photolithography-cleaning.

The present invention performs spatial encoding by de novo sequencing rather than in situ hybridization.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention utilizes light-controlled reaction to in situ synthesize a predetermined nucleic acid tag sequence on a biological macromolecule of a tissue sample. Synthesize nucleic acid tags (and ordered combinations) of specified sequences at any specified spatial location in the tissue sample. Through repeated iterations of the same lithography pattern, a complex time sequence of mask plates is formed, and multi-omics space coding is performed through combination coding, which simplifies the design of lithography pattern sequences. The use of multi-band maskless lithography technology and different reaction substrates caged with multi-color photosensitive components shortens the number of reactions and costs.

2. The imaging system of the present invention can achieve high spatial resolution, which is only limited by the photolithographic resolution and the diffusion range of the photolytic substrate, and can realize the resolution of single cells or even subcellular structures. The resolution can be adjusted. If the resolution is set to a cell diameter scale of about 15 microns, the transcriptome can be captured in a larger range, limited only by the working range of the lithography machine, which can reach a diameter of more than 10 cm.

3. Combined with tissue structure analysis, it is possible to accurately track the transcriptome expression of specific structures in the tissue (such as blood vessels, etc.). Precisely locate the location of different types of mRNA in micron-scale subcellular structures (such as the protrusions of neurons, astrocytes and microglia, and the myelin sheath formed by oligodendrocytes, etc.).

4. The imaging system of the present invention performs spatial encoding through de novo sequencing instead of in situ hybridization, and the sequencing throughput is higher. Multiple tissue samples can be encoded simultaneously. The transcriptome that can be captured by a single cell is more complete and is not limited to where the surface of the tissue section touches the solid-phase probe.

Description of drawings

Figure 1 is the principle of sequencing-based cell imaging technology;

Figure 2 is a schematic diagram of DNA fragment ligation reaction;

Figure 3 is a schematic diagram of light-controlled ATP release based on DMNPE photocleavage groups;

Figure 4 is a flow chart of spatial encoding by 4 x 4 coordinate single-wavelength lithography based on DNA ligation reaction conditions;

Fig. 5 is a schematic diagram of lithography mode iteration under single-wavelength lithography conditions;

Fig. 6 is a schematic diagram of lithography mode iteration under multi-wavelength parallel lithography conditions;

FIG. 7 is a multi-wavelength parallel lithography optical path diagram;

Figure 8 is a working cycle diagram of the microfluidic-lithography integrated system;

Fig. 9 is a schematic diagram of using complementary sequences to neutralize residual fragments in the last round of reactions;

FIG. 10 is a schematic diagram of a photolithography iteration mode;

Fig. 11 is a schematic diagram of photolithography iteration results;

Figure 12 is the design of the segment to be connected-connection template complex;

Figure 13 is a schematic diagram of the annealing temperature of the temperature-regulated junction complex;

Fig. 14 is a schematic diagram of the coding reaction of one-time coding 16 coordinate points;

Figure 15 is the encoding flow chart and equipment diagram, where A is the flow of the encoding reaction for one-time encoding of 16 coordinate points, and B is the connection of the equipment required for the above reaction.

Detailed ways

The present invention will be further described with reference to the accompanying drawings and examples below.

Example 1:

In order to use different nucleic acid sequences to carry out spatial encoding of biological tissue macromolecules and realize sequencing-based biological tissue imaging, the following steps need to be completed:

1. Stabilize biological macromolecules in tissue specimens with fixatives. Stabilization involves immobilizing biomacromolecules in situ (eg, using alcohol-based fixatives) and/or cross-linking different biomacromolecules (eg, using aldehyde-based fixatives).

2. Perform initial index labeling of multiple omics information in tissue samples, including reverse transcription of mRNA into cDNA and connection of common adapters (with known nucleic acid sequences), labeling various proteins with antibodies carrying index tags, etc. The above-mentioned index fragments are short fragments of nucleic acid, at least one type of its 3' end or 5' end or internal sites can be protected by photosensitive components.

3. Fully fill the reaction system required for spatial encoding into the biological tissue sample. In the encoding reaction system, some components are clathrated with photocutting groups.

4. Utilize light of a specific wavelength (such as 355nm) or its combination and a mask with a specific pattern to irradiate a biological tissue sample (such as a two-dimensional slice) at a specific spatial coordinate X, and release the photocutting group cage at the coordinate X The composition is synthesized, the encoding reaction is initiated, and all mRNAs at coordinate X are tagged with an index fragment XN ⁰ of known sequence. Thoroughly wash the tissue sample and inject the next round of coding reaction system (including photosensitive components and nucleic acid markers of known sequence).

5. Switch the mask, change the spatial position of light irradiation, and mark the nucleic acid marker fragment YN ⁰ on another spatial coordinate Y of the biological tissue sample (eg, a two-dimensional slice).

6. Repeat steps 4 and 5 until all coordinate points in the tissue sample space are marked with specific nucleic acid markers.

The encoding reaction can be an enzymatic reaction, including DNA ligase reaction and DNA terminal transferase reaction, or other synthesis methods of DNA fragments that can be regulated by light. The photosensitive component can be a nucleoside triphosphate with a photocleavable group, a nucleic acid fragment protected by a photocleavable group at the 3' end or 5' end or internally, a multivalent metal ion caged by a photocleavable group, etc. Photosensitive substrates or catalysts, and other components carrying photocleavable groups necessary for enzymatic reactions. These components dissociate from the photocleavage group and initiate an enzymatic reaction when exposed to light of a specific wavelength. Commonly used photocleavage groups are 1-(4,5-Dimethoxy-2-Nitrophenyl)ethyl (DMNPE), [7‐(diethylamino)coumarin‐4‐yl]methyl (DEACM), 7-diethylamino-4-hydroxymethyl -thiocoumarin (Thio-DEACM), 7‐methoxycoumarin‐4‐yl]methyl (MCM), etc. The excitation light wavelengths of the four are 365nm, 420nm, 490nm and 325nm respectively, which can be used to cage four nucleoside triphosphates and different nucleic acid fragments respectively. Among them, the use of DMNPE is the most common.

Example 2: Taking the nucleic acid fragment synthesis reaction mediated by DNA ligase as an example, the free component (such as ATP) of the photocleavage group caged coding reaction is used to realize the light-controlled release of ATP, and realize the spatial coding of 16 coordinate points .

(1) The reaction process of nucleic acid fragment synthesis mediated by DNA ligase is shown in FIG. 1 . Figure 2 is a schematic diagram of the DNA fragment ligation reaction. The necessary components include magnesium ions, ATP, the 3' terminal hydroxyl of the index fragment, the 5' terminal phosphate of the last round of encoded product fragments, DNA ligase, and template nucleic acid fragments complementary to the index fragment and the last round of encoded product fragments . If ATP is caged by the photocleavage group DMNPE, ATP can be released only under the condition of 365nm ultraviolet irradiation to activate the ligation reaction, as shown in Figure 3. Since microscopy technology can control the range of ultraviolet irradiation to the submicron level, and maskless lithography technology can irradiate in parallel at any spatial position and realize complex temporal and spatial sequences, therefore, labeling fragments and elution processes by replacing different nucleic acid sequences , which can be used to add specific index labels to any spatial coordinates of tissue samples to achieve high-resolution spatial encoding. The encoding process of 16 coordinate points (4x4) in the tissue sample space is shown in Figure 4.

(2) It can be seen that under the condition of using single-wavelength excitation light (365nm), the light control can produce two states (365nm on, 365nm off), and its illumination mode can be iterated in 2 points according to Figure 5 until the light spot is only Limited to a single coordinate. Therefore, a total of 4 rounds of reactions are required for 16 coordinate points, and it can be obtained by induction that the number of reactions n required to encode N coordinate points is:

n=log ₂ N

(3) To encode 16384 (128x128) coordinate points, 14 rounds of reactions are required. If each round of reaction takes 10 minutes and elution takes 5 minutes, the entire encoding process takes 210 minutes. The coding resolution of this patent can achieve sub-micron level. If the coordinate resolution of a single point is set at 1 micron, the coding system of this patent can cover tissue samples with a diameter range of about 128 microns. If the pixel is increased to 1024x1024 (the range is about 1 mm in diameter, the level of the 10x lens of an ordinary microscope), under the condition of single-wavelength light control, 20 rounds are required, which takes 5 hours.

(4) If the light-controlled intermediary is changed to the free hydroxyl group at the 3' end of the nucleic acid labeling fragment, and multi-band light is used, the encoding process can be further shortened. If in each round of reaction, three kinds of nucleic acid marker fragments with different sequences are simultaneously injected, and each fragment is caged with photocleavage groups with different sensitive bands, then four different photocontrol states (wavelength 1 to 3, and all off), then the number of responses n required to encode N coordinate points is:

n=log ₄ N

(5) To achieve 1024 x 1024 coordinate point encoding, 10 rounds of responses are required, which takes 150 minutes. Its lithography iteration mode is shown in Figure 6. Compared with monochromatic light conditions, the time is shortened by half. (6) In order to realize multi-band illumination space encoding, we need to be equipped with light sources of different bands. Taking three-color encoding as an example, we need to use DMNPE (365nm), DEACM (420nm), and Thio-DEACM (490 nm) The photocleavage group modifies the 5'-terminal phosphate groups of the three coding sequences in a certain round of reaction respectively. In order to realize the parallel lithography of light with three wavelengths, the optical path is designed according to Fig. 7 .

If different photolithographic resolutions are set for adjacent slices of the same sample, tissue imaging at different scales can be achieved. The aforementioned methods are all based on sub-micron lithographic resolution for studying subcellular structures. If the lithographic resolution is set to be above 10 microns, the study of cell types can be realized, and 10,000 pixels can cover a sample of about 1 cm or more.

The invention can also be extended to three-dimensional tissue samples. Using structured light technology, complex volume pixel illumination points are produced in three-dimensional space for initiating fragment labeling reactions; or layer-by-layer scanning is performed on the basis of two-dimensional planes. In order to realize the rapid switching of different labeled fragments between each round of reactions, the microfluidic chip system will be used to automatically clean the previous round of reaction components and inject the next round of reaction components. The microfluidic chip system will be integrated with the photolithography system to realize multiple cycles of injection-lithography-cleaning, as shown in Figure 8.

A possible defect of the present invention is that residual labeled fragments from previous rounds of reactions contaminate the next round of reactions, as shown in Figure 9 . Complementary nucleic acid fragments for the previous round of fragments can be used to prevent cross-labeling caused by contamination. The lithography iteration patterns and results are shown in Figures 10 and 11. Similar to the ligation reaction, the deoxynucleotide terminal transfer reaction can also be used for spatial encoding. Its design includes using a four-color photocleavage group to cage four nucleoside triphosphates for four-color lithography; or using a monochromatic photocleavage group to cage four nucleoside triphosphates (including phosphate and hydroxyl), Perform monochrome lithography; or use DMNPE-EDTA, a photosensitive chelating agent, to control the amount of Mo ²⁺ ions to perform monochrome lithography.

Example 3: Taking the synthesis reaction of nucleic acid fragments mediated by DNA ligase as an example, the components anchored to the biomacromolecule (the 5' end of the index fragment) in the photocutting group protection coding reaction are used to realize the light control reaction, or the light Combined with temperature control to realize the spatial encoding of 16 coordinate points

(1) The necessary components for DNA ligase-mediated synthesis of nucleic acid fragments include magnesium ions, ATP, the 3' terminal hydroxyl of the index fragment, the 5' terminal phosphate of the last round of encoded product fragments, DNA ligase, and the same The template nucleic acid fragments complementary to the junction fragments (index fragments, last round coding product fragments). Among them, the 5' ends of the nucleic acid fragments to be ligated (including the last round of coding products and index fragments) are protected by different photocleavage groups (the excitation lights of different photocleavage groups are different); and the template sequence is the same as that to be ligated Complementary pairing between fragments is controlled by reaction temperature. Since the complementary pairing between the template sequence and the fragments to be ligated is necessary for the encoding reaction, we can adjust the reaction temperature to make the specific index fragments to be ligated be ligated in order to further improve the controllability of the spatial encoding. One of its applications is to complete multi-coordinate encoding in the same reaction system without elution. Although its encoding parallelism is lower than the aforementioned multi-color encoding, its spatial encoding accuracy can be greatly improved. Among them, the necessary intermediate of the coding reaction-the template sequence-the DNA duplex formed by the complementarity of the fragments to be ligated, its design is shown in Figure 12, and the key DNA single strands are marked with letters A to E; each DNA single strand is It has its specific annealing temperature. If the following conditions are met, the directional connection of specific index fragments to be connected can be achieved by adjusting the reaction temperature:

①25℃<Tm(B)<Tm(A)<Tm(C)<Tm(E)<Tm(D)<70℃

②The difference between Tm(A) and Tm(B) enables the existence of the following temperature range: B is completely dissociated into single chains, while A can maintain a double chain state. Similarly, the difference between Tm(C) and Tm(A), Tm(E) and Tm(C), Tm(D) and Tm(E), and so on. Generally speaking, when designing the sequence, keep the annealing temperature difference above 5°C to achieve the above goals. The range of 25°C to 70°C is currently the largest range in which DNA ligase can efficiently connect and avoid high temperature inactivation (for example, a combination of T4 DNA ligase, Hi-T4 DNA ligase, and Taq-DNA ligase can be used. over the above temperature range).

(2) Figure 13 shows how to achieve directional ligation of specific index fragments to be ligated by adjusting the reaction temperature. First, assemble a nucleic acid fragment complex composed of the 3' end protection fragment B, the index fragment D to be ligated, and the ligation template E. In FIG. 13, complex 1 and complex 2 were assembled separately (FIGS. 13A1 and A2), then mixed, and injected into the tissue sample encoding reaction system together with ligase, magnesium ions, and ATP. At 25°C, both complex 1 and complex 2 existed stably, and the ligation reaction could not be initiated due to the presence of protective fragment B. After the temperature is set to 30°C, the protected fragment B in complex 1 dissociates, and the encoding reaction is started, and the index fragment D to be ligated in complex 1 is connected to the 5' end of the fragment F of the last round of encoding reaction product; at this time The protective fragment B of complex 2 has not dissociated and thus cannot participate in the ligation reaction (Figure 13B1 and B2). If the temperature is set to 35°C, complex 1 is completely dissociated into a single strand, unable to participate in the encoding reaction, while the protected fragment B of complex 2 just happens to dissociate, and the ligation reaction is initiated, and the index fragment D to be ligated in complex 2 is , connected to the 5' end of fragment F, the product of the previous round of encoding reaction (Fig. 13C1 and C2). It can be seen from Figure 13 that by setting the reaction temperature, the D segment in complex 1 or the D segment in complex 2 can be artificially selected for connection. Combining light control and temperature control, it is possible to select specific index fragments for directional connection at specific coordinates.

(3) Figure 14 shows how to combine light control and temperature control to complete the encoding of 16 spatial coordinates in the same reaction system without elution. Assume that in the initial state, all biomacromolecules have been labeled with the first index fragment, and the 5' end of this fragment is protected by a 325nm photocleavage group. First, 325nm light is used to irradiate a subset of the first area of the tissue sample (such as the left half of the 4x4 grid), and the encoding response of this area is initiated at 35 degrees. Then inject the complex to be connected (divided into four groups a, b, c, d, the number of complexes in each group is 1, 2, 4, 8 respectively, each group has different annealing temperatures, corresponding to specific reaction temperatures, such as a Group b corresponds to 35°C, group b corresponds to 40°C, group c corresponds to 45°C, group d corresponds to 50°C, and the 5' end of each fragment D has photocleavage groups with different wavelengths) and reaction components such as ligase . First, start the ligation reaction of group a complex in the lighted area at 35°C. Then irradiate the second subset of regions with 365nm light, raise the temperature to 40°C, and start the ligation reaction of group b complex in the illuminated region. Then irradiate the third subset of regions with 425nm light, raise the temperature to 45°C, and start the ligation reaction of group C complex in the illuminated region. Finally, 490nm light is used to irradiate the fourth subset of the region, and the temperature is raised to 50°C to initiate the ligation reaction of the group d complex in the illuminated region. The last step of the encoding reaction can omit the photocleavage group at the 5' end of the d group complex fragment D. After the ligation reaction is completed, all the reaction components are eluted, and a new photocleavage group carrying a 325nm, 365nm or 425nm is added. The complex (8 kinds of sequences, monochromatic photosensitive protection group) and the new DNA ligase and other components perform a new round of ligation, so that the 5' end of the index fragment combination sequence anchored in the biomacromolecule can be restored to the photoinduced The protected state of the cleavage group can enter the next round of coordinate-specific encoding reactions. Fig. 15 shows the flow chart of the coding reaction and the required equipment for coding 16 coordinate points in the same reaction system.

(4) The index segment D to be connected is protected by a reversible light-controlled allosteric molecule (such as a cyclic azobenzene derivative). After ultraviolet light irradiation, the index segment is separated from the template segment and cannot participate in the ligation reaction. After visible light irradiation, the index segment is the same as the template The fragments combine to initiate the ligation reaction, eliminating the need to protect the 5' end of the index fragment D of different ligated complexes with a multicolor photocleavage group.

Claims

A method for imaging biological tissues based on sequencing, characterized in that it comprises the following steps:

(1) Take a tissue sample, and use a fixative to stabilize the biological macromolecules in the tissue sample;

(2) performing initial index marking on the omics information in the tissue specimen of the stabilized biomacromolecule obtained in the above step (1);

(3) fully filling the coding reaction system into the tissue sample obtained through the above step (2), wherein at least one component in the coding reaction system contains one or more photosensitive components caged or protected;

(4) Use a specific shape mask to cover the tissue sample processed in the above step (3), and irradiate part of the spatial coordinate X of the covered tissue sample with light of one or more wavelengths to start the coding reaction, and the light All the omics information at the coordinate X of the irradiation space is marked with the index fragment XN 0 of the known sequence, thoroughly cleaned, and added to the next encoding reaction system, wherein at least one component in the next encoding reaction system contains one or more photosensitive ingredients;

(5) switch the position of the mask plate, change the spatial site of one or more wavelengths of light irradiation, and mark the index segment YN 0 of known sequence for all the omics information at the spatial coordinate Y of the light irradiation;

Optionally, (6) overlapping the tissue samples obtained in step (5) according to the equal proportion of the mask position, and marking ZN 0 for all omics information;

(7) Steps (4)-(5) are repeated until all coordinate points in the space of the tissue sample are marked with specific nucleic acid markers.
The imaging method according to claim 1, characterized in that the stabilization in the step (1) includes immobilizing the biomacromolecules in situ and/or cross-linking different biomacromolecules to fix the biomacromolecules in situ The method includes the use of alcohol fixatives, and the method of cross-linking different biomacromolecules includes the use of aldehyde fixatives.
The imaging method according to claim 1, characterized in that the method for initially indexing the omics information in the step (2) comprises: reverse transcribing the mRNA into cDNA and connecting it to the index segment, and using the index segment to carry the index segment The antibody labeled protein, the index fragment is a nucleic acid fragment, the 5' end and/or internal site of the index fragment has a photosensitive component, and the 5' end to the 3' end of the index fragment are: fixed linker sequence, variable coding sequence , the variable linker sequence.
The imaging method according to claim 1, characterized in that in the steps (3) and (4), the photosensitive component comprises a photocutting group/photosensitive switch group/photochromogenic group, a photosensitive metal ion chelating agent , at least one of a photoinduced nucleic acid binding molecule and a photogenerated proton reagent, preferably a photoinduced cleavage group comprising one or more of DMNPE, DEACM, Thio-DEACM, MCM, respectively used to cage different nucleic acid fragments for single Color or multi-color lithography, preferably photocleavage group cage control ATP for monochrome lithography, photosensitive switch group controls activation or depolymerization of template-junction complex, light-controlled chromophore controls template-junction complex The melting temperature of the body, the photosensitive chelator controls the amount of Mg 2+ ions for monochrome lithography, and the photoinduced nucleic acid binding molecules control the activation or depolymerization of the template-linker complex.
The imaging method according to claim 1, wherein the encoding reaction in the step (4) comprises one of enzymatic reaction and DNA fragment synthesis reaction, preferably enzymatic reaction comprises DNA ligase reaction, DNA terminal transferase Reaction, DNA fragment synthesis reaction includes DNA chemical synthesis reaction.
The imaging method according to claim 1, wherein when the coding reaction in the step (4) is an enzymatic reaction, the coding reaction system in the step (3) includes a substrate carrying a photosensitive component, a substrate carrying a photosensitive component One or more of the catalysts, preferably the substrate carrying the photosensitive component and the catalyst carrying the photosensitive component include nucleoside triphosphate or activated derivatives thereof with a photosensitive component, nucleoside triphosphate activated by light-controlled protons and its Derivatives, 3' end or 5' end or nucleic acid fragments protected by photosensitive components, nucleic acid fragments whose melting temperature is regulated by photosensitive components, multivalent metal ions caged by photosensitive components.
The imaging method according to claim 1, wherein when the coding reaction in the step (4) is a DNA ligase reaction, the components of the coding reaction system in the step (3) include a marker fragment, an index fragment, and a template sequence Preferably, the template sequence includes a sequence fragment complementary to the index fragment variable joint sequence and a sequence fragment complementary to the index fragment fixed variable joint sequence sequentially from the 5' end to the 3' end.
The imaging method according to claim 1, wherein when the encoding reaction in the step (4) is a DNA terminal transferase reaction, the encoding reaction system in the step (3) includes four kinds of polychromatic photosensitive components caged three Nucleoside phosphate or its activated derivatives are used for four-color lithography, or four kinds of nucleoside triphosphates or Mo 2+ are caged in monochrome photosensitive components for monochrome lithography.
The imaging method according to claim 1, wherein when the encoding reaction in the step (4) is a DNA chemical synthesis reaction, the encoding reaction system in the step (3) includes polychromatic photosensitive components caged with four kinds of triphosphoric acid Four-color lithography of nucleosides or their activated derivatives, or single-color photosensitive components cage four nucleoside triphosphates or their activated derivatives for monochrome lithography, or monochromatic photosensitive components locally generate protons to initiate synthesis reactions .
The imaging method according to claim 1, characterized in that the next encoding reaction system in the step (4) includes an index segment different from the previous cycle space encoding, a new template adapted to the index segment and used to prevent the last Contaminated complementary fragments are spatially encoded, and the index fragment has a light-sensitive component at the 3' end.
The imaging method according to claim 1, wherein when the next coding reaction system in the step (4) is a DNA ligase reaction system, preferably the next coding reaction system includes two templates, which are different from the previous cycle space coding The index fragment and DNA ligase, two templates include: the first template is the template complementary to the 3' end sequence and the 5' end sequence of the index fragment, and the second template is the 3' end sequence of the linear fragment of the biomacromolecule , and a template complementary to the 5' end sequence of the index fragment.
The imaging method according to claim 1, characterized in that the annealing temperature of the template sequence-index fragment complex in the encoding reaction in the step (4) is fixed or variable.
The imaging method according to claim 1, characterized in that when the annealing temperature is varied in the step (4), wherein the annealing temperature is 25°C<Tm(B)<Tm(A)<Tm(C)< Tm(E)<Tm(D)<70℃, A is the complementary strand of the 5' end sequence of the last round of coding products, B is the 3' end protection fragment of the index fragment, C is the complementary strand of the 3' end of the index fragment to be connected, D is the index segment to be connected, E is the template sequence, and the difference of the annealing temperature is above 5°C.
A sequencing-based biological tissue imaging system is characterized in that the biological tissue imaging system includes a microfluidic module, a photolithography machine module and a temperature control module.
According to claim 8, a sequencing-based biological tissue imaging system tracks the expression of transcriptomes in tissue structures at the resolution of single cells and subcellular structures, and locates the location of different types of mRNA in micron-scale subcellular structures. application.
A sequencing-based biological tissue imaging system as claimed in claim 8 tracks the expression of transcriptomes in tissue structures at the resolution of single cells and subcellular structures, locates different types of mRNAs in micron-scale subcellular structures, and performs cell classification and subcellular structures as well as applications in multi-omics at sub-1 micron resolution.