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WO2023123065A1 - Système d'imagerie de tissus biologiques fondé sur le séquençage, son procédé d'imagerie et son utilisation - Google Patents

Système d'imagerie de tissus biologiques fondé sur le séquençage, son procédé d'imagerie et son utilisation Download PDF

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WO2023123065A1
WO2023123065A1 PCT/CN2021/142544 CN2021142544W WO2023123065A1 WO 2023123065 A1 WO2023123065 A1 WO 2023123065A1 CN 2021142544 W CN2021142544 W CN 2021142544W WO 2023123065 A1 WO2023123065 A1 WO 2023123065A1
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reaction
fragment
index
sequence
photosensitive
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PCT/CN2021/142544
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Chinese (zh)
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徐臻
吴天准
舒伟良
黄玉斌
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • 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.
  • MERFISH has been commercialized (VizgenMERFISH).
  • 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.
  • 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.
  • 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 ⁇ m, 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.
  • the patent with the international publication number WO 2020/076976 A1 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.
  • WO 2020/076976 A1 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
  • 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.
  • 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.
  • 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.
  • 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.
  • a method for imaging biological tissues based on sequencing comprising the following steps:
  • (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;
  • Steps (4)-(5) are repeated until all coordinate points in the space of the tissue sample are marked with specific nucleic acid markers.
  • 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.
  • 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
  • the 5' end and/or internal site of the index fragment has a photosensitive component
  • 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.
  • 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 DM
  • 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.
  • 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.
  • 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.
  • 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;
  • 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 .
  • 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
  • the next encoding reaction system is a DNA ligase reaction system
  • the next encoding reaction system includes two templates, an index segment and DNA ligase that are different from the previous cycle space encoding
  • 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.
  • the annealing temperature of the template sequence-index fragment complex in the encoding reaction in step (4) is fixed or variable.
  • 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 the difference in the annealing temperature is above 5°C.
  • a biological tissue imaging system based on sequencing 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:
  • 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.
  • Tm(C) and Tm(A), Tm(E) and Tm(C), Tm(D) and Tm(E), and so on are characterized by the difference between Tm(C) and Tm(A), Tm(E) and Tm(C), Tm(D) and Tm(E), and so on.
  • annealing temperature difference above 5°C to achieve the above goals.
  • 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.
  • 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.
  • 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.
  • the present invention has the following beneficial effects:
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • Stabilize biological macromolecules in tissue specimens with fixatives involves immobilizing biomacromolecules in situ (eg, using alcohol-based fixatives) and/or cross-linking different biomacromolecules (eg, using aldehyde-based fixatives).
  • 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.
  • 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.
  • 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.
  • DMNPE 1-(4,5-Dimethoxy-2-Nitrophenyl)ethyl
  • DEACM diethylamino-4-hydroxymethyl -thiocoumarin
  • MCM 7 ⁇ methoxycoumarin ⁇ 4 ⁇ yl]methyl
  • the excitation light wavelengths of the four are 365nm, 420nm, 490nm and 3
  • 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 .
  • the free component such as ATP
  • FIG. 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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
  • 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
  • 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:
  • 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.
  • Tm(C) and Tm(A), Tm(E) and Tm(C), Tm(D) and Tm(E), and so on are characterized by the difference between Tm(C) and Tm(A), Tm(E) and Tm(C), Tm(D) and Tm(E), and so on.
  • 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).
  • Figure 13 shows how to achieve directional ligation of specific index fragments to be ligated by adjusting the reaction temperature.
  • 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.
  • both complex 1 and complex 2 existed stably, and the ligation reaction could not be initiated due to the presence of protective fragment B.
  • 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).
  • 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.
  • all biomacromolecules have been labeled with the first index fragment, and the 5' end of this fragment is protected by a 325nm photocleavage group.
  • 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.
  • 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 .
  • reaction components such as ligase .
  • Fig. 15 shows the flow chart of the coding reaction and the required equipment for coding 16 coordinate points in the same reaction system.
  • 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.
  • a reversible light-controlled allosteric molecule such as a cyclic azobenzene derivative

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Abstract

Système d'imagerie de tissus biologiques fondé sur le séquençage, son procédé d'imagerie et son utilisation, appartenant au domaine technique de la biologie synthétique. Le procédé d'imagerie est fondé sur la technologie de photogravure ou sur une technologie combinée de régulation de la température et de la lumière, une séquence de marquage d'acide nucléique et un anticorps portant la séquence de marquage d'acide nucléique sont reliés à des macromolécules biologiques sur des sites spatiaux prédéfinis, et un codage spatial est effectué sur chaque point de coordonnées d'un échantillon de tissu, afin que tous les points de coordonnées dans l'espace de l'échantillon de tissu soient dotés de marqueurs d'acide nucléique spécifiques. Au moins un des composants de la réaction de codage est encagé ou protégé par un ou plusieurs composants photosensibles, et les marqueurs d'acide nucléique sont bidimensionnels ou tridimensionnels. Le système d'imagerie utilisant le procédé d'imagerie peut atteindre une résolution spatiale élevée, et obtenir la résolution de cellules uniques et de structures sous-cellulaires. En combinaison avec l'analyse de la structure des tissus basée sur le protéome, il est possible de suivre avec précision l'état d'expression du transcriptome d'une structure spécifique dans un tissu et de positionner avec précision différents types d'ARNm dans des structures subcellulaires de taille micrométrique.
PCT/CN2021/142544 2021-12-29 2021-12-29 Système d'imagerie de tissus biologiques fondé sur le séquençage, son procédé d'imagerie et son utilisation WO2023123065A1 (fr)

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