WO2025138284A1 - Array and method for detecting spatial information of nucleic acids - Google Patents

Abstract

The present application provides a nucleic acid array for detecting spatial information of nucleic acids in a sample, a method for detecting spatial information of nucleic acids in a sample on the basis of the array, and a method for producing the nucleic acid array.

Description

Array and detection method for detecting nucleic acid spatial information Technical Field

The present application relates to the field of biomolecule spatial detection. Specifically, the present application provides a nucleic acid array for detecting nucleic acid spatial information in a sample, a method for detecting nucleic acid spatial information in a sample based on the array, and a method for generating the nucleic acid array.

Background Art

Spatial omics technology is a technology that obtains omics information such as transcriptome, genome, epigenome and proteome in cells in situ in tissues. Spatial omics technology was named the technical method of the year by Nature methods in 2020, and was listed as one of Nature's seven most noteworthy technologies of the year in 2022. Spatial omics technology can be divided into four categories based on different principles, namely microdissection, in situ hybridization, in situ sequencing, chip in situ capture and high-throughput sequencing. Spatial omics technology based on chip in situ capture and high-throughput sequencing occupies a dominant position due to its simple operation and high throughput, among which the capture chip is the key to this technology.

Spatial omics technology based on chip capture uses probes with spatial information on the chip to capture nucleic acids and other molecules in tissue sections in situ, uses high-throughput sequencing technology to obtain spatial information on the chip and nucleic acid molecules in cells, and then restores the molecular information in the tissue through algorithm analysis. Since Joakim Lundeberg of the Royal Institute of Technology in Sweden first published spatial transcriptomics technology in Science in 2016, this field has developed rapidly in recent years due to the advantages of high-throughput whole transcriptome technology. Other similar spatial transcriptomics technologies include HDST technology (High-definition spatial transcriptomics) based on silica magnetic beads, slide-seq technology (sequencing by oligonucleotide ligation and detection) based on barcode microbeads, DBiT-seq technology (Deterministic Barcoding in Tissue) based on microfluidics, Seq-Scope technology based on IllμMina sequencing platform, and stereo-seq technology (spatial enhanced resolution omics sequencing) based on DNB sequencing platform. Among them, Joakim's spatial transcriptomics technology was acquired and commercialized by 10x Genomics at the end of 2018, and the VisiμM product was released in 2019. Based on this technology, 10x Genomics has formed another spatial omics product line in addition to the single-cell production line, including instruments, analysis software and reagents and consumables. At the same time, Biomarker Biotech also released spatial transcriptomics products based on this technology in 2022.

The core of the existing spatial omics technology based on chip in situ capture lies in the resolution and capture efficiency of the capture chip. The resolution of the chip is determined by the spatial information density on the chip, which is related to whether the cells on the tissue slice can be finely analyzed with spatial single cell or subcellular resolution, while the capture efficiency is determined by the capture information on the chip, which is related to whether all molecular information in the cell can be captured without bias. In the existing technology that uses sequencing chips as capture chips, the capture function area and spatial information area of the chip are on the same molecule of the nucleic acid array, but it is limited by The resolution of the sequencing chip is affected. The nucleic acid molecules containing the spatial information region in the nucleic acid array need to be separated by a certain distance to be recognized by the sequencer. If the separation distance is too small, each nucleic acid molecule cannot be accurately identified. When the capture function region and the spatial information region are on the same nucleic acid molecule, the resolution is limited, and the capture efficiency is also limited.

In addition, because existing capture chips are based on poly T probes to capture mRNA, in order to simultaneously obtain the expression map and spatial information of the transcripts, they can only capture the 3’ end transcripts of mRNA, while losing the information of the 5’ end transcripts. For the immune repertoire, important molecular information is concentrated at the 5’ end, so the existing technology is lacking in capturing the 5’ end transcripts.

Summary of the invention

In order to achieve both high-resolution and high-capture efficiency spatial omics technology, the present application splits the capture area and spatial information on the capture chip probe in the prior art into two independent molecules, namely the positioning probe and the capture probe, wherein the positioning probe is used to provide the spatial position information of spatial omics, and the capture probe is used to capture molecules in cells, thereby getting rid of the limitation that the number of capture probes is affected by spatial density, so as to improve the capture efficiency of molecules while ensuring high resolution.

In addition, the nucleic acid array (e.g., capture chip) provided in the present application can also realize the acquisition of 5' transcript information.

Nucleic acid array

Therefore, in one aspect, the present application provides a nucleic acid array for detecting spatial information of nucleic acids in a sample, comprising a solid support to which one or more capture probes and at least two localization probes are attached;

The capture probe and the localization probe are each independently connected to the solid support;

Each localization probe occupies a different position on the solid support;

The positioning probe contains a first universal sequence and a positioning sequence, wherein the positioning sequence has a nucleotide sequence corresponding to the position of the positioning probe on the solid support;

The capture probe contains a capture sequence that is capable of annealing to the nucleic acid molecule to be captured and initiating an extension reaction.

In certain embodiments, each of the localization probes consists of one or more localization probe molecules.

In certain embodiments, the localization sequences contained in localization probe molecules belonging to the same type of localization probe are identical to each other, and the localization sequences contained in localization probe molecules belonging to different types of localization probes are different from each other.

It is easy for a person skilled in the art to understand that one or more localization probe molecules of the same localization probe have the same localization sequence, however, it is not necessary for every localization probe molecule of each localization probe to have the same complete sequence.

In certain embodiments, the localization probe molecules and the capture probes of the nucleic acid array are separated from each other.

In certain embodiments, the first universal sequence is located 3' to the localization sequence.

In certain embodiments, the localization probe further comprises a second universal sequence.

In certain embodiments, the second universal sequence is located 5' to the localization sequence.

In certain embodiments, the second universal sequence comprised by different types of localization probes attached to the solid support is the same or different. In certain embodiments, the second universal sequence comprised by different types of localization probes attached to the solid support is the same. In certain embodiments, the second universal sequence comprised by the same type of localization probes attached to the solid support is the same or different.

In certain embodiments, the first universal sequence comprised by the same type of localization probes attached to the solid support is the same or different.

In some embodiments, the localization probe does not contain or further contains a molecular tag sequence (MID, molecular identifier).

In certain embodiments, the localization probe further comprises a MID sequence.

In certain embodiments, the MID sequence is located at the 5' end of the first universal sequence, and/or, the MID sequence is located at the 3' end of the second universal sequence.

In certain embodiments, cognate localization probes comprise MID sequences that are different from each other.

In certain embodiments, the capture sequence is located at the 3' end of the capture probe.

In certain embodiments, the capture sequence has a free hydroxyl group (-OH) at the 3' end.

In certain embodiments, the capture probe further comprises an immobilization sequence.

In certain embodiments, the fixed sequences comprised by different capture probes attached to the solid support are identical or different. In certain embodiments, the fixed sequences comprised by different capture probes attached to the solid support are identical.

In certain embodiments, the fixed sequence is located 5' to the capture sequence.

In certain embodiments, the capture probe contains a single-stranded region comprising the capture sequence.

In certain embodiments, the capture probe exists in a single-stranded form or in a duplex form containing the single-stranded region.

In certain embodiments, the capture probe is in a single-stranded form, and the capture sequence is located at the 3' end of the capture probe; or

The capture probe exists in the form of a double-stranded body containing a single-stranded region, which includes a first chain directly connected to the solid support and a second chain hybridized with the first chain; and the 3' end of the second chain includes a single-stranded region, and the capture sequence is located at the 3' end of the single-stranded region.

In certain embodiments, the second strand comprises the capture sequence and the immobilization sequence.

In certain embodiments, the nucleic acid array has one or more features selected from the following:

(1) the positioning sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35 or 20-45) random nucleotides, and in certain embodiments, each random nucleotide is independently any one of deoxyribonucleotides A, C, G and T;

(2) The solid support is selected from latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes and silicon wafers; in some embodiments, the solid support is a chip; in some embodiments, the solid support can be used in a sequencing platform; in some embodiments, the solid support is a sequencing chip (e.g., a high-throughput sequencing chip), such as a sequencing chip for an Illumina, MGI or Thermo Fisher sequencing platform (e.g., a high-throughput sequencing chip);

(3) The same type of positioning probes (i.e., positioning probes containing the same positioning sequence) occupy the same area on the surface of the solid support, different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (e.g., less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm);

(4) the first universal sequence is capable of annealing with (i) a nucleic acid molecule captured by the capture sequence, or (ii) a nucleic acid molecule derived from (i);

(5) the first universal sequences contained in the different types of localization probes connected to the solid support are the same or different;

(6) The solid support is capable of releasing the localization probe and/or capture probe spontaneously or when exposed to one or more stimuli (e.g., temperature change, pH change, exposure to specific chemicals or phases, exposure to light, reducing agents, etc.).

In certain embodiments, the localization probe and/or the capture probe is covalently or non-covalently attached to the solid support.

In certain embodiments, the localization probe and/or the capture probe is covalently linked to the solid support.

In certain embodiments, the localization probe and the capture probe are each independently linked to the solid support via the same or different click chemistry reactions.

In certain embodiments, the molecule or group pair capable of the click chemistry reaction is selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol. In certain embodiments, the molecule or group pair capable of the click chemistry reaction is azido/alkynyl.

In certain embodiments, the surface of the solid support is modified with a molecule or group X, and the localization probe is modified with There is a molecule or group Y, the molecule or group X can undergo a click chemical reaction with the molecule or group Y, and the positioning probe is connected to the solid support through the click chemical reaction between the molecule or group X and the molecule or group Y; and/or, the surface of the solid support is modified with the molecule or group X', the capture probe is modified with a molecule or group Y', the molecule or group X' can undergo a click chemical reaction with the molecule or group Y', and the capture probe is connected to the solid support through the click chemical reaction between the molecule or group X' and the molecule or group Y'.

In certain embodiments, the molecules or groups X-Y and X'-Y' are each independently selected from the group consisting of: alkynyl/azide, azide/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azide/alkynyl, cyano/azide, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone.

In certain embodiments, the molecule or group X and/or X' is an azide group, and the molecule or group Y and/or Y' is

In certain embodiments, the surface of the solid support is modified with an azide group, and the localization probe and/or the capture probe is modified with The positioning probe and/or the capture probe is connected to the solid support through a click chemistry reaction between the azide group and the DBCO.

It is easy for a person skilled in the art to understand that any part of the positioning probe can be used to connect to the solid support, as long as the connection does not affect the positioning probe to perform its function (for example, annealing with the target sequence and/or the function of initiating an extension reaction at the 3' end). In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is capable of initiating an extension reaction. In this case, any part of the positioning probe can be used to connect to the solid support, as long as the connection does not affect the annealing of the positioning probe to the target sequence and/or the initiation of an extension reaction at the 3' end. In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is closed. In this case, any part of the positioning probe can be used to connect to the solid support, as long as the connection does not affect the annealing of the positioning probe to the target sequence. In certain embodiments, the positioning sequence of the positioning probe is not directly connected to the solid support. In certain embodiments, the first universal sequence of the positioning probe is not directly connected to the solid support. In some embodiments, neither the first universal sequence nor the localization sequence of the localization probe is directly attached to the solid support. In some embodiments, the localization probe is attached to the solid support via the second universal sequence. In some embodiments, the localization probe is attached to the solid support via the 5' end (e.g., the 5' end) Connected to the solid support.

It is easy for a person skilled in the art to understand that any part of the capture probe can be used to connect to the solid support, as long as the connection does not affect the function of the capture probe (e.g., annealing with the target sequence and the function of the 3' end initiation extension reaction). In certain embodiments, the capture probe exists in a single-stranded form. In this case, any part of the capture probe can be used to connect to the solid support, as long as the connection does not affect the annealing of the capture probe to the target sequence and the function of the 3' end initiation extension reaction. In certain embodiments, the capture sequence of the capture probe is not directly connected to the solid support. In certain embodiments, the capture probe is connected to the solid support via its 5' end (e.g., 5' end).

In certain embodiments, the capture probe exists in the form of a duplex containing a single-stranded region, the duplex comprising a first chain directly connected to the solid support and a second chain hybridized to the first chain, in which case any portion of the first chain can be used to connect to the solid support, as long as the connection does not affect the first chain to perform its function (e.g., the function of annealing to the second chain). In certain embodiments, the first chain is connected to the solid support through a portion that is not annealed to the second chain. In certain embodiments, the first chain is connected to the solid support through its end (e.g., 5' end or 3' end). In certain embodiments, the second chain is not directly connected to the solid support.

In certain embodiments, the distribution of the capture probe and the positioning probe on the solid support is configured as follows: the nucleic acid molecule connected to the capture probe (for example, a nucleic acid molecule annealed to the capture probe, or a nucleic acid molecule obtained by extending the capture probe through a nucleic acid polymerization reaction) is able to contact the positioning probe adjacent to it.

In certain embodiments, each of the positioning probes is adjacent to at least 100 capture probes. For example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 capture probes.

In certain embodiments, there are 500-10,000 (eg, 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed adjacent to each of the localization probes.

In certain embodiments, the expression "in the vicinity of each of the positioning probes" refers to an area on the surface of a solid support near the positioning probe, wherein a nucleic acid molecule (e.g., The nucleic acid molecule to which the capture probe is annealed, or the nucleic acid molecule obtained by extending the capture probe through nucleic acid polymerization reaction) can be contacted with the positioning probe.

In certain embodiments, at least 100 capture probes are distributed within a radius of 200nm-300nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support). For example, at least 100, at least 200, at least 300, 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 of the capture probes.

In certain embodiments, there are 500-10000 (e.g., 1000-1500, 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-8000, 1000-10000) capture probes distributed within a radius of 200nm-300nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support).

As used herein, the expression "the area occupied by each of the positioning probes on the surface of the solid support" or similar expressions has the same meaning as "the position occupied by each of the positioning probes on the solid support" and can be used interchangeably.

In certain embodiments, the nucleic acid array comprises one or more of the capture probes.

In certain embodiments, different species of capture probes contain different capture sequences.

In certain embodiments, the nucleic acid molecule to be captured is RNA (e.g., mRNA), and the capture sequence of the capture probe contains a poly (dT) sequence or a random oligonucleotide sequence;

The nucleic acid molecule to be captured is a target nucleic acid (e.g., a target DNA and/or RNA) or a nucleic acid molecule derived from the target nucleic acid, the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has a target nucleotide sequence contained in the target nucleic acid, and the capture sequence of the capture probe comprises a sequence that can specifically anneal to the target nucleotide sequence; or,

The nucleic acid molecules to be captured are RNA (e.g., mRNA) and target nucleic acids (e.g., target DNA and/or RNA), and the nucleic acid array comprises a first capture probe for capturing RNA (e.g., mRNA) and a second capture probe for capturing the target nucleic acid or a nucleic acid molecule derived from the target nucleic acid, wherein the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has a target nucleotide sequence contained in the target nucleic acid; wherein the capture sequence of the first capture probe contains a poly (dT) sequence or a random oligonucleotide sequence, and the capture sequence of the second capture probe contains a sequence that can specifically anneal to the target nucleotide sequence.

It is easy for a person skilled in the art to understand that, unless otherwise specified herein or clearly contradicted by the context, the above definition/description of the capture probe is also applicable to the first capture probe and the second capture probe.

In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and/or abundance of the nucleic acid.

In certain embodiments, the first universal sequence is a nucleotide sequence consisting of 5-80 (e.g., 5-20, 5-30, 5-40, 5-50, 5-70, 10-30, 10-50, 10-70, 20-30, 20-50, 20-70, 25-40, 25-50, 25-70) nucleotides.

In certain embodiments, the second universal sequence is a nucleotide sequence consisting of 5-80 (e.g., 5-20, 5-30, 5-40, 5-50, 5-70, 10-30, 10-50, 10-70, 20-30, 20-50, 20-70, 25-40, 25-50, 25-70) nucleotides.

In certain embodiments, the fixed sequence is a nucleotide sequence consisting of 5-80 (e.g., 5-20, 5-30, 5-40, 5-50, 5-70, 10-30, 10-50, 10-70, 20-30, 20-50, 20-70, 25-40, 25-50, 25-70) nucleotides.

In certain embodiments, the capture sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) nucleotides.

In certain embodiments, the random oligonucleotide sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35 or 20-45) random nucleotides. In certain embodiments, each random nucleotide is independently any one of deoxyribonucleotides A, C, G and T.

In certain embodiments, the poly (dT) sequence consists of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) thymidine deoxyribonucleotide residues.

In certain embodiments, the first universal sequence, the positioning sequence, the second universal sequence, the fixed sequence, and the capture sequence each independently comprise or do not comprise non-natural nucleotide residues (eg, modified nucleotide residues).

Nucleic acid array preparation method

In another aspect, the present application provides a method for preparing a nucleic acid array as described above, comprising the following steps:

(A) attaching and/or synthesizing a localization probe on a solid support, the localization probe being as defined above; and,

(B) attaching and/or synthesizing a capture probe on a solid support, wherein the capture probe is as defined above;

Wherein, (A) and (B) can be carried out in any order or simultaneously (for example, simultaneously in the same reaction system).

In certain embodiments, (A) is performed before (B), and the solid support in (B) It is a solid support to which the localization probe has been attached.

In certain embodiments, (B) is performed before (A), and the solid support in (A) is a solid support to which the capture probe has been attached.

In certain embodiments, (A) and (B) are performed simultaneously, and the solid phase support in (A) and (B) is the same solid phase support.

In certain embodiments, step (A) comprises:

(1) providing: (a) the free positioning probe, wherein the positioning probe is modified with a molecule or group A; and, (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B, wherein the molecule or group A can form a connection (e.g., a covalent or non-covalent connection) with the molecule or group B; and,

(2) contacting the positioning probe with the solid support under conditions suitable for forming a connection between the molecule or group A and the molecule or group B, thereby obtaining a solid support to which the positioning probe is connected.

In certain embodiments, step (A) further comprises step (3): performing bridge amplification on the positioning probes on a solid support to which the positioning probes are attached, thereby obtaining multi-copy clusters of each positioning probe.

It is easy for a person skilled in the art to understand that any part of the positioning probe can be used to modify the molecule or group A, as long as it does not affect the positioning probe to perform its function (e.g., annealing with the target sequence and/or the function of initiating an extension reaction at the 3' end). In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is capable of initiating an extension reaction. In this case, any part of the positioning probe can be used to modify the molecule or group A, as long as it does not affect the annealing of the positioning probe to the target sequence and/or the initiation of an extension reaction at the 3' end. In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is closed. In this case, any part of the positioning probe can be used to modify the molecule or group A, as long as it does not affect the annealing of the positioning probe to the target sequence. In certain embodiments, the positioning sequence of the positioning probe does not modify the molecule or group A. In certain embodiments, the first universal sequence of the positioning probe does not modify the molecule or group A. In certain embodiments, neither the first universal sequence nor the positioning sequence of the positioning probe modifies the molecule or group A. In certain embodiments, the second universal sequence of the localization probe is modified with the molecule or group A. In certain embodiments, the 5' end (e.g., the 5' terminus) of the localization probe is modified with the molecule or group A.

In certain embodiments, the molecule or group A is capable of undergoing a click chemistry reaction with the molecule or group B.

In certain embodiments, the molecules or groups A/B are selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3/diol-aldehyde, 1,3/diol-ketone.

In certain embodiments, the molecules or groups A/B are azide/alkynyl or alkynyl/azide.

In certain embodiments, the molecule or group A is DBCO and the molecule or group B is azide.

In certain embodiments, the localization probe is modified with The surface of the solid support is modified with an azide group, and the positioning probe and the solid support are connected through a click chemical reaction between DBCO and the azide group.

In certain embodiments, step (A) comprises:

(1) providing at least two vectors, each vector comprising at least one copy of a vector sequence, wherein the vector sequence comprises: a complementary sequence of a positioning sequence and a complementary sequence of a first universal sequence; the first universal sequence and the positioning sequence are as defined above; wherein the complementary sequence of the positioning sequence of each vector sequence is different from each other;

(2) placing at least two of the carriers on the surface of the solid support;

(3) providing a fixed primer, and using the vector sequence as a template, performing a nucleic acid polymerization reaction to generate an extension product, wherein the extension product is a positioning probe; wherein the fixed primer can anneal with the vector sequence and initiate an extension reaction; and,

(4) connecting the immobilized primer to the surface of the solid support;

Wherein, steps (3) and (4) are performed in any order (for example, step (3) is performed before or after step (4), or step (3) and step (4) are performed simultaneously).

In certain embodiments, in step (2), the carrier is placed on the surface of the solid support by forming a connection (eg, a non-covalent connection or a covalent connection) with the surface of the solid support.

In certain embodiments, the vector sequence comprises, in order from the 5' end to the 3' end: a complementary sequence to the universal sequence, and a complementary sequence to the positioning sequence.

In certain embodiments, the extension product comprises, in order from 5' to 3', the positioning sequence, and the first universal sequence.

In certain embodiments, each vector is a DNB formed from concatemers of multiple copies of the vector sequence.

In certain embodiments, the method optionally comprises step (5): digesting the vector sequence, and/or separating (eg, melting) the extension product in step (3) from the vector sequence annealed thereto.

In some embodiments, the vector sequence further comprises a cleavage site. In some embodiments, the cleavage is selected from nicking enzyme cleavage, USER cleavage, photoexcision, chemical excision or CRISPR excision. In some embodiments, in step (5), the cleavage site contained in the vector sequence is cut so that the vector sequence and the extension product of step (3) annealed thereto are separated.

In certain embodiments, the vector is provided in step (1) by:

(i) providing a vector template sequence, wherein the vector template sequence comprises a complementary sequence to the vector sequence;

(ii) performing a nucleic acid amplification reaction using the vector template sequence as a template to obtain an amplification product of the vector template sequence, wherein the amplification product comprises at least one copy of the vector sequence; in certain embodiments, performing a rolling circle replication to obtain a DNB formed by concatemers of the vector sequence.

As used herein, "DNB" (DNA nano ball) is a typical RCA (rolling circle amplification, RCA) product, which has the characteristics of an RCA product. The RCA product is a multi-copy single-stranded DNA sequence, which can form a "spherical" structure due to the interaction between the bases of the internal DNA sequence. Typically, the library molecule is circularized to form a single-stranded circular DNA, and then the single-stranded circular DNA can be amplified by multiple orders of magnitude using the rolling circle amplification technique, and the resulting amplification product is called DNB.

In certain embodiments, the vector sequence further comprises a complementary sequence to a second universal sequence or a partial sequence thereof (e.g., the vector sequence further comprises a complementary sequence to the second universal sequence or a partial sequence at its 3' end), and the second universal sequence is as defined above.

In certain embodiments, the complementary sequence of the second universal sequence or a partial sequence thereof is located 3' to the complementary sequence of the positioning sequence.

In certain embodiments, the fixed primer comprises the second universal sequence or a partial sequence thereof (e.g., the fixed primer comprises the second universal sequence or a partial sequence at the 5' end thereof).

In certain embodiments, the extension product comprises, in order from 5' to 3' direction: the second universal sequence, the positioning sequence, and the first universal sequence.

In certain embodiments, the vector sequence does not comprise or further comprises a template sequence for a MID sequence.

In certain embodiments, the vector sequence further comprises a template sequence of a MID sequence. In certain embodiments, the template sequence of the MID sequence is located at the 3' end of the complementary sequence of the first universal sequence, and/or, the template sequence of the MID sequence is located at the 5' end of the complementary sequence of the second universal sequence or a partial sequence thereof. In certain embodiments, the MID sequence consists of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35 or 20-45) degenerate deoxyribonucleotide residues.

In certain embodiments, the immobilized primer is covalently or non-covalently attached to the solid support.

In certain embodiments, the immobilized primer is covalently linked to the solid support.

In certain embodiments, the immobilized primer is linked to the solid support via a click chemistry reaction.

In certain embodiments, the molecule or group pair capable of the click chemistry reaction is selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol. In certain embodiments, the molecule or group pair capable of the click chemistry reaction is azido/alkynyl.

In certain embodiments, the fixed primer is modified with The surface of the solid phase support is modified with an azide group, and the fixed primer and the solid phase support are connected through a click chemical reaction between DBCO and the azide group.

In certain embodiments, the capture probe is present in a single-stranded form comprising a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

(1) providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and, (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent or non-covalent connection) with the molecule or group B'; and,

(2) contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the capture probe is connected.

It is easy for a person skilled in the art to understand that any part of the capture probe can be used to modify the molecule or group A', as long as it does not affect the function of the capture probe (e.g., annealing with the target sequence and the function of the 3' end initiation extension reaction). In some embodiments, the capture sequence of the capture probe does not modify the molecule or group A'. In some embodiments, the 5' end (e.g., 5' end) of the capture probe is modified with the molecule or group A'.

In certain embodiments, the capture sequence is located at the 3' end of the capture probe.

In certain embodiments, the capture probe is present in a duplex form containing a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

(I)(1) providing: (a) a free first strand of the capture probe, the first strand being modified with a molecule or group A'; and, (b) the solid support, the surface of the solid support being modified with a molecule or group B', the molecule or group A' being capable of forming a connection (e.g., covalent and/or non-covalent connection) with the molecule or group B'; and, (c) the free second strand of the capture probe, the second strand comprising the capture sequence and being capable of annealing with the first strand to form a duplex;

(2) contacting the first chain with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the first chain is connected; and,

(3) contacting the free second strand with the solid support connected to the first strand formed in step (2) under conditions allowing annealing, thereby obtaining a solid support connected to the capture probe;

or,

(II)(1) providing: (a) a duplex formed by annealing a first strand and a second strand of the capture probe, wherein the first strand is connected to a molecule or group A', and the second strand contains the capture sequence; and (b) a solid support, wherein the The surface of the solid support is modified with a molecule or group B', and the molecule or group A' can form a connection (eg, covalent and/or non-covalent connection) with the molecule or group B'; and,

(2) contacting the duplex with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support connected with the capture probe.

In certain embodiments, any portion of the first strand can be used to connect to the solid support, as long as the connection does not affect the first strand in performing its function (e.g., the function of annealing with the second strand). In certain embodiments, the portion of the first strand that is not annealed to the second strand is modified with the molecule or group A'. In certain embodiments, the end (e.g., 5' end or 3' end) of the first strand is modified with the molecule or group A'.

In certain embodiments, the second strand is not directly attached to the solid support.

In certain embodiments, in the duplex formed by annealing of the first strand and the second strand of the capture probe, the 3' end of the second strand comprises the capture sequence, and the capture sequence does not anneal to the first strand.

In certain embodiments, the molecule or group A' is capable of undergoing a click chemistry reaction with the molecule or group B'.

In certain embodiments, the molecules or groups A'/B' are selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone.

In certain embodiments, the molecules or groups A'/B' are azide/alkynyl or alkynyl/azide.

In certain embodiments, the molecule or group A' is DBCO and the molecule or group B' is azido.

In certain embodiments, the first strand of the capture probe is modified with The surface of the solid support is modified with an azide group, and the first chain of the capture probe and the solid support are connected through a click chemical reaction between DBCO and the azide group.

In certain embodiments, the solid support has one or more characteristics selected from the group consisting of:

(1) The solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers; in certain embodiments, the solid support is a chip;

(2) The solid support is planar, spherical or porous;

(3) the solid support can be used in a sequencing platform; in certain embodiments, the solid support is a sequencing chip for use in an IllμMina, MGI or Thermo Fisher sequencing platform; and

(4) The solid support is capable of releasing the localization probe and/or capture probe spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to a specific chemical or phase, exposure to light, a reducing agent, etc.). Probe.

In certain embodiments, the same type of positioning probes (i.e., positioning probes comprising the same positioning sequence) occupy the same area on the surface of the solid support, different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (e.g., less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm).

In certain embodiments, the distribution of the capture probe and the positioning probe on the solid support is configured as follows: the nucleic acid molecule connected to the capture probe (for example, a nucleic acid molecule annealed to the capture probe, or a nucleic acid molecule obtained by extending the capture probe through a nucleic acid polymerization reaction) is able to contact the positioning probe adjacent to it.

In certain embodiments, each of the positioning probes is adjacent to at least 100 capture probes. For example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 capture probes.

In certain embodiments, there are 500-10,000 (eg, 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed adjacent to each of the localization probes.

In certain embodiments, the expression "adjacent to each of the positioning probes" refers to an area near the positioning probe on the surface of a solid support, wherein nucleic acid molecules connected to capture probes distributed in the area (e.g., nucleic acid molecules annealed to the capture probes, or nucleic acid molecules obtained by extending the capture probes through nucleic acid polymerization reaction) are able to contact the positioning probes.

In certain embodiments, at least 100 capture probes are distributed within a radius of 200 nm to 300 nm around the center of the position occupied by each positioning probe on the solid support (e.g., the center of the area occupied by each positioning probe on the surface of the solid support). For example, at least 100, at least 200, at least 300, 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or more. At least 10,000 of the capture probes.

In certain embodiments, there are 500-10000 (e.g., 1000-1500, 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-8000, 1000-10000) capture probes distributed within a radius of 200nm-300nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support).

Nucleic acid array preparation method

In another aspect, the present application provides a method for preparing a nucleic acid array as described above, comprising the following steps:

(A) providing a nucleic acid array to be processed, wherein the nucleic acid array to be processed comprises a solid support, and the solid support is connected with at least two oligonucleotide molecules;

Each oligonucleotide molecule occupies a different position on the solid support;

The oligonucleotide molecule contains a positioning sequence and a first universal sequence in sequence from the 5' end to the 3' end, wherein the positioning sequence has a nucleotide sequence corresponding to the position of the oligonucleotide molecule on the solid support; the first universal sequence is as defined above;

and,

(B) attaching and/or synthesizing capture probes on the solid support contained in the nucleic acid array to be processed, wherein the capture probes are as defined above.

In certain embodiments, each oligonucleotide molecule consists of one or more oligonucleotide molecules.

In certain embodiments, the localization sequences contained in the same oligonucleotide molecules are identical to each other, and the localization sequences contained in different oligonucleotide molecules are different from each other.

It is easy for a person skilled in the art to understand that one or more oligonucleotide molecules of the same oligonucleotide molecule have the same positioning sequence, however, it is not necessary for every oligonucleotide molecule of each oligonucleotide molecule to have the same complete sequence.

In certain embodiments, the oligonucleotide molecule does not comprise a capture sequence at the 3' end of the first universal sequence, the capture sequence being as defined above.

In certain embodiments, the oligonucleotide molecule comprises a capture sequence at the 3′ end of the first universal sequence, the capture sequence being as defined above, and the method further comprises step (B′): removing the capture sequence contained in the oligonucleotide molecule;

Wherein, the steps (B) and (B') can be carried out in any order or simultaneously (for example, simultaneously in the same reaction system).

In certain embodiments, step (B') removes the capture sequence contained in the oligonucleotide molecule by the steps comprising:

(i) providing a blocking probe, wherein the blocking probe is capable of binding to the first universal sequence of the oligonucleotide molecule of (a) (a) a sequence or a partial sequence thereof (e.g., a partial sequence at the 3' end of the first universal sequence), or (b) a sequence in the oligonucleotide molecule that is located at the 5' end of the capture sequence and at the 3' end of the first universal sequence, or (c) a combination of (a) and (b), annealing;

(ii) contacting the blocking probe with the nucleic acid array to be processed containing the oligonucleotide molecules under conditions suitable for annealing the blocking probe with the oligonucleotide molecules;

(iii) contacting the product of step (ii) with an exonuclease under conditions that allow the exonuclease to exert its cleavage activity; wherein the exonuclease has exonuclease activity from the 3' to 5' end of a single-stranded nucleic acid.

In some embodiments, the exonuclease is exonuclease I.

In certain embodiments, step (B') further comprises the step of removing the blocking probe (e.g., removing the blocking probe by unzipping the blocking probe from the oligonucleotide molecule).

In certain embodiments, in the method, step (B) is performed after step (B').

In certain embodiments, the oligonucleotide molecule further comprises a second universal sequence, the second universal sequence being as defined above.

In certain embodiments, the second universal sequence is located 5' to the localization sequence.

In certain embodiments, the oligonucleotide molecule does not comprise or further comprises a MID sequence.

In certain embodiments, the oligonucleotide molecule further comprises a MID sequence. In certain embodiments, the MID sequence is located at the 5' end of the first universal sequence, and/or, the MID sequence is located at the 3' end of the second universal sequence. In certain embodiments, the MID sequences comprised by the same oligonucleotide molecule are different from each other.

In certain embodiments, in the nucleic acid array to be processed, the oligonucleotide molecules are covalently or non-covalently linked to the solid support.

In certain embodiments, the oligonucleotide molecule is covalently linked to the solid support.

In certain embodiments, the oligonucleotide molecule is linked to the solid support via a click chemistry reaction.

In certain embodiments, the molecule or group pair capable of the click chemistry reaction is selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol. In certain embodiments, the molecule or group pair capable of the click chemistry reaction is azido/alkynyl.

In certain embodiments, the surface of the solid support is modified with an azide group, and the oligonucleotide molecule is modified with The oligonucleotide molecule is connected to the solid support through a click chemistry reaction between the azide group and the DBCO.

In certain embodiments, the capture probe is present in a single-stranded form comprising a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

(1) providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and, (b) the nucleic acid array to be treated or the nucleic acid array treated in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' can form a connection (e.g., covalent or non-covalent connection) with the molecule or group B'; and,

(2) contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the capture probe is connected.

It is easy for a person skilled in the art to understand that any part of the capture probe can be used to modify the molecule or group A', as long as it does not affect the function of the capture probe (e.g., annealing with the target sequence and the function of the 3' end initiation extension reaction). In some embodiments, the capture sequence of the capture probe does not modify the molecule or group A'. In some embodiments, the 5' end (e.g., 5' end) of the capture probe is modified with the molecule or group A'.

In certain embodiments, the capture sequence is located at the 3' end of the capture probe.

In certain embodiments, the capture probe is present in a duplex form containing a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

(I)(1) providing: (a) a free first strand of the capture probe, wherein the first strand is modified with a molecule or group A'; and, (b) the nucleic acid array to be treated or the nucleic acid array treated in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent and/or non-covalent connection) with the molecule or group B'; and, (c) the free second strand of the capture probe, wherein the second strand comprises the capture sequence and is capable of annealing with the first strand to form a duplex;

(2) contacting the first chain with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the first chain is connected; and,

(3) contacting the free second strand with the solid support connected to the first strand formed in step (2) under conditions allowing annealing, thereby obtaining a solid support connected to the capture probe;

or,

(II)(1) providing: (a) a duplex formed by annealing the first strand and the second strand of the capture probe, wherein the first strand is connected to a molecule or group A', and the second strand contains the capture sequence; and, (b) the nucleic acid array to be treated or the nucleic acid array treated in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' can form a connection (e.g., covalent and/or non-covalent connection) with the molecule or group B'; and,

(2) contacting the duplex with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support connected with the capture probe.

In certain embodiments, any portion of the first strand can be used to bind to the solid support. In some embodiments, the first strand is connected to the second strand, as long as the connection does not affect the first strand to perform its function (e.g., the function of annealing with the second strand). In some embodiments, the portion of the first strand that is not annealed to the second strand is modified with the molecule or group A'. In some embodiments, the end (e.g., 5' end or 3' end) of the first strand is modified with the molecule or group A'.

In certain embodiments, the second strand is not directly attached to the solid support.

In certain embodiments, in the duplex formed by annealing of the first strand and the second strand of the capture probe, the 3' end of the second strand comprises the capture sequence, and the capture sequence does not anneal to the first strand.

In certain embodiments, the molecule or group A' is capable of undergoing a click chemistry reaction with the molecule or group B'.

In certain embodiments, the molecules or groups A'/B' are selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone.

In certain embodiments, the molecules or groups A'/B' are azide/alkynyl or alkynyl/azide.

In certain embodiments, the molecule or group A' is DBCO and the molecule or group B' is azido.

In certain embodiments, the first strand of the capture probe is modified with The surface of the solid support is modified with an azide group, and the first chain of the capture probe and the solid support are connected through a click chemical reaction between DBCO and the azide group.

In certain embodiments, the solid support has one or more characteristics selected from the group consisting of:

(1) The solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers; in certain embodiments, the solid support is a chip;

(2) The solid support is planar, spherical or porous;

(3) the solid support can be used in a sequencing platform; in certain embodiments, the solid support is a sequencing chip for use in an IllμMina, MGI or Thermo Fisher sequencing platform; and

(4) The solid support is capable of releasing the localization probe and/or capture probe spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to specific chemicals or phases, exposure to light, reducing agents, etc.).

In certain embodiments, the same type of positioning probes (i.e., positioning probes comprising the same positioning sequence) occupy the same area on the surface of the solid support, different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (e.g., less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 500 nm, less than 600 nm, less than 700 nm, less than 800 nm, less than 900 nm, less than 10 ... Less than 200nm).

In certain embodiments, the distribution of the capture probe and the positioning probe on the solid support is configured as follows: the nucleic acid molecule connected to the capture probe (for example, a nucleic acid molecule annealed to the capture probe, or a nucleic acid molecule obtained by extending the capture probe through a nucleic acid polymerization reaction) is able to contact the positioning probe adjacent to it.

In certain embodiments, each of the positioning probes is adjacent to at least 100 capture probes. For example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 capture probes.

In certain embodiments, there are 500-10,000 (eg, 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed adjacent to each of the localization probes.

In certain embodiments, the expression "adjacent to each of the positioning probes" refers to an area near the positioning probe on the surface of a solid support, wherein nucleic acid molecules connected to capture probes distributed in the area (e.g., nucleic acid molecules annealed to the capture probes, or nucleic acid molecules obtained by extending the capture probes through nucleic acid polymerization reaction) are able to contact the positioning probes.

In certain embodiments, at least 100 capture probes are distributed within a radius of 200nm-300nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support). For example, at least 100, at least 200, at least 300, 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 of the capture probes.

In certain embodiments, there are 500-10000 (e.g., 1000-1500, 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-8000, 1000-10000) capture probes distributed within a radius of 200nm-300nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support).

Nucleic acid spatial information detection method

In another aspect, the present application provides a method for detecting spatial information of nucleic acids in a sample, comprising the following steps:

(1) providing a nucleic acid array as described above;

(2) contacting the nucleic acid array with a sample to be tested under conditions that allow annealing, so that the nucleic acid derived from the sample to be tested anneals with the capture sequence in the capture probe of the nucleic acid array;

(3) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization to produce a first extension product, wherein the first extension product comprises a capture sequence and a complementary sequence of a nucleic acid molecule or a partial sequence thereof that anneals to the capture sequence, and the first extension product is connected to the solid support via the capture sequence contained therein;

(4) annealing the first extension product connected to the solid support with the adjacent positioning probe of the nucleic acid array under conditions that allow annealing;

wherein the first universal sequence of the positioning probe can anneal with the first extension product; and the first universal sequence is located at the 3' end of the positioning sequence of the positioning probe;

(5) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization to produce a second extension product and/or its complementary chain, wherein the second extension product comprises: (a) the positioning sequence of the positioning probe and the complementary sequence of the first extension product or its partial sequence, or (b) the first extension product sequence or its partial sequence and the complementary sequence of the positioning sequence of the positioning probe; the positioning sequence or its complementary sequence contained in the second extension product serves as its spatial information marker, and the complementary sequence of the positioning sequence or the positioning sequence contained in the complementary chain of the second extension product serves as its spatial information marker, so that the position of the nucleic acid derived from the sample to be tested in the sample to be tested is mapped to the position of the positioning probe corresponding to the positioning sequence; thereby obtaining a nucleic acid molecule containing the spatial information marker; and

(6) Analyzing: (i) the nucleic acid molecule containing the spatial information marker obtained in step (5), and/or, (ii) the sequence of the nucleic acid molecule containing the spatial information marker derived from (i).

It is easy for a person skilled in the art to understand that in steps (4) to (5), the first extension product anneals with the adjacent positioning probe and undergoes nucleic acid polymerization reaction to produce the second extension product, whereby the second extension product contains the positioning sequence or its complementary sequence in the positioning probe, so that the position of the nucleic acid derived from the sample to be tested captured by the capture probe in the sample to be tested is corresponded to the position of the positioning probe on the nucleic acid array that anneals with the first extension product.

In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and/or abundance of the nucleic acid.

In certain embodiments, in step (3), the nucleic acid polymerization reaction uses the nucleic acid molecule that anneals to the capture sequence as a template to extend the capture sequence.

In certain embodiments, the capture sequence is located at the 3' end of the capture probe, and/or the 3' end of the capture sequence has a free hydroxyl group (-OH).

In certain embodiments, in step (5), the polymerization reaction uses the first extension product as a template to extend the positioning probe; and/or uses the positioning probe as a template to extend the first extension product.

In some embodiments, in step (5), the polymerization reaction uses the first extension product as a template to extend the positioning probe to produce a second extension product and/or its complementary chain, wherein the second extension product comprises the positioning sequence of the positioning probe and the complementary sequence of the first extension product or a partial sequence thereof. In some embodiments, the first universal sequence of the positioning probe is located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe has a free hydroxyl group (-OH).

In certain embodiments, in step (5), the polymerization reaction uses the positioning probe as a template to extend the first extension product to produce a second extension product and/or its complementary strand, wherein the second extension product comprises the first extension product sequence or a partial sequence thereof and a complementary sequence to the positioning sequence of the positioning probe. In certain embodiments, the first universal sequence of the positioning probe is located at or not located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe is blocked or unblocked.

In certain embodiments, in step (5), in the polymerization reaction, the first extension product and the positioning probe serve as templates for each other, and the first extension product and the positioning probe are extended to produce a second extension product and/or its complementary chain, the second extension product comprises the positioning sequence of the positioning probe and the complementary sequence of the first extension product or its partial sequence, and the second extension product comprises the first extension product sequence or its partial sequence and the complementary sequence of the positioning sequence of the positioning probe. In certain embodiments, the first universal sequence of the positioning probe is located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe has a free hydroxyl group (-OH).

In certain embodiments, in step (6), the sequence of the nucleic acid molecule containing the spatial information label attached to the solid support of the nucleic acid array is analyzed.

In certain embodiments, before step (6) and after step (5), the method further comprises a step pre-(6): releasing at least a portion of the nucleic acid molecules containing spatial information labels from the surface of the nucleic acid array.

In certain embodiments, in step (6), (i) the nucleic acid molecule containing the spatial information marker released in step pre-(6) and/or, (ii) the sequence of the nucleic acid molecule containing the spatial information marker derived from (i) are analyzed.

In certain embodiments, in step pre-(6), the nucleic acid molecule is released from the solid support surface by: (i) nucleic acid shearing; and/or, (ii) denaturation.

In certain embodiments, after step pre-(6) and before step (6), the method further comprises a step of amplifying the released nucleic acid molecules.

In certain embodiments, before performing step (6), the method further comprises a step of purifying the released nucleic acid molecules.

In certain embodiments, the method has one or more features selected from the group consisting of:

(i) in step (1), providing the nucleic acid array by the method for preparing a nucleic acid array as described above;

(ii) in step (2), the sample to be tested is treated (for example, permeabilized or cell lysed) to release the nucleic acid from the sample to be tested so that the nucleic acid anneals with the capture sequence;

(iii) after step (3) and before step (4), the method further comprises the step of removing the template strand bound to the first extension product (for example, removing the template strand bound to the first extension product by enzyme cleavage or denaturing and melting);

(iv) after step (3) and before step (4), the method further comprises a step of washing the nucleic acid array to remove residual sample (e.g., tissue or cells);

(v) After step (5) and before step (6) (e.g., after step (5) and before step pre-(6)), the method further comprises the step of amplifying (e.g., in situ amplification) the nucleic acid molecule containing the spatial information marker. In certain embodiments, the nucleic acid molecule containing the spatial information marker is connected to the solid support of the nucleic acid array.

In certain embodiments, the sample is a tissue sample (eg, a tissue section) or a single cell sample (eg, a single cell suspension).

In certain embodiments, the tissue sample (eg, tissue section) is prepared from fixed tissue, eg, formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue or fresh tissue.

In certain embodiments, the cell is an immune cell, such as a B cell or a T cell. In certain embodiments, the nucleic acid derived from the sample to be tested comprises a T cell receptor gene or its nucleic acid product (e.g., mRNA), or a B cell receptor gene or its nucleic acid product (e.g., mRNA).

In certain embodiments, the nucleic acid derived from the sample to be tested is selected from: RNA molecules (e.g., mRNA) molecules, target nucleic acid molecules (e.g., target DNA and/or RNA), genomic nucleic acid fragments in open chromatin regions, and any combination thereof.

In certain embodiments, in step (6), the analysis comprises sequencing and/or sequence-specific PCR reaction.

In certain embodiments, before sequencing, the method further comprises the step of constructing a sequencing library for the nucleic acid molecule containing the spatial information marker or its amplified product obtained in step (5).

In certain embodiments, the methods are used to detect the spatial information of RNA (eg, mRNA) of cells in a sample.

In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested is RNA (eg, mRNA) in cells of the sample to be tested, and the capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence.

In certain embodiments, step (3) comprises:

(a) under conditions allowing nucleic acid polymerization, using a nucleic acid molecule annealed to the capture sequence as a template, performing a nucleic acid polymerization reaction, extending the capture sequence, and generating a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the capture sequence as a reverse transcription primer, and a 3' end overhang;

(b) under conditions that allow nucleic acid polymerization, reacting the template switching sequence with the cDNA strand generated in (a) annealing, and continuing nucleic acid polymerization reaction with the template switching sequence as a template to generate the first extension product;

Wherein, the template switching sequence comprises a consensus sequence, a 3'-terminal overhang complementary sequence, and an optional MID sequence; in certain embodiments, the MID sequences contained in each template switching sequence are different from each other;

The first extension product comprises: a capture sequence, the cDNA chain sequence or a partial sequence thereof, and a complementary sequence of the common sequence.

In certain embodiments, the 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides. In certain embodiments, the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

In certain embodiments, the method further comprises, before step (4), a step of removing the template switching sequence bound to the first extension product (eg, removing the template switching sequence bound to the first extension product by enzyme cleavage or denaturing melting).

In certain embodiments, in step (4) of the method, the first universal sequence of the localization probe is capable of annealing to the complement of the consensus sequence.

In certain embodiments, the positioning probe does not contain a MID sequence and the template switch sequence contains a MID sequence; alternatively, the positioning probe contains a MID sequence and the template switch sequence does not contain a MID sequence; alternatively, both the positioning probe and the template switch sequence contain a MID sequence.

In certain embodiments, the method is used to detect the spatial information of a target nucleic acid (eg, target DNA and/or RNA) of a cell in a sample, wherein the target nucleic acid comprises a target nucleotide sequence.

In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested comprises the target nucleotide sequence. In certain embodiments, the nucleic acid derived from the sample to be tested comprises the target nucleic acid and/or a nucleic acid molecule derived from the target nucleic acid, and the nucleic acid molecule derived from the target nucleic acid comprises the target nucleotide sequence.

In certain embodiments, in step (2), the capture sequence comprises a sequence that specifically recognizes the target nucleotide sequence.

In certain embodiments, in step (2), the capture sequence comprises a sequence that specifically recognizes the first segment of the target target nucleotide sequence; in step (4), the first universal sequence of the positioning probe comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target target nucleotide sequence, or the first universal sequence comprises a random oligonucleotide sequence; in certain embodiments, in the target target nucleotide sequence, the first segment is located at the 3’ end of the second segment.

In certain embodiments, in step (2), the first segment is present in a single-stranded region of the nucleic acid molecule derived from the sample to be tested that anneals to the capture sequence.

In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested that anneals to the capture sequence is a single-stranded nucleic acid or a double-stranded nucleic acid containing a single-stranded region comprising the first segment.

In certain embodiments, the method is used to detect spatial information of RNA (eg, mRNA) and target nucleic acid (eg, target DNA and/or RNA) of cells in a sample, wherein the target nucleic acid comprises a target nucleotide sequence.

In certain embodiments, the nucleic acid array contains a first capture probe capable of capturing the RNA (e.g., mRNA) and a second capture probe capable of capturing a nucleic acid molecule containing the target nucleotide sequence; the first capture probe contains a first capture sequence, which comprises a poly (dT) sequence or a random oligonucleotide sequence; and the second capture probe contains a second capture sequence, which comprises a sequence that specifically recognizes the target nucleotide sequence.

It is easy for those skilled in the art to understand that, unless otherwise specified herein or obviously contradictory according to the context, the definition/description of the capture probe above also applies to the first capture probe and the second capture probe, and the definition/description of the capture sequence above also applies to the first capture sequence and the second capture sequence.

In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested comprises RNA (e.g., mRNA) derived from the sample to be tested and a nucleic acid molecule comprising a target nucleotide sequence; in certain embodiments, the nucleic acid molecule comprising a target nucleotide sequence comprises the target target nucleic acid and/or a nucleic acid molecule derived from the target target nucleic acid.

In certain embodiments, step (3) comprises:

(A)(a) Under conditions that allow nucleic acid polymerization, using a nucleic acid molecule that anneals to the first capture sequence as a template, performing a nucleic acid polymerization reaction, extending the first capture sequence, and generating a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the first capture sequence as a reverse transcription primer, and a 3' end overhang;

(b) annealing the template switching sequence with the cDNA chain generated in (a) under conditions that allow nucleic acid polymerization, and continuing nucleic acid polymerization reaction with the template switching sequence as a template to generate a first extension product I;

Wherein, the template switching sequence comprises a consensus sequence, a 3'-terminal overhang complementary sequence, and an optional MID sequence; in certain embodiments, the MID sequences contained in each template switching sequence are different from each other;

as well as,

(B) under conditions allowing nucleic acid polymerization, using the nucleic acid molecule annealed to the second capture sequence as a template, performing a nucleic acid polymerization reaction to extend the second capture sequence to generate a first extension product II, wherein the first extension product II comprises a second capture sequence and a complementary sequence to the nucleic acid molecule annealed to the second capture sequence or a partial sequence thereof;

Wherein, step (B) and step (A) are performed in any order; for example, step (B) is performed before or after step (A), or step (B) and step (A) are performed simultaneously (for example, in the same reaction system).

In certain embodiments, the 3' terminal overhang has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or a length of 2-10 nucleotides. In certain embodiments, the 3'-terminal overhang is an overhang of 2-5 cytosine nucleotides (eg, a CCC overhang).

In certain embodiments, in step (4), the positioning probe contains a first universal sequence I that can anneal to the first extension product I, and a first universal sequence II that can anneal to the first extension product II; in certain embodiments, the first universal sequence I and the first universal sequence II are present in the same positioning probe, or, respectively, in different positioning probes.

In certain embodiments, the nucleic acid array comprises a first positioning probe comprising the first universal sequence I and a second positioning probe comprising the first universal sequence II.

It is easy for those skilled in the art to understand that, unless otherwise specified herein or clearly contradictory according to the context, the definition/description of the positioning probe above also applies to the first positioning probe and the second positioning probe, and the definition/description of the first universal sequence above also applies to the first universal sequence I and the first universal sequence II.

In certain embodiments, in step (4) of the method, the first universal sequence I is capable of annealing to the complementary sequence of the consensus sequence.

In certain embodiments, in step (2) of the method, the second capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence; in step (4), the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence. In certain embodiments, in the target nucleotide sequence, the first segment is located at the 3' end of the second segment.

In certain embodiments, the method further comprises, before step (4), a step of removing the template switching sequence bound to the first extension product I (e.g., removing the template switching sequence bound to the first extension product I by enzyme cleavage or denaturing melting).

In certain embodiments, in step (2), the first segment is present in a single-stranded region of the nucleic acid molecule derived from the sample to be tested that anneals to the second capture sequence.

In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested that anneals to the second capture sequence is a single-stranded nucleic acid or a double-stranded nucleic acid containing a single-stranded region comprising the first segment.

In certain embodiments, the first positioning probe does not contain a MID sequence and the template switch sequence contains a MID sequence; or, the first positioning probe contains a MID sequence and the template switch sequence does not contain a MID sequence, or, both the first positioning probe and the template switch sequence contain a MID sequence.

Reagent test kit

In another aspect, the present application provides a kit comprising the nucleic acid array as described above.

In some embodiments, the kit further comprises instructions for use. In some embodiments, the instructions for use record the method for detecting nucleic acid spatial information as described above.

In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and/or abundance of the nucleic acid.

For mRNA capture

In certain embodiments, the capture sequence of the nucleic acid array comprises a poly(dT) sequence or a random oligonucleotide sequence.

In some embodiments, the kit further comprises a template switching sequence, the template switching sequence comprising a consensus sequence, a cDNA 3' terminal overhang complementary sequence, and an optional MID sequence; in some embodiments, the MID sequences contained in each template switching sequence are different from each other. In some embodiments, the cDNA 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or 2-10 nucleotides. In some embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., CCC overhang).

In certain embodiments, the complement of the consensus sequence of the template switch sequence is capable of annealing to the first universal sequence of the positioning probe of the nucleic acid array.

In certain embodiments, the positioning probe does not contain a MID sequence and the template switch sequence contains a MID sequence; alternatively, the positioning probe contains a MID sequence and the template switch sequence does not contain a MID sequence; alternatively, both the positioning probe and the template switch sequence contain a MID sequence.

For target nucleic acid capture

In certain embodiments, the capture sequence of the nucleic acid array comprises a sequence that specifically recognizes a target nucleotide sequence comprised in a target nucleic acid (eg, a specific target DNA and/or RNA).

In certain embodiments, the first universal sequence comprises a sequence that specifically recognizes a complementary sequence of the target nucleotide sequence of interest.

In certain embodiments, the capture sequence comprises a sequence that specifically recognizes the first segment of the target target nucleotide sequence, the first universal sequence of the positioning probe of the nucleic acid array comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target target nucleotide sequence, or the first universal sequence comprises a random oligonucleotide sequence.

In certain embodiments, in the target nucleotide sequence of interest, the first segment is located at the 3' end of the second segment.

For dual capture of mRNA and target nucleic acid

In certain embodiments, the nucleic acid array contains a first capture probe and a second capture probe; the first capture probe contains a first capture sequence, and the first capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence; the second capture probe contains a second capture sequence, and the second capture sequence comprises a sequence that specifically recognizes a target target nucleotide sequence contained in a target target nucleic acid (e.g., a specific target DNA and/or RNA).

It is easy for those skilled in the art to understand that, unless otherwise specified herein or obviously contradictory according to the context, the definition/description of the capture probe above also applies to the first capture probe and the second capture probe, and the definition/description of the capture sequence above also applies to the first capture sequence and the second capture sequence.

In certain embodiments, the kit further comprises a template conversion sequence, the template conversion sequence comprising a consensus sequence, a cDNA 3' terminal overhang complementary sequence, and an optional MID sequence. In certain embodiments, the MID sequences contained in each template conversion sequence are different from each other. In certain embodiments, the cDNA 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or 2-10 nucleotides. In certain embodiments, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

In certain embodiments, the positioning probe of the nucleic acid array comprises a first universal sequence I and a first universal sequence II; wherein the first universal sequence I is capable of annealing with a complementary sequence to the consensus sequence, the first universal sequence II comprises a sequence that specifically recognizes a complementary sequence to the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence. In certain embodiments, the first universal sequence I and the first universal sequence II are present in the same positioning probe together, or are present in different positioning probes respectively.

In certain embodiments, the nucleic acid array comprises a first positioning probe comprising the first universal sequence I and a second positioning probe comprising the first universal sequence II. In certain embodiments, the first positioning probe does not contain a MID sequence, and the template switch sequence contains a MID sequence; or, the first positioning probe contains a MID sequence, and the template switch sequence does not contain a MID sequence, or, both the first positioning probe and the template switch sequence contain a MID sequence.

It is easy for those skilled in the art to understand that, unless otherwise specified herein or clearly contradictory according to the context, the definition/description of the positioning probe above also applies to the first positioning probe and the second positioning probe, and the definition/description of the first universal sequence above also applies to the first universal sequence I and the first universal sequence II.

In certain embodiments, the second capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence, the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence. In certain embodiments, in the target target nucleotide sequence, the first segment is located at the 3' end of the second segment.

In certain embodiments, the first universal sequence contained in the positioning probe of the nucleic acid array is located at or not located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe of the nucleic acid array is blocked or unblocked.

In certain embodiments, the kit further comprises: reagents for nucleic acid hybridization, reagents for nucleic acid extension, reagents for nucleic acid amplification, reagents for recovering or purifying nucleic acids, reagents for constructing a transfection kit, The invention relates to reagents for sequencing a genome sequencing library, reagents for sequencing (e.g., second-generation sequencing or third-generation sequencing), or any combination thereof.

In another aspect, the present application provides the use of the nucleic acid array or kit as described above for constructing a nucleic acid molecule library, for performing nucleic acid sequencing, or for detecting nucleic acid spatial information in a sample.

In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and/or abundance of the nucleic acid.

As used herein, "target nucleotide sequence" and "target nucleotide sequence" have the same meaning and can be used interchangeably.

Advantageous Effects of the Invention

The capture area and spatial information of the nucleic acid array (e.g., capture chip) provided by the present application exist in two independent molecules, for example, the capture area and spatial information exist in the capture probe and the positioning probe, respectively, wherein the positioning probe is used to provide spatial position information of spatial omics, and the capture probe is used to capture molecules in cells, thereby getting rid of the limitation that the number of capture probes is affected by spatial density, and being able to make full use of all areas on the chip to graft capture probes, thereby improving the capture efficiency of intracellular molecules while ensuring high resolution.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram of an exemplary capture chip of the present application, wherein the capture probe (including the capture domain) and the positioning probe (including spatial information, spatial barcode) on the chip are respectively on two molecules, and the two molecules are independent of each other.

Figure 2: Exemplary implementation process of spatial transcriptomics in this application.

Figure 3: Chip probe detection results.

Figure 4: cDNA Bioanalyzer 2100 test results.

Figure 5: Spatial expression map of mouse brain sections.

Figure 6: Cell types in mouse brain slices.

Figure 7: Gene coverage analysis results of the captured transcriptome.

Sequence information

A description of the sequences involved in this application is provided in the table below.

Table 1: Sequence information

Note: V＝A, C, or G; N＝A, T, C, or G; "r" means the nucleotide at the 3' adjacent position is a ribonucleotide; "iXNA" means LNA locked nucleotide.

DETAILED DESCRIPTION

The present invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention and are not intended to limit the scope of the invention as claimed.

An exemplary implementation process for obtaining spatial transcriptomic information of samples based on the chip provided in this application is shown in FIG2 :

(1) preparing a capture chip as shown in FIG1 ;

(2) mRNA capture: tissue sections are attached to the capture chip, and the tissue is processed to release mRNA molecules;

(3) The released mRNA molecules are captured by the capture probes on the chip and synthesized into cDNA molecules in situ under the action of reverse transcriptase with terminal transferase activity; since the reverse transcriptase with terminal transferase activity will add some additional nucleotides C to the 3' end of the newly synthesized cDNA molecules, forming base complementary pairing with the 3' end of TSO (template switching oligonucleotide), the reverse transcriptase "switches" the template and continues to synthesize cDNA molecules using TSO as a template until the 5' end of TSO;

(4) The 3' end of the cDNA molecule can be captured by the adjacent positioning probe containing position information and further extended, thereby adding spatial information to the cDNA molecule and synthesizing a cDNA molecule with spatial information;

(5) collecting cDNA molecules from the chip and performing cDNA amplification, or performing PCR directly on the chip to amplify cDNA;

(6) Library construction and sequencing;

(7) Data analysis: restore the spatial information and RNA expression to the chip location.

In addition, the applicant wishes to emphasize that based on different designs of the capture zone sequence, it can be designed to target a variety of target sequences, not limited to mRNA. Therefore, the chip provided in this application can not only be used to obtain spatial transcriptomics information, but it can also be used to obtain genomic, epigenomic, multi-omics and other information, and can also be used to obtain information on specific target molecules.

1. Capture Chip Preparation

An exemplary capture chip of the present application comprises a positioning probe and a capture probe, wherein the capture probe comprises a linker sequence and a capture sequence in sequence from the 5’ end to the 3’ end, and the positioning probe comprises a first linker sequence, a spatial position sequence (i.e., a positioning sequence) and a second linker sequence in sequence from the 5’ end to the 3’ end.

This embodiment shows an exemplary method for preparing the capture chip, which comprises:

(a) providing a conventional chip conventionally used in the art that contains a positioning sequence and a capture sequence in the same probe (for example, the conventional chip can be provided by purchase or self-preparation); the probe of the conventional chip sequentially contains a first linker sequence, a spatial position sequence (i.e., a positioning sequence), a second linker sequence, and a capture sequence from the 5' end to the 3' end;

(b) blocking the second linker, for example, hybridizing an oligonucleotide molecule capable of annealing to the second linker sequence with the probe, thereby forming a double-stranded structure at the position of the second linker of the probe;

(c) incubating a nuclease with the product of (b), wherein the nuclease has a 3' to 5' end single-stranded nuclease activity; thereby, the capture sequence of the probe of the conventional chip is cut off by the nuclease, thereby forming a positioning probe that does not contain a capture sequence but contains a positioning sequence;

(d) Connecting capture probes to the chip containing the positioning probes obtained in (c) through a click chemistry reaction, thereby obtaining an exemplary capture chip comprising capture probes and positioning probes of the present application.

Specifically, the capture chip preparation steps are as follows:

(1) Block and remove the conventional chip capture area to form a positioning probe: synthesize an oligo sequence from Bio-Industry: GTCTTAGGAAGACAA (SEQ ID NO: 1), and use 5×SSC (saline sodium citrate) to prepare it into a 1 μM oligo solution; take out the chip in the stereo-seq transcriptomics T kit (BGI, catalog number: 111KT114) (conventional chip, the structure of each nucleic acid molecule from 5' to 3' end is: linker 1-spatial position sequence-linker 2-capture sequence poly T), draw 100 μL of 1 μM oligo solution onto the chip, and hybridize at room temperature for 30 min to block the linker 2 of the nucleic acid molecule on the conventional chip; draw off the above hybridization solution, wash the chip with 0.1×SSC, and add 50 μL Exonuclease I (ThermoFisher: EN0581) reaction solution (5 μL Exonuclease I, 5 μL 10x Reaction buffer, 40 μL ddH ₂ O) react at 37°C for 20 minutes to remove the capture sequence poly T of the nucleic acid molecule on the conventional chip; discard the reaction solution and wash it with 0.1×SSC; the chip only contains "connector 1-spatial position sequence-connector 2".

(2) Chip surface modification: Prepare 0.1% poly-lysine solution (PLL, sigma P8920), add it to the above-treated chip, and incubate at 30°C for 3 h to modify the chip surface with amino groups. Discard the reaction solution, wash the chip with ddH2O, and dry the chip for 1 h. Prepare NHS-PEG-N3 reaction solution (sigma JKA5088) according to the instructions and add it to the above-mentioned chip. Incubate at 37°C for 5 h to modify the chip surface with azide groups.

(3) Oligo dT modification of chip capture probe: Bioengineering synthesized a capture probe with a linker and oligo dT: 5’-CCTCCGACTGTGTGACTTAGACTCCTGCCACCTCCTGATGTGCTTTTTTTTTTTTTTTTTTTTTTV-3’ (SEQ ID NO: 2). The 5’ end of the probe was modified with DBCO (dibenzocyclooctyne). The probe was diluted to 1 μM with PBS and added to the chip for reaction at 37°C overnight. This allowed the azide groups on the chip surface to undergo a click reaction with the DBCO-modified probe, thereby connecting the oligo dT probe to the chip to obtain a chip containing capture probes.

(4) Chip probe detection: The detection probe AAAAAAAAAAAAAAAAGCACA (SEQ ID NO: 3) with cy5 fluorescence modified at the 3' end was used for hybridization photography to detect the synthesis of the capture probe on the chip and the excision of the capture area on the chip in step 1. The results are shown in Figure 3: Figure ① is a photograph of the PLL-modified chip obtained in step (2) after adding the detection probe, and Figure ② is the background of the positioning probe chip obtained in step (1) (no detection probe added); Figure ③ is a photograph of the positioning probe chip obtained in step (1) after adding the detection probe; Figure ④ is the fluorescence of the chip connected to oligo dT obtained in step (3) after adding the detection probe. The results show that Figures ② and ③ show that the original capture area poly T on the commercial chip was successfully excised, and Figure ④ shows that the newly synthesized capture probe oligo dT was successfully modified on the chip.

2. mRNA Molecular Capture

Referring to the instruction manual of the stereo-seq transcriptomics T kit, tissue slices, such as mouse brain slices, are attached to the chip, and the slices are permeabilized to release mRNA, so that the mRNA in the tissue can be captured by the capture probes on the chip.

3. cDNA in situ synthesis

Refer to the instructions of the Stereo-seq Transcriptomics T Kit and configure the following reverse transcription reaction system at 42°C for 5 hours.

4. Add spatial information

After cDNA synthesis, discard the reaction solution, add TR buffer (1000028507) according to the instructions, react for 30 minutes, remove the tissue; prepare formamide solution, add it to the chip surface, and place it at 80℃ for 3 hours, aspirate the reaction solution, remove mRNA, and wash the chip surface with ddH ₂ O. Add 5×SSC reaction solution to the chip surface and react at 37℃ for 30 minutes; remove the reaction solution, use 0.1×SSC to wash the chip surface, and then add the following reaction solution, react at 37℃ for 3 hours to extend the spatial information.

5. cDNA Release and cDNA Amplification

Prepare 100 μL of 100 mM KOH solution, add it to the chip and react at 55°C for 3 h. Collect the reaction supernatant into a PCR tube.

The chip was cleaned with 50 μL ddH ₂ O, and the cleaning solution and supernatant were combined into a PCR tube. The reaction solution was neutralized to pH 8.5 with 0.1 M HCl, and then divided into three tubes for PCR reaction. The reaction solution was as follows. The PCR reaction was carried out according to the instructions. The obtained cDNA was detected by Bioanalyzer 2100. The results are shown in Figure 4.

6. Library Construction and Sequencing

Refer to the instructions of stereo-seq transcriptomics T kit to shear the cDNA library and prepare DNB.

According to the operating instructions of the sequencer MGISEQ 2000, DNB was loaded onto the sequencing chip of MGISEQ2000 for sequencing. The sequencing was set to 25bp for spatial information decoding, followed by a 22bp dark reaction, and then a 5bp sequencing reaction to obtain the molecular tag (MID), and then a 50bp-100bp sequencing reaction to obtain the information of the cDNA 5' end transcript.

7. Data Analysis

(1) Log in to the website http://stereomap.cngb.org/Stereo-Draftsman/report/index and analyze the data according to the website operation guide. Compare the first 25 bp of the read1 sequence (from the first strand sequencing) obtained in step 6 with The 25bp position information of the capture chip in step 1 is compared, and the reads that can be compared to the position information on the chip are retained and matched to the corresponding chip positions. The sequence starting at 46bp corresponding to the reads corresponding to the chip position is found as the cDNA sequence. After reverse complementation of the sequence, it is compared with the mouse brain genome. According to the MID information obtained from 41bp-45bp, duplicate reads are removed to obtain the number of each gene expressed in the mouse brain.

(2) The number of genes expressed was further plotted to obtain the spatial expression map of the mouse brain slice as shown in Figure 5. The expression levels were subjected to spatial cluster analysis to obtain the cell types of the mouse brain slice as shown in Figure 6.

(3) 5’-end transcript capture analysis

The captured transcriptome was subjected to gene coverage analysis, and it can be seen that most of the captured transcriptome is concentrated in the transcription start region (TSS), proving that this scheme can achieve the capture of 5' end transcripts, as shown in Figure 7.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (44)

A nucleic acid array for detecting spatial information of nucleic acids in a sample, comprising a solid support, wherein the solid support is connected with one or more capture probes and at least two positioning probes; The capture probe and the localization probe are each independently connected to the solid support; Each localization probe occupies a different position on the solid support; The positioning probe contains a first universal sequence and a positioning sequence, wherein the positioning sequence has a nucleotide sequence corresponding to the position of the positioning probe on the solid support; The capture probe contains a capture sequence that is capable of annealing to the nucleic acid molecule to be captured and initiating an extension reaction. The nucleic acid array of claim 1, wherein the first universal sequence is located at the 3' end of the positioning sequence; Preferably, the localization probe further comprises a second universal sequence; Preferably, the second universal sequence is located at the 5' end of the positioning sequence; Preferably, the second universal sequences contained in the different types of positioning probes connected to the solid support are the same or different; preferably, the second universal sequences contained in the different types of positioning probes connected to the solid support are the same; Preferably, the localization probe does not contain or further contains a molecular tag sequence (MID, molecular identifier); Preferably, the localization probe further comprises a MID sequence; Preferably, the MID sequence is located at the 5' end of the first universal sequence, and/or, the MID sequence is located at the 3' end of the second universal sequence; Preferably, the MID sequences comprised by the same type of localization probes are different from each other. The nucleic acid array of claim 1 or 2, wherein the capture sequence is located at the 3' end of the capture probe; Preferably, the 3' end of the capture sequence has a free hydroxyl group (-OH); Preferably, the capture probe further comprises a fixed sequence; Preferably, the fixed sequences contained in different capture probes connected to the solid support are the same or different; preferably, the fixed sequences contained in different capture probes connected to the solid support are the same; Preferably, the immobilization sequence is located at the 5' end of the capture sequence. The nucleic acid array of any one of claims 1 to 3, wherein the capture probe comprises a single-stranded region comprising the capture sequence; Preferably, the capture probe is present in a single-stranded form or a duplex form containing the single-stranded region; Preferably, the capture probe exists in a single-stranded form, and the capture sequence is located at the 3' end of the capture probe; or, The capture probe exists in the form of a double-stranded body containing a single-stranded region, which includes a first chain directly connected to the solid support and a second chain hybridized with the first chain; and the 3' end of the second chain contains a single-stranded region, and the capture sequence is located at the 3' end of the single-stranded region. The nucleic acid array according to any one of claims 1 to 4, having one or more characteristics selected from the following: (1) the positioning sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35 or 20-45) random nucleotides, preferably, each random nucleotide is independently any one of deoxyribonucleotides A, C, G and T; (2) The solid support is selected from latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes and silicon wafers; preferably, the solid support is a chip; preferably, the solid support can be used in a sequencing platform; preferably, the solid support is a sequencing chip, such as a sequencing chip for an Illumina, MGI or Thermo Fisher sequencing platform; (3) The same type of positioning probes occupy the same area on the surface of the solid support, and different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (for example, less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm); (4) the first universal sequence is capable of annealing with (i) a nucleic acid molecule captured by the capture sequence, or (ii) a nucleic acid molecule derived from (i); (5) the first universal sequences contained in the different types of localization probes connected to the solid support are the same or different; (6) The solid support is capable of releasing the localization probe and/or capture probe spontaneously or when exposed to one or more stimuli (e.g., temperature change, pH change, exposure to specific chemicals or phases, exposure to light, reducing agents, etc.). The nucleic acid array of any one of claims 1 to 5, wherein the localization probe and/or the capture probe is covalently or non-covalently linked to the solid support; Preferably, the localization probe and/or the capture probe is covalently linked to the solid support; Preferably, the localization probe and the capture probe are each independently connected to the solid support through the same or different click chemistry reactions; Preferably, the molecule or group pair capable of the click chemistry reaction is selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol; preferably, the molecule or group pair capable of the click chemistry reaction is azido/alkynyl; For example, the surface of the solid support is modified with a molecule or group X, the positioning probe is modified with a molecule or group Y, the molecule or group X can undergo a click chemical reaction with the molecule or group Y, and the positioning probe is connected to the solid support through the click chemical reaction between the molecule or group X and the molecule or group Y; and/or, the surface of the solid support is modified with a molecule or group X', the capture probe is modified with a molecule or group Y', the molecule or group X' can undergo a click chemical reaction with the molecule or group Y', and the capture probe is connected to the solid support through the click chemical reaction between the molecule or group X' and the molecule or group Y'; Preferably, the pairs of molecules or groups X/Y and X'/Y' are each independently selected from the group consisting of: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone; Preferably, the surface of the solid support is modified with an azide group, and the localization probe and/or the capture probe is modified with (DBCO), the positioning probe and/or the capture probe is connected to the solid support through a click chemistry reaction between the azide group and the DBCO. The nucleic acid array according to any one of claims 1 to 6, wherein at least 100 capture probes, preferably 500 to 10,000 capture probes, are distributed adjacent to each of the positioning probes; Preferably, at least 100 capture probes, preferably 500-10,000 capture probes, are distributed within a radius of 200nm-300nm around the center of the position occupied by each positioning probe on the solid support (for example, the center of the area occupied by each positioning probe on the surface of the solid support). The nucleic acid array of any one of claims 1 to 7, wherein the nucleic acid array comprises one or more of the capture probes; Preferably, different types of capture probes contain different capture sequences; Preferably, the nucleic acid molecule to be captured is RNA (eg, mRNA), and the capture sequence of the capture probe contains a poly (dT) sequence or a random oligonucleotide sequence; or, The nucleic acid molecule to be captured is a target nucleic acid (e.g., a target DNA and/or RNA) or a nucleic acid molecule derived from the target nucleic acid, the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has a target nucleotide sequence contained in the target nucleic acid, and the capture sequence of the capture probe comprises a sequence that can specifically anneal to the target nucleotide sequence; or, The nucleic acid molecules to be captured are RNA (e.g., mRNA) and target nucleic acids (e.g., target DNA and/or RNA), and the nucleic acid array comprises a first capture probe capable of capturing RNA (e.g., mRNA) and a second capture probe capable of capturing the target nucleic acid or a nucleic acid molecule derived from the target nucleic acid, wherein the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has a target nucleotide sequence contained in the target nucleic acid; wherein the capture sequence of the first capture probe contains a poly (dT) sequence or a random oligonucleotide sequence, and the capture sequence of the second capture probe contains a sequence that can specifically anneal to the target nucleotide sequence. A method for preparing a nucleic acid array according to any one of claims 1 to 8, comprising the following steps: (A) attaching and/or synthesizing a localization probe on a solid support, the localization probe being as defined in claim 1, 2 or 5; and, (B) attaching and/or synthesizing a capture probe on a solid support, wherein the capture probe is as defined in any one of claims 1, 3-5, and 8; Wherein, (A) and (B) can be carried out in any order or simultaneously (for example, simultaneously in the same reaction system); For example, (A) is performed before (B), and the solid support in (B) is a solid support to which the positioning probe has been connected; For example, (B) is performed before (A), and the solid support in (A) is a solid support to which the capture probe has been connected; For example, (A) and (B) are performed simultaneously, and the solid phase support in (A) and (B) is the same solid phase support. The method of claim 9, wherein step (A) comprises: (1) providing: (a) the free positioning probe, wherein the positioning probe is modified with a molecule or group A; and, (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B, wherein the molecule or group A can form a connection (e.g., a covalent or non-covalent connection) with the molecule or group B; and, (2) under conditions suitable for forming a connection between the molecule or group A and the molecule or group B, contacting the positioning probe with the solid support, thereby obtaining a solid support connected with the positioning probe; Preferably, the molecule or group A is capable of undergoing a click chemistry reaction with the molecule or group B; Preferably, the molecules or groups A/B are selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone; Preferably, the molecules or groups A/B are azide/alkynyl or alkynyl/azide; Preferably, the localization probe is modified with (DBCO), the surface of the solid support is modified with an azide group, and the positioning probe and the solid support are connected through a click chemical reaction between DBCO and the azide group. The method of claim 9, wherein step (A) comprises: (1) providing at least two vectors, each vector comprising at least one copy of a vector sequence, wherein the vector sequence comprises: a complementary sequence of a positioning sequence and a complementary sequence of a first universal sequence; the first universal sequence and the positioning sequence are as defined in any one of claims 1, 2, and 5; wherein the complementary sequence of the positioning sequence of each vector sequence is different from each other; (2) placing at least two of the carriers on the surface of the solid support; (3) providing a fixed primer, and using the vector sequence as a template, performing a nucleic acid polymerization reaction to generate an extension product, wherein the extension product is a positioning probe; wherein the fixed primer can anneal with the vector sequence and initiate an extension reaction; and, (4) connecting the immobilized primer to the surface of the solid support; wherein steps (3) and (4) are performed in any order (for example, step (3) is performed before or after step (4), or step (3) and step (4) are performed simultaneously); Preferably, the vector sequence comprises, in order from the 5' end to the 3' end: a complementary sequence to the universal sequence, and a complementary sequence to the positioning sequence; Preferably, the extension product comprises, in order from 5' to 3', the positioning sequence and the first universal sequence; Preferably, each vector is a DNB formed by concatemers of multiple copies of the vector sequence; Preferably, the method optionally comprises step (5): digesting the vector sequence, and/or separating the extension product in step (3) from the vector sequence annealed thereto; Preferably, in step (1), the carrier is provided by the following steps: (i) providing a vector template sequence, wherein the vector template sequence comprises a complementary sequence to the vector sequence; (ii) performing a nucleic acid amplification reaction using the vector template sequence as a template to obtain an amplification product of the vector template sequence, wherein the amplification product comprises at least one copy of the vector sequence; preferably, performing a rolling circle replication to obtain a DNB formed by concatemers of the vector sequence. The method of claim 11, wherein the vector sequence further comprises a complementary sequence to a second universal sequence or a partial sequence thereof (e.g., the vector sequence further comprises a complementary sequence to the second universal sequence or a partial sequence at its 3' end), the second universal sequence being as defined in claim 2; Preferably, the complementary sequence of the second universal sequence or a partial sequence thereof is located at the 3' end of the complementary sequence of the positioning sequence; Preferably, the fixed primer comprises the second universal sequence or a partial sequence thereof (for example, the fixed primer comprises the second universal sequence or a partial sequence at the 5' end thereof); Preferably, the extension product comprises, in order from 5' to 3', the second universal sequence, the positioning sequence, and the first universal sequence; Preferably, the vector sequence does not contain or further contains a template sequence of a MID sequence; Preferably, the vector sequence further comprises a template sequence of a MID sequence; preferably, the template sequence of the MID sequence is located at the 3' end of the complementary sequence of the first universal sequence, and/or, the template sequence of the MID sequence is located at the 5' end of the complementary sequence of the second universal sequence or a partial sequence thereof; preferably, the MID sequence consists of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35 or 20-45) degenerate deoxyribonucleotide residues. The method of claim 11 or 12, wherein the immobilized primer is covalently or non-covalently linked to the solid support; Preferably, the immobilized primer is covalently linked to the solid support; Preferably, the immobilized primer is connected to the solid support via a click chemistry reaction; Preferably, the molecule or group pair capable of the click chemistry reaction is selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol; preferably, the molecule or group pair capable of the click chemistry reaction is azido/alkynyl; Preferably, the fixed primer is modified with (DBCO), the surface of the solid support is modified with an azido group, and the immobilized primer and the solid support are formed by a click chemical reaction between DBCO and the azido group. Into connection. The method of any one of claims 9 to 13, wherein the capture probe is in a single-stranded form comprising a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined in any one of claims 1, 3 to 5, 8, and step (B) comprises: (1) providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and, (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent or non-covalent connection) with the molecule or group B'; and, (2) contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support connected with the capture probe; Preferably, the capture sequence is located at the 3' end of the capture probe. The method of any one of claims 9 to 13, wherein the capture probe is present in a duplex form containing a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined in any one of claims 1, 3 to 5, 8, and step (B) comprises: (I)(1) providing: (a) a free first strand of the capture probe, the first strand being modified with a molecule or group A'; and, (b) the solid support, the surface of the solid support being modified with a molecule or group B', the molecule or group A' being capable of forming a connection (e.g., covalent and/or non-covalent connection) with the molecule or group B'; and, (c) the free second strand of the capture probe, the second strand comprising the capture sequence and being capable of annealing with the first strand to form a duplex; (2) contacting the first chain with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the first chain is connected; and, (3) contacting the free second strand with the solid support connected to the first strand formed in step (2) under conditions allowing annealing, thereby obtaining a solid support connected to the capture probe; or, (II)(1) providing: (a) a duplex formed by annealing a first strand and a second strand of the capture probe, wherein the first strand is connected to a molecule or group A', and the second strand contains the capture sequence; and, (b) a solid support, wherein the surface of the solid support is modified with a molecule or group B', and the molecule or group A' is capable of forming a connection (e.g., covalent and/or non-covalent connection) with the molecule or group B'; and, (2) contacting the duplex with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support connected with the capture probe; Preferably, in the duplex formed by annealing the first strand and the second strand of the capture probe, the second The 3' end of the strand comprises the capture sequence, and the capture sequence does not anneal to the first strand. The method of claim 14 or 15, wherein the molecule or group A' is capable of undergoing a click chemistry reaction with the molecule or group B'; Preferably, the molecules or groups A'/B' are selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone; Preferably, the molecules or groups A'/B' are azide/alkynyl or alkynyl/azide; Preferably, the first strand of the capture probe is modified with (DBCO), the surface of the solid support is modified with an azide group, and the first chain of the capture probe and the solid support are connected through a click chemical reaction between DBCO and the azide group. A method for preparing a nucleic acid array according to any one of claims 1 to 8, comprising the following steps: (A) providing a nucleic acid array to be processed, wherein the nucleic acid array to be processed comprises a solid support, and the solid support is connected with at least two oligonucleotide molecules; Each oligonucleotide molecule occupies a different position on the solid support; The oligonucleotide molecule contains a positioning sequence and a first universal sequence in sequence from the 5' end to the 3' end, wherein the positioning sequence has a nucleotide sequence corresponding to the position of the oligonucleotide molecule on the solid support; the first universal sequence is defined in any one of claims 1, 2, and 5; and, (B) Connecting and/or synthesizing capture probes on the solid support contained in the nucleic acid array to be processed, wherein the capture probes are as defined in any one of claims 1, 3-5, and 8. The method of claim 17, wherein the oligonucleotide molecule does not contain a capture sequence at the 3' end of the first universal sequence, and the capture sequence is as defined in any one of claims 1, 3-5, and 8. The method of claim 17, wherein the oligonucleotide molecule comprises a capture sequence at the 3' end of the first universal sequence, the capture sequence being as defined in any one of claims 1, 3-5, and 8, and the method further comprises step (B'): removing the capture sequence contained in the oligonucleotide molecule; The steps (B) and (B') can be performed in any order or simultaneously (for example, simultaneously in the same reaction system). The method of claim 19, wherein in step (B') the capture sequence contained in the oligonucleotide molecule is removed by the steps comprising: (i) providing a blocking probe, wherein the blocking probe is capable of annealing with (a) the first universal sequence or a partial sequence thereof (e.g., a partial sequence at the 3' end of the first universal sequence) of the oligonucleotide molecule, or (b) a sequence located at the 5' end of the capture sequence and at the 3' end of the first universal sequence in the oligonucleotide molecule, or (c) a combination of (a) and (b); (ii) contacting the blocking probe with the nucleic acid array to be processed containing the oligonucleotide molecules under conditions suitable for annealing the blocking probe with the oligonucleotide molecules; (iii) contacting the product of step (ii) with an exonuclease under conditions that allow the exonuclease to exert its cleavage activity; wherein the exonuclease has exonuclease activity from the 3' to 5' end of a single-stranded nucleic acid; Preferably, the exonuclease is exonuclease I; Preferably, the step (B') further comprises the step of removing the blocking probe (e.g., removing the blocking probe by unzipping the blocking probe from the oligonucleotide molecule); Preferably, in the method, step (B) is performed after step (B'). The method of any one of claims 17 to 20, wherein the oligonucleotide molecule further comprises a second universal sequence, the second universal sequence being as defined in claim 2 or 5; Preferably, the second universal sequence is located at the 5' end of the positioning sequence; Preferably, the oligonucleotide molecule does not contain or further contains a MID sequence; Preferably, the oligonucleotide molecule further comprises a MID sequence; preferably, the MID sequence is located at the 5' end of the first universal sequence, and/or, the MID sequence is located at the 3' end of the second universal sequence; preferably, the MID sequences contained in the same oligonucleotide molecules are different from each other. The method of any one of claims 17 to 21, wherein in the nucleic acid array to be processed, the oligonucleotide molecules are covalently or non-covalently linked to the solid support; Preferably, the oligonucleotide molecule is covalently linked to the solid support; Preferably, the oligonucleotide molecule is linked to the solid support via a click chemistry reaction; Preferably, the molecule or group pair capable of the click chemistry reaction is selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol; preferably, the molecule or group pair capable of the click chemistry reaction is azido/alkynyl; Preferably, the surface of the solid support is modified with an azide group, and the oligonucleotide molecule is modified with (DBCO), the oligonucleotide molecule is connected to the solid support through a click chemistry reaction between the azide group and the DBCO. The method of any one of claims 17 to 22, wherein the capture probe is in a single-stranded form comprising a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined in any one of claims 1, 3 to 5, 8, and step (B) comprises: (1) providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and, (b) the nucleic acid array to be treated or the nucleic acid array treated in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' can form a connection (e.g., covalent or non-covalent connection) with the molecule or group B'; and, (2) contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support connected with the capture probe; Preferably, the capture sequence is located at the 3' end of the capture probe. The method of any one of claims 17 to 23, wherein the capture probe is in the form of a duplex containing a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined in any one of claims 1, 3 to 5, 8, and step (B) comprises: (I)(1) providing: (a) a free first strand of the capture probe, wherein the first strand is modified with a molecule or group A'; and, (b) the nucleic acid array to be treated or the nucleic acid array treated in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent and/or non-covalent connection) with the molecule or group B'; and, (c) the free second strand of the capture probe, wherein the second strand comprises the capture sequence and is capable of annealing with the first strand to form a duplex; (2) contacting the first chain with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the first chain is connected; and, (3) contacting the free second strand with the solid support connected to the first strand formed in step (2) under conditions allowing annealing, thereby obtaining a solid support connected to the capture probe; or, (II)(1) providing: (a) a duplex formed by annealing the first strand and the second strand of the capture probe, wherein the first strand is connected to a molecule or group A', and the second strand contains the capture sequence; and (b) the nucleic acid array to be treated or the nucleic acid array treated in step (B'), wherein the surface of the solid support of the nucleic acid array is modified with a molecule or group A'. or group B', the molecule or group A' is capable of forming a connection (eg, covalent and/or non-covalent connection) with the molecule or group B'; and, (2) contacting the duplex with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support connected with the capture probe; Preferably, in the duplex formed by annealing the first strand and the second strand of the capture probe, the 3' end of the second strand contains the capture sequence, and the capture sequence does not anneal with the first strand. The method of claim 23 or 24, wherein the molecule or group A' is capable of undergoing a click chemistry reaction with the molecule or group B'; Preferably, the molecules or groups A'/B' are selected from: alkynyl/azido, azido/cyano, amine/ene, thiol/ene, thiol/alkyne, aldehyde/1,3-diol, ketone/1,3-diol, azido/alkynyl, cyano/azido, ene/amine, ene/thiol, alkyne/thiol, 1,3-diol/aldehyde, 1,3-diol/ketone; Preferably, the molecules or groups A'/B' are azide/alkynyl or alkynyl/azide; Preferably, the first strand of the capture probe is modified with (DBCO), the surface of the solid support is modified with an azide group, and the first chain of the capture probe and the solid support are connected through a click chemical reaction between DBCO and the azide group. A method for detecting spatial information of nucleic acid in a sample, comprising the following steps: (1) Providing a nucleic acid array according to any one of claims 1 to 8; (2) contacting the nucleic acid array with a sample to be tested under conditions that allow annealing, so that the nucleic acid derived from the sample to be tested anneals with the capture sequence in the capture probe of the nucleic acid array; (3) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization to produce a first extension product, wherein the first extension product comprises a capture sequence and a complementary sequence of a nucleic acid molecule or a partial sequence thereof that anneals to the capture sequence, and the first extension product is connected to the solid support via the capture sequence contained therein; (4) annealing the first extension product connected to the solid support with the adjacent positioning probe of the nucleic acid array under conditions that allow annealing; wherein the first universal sequence of the positioning probe can anneal with the first extension product; and the first universal sequence is located at the 3' end of the positioning sequence of the positioning probe; (5) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization to produce a second extension product and/or its complementary chain, wherein the second extension product comprises: (a) the positioning sequence of the positioning probe and a complementary sequence to the first extension product or a partial sequence thereof, or (b) the first extension product sequence or a partial sequence thereof and the positioning probe; the complementary sequence of the positioning sequence; the positioning sequence or its complementary sequence contained in the second extension product serves as its spatial information marker, and the complementary sequence of the positioning sequence or the positioning sequence contained in the complementary chain of the second extension product serves as its spatial information marker, so that the position of the nucleic acid derived from the sample to be tested in the sample to be tested is corresponded to the position of the positioning probe corresponding to the positioning sequence; thereby obtaining a nucleic acid molecule containing the spatial information marker; and (6) analyzing: (i) the nucleic acid molecule containing the spatial information marker obtained in step (5), and/or, (ii) the sequence of the nucleic acid molecule containing the spatial information marker derived from (i); Preferably, the spatial information of the nucleic acid includes the location, distribution and/or abundance of the nucleic acid. The method of claim 26, wherein in step (3), the nucleic acid polymerization reaction uses a nucleic acid molecule annealed to the capture sequence as a template to extend the capture sequence; Preferably, the capture sequence is located at the 3' end of the capture probe, and/or the 3' end of the capture sequence has a free hydroxyl group (-OH). The method of claim 26 or 27, wherein in step (5), the polymerization reaction uses the first extension product as a template to extend the positioning probe; and/or uses the positioning probe as a template to extend the first extension product; For example, in step (5), the polymerization reaction uses the first extension product as a template to extend the positioning probe to produce a second extension product and/or its complementary chain, wherein the second extension product comprises the positioning sequence of the positioning probe and a complementary sequence to the first extension product or a partial sequence thereof; preferably, the first universal sequence of the positioning probe is located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe has a free hydroxyl group (-OH); For example, in step (5), the polymerization reaction uses the positioning probe as a template to extend the first extension product to produce a second extension product and/or its complementary chain, wherein the second extension product comprises the first extension product sequence or a partial sequence thereof and a complementary sequence to the positioning sequence of the positioning probe; preferably, the first universal sequence of the positioning probe is located at or not located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe is blocked or unblocked; For example, in step (5), in the polymerization reaction, the first extension product and the positioning probe serve as templates for each other, and the first extension product and the positioning probe are extended to produce a second extension product and/or its complementary chain, wherein the second extension product comprises the positioning sequence of the positioning probe and the complementary sequence of the first extension product or a partial sequence thereof, and the second extension product comprises the first extension product sequence or a partial sequence thereof and the complementary sequence of the positioning sequence of the positioning probe; preferably, the first universal sequence of the positioning probe is located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe has a free hydroxyl group (-OH). The method of any one of claims 26 to 28, wherein in step (6), the sequence of the nucleic acid molecule containing the spatial information marker connected to the solid support of the nucleic acid array is analyzed. The method of any one of claims 26 to 28, wherein, before step (6) and after step (5), the method further comprises a step pre-(6): releasing at least a portion of the nucleic acid molecules containing spatial information labels from the surface of the nucleic acid array; Preferably, in step (6), (i) the nucleic acid molecule containing the spatial information marker released in step pre-(6), and/or, (ii) the sequence of the nucleic acid molecule containing the spatial information marker derived from (i) is analyzed; Preferably, in step pre-(6), the nucleic acid molecule is released from the surface of the solid support by: (i) nucleic acid shearing; and/or, (ii) denaturation; Preferably, after step pre-(6) and before step (6), the method further comprises the step of amplifying the released nucleic acid molecule; Preferably, before performing step (6), the method further comprises a step of purifying the released nucleic acid molecules. The method of any one of claims 26 to 30, which has one or more features selected from the following: (i) in step (1), providing the nucleic acid array by the method according to any one of claims 9 to 25; (ii) in step (2), the sample to be tested is treated (for example, permeabilized or cell lysed) to release the nucleic acid from the sample to be tested so that the nucleic acid anneals with the capture sequence; (iii) after step (3) and before step (4), the method further comprises the step of removing the template strand bound to the first extension product (for example, removing the template strand bound to the first extension product by enzyme cleavage or denaturing and melting); (iv) after step (3) and before step (4), the method further comprises a step of washing the nucleic acid array to remove residual sample (e.g., tissue or cells); (v) After step (5) and before step (6) (for example, after step (5) and before step pre-(6)), the method further comprises the step of amplifying (for example, in situ amplification) the nucleic acid molecules containing the spatial information markers; preferably, the nucleic acid molecules containing the spatial information markers are connected to the solid support of the nucleic acid array. The method of any one of claims 26 to 31, wherein the sample is a tissue sample (e.g., a tissue section) or a single cell sample (e.g., a single cell suspension); Preferably, the tissue sample (eg, tissue section) is prepared from fixed tissue, such as formalin-fixed paraffin-embedded (FFPE) tissue, deep-frozen tissue or fresh tissue. The method of any one of claims 26-32, wherein the nucleic acid derived from the sample to be tested is selected from: RNA molecules (e.g., mRNA) molecules, target nucleic acid molecules (e.g., target DNA and/or RNA), genomic nucleic acid fragments in open chromatin regions, and any combination thereof. The method of any one of claims 26 to 33, wherein, in step (6), the analysis comprises sequencing and/or sequence-specific PCR reaction; Preferably, before sequencing, the method further comprises the step of constructing a sequencing library for the nucleic acid molecule containing the spatial information marker or its amplified product obtained in step (5). The method of any one of claims 26-34, wherein the method is used to detect spatial information of RNA (e.g., mRNA) of cells in a sample. The method of claim 35, wherein in step (2), the nucleic acid derived from the sample to be tested is RNA (e.g., mRNA) in cells of the sample to be tested, and the capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence; Preferably, the step (3) comprises: (a) under conditions allowing nucleic acid polymerization, using a nucleic acid molecule annealed to the capture sequence as a template, performing a nucleic acid polymerization reaction, extending the capture sequence, and generating a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the capture sequence as a reverse transcription primer, and a 3' end overhang; (b) annealing the template switching sequence to the cDNA chain generated in (a) under conditions that allow nucleic acid polymerization, and continuing nucleic acid polymerization reaction using the template switching sequence as a template to generate the first extension product; Wherein, the template switching sequence comprises a consensus sequence, a 3'-terminal overhang complementary sequence, and an optional MID sequence; preferably, the MID sequences contained in each template switching sequence are different from each other; The first extension product comprises: a capture sequence, the cDNA chain sequence or a partial sequence thereof, and a complementary sequence of the common sequence; Preferably, the method further comprises, before step (4), a step of removing the template switching sequence bound to the first extension product (for example, removing the template switching sequence bound to the first extension product by enzyme cleavage or denaturing and melting); Preferably, in step (4) of the method, the first universal sequence of the localization probe is capable of annealing to the complementary sequence of the consensus sequence; Preferably, the positioning probe does not contain a MID sequence, and the template switching sequence contains a MID sequence; or, the positioning probe contains a MID sequence, and the template switching sequence does not contain a MID sequence, or, both the positioning probe and the template switching sequence contain a MID sequence. The method of any one of claims 26 to 34, wherein the method is used to detect spatial information of a target nucleic acid (e.g., a target DNA and/or RNA) of a cell in a sample, wherein the target nucleic acid comprises a target nucleotide sequence; Preferably, in step (2), the nucleic acid derived from the sample to be tested comprises the target nucleotide sequence; preferably, the nucleic acid derived from the sample to be tested comprises the target nucleic acid and/or a nucleic acid molecule derived from the target nucleic acid, and the nucleic acid molecule derived from the target nucleic acid comprises the target nucleotide sequence; Preferably, in step (2), the capture sequence comprises a sequence that specifically recognizes the target nucleotide sequence; Preferably, in step (2), the capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence; in step (4), the first universal sequence of the positioning probe comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence comprises a random oligonucleotide sequence; preferably, in the target nucleotide sequence, the first segment is located at the 3' end of the second segment; Preferably, in step (2), the first segment is present in a single-stranded region of a nucleic acid molecule derived from the sample to be tested that anneals to the capture sequence; Preferably, in step (2), the nucleic acid derived from the sample to be tested that anneals to the capture sequence is a single-stranded nucleic acid or a double-stranded nucleic acid containing a single-stranded region including the first segment. The method of any one of claims 26 to 34, wherein the method is used to detect spatial information of RNA (e.g., mRNA) and target nucleic acid (e.g., target DNA and/or RNA) of cells in a sample, wherein the target nucleic acid comprises a target nucleotide sequence; Preferably, the nucleic acid array contains a first capture probe capable of capturing the RNA (e.g., mRNA), and a second capture probe capable of capturing a nucleic acid molecule containing the target nucleotide sequence; the first capture probe contains a first capture sequence, the first capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence; the second capture probe contains a second capture sequence, the second capture sequence comprises a sequence that specifically recognizes the target nucleotide sequence; Preferably, in step (2), the nucleic acid derived from the sample to be tested comprises RNA (e.g., mRNA) derived from the sample to be tested and a nucleic acid molecule comprising a target nucleotide sequence; preferably, the nucleic acid molecule comprising a target nucleotide sequence comprises the target nucleic acid and/or a nucleic acid molecule derived from the target nucleic acid; Preferably, the step (3) comprises: (A)(a) Under conditions that allow nucleic acid polymerization, using a nucleic acid molecule that anneals to the first capture sequence as a template, performing a nucleic acid polymerization reaction, extending the first capture sequence, and generating a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the first capture sequence as a reverse transcription primer, and a 3' end overhang; (b) under conditions that allow nucleic acid polymerization, combining the template switching sequence with the cDNA generated in (a) The strands are annealed, and the nucleic acid polymerization reaction is continued with the template switching sequence as a template to generate a first extension product I; Wherein, the template switching sequence comprises a consensus sequence, a 3'-terminal overhang complementary sequence, and an optional MID sequence; preferably, the MID sequences contained in each template switching sequence are different from each other; as well as, (B) under conditions allowing nucleic acid polymerization, using the nucleic acid molecule annealed to the second capture sequence as a template, performing a nucleic acid polymerization reaction to extend the second capture sequence to generate a first extension product II, wherein the first extension product II comprises a second capture sequence and a complementary sequence to the nucleic acid molecule annealed to the second capture sequence or a partial sequence thereof; Wherein, step (B) and step (A) are performed in any order; for example, step (B) is performed before or after step (A), or step (B) and step (A) are performed simultaneously (for example, in the same reaction system); Preferably, in step (4), the positioning probe contains a first universal sequence I that can anneal to the first extension product I, and a first universal sequence II that can anneal to the first extension product II; preferably, the first universal sequence I and the first universal sequence II are present in the same positioning probe, or are present in different positioning probes respectively; Preferably, the nucleic acid array comprises a first positioning probe comprising the first universal sequence I and a second positioning probe comprising the first universal sequence II; Preferably, in step (4) of the method, the first universal sequence I is capable of annealing with a complementary sequence of the consensus sequence; Preferably, in step (2) of the method, the second capture sequence comprises a sequence that specifically recognizes the first segment of the target target nucleotide sequence; in step (4), the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence; preferably, in the target target nucleotide sequence, the first segment is located at the 3' end of the second segment; Preferably, the method further comprises, before step (4), a step of removing the template switching sequence bound to the first extension product I (for example, removing the template switching sequence bound to the first extension product I by enzyme cleavage or denaturing and melting); Preferably, in step (2), the first segment is present in a single-stranded region of a nucleic acid molecule derived from a sample to be tested that anneals to the second capture sequence; Preferably, in step (2), the nucleic acid derived from the sample to be tested that anneals with the second capture sequence is a single-stranded nucleic acid or a double-stranded nucleic acid containing a single-stranded region including the first segment; Preferably, the first positioning probe does not contain a MID sequence, and the template switching sequence contains a MID sequence; or, the first positioning probe contains a MID sequence, and the template switching sequence does not contain a MID sequence, or, both the first positioning probe and the template switching sequence contain a MID sequence. A kit comprising the nucleic acid array according to any one of claims 1 to 8. The kit of claim 39, wherein the capture sequence of the nucleic acid array comprises a poly(dT) sequence or a random oligonucleotide sequence; Preferably, the kit further comprises a template switching sequence, wherein the template switching sequence comprises a consensus sequence, a cDNA 3' terminal overhang complementary sequence, and an optional MID sequence; preferably, the MID sequences contained in each template switching sequence are different from each other; preferably, the cDNA 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or 2-10 nucleotides; preferably, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., CCC overhang); Preferably, the complementary sequence of the consensus sequence of the template switching sequence can anneal with the first universal sequence of the positioning probe of the nucleic acid array; Preferably, the positioning probe does not contain a MID sequence, and the template switching sequence contains a MID sequence; or, the positioning probe contains a MID sequence, and the template switching sequence does not contain a MID sequence; or, both the positioning probe and the template switching sequence contain a MID sequence. The kit of claim 39, wherein the capture sequence of the nucleic acid array comprises a sequence that specifically recognizes a target target nucleotide sequence comprised in a target nucleic acid (e.g., a specific target DNA and/or RNA); Preferably, the first universal sequence comprises a sequence that specifically recognizes a complementary sequence of the target nucleotide sequence; Preferably, the capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence, the first universal sequence of the positioning probe of the nucleic acid array comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence comprises a random oligonucleotide sequence; Preferably, in the target nucleotide sequence, the first segment is located at the 3’ end of the second segment. The kit of claim 39, wherein the nucleic acid array comprises a first capture probe and a second capture probe; the first capture probe comprises a first capture sequence, the first capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence; the second capture probe comprises a second capture sequence, the second capture sequence comprises a sequence that specifically recognizes a target target nucleotide sequence contained in a target nucleic acid (e.g., a specific target DNA and/or RNA); Preferably, the kit further comprises a template switching sequence, wherein the template switching sequence comprises a consensus sequence, cDNA 3' terminal overhang complementary sequence, and optionally a MID sequence; preferably, the MID sequence contained in each template switch sequence is different from each other; preferably, the cDNA 3' terminal overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or 2-10 nucleotides; preferably, the cDNA 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., CCC overhang); Preferably, the positioning probe of the nucleic acid array comprises a first universal sequence I and a first universal sequence II; wherein the first universal sequence I can anneal with the complementary sequence of the common sequence, and the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence; preferably, the first universal sequence I and the first universal sequence II are present in the same positioning probe together, or, respectively, in different positioning probes; Preferably, the nucleic acid array comprises a first positioning probe containing the first universal sequence I and a second positioning probe containing the first universal sequence II; preferably, the first positioning probe does not contain a MID sequence, and the template switching sequence contains a MID sequence; or, the first positioning probe contains a MID sequence, and the template switching sequence does not contain a MID sequence, or, both the first positioning probe and the template switching sequence contain a MID sequence; Preferably, the second capture sequence comprises a sequence that specifically recognizes the first segment of the target target nucleotide sequence, the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence; preferably, in the target target nucleotide sequence, the first segment is located at the 3’ end of the second segment. The kit of any one of claims 39 to 42, wherein the first universal sequence contained in the positioning probe of the nucleic acid array is located at or not located at the 3' end of the positioning probe, and/or the 3' end of the first universal sequence of the positioning probe of the nucleic acid array is blocked or unblocked; Preferably, the kit further comprises: reagents for nucleic acid hybridization, reagents for nucleic acid extension, reagents for nucleic acid amplification, reagents for recovering or purifying nucleic acids, reagents for constructing a transcriptome sequencing library, reagents for sequencing (e.g., second-generation sequencing or third-generation sequencing), or any combination thereof. Use of the nucleic acid array of any one of claims 1 to 8 or the kit of any one of claims 39 to 43 for constructing a nucleic acid molecule library, for nucleic acid sequencing or for detecting nucleic acid spatial information in a sample.