Oligonucleotides, omics analysis method and omics analysis kit

Abstract

This oligonucleotide includes an added sequence for nucleic acid detection. At least one nucleotide in the added sequence is modified with a photocleavable-type protecting group.

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide, an omics analysis method, and an omics analysis kit. [Background technology]

[0002] Using next-generation sequencers capable of determining large amounts of base sequences, methods have been established to obtain omics information, such as RNA sequence analysis (RNA-seq), comprehensive open chromatin region analysis (ATAC-seq), and genomic methylation region analysis (Bisulfite-seq).

[0003] As an example of RNA-seq specialized for a small number of cells, Non-Patent Document 1 discloses CEL-Seq (Cell Expression by Linear amplification and Sequencing), which uses linear amplification by in vitro transcription (IVT) reaction.

[0004] CEL-Seq2, which is an improvement of CEL-Seq and has improved sensitivity, is disclosed in Non-Patent Document 2. In CEL-Seq2, first, an oligonucleotide having an additional sequence including a T7 promoter sequence and a poly T sequence at the 5' end is hybridized to the poly A end of mRNA in a sample, and a first strand is synthesized by reverse transcriptase. Then, a second strand, which is a complementary strand of the first strand, is synthesized. Next, an IVT reaction is carried out by T7 RNA polymerase. This allows the base sequence on the 3' end side of the T7 promoter sequence to be amplified as RNA. Then, a library is synthesized by reverse transcription and polymerase chain reaction (PCR), and the base sequence is analyzed by a sequencer. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tamar Hashimshony and 3 others, "CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification", Cell Rep., 2012, 2(3), 666-673 [Non-Patent Document 2] Tamar Hashimshony and 12 others, “CEL-Seq2:sensitive highly-multiplexed single-cell RNA-Seq”, Genome Biology, 2016, 17(77) Summary of the Invention [Problem to be solved by the invention]

[0006] CEL-Seq and CEL-Seq2 can obtain gene expression information for individual cells, but require cell isolation, which requires cell sorting or laser microdissection to separate the cells.

[0007] As mentioned above, cell sorting can damage cells. Therefore, the omics information obtained from the sorted cells may differ from the actual state of the living body due to the damage. In addition, there is the inconvenience that the spatial resolution is limited depending on the sorting method.

[0008] The present invention has been made in consideration of the above-mentioned situation, and aims to provide an oligonucleotide, an omics analysis method, and an omics analysis kit that can obtain omics information that more accurately reflects the actual state of the living body with high spatial resolution. [Means for solving the problem]

[0009] The oligonucleotide according to the first aspect of the present invention comprises: Transfer Promoter sequence, a base sequence involved in the initiation of transcription and an identification sequence for identification located on the 3' end side of the promoter sequence. An additional sequence for detecting nucleic acids, and, a poly T sequence at the 3' end of the additional sequence; having At least one nucleotide in the additional sequence is bound to the base of the nucleotide, inhibits the formation of hydrogen bonds between the base and a complementary base, and is decomposed by irradiation with light to release the inhibition of hydrogen bond formation. It is modified with a photocleavable protecting group, The nucleotide is It is located on the 3'-terminal side of the promoter sequence.

[0010] In this case, the photocleavable protecting group is 6 -nitropiperonyloxymethyl group, This may also be the case.

[0011] The oligonucleotide according to the first aspect of the present invention is At the 3' end of the additional sequence, The above Further comprising at least one nucleotide modified with a photocleavable protecting group; This may also be the case.

[0015] The omics analysis method according to the second aspect of the present invention comprises: an exposure step of exposing the oligonucleotide according to the first aspect of the present invention to a tissue section or a cell mass; a first synthesis step of synthesizing a first strand by extending the oligonucleotide using mRNA contained in the tissue section or cell mass as a template; an irradiation step of irradiating a portion of the tissue slice or cell aggregate with light; a second synthesis step of synthesizing a second strand complementary to the first strand using the first strand as a template; an IVT step of synthesizing cRNA from double-stranded DNA consisting of the first strand and the second strand by an in vitro transcription reaction; Using cDNA synthesized from the cRNA determining a base sequence of the first strand or the second strand; Includes.

[0018] The present invention 3 The omics analysis kit according to the above aspect is The present invention comprises an oligonucleotide according to the first aspect of the present invention. Effect of the Invention

[0019] According to the present invention, omics information that more accurately reflects the actual state of a living body can be obtained with high spatial resolution. [Brief description of the drawings]

[0020] [Figure 1] FIG. 2 shows the base sequence of an oligonucleotide according to an embodiment of the present invention and the position of a photocleavable protecting group in the base sequence. [Diagram 2] 1 is a schematic diagram for explaining CEL-Seq2 using oligonucleotides according to an embodiment of the present invention. (A) shows the extension reaction of the first strand. (B) shows the state of the synthesis reaction of the second strand without light irradiation. (C) shows the state of the synthesis reaction of the second strand with light irradiation. [Diagram 3] 1 is a schematic diagram for explaining a genome sequence analysis method using oligonucleotides according to another embodiment of the present invention. (A) shows the structure of the oligonucleotide. (B) shows the oligonucleotide inserted into genome DNA. (C) shows the blunted DNA fragment obtained from the DNA fragment in the light-irradiated region. [Figure 4] FIG. 1 shows the base sequence of an oligodeoxyribonucleic acid (DNA) according to Example 1 and the positions of photocleavable protecting groups in the base sequence. [Diagram 5] FIG. 5 shows the quantification results of the Gapdh gene in quantitative PCR (qPCR) for cDNA obtained by CEL-seq2 of cultured cells using the oligo DNA shown in FIG. 4. [Figure 6] FIG. 5 shows the results of electrophoresis of a library constructed using the oligo DNA shown in FIG. [Figure 7] 1 shows the expression of the Sox2 gene in tissue sections according to Example 2. (A) shows the position of ultraviolet (UV) irradiation in the tissue sections, and (B) shows the results of immunostaining for the Sox2 gene. [Figure 8]1 shows the results of qPCR for the Sox2 gene and Gapdh gene in Example 2. (A) and (B) show the expression levels of the Sox2 gene and Gapdh gene, respectively. [Figure 9] FIG. 1 shows the base sequence of an oligo-DNA according to Example 3 and the position of a photocleavable protecting group in the base sequence. [Figure 10] FIG. 13 shows the results of electrophoresis of the reaction products in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to the accompanying drawings, in which: FIG.

[0022] (Embodiment 1) The oligonucleotide according to the present embodiment has an additional sequence for nucleic acid detection. The additional sequence is a base sequence designed for nucleic acid detection. Preferably, the additional sequence is a base sequence that does not hybridize with a target nucleic acid in a living body to be detected. For example, the additional sequence is a base sequence added to at least one of the 5'-end and 3'-end of a nucleic acid in a living body to be detected or a nucleic acid complementary to the nucleic acid.

[0023] More specifically, examples of the additional sequence include an adapter sequence, which is a common sequence for amplifying or detecting a nucleic acid, a promoter sequence involved in the initiation of transcription, and an identification sequence for identification. The adapter sequence is, for example, a primer binding sequence for PCR and a base sequence for amplification by bridge PCR using a next-generation sequencer or the like. The promoter sequence is, for example, a T7, T3, or SP6 promoter sequence used in IVT or the like for nucleic acid detection.

[0024] Examples of the identification sequence include a barcode sequence and a UMI (Unique Molecular Identifier) ​​sequence. The barcode sequence is an artificial sequence that is determined in advance for each sample to be analyzed. When the oligonucleotide is used for multiple samples, a barcode sequence with a different base sequence is assigned to each sample. The UMI sequence is a random base sequence. The identification sequence may include both the barcode sequence and the UMI sequence, or may be either one of them. When used in CEL-Seq2, the additional sequence preferably includes, from the 5' end to the 3' end, a T7 promoter sequence, an adapter sequence, a UMI sequence, and a barcode sequence.

[0025] In the oligonucleotide according to the present embodiment, at least one nucleotide in the additional sequence is modified with a photocleavable protecting group. The photocleavable protecting group is bonded to a base of a nucleotide in the base sequence of the oligonucleotide. The protecting group is not particularly limited as long as it inhibits hydrogen bond formation between the base and a complementary base and can be decomposed by irradiation with light to release the inhibition of hydrogen bond formation. For example, the protecting group is a 2-(2-nitrophenyl)propyl group, a 2-(2-nitrophenyl)propyloxymethyl group, a 1-(2-nitrophenyl)ethyl group, a 6-nitropiperonyloxymethyl group (NPOM), etc. The photocleavable protecting group is preferably NPOM. The base into which the photocleavable protecting group is introduced is preferably thymine (T), but is not limited to thymine, and may be adenine (A), cytosine (C), or guanine (G) depending on the protecting group used.

[0026] FIG. 1 illustrates the base sequences of oligonucleotides 1 to 6 according to the present embodiment used in CEL-Seq2 and the positions of photocleavable protecting groups. In each of oligonucleotides 1 to 6, the base sequences surrounded by dashed lines are additional sequences. The additional sequences contained in oligonucleotides 1 to 6 all include a T7 promoter sequence, an adapter sequence, a UMI sequence, and a barcode sequence. In FIG. 1, the "t" marked with a black circle is deoxythymidine modified with a photocleavable protecting group. As shown in FIG. 1, the photocleavable protecting group is located in the additional sequence. It is sufficient that the oligonucleotide contains at least one nucleotide modified with a photocleavable protecting group, and preferably contains multiple nucleotides.

[0027] Preferably, when the additional sequence contains a promoter sequence such as a T7 promoter, a nucleotide modified with a photocleavable protecting group, as in oligonucleotides 3 to 6, is located on the 3'-terminal side of the promoter sequence, for example, between the promoter sequence and the adapter sequence.

[0028] In addition to the photocleavable protecting group located in the additional sequence, the oligonucleotide may further have at least one nucleotide modified with a photocleavable protecting group at the 3' end of the additional sequence. For example, like oligonucleotides 2, 4 to 6 shown in Figure 1, the oligonucleotide according to this embodiment has 1 to 3 nucleotides modified with a photocleavable protecting group at the 3' end of the additional sequence.

[0029] The sequence length of the oligonucleotide according to this embodiment is, for example, 10 to 120 bases, 20 to 110 bases, 50 to 100 bases, 60 to 95 bases, or 70 to 90 bases. When used in CEL-Seq2, the sequence length of the oligonucleotide is preferably 80 to 90 bases. The oligonucleotide according to this embodiment can be synthesized by a known method. The oligonucleotide is also called a polynucleotide.

[0030] When the oligonucleotide according to the present embodiment is used as a template for the extension reaction of a nucleic acid, the formation of a hydrogen bond between the base modified with a photocleavable protecting group and the complementary base is inhibited. Therefore, in the extension reaction, the region complementary to the base sequence from the nucleotide modified with a photocleavable protecting group to the 5' end in the oligonucleotide is not extended in the nucleic acid to be extended. On the other hand, when the oligonucleotide is irradiated with light, the photocleavable protecting group is decomposed, and the region complementary to the base sequence from the nucleotide modified with a photocleavable protecting group to the 5' end in the oligonucleotide is extended.

[0031] For example, when the oligonucleotide according to the present embodiment is used in CEL-Seq2, as shown in FIG. 2(A), the oligonucleotide hybridized to the mRNA via polyT is extended by reverse transcriptase to obtain the first strand. When the second strand is extended using the first strand as a template, as shown in FIG. 2(B), the extension reaction stops at the position of the nucleotide modified with a photocleavable protecting group in the additional sequence, and the second strand lacks the region from that position to the 3' end, i.e., a part of the additional sequence. Only when the oligonucleotide is irradiated with light to decompose the photocleavable protecting group, as shown in FIG. 2(C), is the inhibition of the extension reaction by the photocleavable protecting group released, and a second strand having a full-length additional sequence is obtained.

[0032] According to the oligonucleotide of the present embodiment, a nucleic acid having an arbitrary additional sequence can be obtained only when light is irradiated. Since the nucleic acid can be detected via the additional sequence, for example, by irradiating a part of a tissue section or a cell mass with light, the nucleic acid present in the tissue section or the cell mass can be detected only in the part irradiated with light. Therefore, the oligonucleotide is useful for an omics analysis method for acquiring omics information related to a specific area irradiated with light. In the following, an omics analysis method using the oligonucleotide is described using a gene expression analysis method as an example.

[0033] When the oligonucleotide according to the present embodiment is used in a gene expression analysis method, the oligonucleotide is preferably applied to CEL-Seq2. The gene expression analysis method includes an exposure step, a first synthesis step, a second synthesis step, and a determination step. Below, the gene expression analysis method using the above oligonucleotide will be described using CEL-Seq2 as an example. The oligonucleotide has an adapter sequence and a T7 promoter sequence on the 5'-terminal side of the adapter sequence in the additional sequence.

[0034] In the exposure step, the oligonucleotide is exposed to a tissue section or a cell mass. The tissue section is any tissue of an animal or a plant. The tissue section is prepared by a known method such as a paraffin method or a freezing method. The tissue section may be subjected to a known treatment to increase the permeability of the tissue section. The thickness of the section is 3 to 15 μm, 4 to 12 μm, or 5 to 10 μm. The size of the section is not limited, but is, for example, 1 mm x 1 mm or more in consideration of ease of operation. The cell mass is any cell population of an animal or a plant. To expose the oligonucleotide to the tissue section or the cell mass, a reagent containing the oligonucleotide may be dropped onto the tissue section or the cell mass. Preferably, the oligonucleotide is dropped onto the tissue section or the cell mass together with the reagent used for elongating the oligonucleotide in the first synthesis step.

[0035] In the first synthesis step, the oligonucleotide is elongated using the mRNA contained in the tissue section or cell mass as a template to synthesize the first strand. The extension reaction of the oligonucleotide using the mRNA as a template may be performed by a known method. For example, in the first synthesis step, the tissue section or cell mass is exposed to a synthesis reagent for synthesizing the first strand. The synthesis reagent includes dNTP and reverse transcriptase, and may further include an RNase inhibitor. The reaction conditions for the synthesis of the first strand are appropriately set depending on the synthesis reagent used.

[0036] In the irradiation step, a part of the tissue section or cell aggregate is irradiated with light. Depending on the size of the region to be analyzed in the tissue section, a part of the tissue section may be irradiated with light under a microscope. When a microscope is used, the magnification of the visual field is, for example, 2 to 500 times, 10 to 300 times, 20 to 200 times, or 50 to 150 times. The wavelength of the light is adjusted according to the protecting group of the oligonucleotide. When the protecting group is NPOM, the light is UV, and the wavelength of the UV is preferably 340 to 380 nm. The light irradiation time is set according to the protecting group and the wavelength of the light. When the protecting group is NPOM, the light irradiation time is, for example, 10 to 30 minutes, 12 to 20 minutes, or 13 to 18 minutes. Before irradiating a part of the tissue section or cell aggregate with light, the tissue section or cell aggregate may be washed with phosphate buffered saline (PBS) or the like.

[0037] In the second synthesis step, a second strand complementary to the first strand is synthesized using the first strand as a template. For example, in the second synthesis step, all cDNA contained in the tissue section dissolved using Proteinase K or the like may be used as a template to synthesize a complementary strand by a known method. Synthesis reagents for synthesizing the second strand include, for example, dNTP, RNase H, DNA ligase, and DNA polymerase. The reaction conditions for synthesizing the second strand are appropriately set depending on the synthesis reagent used.

[0038] In the determination step, the base sequence of the first strand or the second strand is determined. In the determination step, the double-stranded DNA consisting of the first strand and the second strand obtained in the second synthesis step is purified, and an IVT reaction is performed to transcribe the sequence downstream of the T7 promoter and synthesize cRNA. The purification of the double-stranded cDNA and the IVT reaction can be performed by a known method or a commercially available kit, etc.

[0039] Next, cDNA is synthesized from the cRNA synthesized by IVT using reverse transcriptase. The cDNA is amplified by PCR and purified to obtain a library. The cDNA contained in the library has additional sequences at its ends. Therefore, sequence analysis can be performed via the additional sequences.

[0040] In particular, when the additional sequence contains a barcode sequence as an identification sequence, the samples can be identified via the barcode sequence even when multiple samples are mixed. Also, when the additional sequence contains a UMI sequence as an identification sequence, the amplification products amplified in duplicate during library amplification in PCR, IVT, or the like can be distinguished via the UMI sequence.

[0041] As described above in detail, in the oligonucleotide according to the present embodiment, the photocleavable protecting group in the additional sequence is decomposed only in the area of ​​the tissue section or the like that is irradiated with light, and a nucleic acid having a complete additional sequence that has been designed in advance is synthesized. By using an extension reaction such as PCR, the target nucleic acid in the area of ​​the tissue section or the like that is irradiated with light can be detected via the additional sequence. Light such as UV has a wavelength of 1 μm. 2 Since irradiation of the tissue or cell aggregate can be limited to the following regions, omics information of the tissue or cell aggregate can be obtained with high spatial resolution. This eliminates the need to separate cells by cell sorting or cutting out cell aggregates by laser microdissection, and allows the acquisition of omics information that more closely reflects the actual state of the living body without damaging the cells.

[0042] Although the oligonucleotide according to this embodiment may have nucleotides modified with 1 to 3 photocleavable protecting groups at the 3'-end of the additional sequence, the additional sequence may have a poly-T sequence at the 3'-end, and 1 to 3 deoxythymidines at the 5'-end of the poly-T sequence may be modified with photocleavable protecting groups. The 3'-end side of the oligonucleotide according to this embodiment may be a random sequence or a sequence complementary to a part of mRNA or genomic DNA other than the poly-T sequence, and the deoxythymidines contained therein may be modified with photocleavable protecting groups.

[0043] In addition, the oligonucleotide according to the present embodiment may contain an identification sequence in the additional sequence. This allows identification sequences with different base sequences, particularly barcode sequences, to be associated with each of a plurality of samples, thereby making it possible to identify samples even when a plurality of samples are mixed. Furthermore, by including a UMI sequence in the identification sequence, it is possible to distinguish duplicated libraries in library amplification by PCR, IVT reaction, or the like.

[0044] In another embodiment, a genome sequence analysis method is provided as an omics analysis method. The genome sequence analysis method differs from the above-mentioned gene expression analysis method in the first synthesis step, and the other steps are the same as those of the gene expression analysis method. In the first synthesis step of the genome sequence analysis method, the above-mentioned oligonucleotide is elongated using DNA contained in a tissue slice or a cell mass as a template to synthesize a first strand. In the genome sequence analysis method, the double-stranded DNA consisting of the first strand and the second strand obtained in the second synthesis step is subjected to PCR to construct a library. Since the DNA contained in the library has an additional sequence at its end, the base sequences of the first strand and the second strand can be determined by performing sequence analysis or the like via the additional sequence.

[0045] In addition, if DNA contained in a tissue section or a cell mass is subjected to bisulfite treatment by a known method prior to the first synthesis step in the genome sequence analysis method, genome methylation information can also be obtained by the genome sequence analysis method. Furthermore, the oligonucleotide according to the present embodiment used in the genome sequence analysis method does not need to have a promoter sequence in the additional sequence.

[0046] In another embodiment, the oligonucleotide can be applied to a genome sequence analysis method using transposase. The genome sequence analysis method using transposase includes an exposure step, an irradiation step, a reaction step, and a determination step. In the following, the differences between each step of the genome sequence analysis method using transposase and the above-mentioned gene expression analysis method using mRNA as a template or the genome sequence analysis method using genomic DNA as a template will be mainly described.

[0047] As shown in FIG. 3(A), the oligonucleotide used in the genome sequence analysis method using transposase further has a transposon sequence on the 3'-end side of the additional sequences X and Y containing NPOM-modified nucleotides (indicated by ●). The additional sequence may include a T7 promoter sequence, an adapter sequence, a UMI sequence, a barcode sequence, and the like. The transposon sequence is a base sequence that can transfer a position on the genome in a cell by the action of a transposase. The transposon sequence is a recognition sequence for Tn5 transposase. When the double-stranded oligonucleotide solution shown in FIG. 3(A) is mixed with Tn5 transposase, the double-stranded oligonucleotide and Tn5 transposase form a complex.

[0048] In the exposure step, the above-mentioned complex, i.e., the oligonucleotide and transposase according to the present embodiment, are exposed to a tissue section or a cell mass. When the complex is exposed to genomic DNA, the oligonucleotide has a transposon sequence, so that the oligonucleotide is inserted into various regions of the genomic DNA (tagmentation) as shown in FIG. 3(B). As a result, the additional sequences X and Y are inserted into various genomic regions together with the transposon sequence.

[0049] After the irradiation step, the 5' overhanging ends of the DNA fragments recovered from the tissue section or cell mass are blunted (filled in). The fill-in reaction occurs only when the protecting group is cleaved by light irradiation. As a result, the 5' overhanging ends of only the DNA fragments obtained from the light-irradiated area of ​​the tissue section, etc. are blunted and the full-length additional sequence X or Y is added to the end, as shown in Figure 3(C).

[0050] In the determination step, the base sequence of the blunted DNA fragment is determined. Since the blunted DNA fragment has a double-stranded additional sequence X or Y at its end, even a small amount of nucleic acid such as genomic DNA can be amplified by PCR using a primer for the additional sequence X or Y. This allows the amplification and analysis of various genomic regions.

[0051] When the complex is exposed to unfixed cell nuclei, the oligonucleotides are selectively inserted into open chromatin regions. After an irradiation step, the ends of the recovered DNA fragments are blunted, and the DNA fragments are amplified by PCR and sequenced to identify open chromatin regions (ATAC-seq).

[0052] The transposon sequence is not particularly limited, and known transposons such as Tn5 transposon, Tn7 transposon, Tn9 transposon, etc. The transposase used may be one that corresponds to the transposon sequence.

[0053] The oligonucleotide according to the present embodiment can be used in various techniques that can detect a target nucleic acid via an additional sequence. Such techniques include qPCR. The qPCR may be a probe method or an intercalator method. The oligonucleotide according to the present embodiment can also be applied to the FISH (fluorescence in situ hybridization) method.

[0054] (Embodiment 2) The omics analysis kit according to this embodiment includes the oligonucleotide described in the first embodiment. Preferably, the omics analysis kit further includes a reagent for extending the oligonucleotide. In the case of the above-mentioned CEL-Seq2 and genome sequence analysis method, the reagent for extending the oligonucleotide using a nucleic acid as a template is a reagent for synthesizing the first strand. In the case of the genome sequence analysis method using a transposase, the reagent includes a transposase for insertion into the region, and a reagent for fill-in of the DNA fragment after insertion and PCR amplification. Specifically, the reagent includes a transposase such as Tn5, a DNA polymerase, dNTP, a buffer, and the like. The reagent may further include a reducing agent such as DTT (dithiothreitol).

[0055] The omics analysis kit may further include a reagent for synthesizing the second strand. Examples of the reagent for synthesizing the second strand include dNTP, RNaseH, DNA ligase, and DNA polymerase. Preferably, the omics analysis kit according to this embodiment includes a plurality of types of oligonucleotides, each of which includes a different identification sequence in the additional sequence. More specifically, the plurality of types of oligonucleotides include a barcode sequence having a different base sequence in the additional sequence as an identification sequence. For example, the omics analysis kit includes a first oligonucleotide including a first barcode sequence in the additional sequence as an identification sequence, and a second oligonucleotide including a second barcode sequence having a base sequence different from the first barcode sequence in the additional sequence as an identification sequence. The number of types of oligonucleotides each having a different barcode sequence is not particularly limited, and may be two or more types.

[0056] The omics analysis kit according to the present embodiment is suitable for an omics analysis method using the oligonucleotide according to the above-mentioned embodiment 1. Since the omics analysis kit includes reagents used in the above-mentioned omics analysis method, omics information that more closely reflects the actual state of a living body can be efficiently obtained with high spatial resolution.

[0057] In the omics analysis kit according to the present embodiment, the additional sequence includes an identification sequence, and the oligonucleotide may be a plurality of types having different identification sequences. Even if libraries derived from a plurality of samples are mixed, the identification sequence makes it possible to identify or restore sample information using a next-generation sequencer. Furthermore, by arranging a UMI sequence in the additional sequence, it is possible to distinguish duplicate libraries by amplifying the library through PCR or IVT reaction or the like. EXAMPLES

[0058] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0059] Example 1 (Synthesis of Oligo DNA) Oligo DNAs B to H shown in FIG. 4 were synthesized using NPOM Caged-dT-CE Phosphoramidite (5'-Dimethoxytrityl-N3-[[1-(6-nitro-1,3-benzodioxol-5-yl)ethoxy]methyl]-2'-deoxyThymidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; Glen Research, catalog number 10-1534-95). The base sequences of oligo DNAs B to D and oligo DNAs F to H are shown in SEQ ID NOs: 1 to 3 and 4 to 6, respectively. The base sequence of oligo DNA E is the same as that of oligo DNA D. In FIG. 4, deoxythymidine modified with NPOM is shown as a marked "t".

[0060] (CEL-seq2 in cultured cells) Total RNA from NIH3T3 cells was purified using RNeasy Mini Kit (Qiagen) and adjusted to 10ng / μl. 0.5μl of oligo DNA A-H (500ng / μl) and 0.5μl of dNTP Mix (10mM, New England BioLabs (NEB)) were added to 5μl of the obtained total RNA to prepare a sample. To unravel the higher-order structure of the RNA, the sample was heated at 65℃ for 5 minutes and then rapidly cooled. A reverse transcriptase solution was added to the sample. The composition of the reverse transcriptase solution is as follows: 0.5 μl of RNase OUT (40 U / μl, Invitrogen) 0.5 μl of Superscript II Reverse Transcriptase (200 U / μl, Invitrogen) 5x First Strand Buffer (2μl) and DTT (1μl) included with the above enzyme

[0061] The first strand was synthesized by reacting at 42°C for 60 minutes, followed by 10 minutes at 70°C. The sample was irradiated with UV light for 15 minutes using the following equipment. Inverted microscope (Nikon, Eclipse Ti-U, 10x objective lens) 130W mercury lamp device (Nikon, INTENSILIGHT C-HGFI) Fluorescence filter cube (Semrock, DAPI-5060C; 352-402 nm excitation wavelength)

[0062] After UV irradiation, a second strand synthesis reagent was added. The composition of the second strand synthesis reagent is as follows: 7 μl ultrapure water 2.31 μl of 5×Second strand buffer (Invitrogen) 0.23 μl of dNTP Mix (10 mM, NEB) 0.08 μl of E. coli DNA ligase (10 U / μl; Invitrogen) 0.3 μl of E. coli DNA Polymerase I (10 U / μl; BioLabs) 0.08 μl of E. coli Rnase H (2 U / μl, Invitrogen)

[0063] The second strand was synthesized at 16° C. for 2 hours. DNA was purified using Agencourt AMPure XP (Beckman Coulter) and eluted with 6.4 μl of ultrapure water. 1.6 μl of each of the following reagents included in the MEGAscript T7 Transcription Kit (Ambion) was added. 10×T7 Reaction buffer A / G / C / UTP Solution T7 Enzyme Mix

[0064] The IVT reaction was carried out for 17 hours at 37° C. To remove excess nucleotides and primers, 6 μl of ExoSAP-IT Express PCR Cleanup Reagents (Applied Biosystems) was added and incubated at 37° C. for 15 minutes.

[0065] To shorten the cRNA synthesized by IVT, Fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) was added and heated at 94°C for 3 minutes. The sample was purified using Agencourt RNAClean XP (Beckman Coulter) and eluted with 7 μl of ultrapure water to obtain a sample. The following was added to the sample. 1 μl of randomhexRT oligodeoxynucleotide (5'-GCCTTGGCACCCGAGAATTCCANNNNNN-3' (SEQ ID NO: 7); 250 ng / μl) 0.5 μl dNTP Mix (10 mM)

[0066] To unfold the higher-order structure of cRNA, the sample was heated at 65°C for 5 min and then rapidly cooled. The same reverse transcriptase solution as above was added to the sample. cDNA synthesis was carried out at 25°C for 10 min, followed by 42°C for 60 min, and the following PCR reagents were added: 9.9 μl ultrapure water 22.5 μl Phusion High-Fidelity PCR Master Mix (NEB) 0.23 μl of dNTP Mix (10 mM, NEB) 1.8 μl of RNA PCR Primer [RP1] (Illumina) 1.8 μl of RNA PCR Primer Index [RPIX] (Illumina)

[0067] PCR was performed for up to 11 cycles, and RNase A (10 mg / ml, Nacalai) was added to the PCR product and incubated at 37°C for 30 minutes. DNA was purified using Agencourt AMPure XP and eluted with 25 μl of ultrapure water. Electrophoresis was performed on an E-Gel precast agarose gel, and the 250-300 bp fraction was collected. Next, DNA was purified using the QIAGEN MinElute PCR Purification kit, and a library was constructed.

[0068] The library constructed as described above was electrophoresed from the sample obtained by adding oligo DNA D or oligo DNA G.

[0069] In addition, qPCR (SYBR Green method) of the Gapdh gene was performed using the reaction product of the above cDNA synthesis as a template. The nucleotide sequences of the forward primer and reverse primer used in the qPCR are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0070] (result) As shown in FIG. 5, the expression of the Gapdh gene was detected by oligo DNAs B to H when irradiated with UV light, whereas a significant decrease in the signal was observed when not irradiated with UV light. In particular, the expression of the Gapdh gene was hardly detected when not irradiated with UV light for oligo DNAs F to H, which have at least one deoxythymidine modified with NPOM at the 3' end of the additional sequence. The signal detected by oligo DNAs B to E was higher when not irradiated with UV light than for oligo DNAs F to G. However, since there was a difference in the signal detected between UV irradiation and non-irradiation for oligo DNAs B to E, it is considered that if a wider area of ​​the tissue section is irradiated with UV light, the expression of the Gapdh gene will hardly be detected in the non-irradiated area.

[0071] When a library for sequencing was synthesized using oligo DNA G, the library was not detected without UV irradiation, but was detected only with UV irradiation, as shown in FIG. 6.

[0072] Example 2 (CEL-seq2 of tissue sections) 14-day-old mouse embryos collected from pregnant mice were embedded in OCT compound (Sakura Finetech). Mouse embryos were rapidly frozen by immersion in isopentane containing dry ice. Mouse embryo tissue sections 10 μm thick were prepared using a cryostat and air-dried. After washing with PBS, the sections were fixed in 3.7% formalin solution for 10 minutes. The following treatments were performed to increase the permeability of the tissue sections.

[0073] The tissue sections were immersed in 5% Triton / PBS for 3 minutes and then in 0.1N HCl for 5 minutes. To unravel the higher-order structure of RNA, the sections were neutralized with 1M Tris-HCl (pH 8.0) and rapidly cooled by immersing in 65°C PBS for 5 minutes. The first strand synthesis reagent was dropped onto the tissue sections. The composition of the first strand synthesis reagent is as follows: 0.5 μl of Oligo DNA G (500 ng / μl) 0.5 μl dNTP Mix (10 mM) 5 μl ultrapure water 0.5 μl RNase OUT 0.5 μl Superscript II Reverse Transcriptase 5x First Strand Buffer (2μl) and DTT (1μl) included with the above enzyme

[0074] The reaction was continued at 42°C for 60 minutes, followed by 70°C for 10 minutes to synthesize the first strand. The tissue sections were washed with PBS. The neural tube and hind limb of the tissue section shown in Figure 7(A) were irradiated with UV light for 15 minutes in a circular area with a diameter of approximately 1.2 mm using the following equipment. Upright microscope (Leica, DM5000B, 20x objective lens) 120W mercury lamp device (EL6000) Fluorescence filter cube (Leica, 340-380 nm excitation wavelength)

[0075] Here, the tissue sections were immunostained for Sox2. In the primary antibody reaction in the immunostaining, rabbit anti-Sox2 antibody (clone D9B8N, Cell Signaling) was dropped onto the tissue sections and allowed to react for 1 hour. In the secondary antibody reaction, Alexa-488-labeled anti-rabbit IgG antibody (Invitrogen) was dropped onto the tissue sections and allowed to react for 1 hour. In the nuclear staining, TO-PRO-3 (Invitrogen) was dropped onto the tissue sections and allowed to react for 10 minutes.

[0076] 80 μl of Proteinase K (400 μg / ml, manufactured by Nacalai) was dropped onto the tissue sections and dissolved at 55° C. for 90 minutes.

[0077] (Quantitative PCR) qPCR (TaqMan™ probe method) for the Sox2 gene and the Gapdh gene was performed using as a template a lysate of the entire tissue section obtained with Proteinase K. The nucleotide sequences of the forward primer, reverse primer, and probe for the Sox2 gene are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The nucleotide sequences of the forward primer, reverse primer, and probe for the Gapdh gene are shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.

[0078] (result) The results of immunostaining are shown in Figure 7(B). The Sox2 gene is mainly expressed in the neural tube, but not in the hind limbs. The results of qPCR for the Sox2 gene and the Gapdh gene are shown in Figure 8(A) and Figure 8(B), respectively. In Figure 8(A) and Figure 8(B), one circle indicates a signal from one tissue section. When the tissue section was not irradiated with UV, neither the Sox2 gene nor the Gapdh gene was detected. On the other hand, when the neural tube was irradiated with UV, both the Sox2 gene and the Gapdh gene were detected, and when the hind limbs were irradiated with UV, only the Gapdh gene was detected. This shows that it is possible to analyze gene expression limited to the UV-irradiated area.

[0079] Example 3 (OligoDNA-Tn5 complex formation) As in Example 1, oligo DNAs I to M shown in FIG. 9 were synthesized using NPOM Caged-dT-CE Phosphoramidite. The base sequences of oligo DNAs I, J, and K are shown in SEQ ID NOs: 16, 17, and 18, respectively. The base sequences of oligo DNAs L and M are the same as those of oligo DNAs I and K, respectively, but some deoxythymidines are modified with NPOM. In FIG. 9, the deoxythymidines modified with NPOM in oligo DNAs L and M are shown as a marked "t".

[0080] Oligo DNA J and oligo DNA I, K, L or M were diluted in NTE buffer (20 mM Tris-HCl (pH 7.5), 20 mM NaCl and 1 mM ethylenediaminetetraacetic acid (EDTA)) to a final concentration of 100 μM, heated at 98°C for 15 minutes, and then annealed while slowly lowering the temperature at a rate of -1°C / min to 4°C. The sample containing the annealed oligo DNA was mixed with the following reagents and incubated at 23°C for 1 hour to obtain an oligo DNA-Tn5 complex. 0.625 μl of annealed oligo DNA 10 μl 100% glycerol 5.375 μl of 2× dialysis buffer (100 mM HEPES-KOH (pH 7.2), 0.2 M NaCl, 0.2 mM EDTA, 2 mM dithiothreitol, 0.2% Triton X-100 and 20% glycerol) 9 μl Tn5 (11.56 μM)

[0081] (Pretreatment of tissue sections) As in Example 2, frozen sections of 14-day-old mouse embryos were prepared and fixed in 3.7% formalin solution for 10 minutes. The sections were permeabilized by immersing in 1% Triton / PBS for 10 minutes. Blocking was performed for 10 minutes in a solution containing equal amounts of Blocking OneP (manufactured by Nacalai Tesque) and TBST (Tris-buffered saline and 0.1% Tween 20). The tissue sections obtained were washed with PBS.

[0082] (Transposon insertion) 5 μl of the oligo DNA-Tn5 complex prepared above was mixed with 45 μl of 1×TAPS-DMF buffer (10 mM TAPS-NaOH (pH 8.5), 5 mM MgCl2, 10% N,N-dimethylformamide) and added dropwise to the tissue section prepared above. The tissue section was left to stand at 37°C for 1 hour. The tissue section was washed with 0.2% sodium dodecyl sulfate (SDS) for 10 minutes and then with PBS.

[0083] (UV irradiation) The tissue sections were irradiated with UV light in a circular area of ​​approximately 5 mm in diameter using the following equipment. Upright microscope (Leica, DM2500, 5x objective lens) 103W mercury lamp device (Leica, ebq 100) A Fluorescence filter cube (Leica, 340-380 nm excitation wavelength)

[0084] (DNA extraction and purification) 80 μl of Proteinase K (400 μg / ml, Nacalai) was dropped onto the tissue section, and dissolved at 55° C. for 90 minutes. The tissue was purified using the QIAGEN MinElute PCR Purification kit and eluted with 22 μl of ultrapure water.

[0085] (fill-in and PCR amplification) The following PCR mix was prepared: 1.5 μl of the above eluate 0.375 μl of forward primer (SEQ ID NO: 19) 0.375 μl of reverse primer (SEQ ID NO: 20) 3.75μl of NEBnext 2xPCR mix (NEB) 1.35 μl ultrapure water In addition, "n" in the base sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20 is an index sequence defined by Illumina.

[0086] Fill-in was performed in step 1 below, and after step 2, an amplification reaction (25 or 31 cycles of steps 3 to 5) was performed. Step 1: 72°C, 5 min Step 2: 95°C, 30 seconds Step 3: 95°C, 10 seconds Step 4: 63°C, 30 seconds Step 5: 72°C, 60 seconds

[0087] The reaction products were electrophoresed on an agarose gel and stained with SYBR Gold (Invitrogen).

[0088] (result) Figure 10 shows the results of electrophoresis. When UV irradiation was not performed, genomic DNA was not amplified due to the presence of NPOM (lanes 5 and 6). On the other hand, when UV irradiation was performed, NPOM was decomposed and amplification of genomic DNA was observed (lanes 7 and 8).

[0089] The above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not by the embodiment. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0090] This application is based on Japanese Patent Application No. 2019-094216, filed on May 20, 2019. The entire specification, claims, and drawings of Japanese Patent Application No. 2019-094216 are incorporated herein by reference. [Industrial Applicability]

[0091] The present invention is suitable for omics analysis, which requires high spatial resolution.

Claims

1. An additional sequence for detecting a nucleic acid, the additional sequence including a promoter sequence which is a base sequence involved in the initiation of transcription and an identification sequence for identification located on the 3'-terminal side of the promoter sequence; a poly T sequence at the 3' end of the additional sequence; having At least one nucleotide in the additional sequence is modified with a photocleavable protecting group that is bound to a base of the nucleotide, inhibits hydrogen bond formation between the base and a complementary base, and is decomposed by irradiation with light to release the inhibition of hydrogen bond formation; The nucleotide is Located on the 3' end side of the promoter sequence Oligonucleotides.

2. The photocleavable protecting group is A 6-nitropiperonyloxymethyl group. The oligonucleotide of claim 1.

3. The additional sequence further comprises at least one nucleotide modified with the photocleavable protecting group at the 3' end.

3. The oligonucleotide according to claim 1 or 2.

4. An exposure step of exposing a tissue section or a cell mass to the oligonucleotide according to any one of claims 1 to 3; a first synthesis step of synthesizing a first strand by extending the oligonucleotide using mRNA contained in the tissue section or cell mass as a template; an irradiation step of irradiating a portion of the tissue slice or cell aggregate with light; a second synthesis step of synthesizing a second strand complementary to the first strand using the first strand as a template; an IVT step of synthesizing cRNA from a double-stranded DNA consisting of the first strand and the second strand by an in vitro transcription reaction; a determining step of determining a base sequence of the first strand or the second strand using cDNA synthesized from the cRNA; The omics analysis method includes:

5. 4. The method according to claim 1, further comprising: Omics analysis kit.

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