JP-2026514330-A - Analytical method, method for producing cyclic nucleic acid, and nucleic acid

Abstract

【assignment】 This disclosure aims to shorten the sequence anointed on the target nucleic acid. In addition to shortening the sequence anointed on the target nucleic acid, this disclosure also aims to reduce the aforementioned bias. [Solution] This disclosure provides an analytical method comprising: a complementary nucleic acid generation step of generating complementary strands of one or more target nucleic acids, each with a nucleic acid linker attached to one end of the aforementioned target nucleic acids; a cyclic nucleic acid generation step of linking two or more of the generated complementary strands via the nucleic acid linkers to form a cyclic nucleic acid; and an analytical step of performing an analysis using the cyclic nucleic acid. This disclosure also provides nucleic acids used in the analytical method. [Selection Diagram] Figure 2A

Inventors

• 松本 真寛
• 日高 伊佐夫
• 佐藤 乃愛
• 町田 賢三

Assignees

• ソニーグループ株式会社

Dates

Publication Date

20260511

Application Date

20240328

Priority Date

20230330

Claims (20)

1. A complementary nucleic acid generation step in which a nucleic acid linker is attached to one end of each of one or more target nucleic acids, and a complementary chain of the one or more target nucleic acids is generated. A cyclic nucleic acid generation step, which involves linking two or more generated complementary strands via the nucleic acid linking portion to form a cyclic nucleic acid, and An analytical step in which an analysis is performed using the aforementioned cyclic nucleic acid, Analytical methods including those mentioned.
2. The analytical method according to claim 1, wherein the nucleic acid binding portion has a target nucleic acid capture portion configured to capture the 3' terminal region of the target nucleic acid.
3. The analytical method according to claim 1, wherein, in the complementary chain generation step, the nucleic acid linkage portion is used as a primer to generate a complementary chain of the target nucleic acid.
4. The analytical method according to claim 1, wherein, in the complementary strand generation step, a double strand is formed between each target nucleic acid and its complementary strand.
5. The analytical method according to claim 4, wherein in the cyclic nucleic acid generation step, the double strands are linked via the nucleic acid linking portion.
6. The analytical method according to claim 1, wherein the nucleic acid linker has a complementary strand capture portion configured to capture the 3' terminal region of the complementary strand.
7. In the cyclic nucleic acid generation step, the 5' end of one complementary strand and the 3' end of another complementary strand are linked. The ligation is performed with the complementary strand capture portion of the nucleic acid ligation portion attached to one complementary strand bound to the 3' terminal region of the other complementary strand. The analytical method according to claim 1.
8. The analytical method according to claim 1, wherein, in the cyclic nucleic acid generation step, a double-stranded cyclic nucleic acid is formed, and then the target nucleic acid is removed from the double-stranded cyclic nucleic acid to obtain a single-stranded cyclic nucleic acid with a complementary strand linked.
9. The analytical method according to claim 1, wherein a nucleic acid amplification reaction using the cyclic nucleic acid is performed in the analytical step.
10. The analytical method according to claim 9, wherein the nucleic acid amplification reaction is RCA or PCR.
11. The nucleic acid linking portion is A target nucleic acid capture unit configured to capture the 3' terminal region of the target nucleic acid, A complementary chain capturing unit configured to capture the 3' terminal region of the complementary chain generated in the complementary chain generation step, A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion, The analytical method according to claim 1, comprising:
12. The target nucleic acid capture portion has a poly-T sequence, and the complementary strand capture portion has a base sequence complementary to the base sequence added to the 3' end during reverse transcription by reverse transcriptase. The analytical method according to claim 11.
13. The analytical method according to claim 11, wherein the double-stranded portion has a restriction enzyme recognition sequence.
14. The analytical method according to claim 11, wherein the double-stranded portion has a non-natural base sequence.
15. The analytical method according to claim 11, wherein the double-stranded portion has a base sequence having an error correction function.
16. The aforementioned analytical method is an analytical method for performing single-cell analysis. A nucleic acid ligator having a different double-stranded portion for each cell is used. The analytical method according to claim 11.
17. The aforementioned analytical method includes a cell disruption step that destroys cells, The complementary nucleic acid generation step is performed on the target nucleic acid contained in the cell. The analytical method according to claim 1.
18. The analytical method according to claim 17, wherein the cell disruption step is performed within a space partitioned for each cell.
19. The analytical method according to claim 1, wherein the nucleic acid linkage portion is immobilized on a substrate.
20. A complementary nucleic acid generation step involves generating complementary strands of one or more target nucleic acids, each with a nucleic acid linker attached to one end; and a cyclic nucleic acid generation step involves linking the two or more generated complementary strands via the nucleic acid linkers to form a cyclic nucleic acid. A method for producing cyclic nucleic acids, including

Description

This application claims priority to Japanese Patent Application JP2023-056365, filed on March 30, 2023, the entire contents of which are incorporated herein by reference. This disclosure relates to analytical methods, methods for producing cyclic nucleic acids, and nucleic acids. More specifically, this disclosure relates to analytical methods for cell analysis, methods for producing cyclic nucleic acids used for cell analysis, and nucleic acids used for cell analysis. Single-cell analysis is a highly useful technique for analyzing cells and their intracellular components. In single-cell analysis, for example, nucleic acids, particularly mRNA, contained in each cell are analyzed. Several techniques have been proposed to perform single-cell analysis. Non-Patent Document 1 below discloses a method for analyzing biological particles, which includes a capture step of capturing biological particles via the biological particle capture portion on a surface on which a molecule containing a biological particle capture portion, a barcode sequence, and a cleavable linker is fixed via the linker; a cleavage step of cleaving the linker to release the biological particles from the surface; and an isolation step of isolating the biological particles within a microspace. International Publication No. 2022/009642 This is a schematic diagram showing an example of the structure of a nucleic acid linkage.This figure shows an example of a set of sequences that have an error correction function.This figure shows an example of the structure of a nucleic acid linkage.This figure shows an example of the structure of a nucleic acid linkage.This is a schematic diagram showing a modified example of the structure of a nucleic acid linkage.This is a schematic diagram showing a modified example of the structure of a nucleic acid linkage.This is a schematic diagram illustrating an example of a process for generating cyclic nucleic acids using nucleic acid ligators.This is a schematic diagram illustrating the structure of mRNA.This is a schematic diagram illustrating an example of sequencing.This is an illustrative diagram of the nucleic acid linkage and other components used in the first modified example of the cyclic nucleic acid production process.This is a schematic diagram illustrating the first modified example of the process for generating cyclic nucleic acids using nucleic acid ligators.This is an illustrative diagram of the nucleic acid linkage and other components used in a second modified example of the cyclic nucleic acid generation process.This is a schematic diagram illustrating a second modified example of the process for generating cyclic nucleic acids using nucleic acid linkers.This is an illustrative diagram of the nucleic acid linkage and other components used in a third modified example of the cyclic nucleic acid generation process.This is a schematic diagram illustrating a third modified example of the process for generating cyclic nucleic acids using nucleic acid ligators.This is a schematic diagram illustrating the bias in exponential amplification.This figure shows an example of a restricted area.This is a schematic diagram illustrating an example of tagged short read generation.This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis.This is a schematic diagram showing an example of the complex's structure.This is a schematic diagram showing an example of the complex's structure.This is a schematic diagram showing an example of the manufacturing process of the composite.This is a schematic diagram illustrating an example of the procedure performed in a modified single-cell analysis method.This is an illustrative diagram of the nucleic acid ligation site used in a modified version of the single-cell analysis method.This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis.This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis.This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis.This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis.This is a schematic diagram showing an example of nucleic acids produced by RCA processing.This is a schematic diagram of an example of a well used to perform a particle isolation process.This is a schematic diagram showing an example of equipment used to perform a particle segregation process.This is a schematic diagram showing an example of equipment used to perform a particle segregation process.This is a schematic diagram illustrating an example of an apparatus for forming an emulsion.This is a schematic diagram illustrating an example of a tip for forming an emulsion.This is a diagram illustrating the antibody-nucleic acid molecule complex used when simultaneously performing single-cell analysis and analysis of secreted molecules on the cell surf