JP-2026514381-A - Orthogonal Section Ligation Sequencing (OCLS)

Abstract

This disclosure relates to a method and system for spatial coding/decoding features. The provided orthogonal section-ligation sequencing (OCLS) system encodes two or more orthogonal recognition sites for restriction enzymes, such as IIS-type enzymes, for decoding visual barcodes. Decoding employs specific orthogonal ligation of differentially labeled probes, which enables visual distinction of barcodes at each feature. This process can be repeated over multiple cycles and in parallel.

Inventors

• ダンバッカー コリー エム．

Assignees

• オレゴン ヘルス アンド サイエンス ユニバーシティ

Dates

Publication Date

20260511

Application Date

20240328

Priority Date

20230328

Claims (20)

1. Visual barcodes including the following: At least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, wherein each of the dsDNA segments contains, Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or The dsDNA oligonucleotide segment (cassette) containing both RS and CS; Furthermore, The unfixed end of one end of the visual barcode.
2. The visual barcode according to claim 1, wherein the visual barcode is fixed to a solid substrate by a flexible linker attached to or near the end of the dsDNA of the visual barcode, which is not the unfixed end.
3. A visual barcode according to claim 1, which does not include visually detectable markers.
4. The visual barcode according to claim 1, wherein the RE is an IIS-type restricted endonuclease, and the CS does not overlap with the corresponding RS.
5. The visual barcode according to claim 1, wherein at least one of the dsDNA segments includes a designed CS, and the visual barcode includes an RS specific to the IIS-type RE, which is appropriately positioned so that the homologous IIS-type RE can cut the designed CS based on its position relative to the RS.
6. A visual barcode as described in any one of claims 1 to 5, as shown in Figure 1A or Figure 1B or essentially as described herein.
7. A set of visual barcodes according to any one of claims 1 to 6, wherein the set comprises a plurality of visual barcodes, each comprising a different set of dsDNA segments having different recognition sites (RS), designed cleavage sites (CS), or both, for specific restriction endonucleases (REs).
8. The set of visual barcodes according to claim 7, wherein at least two different visual barcodes are fixed to the same solid substrate.
9. An orthogonal cleavage-ligation sequencing (OCLS) barcode comprising two or more dsDNA segments (cassettes), wherein each dsDNA segment contains a recognition site (RS) for a specific restriction endonuclease (RE) and one or more overlapping regions configured to allow ligation to adjacent dsDNA segments to form a segmental strand, and the strand of the segment constitutes the OCLS barcode.
10. Visually detectable orthogonal ligation probes, including the following: A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein its sequence is Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or The complete or partial double-stranded DNA oligonucleotide, comprising both RS and CS; Furthermore, A visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide.
11. The visually detectable orthogonal ligation probe according to claim 10, wherein the complete or partially double-stranded DNA oligonucleotide comprises: Linear double-stranded DNA oligonucleotides with a 5'overhang; Linear double-stranded DNA oligonucleotides with a 3'overhang; A single-stranded DNA oligonucleotide with a 5' overhang and a hairpin stem-loop configuration; or A single-stranded DNA oligonucleotide with a hairpin stem-loop structure and a 3' overhang.
12. The visually detectable orthogonal ligation probe according to claim 10, wherein the visually detectable label is attached to the oligonucleotide via a flexible linker.
13. The visually detectable orthogonal ligation probe according to claim 10, wherein the visually detectable label comprises one or more fluorescent labels, bioluminescent labels, chemiluminescent labels, chromophores, quantum dots, Raman labels, biotin moieties, or radioisotopes.
14. The visually detectable orthogonal ligation probe according to claim 10, wherein the RE is an IIS-type restriction endonuclease, and the RS is specific to the IIS-type RE.
15. A visually detectable orthogonal ligation probe according to claim 10, comprising an RS specific to an IIS-type RE, which is appropriately positioned so that a cognate IIS-type RE can cut a designed CS based on its position relative to the RS.
16. A visually detectable orthogonal ligation probe as described in any one of claims 10 to 15, as shown in Figure 3A or Figure 3B or essentially as described herein.
17. A set of visually detectable orthogonal ligation probes according to any one of claims 10 to 16, wherein the set comprises a plurality of visually detectable orthogonal ligation probes, each comprising a different recognition site (RS) for specific restriction endonucleases (REs), or both a different RS and a different cleavage site (CS).
18. The set of visually detectable orthogonal ligation probes according to claim 17, wherein at least two different detectable orthogonal ligation probes include visually distinguishable detectable labels.
19. A set of visually detectable orthogonal ligation probes according to claim 18, comprising multiple different probes, each configured such that the cutting of the probe by the RE generates an overhang having a different arrangement from at least 5, at least 7, at least 10, at least 12, at least 15, or more than 15 other probes in the set.
20. Orthogonal cleavage-ligation sequencing (OCLS) oligonucleotide pairs, the following: At least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, wherein each of the dsDNA segments contains, Recognition sites (RS) for specific restriction endonucleases (REs), Designed cutting site (CS), or The dsDNA oligonucleotide segment (cassette) including both RS and CS; and, A visual barcode including an unfixed end at one end of the visual barcode, A complete or partial double-stranded DNA oligonucleotide having 3' or 5' overhangs of at least two nucleotides, wherein its sequence is Recognition sites (RS) for specific restriction endonucleases (REs), or A complete or partial double-stranded DNA oligonucleotide comprising both the RS and the cleavage site (CS); and, A visually detectable orthogonal ligation probe comprising a visually detectable label covalently attached to the complete or partial dsDNA oligonucleotide, The cutting of the RS or CS in the visual barcode generates a single-stranded "sticky end" overhang having an arrangement that is perfectly complementary to the sticky end (overhang) generated by the cutting of the RS in the visually detectable orthogonal ligation probe. The aforementioned OCLS oligonucleotide pair.

Description

Cross-reference of related applications: This application claims priority to U.S. Provisional Application No. 63/492,777 dated 28 March 2023 and the benefits of its early filing, which is incorporated herein by reference in its entirety. A computer-readable text file named "O046-0082PCT.xml," with a file size of 28,237 bytes and created around March 26, 2024, contains the sequence listing of this application, and its entirety is incorporated herein by reference. Area of Disclosure This disclosure generally relates to molecular barcodes, such as visual barcodes, and related methods and systems. More specifically, it relates to labeling compositions, methods, and workflows that overcome the limitations of previously described visual barcoding. Background of Disclosure: Determining the identity and/or location of target molecules (such as proteins or nucleic acids) in a sample can be essential for clinical application, diagnosis, and biomedical research. In-situ hybridization (ISH), immunohistochemistry, laser capture microscopy, and similar techniques enable visualization of the location of target molecules within samples, such as biological samples. The identity of target molecules can also be determined by methods such as labeling them and tracking them through amplification and/or sequencing processes (e.g., probabilistic barcoding). However, research is still ongoing to develop new tools for improved spatial tracking for its applications in single-cell biology and spatial biology. Therefore, there is a need for methods and systems that reliably correlate the identity of target molecules with their positions within a sample, such as a substantially two-dimensional (2D) biological sample. Next-generation sequencing (NGS) technology can be used to determine the sequences of nucleic acid-coated barcoded beads. However, conventional NGS sequencing requires expensive reagents for accurate determination of DNA sequences, including nucleotides with and/or labeled reversible terminators; high temperatures (typically 65°C) are required during decoding; multiple different reagents are introduced during each decoding cycle, and these reagents do not function properly when mixed (e.g., the cleavage solution cannot be mixed with the incorporation solution), which requires the use of advanced fluid dynamics; and the NGS cycle time is significantly longer than 5 minutes each. To read a large surface area of 1 μm beads via NGS, for example, a 1.75 cm × 1.75 cm surface composed of densely packed 1 μm beads with approximately 10 billion (10B) individual 1 μm beads present, the length of the NGS position barcode must be greater than 24 bp to reach the diversity of at least 200 trillion different beads in the library (possible combinations of position barcodes) to reduce the possibility of barcode redundancy within the large surface area. This necessitates a large number of decoding cycles, more than six. For example, there is still a strong need in this field for developing further methods for visually barcoding targets in the analysis of biological molecules. Summary of Disclosure Disclosed herein are methods and systems for visual barcoding with orthogonal cleavage-ligation sequencing (OCLS), and various components used in such methods and systems, including double-stranded DNA oligonucleotides and hairpin-loop single-stranded oligonucleotides. The typical barcodes used for the OCLS embodiments described herein are made from dsDNA. The visually decoded barcodes used in the OCLS workflow are constructed across multiple rounds of splitting and pooling (similar to those described in WO2022/187719), but the segments constituting the OCLS barcode are not labeled. However, similar to the OCS method in WO2022/187719, each segment of the OCLS library can be bound to a bead (or other solid support) or ligated to a previous segment to construct a strand, and these segments can be co-coded together with NGS-captured oligo (CO) barcodes. This disclosure further provides a method for encoding and decoding (sequencing) visual barcodes. Methods including orthogonal cleavage-ligation sequencing (OCLS) of visual barcodes can be implemented to overcome problems associated with other sequencing methods, such as orthogonal cleavage sequencing (OCS) for identimere strand decoding. This OCLS method is disclosed herein, including in Figures 1–6. While the OCS identimere strand may consist of non-nucleic acid polymers for alternative decoding (such as peptide linkers using orthogonal proteases or chemical linkers using orthogonal chemical cleavage agents), the barcodes used for OCLS described herein must consist of double-stranded or partially double-stranded DNA (dsDNA). Briefly, visually decoded barcodes used in the OCLS workflow are also constructed across multiple rounds of splitting and pooling, but the segments that make up the OCLS barcodes are not labeled (because they utilize OCS identimeres). Each segment in the OCLS library is ligated