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JP-2026514410-A - High-performance spatial mapping of individual targets using releaseable handshake sequences

JP2026514410AJP 2026514410 AJP2026514410 AJP 2026514410AJP-2026514410-A

Abstract

Systems, methods, and compositions for generating a spatial map of the distribution of targets in a sample are described. The system for mapping targets may include a substrate and a distribution of functionalized features associated with the substrate, wherein a representative feature of the distribution of functionalized features includes one or more functionalized molecules bound to the representative feature, and the one or more molecules include a handshake sequence comprising at least a reactive moiety, a barcode segment that functions as a spatial address, and a cleavage linker configured to allow the handshake sequence to be released from the representative feature in response to a stimulus. Using this system, nuclei, cells, and/or other target components of a sample can be tagged.

Inventors

  • クリスティーナ チャン
  • ステフェン ピー．エー．フォーダー
  • ヘイ ムン クリスティーナ ファン
  • アナラム シャーラヴァン
  • ジュリー ウィレルミー
  • バートランド ヨング

Assignees

  • タカラ バイオ ユーエスエー， インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20240328
Priority Date
20230329

Claims (20)

  1. It is a method, The method involves tagging the distribution of nuclei with a set of handshake sequences paired with a set of barcode sequences that function as spatial addresses, generating a spatial map of the distribution of nuclei isolated from a tissue sample, wherein generating the spatial map includes recovering and mapping the locations of more than 30% of the nuclei initially present in the tissue sample.
  2. It is a method, The method includes generating a spatial map of the distribution of nuclei isolated from a tissue sample, where the tagging of the distribution of nuclei is performed with a set of handshake sequences paired with a set of barcode sequences that function as spatial addresses, wherein the generation of the spatial map is performed within two hours.
  3. The method according to claim 2, further comprising decoding the set of barcode sequences bonded to a substrate by performing a set of sequencing iterations with error reduction by dynamic annealing and ligation (SEDAL), wherein the decoding of the set of barcode sequences has a pass rate of more than 90% as determined by a set of criteria, the set of criteria including criteria relating to substrate coating and the amount of empty space on the substrate.
  4. It is a method, The method comprising generating a spatial map of the distribution of nuclei in a tissue sample, upon releasing a set of handshake sequences paired with a set of barcode sequences for diffusion toward the distribution of nuclei.
  5. It is a method, The method involves generating a spatial map of the distribution of nuclei in a tissue sample by processing the tissue sample containing the distribution of nuclei with a substrate containing the distribution of functionalized molecules, wherein a representative functionalized molecule is: A handshake sequence configured to bind to one of the nuclei in the set of nuclei, The generation includes a barcode array that functions as a spatial address, and an emittable linker, When stimulating the releaseable linker of the distribution of the functionalized molecules, the set of nuclei is tagged with the handshake sequence of the distribution of the functionalized molecules, thereby tagging the nuclei of the set of nuclei with the barcode sequence of the distribution of the functionalized molecules, To isolate the set of nuclei from the tissue sample, Determine the set of molecular sequences resulting from the set of nuclei and the distribution of the functionalized molecules, thereby determining the set of spatial positions of the set of nuclei based on the barcode sequence associated with each of the set of nuclei. The method comprising generating the spatial map of the distribution of the nuclei from the set of spatial locations.
  6. The method according to claim 5, wherein the generation of the spatial map is performed within two hours.
  7. The method according to claim 5, wherein isolating the set of nuclei comprises recovering more than 30% of the nuclei initially present in the tissue sample.
  8. The method according to claim 7, wherein isolating the set of nuclei comprises grinding the tissue sample with an isolation buffer.
  9. The method according to claim 7, wherein isolating the set of nuclei includes isolating the set of nuclei using an irrigator.
  10. The method according to claim 5, wherein the emittable linker responds to a light-cutting mechanism.
  11. The method according to claim 5, wherein the distribution of the functionalized molecule includes a first subset of linkers configured to cleave in response to a first stimulus, and a second subset of linkers configured to cleave in response to a second stimulus.
  12. The method according to claim 11, wherein the first stimulus is a first wavelength of light, and the second stimulus is a second wavelength of light.
  13. The method according to claim 5, wherein determining the set of spatial positions includes determining the position of the nucleus from the centroid of a subset of spatial positions of a subset of barcode sequences of a subset of functionalized molecules tagged to the nucleus.
  14. The method according to claim 5, wherein the method is performed at a temperature of 4°C or lower.
  15. The method according to claim 5, wherein the distribution of the functionalized molecule is distributed across the distribution of particles bonded to the substrate by the adhesive.
  16. The method according to claim 5, further comprising: a) tagging the set of nuclei in a handshake sequence of the distribution of the functionalized molecules; and b) isolating the set of nuclei from the tissue sample; and covering the tissue sample with a layer of an optimal cleavage temperature (OCT) compound.
  17. The method according to claim 5, further comprising covering the distribution of the functionalized molecules on the substrate with a masking layer, and removing the masking layer before applying the stimulus.
  18. It is a method, When tagging a set of nuclei with a first set of oligonucleotides, the goal is to generate a labeled set of nuclei, The process involves attaching the labeled nuclei set in the interstitial space of the distribution of functionalized particles bonded to the substrate, To generate a single-nucleus sequencing library from an amplicon produced from a set of reactions involving the labeled nucleus and the set of molecules of the functionalized particle distribution, The method comprising: returning a single-nucleus analysis for a set of nuclei when sequencing the single-nucleus sequencing library.
  19. The method according to claim 18, wherein each functionalized particle comes into contact with at most one labeled nucleus from the set of labeled nuclei.
  20. It is a system, Substrate and The distribution of functionalized features associated with the said substrate, A representative feature of the distribution of the functionalized features is: The system comprises one or more functionalized molecules bound to the representative feature, the one or more molecules comprising at least a reactive portion, a barcode segment functioning as a spatial address, and a cleavage linker configured to allow the handshake sequence to be released from the representative feature in response to a stimulus, the handshake sequence.

Description

Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63/455,502, filed on 29 March 2023, and U.S. Provisional Patent Application No. 63/557,828, filed on 26 February 2024, both of which are incorporated herein by this reference in their entirety. This invention generally relates to the field of sample characterization, and more specifically to novel and useful systems, methods, and compositions for characterizing sample targets including single cells and single nuclei. With growing interest in understanding the distribution of specific target analytes within biological samples, improved compositions, methods, and systems enabling analyte mapping are becoming increasingly valuable. Current techniques are limited by resolution (e.g., with respect to the location of the target analyte), the ability to characterize location in multiple dimensions, the ability to characterize location across scales, the ability to characterize different types of analytes, the ability to characterize target locations in situ, and/or other limitations. Furthermore, a streamlined approach for spatial mapping is needed, equipped with the ability to process and recover sample targets related to single cells and single nuclei. Therefore, the field of sample characterization requires novel and useful systems, methods, and compositions for characterizing sample targets with spatial mapping capabilities. Currently, methods and systems for spatially characterizing analytes in a sample (e.g., in situ, in vitro, etc.) are limited with respect to resolution (e.g., with respect to the potential number of target analytes that can be characterized per unit area or volume), low signal-to-noise ratio (e.g., due to high levels of background noise), recovery rate of material from single nuclei and/or single cells of the sample being spatially characterized, diffusion of targets intended to be characterized from their origin within a tissue sample, underutilization of space between interaction sites due to manufacturing or physical constraints, the ability to characterize target samples in multiple dimensions, the ability to characterize different types of analytes simultaneously (e.g., whole transcriptome characterization capability), and/or other methods. Therefore, this disclosure describes embodiments, modifications, and examples of systems, methods, and compositions for performing spatial biology (e.g., spatial transcriptomics, spatial proteomics, spatial multiomics, etc.) in a manner that provides broader transcriptome applications while achieving a high level of spatial resolution. This disclosure describes embodiments, modifications, and examples of methods and systems for spatially localizing individual nuclei and/or individual cells of a sample being processed. This disclosure also describes embodiments, modifications, and examples of methods and systems for spatially localizing other sample targets (e.g., proteins, etc.) in space based on the reactive sequences of the functionalizing molecules involved. Embodiments of this disclosure provide embodiments, variations, and examples of systems, methods, and compositions for efficiently tagging target substances (e.g., DNA, RNA, miRNA, proteins, small molecules, single analytes, multiple analytes, etc.) to enable analysis for characterizing the localization of target substances in space. For nucleic acid targets, the handshake sequences of the described compositions may include molecules complementary to the nucleic acid target (e.g., complementarity based on poly-A/poly-T interactions, complementarity based on interactions between polyadenylated mRNA and other nucleic acids incorporating U bases and/or T (e.g., sequentially, discontinuously, with patterns, without patterns), complementarity based on the sequence of the specific target being tagged, complementarity based on the sequence of a platform for the tagging molecule, etc.). For protein or small molecule targets, the handshake sequences, described in more detail below, may include antibodies or aptamers conjugated with specific nucleic acid sequences for detection. Functionalized molecules incorporating a handshake sequence may further include one or more modifications that enhance diffusion to a sample target (e.g., the nucleus). For example, functionalized molecules incorporating a handshake sequence may include one or more lipid moieties that enhance diffusion to the nucleus and/or other intracellular components. Targets may include cytoplasmic targets, intracellular targets, or other targets on the surface or inside cells (or cellular components). For example, targets may include cytoplasmic targets and cell targets or nucleus-related targets derived from the same tissue, thereby enabling simultaneous mapping of multiple target types in a highly parallel manner. Alternatively, the generated maps of nuclear/cellular targets can be integrated with other maps characte