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EP-4735630-A1 - SIGNAL AMPLIFICATION METHODS AND COMPOSITIONS FOR MOLECULAR TARGET DETECTION BY ITERATIVE PROBE DEPOSITION

EP4735630A1EP 4735630 A1EP4735630 A1EP 4735630A1EP-4735630-A1

Abstract

The present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field.

Inventors

  • COLON, Katsuya
  • TISCHBIREK, CARSTEN
  • CAI, LONG
  • LU, Chuqi

Assignees

  • CALIFORNIA INSTITUTE OF TECHNOLOGY

Dates

Publication Date
20260506
Application Date
20240701

Claims (20)

  1. 1. A method comprising: (a) contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe in the plurality of primary probes interacts with at least one target analyte; (b) contacting each of a plurality of the primary probes, each interacting with at least one analyte, with one or more amplifier probes; (c) optionally, cross-linking one or more amplifier probes to a cellular component; (d) optionally, separating each of one or more amplifier probes from its primary probe or from another amplifier probe; (e) optionally, repeating steps (b), (c), and/or (d); (f) detecting one or more target analytes.
  2. 2. The method claim 1, further comprising contacting one or more amplifier probes with one or more readout probes.
  3. 3. The method of claim 1 , wherein signals are detected by imaging or sequencing.
  4. 4. The method of claim 1, wherein the method comprises at least one step of crosslinking one or more amplifier probes to a cellular component.
  5. 5. The method of claim 4, wherein the step of cross-linking is by click chemistry.
  6. 6. The method of claim 5, wherein the click chemistry is selected from: strain-promoted azide-alkyne cycloadditions (SPAACs), tetrazine-trans-cyclooctene ligations (TCO- Tz), thiol-ene reactions, thiol-yne reactions, oxime ligations, hydrazone ligations, and Diels-Alder reactions; and inverse-electron demand Diels-Alder reactions (lEDDAs).
  7. 7. The method of claim 1, wherein the method comprises at least one step of separating each of one or more amplifier probes from its primary probe or from another amplifier probe.
  8. 8. The method of claim 1, wherein one or more target analytes are detected by signals or absence of signals.
  9. 9. The method of claim 1, further comprising contacting one or more amplifier probes to another amplifier probe.
  10. 10. The method of claim 1, wherein one or more amplifier probes comprises a secondary, tertiary, and quaternary amplifier probe.
  11. 11. The method of claim 10, wherein, the quaternary' amplifier probe is identical to the secondary’ amplifier probe.
  12. 12. The method of claim 10, wherein the quaternary' amplifier probe interacts with the secondary’ amplifier probe.
  13. 13. The method of claim 10, wherein the quaternary amplifier probe comprises one or more binding sites for the secondary’ amplifier probe, one or more binding sites for the tertiary amplifier probe, or any combination thereof.
  14. 14. The method of claim 10, wherein the tertiary' amplifier probe comprises one or more binding sites for the secondary- amplifier probe, one or more binding sites for the quaternary’ amplifier probe, or any combination thereof.
  15. 15. The method of claim 10. wherein the secondary amplifier probe comprises one or more binding sties for the tertiary amplifier probe, one or more binding sites for the quaternary' amplifier probe, one or more binding sites for the primary’ probe, or any combination thereof.
  16. 16. The method of claim 1, yvherein the primary probe is amplified by: (a) contacting the primary' probe with a secondary amplifier probe, (b) contacting the secondary amplifier probe with a tertiary amplifier probe, and (c) contacting the tertiary amplifier probe with a quaternary amplifier probe, wherein the quaternary amplifier probe is the same as the secondary amplifier probe.
  17. 17. The method of claim 1, wherein the samples are imaged after contacting one or more amplifier probes with one or more readout probes.
  18. 18. The method of claim 1 , further comprising amplifying one or more amplifier probes by contacting one or more amplifier probes from a previous contacting step with a new plurality of amplifier probes.
  19. 19. The method of claim 18, wherein the new plurality of amplifier probes is the same as a previous plurality of amplifier probes.
  20. 20. The method of claim 1 , further comprising: (a) repeating the contacting one or more amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes; and (b) optionally, separating one or more amplifier probes from the primary probe or another amplifier probe after imaging the sample. (c) optionally, separating the readout probes from the amplifier probes.

Description

SIGNAL AMPLIFICATION METHODS AND COMPOSITIONS FOR MOLECULAR TARGET DETECTION BY ITERATIVE PROBE DEPOSITION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/524,408, filed June 30, 2023. The contents of the above-referenced application are hereby incorporated by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] The U.S. Government has certain rights in this disclosure pursuant to Grant No NS 131408 awarded by the National Institute of Health. FIELD [0003] The present disclosure provides methods, compositions, and kits for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This invention can be implemented for multiplex profiling of one or multiple ty pes of analytes such as, but not limited to, RNAs, DNAs, proteins, small molecule inhibitors, sugars, lipids, organelle, synthetic barcodes, in biological or clinical samples. BACKGROUND [0004] The spatial profiling of cellular components, including DNA regions, proteins, and mRNAs, plays an important role in understanding cellular functions and the molecular mechanisms in health and disease. Microscopy, capable of resolving these targets in individual cells, provides crucial insights into the abundance and localization of these components. [0005] Spatial transcriptomics has emerged as a powerful tool for understanding the spatial organization of gene expression within tissues and cells, providing valuable insights into cellular heterogeneity and tissue function (1). A major limitation for imaging-based spatial transcriptomics methods is the low signal-to-noise ratio, particularly in thick tissue samples or non-ideal tissue samples such as FFPE or human derived tissue samples. Generally, dim signals can be circumvented by using a larger number of hybridization probes per gene. Further, this approach will not work when targeting short RNAs species. [0006] Various methods have been developed to address these challenges such as ClampFISH (2, 3). RollFISH (4), STARmap (5. 6), HyblSS (Ref 7), FISSEQ (8). hybridization chain reaction (9, 10), and branched DNA (1 1, 12). However, these methods often exhibit several deficiencies such as poor probe diffusion, non-specific signal, low detection efficiency, inability' to scale to near transcriptome-level profiling, or the necessity to extensively screen amplifier probes. Most of these approaches require enzymes which can be costly and require special storage conditions, rely on highly purified and bi-functionalized probes which can also be costly and time consuming to generate, depend on unstable reagents which can limit reproducibility and scalability, or require pre-programed probes to assemble with a defined amplification factor which does not offer a modularity to the overall method. SUMMARY [0007] The present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field. [0008] In some embodiments, there is provided a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample, comprising contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe in the plurality of primary probes interacts with at least one target analyte. In some embodiments, the method comprises contacting each of a plurality of the primary probes, each interacting wi th at least one analyte, with one or more amplifier probes. In some embodiments, the method comprises optionally, cross-linking one or more amplifier probes to a cellular component. In some embodiments, the method comprises optionally, separating each of one or more amplifier probes from its primary’ probe or from another amplifier probe. In some embodiments, the method comprises optionally, repeating any of the previous embodiments either alone or in combination. In some embodiments, the method comprises detecting one or more target analytes. [0009] In some embodiments, the method comprises contacting a plurality of the amplifier probes each with one or more readout probes. In some embodiments, the method comprises imaging the sample so that the interaction of the readout probes with their target analytes is detected. [0010] In some embodiments, the method comprises imaging the samples after contacting the amplifier probes with one or more readout probes. [0011] In some embodiments, the method further comprises amplifying the amplifier probes by contacting the amplifier probes from a previous contacting step with anew plurality of amplifier probes. [0012] In some embodiments, the method further comprises repeating the