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US-20260125743-A1 - COMPOSITIONS AND METHODS FOR TARGET DETECTION

US20260125743A1US 20260125743 A1US20260125743 A1US 20260125743A1US-20260125743-A1

Abstract

Provided herein are methods for identifying subsequences in a sample. The methods may comprise contacting an analyte with a binding moiety, bringing the analyte in contact with a first detection probe and second detection probe, wherein the first detection probe and second detection probe are adjacent and different from one another, detecting the corresponding detection moieties, and using the identified subsequences to identify the analyte.

Inventors

  • Ye Fu
  • Mark Pratt

Assignees

  • STELLAROMICS, INC.

Dates

Publication Date
20260507
Application Date
20250929

Claims (20)

  1. 1 . A method for identifying an analyte in a sample, comprising: (a) contacting an analyte with a binding moiety, wherein said binding moiety comprises (i) a probe that couples to said analyte and (ii) a sequence, wherein said sequence comprises a plurality of subsequences; (b) bringing said sample in contact with a first detection probe and a second detection probe, wherein: (i) said first detection probe couples to a first subsequence of said sequence or derivative thereof, and wherein said first detection probe comprises a first detection moiety; (ii) said second detection probe couples to a second subsequence of said sequence or derivative thereof, and wherein said second detection probe comprises a second detection moiety; and (iii) said first subsequence and said second subsequence are adjacent to each other; (c) detecting said first detection moiety and said second detection moiety to identify at least said first subsequence and said second subsequence; and (d) using at least said first subsequence and said second subsequence identified in (c) to identify said analyte.
  2. 2 . The method of claim 1 , wherein said sample is a tissue.
  3. 3 . The method of claim 1 , wherein said sample is embedded in a hydrogel.
  4. 4 . The method of claim 1 , wherein said analyte comprises a nucleic acid.
  5. 5 . The method of claim 4 , wherein said nucleic acid is ribonucleic acid.
  6. 6 . The method of claim 1 , wherein said probe that couples to said analyte comprises a nucleic acid.
  7. 7 . The method of claim 1 , wherein a subsequence of said plurality of subsequences comprises a nucleic acid.
  8. 8 . The method of claim 1 , wherein said plurality of subsequences comprises subsequences of the same length.
  9. 9 . The method of claim 1 , wherein said plurality of subsequences comprises subsequences of different lengths.
  10. 10 . The method of claim 1 , wherein said first detection probe comprises a nucleic acid.
  11. 11 . The method of claim 1 , wherein said second detection probe comprises a nucleic acid.
  12. 12 . The method of claim 1 , wherein said first detection moiety comprises a fluorescent dye.
  13. 13 . The method of claim 1 , wherein said second detection moiety comprises a fluorescent dye.
  14. 14 . The method of claim 1 , wherein (c) comprises imaging said analyte.
  15. 15 . The method of claim 14 , wherein said imaging comprises using a microscope.
  16. 16 . The method of claim 1 , further comprising amplifying said sequence in (a) to generate an amplified sequence, wherein said amplified sequence comprises multiple copies of said sequence or derivative thereof.
  17. 17 . The method of claim 1 , further comprising ligating said first detection probe and said second detection probe in (b).
  18. 18 . The method of claim 17 , wherein said ligating comprises using a ligase.
  19. 19 . The method of claim 1 , wherein (c) further comprises detecting a ratio of said first detection moiety and said second detection moiety.
  20. 20 . The method of claim 19 , further comprising using said ratio to identify said analyte.

Description

CROSS-REFERENCE This application is a continuation of Int'l Application No. PCT/US2025/030656, filed May 22, 2025, which claims priority to U.S. Provisional Patent Application No. 63/651,318, filed May 23, 2024, each of which is entirely incorporated herein by reference. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 29, 2025, is named 68135-718.301_SL.xml and is 35,803_bytes in size. BACKGROUND Targets can be detected in situ within a biological sample. Multiple methods of detection can be used. SUMMARY In an aspect, the present disclosure provides methods for identifying an analyte in a sample, comprising: (a) contacting the sample with a binding moiety, wherein the binding moiety comprises (i) a probe that couples to the analyte and (ii) a sequence, wherein the sequence comprises a plurality of subsequences; (b) bringing the sample in contact with a first detection probe and a second detection probe, wherein: (i) the first detection probe couples to a first subsequence of the sequence or derivative thereof, and wherein the first detection probe comprises a first detection moiety; (ii) the second detection probe couples to a second subsequence of the sequence or derivative thereof, and wherein the second detection probe comprises a second detection moiety; and (iii) the first subsequence and the second subsequence are adjacent to each other; (c) detecting the first detection moiety and the second detection moiety to identify at least the first subsequence and the second subsequence; and (d) using at least the first subsequence and the second subsequence identified in (c) to identify the analyte. In some embodiments, the first detection moiety and the second detection moiety are different. In some embodiments, the first detection moiety and the second detection moiety are the same. In some embodiments, the sample is a tissue. In some embodiments, the tissue is a fresh-frozen tissue. In some embodiments, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiments, the sample comprises a cell. In some embodiments, the sample is embedded in a hydrogel. In some embodiments, the analyte comprises a protein or a polypeptide. In some embodiments, the analyte comprises a nucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the probe that couples to the analyte comprises an antibody or antibody fragment. In some embodiments, the probe that couples to the analyte comprises a nucleic acid. In some embodiments, the probe that couples to the analyte comprises an aptamer. In some embodiments, a subsequence of the plurality of subsequences comprises a nucleic acid. In some embodiments, the nucleic acid comprises one or more nucleotides. In some embodiments, the nucleic acid comprises two or more nucleotides. In some embodiments, the nucleic acid comprises three or more nucleotides. In some embodiments, the plurality of subsequences comprises subsequences of the same length. In some embodiments, the plurality of subsequences comprises subsequences of different lengths. In some embodiments, the first detection probe comprises a nucleic acid. In some embodiments, the second detection probe comprises a nucleic acid. In some embodiments, the first subsequence is a nucleic acid. In some embodiments, the nucleic acid is at least one nucleotide in length. In some embodiments, the nucleic acid is at least two nucleotides in length. In some embodiments, the nucleic acid is at least three nucleotides in length. In some embodiments, the second subsequence is a nucleic acid. In some embodiments, the nucleic acid is at least one nucleotide in length. In some embodiments, the nucleic acid is at least two nucleotides in length. In some embodiments, the nucleic acid is at least three nucleotides in length. In some embodiments, the first detection moiety comprises a fluorescent dye. In some embodiments, the first detection moiety comprises a linker. In some embodiments, the first detection moiety is coupled to the nucleic acid of the first detection probe via the linker. In some embodiments, the first detection moiety is coupled to a 5′ end of the nucleic acid of the first detection probe. In some embodiments, the first detection moiety is coupled to a 3′ end of the nucleic acid of the first detection probe. In some embodiments, the second detection moiety comprises a fluorescent dye. In some embodiments, the second detection moiety comprises a linker. In some embodiments, the second detection moiety is coupled to the nucleic acid of the second detection probe via the linker. In some embodiments, the second detection moiety is coupled to a 3′ end of the nucleic acid of the second detection probe. In some embodiments, the second detection moiety is coupled to