EP-4739794-A1 - METHODS, COMPOSITONS, AND SYSTEMS FOR ANALYTE DETECTION

Abstract

Provided herein are methods and systems for identifying a proximity of analytes in a sample. The method may comprise contacting one or more analytes with one or more probes. The proximity of the one or more analytes to each other may cause a ligation event between the one or more probes. An amplification reaction may be performed comprising one or more copies of a barcode or derivative thereof. The one or more copies of the barcode or derivative thereof may be detected to identify a proximity between the one or more analytes.

Inventors

• FU, YE

Assignees

• Stellaromics, Inc.

Dates

Publication Date

20260513

Application Date

20240705

Claims (20)

1. 1. A method of detecting analytes in a sample, said method comprising: a) providing a first probe and a second probe, wherein said first probe comprises: (i) a first binding site configured to couple to a first analyte at a first portion; (ii) a second binding site configured to couple to said first analyte at a second portion, wherein said first portion is adjacent to said second portion; (iii) a third binding site configured to couple to said second probe; (iv) a barcode; (v) a first end; and (vi) a second end; wherein said second probe comprises: (i) a fourth binding site configured to couple to said first probe; and (ii) a fifth binding site configured to couple to said second analyte; b) contacting a sample comprising a plurality of analytes comprising said first analyte and said second analyte with said first probe and said second probe, such that: (i) said first probe is coupled to said first analyte; (ii) said second probe is coupled to said second analyte; and (iii) said first probe is coupled to said second probe; c) ligating said first end and said second end to form a circular oligonucleotide; d) amplifying said circular oligonucleotide to generate an amplicon, wherein said amplicon comprises a complement of said barcode; and e) detecting said complement of said barcode or a derivative thereof using a plurality of detection probes, thereby determining a proximity of said first analyte to said second analyte.
2. 2. The method of claim 1, wherein said sample is a tissue sample.
3. 3. The method of claim 1, wherein said tissue sample is a fresh-frozen tissue sample.
4. 4. The method of claim 1, wherein said tissue sample is a formalin-fixed paraffin embedded tissue sample.
5. 5. The method of any one of claims 1-4, wherein said sample is 5-250pm thick.
6. 6. The method of any one of claims 1-4, wherein said sample is 10-200pm thick.
7. 7. The method of any one of claims 1-4, wherein said sample is 25-150pm thick.
8. 8. The method of any one of claims 1-4, wherein said first analyte comprises a nucleic acid.
9. 9. The method of claim 8, wherein said nucleic acid is a ribonucleic acid.
10. 10. The method of claim 9, wherein said ribonucleic acid is a messenger ribonucleic acid.
11. 11. The method of claim 9, wherein said ribonucleic acid is a ribosomal ribonucleic acid.
12. 12. The method of claim 8, wherein said nucleic acid is a deoxyribonucleic acid.
13. 13. The method of any one of claims 1-8, wherein said first analyte comprises a polypeptide.
14. 14. The method of claim 13, wherein said polypeptide comprises a ribosomal protein.
15. 15. The method of any one of claims 1-14, wherein said first analyte comprises a chemical modification.
16. 16. The method of any one of claims 1-15, wherein said second analyte comprises a nucleic acid.
17. 17. The method of claim 16, wherein said nucleic acid is a ribonucleic acid.
18. 18. The method of claim 17, wherein said ribonucleic acid is a messenger ribonucleic acid.
19. 19. The method of claim 17, wherein said ribonucleic acid is a ribosomal ribonucleic acid.
20. 20. The method of claim 16, wherein said nucleic acid is a deoxyribonucleic acid.

Description

METHODS, COMPOSITONS, AND SYSTEMS FOR ANALYTE DETECTION CROSS-REFERENCE [0001] This application claims priority to U.S. Provisional Patent Application No. 63/512,502, filed July 7, 2023, which is entirely incorporated herein by reference. BACKGROUND [0002] The proximity of analytes within a sample have been analyzed and used for determining the state of the sample. Methods have been developed for analyzing the proximity of analytes within a sample. SUMMARY [0003] Aspects disclosed herein provide methods of detecting analytes in a sample, the method comprising: a) providing a first probe and a second probe, wherein the first probe comprises: (i) a first binding site configured to couple to a first analyte at a first portion of the first analyte; (ii) a second binding site configured to couple to the first analyte at a second portion of the first analyte, wherein the first portion of the first analyte is adjacent to the second portion of the first analyte; (iii) a third binding site configured to couple to the second probe; (iv) a barcode; (v) a first end; and (vi) a second end; wherein the second probe comprises: (i) a fourth binding site configured to couple to the first probe; and (ii) a fifth binding site configured to couple to the second analyte; b) contacting a sample comprising a plurality of analytes comprising the first analyte and the second analyte with the first probe and the second probe, such that: (i) the first probe is coupled to the first analyte; (ii) the second probe is coupled to the second analyte; and (iii) the first probe is coupled to the second probe; c) ligating the first end and the second end to form a circular oligonucleotide; d) amplifying the circular oligonucleotide to generate an amplicon, wherein the amplicon comprises a complement of the barcode; and e) detecting the complement of the barcode or a derivative thereof using a plurality of detection probes, thereby determining a proximity of the first analyte to the second analyte. [0004] In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a fresh-frozen tissue sample. In some embodiments, the tissue sample is a formalin- fixed paraffin embedded tissue sample. In some embodiments, the sample is 5-250pm thick. In some embodiments, the sample is 10-200pm thick. In some embodiments, the sample is 25- 150pm thick. [0005] In some embodiments, the first analyte comprises a nucleic acid. In some embodiments, the nucleic acid is a ribonucleic acid. In some embodiments, the ribonucleic acid is a messenger ribonucleic acid. In some embodiments, the ribonucleic acid is a ribosomal ribonucleic acid. In some embodiments, the nucleic acid is a deoxyribonucleic acid. In some embodiments, the first analyte comprises a polypeptide. In some embodiments, the comprises a ribosomal protein. In some embodiments, the first analyte comprises a chemical modification. In some embodiments, the second analyte comprises a nucleic acid. In some embodiments, the nucleic acid is a ribonucleic acid. In some embodiments, the ribonucleic acid is a messenger ribonucleic acid. In some embodiments, the ribonucleic acid is a ribosomal ribonucleic acid. In some embodiments, the nucleic acid is a deoxyribonucleic acid. In some embodiments, the second analyte comprises a polypeptide. In some embodiments, the polypeptide comprises a ribosomal protein. [0006] In some embodiments, the first probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modifications. In some embodiments, the one or more modifications comprise a 5’ phosphate modification. In some embodiments, the one or more modification comprise an intemucleotide linkage. In some embodiments, the intemucleotide linkage is a phosphorothioate. In some embodiments, the intemucleotide linkage is a phosphodiester. In some embodiments, the first probe recognizes a ribonucleic acid. In some embodiments, the nucleic acid comprises a single nucleotide polymorphism. In some embodiments, the first probe recognizes the single nucleotide polymorphism and wherein the ligating in c) does not occur if the first analyte does not comprise the single nucleotide polymorphism. In some embodiments, the first probe does not recognize the single nucleotide polymorphism and wherein the ligating in c) does not occur if the first analyte comprises the single nucleotide polymorphism. In some embodiments, the nucleic acid comprises a modification. In some embodiments, the modification is selected from the group consisting of N 6-methyladenosine, 5-methylcytosine, N 1- methyladenosine, N 7-m ethylguanosine, N 4-acetylcytosine, pseudouridine, and Nl- methylpseudouridine. In some embodiments, the first probe recognizes the modification and wherein the ligating in c) does not occur if the first analyte does not comprise the modification. In some embodiments, the first probe does not recognize th