EP-4741513-A2 - METHODS AND COMPOSITIONS FOR SYNCHRONIZING REACTIONS IN SITU

Abstract

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.

Inventors

• HERNÁNDEZ NEUTA, Jorge Iván
• KÜHNEMUND, Malte
• MARKS, PATRICK J.

Assignees

• 10X Genomics, Inc.

Dates

Publication Date

20260513

Application Date

20220729

Claims (15)

1. A kit for in situ detection of a polynucleotide in a sample, comprising: a polymerase for performing rolling circle amplification (RCA); a circular nucleic acid comprising a hybridization region or a circularizable nucleic acid comprising a hybridization region, said circularizable nucleic acid configured to form the circular nucleic acid comprising the hybridization region, wherein the hybridization region is complementary to a sequence of the polynucleotide; a first reaction mixture comprising a non-catalytic metal cofactor of the polymerase; and a second reaction mixture comprising a catalytic metal cofactor of the polymerase.
2. The kit of claim 1, wherein a pH of the first reaction mixture is about 7.5.
3. The kit of claim 1 or claim 2, wherein the circularizable nucleic acid is a padlock probe.
4. The kit of any one of claims 1-3, further comprising a cell or tissue sample comprising the polynucleotide, optionally comprising the circular nucleic acid in the cell or tissue sample, wherein the hybridization region of the circular nucleic acid is hybridized to the polynucleotide and the cell or tissue sample is in contact with the non-catalytic metal cofactor and the polymerase.
5. The kit of claim 4, wherein the polymerase is in the cell or tissue sample and bound to the polynucleotide.
6. The kit of claim 4 or claim 5, additionally comprising a wash buffer configured to remove the first reaction mixture from the cell or tissue sample.
7. The kit of any one of claims 1-6, wherein the first reaction mixture is substantially free of deoxynucleoside triphosphates (dNTPs) and/or nucleoside triphosphates (NTPs).
8. The kit of any one of claims 1-7, wherein the non-catalytic metal cofactor is calcium, barium, strontium, iron, cobalt, nickel, tin, zinc, or europium, optionally wherein the non-catalytic metal cofactor is Ca 2+ or Sr 2+ .
9. The kit of any one of claims 1-8, wherein the first reaction mixture comprises a chelating agent, optionally wherein the chelating agent comprises EDTA, EGTA, BAPTA, DTPA, or a combination thereof.
10. The kit of any one of claims 1-9, wherein the first reaction mixture is substantially free of the catalytic metal cofactor of the polymerase, optionally wherein the catalytic metal cofactor comprises Mg 2+ , Co 2+ , Zn 2+ , Mn 2+ , or any combination thereof.
11. The kit of any one of claims 1-10, wherein the second reaction mixture comprises a deoxynucleoside triphosphate (dNTP) and/or is substantially free of the polymerase and/or other polymerases.
12. The kit of any one of claims 1-11, wherein the components of the kit are present in separate containers.
13. The kit of any one of claims 1-12, additionally comprising a ligase.
14. The kit of any one of claims 1-13, additionally comprising reagents for fixing, embedding, and/or permeabilizing the sample.
15. The kit of any one of claims 1-14, additionally comprising: a plurality of detectably-labeled probes configured to directly or indirectly bind to a rolling circle amplification product generated by the polymerase; or a plurality of intermediate probes configured to directly or indirectly hybridize to a rolling circle amplification product generated by the polymerase, wherein the intermediate probes are detectable using one or more detectably-labeled probes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63/227,830 filed July 30, 2021, entitled "METHOD AND COMPOSITIONS FOR SYNCHRONIZING REACTIONS IN SITU," which is herein incorporated by reference in its entirety for all purposes. FIELD The present disclosure generally relates to methods and compositions for in situ detection of a plurality of molecules of one or more analytes in a sample. BACKGROUND Genomic, transcriptomic, and proteomic profiling of cells and tissue samples using microscopic imaging can resolve multiple analytes of interest at the same time, thereby providing valuable information regarding analyte abundance and localization in situ. Thus, these in situ assays are important tools, for example, for understanding the molecular basis of cell identity and developing treatment for diseases. In multiplex assays where multiple signals are detected simultaneously, it is important that as much information as possible is collected. However, due to the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample, there can be a wide and heterogeneous size and intensity distribution of signal "spots" in the sample. Large signal spots may overlap with one another and/or mask adjacent smaller signal spots, rendering the smaller spots unresolvable. In addition, some analytes may be associated with bright signal spots (e.g., due to high analyte abundance and/or preferential signal amplification), while other analytes may be associated with signal spots that are too dim to be detected simultaneously (e.g., in the same field of view (FOV) during microscopy) with the bright spots. There is a need for new and improved methods for in situ assays. The present disclosure addresses these and other needs. SUMMARY In some embodiments, provided herein is a method for analyzing a biological sample, comprising contacting the biological sample with a first reaction mixture, wherein the biological sample comprises a circular nucleic acid comprising a hybridization region, the first reaction mixture comprises a polymerase, the circular nucleic acid or the polymerase is prebound to a polynucleotide comprising a sequence complementary to the hybridization region, and the polymerase activity of the polymerase is inhibited. In some embodiments, the method further comprises contacting the biological sample with a second reaction mixture to allow the polymerase to extend the polynucleotide hybridized to the hybridization region using the circular nucleic acid as a template. In any of the preceding embodiments, a rolling circle amplification product of the circular nucleic acid can be generated in the biological sample, for instance, for in situ analysis of the circular nucleic acid and/or one or more analytes of interest associated therewith. In any of the preceding embodiments, the first reaction mixture can stabilize the polymerase and/or inhibit an activity of the polymerase, such as a polymerase activity and/or a nuclease activity. In any of the preceding embodiments, the first reaction mixture can comprise one or more deoxynucleoside triphosphates (dNTPs) and/or nucleoside triphosphates (NTPs). In any of the preceding embodiments, the first reaction mixture can comprise dATP, dTTP, dCTP, and/or dGTP. Alternatively, in any of the preceding embodiments, the first reaction mixture can be substantially free of dNTPs and/or NTPs. In any of the preceding embodiments, the first reaction mixture can comprise a di-cation that is not a cofactor of the polymerase. In some embodiments, the di-cation is Ca2+. In any of the preceding embodiments, the di-cation can stabilize the polymerase. In any of the preceding embodiments, the di-cation can stabilize a preformed complex comprising the polymerase and the polynucleotide. In any of the preceding embodiments, the first reaction mixture can be substantially free of a cofactor of the polymerase. In any of the preceding embodiments, the first reaction mixture can be substantially free of Mg2+, Co2+, and/or Mn2+. In any of the preceding embodiments, the first reaction mixture can comprise a chelating agent. For instance, the chelating agent can chelate a di-cation such as Mg2+ from one or more prior reactions. As such, the chelating agent can chelate residual amounts of the di-cation in the biological sample, such as a tissue slice which has been contacted with a reaction mixture containing the di-cation (e.g., a ligation reaction mixture to circularize a padlock probe to form the circular nucleic acid). In any of the preceding embodiments, the first reaction mixture can comprise EDTA, EGTA, BAPTA, DTPA, or a combination thereof. In any of the preceding embodiments, the first reaction mixture can inhibit the polymerase activity and/or an exonuclease activity of the polymerase. In any of the preceding embodiments, the 3'-5' exonuclease activity and/or the 5'