De-crosslinking for multi analyte analysis

Abstract

The present disclosure relates in some aspects to methods and compositions for in situ analysis involving preparation and treatment (e.g., de-crosslinking) of biological samples. Preparation of biological samples comprising nucleic acid analytes and protein analytes for detection are provided. Methods, in which the sample with bound probes is contacted with a buffer at a temperature above 80°C, improve the detection of optical signals associated with analytes in a fixed biological sample.

Description

202412025040DE-CROSSLINKING FOR MULTI ANALYTE ANALYSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 761,091, filed February 20, 2025, entitled “SAMPLE TREATMENTS FOR MULTI ANALYTE ANALYSIS,” which is herein incorporated by reference in its entirety for all purposes.FIELD

[0002] The present disclosure relates in some aspects to compositions and methods for de-crosslinking of a fixed biological sample and for preparing the biological sample for in situ analysis. In some aspects, the biological samples comprise nucleic acid analytes and protein analytes (e.g., polypeptides) for detection. In some aspects, the methods are for improving detection of optical signals associated with analytes in a fixed biological sample.BACKGROUND

[0003] Methods are available for analyzing analytes such as nucleic acids and proteins present in a biological sample, e.g., a cell or tissue sample. Current methods for analyzing analytes in situ can have low sensitivity and specificity, have high background and / or low signal-to-noise ratio (e.g., due to autofluorescence), have limited plexity, or be timeconsuming, labor-intensive, and / or error-prone. Improved methods for preparing biological samples for analyzing analytes are needed. Provided herein are methods and compositions that meet such and other needs.SUMMARY

[0004] Provided herein is a method comprising contacting a biological sample comprising cells with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample; after de-crosslinking with the catalyst, contacting the biological sample with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells; after binding the plurality of nucleic acid probes to the plurality of RNAs, incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes; contacting the biological sample with a labelling agent that directly or indirectly binds to an analyte at a location in the biological sample; and detecting a plurality of 1MF-367036897202412025040optical signals associated with the plurality of nucleic acid probes or products thereof associated with the plurality of RNAs in the biological sample, and an optical signal associated with the labelling agent or a product thereof in the biological sample.

[0005] In some embodiments, the molecular crosslinks are products of one or more crosslinking agents. In some embodiments, the one or more crosslinking agents comprise an aldehyde, optionally wherein the crosslinking agent comprises formaldehyde. In some embodiments, the molecular crosslinks are on RNA, DNA, protein, carbohydrate, lipid, and / or other molecules in the biological sample.

[0006] In some embodiments, the second buffer comprises Tris and a chelating agent and / or a buffering agent. In some instances, the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE).

[0007] In some embodiments, the catalyst is a water-soluble catalyst. In some instances, the catalyst is an organic molecule. In some instances, the catalyst is a transimination catalyst. In some instances, the catalyst catalyzes de-crosslinking of aminal crosslinks in the biological sample. In some instances, the catalyst catalyzes breakdown of hemi- aminal adducts and / or aminal adducts in the biological sample. In some embodiments, the catalyst is a compound of formula (I),'2(I),or a salt, zwitterion, or solvate thereof, wherein: A is selected from the group consisting of -COOH, -P(=O)(OH)2, and S(=O)2OH; X1, X2, X3, and X4are each independently selected from the group consisting of: CH, CRa, and N; each occurrence of Rais independently selected from the group consisting of C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, -NO2, -NR’R”, and -C(=O)NR’R”; and each occurrence of R’ and R” is independently selected from the group consisting of H and2MF-367036897202412025040[°^ O'CH»C1-6 alkyl which is optionally substituted with [PEG structure image], wherein n1 is an integer from 12 to 16.

[0008] In some embodiments, the catalyst comprises one or more compoundsselected from the group consisting of, or a salt, zwitterion, or solvate thereof. In someinstances, the catalyst comprises, or a salt, zwitterion, or solvate thereof. In3MF-3670368972024120250400ifHO”P^HO Jj ' Nsome instances, the catalyst comprises, or a salt, zwitterion, or solvate thereof.In some instances, the catalyst comprises, or a salt, zwitterion, or solvatethereof. In some instances, the catalyst comprises, or a combination thereof, or a salt, zwitterion, or solvate thereof. In some instances, the catalyst is a compound of formula (II),or a salt, zwitterion, or solvate thereof, wherein: L1is selected from the group consisting of -O-, -N(H)-, -N(C1-3 alkyl)-, -N(CH2CH2O)1-10-CH3-, -S(0)o-2-, -CH2-, and a bond; R1is selected from the group consisting of: H; C1-6 alkyl; C1-6 haloalkyl; C6-10 aryl optionally substituted with 1-4 Rb; and 5- to 10-membered heteroaryl, wherein 1-4 ring atoms are heteroatoms each independently selected from the group consisting of: N, N(H), N(CI-3 alkyl), O, and S, wherein the heteroaryl is optionally substituted with 1-4 independently selected Rb; and each Rbis independently selected from the group consisting of: halo, cyano, -OH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, C1-6 alkyl, C1-6 haloalkyl, C1-4 alkoxy, and C1-4 haloalkoxy.4MF-367036897202412025040HQ z

[0009] In some instances, the catalyst comprisesOsolvate thereof.

[0010] In some embodiments, the biological sample is on a substrate with a planar surface. In some instances, the substrate is transparent. In some examples, the substrate is a glass slide or a plastic slide. In some instances, the substrate does not comprise nucleic acids immobilized thereon prior to contacting the biological sample.

[0011] In some embodiments, the biological sample is a tissue section. In some instances, the biological sample comprise cells immobilized on the substrate. In some instances, the cells are dissociated cells, cultured cells, and / or cells isolated from a subject.

[0012] In some embodiments, the biological sample is a fixed and / or permeabilized biological sample. In some instances, the biological sample is an aldehyde-fixed biological sample. In some instances, the biological sample is a formaldehyde-fixed biological sample. In some instances, the biological sample is a paraffinized biological sample. In some instances, the biological sample is a formaldehyde-fixed paraffin-embedded (FFPE) biological sample. In some instances, the biological sample is a fresh frozen biological sample that has been crosslinked. In some instances, the method comprises dehydrating the biological sample prior to incubating the biological sample with the second buffer.

[0013] In some instances, the biological sample is dehydrated between contacting the biological sample with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells and incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes. In some instances, dehydrating comprises drying the biological sample at 42°C for 3 hours or drying the biological sample at room temperature overnight.5MF-367036897202412025040

[0014] In some embodiments, the biological sample is baked after contacting the biological sample with a plurality of nucleic acid probes and prior to incubating the biological sample with the second buffer. In some instances, the baking is performed at 37°C for at least 5 minutes.

[0015] In some embodiments, the biological sample is de- paraffinized prior to contacting the biological sample with the catalyst. In some instances, the de-paraffinizing comprises contacting the biological sample with xylene, ethanol, and water, or, sequentially contacting the biological sample with xylene, absolute ethanol, about 96% ethanol, and about 70% ethanol.

[0016] In some embodiments, prior to contacting the biological sample with the catalyst, the biological sample is re-hydrated. In some instances, the re-hydrating comprises sequentially contacting the biological sample with 100% ethanol, 100% ethanol, 96% ethanol, 70% ethanol, each for 3 minutes, followed by contacting the biological sample with nuclease free water for 20 seconds. In some embodiments, prior to contacting the biological sample with the catalyst, the biological sample is pretreated with a proteinase. In some embodiments, prior to contacting the biological sample with the catalyst, the biological sample is permeabilized.

[0017] In some embodiments, the catalyst is contacted with the biological sample at a concentration between about 5 mM and about 500 mM. In some instances, the catalyst is contacted with the biological sample at a concentration between about 100 mM and about 200 mM. In some embodiments, the biological sample is catalytically de-crosslinked at a temperature between about 70 °C and about 90 °C. In some embodiments, the catalyst is contacted with the biological sample for between 10 minutes to 60 minutes. In some embodiments, the catalyst is contacted with the biological sample at about 80°C for 30 minutes.

[0018] In some embodiments, the biological sample is incubated with the second buffer for at least 15 minutes. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 20 minutes. In some embodiments, the second buffer has a pH between about 8 and about 9. In some embodiments, the second buffer has a pH of at least 8. In some embodiments, the second buffer has a pH of about 9.

[0019] In some embodiments, the first buffer comprises citrate, tris(hydroxymethyl)aminomethane (Tris), phosphate-buffered saline (PBS), 2-[4-(2- 6MF-367036897202412025040hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof. In some instances, the first buffer comprises dimethyl sulfoxide (DMSO). In some instances, the first buffer comprises citrate and the second buffer is a Tris-EDTA (TE) buffer. In some instances, the first buffer has a pH of less than 8. In some instances, the first buffer is a citrate buffer between pH 5 and pH 7.

[0020] In some embodiments, the method comprises washing the biological sample after contacting the biological sample with the catalyst and / or after incubating with the second buffer. In some embodiments, the method comprises washing the biological sample after contacting the biological sample with the labelling agent. In some embodiments, the washing comprises washing the biological sample in phosphate-buffered saline with Tween detergent (PBST).

[0021] In some embodiments, the biological sample is contacted with a blocking buffer prior to contacting the biological sample with the labelling agent. In some embodiments, the labelling agent comprises a binding moiety, wherein the binding moiety comprises an antibody or epitope binding fragment thereof. In some embodiments, the biological sample is contacted with a plurality of labelling agents. In some embodiments, the biological sample is contacted with a fixative after contacting the biological sample with the labelling agent.

[0022] In some embodiments, the biological sample is contacted with a quencher after contacting the biological sample with the labelling agent and before the detecting. In some instances, the quencher comprises a quencher dye. In some instances, the quencher dye comprises: at least three aromatic residues, wherein each aromatic residue is independently an un substituted aryl, a substituted aryl, an un substituted heteroaryl, or a substituted heteroaryl, wherein at least one of said aromatic residues is covalently linked to two other aromatic residues via two exocyclic azo bonds; or at least two aromatic residues, wherein each aromatic residue is independently an un substituted aryl, a substituted aryl, an unsubstituted heteroaryl, or a substituted heteroaryl, wherein at least two of said aromatic residues are covalently linked via an exocyclic azo bond, and wherein at least one said aromatic residue is an unsubstituted polycyclic and, a substituted polycyclic aryl, an unsubstituted polycyclic heteroaryl group, or a substituted polycyclic heteroaryl group. In some instances, the quencher comprises a targeting moiety, wherein the targeting moiety binds and / or reacts with a biological moiety. In some instances, the 7MF-367036897202412025040biological moiety is endogenous to the biological sample. In some instances, the biological moiety is a polypeptide or a lipid.

[0023] In some embodiments, the labelling agent comprises a detectable label. In some embodiments, the labelling agent is bound to a nucleic acid comprising a detectable label.In some embodiments, the labelling agent comprises a reporter oligonucleotide. In some instances, the reporter oligonucleotide comprises a barcode sequence. In some instances, the barcode sequence or complement thereof is detected by hybridizing an intermediate probe to the barcode sequence or complement thereof, hybridizing a detectably labeled probe to the intermediate probe, and detecting the detectably labeled probe.

[0024] In some embodiments, the plurality of RNAs comprise a plurality of mRNA. In some instances, the plurality of nucleic acid probes each comprise a barcode sequence. In some instances, the barcode sequence or complement thereof is detected by hybridizing an intermediate probe to the barcode sequence or complement thereof, hybridizing a detectably labeled probe to the intermediate probe, and detecting the detectably labeled probe. In some instances, the barcode sequence or complement thereof is detected by contacting the biological sample with a universal pool of detectably labeled probes and a first pool of intermediate probes, wherein an intermediate probe of the first pool of intermediate probes comprises a hybridization region complementary to the barcode sequence or complement thereof and a reporter region complementary to a detectably labeled probe of the universal pool of detectably labeled probes; detecting a complex formed between the barcode sequence or complement thereof, the intermediate probes of the first pool of intermediate probes, and the detectably labeled probe; and removing the intermediate probe of the first pool of intermediate probes and the detectably labeled probe. In some instances, detecting the barcode sequence or complement thereof further comprises contacting the biological sample with the universal pool of detectably labeled probes and a second pool of intermediate probes, wherein an intermediate probe of the second pool of intermediate probes comprises a hybridization region complementary to the barcode sequence or complement thereof and a reporter region complementary to a detectably labeled probe of the universal pool of detectably labeled probes; and detecting a complex formed between the barcode sequence or complement thereof, the intermediate probe of the second pool of intermediate probes, and the detectably labeled probe.8MF-367036897202412025040

[0025] In some embodiments, the plurality of nucleic acid probes comprise a plurality of circularizable probes. In some instances, the plurality of circularizable probes is ligated to form a plurality of circularized probes prior to incubating the biological sample with the second buffer. In some instances, the plurality of circularizable probes comprise a plurality of padlock probes.

[0026] In some embodiments, the method comprises amplifying the plurality of circularized probes. In some embodiments, the amplifying is performed using the probes of the plurality of nucleic acid probes bound to RNAs in the biological sample and is before incubating the biological sample with a second buffer. In some embodiments, the amplifying is performed after binding the plurality of nucleic acid probes to the plurality of RNAs and before incubating the biological sample with a second buffer. In some instances, the plurality of circularized probes are enzymatically amplified in situ in the biological sample. In some instances, the enzymatic amplification comprises performing rolling circle amplification (RCA) to generate a plurality of RCA products prior to incubating the biological sample with the second buffer. In some instances, the plurality of nucleic acid probes is used to perform a hybridization chain reaction (HCR) or a primer exchange reaction (PER) product.

[0027] In some embodiments, the plurality of nucleic acid probes comprises a plurality of linear probes comprising a 3’ overhang and a 5’ overhang upon hybridization to the an RNA of the plurality of RNAs in the cells. In some instances, the 3’ overhang and the 5’ overhang each independently comprises one or more barcode sequences.

[0028] In some embodiments, the analyte comprises a non-nucleic acid moiety, wherein the non-nucleic acid moiety is a protein, a carbohydrate, a lipid, a small molecule, or a complex thereof. In some embodiments, the protein is an intracellular protein, a membrane¬ bound protein, or an extracellular protein.

[0029] In some embodiments, the detecting is performed by imaging the biological sample. In some embodiments, detecting the plurality of optical signals associated with the plurality of nucleic acid probes or products thereof comprises performing sequential cycles of binding detectably labeled probes and imaging the biological sample. In some instances, the imaging comprises fluorescent microscopy. In some embodiments, the method comprises9MF-367036897202412025040performing cell segmentation. In some instances, cell segmentation is performed using the detected optical signals.

[0030] Provided herein is a method comprising contacting a biological sample comprising cells with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample; contacting the biological sample with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells and generating a plurality of amplification products using the plurality of nucleic acid probes; baking comprises baking the biological sample; incubating the biological sample after baking with a second buffer at a temperature of at least 80°C for at least 10 minutes; contacting the biological sample with a labelling agent that directly or indirectly binds to a protein analyte at a location in the biological sample; and detecting a plurality of optical signals associated with the plurality of amplification products with the plurality of RNAs in the biological sample; and an optical signal associated with the labelling agent or a product thereof in the biological sample.

[0031] In some embodiments, the second buffer comprises Tris and a chelating agent. In some instances, the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE). In some embodiments, the baking is performed at a temperature of 30°C or higher for at least 5 minutes.

[0032] In some embodiments, the catalyst is a compound of formula (I),'2(I),or a salt, zwitterion, or solvate thereof, wherein A is selected from the group consisting of -COOH, -P(=O)(OH)2, and S(=O)2OH; X1, X2, X3, and X4are each independently selected from the group consisting of: CH, CRa, and N; each occurrence of Rais independently selected from the group consisting of C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, -NO2, -NR’R”, and -C(=O)NR’R”; and each occurrence of R’ and R” is independently selected from the group consisting of H and10MF-367036897202412025040[°^ O'CH»C1-6 alkyl which is optionally substituted with [PEG structure image], wherein n1 is an integer from 12 to 16. In some instances, the catalyst comprises one or more compounds selected from thegroup consisting of

[0033] In some embodiments, the biological sample is incubated with the second buffer for at least 20 minutes. In some instances, the second buffer has a pH of at least 8. In some instances, the plurality of amplification products are generated by performing rolling circle amplification (RCA). In some instances, the detecting is performed by imaging the biological sample. In some instances, the method comprises performing cell segmentation. In some11MF-367036897202412025040embodiments, the detected optical signal associated with the labelling agent or a product thereof in the biological sample is used to perform cell segmentation.

[0034] Provided herein is a system comprising a biological sample comprising cells on a solid support; a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks; a second buffer comprising Tris and a chelating agent; a plurality of circularizable probes configured to bind a plurality of RNAs; and a plurality of labelling agents configured to bind to a plurality of protein analytes. In some instances, the system comprises a heater configured to receive the biological sample. In some instances, the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE). In some instances, the catalyst is a compound of formula (I). In some instances, the catalyst comprisesone or more compounds selected from the group consisting of12MF-367036897202412025040OH HOOC OH NH2H2N H2N, and, or a salt, zwitterion, or solvate thereof.

[0035] In some embodiments, the system comprises a polymerase and dNTPs for performing rolling circle amplification (RCA) using the plurality of circularizable probes. In some embodiments, the system comprises reagents for detecting one or more sequence of the plurality of circularizable probes. In some instances, the reagents for detecting one or more sequences of the plurality of circularizable probes comprises a plurality of detectably labeled probes for binding to a sequence of an amplification product generated using RCA. In some embodiments, the system comprises reagents for sequencing. In some embodiments, the system comprises a ligase for forming a plurality of circularized probes from the plurality of circularizable probes. In some embodiments, the system comprises reagents for detecting the plurality of labelling agents. In some instances, the reagents for detecting the plurality of labelling agents comprises a plurality of detectably labeled probes for binding to a reporter oligonucleotide of the plurality of labelling agents.

[0036] Provided herein is a kit comprising a solid support; a first buffer comprising a catalyst that catalyzes de-crosslinking of molecular crosslinks; a second buffer comprising Tris and a chelating agent; and a plurality of circularizable probes configured to bind a plurality of RNAs. In some embodiments, the kit comprises a plurality of labelling agents configured to bind a plurality of protein analytes. In some embodiments, the kit comprises a plurality of detectably labeled probes for binding to a sequence of a reporter oligonucleotide of the plurality of labelling agents.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

[0038] FIG. 1 depicts an example of a workflow including preparation and treatment (e.g., de-crosslinking) of biological samples for analyte detection.13MF-367036897202412025040

[0039] FIGS. 2A-2B show representative images of CD68 antibody staining in FFPE tonsil tissue samples and HER2 antibody staining on human breast cancer tissue samples, respectively, to compare conditions with and without performing a secondary treatment with TE buffer.

[0040] FIG. 3 shows the median transcripts detected per cell as a metric for assay sensitivity (left) and detected rolling circle amplification product (RCP) signal brightness (right) in samples to compare secondary treatments with TE buffer under various conditions (e.g., temperature and pH).

[0041] FIG. 4 shows the estimated brightness decay per cycle of detected RCPs (left) and detected genomic control signals (right) in samples to compare secondary treatments with TE buffer under various conditions (e.g., temperature and pH).DETAILED DESCRIPTION

[0042] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0043] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. OVERVIEW

[0044] Compositions and methods are needed for analyzing the spatial localization of analytes in a biological sample, such as various archived and preserved tissue materials comprising molecular crosslinks. Formalin / formaldehyde fixation is a dominant method for preserving tissue samples for pathology and life science research due to its robustness in maintaining tissue architecture at even ambient temperatures. However, some methods of analyte detection are not compatible with fixed tissues without specific sample preparation to clear crosslinking and render analytes accessible to biochemical reactions, including those amenable to 14MF-367036897202412025040signal detection and / or amplification. In some cases, optimizing a sample preparation workflow for RNA detection assays results in suboptimal treatment of the tissue sample for other analysis such as protein detection. For example, fixation process may result in masked antigens on proteins targeted for detection. In some aspects, a single de-crosslinking treatment using a catalytic de-crosslinker is not sufficient for antigen retrieval. In some cases, undesired tissue detachment from the solid support (e.g., glass slide) or reduction in sensitivity of the assay(s) may result from certain treatments during sample preparation. Improved methods for preparing biological samples are needed to enable effective detection and analysis of multiple analyte types.

[0045] In some embodiments, provided herein are compositions and methods that involve a treatment process (e.g., de-crosslinking and / or antigen retrieval) for preparing samples, such as FFPE cell and tissue samples. In some embodiments, a sample such as an otherwise inaccessible fixed or FFPE sample is treated with multiple and separate treatment (e.g., decrosslinking) steps to prepare the biological sample. For example, a biological sample is incubated with a first buffer comprising a catalyst or a precursor thereof followed by additional treatment with a second buffer to provide accessibility to target analyte molecules (e.g., proteins) in the sample for in situ analysis. In some embodiments, a sample such as an FFPE sample is deparaffinized. In some embodiments, the de-paraffinized sample is contacted with a first buffer comprising an effective concentration of a catalyst or a precursor thereof for a period of time. In some embodiments, the buffer facilitates and / or promote catalytic de-crosslinking by the catalyst. After the de-crosslinking incubation with the first buffer comprising the catalyst, the sample is contacted with a plurality of nucleic acid probes for binding to a plurality of target RNAs in the biological sample. In some instances, the plurality of nucleic acid probes are used in an amplification reaction to generate a plurality of amplification products in the biological sample. After introducing the nucleic acid probes to the biological sample, a secondary treatment (e.g., de-crosslinking and / or antigen retrieval) is performed by incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes. In some instances, the second buffer is a different buffer from the first buffer. After the secondary treatment, the sample is contacted with a labelling agent that directly or indirectly binds to an analyte in the biological sample (e.g., an analyte labelling agent). In some instances, the labelling agent is used 15MF-367036897202412025040to detect a non-nucleic acid analyte. In some instances, the labelling agent is used to detect an analyte that is a different type of analyte than those targeted by the plurality of nucleic acid probes. In some instances, the labelling agent binds to a protein analyte. In some instances, a plurality of labelling agents are provided to bind a plurality of protein analytes.

[0046] In some embodiments, provided herein are various catalysts and buffer compositions for sample treatment. In some embodiments, provided herein are methods involving catalytic de-crosslinking using any one or more of the compositions disclosed herein as a primary de-crosslinking step and a separate and additional incubation with a second buffer as a secondary treatment step. In some embodiments, provided herein are methods comprising one or more treatments for de-crosslinking and / or antigen retrieval and one or more posttreatment washes.

[0047] FIG. 1 shows an example of a workflow where a fixed sample immobilized on a substrate is provided in 101, and if the sample is paraffin-embedded, a de-paraffinization and re-hydration step is performed in 102 to prepare the sample for primary de-crosslinking with a catalyst in 103. After catalytic de-crosslinking in 103, the sample is contacted with a plurality of nucleic acid probes in 104 that directly or indirectly binds to a plurality of RNAs in the sample and incubated with regents for generating a RNA library (e.g., ligation of circularizable probes, amplification of circularized probes). Following RNA library preparation, the sample is baked in 105 and then incubated with a second buffer at 80°C or higher in 106. After treatment with the second buffer in 106, the sample is contacted with a labelling agent in 107 that directly or indirectly binds to a protein analyte followed by an optional wash and / or treatment with a fixative. In 108, signals associated with the plurality of nucleic acids or products thereof are detected in sequential cycles using detection reagents (e.g., detectably labeled probes) and a signal associated with the labelling agent are detected at locations in the biological sample.

[0048] Treatment of biological samples (e.g., tissue samples) with the distinct and separate primary treatment with the catalytic de-crosslinker and secondary treatment disclosed herein improve detection of signals associated with nucleic acid analytes and non-nucleic acid analytes (e.g., protein analytes) in a fixed biological sample immobilized on a substrate (for instance, as demonstrated in FIG. 2A, FIG. 2B, FIG. 3 and FIG. 4) while substantially maintaining or improving sample integrity and / or adhesion to the substrate as compared to 16MF-367036897202412025040sample treatment (e.g., de-crosslinking and / or antigen retrieval) without the incubation with the second buffer as described herein.II. SAMPLE TREATMENT AND ASSAY

[0049] In some embodiments, provided herein are methods and compositions for providing a fixed biological sample comprising cells and contacting the sample with a first buffer comprising a catalyst or a precursor thereof followed by an additional treatment with a second buffer to provide accessibility of target analyte molecules in the sample for in situ analysis. In some instances, the in situ assay comprises RNA detection and protein detection.

[0050] Provided herein is a method comprising contacting a biological sample comprising cells with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample; contacting the biological sample with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells; after contacting the sample with the plurality of nucleic acid probes, incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes; after incubation with the second buffer, contacting the biological sample with a labelling agent that directly or indirectly binds to an analyte at a location in the biological sample; and detecting a plurality of optical signals associated with the plurality of nucleic acid probes or products thereof associated with the plurality of RNAs in the biological sample and an optical signal associated with the labelling agent or a product thereof in the biological sample. In some embodiments, the analytes or products thereof remains in the biological sample during the contacting with the catalyst, during the incubation with the second buffer, and during detecting of the optical signals. In some instances, the analytes or products thereof substantially remains at the locations during the assay.

[0051] In some embodiments, the de-crosslinking catalyzed by the catalyst is performed separately from the de-crosslinking by incubating with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some embodiments, the de-crosslinking catalyzed by the catalyst is performed before incubation with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some embodiments, the de-crosslinking catalyzed by the catalyst is completed before incubation with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some embodiments, the catalyst is removed from the biological sample before incubation with the second buffer at a temperature of at least 80°C for at least 1017MF-367036897202412025040minutes. In some embodiments, the incubation with the second buffer at a temperature of at least 80°C for at least 10 minutes is performed after the plurality of nucleic acid probes are bound to the plurality of RNAs in the biological sample and before the labelling agents are contacted with the biological sample.

[0052] In some embodiments, the biological sample comprises an analyte endogenous to a biological sample. In some instances, the target analytes are cellular nucleic acid analytes and non-nucleic acid analytes. Methods, probes, and kits disclosed herein can be used to analyze nucleic acid analytes (e.g., using a nucleic acid probes that directly or indirectly hybridizes to a nucleic acid analyte) and / or non-nucleic acid analytes (e.g., using a labelling agent that comprises a reporter oligonucleotide and binds directly or indirectly to a non-nucleic acid analyte). The methods, probes, and kits disclosed herein can be used to detect and analyze a wide variety of different analytes. In some aspects, an analyte can include any biological substance, structure, moiety, or component to be analyzed. In some aspects, a target disclosed herein may similarly include any analyte of interest. In some examples, a target or analyte can be directly or indirectly detected.

[0053] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte is inside a cell or on a cell surface, such as a transmembrane analyte or one that is attached to the cell membrane. In some embodiments, the analyte can be an organelle (e.g., nuclei or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Examples of analytes include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation,18MF-367036897202412025040glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.

[0054] Examples of nucleic acid analytes include DNA analytes such as singlestranded DNA (ssDNA), double- stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. The DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample.

[0055] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), including a nascent RNA, a pre-mRNA, a primary-transcript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5’ 7-methyl guanosine cap), a polyadenylated mRNA (poly- A tail at the 3’ end), and a spliced mRNA in which one or more introns have been removed. Also included in the analytes disclosed herein are non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA) present in a tissue sample. Examples of a non-coding RNAs (ncRNA) that is not translated into a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body-specific RNAs (scaRNAs), and the long ncRNAs such as Xist and HOTAIR. The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single- stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0056] In some embodiments, an analyte comprises a denatured nucleic acid, wherein the resulting denatured nucleic acid is single- stranded. The nucleic acid may be denatured, for example, optionally using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured for use in a method disclosed herein.19MF-367036897202412025040

[0057] Methods, probes, and kits disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes.

[0058] Analytes can be derived from a specific type of cell and / or a specific sub-cellular region. For example, analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. The analyte may include any biomolecule, macromolecule, or chemical compound, including a protein or peptide, a lipid or a nucleic acid molecule, or a small molecule, including organic or inorganic molecules. The analyte may be a cell or a microorganism, including a virus, or a fragment or product thereof.

[0059] Analytes of interest may include nucleic acid molecules (e.g., cellular nucleic acids), such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g. mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and / or modified nucleic acid molecules, (e.g. including nucleic acid domains comprising or consisting of synthetic or modified nucleotides such as LNA, PNA, morpholino, etc.), proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which comprises a protein or polypeptide component, etc., or fragments thereof, or a lipid or carbohydrate molecule, or any molecule which comprise a lipid or carbohydrate component. In some embodiments, the analyte is a single molecule or a complex that contains two or more molecular subunits, e.g. including but not limited to protein-DNA complexes, which may or may not be covalently bound to one another, and which may be the same or different. Thus in addition to cells or microorganisms, such a complex analyte may also be a protein complex or protein interaction. Such a complex or interaction may thus be a homo- or hetero -multimer. Aggregates of molecules, e.g. proteins may also be target analytes, for example aggregates of the same protein or different proteins. The analyte may also be a complex between proteins or peptides and20MF-367036897202412025040nucleic acid molecules such as DNA or RNA, e.g. interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNA or RNA.A. Fixed Biological Samples

[0060] A biological sample disclosed herein can include any sample comprising a cell, a tissue, or a derivative of a cell or a tissue. In some embodiments, a biological sample herein includes a fixed cell or tissue sample comprising molecular crosslinks. The ability to use a fixed biological sample in an analytical method, such as in situ analysis of biological molecules (e.g., genomic DNA, RNA, cDNA, and / or proteins), is enhanced if the cross-links established during fixation of the biological sample are reversed so that an assay can be carried out before sample degradation occurs. In some aspects, data obtained from a de-crosslinked biological sample are similar to that obtained from a fresh sample (e.g., a sample that is not fixed and / or crosslinked). In some instances, the molecular crosslinks are products of one or more crosslinking agents. In some instances, the one or more crosslinking agents comprise an aldehyde. In some instances, the one or more crosslinking agents comprise formaldehyde. In some cases, the biological sample has been treated with aldehyde or formaldehyde. In some instances, the molecular crosslinks are on RNA, DNA, protein, carbohydrate, lipid, and / or other molecules in the biological sample.

[0061] A fixed biological sample can be any appropriate fixed biological sample. In some embodiments, a fixed biological sample is a fixed tissue sample (e.g., a fixed tissue section). In some embodiments, a sample herein is not and does not comprise a dissociated tissue / cell suspension. Molecules (e.g., analytes, labelling agents, nucleic acid probes, etc., or products generated in situ in the sample) may but do not need to be removed from a sample herein for analysis before, during, or after de-crosslinking of the sample. In some embodiments, molecules (e.g., analytes, labelling agents, nucleic acid probes, etc.) are not removed from a sample herein for analysis. In some embodiments, signals associated with the molecules (e.g., analytes, labelling agents, nucleic acid probes, etc., or products generated in situ in the sample) are detected at multiple locations in the de-crosslinked sample (e.g., treated with the catalyst in the first buffer and separately incubated with the second buffer at a temperature of at least 80°C for at least 10 minutes). In some instances, the signals are detected in situ in the de-crosslinked tissue section.21MF-367036897202412025040

[0062] In some embodiments, the method does not comprise migrating molecules (e.g., analytes, labelling agents, nucleic acid probes, etc., or products generated in situ in the sample) outside of the permeabilized biological sample. In some embodiments, the method does not comprise migrating molecules (e.g., analytes, labelling agents, nucleic acid probes, etc., or products generated in situ in the sample) towards the substrate, optionally wherein the migration is passive migration or active migration. In some embodiments, the method does not comprise capturing molecules (e.g., analytes, labelling agents, nucleic acid probes, etc., or products generated in situ in the sample) by a capture agent immobilized on the substrate.

[0063] In some embodiments, the biological sample is fixed and the fixation comprises contacting the sample with one or more agents that react with one another and / or with molecules in the biological sample. In some embodiments, the reaction creates molecular crosslinks between molecules of the one or more agents, between molecules in the biological sample, and / or between molecules of the one or more agents and molecules in the biological sample. In some embodiments, the one or more agents are crosslinking agents, and the molecular crosslinks are products of one or more reactions between a crosslinking agent and a molecule in the biological sample.

[0064] In some embodiments, a biological sample is fixed using one or more crosslinking agents comprising an aldehyde. In some embodiments, an aldehyde includes a compound containing one or more aldehyde (-CHO) groups, where the aldehyde groups are capable of reacting with an amine (e.g., a primary amine, a secondary amine, or a tertiary amine) or with an amide. Amines are derivatives of ammonia, wherein one or more hydrogen atoms in amines have been replaced by a substituent such as an alkyl or aryl group. These may respectively be called alkylamines and arylamines, and amines in which both types of substituent are attached to one nitrogen atom may be called alkylarylamines. Examples of amines include amino acids (including amino acid residues of a protein having side chains that can react with an aldehyde), biogenic amines, trimethylamine, and aniline. In some embodiments, molecular crosslinks in a fixed sample are formed via condensation between an aldehyde and an amine, and in some aspects, the condensation does not require heating and / or an acidic condition. Amides having the structure R-CO-NR'R" in which a nitrogen atom is attached to a carbonyl group. In some embodiments, molecular crosslinks in a fixed sample are formed via condensation between 22MF-367036897202412025040an aldehyde and an amide, e.g., under heating and / or acidic conditions. Examples of aldehydes can include formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, and the like.

[0065] In some embodiments, fixing a biological sample comprises treating the sample with a crosslinking agent. In some embodiments, the crosslinking agent comprises formaldehyde. Paraformaldehyde (PFA) is a polymer of formaldehyde. While paraformaldehyde itself is not a fixing agent, it can be heated and / or treated under basic conditions until it becomes solubilized and broken down to formaldehyde molecules.

[0066] In some embodiments, the molecular crosslinks are on RNA, DNA, protein, carbohydrate, lipid, and / or other molecules in the biological sample. In some embodiments, the molecular crosslinks comprise one or more aminal crosslinks such as aminal bridges. In some embodiments, a fixed biological sample can comprise aminal crosslinks among nucleic acids (e.g., genomic DNA, RNA such as mRNA, and / or cDNA), proteins, carbohydrates, lipids, and / or other molecules in the biological sample. Aminal crosslinks can be made, for example, by fixing a sample with formaldehyde.

[0067] In some embodiments, the fixative or fixation agent is formaldehyde.Formaldehyde as fixative comprises paraformaldehyde (or “PFA”) and formalin, both of which relate to the formaldehyde composition (e.g., formalin is a mixture of formaldehyde and methanol). Thus, a formaldehyde-fixed biological sample may be formalin-fixed or PFA-fixed. In some embodiments, a biological sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample (e.g., an FFPE tissue section).

[0068] In some embodiments, an aldehyde fixation method is combined with other tissue preservation methods. For example, aldehyde fixation can be combined with fresh frozen preservation of tissues, e.g., fresh frozen tissues can be fixed using an aldehyde. Aldehyde fixation can also be combined with alcohol fixation, or with any number of commercially available fixation / preservation techniques. For example, aldehyde fixation can be combined with salt-rich buffer solutions such as RNAlater™, low-temperature preservation buffers such as HypoThermosol, alcohol- PEG fixation (e.g., Neo-Rix, STATFIX, PAGA, UMFIX), PAXGene, Allprotect / Xprotect, CellCover, RN Assist, and / or zinc buffers.

[0069] In some embodiments, preparing fixed (e.g., aldehyde-fixed) biological samples for in situ analysis comprises de-crosslinking disclosed herein in combination with 23MF-367036897202412025040additional sample processing steps and / or conditions before, during, and / or after each of the decrosslinking steps.

[0070] In some embodiments, provided herein are methods of de-crosslinking aminal crosslinks in a fixed biological sample. In some embodiments, provided herein are methods of in situ analysis using such a de-crosslinked sample. The methods described herein are not limited to any particular fixation reagent that results in crosslinks (e.g., aminal crosslinks) and are equally amenable with any fixation method that results in intra-tissue crosslinking events (e.g., aminal intra tissue crosslinking events). In some aspects, molecular crosslinks from fixation could lead to antigen masking and / or background autofluorescence in the sample. For example, PFA induced crosslinks are known to be responsible for increased autofluorescence in FFPE tissues. In some cases, molecular crosslinks block or restrict biochemical reactions such as nucleic acid hybridization or methods of signal amplification utilized for analyte detection. Conventional methods for antigen retrieval may not sufficiently retrieve the masked antigens. The process (e.g., de-crosslinking and / or antigen retrieval) methods disclosed herein with the distinct and separate primary and secondary treatments address these and other issues with conventional methods.B. Preparing Samples

[0071] In some instances, the biological sample is immobilized on a substrate before, during, and / or after the de-crosslinking treatments disclosed herein. In some embodiments, the biological sample is immobilized on the substrate before the de-crosslinking treatments disclosed herein. In some embodiments, the biological sample is immobilized on the substrate before contacting with the catalyst. In some embodiments, the biological sample remains immobilized on the substrate during and after contacting with the catalyst. In some embodiments, the biological sample is immobilized on the substrate after contacting with the catalyst and during incubation with the second buffer (e.g., TE incubation at 80°C or higher for at least 10 minutes). In some embodiments, the biological sample is immobilized on the substrate during contacting with the catalyst and during contacting with the catalyst and during incubation with the second buffer (e.g., TE incubation at 80°C or higher for at least 10 minutes). In some embodiments, a biological sample is provided in a fixed state. In some embodiments, a fixed biological sample undergoes one or more preparation steps before it is further treated and / or de-crosslinked.24MF-367036897202412025040

[0072] In some embodiments, the substrate comprises a planar surface configured to contact the biological sample and does not comprises a bead, particle, or microwell. In some instances, the substate provides support to a biological sample, particularly, for example, a thin tissue section. Accordingly, a “substrate” is a support that is insoluble in aqueous liquid and which allows for positioning of biological samples, on the substrate. In general, a substrate can be any suitable support material. Exemplary substrates include, but are not limited to, glass, modified and / or functionalized glass, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene and polycarbonate.

[0073] In some embodiments, the substrate is a glass slide or a plastic slide. In some embodiments, the substrate is transparent. In some embodiments, the substrate is suitable for imaging using fluorescent microscopy, for instance, for in situ analyte detection, e.g., in situ sequencing or in situ sequential hybridization. In some embodiments, the substrate does not comprises nucleic acid immobilized thereon prior to contacting the biological sample. In some embodiments, the biological sample is a tissue section. In some embodiments, the biological sample comprises a plurality of cells in a tissue sample. In some embodiments, the biological sample comprise cells immobilized on the substrate. In some embodiments, the cells are dissociated cells, cultured cells, and / or cells isolated from a subject. In some embodiments, the biological sample is an aldehyde-fixed biological sample. In some embodiments, the biological sample is a formaldehyde-fixed biological sample. In some embodiments, the biological sample is a paraffinized biological sample. In some embodiments, the biological sample is a formaldehyde-fixed paraffin-embedded (FFPE) biological sample. In some embodiments, the biological sample is a fresh frozen biological sample that has been crosslinked.

[0074] In some embodiments, prior to contacting the biological sample with the catalyst, the method comprises a step of pre-warming the biological sample. In some embodiments, a fixed biological sample (e.g., an FFPE tissue section) is pre-warmed to between 25MF-367036897202412025040about 20°C and about 60°C, e.g., about 30°C to about 50°C, about 35°C to about 45°C, or about 40°C to about 43°C. In some embodiments, the fixed biological sample is pre-warmed by incubation in a water bath. In some embodiments, the fixed biological sample is a block of embedded tissue (e.g., formalin fixed and paraffin embedded) that is sliced using a microtome to generate embedded tissue sections, e.g., about 5 pm in thickness. In some embodiments, the microtome is pre- warmed to between about 40 °C and about 43 °C for slicing the fixed biological sample.

[0075] In some embodiments, prior to de -crosslinking (e.g., treating the sample with the catalytic de-crosslinker ), the method comprises dehydrating the biological sample. In some such embodiments, the fixed biological is dehydrated by drying at a temperature higher than room temperature, e.g., at about 20°C to about 60°C, about 30°C to about 50°C, about 35°C to about 45°C, or about 40°C to about 43°C, such as at about 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C, for a period of time (e.g., about 30 minutes to about 6 hours, about 1 hour to about 5 hours, about 2 hours to about 4 hours, or about 3 hours). In some such embodiments, the fixed biological is dried at room temperature for a period of time (e.g., about 2 hours to about 24 hours, about 5 hours to about 20 hours, about 8 hours to about 16 hours, or overnight), for example, in a desiccator. In some such embodiments, the fixed biological sample is dried at a temperature higher than room temperature, followed by drying at room temperature.

[0076] In some such embodiments, for paraffin-embedded biological samples (e.g., FFPE samples), the sample is de-paraffinized (e.g., to produce a de-paraffinized fixed biological sample) and re-hydrated. In some embodiments, de-paraffinizing comprises contacting the biological sample with xylene, ethanol, and water, or, sequentially contacting the biological sample with xylene and an alcohol (e.g.. ethanol) series such as absolute ethanol, about 96% ethanol, and about 70% ethanol. In some embodiments, de-paraffinizing comprises treating with xylene and ethanol (e.g., absolute ethanol, about 96% ethanol, and or about 70% ethanol). In some embodiments, de-paraffinization comprises, sequentially, treating with xylene (e.g., once, twice, or more times, each for about 5 minutes to about 15 minutes, such as about 10 minutes each), treating with absolute ethanol (e.g., once, twice, or more times, each for about 1 minute to about 10 minutes, such as about 2 minute to about 5 minutes, e.g., about 3 minutes each), treating with about 96% ethanol (e.g., once, twice, or more times, each for about 1 minute to 26MF-367036897202412025040about 10 minutes, such as about 2 minute to about 5 minutes, e.g., about 3 minutes each), and treating with about 70% ethanol (e.g., once, twice, or more times, each for about 1 minute to about 10 minutes, such as about 2 minute to about 5 minutes, e.g., about 3 minutes each). In some embodiments, the sample is treated with water for re-hydration (e.g., in nuclease free water), e.g., once, twice, or more times, each for about 5 seconds to about 1 minute, such as 10 seconds to about 30 seconds, e.g., about 20 seconds each.

[0077] In some embodiments, a fixed biological sample is pretreated with a proteinase prior to delivery or application of a de-crosslinking agent (e.g., a catalyst disclosed herein). For example, a fixed biological sample is pretreated with a proteinase at about 0.005 to about 0.5 U / pL (e.g., about 0.01 to about 0.5 U / pL, about 0.05 to about 0.5 U / pL, about 0.1 to about 0.5 U / pL, about 0.1 to about 0.3 U / pL, or about 0.2 U / pL). In some embodiments, a proteinase comprises pepsin, Proteinase K, or an ArcticZymes Proteinase (an unspecific endopeptidase that can be inactivated after use). The proteinase can optionally be applied with a buffer, such as Hank’s Balanced Salt Solution (HBSS) buffer. In some embodiments, if pepsin is used for permeabilization, a pretreating reagent can include a proteinase (e.g., a second proteinase or a proteinase other than pepsin). In some embodiments, if Proteinase K is used for permeabilization, a pretreating reagent may but does not need to include a proteinase. In some instances, the proteinase is a collagenase.

[0078] In some embodiments, a fixed biological sample is pretreated with a detergent. For example, a fixed biological sample is pretreated with a detergent at about 0.05% to about 2% (v / v), about 0.1% to about 1% (v / v), about 0.1% (v / v), or about 0.5% (v / v)). In some embodiments, the detergent is a non-ionic detergent. In some embodiments, the detergent comprises TRITON™ X-100. In some embodiments, the detergent is in a buffer. In some embodiments, the buffer comprises, for example, tris(hydroxymethyl)aminomethane-Ethylenediaminetetraacetic acid (TE), phosphate-buffered saline (PBS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and / or 2-(N-morpholino)ethanesulfonic acid (MES), with a pH of about 7.0 to about 9.0 (e.g., about 7.5 to about 8.5, or about 8.0).

[0079] In some embodiments, a fixed biological sample is pretreated with a detergent and / or a proteinase for between about 1 minute and about 60 minutes. In some embodiments, a 27MF-367036897202412025040fixed biological sample is pretreated with a detergent and / or a proteinase for between about 1 minute and about 55 minutes, about 1 minute and about 50 minutes, about 1 minute and about 45 minutes, about 1 minute and about 40 minutes, about 1 minute and about 35 minutes, about 1 minute and about 30 minutes, about 1 minute and about 25 minutes, about 1 minute and about 20 minutes, about 5 minutes and about 60 minutes, about 10 minutes and about 60 minutes, about 10 minutes and about 50 minutes, about 10 minutes and about 40 minutes, or about 10 minutes and about 30 minutes. In some embodiments, a fixed biological sample is pretreated with a detergent and / or a proteinase for about 20 minutes.

[0080] In some embodiments, a fixed biological sample is pretreated with a detergent and / or a proteinase at a temperature of about 30°C and about 45°C during pretreatment. In some embodiments, a fixed biological sample is pretreated with a detergent and / or a proteinase at a temperature of about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C during pretreatment. In some embodiments, a fixed biological sample is pretreated with a detergent and / or a proteinase at a temperature of about 37°C.

[0081] In some embodiments, a de-paraffinized and re-hydrated biological is not pretreated by permeabilizing the sample (e.g., using a protease or detergent) prior to contacting the sample with a de-crosslinking agent. In some embodiments, de-paraffinization and / or rehydration is performed on a fixed biological prior to delivery or application of a de-crosslinking agent (e.g., a catalyst provided in a first buffer).C. Catalytic De-crosslinking in a First Buffer

[0082] In some embodiments, a biological sample is contacted with a catalyst, wherein catalyst is a de-crosslinking agent or un-fixing agent. In some aspects, the catalyst reverses fixation and / or removes the crosslinks within or between biomolecules (e.g., analytes for analytical methods, such as those described herein) in a sample caused by previous use of a fixation reagent. In some embodiments, the catalyst is provided in a first buffer. In some embodiments, de-crosslinking agents are compounds that act catalytically in removing crosslinks in a fixed sample. In some embodiments, de-crosslinking agents are compounds that act catalytically in removing aminal crosslinks in a fixed sample. In some embodiments, decrosslinking agents can act on biological samples fixed with an aldehyde (e.g., formaldehyde), an N-hydroxy succinimide (NHS) ester, an imidoester, or a combination thereof. In some28MF-367036897202412025040embodiments, provided herein are catalysts that catalyze de-crosslinking of inter-molecular crosslinks and / or intra-molecular crosslinks in the biological sample. In some embodiments, provided herein are catalysts that catalyze the cleavage of aminal bridges, thereby decrosslinking the inter-molecular crosslinks and / or intra-molecular crosslinks.

[0083] In some embodiments, the catalyst is a water-soluble catalyst. In some embodiments, the catalyst is an organic molecule. In some embodiments, the catalyst is a transimination catalyst. In some embodiments, the catalyst is a bifunctional transimination catalyst that accelerates hydrazone and oxime formation. In some embodiments, the catalyst catalyzes de-crosslinking of aminal crosslinks in the biological sample. In some embodiments, the catalyst catalyzes breakdown of hemi- aminal adducts and / or aminal adducts in the biological sample. Aminal crosslinks (e.g., aminal bridges) can be catalytically reversed using one or more organocatalyst. In some embodiments, in catalytic reversal of aminal crosslinks, a first C-N bond of the aminal bridge can be broken in an acid-base reaction, and the second C-N bond of the aminal can be broken to generate repaired NH2 groups on the first and second molecules.

[0084] In some embodiments, the catalyst is a compound of formula (I),'2X4■2(I),or a salt, zwitterion, or solvate thereof, wherein:A is selected from the group consisting of -COOH, -P(=O)(OH)2, and -S(=O)2OH;X1, X2, X3, and X4are each independently selected from the group consisting of: CH, CRa, and N;each occurrence of Rais independently selected from the group consisting of C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, -NO2, -NR’R”, and -C(=O)NR’R”; and29MF-367036897202412025040each occurrence of R’ and R” is independently selected from the group consisting of H and Ci-6O'alkyl which is optionally substituted withn1, wherein nl is an integer from 12 to 16.

[0085] In some embodiments of formula (I), it is provided that when A is -P(=O)(OH)2 and X1, X2, and X4are CH, then X3is other than C-CH3.

[0086] In some embodiments of formula (I), A is -COOH. In some embodiments of formula (I), A is -P(=O)(OH)2. In some embodiments of formula (I), A is -S(=O)2OH.

[0087] In some embodiments of Formula (I), X1is CH. In some embodiments of Formula (I), X1is CRa. In certain of these embodiments, X1is C-CH3. In some embodiments of Formula (I), X2is CH. In some embodiments of Formula (I), X2is N. In some embodiments of Formula (I), X4is CH. In some embodiments of Formula (I), X4is N.

[0088] In some embodiments of Formula (I), X3is N. In some embodiments of Formula (I), X3is CH. In some embodiments of Formula (I), X3is CRa. In certain of these embodiments, Rais C1-6 alkyl (e.g., methyl). In certain embodiments, Rais NO2. In certain embodiments, Rais NR’R” (e.g., NH2). In certain embodiments, Rais C(=O)NR’R”. As a non¬limiting example of the foregoing embodiments, Rais

[0089] In some embodiments of Formula (I), X2and X4are CH. In some embodiments of Formula (I), X1, X2, and X4are CH. In certain of these embodiments, X3is CRa(e.g., C-CH3). In certain other embodiments, X3is N. In certain of the foregoing embodiments (when X2and X4are CH; or when X1, X2, and X4are CH), A is -COOH or - P(=O)(OH)2.

[0090] In some embodiments, the compound of Formula (I) is a compound of Formula (IA):>2Ra(IA),30MF-367036897202412025040or a salt, zwitterion, or solvate thereof.

[0091] In some embodiments of Formula (IA), A is -COOH. In some of these embodiments of Formula (IA), Rais Ci-6 alkyl. In certain of these embodiments, Rais C1-3 alkyl. For example, in some embodiments, Rais methyl. In other of these embodiments, Rais methoxy. In other of these embodiments, Rais -NH2. In other of these embodiments, Rais -N(CH3)2.

[0092] In some embodiments of Formula (IA), A is -P(=O)(OH)2. In some of these embodiments of Formula (IA), Rais C1-6 alkyl. In certain of these embodiments, Rais C1-3 alkyl. For example, in some embodiments, Rais methyl. In other of these embodiments, Rais methoxy. In other of these embodiments, Rais -NH2. In other of these embodiments, Rais -N(CH3)2.

[0093] In some embodiments of Formula (IA), A is -S(=O)2OH. In some of these embodiments of Formula (IA), Rais C1-6 alkyl. In certain of these embodiments, Rais C1-3 alkyl. For example, in some embodiments, Rais methyl. In other of these embodiments, Rais methoxy. In other of these embodiments, Rais -NH2. In other of these embodiments, Rais -N(CH3)2.

[0094] In some embodiments, the compound of Formula (I) is a compound of Formula (IB):l2or a salt, zwitterion, or solvate thereof, wherein: X3is CH or N.

[0095] In some embodiments of Formula (IB), A is -P(=O)(OH)2. In some embodiments of Formula (IB), X3is N.

[0096] In some embodiments, the compound of Formula (I) is a compound of Formula (IC):31MF-367036897202412025040NH2ARC(io.or a salt, zwitterion, or solvate thereof, wherein Rcis an electron releasing group. In some of these embodiments, the electron releasing group (Rc) is selected from the group consisting of alkyl, alkoxy, hydroxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, silyloxy, aryloxy, and alkylthio. In some of these embodiments, the electron releasing group is lower alkyl or lower alkoxy. In other of these embodiments, the electron releasing group is -NH2. In still other of these embodiments, the electron releasing group is -N(CH₃)₂.

[0097] In some embodiments, the compound of Formula (I) is a compound of Formula (IC’):or a salt, zwitterion, or solvate thereof, wherein Rcis an electron releasing group. In some of these embodiments, the electron releasing group (Rc) is selected from the group consisting of alkyl, alkoxy, hydroxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, silyloxy, aryloxy, and alkylthio. In some of these embodiments, the electron releasing group is lower alkyl or lower alkoxy. In other of these embodiments, the electron releasing group is -NH2. In still other of these embodiments, the electron releasing group is -N(CH₃)₂.

[0098] In some embodiments, the sample is contacted with a compound (e.g., in a solution or suspension) for catalytic de-crosslinking selected from the group consisting of 2-amino-5-methylbenzoic acid, 2-amino-5-nitrobenzoic acid, (2-amino-5-methylphenyl)phosphonic acid, 2-amino-5-methylbenzenesulfonic acid, 2,5-diaminobenzenesulfonic acid, 2-amino-3,5-dimethylbenzenesulfonic acid, (2-amino-5-nitrophenyl)phosphonic acid, (4-aminopyridin-3-yl)phosphonic acid, (3-aminopyridin-2-yl)phosphonic acid, (5-aminopyrimidin-4-yl)phosphonic acid, (2-amino-5-{[2-poly- 32MF-367036897202412025040ethoxy]ethyl}carbamoyl)phenyl)phosphonic acid, 4-aminonicotinic acid, 3-aminoisonicotinic acid, 2-aminonicotinic acid, and (2-aminophenyl)phosphonic acid. In some embodiments, the sample is contacted with Compound 1 (2-amino-5-methylbenzoic acid) in a solution or suspension for catalytic de-crosslinking. In some embodiments, the sample is contacted with Compound 8 ((4-aminopyridin-3-yl)phosphonic acid) in a solution or suspension for catalytic decrosslinking. In some embodiments, the sample is contacted with Compound 15 ((2-aminophenyl)phosphonic acid) in a solution or suspension for catalytic de-crosslinking.

[0099] In some embodiments, the catalyst of formula (I) is selected from the group consisting of:Compound No. Compound Structure102■ 1 1NH023Ix:>' HbCA. OH41 JbP. OH5I, XNH0233MF-367036897202412025040NH-, O it P~OH NO2HOH2NHO10NH20 X,,^-OH HNU:n 034MF-367036897202412025040A Z~12 / / ( D->—z\ / Q _ Z / / HO \ / _ / / \ OO O -xC.O O '13 XxH2IXTHOOCX14H2NX1516oy0HT'OH171or a salt, zwitterion, or solvate thereof.

[0100] In some embodiments, the catalyst comprises or a salt, zwitterion, or solvate thereof. In some embodiments, the catalyst comprises35MF-367036897202412025040, or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises, or a salt, zwitterion, or solvate thereof. In someembodiments, the catalyst comprises, or a salt, zwitterion, or solvate thereof.In some embodiments, the catalyst comprises, or a salt, zwitterion, orsolvate thereof. In some embodiments, the catalyst comprisesNH-. 0!4y JohX zwitterion, or solvate thereof. In some embodiments, the catalyst comprises2, or a salt, zwitterion, or solvate thereof. In some embodiments, the catalyst comprises0HO A.HO' J, or a salt, zwitterion, or solvate thereof. In some embodiments, the catalyst36MF-367036897202412025040NH20X, R“OHx J °HHN^'Ocomprises', or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises, or a salt, zwitterion, or solvate thereof. In some HOOC|_| KIembodiments, the catalyst comprises2, or a salt, zwitterion, or solvate thereof.H NIn some embodiments, the catalyst comprises2, or a salt, zwitterion, or solvatethereof. In some embodiments, the catalyst comprises, or a salt, zwitterion, or O.p'OH7" OH ^kxNH2solvate thereof. In some embodiments, the catalyst comprisesO, or a salt,37MF-367036897202412025040zwitterion, or solvate thereof. In some embodiments, the catalyst comprisesa salt, zwitterion, or solvate thereof.

[0101] In some embodiments, a compound disclosed herein catalytically breaks down the aminal and hemi-aminal adducts that form in RNA treated with formaldehyde, and is compatible with many RNA extraction and detection conditions. Examples of compounds include those described in Karmakar et al., “Organocatalytic removal of formaldehyde adducts from RNA and DNA bases,” Nature Chemistry, 7: 752-758 (2015); US 2017 / 0283860; and US 2019 / 0135774, each of which is incorporated by reference herein in its entirety.

[0102] In some embodiments, the catalyst is a compound of formula (II):or a salt, zwitterion, or solvate thereof, wherein:L1is selected from the group consisting of -O-, -N(H)-, -N(CI-3 alkyl), -N(CH2CH2O)1-10-CH3-, -S(0)o-2-, -CH2-, and a bond;R1is selected from the group consisting of: H; C1-6 alkyl; C1-6 haloalkyl; C6-10 aryl optionally substituted with 1-4 Rb; and 5- to 10-membered heteroaryl, wherein 1-4 ring atoms are heteroatoms each independently selected from the group consisting of: N, N(H), N(CI-3 alkyl), O, and S, wherein the heteroaryl is optionally substituted with 1-4 independently selected Rb; andeach Rbis independently selected from the group consisting of: halo, cyano, -OH, -NH2, -NH(Ci-3 alkyl), -N(CI-3 alkyl)2, C1-6 alkyl, C1-6 haloalkyl, C1-4 alkoxy, and C1-4 haloalkoxy.

[0103] In some embodiments of formula (II), -L'-R1and the -COOH group are cis to one another. In some embodiments of formula (II), -L'-R1and the -COOH group are trans to one another.38MF-367036897202412025040

[0104] In some embodiments of formula (II), the catalyst is a compound of formula (II-a):,0N OHH (II-a),or a salt, zwitterion, or solvate thereof.

[0105] In some embodiments of formula (II), the catalyst is a compound of formula (II-al):. L1R1NH (II-al),or a salt, zwitterion, or solvate thereof.

[0106] In some embodiments of formula (II), the catalyst is a compound of formula (II- a2):,0R1NH (II-a2),or a salt, zwitterion, or solvate thereof.

[0107] In some embodiments of formula (II), the catalyst is a compound of formula (Il-b):A1- R1N OH(II-b).or a salt, zwitterion, or solvate thereof.

[0108] In some embodiments of formula (II), the catalyst is a compound of formula (Il-bl):Al,0R1N OHH (Il-bl),or a salt, zwitterion, or solvate thereof.39MF-367036897202412025040

[0109] In some embodiments of formula (II), the catalyst is a compound of formula (II-b2):or a salt, zwitterion, or solvate thereof.

[0110] In some embodiments of formula (II), (II-a), (II-a1), (II-a2), (II-b), (II-b1), or (II-b2), L¹ is -O-. In some embodiments of formula (II), (II-a), (II-a1), (II-a2), (II-b), (II-b1), or (II-b2), L¹ is -N(H)- or -N(C₁₋₃ alkyl)-. In certain of these embodiments, L¹ is -N(H)-.

[0111] In some embodiments of formula (II), (Il-a), (Il-al), (II-a2), (Il-b), (Il-bl), or (II-b2), R1is H.

[0112] In some embodiments of formula (II), (Il-a), (Il-al), (II-a2), (Il-b), (Il-bl), or (II-b2), R1is a heteroaryl containing 5-10 ring atoms, wherein 1-4 ring atoms are heteroatoms each independently selected from the group consisting of: N, N(H), N(CI-3 alkyl), O, and S; and wherein the heteroaryl is optionally substituted with 1-4 independently selected Rb.

[0113] In certain of these embodiments, R1is a heteroaryl containing 5-6 ring atoms, wherein 1-4 ring atoms are heteroatoms each independently selected from the group consisting of: N, N(H), N(C₁₋₃ alkyl), O, and S; and wherein the heteroaryl is optionally substituted with 1-2 independently selected Rᵇ.

[0114] In certain of the foregoing embodiments, R1is a heteroaryl containing 6 ring atoms, wherein 1-2 ring atoms are ring nitrogen atoms, and wherein the heteroaryl is optionally substituted with 1-2 independently selected Rb.

[0115] As a non-limiting example of the foregoing embodiments, R1can be pyridyl, which is optionally substituted with 1-2 independently selected Rb. For example, R1can be 3-pyridyl, which is optionally substituted with 1-2 independently selected Rb(e.g., unsubstituted 3-pyridyl, 3-pyridyl substituted with one Rb, or 3-pyridyl substituted with two Rb). As another non-limiting example, R1can be 4-pyridyl which is optionally substituted with 1-2 Rb(e.g., unsubstituted 4-pyridyl, 4-pyridyl substituted with one Rb, or 4-pyridyl substituted with two Rb).

[0116] In some embodiments of formula (II), the catalyst is a compound of formula (II-a1); L¹ is -O-; and R¹ is heteroaryl containing 6 ring atoms, wherein 1-2 ring atoms are ring40MF-367036897202412025040nitrogen atoms, and wherein the heteroaryl is optionally substituted with 1-2 independently selected Rb. In certain of these embodiments, R1is pyridyl which is optionally substituted with 1-2 independently selected Rb. For example, R1can be 3-pyridyl which is optionally substituted with 1-2 independently selected Rb(e.g., unsubstituted 3-pyridyl). As another non-limiting example, R1can be 4-pyridyl which is optionally substituted with 1-2 Rb(e.g., unsubstituted 4-pyridyl).

[0117] In some embodiments of formula (II), the catalyst is a compound of formula (Il-al); L1is -O-, -N(H)-, or -N(C1-3 alkyl)-; and R1is H.

[0118] In some embodiments, the catalyst is selected from the group consisting of (2S,4R)-4-hydroxyproline, (2R,4S)-4-hydroxyproline, (2S,4S)-4-hydroxyproline, (2R,4R)-4-hydroxyproline, (2S,4R)-4-aminoproline, (2R,4S)-4-aminoproline, (2S,4S)-4-aminoproline, and (2R,4R)-4-aminoproline.

[0119] In some embodiments, the catalyst of formula (II) is selected from the group consisting of:41MF-367036897202412025040Compound No. Compound Structure HO,18 / \ OH H o HO19U vOHH I HO20 O _0HH O HQ,21 O„. z0HH 0 H~N22 / \.. OH H 023H X.*N A24H O42MF-36703689720241202504025 / \,0HN H OP26 L / AN OHH27N OHHor a salt, zwitterion, or solvate thereof.

[0120] In some embodiments, the catalyst compriseszwitterion, or solvate thereof. In some embodiments, the catalyst comprisesOH, or a salt, zwitterion, or solvate thereof. In some embodiments, the HOcatalyst comprises, or a salt, zwitterion, or solvate thereof. In some HQ,. OHembodiments, the catalyst comprises, or a salt, zwitterion, or solvate43MF-367036897202412025040z X x) X 0£.:< o Xt>. _ / \^ O'e f Iz^jy°ther o. n some embodiments, the catalyst comprises H, or a salt, / \ / %zwitterion, or solvate thereof. In some embodiments, the catalyst c o / omprisesH, or a salt, zwitterion, or solvate thereof.

[0121] In some embodiments, the catalyst is selected from the group consisting of:Compound No. Compound Structure10O2N.V / 0 JL,221 T()H0u3 X W ‘ 'OH|| J0H4. OHH -> N,, S' '5'~S' ' NH244MF-367036897202412025040NH-, O it P~OH NO2HOH2NHO10NH20 X,,^-OH HNU:n 045MF-367036897202412025040I CX A Z~ 12 / / \ / Q _ Z / / HO \ OO ° - o C.13 XH2I\T HOOCX14H2NXO*?'OH 1516o.p'OHT'OH 171HQ.18H O46MF-367036897202412025040HO19 / \ OH Sr 'Y H 0 HO20HN' \\ H o HQ21 O„, z0HH 0 H2N22 / \ OHH, N- 23 / .. OHXNZH 0 H2M^24H 0 l-'UH<f— <25 / V zOH H 047MF-36703689720241202504026Ao127or a salt, zwitterion, or solvate thereof.

[0122] In some embodiments, a catalyst is contacted with (e.g., applied to) a biological sample in a solution or suspension with a concentration of about 5 mM to about 500 mM (e.g., about 10 mM to about 100 mM, about 10 mM to about 200 mM, about 10 mM to about 300 mM, about 10 mM to about 400 mM, about 100 mM to about 200 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 100 mM to about 500 mM, about 200 omM to about 300 mM, about 200 mM to about 400 mM, about 200 mM to about 500 mM, about p300 mM to about 400 mM, about 300 mM to about 500 mM, or about 400 mM to about 500 mM). In some embodiments, a catalyst is contacted with (e.g., applied to) a biological sample in a solution or suspension with a concentration of about 10 mM to about 100 mM (e.g., about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 40 mM, cr about 10 ImM to about 50 mM, about 10 mM to about 60 mM, about 10 mM to about 70 mM, about 10 mM to about 80 mM, about 10 mM to about 90 mM, about 20 mM to about 30 mM, about 20 mM to about 40 mM, about 20 mM to about 50 mM, about 20 mM to about 60 mM, about 20 mM to about 70 mM, about 20 mM to about 80 mM, about 20 mM to about 90 mM, about 20 mM to about 100 mM, about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 30 mM to about 80 mM, about 30 mM to about 90 mM, about 30 mM to about 100 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM, about 40 mM to about 80 mM, about 40 mM to about 90 mM, about 40 mM to about 100 mM, about 50 mM to about 60 mM, about 50 mM to about 70 mM, about 50 mM to about 80 mM, about 50 mM to about 90 mM, about 50 mM to about 100 mM, about 60 mM to about 70 mM, about 60 mM to about 80 mM, about 60 mM to about 90 mM, about 60 mM to about 100 mM, about 70 mM to about 80 mM, about 7048MF-367036897202412025040mM to about 90 mM, about 70 mM to about 100 mM, about 80 mM to about 90 mM, about 80 mM to about 100 mM, or about 90 mM to about 100 mM) of the catalyst. In some embodiments, a catalyst is contacted with (e.g., applied to) a biological sample in a solution or suspension with a concentration of about 30 mM to about 70 mM of the catalyst. In some embodiments, a catalyst is contacted with (e.g., applied to) a biological sample in a solution or suspension with a concentration of about 40 mM to about 60 mM of the catalyst. In some embodiments, a catalyst is contacted with (e.g., applied to) a biological sample in a solution or suspension with a concentration of about 50 mM of the catalyst. In some embodiments, a catalyst is contacted with (e.g., applied to) a biological sample in a solution or suspension with a concentration of about 150 mM of the catalyst.

[0123] In some embodiments, the catalyst is contacted with the biological sample at a concentration between about 5 mM and about 500 mM. In some embodiments, the catalyst is contacted with the biological sample at a concentration between about 10 mM and about 400 mM. In some embodiments, the catalyst is contacted with the biological sample at a concentration between about 50 mM and about 300 mM. In some embodiments, the catalyst is contacted with the biological sample at a concentration between about 75 mM and about 250 mM. In some embodiments, the catalyst is contacted with the biological sample at a concentration between about 100 mM and about 200 mM, such as about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

[0124] In some embodiments, the catalyst is contacted with the biological sample for between 1 minute and 1 day (e.g., between 1 minute and 1 hour, 1 minute and 2 hours, 1 minute and 4 hours, 1 minute and 6 hours, 1 minute and 12 hours, 1 minute and 18 hours, 1 hour and 2 hours, 1 hour and 4 hours, 1 hour and 6 hours, 1 hour and 12 hours, 1 hour and 18 hours, 1 hour and 1 day, 2 hours and 4 hours, 2 hours and 6 hours, 2 hours and 12 hours, 2 hours and 18 hours, 2 hours and 1 day, 4 hours and 6 hours, 4 hours and 12 hours, 4 hours and 18 hours, 4 hours and 1 day, 6 hours and 12 hours, 6 hours and 18 hours, 6 hours and 1 day, 12 hours and 18 hours, 12 hours and 1 day, or 18 hours and 1 day). In some embodiments, the catalyst is contacted with the biological sample for about 1 minute to about 150 minutes. In some embodiments, the catalyst is contacted with the biological sample for about 5 minutes to about 100 minutes. In some49MF-367036897202412025040embodiments, the catalyst is contacted with the biological sample for about 10 minutes to about 50 minutes. In some embodiments, the catalyst is contacted with the biological sample for about 15 minutes to about 30 minutes. In some embodiments, the catalyst is contacted with the biological sample for about 30 minutes.

[0125] In some embodiments, the catalyst is contacted with the biological sample at a temperature between about 5°C and about 100°C. In some embodiments, the catalyst is contacted with the biological sample at a temperature between about 50°C and about 95°C. In some embodiments, the catalyst is contacted with the biological sample at a temperature between about 60°C and about 90°C, such as about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, or about 90°C. In some embodiments, the catalyst is contacted with the biological sample at a temperature between about 75°C and about 85°C. In some embodiments, the catalyst is contacted with the biological sample at a temperature of about 80°C or higher. In some embodiments, the catalyst is contacted with the biological sample at a temperature of about 80°C.

[0126] In some embodiments, the biological sample is incubated with the catalyst between about 25°C and about 100°C. In some embodiments, the biological sample is incubated with the catalyst between about 25°C and about 40°C, about 37°C and about 60°C, about 45°C and about 95°C, about 50°C and about 90°C, about 55°C and about 85°C, about 60°C and about 85°C, about 75°C and about 85°C. In some embodiments, the catalyst is incubated with the biological sample at a temperature between about 75°C and about 85°C. In some embodiments, the catalyst is incubated with the biological sample at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the catalyst at about 80 °C. In some instances, the biological sample is incubated with the catalyst at about 80 °C or higher.

[0127] In some embodiments, the catalyst agent is applied to the biological sample for about 10 minutes, about 20, 30, 40, 50, 60, 70, 80, 90, 110, or about 120 minutes, and at a temperature between about 70°C and about 95°C, such as about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, or 95°C. In some embodiments, the catalyst is contacted to the biological sample for approximately 30 minutes at a temperature between about 75°C and about 85°C, such as about 80°C.50MF-367036897202412025040

[0128] A catalyst can be delivered to a biological sample using any appropriate method. In some embodiments, a catalyst can be delivered as a solution (e.g., a first buffer) or a suspension. In some embodiments, a catalyst can be delivered as a solution or a suspension in a buffer (e.g., a first buffer). In some embodiments, the buffer is citrate, tris(hydroxymethyl)aminomethane (Tris), Tris-EDTA, phosphate-buffered saline (PBS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof. In some embodiments, the buffer is Tris. In some embodiments, the buffer comprises Tris and a chelating agent, optionally wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the buffer is Tris-EDTA (TE). In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises citrate and dimethyl sulfoxide (DMSO). In some embodiments, the buffer comprises 1%-5% (v / v) DMSO. In some embodiments, the buffer comprises 2% (v / v) DMSO. In some embodiments, the buffer comprises citrate but no DMSO. A buffer can have any appropriate concentration. For example, in some embodiments, a buffer has a concentration of about 5 mM to about 60 mM (e.g., about 10 mM to about 50 mM, about 20 mM to about 40 mM, or about 30 mM). In some embodiments, the catalyst is formulated with DMSO and combined with the buffer (e.g., a citrate buffer, a PBS buffer, or a TE buffer) or before contacting the biological sample.

[0129] In some embodiments, the buffer (e.g.. first buffer) is at a concentration between about 5 mM and about 300 mM. In some embodiments, the buffer is at a concentration between about 10 mM and about 250 mM, such as between about 100 mM and about 200 mM, such as about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

[0130] In some embodiments, the first buffer has a pH between about 4 and about 10. In some embodiments, the first buffer has a pH between about 6 and about 9. In some embodiments, the first buffer has a pH between about 6.5 and about 8, such as between about 6.8 and about 7.4. In some embodiments, the first buffer is present at a concentration between about 100 mM and about 200 mM and has a pH between about 6.5 and about 8, such as pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, or pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9. In some embodiments, the first buffer is an acidic buffer. In some embodiments, the first buffer has a pH of 8 or below. In some embodiments, the first buffer has a pH of about 7.51MF-367036897202412025040

[0131] In some embodiments, the first buffer comprises sodium dodecyl sulfate (SDS), urea, and / or a proteinase, optionally wherein the proteinase is proteinase K. In some embodiments, the first buffer comprises SDS and proteinase K. In some embodiments, the first buffer comprises urea and proteinase K. In some embodiments, the urea concentration is between about 0.01 M and about 1 M, such as 0.01 M, 0.02 M, 0.05 M, 0.1 M, 0.2 M, 0.5 M, 0.75 M, or 1 M, or any concentration in between the aforementioned values. In some embodiments, the proteinase K concentration is between about 0.1 μg / mL and about 2 μg / mL, such as 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 0.75 μg / mL, 1 μg / mL, 1.25 μg / mL, 1.5 μg / mL, 1.75 μg / mL, or 2 μg / mL, or any concentration in between the aforementioned values. In some embodiments, the SDS concentration (w / v) is between about 0.01% and about 1%, such as 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, or 1%, or any concentration in between the aforementioned values. In some embodiments, the first buffer comprises 0.05% SDS, 0.2% SDS, 0.5% SDS, 0.05 M urea, 0.5 M urea, 0.2 μg / ml proteinase K, 0.5 μg / ml proteinase K, 1 μg / ml proteinase K, or any combination thereof.

[0132] In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a phosphate buffered saline (PBS), e.g., a PBS buffer solution having a pH between about 6.5 and about 8, such as pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, or pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9. In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a PBS buffer solution having a pH of about 7.4. In some embodiments, disclosed herein is a compound of formula (I), such as 2-amino-5-methylbenzoic acid, (2-aminophenyl)phosphonic acid, and / or (4-aminopyridin-3-yl)phosphonic acid, as well as a PBS buffer solution having a pH of about 7.4 comprising the compound of formula (I).

[0133] In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a first buffer comprising citrate, e.g., a citrate solution having a pH between about 5 and about 8, such as about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, or about pH 7.5. In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a citrate buffer solution having a pH of about 6.0. In some embodiments, disclosed herein is a compound of formula (I), such as 2-amino-5-methylbenzoic acid, (2-aminophenyl)phosphonic acid, and / or (4-aminopyridin-3- 52MF-367036897202412025040yl)phosphonic acid, as well as a citrate buffer solution having a pH of about 6.0 comprising the compound of formula (I). In some embodiments, disclosed herein is a compound of formula (I), such as 2-amino-5-methylbenzoic acid, (2-aminophenyl)phosphonic acid, and / or (4-aminopyridin-3-yl)phosphonic acid, in a citrate buffer solution having a pH of about 7.0.

[0134] In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a first buffer comprising Tris, e.g., a Tris-EDTA solution having a pH between about 8 and about 10, such as about pH 8.5, about pH 9.0, about pH 9.5, or about pH 10.0. In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a Tris-EDTA buffer solution having a pH of about 9.0. In some embodiments, disclosed herein is a compound of formula (I), such as 2-amino-5-methylbenzoic acid, (2-aminophenyl)phosphonic acid, and / or (4-aminopyridin-3-yl)phosphonic acid, as well as a Tris-EDTA buffer solution having a pH of about 9.0 comprising the compound of formula (I).

[0135] A catalyst can be contacted with (e.g., applied to) the biological sample (e.g., a cell or tissue sample such as a tissue section) in any number of ways. In some embodiments, a catalyst is delivered in the first buffer. In some embodiments, the biological sample is soaked in a solution or suspension comprising the catalyst. In some embodiments, the catalyst is sprayed onto the biological sample, e.g., as a solution or suspension. In some embodiments, the catalyst is supplied to the biological sample via a microfluidic system (e.g., as a solution or suspension). In some embodiments, a catalyst is pipetted or otherwise aliquoted onto the biological sample. In some embodiments, the biological sample is dipped into a solution or suspension of a catalyst, wherein excess solution or suspension is removed from the biological sample. In some embodiments, a catalyst is delivered to the biological sample via a hydrogel, wherein the hydrogel is contacted with the biological sample.D. Nucleic Acid Detection

[0136] In some embodiments, after contacting a biological sample comprising cells with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample, the biological sample is contacted with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells. In some aspects, the de-crosslinking is performed prior to contacting the biological sample with the plurality of 53MF-367036897202412025040nucleic acid probes. In some aspects, the de-crosslinking allows target RNAs in the biological sample to become de-crosslinked and available for binding to the nucleic acid probes.

[0137] In some embodiments, provided herein are methods, probes, and kits for analyzing one or more endogenous nucleic acid analytes in a biological sample. In some embodiments, an endogenous analyte (e.g., a viral or cellular DNA or RNA) or a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, or a transcription / reverse transcription product thereof is analyzed. In some instances, the provided methods are for gene expression analysis for RNA transcripts and protein analysis in the same intact biological sample (e.g., a tissue section).

[0138] Disclosed herein in some aspects are a plurality of nucleic acid probes that are introduced into a cell or used to otherwise contact a biological sample such as a tissue sample. The plurality of nucleic acid probes may comprise any of a variety of entities that can hybridize to a nucleic acid, typically by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc. In some aspects, a nucleic acid probe comprises a hybridization region that is able to directly or indirectly bind to at least a portion of a target sequence in a target nucleic acid. In some aspects, a nucleic acid probe binds to a specific target nucleic acid (e.g., an mRNA, or other nucleic acids disclosed herein). In some aspects, a nucleic acid probe is detected using a detectable label, and / or by using other detectably labeled probes that are able to bind to the nucleic acid probes or a product thereof. In some embodiments, the nucleic acid probes are compatible with one or more biological and / or chemical reactions. For instance, a nucleic acid probe disclosed herein can serve as a template for a polymerase, a template or substrate for a ligase, a substrate for a click chemistry reaction, and / or a substrate for a nuclease (e.g., endonuclease or exonuclease for cleavage or digestion).

[0139] In some aspects, “binding” as used herein refers to the coupling between two or more nucleic acids, e.g., oligonucleotides and / or polynucleotides. In some embodiments, the binding is indirect binding. In some embodiments, the binding is direct (e.g., binding comprising direct hybridization of nucleic acid sequences). The nature of the binding may vary. In some instances, a first nucleic acid sequence directly binds to a second nucleic acid sequence via hybridization of complementary sequences. In some instances, a first nucleic acid sequence indirectly binds to a second nucleic acid sequence via one or more intermediate nucleic acids.54MF-367036897202412025040For example, an intermediate nucleic acid comprises a first region that binds to the first nucleic acid sequence and a second region for binding to the second nucleic acid sequence, thereby forming a complex comprising the first and second nucleic acid sequences.

[0140] The hybridization region of a probe of the plurality of nucleic acid probes is a target-binding sequence (sometimes also referred to as the targeting region / sequence or the recognition region / sequence) that can be positioned anywhere within the probe. For instance, the target-binding sequence of a nucleic acid probe that binds to a target nucleic acid (e.g., target RNA) can be 5’ or 3’ to any barcode sequence in the probe. In some embodiments, the targetbinding sequence comprises a sequence that is substantially complementary to a portion of a target nucleic acid. In some embodiments, the portions may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.

[0141] In some embodiments, the hybridization region of the nucleic acid probe is to identify a particular analyte comprising or associated with a target (e.g., an RNA comprising a target sequence). In some cases, multiple probes can be used, sequentially and / or simultaneously, that can bind to (e.g., hybridize to) different regions of the same target nucleic acid. In other examples, a probe may comprise target-binding sequences (e.g., hybridization regions) that can bind to different target nucleic acid sequences, e.g., various intron and / or exon sequences of the same gene (for detecting splice variants, for example), or sequences of different genes, e.g., for detecting a product that comprises the different target nucleic acid sequences, such as a genome rearrangement (e.g., inversion, transposition, translocation, insertion, deletion, duplication, and / or amplification).

[0142] In some instances, a plurality of nucleic acid probes comprises a plurality of circular probes. In some instances, a plurality of nucleic acid probes comprises a plurality of circularizable probes. In some embodiments, a circularizable probe is ligated to form a circularized template for rolling circle amplification (RCA). In some instances, each of the circularizable probes comprises a barcode region comprising one or more barcode sequences. In some instances, a plurality of nucleic acid probes comprises a plurality of padlock probes. In some instances, a plurality of nucleic acid probes comprises a plurality of gapped padlock probes. In some instances, a circularizable probe is provided in the form of a linear molecule 55MF-367036897202412025040having ligatable ends which are circularized by ligating the ends together directly or indirectly, e.g. to each other, or to the respective ends of an intervening ("gap") oligonucleotide or to an extended 3' end of the circularizable RCA template. In some instances, the circularizable probe is provided in two or more parts, namely two or more molecules (e.g. oligonucleotides) which may be ligated together to form the circular nucleic acid. In some aspects, the circularizable probe is circularized by ligation prior to RCA. In some instances, the ligation is templated using a ligation template. In some instances, the target analyte is the ligation template, or a ligation template is separately provided. The circularizable RCA template (or template part or portion) will comprise at its respective 3' and 5' ends regions of complementarity to corresponding cognate complementary regions (or binding sites) in the ligation template, which may be adjacent where the ends are directly ligated to each other, or non-adjacent, with an intervening "gap" sequence, where indirect ligation is to take place.

[0143] In some instances, the circularizable probes are padlock probes, and in one embodiment, the ends of the padlock probe are brought into proximity to each other by hybridization to adjacent sequences on a target nucleic acid molecule (such as a target nucleic acid analyte), which acts as a ligation template, thus allowing the ends to be ligated together to form a circular nucleic acid molecule. In some aspects, the hybridization and subsequent ligation allows the circularized padlock probe to form a circular nucleic acid that acts as a template for an RCA reaction. In some aspects, the 5’ end and 3’ end of the padlock probe is ligated to form a circular nucleic acid molecule. In such an example, the terminal sequences of the padlock probe which hybridize to the target nucleic acid molecule will be specific to the target nucleic acid analyte in question, and will be replicated repeatedly in the RCA product. They may therefore act as a marker sequence indicative of that target analyte. Accordingly, it can be seen that the marker sequence in the RCA product may be equivalent to a sequence present in the target analyte itself. Alternatively, in some embodiments, a marker sequence (e.g., tag or barcode sequence) is provided in the non-target complementary parts of the circularizable probe (e.g., padlock probe). In some embodiments, a marker sequence is between the respective hybridized ends of the padlock probe, where they are hybridized to non-adjacent sequences in the target molecule. In some embodiments, circular probes or circularizable probes (e.g., a padlock probe) comprises one or more barcodes. For example, the circular nucleic acid for amplification 56MF-367036897202412025040comprises a target hybridization region and a hybridization region complementary to a primer. In some embodiments, the barcodes of the circular nucleic acids or complements thereof in the generated RCA product are targeted by detectably labeled oligonucleotides, such as fluorescently labeled oligos. In some embodiments, one or more decoding schemes are used to decode the signals, such as fluorescence, for sequence determination.

[0144] Various probes can be hybridized to an endogenous analyte. In some embodiments, each probe of the plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the sample comprises one or more barcode sequences. In some instances, various probes (e.g., circularized probes) are used to generate a product comprising a target sequence that can be hybridized directly or indirectly by one or more detectable probes. In some instances, a plurality of nucleic acid probes comprises a plurality of RNA-templated ligation probes. The specific probe design can vary. In some embodiments, a plurality of nucleic acid probes comprises circular or circularizable probes. In some embodiments, the plurality of nucleic acid probes are analyzed using in situ hybridization (e.g., sequential hybridization) or in situ sequencing (e.g., using circular or circularizable probes and rolling circle amplification of circular or circularized probes).a. Hybridization and Ligation

[0145] In some embodiments, the plurality of nucleic acid probes are used to detect an endogenous analyte or a product or derivative thereof via pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. In some embodiments, the hybridization of complementary nucleic acid sequences are between an endogenous molecule and an exogenous molecule such as a nucleic acid probe. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another.

[0146] In some instances, a probe of the plurality of nucleic acid probes comprises ends which are brought into proximity to each other by hybridization to adjacent sequences on a target nucleic acid molecule (such as a target RNA), thus allowing the ends to be positioned for 57MF-367036897202412025040ligation to form a circular nucleic acid molecule. In some aspects, the hybridization and subsequent ligation allows the circularized nucleic acid probe to form a circular nucleic acid that acts as a template for an RCA reaction. In some aspects, a 5’ end and 3’ end of a padlock probe is ligated to form a circular nucleic acid molecule. In some embodiments, a plurality of ligation products is formed using the plurality of nucleic acid probes. In some embodiments, the ligation product is from an intramolecular ligation of a nucleic acid probe, for example, the circularization of a circularizable probe upon hybridization to a target sequence. The target sequence of a nucleic acid probe can be comprised in an endogenous analyte (e.g., nucleic acid such as genomic DNA or mRNA) or a product thereof (e.g., cDNA from a cellular mRNA transcript.

[0147] In some embodiments, provided herein is a plurality of nucleic acid probes capable of DNA-templated ligation, such as from a cDNA molecule. See, e.g., U. S. Pat.8,551,710, which is hereby incorporated by reference in its entirety. In some embodiments, provided herein is a plurality of nucleic acid probes capable of RNA-templated ligation. See, e.g., U. S. Pat. Pub. 2020 / 0224244 which is hereby incorporated by reference in its entirety. In some embodiments, the plurality of nucleic acid probes comprise a SNAIL probe set. See, e.g., U. S. Pat. Pub. 20190055594, which is hereby incorporated by reference in its entirety.

[0148] In some embodiments, the ligation involves chemical ligation. In some embodiments, the ligation involves template dependent ligation. In some embodiments, the ligation involves template independent ligation. In some embodiments, the ligation involves enzymatic ligation. In some embodiments, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases comprise ATP-dependent double-strand polynucleotide ligases, NAD+-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase™ (Epicentre Biotechnologies)58MF-367036897202412025040and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some embodiments, the ligase is a T4 RNA ligase. In some embodiments, the ligase is a splintR ligase. In some embodiments, the ligase is a single stranded DNA ligase. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some embodiments, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0149] In some embodiments, the ligation herein is a direct ligation. In some embodiments, the ligation herein is an indirect ligation. " Direct ligation" means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize non- adjacently to one another, e.g., separated by one or more intervening nucleotides or "gaps". In some embodiments, the ends are not ligated directly to each other, but instead occurs either via the intermediacy of one or more intervening (so-called "gap" or "gap-filling" (oligo)nucleotides) or by the extension of the 3' end of a probe to "fill" the "gap" corresponding to said intervening nucleotides (intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be "filled" by one or more "gap" (oligo)nucleotide(s) which are complementary to a splint, padlock probe, or target nucleic acid. In some instances, the gap is a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides In some instances, the gap is a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap between said terminal regions is filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some embodiments, the ligation herein is preceded by gap filling. In other embodiments, the ligation herein does not require gap filling.

[0150] In some embodiments, ligation of the polynucleotides produces polynucleotides with melting temperature higher than that of unligated polynucleotides. Thus, in some aspects, ligation stabilizes the hybridization complex (e.g., between the circularizable59MF-367036897202412025040probe and its target RNA) containing the ligated polynucleotides prior to subsequent steps, comprising amplification and detection.

[0151] In some aspects, a high fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tmaround the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0152] In some embodiments, the ligation herein is a proximity ligation of ligating two (or more) nucleic acid sequences that are in proximity with each other, e.g., through enzymatic means (e.g., a ligase). In some embodiments, proximity ligation can include a “gapfilling” step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic acid molecules of interest (see, e.g., U. S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference). A wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt-end” ligations. Additionally, single-stranded ligation can be used to perform proximity ligation on a single-stranded nucleic acid molecule. Sticky-end proximity ligations involve the hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be joined, prior to the ligation event itself. Blunt-end proximity ligations generally do not include hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack a single-stranded overhang at the site of ligation.b. Amplification

[0153] In some embodiments, an extension product is generated using a plurality of nucleic acid probes bound to the analytes (e.g., bound to corresponding RNAs). In some embodiments, the plurality of nucleic acid probes is used as a template for extension of a primer or other nucleic acid molecule. A primer is generally a single- stranded nucleic acid sequence having a 3’ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid 60MF-367036897202412025040extension reaction. RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases. A primer, may in some cases, refer to a primer binding sequence. A primer extension reaction generally refers to any method where two nucleic acid sequences become linked (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (e.g., for example, 3’ termini). Such linking can be followed by nucleic acid extension (e.g., an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for extension. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and / or a reverse transcriptase.

[0154] In some embodiments, a circular nucleic acid probe or circularized nucleic acid probe is used as template for extension in an amplification reaction. In some embodiments, the amplifying is achieved by performing rolling circle amplification (RCA). In other embodiments, a primer that hybridizes to the circular nucleic acid probe or circularized nucleic acid probe is added and used as such for amplification. In some embodiments, the RCA comprises a linear RCA, a branched RCA, a dendritic RCA, or any combination thereof.

[0155] In some embodiments, the amplification is performed at a temperature between or between about 20°C and about 60°C. In some embodiments, the amplification is performed at a temperature between or between about 30°C and about 40°C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, or 49°C.

[0156] In some embodiments, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, a primer is elongated to produce multiple copies of the circular template. This amplification step can utilize isothermal amplification or 61MF-367036897202412025040non-isothermal amplification. In some embodiments, after the formation of the hybridization complex and association of the amplification probe, the hybridization complex is rolling-circle amplified to generate a cDNA nanoball (e.g., amplicon) containing multiple copies of the cDNA. Techniques for rolling circle amplification (RCA) include linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. See, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res.2016 November 15; 49(11): 2540-2550; Schweitzer et al. Proc. Natl Acad. Sci. USA 97(18): 10113-9, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:e118, 2001; Dean et al. Genome Res. 11:1095-1099, 2001; Schweitzer et al, Nature Biotech.20:359-365, 2002; U. S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801, all of which are incorporated by reference. Examples of polymerases for use in RCA comprise DNA polymerase such phi29 (φ29) polymerase, Klenow fragment, Bacillusstearothe rmophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some aspects, DNA polymerases that have been engineered or mutated to have desirable characteristics can be employed. In some embodiments, the polymerase is phi29 DNA polymerase.

[0157] In some aspects, during the amplification step, modified nucleotides are added to the reaction to incorporate the modified nucleotides in the amplification product (e.g., nanoball). In some examples, the modified nucleotides comprise amine-modified nucleotides. In some aspects of the methods, for example, for anchoring or cross-linking of the generated amplification product (e.g., nanoball) to a scaffold, to cellular structures and / or to other amplification products (e.g., other nanoballs). In some aspects, the amplification product comprises a modified nucleotide, such as an amine-modified nucleotide. In some embodiments, the amine-modified nucleotide comprises an acrylic acid N-hydroxy succinimide moiety modification. Examples of other amine-modified nucleotides comprise, but are not limited to, a 5-Aminoallyl-dUTP moiety modification, a 5-Propargylamino-dCTP moiety modification, a N6- 6-Aminohexyl-dATP moiety modification, or a 7-Deaza-7-Propargylamino-dATP moiety modification.

[0158] In some aspects, the polynucleotides and / or amplification product (e.g., amplicon) are anchored to a polymer matrix. For example, the polymer matrix can be a62MF-367036897202412025040hydrogel. In some embodiments, one or more of the polynucleotide probe(s) are modified to contain functional groups that can be used as an anchoring site to attach the polynucleotide probes and / or amplification product to a polymer matrix. Examples of a modification and polymer matrix that can be employed in accordance with the provided embodiments comprise those described in, for example, US 2016 / 0024555, US 2018 / 0251833, US 2017 / 0219465, US 10,138,509, US 10,494,662, US 11,078,520, US 11,299,767, US 10,266,888, US 11,118,220, US 2021 / 0363579, US 2021 / 0324450, and US 2021 / 0215581, all of which are herein incorporated by reference in their entireties. In some examples, the scaffold also contains modifications or functional groups that can react with or incorporate the modifications or functional groups of the probe or amplification product. In some examples, the scaffold can comprise oligonucleotides, polymers or chemical groups, to provide a matrix and / or support structures.

[0159] The amplification products (e.g., RCA products) may be immobilized within the matrix generally at the location of the nucleic acid being amplified, thereby creating a localized colony of amplicons. In some aspects, the amplification products are immobilized within the matrix by steric factors. In some aspects, the amplification products are immobilized within the matrix by covalent or noncovalent bonding. In this manner, the amplification products may be considered to be attached to the matrix. By being immobilized to the matrix, such as by covalent bonding or cross -linking, the size and spatial relationship of the original amplicons is maintained. By being immobilized to the matrix, such as by covalent bonding or cross -linking, the amplification products are resistant to movement or unraveling under mechanical stress.

[0160] In some aspects, the amplification products are copolymerized and / or covalently attached to the surrounding matrix thereby preserving their spatial relationship and any information inherent thereto. For example, if the amplification products are those generated from DNA or RNA within a cell embedded in the matrix, the amplification products can also be functionalized to form covalent attachment to the matrix preserving their spatial information within the cell thereby providing a subcellular localization distribution pattern. In some embodiments, the provided methods involve embedding the one or more nucleic acid probes and / or the amplification products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the hydrogel-tissue chemistry described comprises covalently attaching nucleic acids to in situ synthesized hydrogel for tissue 63MF-367036897202412025040clearing, enzyme diffusion, and multiple-cycle sequencing while an existing hydrogel-tissue chemistry method cannot. In some embodiments, to enable amplification product embedding in the tissue-hydrogel setting, amine-modified nucleotides are comprised in the amplification step (e.g., RCA), functionalized with an acrylamide moiety using acrylic acid N-hydroxy succinimide esters, and copolymerized with acrylamide monomers to form a hydrogel.

[0161] In some embodiments, the RCA template comprises the target analyte, or a part thereof (e.g., a sequence thereof), where the target analyte is a nucleic acid, or it may be provided or generated as a proxy, or a marker, for the analyte. As noted above, the detection of numerous different analytes may use a RCA-based detection system, e.g., where the signal is provided by generating a target sequence from a circular RCA template which is provided or generated in the assay, and the target sequence is detected to detect the corresponding analyte. The target sequence may thus be regarded as a reporter which is detected to detect the target analyte. However, the RCA template may also be regarded as a reporter for the target analyte; the target sequence is generated based on the RCA template, and comprises complementary copies of the RCA template. The RCA template determines the signal which is detected, and is thus indicative of the target analyte. In some instances, the RCA template is a circularized probe, which is used as a reporter for the assay, or a part of a reporter, or signal-generation system.E. Treatment with a Second Buffer

[0162] In some embodiments, a biological sample comprising cells is treated with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample; then the biological sample is contacted with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells; and after contacting the sample with the plurality of nucleic acid probes, the biological sample is incubated with a second buffer at a temperature of at least 80°C for at least 10 minutes. In some instances, the treatment with the second buffer provides accessibility of target analyte molecules in the sample for detection. In some instances, the treatment with the second buffer provides accessibility to antigens in the biological sample (e.g., tissue sample). In some instances, antibodies for binding to antigens are provided after treatment with a second buffer. In some examples, antigens refer to moieties that may bind an antibody, an antibody fragment and / or an aptamer. In some instances, the antigens are endogenous to the biological sample. In some aspects, harsh treatment (e.g., at 64MF-367036897202412025040high temperatures and / or high pH) of the biological sample may need to be avoided to preserve the RNA for binding the plurality of nucleic acid probes. In some aspects, performing a secondary treatment (e.g., antigen retrieval and / or de-crosslinking) in a second buffer at a temperature of at least 80°C for at least 10 minutes after binding the plurality of nucleic acid probes minimizes impact on quality of the RNA detection assay. In some aspects, the amplification products generated using the bound plurality of nucleic acid probes are stable and less susceptible to damage by high temperatures and / or high pH. In some aspects, the secondary treatment in a second buffer at a temperature of at least 80°C for at least 10 minutes is performed after RNA library preparation to avoid degrading RNA needed for RNA detection. In some aspects, the additional treatment with the second buffer improves detection using labelling agents comprising a binding moiety (e.g., for protein detection). In some aspects, the additional treatment with the second buffer is for antigen retrieval and improves protein detection. In some aspects, the sample treatment process described herein is optimized for a combined assay (e.g., for detecting nucleic acid analytes and non-nucleic acid analytes).

[0163] In some embodiments, the method comprises baking the biological sample. In some embodiments, the biological sample is baked prior to treatment with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some embodiments, the biological sample is baked after the biological sample has been contacted with the plurality of nucleic acid probes and prior to treatment with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some embodiments, the biological sample is baked at a temperature, e.g., at about 30°C to about 80°C, about 45°C to about 75°C, about 50°C to about 70°C, or about 55°C to about 65°C, such as at about 35°C, 37°C, 40°C, 45°C, or 50°C, for a period of time. In some embodiments, the biological sample is baked for about 2 minutes to about 1 hour, about 2 minutes to about 30 minutes, about 2 minutes to about 10 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 10 minutes, or about 5 minutes). In some instances, the baking is performed at a temperature of 30°C or higher for at least 5 minutes. In some embodiments, the biological sample is baked uncovered in an oven. In some embodiments, the baked biological sample is calibrated to room temperature for a period of time (e.g., about 3 minutes to about 30 minutes, about 5 minutes to about 20 minutes, or about 7 minutes).65MF-367036897202412025040

[0164] In some embodiments, the method comprises dehydrating the biological sample. In some embodiments, the biological sample is dehydrated prior to treatment with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some embodiments, the biological sample is dehydrated after the plurality of nucleic acid probes binds to a plurality of RNAs in the biological sample and prior to treatment with the second buffer at a temperature of at least 80°C for at least 10 minutes. In some instances, the dehydrating is performed using an ethanol series (e.g., 70% ethanol followed by 100% ethanol).

[0165] In some embodiments, after contacting the nucleic acid probes with the biological sample, the biological sample is incubated with a second buffer at a temperature of at least 80°C for at least 10 minutes. In some instances, the biological sample is not contacted twice with the catalytic de-crosslinker. In some embodiments, the second buffer contacted with the biological sample does not comprise a catalyst compound (e.g., Compounds of formula (I) or formula (II) provided herein).

[0166] In some embodiments, the second buffer comprises Tris. In some embodiments, the second buffer comprises a chelating agent. In some embodiments, the second buffer comprises a buffering agent. In some instances, the second buffer comprises Tris and a chelating agent. In some instances, the second buffer comprises 'Tris and a buffering agent. In some instances, the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the buffer is tris(hydroxymethyl)aminomethane-Ethylenediaminetetraacetic acid (TE). In some instances, the buffering agent is hydrochloride (HC1) and the second buffer is Tris-HCl In some embodiments, the second buffer is different from the first buffer used to deliver the catalyst to the biological sample. In some embodiments, the second buffer is a citrate buffer.

[0167] In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 30 minutes. In some embodiments, the biological sample is incubated with the second buffer for about 15 minutes to about 30 minutes. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes to about 30 minutes. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 20 minutes. In some embodiments, the biological sample is incubated with the second buffer for no more than 30 minutes. In some embodiments, the biological sample is incubated with the second buffer for no more than 20 minutes. In some 66MF-367036897202412025040embodiments, the biological sample is incubated with the second buffer for no more than 15 minutes. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes.

[0168] In some embodiments, the biological sample is incubated with the second buffer at a temperature between about 80°C and about 100°C. In some embodiments, the biological sample is incubated with the second buffer at a temperature between about 80°C and about 90 C. In some embodiments, the biological sample is incubated with the second buffer at a temperature between about 85°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer at a temperature between about 75°C and about 85°C. In some embodiments, the biological sample is incubated with the second buffer at a temperature between about 80°C and about 85°C. In some embodiments, the biological sample is incubated with the second buffer at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer at a temperature of about 80°C.

[0169] In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 30 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 20 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for about 15 minutes to about 30 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes to about 30 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for no more than 30 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for no more than 20 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for no more than 15 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes at a temperature of about 80°C or higher. In some embodiments, the biological sample is incubated with the second buffer for about 15 minutes at a temperature of about 80°C67MF-367036897202412025040or higher. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes at a temperature of about 80°C or higher.

[0170] In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 30 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes to about 20 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 15 minutes to about 30 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes to about 30 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for no more than 20 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for no more than 30 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for no more than 15 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes at a temperature between about 80°C and about 90 C. In some embodiments, the biological sample is incubated with the second buffer for about 15 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 10 minutes at a temperature of about 90°C. In some embodiments, the biological sample is incubated with the second buffer for about 20 minutes at a temperature of about 90 C.

[0171] In some embodiments, the second buffer has a concentration of about 5 mM to about 60 mM (e.g., about 10 mM to about 50 mM, about 20 mM to about 40 mM, or about 30 mM). In some embodiments, the second buffer is at a concentration between about 5 mM and about 300 mM. In some embodiments, the second buffer is at a concentration between about 10 mM and about 250 mM, such as between about 100 mM and about 200 mM, such as about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.68MF-367036897202412025040

[0172] In some embodiments, the second buffer comprises about 5mM to about 50mM, about 10 mM to about 50 mM, about 20 mM to about 40 mM, or about 30 mM Tris. In some embodiments, the second buffer comprises about 5 M to about 25 mM Tris. In some embodiments, the second buffer comprises no more than about 50 mM, no more than about 40 mM, no more than about 30 mM Tris, or no more than about 20 mM Tris. In some embodiments, the second buffer comprises about 10 mM Tris.

[0173] In some embodiments, the second buffer comprises about 0.1 mM to about 50mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 50mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, or about 1 mM to about 5 mM EDTA. In some embodiments, the second buffer comprises about 0.5 mM to about 50mM, about 0.5 mM to about 40 mM, about 0.5 mM to about 30 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 10 mM, or about 0.5 mM to about 5 mM EDTA. In some embodiments, the second buffer comprises about 0.1 mM to about 10 mM EDTA. In some embodiments, the second buffer comprises no more than about lOmM, no more than about 5 mM, no more than about 5 mM, or no more than about 2 mM EDTA. In some embodiments, the second buffer comprises about 1 mM EDTA.

[0174] In some embodiments, the second buffer comprises no more than about 50 mM, no more than about 40 mM, no more than about 30 mM Tris, or no more than about 20 mM Tris and no more than about lOmM, no more than about 5 mM, no more than about 5 mM, or no more than about 2 mM EDTA. In some embodiments, the second buffer comprises about 0.1 mM to about 10 mM EDTA and about 5 mM to about 25 mM Tris.. In some embodiments, the second buffer comprises about 5 mM to about 25 mM Tris and about 10 mM Tris to about 1 mM EDTA.

[0175] In some embodiments, the second buffer has a pH between about 8 and about 10. In some embodiments, the first buffer has a pH between about 8 and about 9. In some embodiments, the first buffer has a pH between about 8 and about 9.5, between about 8 and about 9, between about 8.5 and about 9, or between about 8.5 and about 9.5. In some embodiments, the second buffer comprises about 5 mM to about 25 mM Tris and about 10 mM Tris to about 1 mM EDTA and has a pH between about 8 and about 9, such as pH 8.1, pH 8.2,69MF-367036897202412025040pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9. In some embodiments, the second buffer has a pH of at least 8. In some embodiments, the second buffer has a pH of between about 8.5 and about 9.5. In some embodiments, the second buffer has a pH of about 9. In some embodiments, the second buffer at a higher pH than the first buffer used to deliver the catalyst to the biological sample. In some embodiments, the first buffer used to deliver the catalyst to the biological sample has a pH of no greater than 7.5 and the second buffer has a pH of at least 8. In some embodiments, disclosed herein is a catalyst (e.g., a compound of formula (I) or (II) disclosed herein) in a first buffer comprising citrate having a pH of no greater than 7.5 and treatment with a second buffer comprising Tris and EDTA with a pH between about 8 and about 9. In some embodiments, the first buffer used to deliver the catalyst to the biological sample has a pH of no greater than 7.5 and the second buffer has a pH of at least 8 and comprises about 0.1 mM to about 10 mM EDTA and about 5 mM to about 25 mM Tris.

[0176] In some aspects, catalytic de-crosslinking with the first buffer is performed under less stringent conditions than the treatment with the second buffer. In some aspects, the first buffer is a citrate buffer and the second buffer is a TE buffer.

[0177] In some embodiments, the biological sample is incubated with a TE buffer of about pH 8.0 for about 10 minutes to about 30 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with a TE buffer of about pH 9.0 for about 10 minutes to about 20 minutes at a temperature between about 80°C and about 90°C. In some embodiments, the biological sample is incubated with the TE buffer of about pH 8.0 for about 10 minutes at a temperature of about 90°C. In some embodiments, the biological sample is incubated with the TE buffer of about pH 8.0 for about 20 minutes at a temperature of about 90°C. In some embodiments, the biological sample is incubated with the TE buffer of about pH 9.0 for about 20 minutes at a temperature of about 80°C. In some embodiments, the biological sample is incubated with the TE buffer of about pH 9.0 for about 10 minutes at a temperature of about 90°C.

[0178] In some embodiments, the biological sample is incubated with a second buffer comprising Tris and EDTA for about 10 minutes at a temperature of about 90°C. In some embodiments, the biological sample is incubated with a second buffer comprising Tris and EDTA for about 20 minutes at a temperature of about 80°C. In some embodiments, the70MF-367036897202412025040biological sample is incubated with a second buffer comprising Tris and EDTA for about 15 minutes at a temperature of about 80°C. In some embodiments, the biological sample is incubated with a second buffer of about pH 9 comprising Tris and EDTA for about 10 minutes at a temperature of about 90°C. In some embodiments, the biological sample is incubated with a second buffer of about pH 9 comprising Tris and EDTA for about 20 minutes at a temperature of about 80°C. In some embodiments, the biological sample is incubated with a second buffer of about pH 9 comprising Tris and EDTA for about 15 minutes at a temperature of about 80°C.

[0179] In some embodiments, the biological sample is washed after the incubation with the second buffer. In some embodiments, the biological sample is washed prior to and after the incubation with the second buffer. In some embodiments, the biological sample is washed after the incubation with the second buffer and before blocking for labelling agent incubation. In some embodiments, the biological sample is washed in a phosphate buffered saline with Tween detergent (PBST). In some instances, the biological sample is washed for one minute or more using three or more washes.F. Analyte Detection with Labelling Agent

[0180] Disclosed herein in some aspects are labelling that are introduced into a cell or used to otherwise contact a biological sample such as a tissue sample. In some embodiments, a biological sample comprising cells is treated with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample; then the biological sample is contacted with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells; and after contacting the sample with the plurality of nucleic acid probes, the biological sample is incubated with a second buffer at a temperature of at least 80°C for at least 10 minutes prior to introducing a labelling agent to the biological sample. In some aspects, additional treatment after de-crosslinking with the catalyst is needed for detection of an additional analyte (e.g., protein in addition to RNA detection). In some aspects, incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes after introducing the plurality of nucleic acid probes for RNA binding and before providing a labelling agent to the sample improves protein detection. In some instances, the treatment with the second buffer provides accessibility to antigens in the biological sample (e.g., tissue sample). In some instances, antibodies for binding to antigens are provided after treatment with a second 71MF-367036897202412025040buffer. In some examples, a plurality of labelling agents (e.g., antibodies, antibody fragments and / or aptamers) are provided to detect one or more analytes endogenous to the biological sample.

[0181] In some embodiments, the method comprises, after incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes, contacting the sample with a labelling agent that directly or indirectly binds to an analyte at a location in the biological sample, e.g., for analyte detection using labelled antibodies for protein detection. In some embodiments, provided herein are methods, probes, and kits for analyzing endogenous analytes (e.g., cell surface or intracellular proteins and / or metabolites) using one or more labelling agents. In some embodiments, the labelling agent is an immunohistochemistry (IHC) probe that is excited at various different wavelengths. In some embodiments, an analyte labelling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labelling agents comprise a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the labelling agent. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. In some cases, the sample contacted by the labelling agent is further contacted with a probe (e.g., a single- stranded probe sequence), that hybridizes to a reporter oligonucleotide of the labelling agent, in order to identify the analyte associated with the labelling agent. In some embodiments, the analyte labelling agent comprises an analyte binding moiety and a labelling agent barcode domain comprising one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety and / or the analyte. An analyte binding moiety barcode comprises to a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds can also be identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or sequence that is associated with the analyte binding moiety. An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein.

[0182] In the methods described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes,72MF-367036897202412025040cells and / or cell features. In some instances, cell features include cell surface features. Analytes may include, but are not limited to, a protein, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof.

[0183] In some embodiments, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). A labelling agent may include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi- specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a probody, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) may have coupled thereto a first reporter oligonucleotide, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) may have a different reporter oligonucleotide coupled thereto. For examples of labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U. S. Pat. 10,550,429; U. S. Pat. Pub.20190177800; and U. S. Pat. Pub. 20190367969, which are each incorporated by reference herein in their entirety.

[0184] In some embodiments, an analyte binding moiety comprises one or more antibodies or antigen binding fragments thereof. The antibodies or antigen binding fragments including the analyte binding moiety can specifically bind to a target analyte. In some73MF-367036897202412025040embodiments, the analyte is a protein (e.g., a protein on a surface of the biological sample (e.g., a cell) or an intracellular protein). In some embodiments, a plurality of analyte labelling agents comprising a plurality of analyte binding moieties bind a plurality of analytes present in a biological sample. In some embodiments, the plurality of analytes comprises a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which the plurality of analytes comprises a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the same. In some embodiments in which the plurality of analytes comprises a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the different (e.g., members of the plurality of analyte labelling agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites). In some embodiments, the plurality of analytes comprises multiple different species of analyte (e.g., multiple different species of polypeptides).

[0185] In other instances, e.g., to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature may have a first plurality of the labelling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent is coupled to a second reporter oligonucleotide.

[0186] In some aspects, these reporter oligonucleotides comprises nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The selection of oligonucleotides as the reporter may provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected.

[0187] Attachment (coupling) of the reporter oligonucleotides to the labelling agents may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of a labelling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and74MF-367036897202412025040oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U. S. Pat. No. 6,265,552, which is entirely incorporated herein by reference for all purposes. Furthermore, click reaction chemistry may be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art may be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide comprising a barcode sequence that identifies the label agent. For instance, the labelling agent may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that comprises a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be attached to the labelling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein.

[0188] In some cases, the labelling agent comprises a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. In some instances, the label is conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to a first oligonucleotide that is complementary (e.g., hybridizes) to a sequence of the reporter oligonucleotide.

[0189] In some instances, the biological sample is contacted with a blocking buffer prior to contacting the biological sample with the labelling agent. In some instances, the biological sample is incubated with a blocking buffer comprising BSA. In some instances, the75MF-367036897202412025040biological sample is incubated with a blocking buffer for at least 30 minutes, at least 1 hour, or at least 2 hours.

[0190] In some embodiments, the method comprises incubating the sample with a plurality of labelling agents simultaneously. In some instances, the plurality of labelling agents are for detecting a plurality of different analytes (e.g., different proteins) in a biological sample. Methods and compositions disclosed herein can be used to detect any number of analytes with the labelling agents. In some instances, the plurality of labelling agents are for detecting a plurality of different analytes and the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, or more different analytes in the biological sample. In some instances, the biological sample is contacted with a plurality of labelling agents for detecting panel of proteins comprising at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, or at least about 25 different proteins. In some instances, the biological sample is contacted with a plurality of labelling agents for detecting a panel of analytes (e.g., proteins) comprising at least about 10 different target analytes. In some instances, the biological sample is contacted with a plurality of labelling agents for detecting a panel of analytes (e.g., proteins) comprising at least about 20 different target analytes.

[0191] In some embodiments, the sample is washed after being incubated with a labelling agent. In some instances, the washing comprises washing the biological sample in phosphate-buffered saline with Tween detergent ( PBST). In some embodiments, the sample is incubated with a labelling agent for no more than about 24 hours, no more than about 20 hours, no more than about 18 hours, no more than about 16 hours, or no more than about 14 hours before optionally performing wash steps and / or imaging the biological sample. In some embodiments, the method comprises incubating the sample with the labelling agent(s) at a temperature between about 4°C and about 40°C. In some embodiments, the method comprises incubating the sample with the labelling agent(s) at room temperature (e.g., between about 2076MF-367036897202412025040°C and about 25 °C). In some embodiments, the sample is incubated with the labelling agent(s) at a temperature between about 4°C and about 6°C. In some embodiments the method comprises incubating the sample with the labelling agent(s) at a temperature of about 4°C. In some embodiments, the method comprises incubating the sample with the labelling agent(s) at a temperature of about 4°C for between 16-18 hours.

[0192] In some embodiments, the method comprises one or more post-fixing (also referred to as post-fixation) steps after contacting the sample with one or more labelling agents. In some instances, the biological sample is contacted with a fixative after contacting the biological sample with the labelling agent. In some instances, the biological sample is contacted with a quencher after contacting the biological sample with the labelling agent and before the detecting. In some examples, the quencher comprises a quencher dye. In some embodiments, the quencher dye comprises at least three aromatic residues, wherein each aromatic residue is independently an unsubstituted aryl, a substituted aryl, an unsubstituted heteroaryl, or a substituted heteroaryl, wherein at least one of said aromatic residues is covalently linked to two other aromatic residues via two exocyclic azo bonds: or at least two aromatic residues, wherein each aromatic residue is independently an unsubstituted aryl, a substituted aryl, an unsubstituted heteroaryl, or a substituted heteroaryl, wherein at least two of said aromatic residues are covalently linked via an exocyclic azo bond, and wherein at least one said aromatic residue is an unsubstituted polycyclic aryl, a substituted polycyclic aryl, an unsubstituted polycyclic heteroaryl group, or a substituted polycyclic heteroaryl group. An example of a quencher is described in U. S. Pat. Pub. 2024 / 0043914, which is hereby incorporated by reference in its entirety.

[0193] In some embodiments, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes can be associated with locations of the analytes in the biological sample. Such information (e.g., proteomic information when the analyte binding moiety(ies) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (e.g., sequences of transcripts), or both). For example, a cell surface protein of a cell 77MF-367036897202412025040can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte labelling agent comprising an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety. Results of protein analysis in a sample (e.g., a tissue sample or a cell) can be associated with DNA and / or RNA analysis in the sample.III. DETECTION

[0194] In some embodiments, provided herein are methods and compositions for sample analysis comprising contacting a fixed biological sample that has been catalytically decrosslinked, contacted with a plurality of nucleic acid probes, subsequently incubated with a second buffer, then contacted with a labelling agent that binds to an analyte at a location in the biological sample, and detecting optical signals associated with the plurality of nucleic acid probes or products thereof associated with the plurality of RNAs in the biological sample and an optical signal associated with the labelling agent or a product thereof, thereby detecting the multiple analytes at the respective locations in the biological sample. In some embodiments, a plurality of detectably labeled probes are used to provide the optical signals. A biological sample may comprise one or a plurality of analytes of interest. Methods for performing multiplexed assays to detect signals associated with two or more different analytes in a single biological sample are provided.

[0195] In some instances, the plurality of nucleic acid probes (e.g., as described in section II.D) are used to generate an amplification product to be detected. In some instances, the amplification product comprises a RCA product. In some instances, a amplification product associated with a probe of the plurality of nucleic acid probes is generated that comprises one or more probe hybridizations. For example, a signal amplification method comprises targeted assembly of branched structures (e.g., bDNA). In some instances, detection of nucleic acids sequences in situ includes combination of the sequential decoding methods described herein with an assembly for branched signal amplification using the nucleic acid probes provided herein. In some instances, the assembly complex comprises an amplifier hybridized directly or indirectly (via one or more oligonucleotides) to a sequence of a cellular nucleic acid. In some instances, a sequence of the generated amplification product is detected using detectable probes (e.g.,78MF-367036897202412025040detectably labeled probes). Higher order probes that directly or indirectly bind to the generated amplification product may also be used, and the higher order probes or products thereof can then be detected using detectably labeled probes.

[0196] After contacting the biological sample with a plurality of labelling agents (e.g., as described in section II.F), the labelling agents are directly detected by determining detectable labels (if present), and / or detected by using one or more other probes that bind directly or indirectly to the reporter oligonucleotides of the labelling agents. In some instances, the labelling agent comprises a reporter oligonucleotide and the reporter oligonucleotide comprises a barcode sequence. In some instances, the labelling agent comprises a detectable label. In some instances, a sequence of the reporter oligonucleotide is detected using detectable probes (e.g., detectably labeled probes). Higher order probes that directly or indirectly bind to the reporter oligonucleotide may also be used, and the higher order probes or products thereof can then be detected using detectably labeled probes.

[0197] In some embodiments, a plurality of labelling agents contacted with the biological sample for detecting a first analyte and a second analyte are sequentially detected in a biological sample. In some instances, the first and second analytes are detected using corresponding labelling agents, wherein the labelling agents each comprise a reporter oligonucleotide. In some instances, a first labelling agent is bound directly or indirectly to a first analyte in the biological sample, wherein the first labelling agent comprises a first reporter oligonucleotide, and a second labelling agent is bound directly or indirectly to a second analyte in the biological sample, wherein the second labelling agent comprises a second reporter oligonucleotide. In some embodiments, a first detectably labeled probe binds directly or indirectly to the first reporter oligonucleotide and the first detectably labeled probe is subsequently from the biological sample after it is detected. In some instances, a second detectably labeled probe binds directly or indirectly to the second reporter oligonucleotide. In some instances, the second detectably labeled probe is labeled with a different label (e.g., generates a different optical signal) from the first detectably labeled probe. In some cases, the first and second detectably labeled probes are detected in the same cycle (e.g., using different channels). In some cases, the first and second detectably labeled probes are detected in different detection cycles.79MF-367036897202412025040

[0198] In some embodiments, multiple different species of analytes (e.g., polypeptides) from the biological sample are subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes are associated with locations of the analytes in the biological sample. Such information (e.g., proteomic information when the analyte binding moiety(ies) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (e.g., sequences of transcripts), or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell). In some instances, the one or more physical properties are characterized by imaging the cell. In some instances, the cell is bound by an analyte labelling agent comprising an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety. In some instances, the results of protein analysis in a sample (e.g., a cell or tissue sample) are associated with DNA and / or RNA analysis in the sample. In some embodiments, RNA analysis is performed prior to protein analysis in the same sample.

[0199] In some embodiments, the methods provided herein comprise detecting one or more analytes, labelling agents, and / or products thereof in the biological sample. In some embodiments, one or more analytes and the labelling agents (e.g., as described in Section II) or products are detected in the biological sample. In some embodiments, a plurality of RNA analytes and a plurality of protein analytes (e.g., using the labelling agents as described in Section II. F) in the biological sample are detected. In some embodiments, optical signals associated with a plurality of RNA analytes are detected prior to detecting optical signals associated with a plurality of protein analytes. In some embodiments, optical signals associated with a plurality of RNA analytes are detected subsequent to detecting optical signals associated with a plurality of protein analytes.

[0200] In some embodiments, to detect the optical signals associated with the plurality of nucleic acid probes or products thereof associated with the plurality of RNAs in the biological sample, detectably labeled probes are used. In some instances, detectably labeled probes directly bind to a nucleic acid probe or a product thereof (e.g., an RCA product) at a barcode sequence or portion(s) or a complement thereof. In some instances, detectably labeled 80MF-367036897202412025040probes indirectly bind to a nucleic acid probe or a product thereof (e.g., an RCA product) at a barcode sequence or portion(s) or a complement thereof. In some embodiments, a combination of barcode sequences (which may be continuous or spaced from one another) in the nucleic acid probe or a product thereof is detected. In some embodiments, the binding is specific, or the binding may be such that a recognition sequence preferentially binds to or hybridizes with only one of the barcode sequences or complements thereof that are present.

[0201] In some instances, detectably labeled probes directly or indirectly bind to a nucleic acid probe or a product thereof (e.g., an RCA product) at a recognition sequence. The recognition sequences may be of any length, and multiple recognition sequences in the same or different detectably labeled probes may be of the same or different lengths. If more than one recognition sequence is used, the recognition sequences may independently have the same or different lengths. For instance, the recognition sequence may be at least 4, at least 5, least 6, least 7, least 8, least 9, at least 10, least 11, least 12, least 13, least 14, at least 15, least 16, least 17, least 18, least 19, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 nucleotides in length. In some embodiments, the recognition sequence may be no more than 48, no more than 40, no more than 32, no more than 24, no more than 16, no more than 12, no more than 10, no more than 8, or no more than 6 nucleotides in length. Combinations of any of these are also possible, e.g., the recognition sequence may have a length of between 5 and 8, between 6 and 12, or between 7 and 15 nucleotides, etc. In some embodiments, the recognition sequence is of the same length as a barcode sequence or complement thereof of a primary nucleic acid probe or a product thereof. In some embodiments, the recognition sequence may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to the barcode sequence or complement thereof.

[0202] In some embodiments, the nucleic acid probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more barcode sequences. As an illustrative example, a first nucleic acid probe may contain a first target-binding sequence, a first barcode sequence, and a second barcode sequence, while a second, different nucleic acid probe may contain a second target-binding sequence (that is different from the first target-binding sequence in the first probe), the same first barcode sequence as in the first probe, but a third barcode sequence instead 81MF-367036897202412025040of the second barcode sequence. Such probes may thereby be distinguished by determining the various barcode sequence combinations present or associated with a given probe at a given location in a sample.

[0203] In some embodiments, the nucleic acid probes disclosed herein may be made using only 2 or only 3 of the 4 bases, such as leaving out all the “G”s and / or leaving out all of the “C”s within the probe. Sequences lacking either “G”s or “C”s may form very little secondary structure, and can contribute to more uniform, faster hybridization in certain embodiments.

[0204] In some embodiments, a nucleic acid probe disclosed herein may contain a detectable label such as a fluorophore. In some embodiments, one or more probes of a plurality of nucleic acid probes used in an assay may lack a detectable label, while one or more other probes in the plurality each comprises a detectable label selected from a limited pool of distinct detectable labels (e.g., red, green, yellow, and blue fluorophores), and the absence of detectable label may be used as a separate “color.” As such, detectable labels are not required in all cases. In some embodiments, a nucleic acid probe disclosed herein lacks a detectable label. While a detectable label may be incorporated into an amplification product of a probe, such as via incorporation of a modified nucleotide into an RCA product of a circularized probe, the amplification product itself in some embodiments is not detectably labeled. In some embodiments, a probe that binds to the nucleic acid probe or a product thereof comprises a detectable label and may be used to detect the nucleic acid probe or product thereof. In some embodiments, an intermediate probe disclosed herein lacks a detectable label, and a detectably labeled probe that binds to the intermediate probe is used to detect the intermediate probe bound to the amplification product.

[0205] In some aspects, intermediate probes are selected from the group consisting of a circular probe, a circularizable probe, and a linear probe. In some aspects, intermediate probes are linear probes. In some embodiments, a linear probe comprises a target recognition sequence and a sequence that does not hybridize to a target nucleic acid, such as a 5’ overhang, a 3’ overhang, and / or a linker or spacer (which may comprise a nucleic acid sequence or a non-nucleic acid moiety). In some embodiments, the sequence (e.g., the 5’ overhang, 3’ overhang, and / or linker or spacer) is non-hybridizing to the target nucleic acid but may hybridize to one another and / or one or more other probes, such as detectably labeled probes. In some82MF-367036897202412025040embodiments, an intermediate probe comprise a sequence that hybridizes to a sequence of an amplification product generated using the nucleic acid probe as template, and a region that is non-hybridizing to the amplification product but is configured to hybridize to one another and / or one or more other probes, such as detectably labeled probes.

[0206] In some embodiments, signals associated with the detectably labeled is used to detect one or more barcode sequences in the nucleic acid probe bound to the RNA analyte, e.g., by using sequential hybridization of detectably labeled probes, sequencing by hybridization, sequencing by ligation, and / or in situ sequencing e.g., sequencing-by-synthesis (SBS), sequencing-by-avidity (SBA) or sequencing-by-binding (SBB).

[0207] In some embodiments, the nucleic acid probes or products thereof are detected with a method that comprises signal amplification by performing a primer exchange reaction (PER). In various embodiments, a primer with domain on its 3’ end binds to a catalytic hairpin, and is extended with a new domain by a strand displacing polymerase. For example, a primer with domain 1 on its 3’ ends binds to a catalytic hairpin, and is extended with a new domain 1 by a strand displacing polymerase, with repeated cycles generating a concatemer of repeated domain 1 sequences. In various embodiments, the strand displacing polymerase is Bst. In various embodiments, the catalytic hairpin includes a stopper which releases the strand displacing polymerase. In various embodiments, branch migration displaces the extended primer, which can then dissociate. In various embodiments, the primer undergoes repeated cycles to form a concatemer primer. In various embodiments, a plurality of concatemer primers is contacted with a sample comprising the plurality of nucleic acid probes described herein. In various embodiments, the plurality of nucleic acid probes may be contacted with a plurality of concatemer primers and a plurality of labeled probes, see e.g., U. S. Pat. Pub. No.US20190106733, which is incorporated herein by reference, examples of molecules and PER reaction components.

[0208] In some embodiments, more than one type of nucleic acid probes is contacted with a sample, e.g., simultaneously or sequentially in any suitable order, such as in sequential probe hybridization / unhybridization cycles. In some embodiments, an intermediate probe that binds to a sequence of the nucleic acid probe or a complement thereof (e.g., in an amplification product) is contacted with a sample, e.g., simultaneously or sequentially in any suitable order,83MF-367036897202412025040such as in sequential probe hybridization / unhybridization cycles. In some embodiments, more than one type of detectably labeled nucleic acid probes are contacted with a sample, e.g., simultaneously or sequentially in any suitable order, such as in sequential probe hybridization / unhybridization cycles. In some embodiments, the detectably labeled nucleic acid probes are used to bind to one or more nucleic acid probes bound to the target analyte, one or more higher order probes, one or more intermediate probes, and / or one or more detectably or non-detectably labeled probes (e.g., as in the case of a hybridization chain reaction (HCR), a branched DNA reaction (bDNA), or the like).

[0209] In some instances, the disclosed methods may comprise the use of a hybridization chain reaction (HCR) approach to amplify signals. In a hybridization chain reaction, two fluorescently-labeled metastable hairpin oligonucleotides self-assemble into long fluorescent polymers starting from an initiator sequence present on each probe molecule. The degree of amplification achieved through HCR can be tuned by changing the hybridization or polymerization times, and can be adjusted to achieve highly amplified signals (which may, however, increase the size of the fluorescent spots generated and / or lead to variable degrees of amplification for different copies of the same target molecule).

[0210] In some embodiments, provided herein are methods and compositions for analyzing analytes in a sample using concatemer primers and labelling agents. In various embodiments, a primer with domain on its 3' end binds to a catalytic hairpin, and is extended with a new domain by a strand displacing polymerase. For example, a primer with domain 1 on its 3 ends binds to a catalytic hairpin, and is extended with a new domain 1 by a strand displacing polymerase, with repeated cycles generating a concatemer of repeated domain 1 sequences. In various embodiments, the strand displacing polymerase is Bst. In various embodiments, the catalytic hairpin includes a stopper which releases the strand displacing polymerase. In various embodiments, branch migration displaces the extended primer, which can then dissociate. In various embodiments, the primer undergoes repeated cycles to form a concatemer primer.

[0211] In various embodiments, a plurality of concatemer primers is contacted with a sample. In various embodiments, an assembly include a plurality of concatemer primers, a plurality of labeled probes, and a sample including nucleic acids. In various embodiments, each 84MF-367036897202412025040the plurality of concatemer primers each includes domain 1, 2, 3, etc. In various embodiments, each the plurality of labeled probes each include domain 1', 2', 3', etc., with each corresponding domain 1', 2', 3' being complementary to domain 1, 2, 3, etc., respectively. In various embodiments, the assembly includes the plurality of concatemer primers, which are capable of hybridizing to target nucleic acid sequences in the sample. Described herein is a method using the aforementioned assembly, including contacting the sample including target nucleic acids with the plurality of concatemer primers, then contacting the sample and plurality of concatemer primers with the plurality of labeled probes, thereby labelling the target nucleic acid sequences with a plurality of labeled probes. See e.g., U. S. Pat. Pub. No. 2021 / 0147902 and 2020 / 0362398, each of which is fully incorporated by reference herein.

[0212] In some embodiments, the plurality of nucleic acid probes comprises at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 250,000, at least 500,000, or at least 1,000,000 distinguishable nucleic acid probes that is contacted with a sample, e.g., simultaneously or sequentially in any suitable order. Between any of the probe contacting steps disclosed herein, the method may comprise one or more intervening reactions and / or processing steps, such as modifications of a target nucleic acid, modifications of a probe or product thereof (e.g., via hybridization, ligation, extension, amplification, cleavage, digestion, branch migration, primer exchange reaction, click chemistry reaction, crosslinking, attachment of a detectable label, activating photo-reactive moieties, etc.), removal of a probe or product thereof (e.g., cleaving off a portion of a probe and / or unhybridizing the entire probe), signal modifications (e.g., quenching, masking, photo-bleaching, signal enhancement (e.g., via FRET), signal amplification, etc.), signal removal (e.g., cleaving off or permanently inactivating a detectable label), crosslinking, de-crosslinking, and / or signal detection.

[0213] In some embodiments, the barcode sequences are used to combinatorially encode a plurality of analytes of interest. As such, signals associated with the detectably labeled probes at particular locations in a biological sample can be used to generate distinct signal signatures that each corresponds to an analyte in the sample, thereby identifying the analytes at the particular locations, e.g., for in situ spatial analysis of the sample.85MF-367036897202412025040

[0214] In some aspects, targets (e.g., analytes) are barcoded through the incorporation of one or more barcode sequences (e.g., sequences that can be detected or otherwise “read”) in the nucleic acid probe that binds the targeted analyte. In some aspects, the nucleic acid probes or amplification products thereof described herein are in turn targeted by intermediate probes, which are also barcoded through the incorporation of one or more barcode sequences that are separate from a recognition sequence in the intermediate probe that directly or indirectly binds the nucleic acid probes described herein or a product thereof. In some embodiments, an intermediate probe binds to a barcode sequence in the primary probe. In some aspects, a detectably labeled probe binds to the intermediate probe e.g., at a barcode sequence or complement thereof in the intermediate probe. In some embodiments, through the detection of signals associated with detectably labeled probes in a sample, the location of one or more analytes in the sample and the identity of the analyte(s) can be determined. In some embodiments, the presence / absence, absolute or relative abundance, an amount, a level, a concentration, an activity, and / or a relation with another analyte of a particular analyte can be analyzed in situ in the sample.

[0215] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, one or more sequences present in the probes or products thereof (e.g., rolling circle amplification products thereof). In some embodiments, the detecting is performed at one or more locations in the biological sample. In some embodiments, the locations are the locations of RNA transcripts in the biological sample. In some embodiments, the locations are the locations at which the probes hybridize to the RNA transcripts in the biological sample, and are ligated and amplified by rolling circle amplification. Following amplification, a sequence of the RCA product or a portion thereof, is determined or otherwise analyzed, for example detected by imaging.

[0216] In some embodiments, the detecting comprises a plurality of repeated cycles of hybridization and removal of probes (e.g., detectably labeled probes, or intermediate probes that bind to detectably labeled probes) to the RCA product generated (e.g., as described in Section II. D).86MF-367036897202412025040

[0217] In some aspects, the sequencing or analysis of the amplification products comprise sequencing by hybridization, sequencing by ligation, and / or in situ sequencing e.g., sequencing-by- synthesis (SBS), sequencing-by-avidity (SBA) or sequencing-by-binding (SBB). In some instances, in situ hybridization comprises sequential fluorescent in situ hybridization.

[0218] In some embodiments, the detection is spatial, e.g., in two or three dimensions. In some embodiments, detectably labeled probes comprise any of a variety of entities able to hybridize a nucleic acid, e.g., DNA, RNA, LNA, and / or PNA, etc., depending on the application. In some embodiments, the detecting comprises binding an intermediate probe directly or indirectly to nucleic acid probes, binding a detectably labeled probe directly or indirectly to a detection region of the intermediate probe, and detecting a signal associated with the detectably labeled probe. In some embodiments, the method comprises detecting a rolling circle amplification product (RCP) generated using a circular or circularized probe as a template. In some embodiments, detecting the RCP comprises binding an intermediate probe directly or indirectly to the RCP, binding a detectably labeled probe directly or indirectly to a detection region of the intermediate probe, and detecting a signal associated with the detectably labeled probe. In some embodiments, the method comprises performing one or more wash steps to remove unbound and / or nonspecifically bound intermediate probe molecules from the RCPs.

[0219] In some embodiments, the detecting comprises detecting signals associated with detectably labeled probes that are hybridized to barcode regions or complements thereof in a generated amplification product (e.g., an RCP); and / or detecting signals associated with detectably labeled probes that are hybridized to intermediate probes which are in turn hybridized to the barcode regions or complements thereof. In some embodiments, the detectably labeled probes are fluorescently labeled.

[0220] In some embodiments, the methods comprise detecting the sequence in all or a portion of an RCP, such as one or more barcode sequences present in the RCP. In some embodiments, the sequence of the RCP, or barcode thereof, is indicative of a sequence of the target nucleic acid to which the circular nucleic acid is hybridized. In some embodiments, the detection step involves sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, and / or fluorescent in situ sequencing (FISSEQ), and / or87MF-367036897202412025040hybridization-based in situ sequencing. In some embodiments, the detection step is by sequential fluorescent in situ hybridization (e.g., for combinatorial decoding of the barcode sequence or complement thereof).

[0221] In some embodiments, the detection or determination comprises using a detection oligonucleotide labeled with a fluorophore, an isotope, a mass tag, or a combination thereof. In some embodiments, the detection or determination comprises imaging one or more detectably labeled probes hybridized directly or indirectly to an RCP. In some embodiments, the target nucleic acid is an mRNA in a tissue sample, and the detection or determination is performed when the amplification product is in situ in the tissue sample. In some embodiments, the target nucleic acid is an amplification product (e.g., RCP).

[0222] In some aspects, the provided methods comprise imaging a detectably labeled probe bound directly or indirectly to the nucleic acid probe or product thereof and detecting the detectable label. In some aspects, the provided methods comprise imaging a detectably labeled probe bound directly or indirectly to the reporter oligonucleotide of the labelling agent and detecting the detectable label. In some embodiments, the detectably labeled probe comprises a detectable label that can be measured and quantitated. The label or detectable label can comprise a directly or indirectly detectable moiety, e.g., any fluorophores, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.

[0223] A fluorophore can comprise a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range. Particular examples of labels that may be used in accordance with the provided embodiments comprise, but are not limited to phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase,88MF-367036897202412025040horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenical acetyl transferase, and urease.

[0224] Fluorescence detection in tissue samples can often be hindered by the presence of strong background fluorescence. Background fluorescence can include autofluorescence (that can arise from a variety of sources, including aldehyde fixation, extracellular matrix components, red blood cells, lipofuscin, and the like), as opposed to the desired immunofluorescence from the fluorescently labeled antibodies or probes. Tissue autofluorescence can lead to difficulties in distinguishing the signals due to fluorescent antibodies or probes from the general background. In some embodiments, a method disclosed herein utilizes one or more agents to reduce tissue autofluorescence, for example, Autofluorescence Eliminator (Sigma / EMD Millipore), TrueBlack Lipofuscin Autofluorescence Quencher (Biotium), MaxBlock Autofluorescence Reducing Reagent Kit (MaxVision Biosciences), and / or a very intense black dye (e.g., Sudan Black, or comparable dark chromophore).

[0225] Examples of detectable labels comprise but are not limited to various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs and protein-antibody binding pairs. Examples of fluorescent proteins comprise, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin.

[0226] Examples of bioluminescent markers comprise, but are not limited to, luciferase (e.g., bacterial, firefly and click beetle), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals comprise, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases and cholinesterases. Identifiable markers also comprise radioactive compounds such as125I,35S,14C, or3H. Identifiable markers are commercially available from a variety of sources.

[0227] In some embodiments, one or more fluorescent dyes are used as detectable labels. Commercially available fluorescent dyes include, but are not limited to 4,7- 89MF-367036897202412025040dichlorofluorescein dyes, spectrally resolvable rhodamine dyes, 4,7- dichlororhodamine dyes, cyanine dyes, ether- substituted fluorescein dyes, energy transfer dyes, and xanthine dyes.Labelling can also be carried out with quantum dots. In some embodiments, a fluorescent label comprises a signaling moiety that conveys information through the fluorescent absorption and / or emission properties of one or more molecules. Examples of fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer.

[0228] Examples of commercially available fluorescent nucleotide analogues readily incorporated into nucleotide and / or polynucleotide sequences comprise, but are not limited to, Cy3™-dCTP (cyanine 3-dCTP), Cy3™-dUTP (cyanine 3-dUTP), Cy5™-dCTP (cyanine 5-dCTP), Cy5™-dUTP (cyanine 5 dUTP) (Amersham Biosciences, Piscataway, N. J.), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED®-5-dUTP (red fluorescent dye-dUTP), CASCADE® BLUE-7-dUTP (blue fluorescent dye - dUTP), BODIPY™ FL-14-dUTP (green fluorescent dye-dUTP), BODIPY™ TMR-14-dUTP (orange fluorescent dye-dUTP), BODIPY™ TR-14-dUTP (red fluorescent dye-dUTP), RHODAMINE GREEN™-5-dUTP (green fluorescent dye-dUTP), OREGON GREEN™ 488-5-dUTP (green fluorescent dye-dUTP), TEXAS RED™-12-dUTP (red fluorescent dye-dUTP), BODIPY™ 630 / 650- 14-dUTP (far red fluorescent dye-dUTP), BODIPY™ 650 / 665- 14-dUTP (far red fluorescent dye-dUTP), ALEXA FLUOR™ 488-5-dUTP (green fluorescent dye-dUTP), ALEXA FLUOR™ 532-5-dUTP (yellow fluorescent dye-dUTP), ALEXA FLUOR™ 568-5-dUTP (red / orange fluorescent dye-dUTP), ALEXA FLUOR™ 594-5-dUTP (red fluorescent dye-dUTP), ALEXA FLUOR™ 546- 14-dUTP (orange fluorescent dye-dUTP), fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP (red fluorescent dye-UTP), mCherry, CASCADE® BLUE-7-UTP (blue fluorescent dye-UTP), BODIPY™ FL-14-UTP (green fluorescent protein-UTP), BODIPY™ TMR- 14-UTP (orange fluorescent dye-UTP), BODIPY™ TR-14-UTP (red fluorescent dye-UTP), RHODAMINE GREEN™-5-UTP (green fluorescent dye-UTP), ALEXA FLUOR™ 488-5-UTP (green fluorescent dye-UTP), and ALEXA FLUOR™ 546- 14-UTP (orange fluorescent dye-UTP) (Molecular Probes, Inc. Eugene, Oreg.). Methods are known for custom synthesis of nucleotides having other fluorophores.90MF-367036897202412025040

[0229] Other fluorophores available for post-synthetic attachment comprise, but are not limited to, ALEXA FLUOR™ dyes (fluorescent dyes) such as ALEXA FLUOR™ 350 (blue fluorescent dye), ALEXA FLUOR™ 594 (red fluorescent dye), and ALEXA FLUOR™ 647 (far red fluorescent dye); BODIPY™ dyes (fluorescent dyes) such as BODIPY™ FL (green fluorescent dye), BODIPY™ TMR (orange fluorescent dye), and BODIPY™ 650 / 665 (far red fluorescent dye); Cascade® Blue (blue fluorescent dye), Cascade® Yellow (yellow fluorescent dye), Dansyl, lissamine rhodamine B, Marina Blue™ (blue fluorescent dye), Oregon Green™ 488, Oregon Green™ 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red® (red fluorescent dye) (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2™ (cyanine 2), Cy3.5™ (cyanine 3.5), Cy5.5™ (cyanine 5.5), and Cy7™ (cyanine 7) (Amersham Biosciences, Piscataway, N. J.). FRET tandem fluorophores may also be used, comprising, but not limited to, PerCP-Cy™5.5 (far red fluorescent tandem fluorophore), PE-Cy™5 (red fluorescent tandem fluorophore), PE-Cy™5.5 (red fluorescent tandem fluorophore), PE-Cy™7 (far red fluorescent tandem fluorophore), PE-Texas Red® (red fluorescent tandem fluorophore), APC-Cy™7 (far red fluorescent tandem fluorophore), PE-Alexa™ dyes (e.g., 610, 647, 680), and APC-Alexa™ dyes.

[0230] In some cases, metallic silver or gold particles may be used to enhance signal from fluorescently labeled nucleotide and / or polynucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62).

[0231] Biotin, or a derivative thereof, may also be used as a label on a nucleotide and / or a polynucleotide sequence, and subsequently bound by a detectably labeled avidin / streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin). An aminoallyl-dUTP residue may be incorporated into a polynucleotide sequence and subsequently coupled to an N-hydroxy succinimide (NHS) derivatized fluorescent dye. In general, any member of a conjugate pair may be incorporated into a detection polynucleotide provided that a detectably labeled conjugate partner can be bound to permit detection.91MF-367036897202412025040

[0232] Other suitable labels for a polynucleotide sequence may comprise fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and phosphor-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments the following hapten / antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin / a-biotin, digoxigenin / a- digoxigenin, dinitrophenol (DNP) / a-DNP, 5-Carboxyfluorescein (FAM) / a-FAM.

[0233] In some embodiments, a polynucleotide sequence is indirectly labeled, such as with a hapten that is then bound by a capture agent. Many different hapten-capture agent pairs are available for use. Examples of haptens comprise, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, cyanine dyes (e.g., Cy5™, and digoxigenin. For biotin, a capture agent may be avidin, streptavidin, or antibodies. Antibodies may be used as capture agents for the other haptens (many dye-antibody pairs being commercially available, e.g., Molecular Probes, Eugene, Oreg.).

[0234] In some embodiments, detection of the barcode sequences of the nucleic acid probes or complements thereof is performed by sequential hybridization of oligonucleotide probes to the barcode sequences or complements thereof and detecting complexes formed by the probes and barcode sequences or complements thereof. In some cases, each barcode sequence or complement thereof is assigned a sequence of signal codes that identifies the barcode sequence or complement thereof (e.g., a temporal signal signature or code that identifies the analyte), and detecting the barcode sequences or complements thereof can comprise decoding the barcode sequences of complements thereof by detecting the corresponding sequences of signal codes detected from sequential hybridization, detection, and removal of sequential pools of oligonucleotide probes and the universal pool of detectably labeled probes. In some cases, the sequences of signal codes are fluorophore sequences assigned to the corresponding barcode sequences or complements thereof. In some embodiments, the detectably labeled probes are fluorescently labeled. In some embodiments, the barcode sequence or complement thereof is performed by sequential probe hybridization as described in US 2021 / 0340618, the content of which is herein incorporated by reference in its entirety.92MF-367036897202412025040

[0235] In some embodiments, the detecting comprises contacting the biological sample with one or more detectably labeled probes that directly or indirectly hybridize to the barcode sequences or complements thereof (e.g., in RCA products generated using the probes), and dehybridizing the one or more detectably labeled probes. In any of the embodiments herein, the contacting and dehybridizing steps are repeated with the one or more detectably labeled probes and / or one or more other detectably labeled probes that directly or indirectly hybridize to the barcode sequences or complements thereof. In some aspects, the method comprises sequential hybridization of detectably labeled probes to create a spatiotemporal signal signature or code that identifies the analyte.

[0236] In some embodiments, analyzing, e.g., detecting or determining, one or more sequences present in the biological sample is performed using a base-by-base sequencing method, e.g., sequencing-by-synthesis (SBS), sequencing-by-avidity (SBA) or sequencing-by-binding (SBB). In some embodiments, the biological sample is contacted with a sequencing primer and base-by-base sequencing using a cyclic series of nucleotide incorporation or binding, respectively, thereby generating extension products of the sequencing primer is performed followed by removing, cleaving, or blocking the extension products of the sequencing primer.

[0237] Generally in sequencing-by-synthesis methods, a first population of detectably labeled nucleotides (e.g., dNTPs) are introduced to contact a template nucleotide (e.g., a barcode sequence in the RCP) hybridized to a sequencing primer, and a first detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by a polymerase to extend the sequencing primer in the 5’ to 3’ direction using a complementary nucleotide (a first nucleotide residue) in the template nucleotide as template. A signal from the first detectably labeled nucleotide can then be detected. The first population of nucleotides may be continuously introduced, but in order for a second detectably labeled nucleotide to incorporate into the extended sequencing primer, nucleotides in the first population of nucleotides that have not incorporated into a sequencing primer are generally removed (e.g., by washing), and a second population of detectably labeled nucleotides are introduced into the reaction. Then, a second detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by the same or a different polymerase to extend the already extended sequencing primer in the 5’ to 3’ direction using a complementary nucleotide (a93MF-367036897202412025040second nucleotide residue) in the template nucleotide as template. Thus, in some embodiments, cycles of introducing and removing detectably labeled nucleotides are performed.

[0238] In some embodiments, the base-by-base sequencing comprises using a polymerase that is fluorescently labeled. In some embodiments, the base-by-base sequencing comprises using a polymerase-nucleotide conjugate comprising a fluorescently labeled polymerase linked to a nucleotide moiety that is not fluorescently labeled. In some embodiments, the base-by-base sequencing comprises using a multivalent polymer-nucleotide conjugate comprising a polymer core, multiple nucleotide moieties, and one or more fluorescent labels.

[0239] In some embodiments, sequencing is performed by sequencing-by-synthesis (SBS). In some embodiments, a sequencing primer is complementary to sequences at or near the one or more detection sequence or amplification sequences (e.g., barcode(s)). In such embodiments, sequencing-by-synthesis can comprise reverse transcription and / or amplification in order to generate a template sequence from which a primer sequence can bind. In some embodiments, the SBS methods comprise incorporation and / or imaging such as those described in US 2013 / 0079232; use reagents including, for example, modified and / or labelled nucleotides such as those described in US 2007 / 0166705 and US 7,057,026; polymerases such as those described in US 2006 / 0281109, all of which are herein incorporated by reference in their entireties.

[0240] In some embodiments, sequencing is performed by sequencing-by-binding (SBB). Various aspects of SBB are described in U. S. Pat. No. 10,655,176 B2, the content of which is herein incorporated by reference in its entirety. In some embodiments, SBB comprises performing repetitive cycles of detecting a stabilized complex that forms at each position along the template nucleic acid to be sequenced (e.g. a ternary complex that includes the primed template nucleic acid, a polymerase, and a cognate nucleotide for the position), under conditions that prevent covalent incorporation of the cognate nucleotide into the primer, and then extending the primer to allow detection of the next position along the template nucleic acid. In the sequencing-by-binding approach, detection of the nucleotide at each position of the template occurs prior to extension of the primer to the next position. Generally, the methodology is used to distinguish the four different nucleotide types that can be present at positions along a nucleic94MF-367036897202412025040acid template by uniquely labelling each type of ternary complex (i.e. different types of ternary complexes differing in the type of nucleotide it contains) or by separately delivering the reagents needed to form each type of ternary complex. In some instances, the labelling may comprise fluorescence labelling of, e.g., the cognate nucleotide or the polymerase that participate in the ternary complex.

[0241] In some embodiments, sequencing is performed by sequencing-by-avidity (SBA). Some aspects of SBA approaches are described in U. S. Pat. No. 10,768,173 B2, the content of which is herein incorporated by reference in its entirety. In some embodiments, SBA comprises detecting a multivalent binding complex formed between a fluorescently-labeled polymer-nucleotide conjugate, and a one or more primed target nucleic acid sequences (e.g., barcode sequences). Fluorescence imaging is used to detect the bound complex and thereby determine the identity of the N+l nucleotide in the target nucleic acid sequence (where the primer extension strand is N nucleotides in length). Following the imaging step, the multivalent binding complex is disrupted and washed away, the correct blocked nucleotide is incorporated into the primer extension strand, and the sequencing cycle is repeated.

[0242] In some embodiments, detection of the barcode sequences is performed by sequential hybridization of probes to the barcode sequences or complements thereof and detecting complexes formed by the probes and barcode sequences or complements thereof. In some cases, each barcode sequence or complement thereof is assigned a sequence of signal codes that identifies the barcode sequence or complement thereof (e.g., a temporal signal signature or code that identifies the analyte), and detecting the barcode sequences or complements thereof can comprise decoding the barcode sequences of complements thereof by detecting the corresponding sequences of signal codes detected from sequential hybridization, detection, and removal of sequential pools of intermediate probes and the universal pool of detectably labeled probes. In some cases, the sequences of signal codes comprise fluorophore sequences assigned to the corresponding barcode sequences or complements thereof. In some embodiments, the detectably labeled probes are fluorescently labeled. In some embodiments, the barcode sequence or complement thereof is performed by sequential probe hybridization as described in US 2021 / 0340618, the content of which is herein incorporated by reference in its entirety.95MF-367036897202412025040

[0243] In some embodiments, the detecting comprises contacting the biological sample with one or more detectably labeled probes that directly or indirectly hybridize to the barcode sequences or complements thereof (e.g., in amplification products generated using the nucleic acid probes), and dehybridizing the one or more detectably labeled probes. In some embodiments, the contacting and dehybridizing steps are repeated with the one or more detectably labeled probes and / or one or more other detectably labeled probes that directly or indirectly hybridize to the barcode sequences or complements thereof. In some aspects, the method comprises sequential hybridization of detectably labeled probes to create a spatiotemporal signal signature or code that identifies the analyte.

[0244] In some embodiments, detecting a nucleic acid sequence (e.g., a barcode sequence or barcode subunit) comprises contacting the biological sample with one or more first detectably labeled probes that directly hybridize to the nucleic acid sequence. In some instances, detecting a nucleic acid sequence comprises contacting the biological sample with one or more first detectably labeled probes that indirectly bind to the nucleic acid sequence (e.g., via binding to an intermediate probe that binds to the nucleic acid sequence).

[0245] In any of the embodiments herein, the detecting comprises contacting the biological sample with one or more intermediate probes that directly or indirectly hybridize to the barcode sequences or complements thereof (e.g., of the plurality of nucleic acid probes or rolling circle amplification product generated using the plurality of nucleic acid probes), wherein the one or more intermediate probes are detectable using one or more detectably labeled probes. In any of the embodiments herein, the detecting step can further comprise dehybridizing the one or more intermediate probes and / or the one or more detectably labeled probes from the barcode sequences or complements thereof (e.g., of the plurality of nucleic acid probes or rolling circle amplification product generated using the plurality of nucleic acid probes). In any of the embodiments herein, the contacting and dehybridizing steps can be repeated with the one or more intermediate probes, the one or more detectably labeled probes, one or more other intermediate probes, and / or one or more other detectably labeled probes. In some cases, the repeated contacting, detection and dehybridizing steps allows detection of barcode sequences or complements thereof and identification of the corresponding sequences of signal codes (e.g.,96MF-367036897202412025040fluorophore sequences assigned to the corresponding barcode sequences or complements thereof).

[0246] In some embodiments, the methods provided herein comprise detecting one or more analytes, labelling agents, or products in the biological sample. In some embodiments, one or more analytes and the labelling agents (e.g., as described in Section II) or products are detected in the biological sample. In some embodiments, a plurality of RNA analytes and a plurality of protein analytes are detected in the biological sample. In some embodiments, signals associated with a plurality of RNA analytes are detected prior to detecting signals associated with a plurality of protein analytes. In some embodiments, signals associated with a plurality of RNA analytes are detected subsequent to detecting signals associated with a plurality of protein analytes are detected in the biological sample. In some cases, a labelling agent is associated with an analyte by binding directly to the analyte. In some cases, a labelling agent is associated with an analyte by binding indirectly to the analyte.

[0247] In some embodiments, a plurality of delectably labeled probes for RNA detection are added to a biological sample, followed by addition of a plurality of detectably labeled probes for detecting labelling agents. In some instances, multiple cycles of detection are performed to detect a plurality of signals associated with different labelling agents.

[0248] In some embodiments, detecting a protein in the biological sample comprises contacting the biological sample with a labelling agent that binds to the protein (e.g., as described in Sections II), and detecting the labelling agent. In some embodiments, the labelling agent is an antibody or antigen-binding fragment thereof comprising a reporter oligonucleotide. In some embodiments, a first labelling agent associated with a first analyte and a second labelling agent associated with a second analyte are detected in the biological sample sequentially. In some instances, an optical signal associated with the first labelling agent is detected prior to removing the first detectably labeled probe from the biological sample. In some embodiments, a first labelling agent associated with a first analyte and a second labelling agent associated with a second analyte are detected in the biological sample sequentially using different intermediate probes. In some embodiments, the different intermediate probes share a common sequence for binding to detectably labeled probes. In some instances, an optical signal97MF-367036897202412025040associated with the second labelling agent is detected after contacting the biological sample with a second intermediate probe and a second detectably labeled probe to detect the second labelling agent. In some embodiments, the detecting is performed at one or more locations in the biological sample. For example, the locations are the locations of protein analytes bound by the labelling agents in the biological sample. In some embodiments, the locations are the locations at which the intermediate probes hybridize to the reporter oligonucleotide of the labelling agents in the biological sample.

[0249] In some embodiments, the detecting comprises a plurality of repeated cycles of hybridization and removal of probes (e.g., detectably labeled probes and / or intermediate probes bound by detectably labeled probes). In some embodiments, the detecting comprises a plurality of repeated cycles of hybridization and removal of probes (e.g., detectably labeled probes and / or intermediate probes bound by detectably labeled probes) to the reporter oligonucleotides of labelling agents in the biological sample. In some embodiments, a cycle of protein detection comprises detecting a plurality of different protein analytes each in a different channel (e.g., each is excited by a different color channel and imaged for detection). For example, in one cycle, 4 different proteins are detected in 4 different channels. In some instances, in a subsequent cycle, 4 additional different proteins are detected in the 4 different channels. In some embodiments, the subsequent cycle uses detectably labeled probes with the same sequence and labels as used in a previous cycle.

[0250] In some embodiments, sequencing is performed using single molecule sequencing by ligation. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. In some embodiments, nucleic acid hybridization is used for sequencing. These methods utilize labeled nucleic acid decoder probes that are complementary to at least a portion of a barcode sequence. Multiplex decoding can be performed with pools of many different probes with distinguishable labels.

[0251] In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporations can be detected through98MF-367036897202412025040fluorescence resonance energy transfer (FRET), as described for example in Levene et al., Science (2003), 299, 682-686, Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al., Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181.

[0252] In some embodiments, the analysis and / or sequence determination involves washing to remove unbound polynucleotides, thereafter revealing a fluorescent product for imaging.

[0253] In some embodiments, at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 500, at least 800, at least 1,000, at least 3,000, at least 5,000, at least 8,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 250,000, at least 500,000, or at least 1,000,000 distinguishable nucleic acid probes are contacted with a sample, e.g., simultaneously or sequentially in any suitable order. Between any of the probe contacting steps disclosed herein, the method may comprise one or more intervening reactions and / or processing steps, such as modifications of a target nucleic acid, modifications of a probe or product thereof (e.g., via hybridization, ligation, extension, amplification, cleavage, digestion, branch migration, primer exchange reaction, click chemistry reaction, crosslinking, attachment of a detectable label, activating photo-reactive moieties, etc.), removal of a probe or product thereof (e.g., cleaving off a portion of a probe and / or unhybridizing the entire probe), signal modifications (e.g., quenching, masking, photo-bleaching, signal enhancement (e.g., via FRET), signal amplification, etc.), signal removal (e.g., cleaving off or permanently inactivating a detectable label), crosslinking, de-crosslinking, and / or signal detection.

[0254] In some aspects, the detection (comprising imaging) is carried out using any of a number of different types of microscopy, e.g., confocal microscopy, two-photon microscopy, light-field microscopy, intact tissue expansion microscopy, and / or CLARITY™-optimized light sheet microscopy (COLM).

[0255] In some embodiments, fluorescence microscopy is used for detection and imaging. In some aspects, a fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. In fluorescence microscopy, a sample is illuminated with light of a wavelength which excites fluorescence in the sample. The fluoresced 99MF-367036897202412025040light, which is usually at a longer wavelength than the illumination, is then imaged through a microscope objective. Two filters may be used in this technique; an illumination (or excitation) filter which ensures the illumination is near monochromatic and at the correct wavelength, and a second emission (or barrier) filter which ensures none of the excitation light source reaches the detector. Alternatively, these functions may both be accomplished by a single dichroic filter. The "fluorescence microscope" comprises any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope, or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image.

[0256] In some embodiments, confocal microscopy is used for detection and imaging. Confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity - so long exposures are often required. As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e., a rectangular pattern of parallel scanning lines) in the specimen. The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples. CLARITY™-optimized light sheet microscopy (COLM) provides an alternative microscopy for fast 3D imaging of large clarified samples. COLM interrogates large immunostained tissues, permits increased speed of acquisition and results in a higher quality of generated data.

[0257] Other types of microscopy that can be employed comprise bright field microscopy, oblique illumination microscopy, dark field microscopy, phase contrast, differential interference contrast (DIC) microscopy, interference reflection microscopy (also known as reflected interference contrast, or RIC), single plane illumination microscopy (SPIM), superresolution microscopy, laser microscopy, electron microscopy (EM), Transmission electron 100MF-367036897202412025040microscopy (TEM), Scanning electron microscopy (SEM), reflection electron microscopy (REM), Scanning transmission electron microscopy (STEM) and low- voltage electron microscopy (LVEM), scanning probe microscopy (SPM), atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C- AFM), electrochemical scanning tunneling microscope (ECSTM), electrostatic force microscopy (EFM), fluidic force microscope (FluidFM), force modulation microscopy (FMM), feature- oriented scanning probe microscopy (FOSPM), kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscopy, SNOM, Piezoresponse Force Microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microspectroscopy / microscopy (PTMS), SCM, scanning capacitance microscopy (SCM), SECM, scanning electrochemical microscopy (SECM), SGM, scanning gate microscopy (SGM), SHPM, scanning Hall probe microscopy (SHPM), SICM, scanning ion-conductance microscopy (SICM), SPSM spin polarized scanning tunneling microscopy (SPSM), SSRM, scanning spreading resistance microscopy (SSRM), SThM, scanning thermal microscopy (SThM), STM, scanning tunneling microscopy (STM), STP, scanning tunneling potentiometry (STP), SVM, scanning voltage microscopy (SVM), and synchrotron x-ray scanning tunneling microscopy (SXSTM), and intact tissue expansion microscopy (exM).

[0258] In some aspects, the provided embodiments can be applied to an in situ method of analyzing target nucleic acid sequences (e.g., RNAs) and / or other targets (e.g., proteins) in intact tissues or samples in which the spatial information has been preserved. In some aspects, the embodiments can be applied in an imaging or detection method for multiplexed analysis of nucleic acids and / or other targets (e.g., proteins). In some aspects, the provided embodiments can be used to identify or detect regions and / or sequences of interest in target nucleic acids.

[0259] In some cases, analysis is performed on one or more images captured, and may comprise processing the image(s) and / or quantifying signals observed. In some embodiments, a method disclosed herein comprises multiplexed analysis of a biological sample comprising consecutive cycles of probe hybridization, fluorescence imaging, and probe removal. In some 101MF-367036897202412025040embodiments, images of signals from different fluorescent and / or non-fluorescent channels and / or detectable probe hybridization cycles can be compared and analyzed. In some embodiments, images of signals (or absence thereof) at a particular location in a sample from different fluorescent channels and / or sequential detectable probe hybridization cycles can be aligned to analyze an analyte at the location. For instance, a particular location in a sample can be tracked and signal spots from sequential hybridization cycles can be analyzed to detect a target polynucleotide sequence (e.g., an associated barcode sequence or subsequence thereof) at the location. The analysis may comprise processing information of one or more cell types, one or more types of analytes, a number or level of analyte, and / or a number or level of cells detected in a particular region of the sample. In some embodiments, the analysis comprises detecting a sequence e.g., a barcode sequence present in an amplification product at a location in the sample. In some embodiments, the analysis comprises determining a sequence e.g., a barcode sequence present in an amplification product at a location in the sample.

[0260] In some instances, the analysis includes using single cell segmentation. In some instances, cell membranes are identified to perform cell segmentation analysis. In some instances, cell boundaries are identified and a cell segmentation mask (outlines of cell boundaries) is generated. In some embodiments, the detected optical signal associated with the labelling agent or a product thereof in the biological sample is used to perform cell segmentation analysis. In some embodiments, the detected optical signal associated with the labelling agent or a product thereof in the biological sample is used to identify cell membranes. In some embodiments, the detected optical signal associated with the labelling agent or a product thereof in the biological sample is used to identify cell boundaries. In some embodiments, one or more cell boundaries are defined based at least in part on the detected signal associated with the labelling agent or a product thereof in one or more images of the biological sample.

[0261] In some instances, a two-dimensional cell segmentation mask comprising the defined cell boundaries is generated. In some instances, the two-dimensional cell segmentation mask is a flattened two-dimensional cell segmentation mask based on two or more Z-stack images of one or more detected signals associated with one or more labelling agents or products thereof in one or more images of the biological sample. In some instances, detected nucleic 102MF-367036897202412025040acid(s) and / or protein(s) in the biological sample are assigned to distinct cells in the biological sample based on the defined cell boundaries. In some instances, assigning the detected nucleic acid(s) and / or protein(s) in the biological sample to distinct cells in the biological sample comprises aligning an image of the biological sample or a detected feature mask representing the identities of the detected nucleic acid(s) and / or protein(s) at their respective location(s) with the cell segmentation mask comprising the defined cell boundaries.

[0262] In some instances, the analysis includes determining cell type frequencies in a region of interest of a sample. In some embodiments, the obtained information may be compared to a positive and negative control, to another selected region of interest, or to a threshold of a feature to determine if the region of interest exhibits a certain feature or phenotype. In some cases, the information may comprise signals from a cell, a region, and / or comprise readouts from multiple detectable labels. In some case, the analysis further includes displaying the information from the analysis or detection. In some embodiments, software may be used to automate the processing, analysis, and / or display of data. In some embodiments, cells in the sample are segmented using one or more images taken of the biological sample. In some embodiments, cells in the sample are segmented using one or more images of the labelling agents detected in the biological sample.IV. SAMPLES, ANALYTES, AND TARGET SEQUENCES

[0263] A sample disclosed herein can be or derived from any biological sample. Methods and compositions disclosed herein may be used for analyzing a biological sample, which may be obtained from a subject using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In addition to the subjects described above, a biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample, a patient derived organoid (PDO) or patient derived xenograft (PDX). A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms,103MF-367036897202412025040in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a predisposition to a disease, and / or individuals in need of therapy or suspected of needing therapy.

[0264] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can include nucleic acids (such as DNA or RNA), proteins / polypeptides, carbohydrates, and / or lipids. In some embodiments, the biological sample is obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. In some embodiments, the biological sample is or comprise a cell pellet or a section of a cell pellet. In some embodiments, the biological sample is or comprise a cell block or a section of a cell block. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions. In some embodiments, the biological sample comprises cells which are deposited on a surface.

[0265] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms. Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

[0266] In some embodiments, a substrate herein can be any support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and / or reagents (e.g., probes) on the support. In some embodiments, a biological sample is attached to a substrate. Attachment of the biological sample can be irreversible or reversible,104MF-367036897202412025040depending upon the nature of the sample and subsequent steps in the analytical method. In certain embodiments, the sample is attached to the substrate reversibly by applying a suitable polymer coating to the substrate, and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers that are suitable for this purpose. In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.

[0267] A variety of steps can be performed to prepare or process a biological sample for and / or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.(i) Preparation

[0268] A biological sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro on a growth substrate or culture dish as a population of cells, and prepared for analysis as a tissue slice or tissue section. Grown samples may be sufficiently thin for analysis without further processing steps. Alternatively, grown samples, and samples obtained via biopsy or sectioning, can be prepared as thin tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome. As another alternative, in some embodiments, a thin tissue section can be prepared by applying a touch imprint of a biological sample to a suitable substrate material.

[0269] The thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell. However, tissue sections having a thickness that is larger than the maximum cross-section cell dimension can also be used. For example, cryostat sections can be used, which can be, e.g., 10-20 pm thick. More generally, the thickness of a tissue section typically depends on the method used to prepare the section and the physical characteristics of the tissue, and therefore sections having a wide variety of different thicknesses can be prepared and used. For example, the thickness of the105MF-367036897202412025040tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 μm. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 μm or more. Typically, the thickness of a tissue section is between 1-100 μm, 1-50 μm, 1-30 μm, 1-25 μm, 1-20 μm, 1-15 μm, 1-10 μm, 2-8 μm, 3-7 μm, or 4-6 μm, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analysed.

[0270] Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can be analysed successively to obtain three-dimensional information about the biological sample.

[0271] In some embodiments, the biological sample (e.g., a tissue section as described above) is prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (e.g., the physical characteristics) of the tissue structure. The frozen tissue sample can be sectioned, e.g., thinly sliced, onto a substrate surface using any number of suitable methods. For example, a tissue sample can be prepared using a chilled microtome (e.g., a cryostat) set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. Such a temperature can be, e.g., less than -15°C, less than -20°C, or less than -25°C.

[0272] In some embodiments, the biological sample are prepared using formalinfixation and paraffin-embedding (FFPE). In some embodiments, cell suspensions and other nontissue samples can be prepared using formalin-fixation and paraffin-embedding. Following fixation of the sample and embedding in a paraffin or resin block, the sample can be sectioned as described above. Prior to analysis, the paraffin-embedding material can be removed from the tissue section (e.g., de-paraffinization) by incubating the tissue section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes). In some embodiments, the biological sample (e.g., FFPE sample) is permeable after de-paraffinization. In some embodiments, processing of the biological sample, such as de-waxing, allows the biological sample to become permeabilized.106MF-367036897202412025040

[0273] As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0274] In some embodiments, the methods provided herein comprises one or more post-fixing (also referred to as postfixation) steps. In some embodiments, one or more postfixing step is performed after contacting a sample with a polynucleotide disclosed herein, e.g., one or more probes such as a circular or padlock probe. In some embodiments, one or more post-fixing step is performed after a hybridization complex comprising a probe and a target is formed in a sample. In some embodiments, one or more post-fixing step is performed prior to a ligation reaction disclosed herein.

[0275] In some embodiments, a method disclosed herein comprises de-crosslinking the reversibly cross-linked biological sample. The de-crosslinking does not need to be complete. In some embodiments, only a portion of crosslinked molecules in the reversibly cross-linked biological sample are de-crosslinked and allowed to migrate.

[0276] In some embodiments, a biological sample is permeabilized to facilitate transfer of species (such as probes) into the sample. If a sample is not permeabilized sufficiently, the transfer of species (such as probes) into the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable.

[0277] In general, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100™ or Tween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample is incubated with a cellular permeabilizing agent to facilitate permeabilization of the sample. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63- 107MF-36703689720241202504066, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in connection with the samples described herein.

[0278] In some embodiments, the biological sample can be permeabilized by any suitable methods. In some embodiments, the biological sample is a permeable biological sample. For example, one or more lysis reagents can be added to the sample. Examples of suitable lysis agents include, but are not limited to, bioactive reagents such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be added to the biological sample to facilitate permeabilization. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chao tropic agents.

[0279] Additional reagents can be added to a biological sample to perform various functions prior to analysis of the sample. In some embodiments, DNase and RNase inactivating agents or inhibitors such as proteinase K, and / or chelating agents such as EDTA, is added to the sample. For example, a method disclosed herein may comprise a step for increasing accessibility of a nucleic acid for binding, e.g., a denaturation step to open up DNA in a cell for hybridization by a probe. For example, proteinase K treatment may be used to free up DNA with proteins bound thereto.(ii) Embedding

[0280] In some embodiments, the biological sample is embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.108MF-367036897202412025040Biological samples can include analytes (e.g., protein, RNA, and / or DNA) embedded in a 3D matrix. In some embodiments, amplicons (e.g., rolling circle amplification products) derived from or associated with analytes (e.g., protein, RNA, and / or DNA) can be embedded in a 3D matrix. In some embodiments, a 3D matrix may comprise a network of natural molecules and / or synthetic molecules that are chemically and / or enzymatically linked, e.g., by crosslinking. In some embodiments, a 3D matrix may comprise a synthetic polymer. In some embodiments, a 3D matrix comprises a hydrogel.

[0281] In some aspects, a biological sample can be embedded in any of a variety of other embedding materials to provide structural substrate to the sample prior to sectioning and other handling steps. In some cases, the embedding material is removed e.g., prior to analysis of tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylate resins), epoxies, and agar.

[0282] In some embodiments, the biological sample is embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.

[0283] In some embodiments, the biological sample is immobilized in the hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and / or photochemically, or alternatively by any other suitable hydrogelformation method.

[0284] In some embodiments, the biological sample is reversibly cross-linked prior to or during an in situ assay. In some aspects, the analytes, polynucleotides and / or amplification product (e.g., amplicon) of an analyte or a probe bound thereto can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more of the polynucleotide probe(s) and / or amplification product (e.g., amplicon) thereof can be modified to contain functional groups that can be used as an anchoring site to attach the polynucleotide probes and / or amplification product to a polymer matrix. In some embodiments,109MF-367036897202412025040a modified probe comprising oligo dT may be used to bind to mRNA molecules of interest, followed by reversible or irreversible crosslinking of the mRNA molecules.

[0285] In some embodiments, the biological sample is immobilized in a hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and / or photochemically, or alternatively by any other suitable hydrogel-formation method. A hydrogel may include a macromolecular polymer gel including a network. Within the network, some polymer chains can optionally be cross-linked, although cross-linking does not always occur.

[0286] In some embodiments, a hydrogel can include hydrogel subunits, such as, but not limited to, acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, polyethylene glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly (hydroxy ethyl acrylate), and poly (hydroxy ethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof.

[0287] In some embodiments, a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in U. S. Patent Nos. 6,391,937 and materials for sample expansion as described, for example, in U. S. Patent Application Publication Nos.2017 / 0253918 and 2018 / 0052081, the entire contents of each of which are incorporated herein by reference.

[0288] The composition and application of the hydrogel-matrix to a biological sample typically depends on the nature and preparation of the biological sample (e.g., sectioned, non- sectioned, type of fixation). As one example, where the biological sample is a tissue section, the hydrogel-matrix can include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, where the biological sample consists of cells (e.g., cultured cells or cells disassociated from a tissue 110MF-367036897202412025040sample), the cells can be incubated with the monomer solution and APS / TEMED solutions. For cells, hydrogel-matrix gels are formed in compartments, including but not limited to devices used to culture, maintain, or transport the cells. For example, hydrogel-matrices can be formed with monomer solution plus APS / TEMED added to the compartment to a depth ranging from about 0.1 pm to about 2 mm.

[0289] Additional methods and aspects of hydrogel embedding of biological samples are described for example in Chen et al., Science 347(6221):543–548, 2015, the entire contents of which are incorporated herein by reference.

[0290] In some embodiments, the hydrogel can form the substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on top of one or more second materials. For example, the hydrogel can be pre-formed and then placed on top of, underneath, or in any other configuration with one or more second materials. In some embodiments, hydrogel formation occurs after contacting one or more second materials during formation of the substrate. Hydrogel formation can also occur within a structure (e.g., wells, ridges, projections, and / or markings) located on a substrate.

[0291] In some embodiments, hydrogel formation on a substrate occurs before, contemporaneously with, or after probes are provided to the sample. For example, hydrogel formation can be performed on the substrate already containing the probes.

[0292] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., tissue section) is embedded in a hydrogel. In some embodiments, hydrogel subunits are infused into the biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

[0293] In embodiments in which a hydrogel is formed within a biological sample, functionalization chemistry can be used. In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC). Any hydrogel-tissue backbone (e.g., synthetic or native) suitable for HTC can be used for anchoring biological macromolecules and modulating functionalization. Non-limiting examples of methods using HTC backbone variants include CLARITY, PACT, ExM, SWITCH and ePACT. In some embodiments, hydrogel formation 111MF-367036897202412025040within a biological sample is permanent. For example, biological macromolecules can permanently adhere to the hydrogel allowing multiple rounds of interrogation. In some embodiments, hydrogel formation within a biological sample is reversible. In some embodiments, HTC reagents are added to the hydrogel before, contemporaneously with, and / or after polymerization. In some embodiments, a cell labelling agent is added to the hydrogel before, contemporaneously with, and / or after polymerization. In some embodiments, a cellpenetrating agent is added to the hydrogel before, contemporaneously with, and / or after polymerization.

[0294] In some embodiments, additional reagents are added to the hydrogel subunits before, contemporaneously with, and / or after polymerization. For example, additional reagents can include but are not limited to oligonucleotides (e.g., probes), endonucleases to fragment DNA, fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used to amplify the nucleic acid and to attach the barcode to the amplified fragments. Other enzymes can be used, including without limitation, RNA polymerase, ligase, proteinase K, and DNAse. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers, and oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, contemporaneously with, and / or after polymerization.

[0295] Hydrogels embedded within biological samples can be cleared using any suitable method. For example, electrophoretic tissue clearing methods can be used to remove biological macromolecules from the hydrogel-embedded sample. In some embodiments, a hydrogel-embedded sample is stored before or after clearing of hydrogel, in a medium (e.g., a mounting medium, methylcellulose, or other semi-solid mediums).

[0296] In some embodiments, a biological sample embedded in a matrix (e.g., a hydrogel) is isometrically expanded. Isometric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in, e.g., Chen et al., Science 347(6221):543–548, 2015 and U. S. Pat. 10,059,990, which are herein incorporated by reference in their entireties. Isometric expansion of the sample can increase the spatial resolution of the subsequent analysis of the sample. The increased resolution in spatial profiling can be determined by comparison of an isometrically expanded sample with a sample that has not been112MF-367036897202412025040isometrically expanded. In some embodiments, a biological sample is isometrically expanded to a size at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its nonexpanded size. In some embodiments, the sample is isometrically expanded to at least 2x and less than 20x of its non-expanded size.(iii) Staining and Immunohistochemistry (IHC)

[0297] To facilitate visualization, biological samples can be stained using a wide variety of stains and staining techniques. In some embodiments, for example, a sample can be stained using any number of stains and / or immunohistochemical reagents. One or more staining steps may be performed to prepare or process a biological sample for an assay described herein or may be performed during and / or after an assay. In some embodiments, the sample is contacted with one or more nucleic acid stains, membrane stains (e.g., cellular or nuclear membrane), cytological stains, or combinations thereof. In some examples, the stain may be specific to proteins, phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, an organelle or compartment of the cell. The sample may be contacted with one or more labeled antibodies (e.g., a primary antibody specific for the analyte of interest and a labeled secondary antibody specific for the primary antibody). In some embodiments, cells in the sample is segmented using one or more images taken of the stained sample.

[0298] In some embodiments, the stain is performed using a lipophilic dye. In some examples, the staining is performed with a lipophilic carbocyanine or aminostyryl dye, or analogs thereof (e.g, Dil, DiO, DiR, DiD). Other cell membrane stains may include FM and RH dyes or immunohistochemical reagents specific for cell membrane proteins. In some examples, the stain may include but is not limited to, acridine orange, acid fuchsin, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamine B), or safranine, or derivatives thereof. In some embodiments, the sample may be stained with haematoxylin and eosin (H& E).113MF-367036897202412025040

[0299] The sample can be stained using hematoxylin and eosin (H& E) staining techniques, using Papanicolaou staining techniques, Masson’s trichrome staining techniques, silver staining techniques, Sudan staining techniques, and / or using Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some embodiments, the sample can be stained using Romanowsky stain, including Wright’s stain, Jenner’s stain, Can-Grunwald stain, Leishman stain, and Giemsa stain.

[0300] In some embodiments, biological samples is destained. Any suitable methods of destaining or discoloring a biological sample may be utilized and generally depend on the nature of the stain(s) applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567–75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899–905, the entire contents of each of which are incorporated herein by reference.V. COMPOSITIONS, SYSTEMS AND KITS

[0301] In some embodiments, provided herein are compositions, systems or kits, for example comprising a catalyst compound provided in a first buffer and a second buffer (e.g., as described in Section II) for de-crosslinking of a fixed biological sample. In some embodiments, the compound is provided in a composition (e.g., a composition comprising DMSO) and a kit comprises one or more other compositions, e.g., a buffer for the compound. In some examples, a solution or a suspension comprising the catalyst and a plurality of buffers are provided. In some embodiments, provided herein are compositions, systems or kits comprising one or more reagents required for one or more steps comprising hybridization, ligation, extension, detection, and / or sample preparation as described herein. In some embodiments, the compositions, systems or kits, comprises one or more labelling agents and a plurality of nucleic acid probes (e.g., disclosed in Section II). In some embodiments, the compositions, systems or kits comprises detectably labeled probes for detecting one or more nucleic acid analytes and / or one or more 114MF-367036897202412025040non-nucleic acid analytes. In some embodiments, the compositions, systems or kits, comprises one or more antibodies (e.g., for detecting protein analytes) which can be optionally labelled with a detectable label such as a fluorophore and / or a reporter oligonucleotide.

[0302] In some aspects, provided herein are compositions comprising any of reagents for detecting labelling agents described herein (e.g., detectably labeled probes). Also provided herein are kits and systems for detecting labelling agents and stripping of associated reagents according to any of the methods described herein. In some embodiments, the kit or system is for multiplexed RNA and protein detection in the biological sample. In some aspects, the system or kit also comprises reagents for removal of reagents for detecting labelling agents and amplification products associated with RNA analytes, e.g., a wash buffer.

[0303] Provided herein is a system comprising a biological sample comprising cells on a solid support; a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks; a second buffer comprising Tris and a chelating agent; a plurality of circularizable probes configured to bind a plurality of RNAs; and a plurality of labelling agents configured to bind to a plurality of protein analytes. In some instances, the system further comprises a heater configured to receive the biological sample. In some instances, the chelating agent in the second buffer is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE).

[0304] In some instances, the system further comprises a instrument having integrated optics and fluidics modules (an “opto-fluidic instrument” or “opto-fluidic system”) for detecting target molecules (e.g., nucleic acids and proteins) in biological sample as described herein. In some aspects, the instrument is for generating and / or detecting optical signals as described in Section II at locations in the biological sample. In some embodiments, the fluidics module of the opto-fluidic instrument is configured to deliver one or more reagents (e.g., detectably labeled probes) to the biological sample and / or remove spent reagents therefrom. Additionally, the optics module of the opto-fluidic instrument is configured to illuminate the biological sample with light having one or more spectral emission curves (over a range of wavelengths) and subsequently capture one or more images of emitted light signals from the biological sample during one or more probing cycles (e.g., as described in Section II). In various embodiments, the captured images may be processed in real time and / or at a later 115MF-367036897202412025040time to determine the presence of the one or more target molecules in the biological sample, as well as three-dimensional position information associated with each detected target molecule. Additionally, the opto-fluidics instrument includes a sample module configured to receive (and, optionally, secure) one or more biological samples. In some instances, the sample module includes an X-Y stage configured to move the biological sample along an X-Y plane (e.g., perpendicular to an objective lens of the optics module). In some instances, the opto-fluidic instrument is an in-situ analysis system used to analyze a biological sample and detect target molecules including but not limited to DNA, RNA, proteins, antibodies, and / or the like. In some embodiments, the in situ analysis system is used to detect one or more amplification products generated according to the methods disclosed herein.

[0305] In some instances, the catalyst is a compound of formula (I) as described in Section II. C. In some instances, the catalyst comprises one or more compounds selected from thegroup consisting of116MF-367036897202412025040

[0306] In some embodiments, the compositions, systems or kits comprise a polymerase and dNTPs for performing rolling circle amplification (RCA) using the plurality of circularizable probes. In some instances, the compositions, systems or kits comprises reagents for detecting one or more sequence of the plurality of circularizable probes. In some cases, the reagents for detecting one or more sequences of the plurality of circularizable probes comprises a plurality of detectably labeled probes for binding to a sequence of an amplification product generated using RCA. In some embodiments, the compositions, systems or kits comprise reagents for detecting the plurality of labelling agents. In some instances, the reagents for detecting the plurality of labelling agents comprises a plurality of detectably labeled probes for binding to a reporter oligonucleotide of the plurality of labelling agents. In some instances, the reagents for detecting the plurality of labelling agents comprises a plurality of intermediate probes, wherein the intermediate probes comprise a sequence for binding to detectably labeled probes.

[0307] In some instances, the compositions, systems or kits comprises reagents for sequencing. In some instances, the compositions, systems or kits comprise a ligase for forming a plurality of circularized probes from the plurality of circularizable probes.

[0308] Provided herein is a kit comprising a solid support; a first buffer comprising a catalyst that catalyzes de-crosslinking of molecular crosslinks; a second buffer comprising Tris 117MF-367036897202412025040and a chelating agent; and a plurality of circularizable probes configured to bind a plurality of RNAs. In some embodiments, the kit comprises a plurality of labelling agents configured to bind a plurality of protein analytes. In some embodiments, the kit comprises a plurality of detectably labeled probes for binding to a sequence of a reporter oligonucleotide of the plurality of labelling agents.

[0309] The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the kits further contain instructions for using the components of the kit to practice the provided methods.

[0310] In some embodiments, the kits can contain reagents and / or consumables required for performing one or more steps of the provided methods. In some embodiments, the kits contain reagents for fixing (e.g., crosslinking), embedding, and / or permeabilizing the biological sample. In some embodiments, the kits contain reagents, such as enzymes and buffers. In some aspects, the kit can also comprise any of the reagents described herein, e.g., wash buffer. In some embodiments, the kits contain reagents for detection and / or sequencing, such as barcode detection probes or detectable labels. In some embodiments, the kits optionally contain other components, for example nucleic acid primers, enzymes and reagents, buffers, nucleotides, and reagents for additional assays.VI. APPLICATIONS

[0311] In some aspects, the provided embodiments can be applied in an in situ method of analyzing nucleic acid sequences, such as fluorescent in situ hybridization (FISH)-based methods, in situ transcriptomic analysis or in situ sequencing, for example from intact tissues or samples in which the spatial information has been preserved. In some aspects, the embodiments can be applied in an imaging or detection method for multiplexed nucleic acid analysis. In some aspects, the provided embodiments can be used to detect a signal associated with a detectable label of a nucleic acid probe that is hybridized to a target sequence of a target nucleic acid in a biological sample.

[0312] In some embodiments, the target nucleic acid comprises a single-nucleotide polymorphism (SNP). In some embodiments, the target nucleic acid comprises is a single-nucleotide variant (SNV). In some embodiments, the target nucleic acid comprises a single- 118MF-367036897202412025040nucleotide substitution. In some embodiments, the target nucleic acid comprises a point mutation. In some embodiments, the target nucleic acid comprises a single-nucleotide insertion.

[0313] In some aspects, the embodiments can be applied in investigative and / or diagnostic applications, for example, for characterization or assessment of particular cell or a tissue from a subject. Applications of the provided method can comprise biomedical research and clinical diagnostics. For example, in biomedical research, applications comprise, but are not limited to, spatially resolved gene expression analysis for biological investigation or drug screening. In clinical diagnostics, applications comprise, but are not limited to, detecting gene markers such as disease, immune responses, bacterial or viral DNA / RNA for patient samples.

[0314] In some aspects, the embodiments can be applied to visualize the distribution of genetically encoded markers in whole tissue at subcellular resolution, for example, chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of gene alleles indicative of a predisposition towards disease or good health, likelihood of responsiveness to therapy, or in personalized medicine or ancestry.VII. TERMINOLOGY

[0315] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0316] The terms "polynucleotide" and "nucleic acid molecule", used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term comprises, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.119MF-367036897202412025040

[0317] A “primer” as used herein, in some embodiments, is an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers usually are extended by a DNA polymerase.

[0318] In some instances, “ligation” refers to the formation of a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and / or polynucleotides, in a template-driven reaction. The nature of the bond or linkage may vary widely and the ligation, in some embodiments, is carried out enzymatically or chemically. As used herein, ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon terminal nucleotide of one oligonucleotide with a 3' carbon of another nucleotide.

[0319] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein comprises (and describes) embodiments that are directed to that value or parameter per se.

[0320] As used herein, the singular forms "a," "an," and "the" comprise plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0321] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be comprised in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any 120MF-367036897202412025040specifically excluded limit in the stated range. Where the stated range comprises one or both of the limits, ranges excluding either or both of those comprised limits are also comprised in the claimed subject matter. This applies regardless of the breadth of the range.

[0322] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.VIII. EXAMPLES

[0323] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.Example 1: FFPE Sample Preparation and Treatment

[0324] Formalin-fixed, paraffin-embedded (FFPE) tissue samples including breast and tonsil tissue samples were cut into thin tissue sections. The sections were placed on slides and dried at a temperature higher than room temperature to dehydrate the tissue samples. The sections were de-paraffinized using xylene and re-hydrated using an ethanol series (e.g., 96% ethanol followed by 70% ethanol) and nuclease free water.

[0325] De-crosslinking of the tissue sections was performed using a catalyst disclosed herein in a first buffer solution. Specifically, 2-amino-5-methylbenzoic acid (Compound 1 at 150 mM) was used as the de-crosslinking agent and applied to de-paraffinized and re-hydrated samples in a first buffer solution (pH 7 citrate buffer comprising DMSO) at 80°C for 30 minutes. Then, circularizable probes (e.g., padlock probes) targeting a panel of more than 300 RNA transcripts were added in hybridization buffers (e.g., including Saline-Sodium Citrate buffer (SSC) and formamide) and incubated with the samples to allow hybridization of the circularizable probes to their target nucleic acids. In addition to a target hybridization region, each circularizable probe also contained a barcode region. Then, the probe 121MF-367036897202412025040hybridization mixture was removed and the samples were washed. For ligation of the circularizable probes hybridized to their target nucleic acids, a ligation reaction mix (e.g., containing a ligase buffer, RNase inhibitor and ligase) and rolling circle amplification (RCA) primers were added to the samples and incubated for probe circularization and RCA primer hybridization to the probes. The samples were washed and an RCA reaction mixture (containing Phi29 reaction buffer, dNTPs, Phi29 polymerase) was added and incubated for RCA of the circularized probes.

[0326] After generating RCA products, the tissue samples were dehydrated and baked at 37°C for 5 minutes. It was shown in a previous experiment that baking prevented the tissue sections from detaching from the slides. After the baking, treatment with a second buffer (“+ Second buffer treatment”) at 90°C for 10 minutes in a second buffer (TE buffer pH 9.0) was performed. For control, some samples (“no second buffer treatment”) did not undergo the treatment with TE at 90°C for 10 minutes. Another condition (TE with Compound 1) was performed to test incubation with TE buffer (90°C for 10 minutes), except provided with the catalytic de-crosslinking with Compound 1 before hybridizing circularizable probes and generating RCA products. After de-crosslinking and / or treatment with the TE buffer, tissue samples were blocked with a buffer (1% BSA) for 1 hour at room temperature. Labelling agents conjugated to reporter oligonucleotides for detecting 4 different protein analytes were added to the tissue samples and incubated overnight at 4°C.

[0327] The samples were washed (e.g., in PBST) and treated with a fixative, and detectable probes in a hybridization buffer (e.g., containing SSC and formamide) were hybridized to RCA products (RCPs) in the sample. The detectable probes included probes that hybridize to sequences (e.g., barcode sequences) in the RCPs and comprise overhangs for hybridization of fluorescently labelled detection oligonucleotides. The samples were imaged in fluorescent microscope with 20x objective and the signals associated with the RCPs were quantified using a software. After detection of the RCPs, the samples were imaged to detect signals of detectably labeled probes (e.g., fluorescently labeled) bound to reporter oligonucleotides conjugated to the antibodies.

[0328] FIG. 2A and 2B shows images of signals detected for labelling agents associated with CD68 in tonsil tissue sample and HER2 in breast cancer tissue sample,122MF-367036897202412025040respectively. Signals detected from probes associated with labelling agents bound to proteins were greater in intensity in tissue samples treated with the second buffer (“+ Second buffer treatment”) compared to samples that did not undergo incubation with the TE buffer at 90°C for 10 minutes (“no second buffer treatment”). In addition, comparable sensitivity for RNA detection was observed and no significant differences were observed in genomic DNA control metrics between the two de-crosslinking conditions. Decreased RCP brightness and increased brightness decay between cycles was observed associated with the RNAs detected. Signals detected from probes associated with labelling agents bound to proteins were also greater in intensity in tissue samples treated with the second buffer (“+ Second buffer treatment”) compared to samples incubated with TE simultaneously during catalytic de-crosslinking with Compound 1. This data supports that the separate and additional treatment with TE as the second buffer after hybridizing circularizable probes and generating RCA products improved protein detection.

[0329] Together these results demonstrate that catalytic de-crosslinking (e.g., at 80°C for 20 minutes) combined with a treatment with a TE buffer of pH 9 (e.g., at 90°C for 10 minutes) post RCP generation rescued dim protein staining signals detected in FFPE human tissues samples in situ without compromising tissue integrity and / or adhesion.Example 2: Analyte Detection in FFPE Samples using Various Treatment Conditions

[0330] FFPE human tonsil tissues were processed essentially as described in Example 1. For the treatment with a second buffer performed after generating the RCA products, five different treatments were tested to optimize the conditions for RNA detection, including: Condition A) 20 minute incubation at 80°C with TE buffer pH 9; Condition B) 20 minute incubation at 80°C with TE buffer pH 8; Condition C) 10 minute incubation at 90°C with TE buffer pH 9; Condition D) 10 minute incubation at 90°C with TE buffer pH 8; and Condition E) 10 minute incubation at 90°C with citrate buffer pH 6.

[0331] A panel of 1,000 RNA analytes were targeted by circularizable probes and a panel of 16 protein analytes were targeted by labelling agents (e.g., antibodies conjugated to reporter oligonucleotides). The protein panel targeted CD4, CD3E, CD8a, CDllc, CD20, CD68, CD31, CD138, Granzyme B, PCNA, CD45RA, CD45R0, PanCK, PTEN, HLA-DR, PD-1. The 123MF-367036897202412025040samples were first imaged to detect signals of detectably labeled probes (e.g., fluorescently labeled) bound to reporter oligonucleotides conjugated to the antibodies. Between cycles of protein detection, the samples were stripped (e.g., using a denaturing agent) and contacted with additional detectably labeled probes for the next imaging round (4 were detected in each cycle, 4 detection cycles total). After protein detection, generated RCPs associated with RNA analytes in the de-crosslinked tissue samples were detected essentially as described in Example 1.

[0332] In summary, a 20 minute incubation at 80°C with TE buffer pH 9 (Condition A) showed superior performance across the tested antibodies compared to any of the other tested conditions for the treatment with the second buffer. Weaker signals associated with protein staining was observed in conditions using citrate buffer pH 6 at 90°C and TE buffer pH 8 at 80°C. In particular, sensitivity (30% higher) and brightness (50% higher) of signals associated with the RCPs was highest in Condition A (FIG. 3), and negative controls including observed genomic DNA metrics and signal decay were lowest in Condition A (FIG.4). This experiment showed that the 20 minute incubation at 80°C with TE buffer pH 9 enhanced protein detection without having negative impact on RNA.

[0333] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.124MF-367036897

Claims

202412025040CLAIMS1. A method, comprising:(a) contacting a biological sample comprising cells with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample;(b) after de-crosslinking with the catalyst, contacting the biological sample with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells;(c) after binding the plurality of nucleic acid probes to the plurality of RNAs, incubating the biological sample with a second buffer at a temperature of at least 80°C for at least 10 minutes;(d) contacting the biological sample with a labelling agent that directly or indirectly binds to an analyte at a location in the biological sample; and(e) detecting:(i) a plurality of optical signals associated with the plurality of nucleic acid probes or products thereof associated with the plurality of RNAs in the biological sample, and (ii) an optical signal associated with the labelling agent or a product thereof in the biological sample.

2. The method of claim 1, wherein the molecular crosslinks are products of one or more crosslinking agents.

3. The method of claim 2, wherein the one or more crosslinking agents comprise an aldehyde, optionally wherein the crosslinking agent comprises formaldehyde.

4. The method of any one of claims 1-3, wherein the molecular crosslinks are on RNA, DNA, protein, carbohydrate, lipid, and / or other molecules in the biological sample.

5. The method of any one of claims 1-4, wherein the second buffer comprises Tris and a chelating agent and / or a buffering agent.

6. The method of claim 5, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE).MF-3670368972024120250407. The method of any one of claims 1-6, wherein the catalyst is a water-soluble catalyst.

8. The method of any one of claims 1-7, wherein the catalyst is an organic molecule.

9. The method of any one of claims 1-8, wherein the catalyst is a transimination catalyst.

10. The method of any one of claims 1-9, wherein the catalyst catalyzes de-crosslinking of aminal crosslinks in the biological sample.

11. The method of any one of claims 1-10, wherein the catalyst catalyzes breakdown of hemi-aminal adducts and / or aminal adducts in the biological sample.

12. The method of any one of claims 1-11, wherein the catalyst is a compound of formula (I),'24 2(I).or a salt, zwitterion, or solvate thereof, wherein:A is selected from the group consisting of -COOH, -P(=O)(OH)2, and S(=O)2OH;X1, X2, X3, and X4are each independently selected from the group consisting of: CH, CRa, and N;each occurrence of Rais independently selected from the group consisting of C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, -NO2, -NR’R”, and -C(=O)NR’R”; andeach occurrence of R’ and R” is independently selected from the group consisting of HO'and Ci-6 alkyl which is optionally substituted withn1, wherein nl is an integer from 12 to 16.126MF-36703689720241202504013. The method of any one of claims 1-12, wherein the catalyst comprises one or more 0. A J d'" " OH compounds selected from the group consisting of014. The method of any one of claims 1-12, wherein the catalyst comprises, or a salt, zwitterion, or solvate thereof.127MF-3670368972024120250400 if HO~P^ HO' |j H'N 15. The method of any one of claims 1-12, wherein the catalyst comprises2or a salt, zwitterion, or solvate thereof.

16. The method of any one of claims 1-12, wherein the catalyst comprisesor a salt, zwitterion, or solvate thereof.

17. The method of any one of claims 1-12, wherein the catalyst comprises ^^NH2, or a combination thereof, or a salt, zwitterion, or18. The method of airy one of claims 1-11, wherein the catalyst is a compound of formula (II),R1or a salt, zwitterion, or solvate thereof, wherein:L1is selected from the group consisting of -O-, -N(H)-, -N(CI-3alkyl)-, -N(CH2CH2O)1-10-CH3-, -S(0)O-2-, -CH2-, and a bond;128MF-367036897202412025040R1is selected from the group consisting of: H; Ci-6 alkyl; Ci-6 haloalkyl; C6-10 aryl optionally substituted with 1-4 Rb; and 5- to 10-membered heteroaryl, wherein 1-4 ring atoms are heteroatoms each independently selected from the group consisting of: N, N(H), N(CI-3 alkyl), O, and S, wherein the heteroaryl is optionally substituted with 1-4 independently selected Rb; andeach Rbis independently selected from the group consisting of: halo, cyano, -OH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, C1-6 alkyl, C1-6 haloalkyl, C1-4 alkoxy, and C1-4 haloalkoxy.HQ,O^°H19. The method of claim 16, wherein the catalyst comprisesOHO HO HO,>O / 0HO ^ / 0H / 0Hy to Y ■"<H O H Q H O, or a salt, zwitterion, or solvate thereof.

20. The method of any one of claims 1-19, wherein the biological sample is on a substrate with a planar surface.

21. The method of claim 19 or claim 20, wherein the substrate is transparent.

22. The method of any one of claims 19-21, wherein the substrate is a glass slide or a plastic slide.

23. The method of any one of claims 19-22, wherein the substrate does not comprise nucleic acids immobilized thereon prior to contacting the biological sample.

24. The method of any one of claims 1-23, wherein the biological sample is a tissue section.

25. The method of any one of claims 1-23, wherein the biological sample comprise cells immobilized on the substrate.MF-36703689720241202504026. The method of claim 25, wherein the cells are dissociated cells, cultured cells, and / or cells isolated from a subject.

27. The method of any one of claims 1-26, wherein the biological sample is a fixed and / or permeabilized biological sample.

28. The method of claim 27, wherein the biological sample is an aldehyde-fixed biological sample.

29. The method of any one of claims 1-28, wherein the biological sample is a formaldehyde- fixed biological sample.

30. The method of any one of claims 1-29, wherein the biological sample is a paraffinized biological sample.

31. The method of claim 30, wherein the biological sample is a formaldehyde-fixed paraffin- embedded (FFPE) biological sample.

32. The method of any one of claims 1-29, wherein the biological sample is a fresh frozen biological sample that has been cross linked.

33. The method of any one of claims 1-32, wherein the method comprises dehydrating the biological sample prior to incubating the biological sample with the second buffer.

34. The method of claim 33, wherein the method comprises dehydrating the biological sample between (b) and (c).

35. The method of claim 34, wherein the dehydrating comprises drying the biological sample at 42°C for 3 hours or drying the biological sample at room temperature overnight.

36. The method of any one of claims 1-35, wherein the method comprises baking the biological sample after contacting the biological sample with a plurality of nucleic acid probes and prior to incubating the biological sample with the second buffer.

37. The method of claim 36, wherein the baking is performed at 37°C for at least 5 minutes.MF-36703689720241202504038. The method of any one of claims 1-37, wherein the method comprises de-paraffinizing the biological sample prior to contacting the biological sample with the catalyst.

39. The method of claim 38, wherein the de-paraffinizing comprises contacting the biological sample with xylene, ethanol, and water, or, sequentially contacting the biological sample with xylene, absolute ethanol, about 96% ethanol, and about 70% ethanol.

40. The method of any one of claims 1-39, wherein the method comprises, prior to contacting the biological sample with the catalyst, a step of re-hydrating the biological sample, optionally wherein the re- hydrating comprises sequentially contacting the biological sample with 100% ethanol, 100% ethanol, 96% ethanol, 70% ethanol, each for 3 minutes, followed by contacting the biological sample with nuclease free water for 20 seconds.

41. The method of any one of claims 1-40, wherein the method comprises, prior to contacting the biological sample with the catalyst, pretreating the biological sample with a proteinase.

42. The method of any one of claims 1-41, wherein the method comprises, prior to contacting the biological sample with the catalyst, permeabilizing the biological sample.

43. The method of any one of claims 1-42, wherein the catalyst is contacted with the biological sample at a concentration between about 5 mM and about 500 mM.

44. The method of any one of claims 1-42, wherein the catalyst is contacted with the biological sample at a concentration between about 100 mM and about 200 mM.

45. The method of any one of claims 1-44, wherein the biological sample is catalytically decrosslinked at a temperature between about 70 °C and about 90 °C.

46. The method of any one of claims 1-45, wherein the catalyst is contacted with the biological sample for between 10 minutes to 60 minutes.

47. The method of any one of claims 1-46, wherein the catalyst is contacted with the biological sample at about 80°C for 30 minutes.131MF-36703689720241202504048. The method of any one of claims 1-47, wherein the biological sample is incubated with the second buffer for at least 15 minutes.

49. The method of any one of claims 1-47, wherein the biological sample is incubated with the second buffer for about 10 minutes to about 20 minutes.

50. The method of any one of claims 1-49, wherein the second buffer has a pH between about 8 and about 9.

51. The method of any one of claims 1-49, wherein the second buffer has a pH of at least 8.

52. The method of any one of claims 1-51, wherein the second buffer has a pH of about 9.

53. The method of any one of claims 1-52, wherein the first buffer comprises citrate, tris(hydroxymethyl)aminomethane (Tris), phosphate-buffered saline (PBS), 2-[4~(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof.

54. The method of any one of claims 1-53, wherein the first buffer comprises dimethyl sulfoxide ( DMSO).

55. The method of any one of claims 1-54, wherein the first buffer comprises citrate and the second buffer is a Tris-EDTA (TE) buffer.

56. The method of any one of claims 1-55, wherein the first buffer has a pH of less than 8.

57. The method of claim 56, wherein the first buffer is a citrate buffer between pH 5 and pH 7.

58. The method of any one of claims 1-57, wherein the method comprises washing the biological sample after contacting the biological sample with the catalyst and / or after incubating with the second buffer.

59. The method of any one of claims 1-58, wherein the method comprises washing the biological sample after contacting the biological sample with the labelling agent.MF-36703689720241202504060. The method of claim 59, wherein the washing comprises washing the biological sample in phosphate-buffered saline with Tween detergent (PBST).

61. The method of any one of claims 1-60, wherein the biological sample is contacted with a blocking buffer prior to contacting the biological sample with the labelling agent.

62. The method of any one of claims 1-61, wherein the labelling agent comprises a binding moiety, wherein the binding moiety comprises an antibody or epitope binding fragment thereof.

63. The method of any one of claims 1-62, wherein the biological sample is contacted with a plurality of labelling agents in (d)64. The method of any one of claims 1-63, wherein the biological sample is contacted with a fixative after contacting the biological sample with the labelling agent.

65. The method of any one of claims 1-64, wherein the biological sample is contacted with a quencher after contacting the biological sample with the labelling agent and before the detecting.

66. The method of claim 65, wherein the quencher comprises a quencher dye.

67. The method of claim 66, wherein the quencher dye comprises:(a) at least three aromatic residues, wherein each aromatic residue is independently an unsubstituted aryl, a substituted aryl, an unsubstituted heteroaryl, or a substituted heteroaryl, wherein at least one of said aromatic residues is covalently linked to two other aromatic residues via two exocyclic azo bonds; or(b) at least two aromatic residues, wherein each aromatic residue is independently an unsubstituted aryl, a substituted aryl, an unsubstituted heteroaryl, or a substituted heteroaryl, wherein at least two of said aromatic residues are covalently linked via an exocyclic azo bond, and wherein at least one said aromatic residue is an unsubstituted polycyclic aryl, a substituted polycyclic aryl, an unsubstituted polycyclic heteroaryl group, or a substituted polycyclic heteroaryl group.

68. The method of claim 65 or claim 66. wherein the quencher comprises a targeting moiety, wherein the targeting moiety binds and / or reacts with a biological moiety.MF-36703689720241202504069. The method of claim 68, wherein the biological moiety is endogenous to the biological sample.

70. The method of claim 69, wherein the biological moiety is a polypeptide or a lipid.

71. The method of any one of claims 1-70, wherein the labelling agent comprises a detectable label detected in (e).

72. The method of any one of claims 1-70, wherein the labelling agent is bound to a nucleic acid comprising a detectable label.

73. The method of any one of claims 1-72, wherein the labelling agent comprises a reporter oligonucleotide, optionally wherein the reporter oligonucleotide comprises a barcode sequence.

74. The method of claim 73, wherein the barcode sequence or complement thereof is detected by hybridizing an intermediate probe to the barcode sequence or complement thereof, hybridizing a detectably labeled probe to the intermediate probe, and detecting the detectably labeled probe.

75. The method of any one of claims 1-74, wherein the plurality of RNAs comprise a plurality of mRNA.

76. The method of any one of claims 1-75, wherein the plurality of nucleic acid probes each comprise a barcode sequence.

77. The method of claim 76, wherein the barcode sequence or complement thereof is detected by hybridizing an intermediate probe to the barcode sequence or complement thereof, hybridizing a detectably labeled probe to the intermediate probe, and detecting the detectably labeled probe.

78. The method of claim 76 or claim 77, wherein the barcode sequence or complement thereof is detected by:contacting the biological sample with a universal pool of detectably labeled probes and a first pool of intermediate probes, wherein an intermediate probe of the first pool of intermediate 134MF-367036897202412025040probes comprises a hybridization region complementary to the barcode sequence or complement thereof and a reporter region complementary to a detectably labeled probe of the universal pool of detectably labeled probes;detecting a complex formed between the barcode sequence or complement thereof, the intermediate probes of the first pool of intermediate probes, and the detectably labeled probe: andremoving the intermediate probe of the first pool of intermediate probes and the detectably labeled probe.

79. The method of claim 78, wherein detecting the barcode sequence or complement thereof further comprises:contacting the biological sample with the universal pool of detectably labeled probes and a second pool of intermediate probes, wherein an intermediate probe of the second pool of intermediate probes comprises a hybridization region complementary to the barcode sequence or complement thereof and a reporter region complementary to a detectably labeled probe of the universal pool of detectably labeled probes; anddetecting a complex formed between the barcode sequence or complement thereof, the intermediate probe of the second pool of intermediate probes, and the detectably labeled probe.

80. The method of any one of claims 1-79, wherein the plurality of nucleic acid probes comprise a plurality of circularizable probes.

81. The method of claim 80, wherein the plurality of circularizable probes is ligated to form a plurality of circularized probes prior to incubating the biological sample with the second buffer.

82. The method of claim 80 or claim 81, wherein the plurality of circularizable probes comprise a plurality of padlock probes.

83. The method of claim 81 or claim 82, wherein in the method further comprises amplifying the plurality of circularized probes.135MF-36703689720241202504084. The method of claim 83, wherein the plurality of circularized probes are enzymatically amplified in situ in the biological sample.

85. The method of claim 84, wherein the enzymatic amplification comprises performing rolling circle amplification (RCA) to generate a plurality of RCA products prior to incubating the biological sample with the second buffer.

86. The method of any one of claims 1-83, wherein the plurality of nucleic acid probes is used to perform a hybridization chain reaction (HCR) or a primer exchange reaction (PER) product.

87. The method of any one of claims 1-86, wherein the plurality of nucleic acid probes comprises a plurality of linear probes comprising a 3’ overhang and a 5’ overhang upon hybridization to the an RNA of the plurality of RNAs in the cells, optionally wherein the 3’ overhang and the 5’ overhang each independently comprises one or more barcode sequences.

88. The method of any one of claims 1-87, wherein the analyte comprises a non-nucleic acid moiety, wherein the non-nucleic acid moiety is a protein, a carbohydrate, a lipid, a small molecule, or a complex thereof.

89. The method of claim 88, wherein the protein is an intracellular protein, a membranebound protein, or an extracellular protein.

90. The method of any one of claims 1-89, wherein the detecting in (e) is performed by imaging the biological sample.

91. The method of any one of claims 1-90, wherein detecting the plurality of optical signals associated with the plurality of nucleic acid probes or products thereof comprises performing sequential cycles of binding detectably labeled probes and imaging the biological sample.

92. The method of claim 90 or claim 91, wherein the imaging comprises fluorescent microscopy.136MF-36703689720241202504093. The method of any one of claims 90-92, further comprising performing cell segmentation.

94. A method, comprising:(a) contacting a biological sample comprising cells with a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks in the biological sample;(b) contacting the biological sample with a plurality of nucleic acid probes that directly or indirectly binds to a plurality of RNAs in the cells and generating a plurality of amplification products using the plurality of nucleic acid probes;(c) after (b), baking comprises baking the biological sample;(d) incubating the biological sample after baking with a second buffer at a temperature of at least 80°C for at least 10 minutes;(e) contacting the biological sample with a labelling agent that directly or indirectly binds to a protein analyte at a location in the biological sample; and(f) detecting:(i) a plurality of optical signals associated with the plurality of amplification products with the plurality of RNAs in the biological sample; and(ii) an optical signal associated with the labelling agent or a product thereof in the biological sample.

95. The method of claim 94, wherein the second buffer comprises Tris and a chelating agent.

96. The method of claim 95, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE).

97. The method of any one of claims 94-96, wherein the baking is performed at a temperature of 30°C or higher for at least 5 minutes.

98. The method of any one of claims 94-97, wherein the catalyst is a compound of formula (I),137MF-367036897202412025040or a salt, zwitterion, or solvate thereof, wherein:A is selected from the group consisting of -COOH, -P(=O)(OH)2, and S(=O)2OH; X1, X2, X3, and X4are each independently selected from the group consisting of: CH, CRa, and N;each occurrence of Rais independently selected from the group consisting of C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, -NO2, -NR’R”, and -C(=O)NR’R”; andeach occurrence of R’ and R” is independently selected from the group consisting of Hand C1-6 alkyl which is optionally substituted withn1, wherein nl is an integer from 12 to 16.

99. The method of any one of claims 94-98, wherein the catalyst comprises one or morecompounds selected from the group consisting of138MF-367036897202412025040, or a salt, zwitterion, or solvate thereof.

100. The method of any one of claims 94-99, wherein the biological sample is incubated with the second buffer for at least 20 minutes.

101. The method of any one of claims 94-100, wherein the second buffer has a pH of at least 8.

102. The method of any one of claims 94- 101, wherein the plurality of amplification products are generated by performing rolling circle amplification (RCA).

103. The method of any one of claims 94-102, wherein the detecting in (f) is performed by imaging the biological sample.

104. The method of claim 103, further comprising performing cell segmentation.

105. The method of claim 93 or claim 104, wherein the detected optical signal associated with the labelling agent or a product thereof in the biological sample is used to perform cell segmentation.

106. A system, comprising:a biological sample comprising cells on a solid support;139MF-367036897202412025040a catalyst in a first buffer, wherein the catalyst catalyzes de-crosslinking of molecular crosslinks;a second buffer comprising Tris and a chelating agent;a plurality of circularizable probes configured to bind a plurality of RNAs; and a plurality of labelling agents configured to bind to a plurality of protein analytes.

107. The system of claim 106, further comprising a heater configured to receive the biological sample.

108. The system of claim 106 or claim 107, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) and the second buffer is Tris-EDTA (TE).

109. The system of any one of claims 106-108, wherein the catalyst is a compound of formula (I),'2or a salt, zwitterion, or solvate thereof, wherein:A is selected from the group consisting of -COOH, -P(=O)(OH)2, and S(=O)2OH;X1, X2, X3, and X4are each independently selected from the group consisting of: CH, CRa, and N;each occurrence of Rais independently selected from the group consisting of C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, -NO2, -NR’R”, and -C(=O)NR’R”; andeach occurrence of R’ and R” is independently selected from the group consisting of HO'and C1-6 alkyl which is optionally substituted withn1, wherein nl is an integer from 12 to 16.140MF-367036897202412025040110. The system of any one of claims 106-109, wherein the catalyst comprises one or morecompounds selected from the group consisting of, or a salt, zwitterion, or solvate thereof.

111. The system of any one of claim 106-110, further comprising a polymerase and dNTPs for performing rolling circle amplification (RCA) using the plurality of circularizable probes.

112. The system of any one of claim 106-111, further comprising reagents for detecting one or more sequence of the plurality of circularizable probes.

113. The system of claim 112, wherein the reagents for detecting one or more sequences of the plurality of circularizable probes comprises a plurality of detectably labeled probes for binding to a sequence of an amplification product generated using RCA.MF-367036897202412025040114. The system of any one of claim 106-113, further comprising reagents for sequencing.

115. The system of any one of claim 106-114, further comprising a ligase for forming a plurality of circularized probes from the plurality of circularizable probes.

116. The system of any one of claim 106-115, further comprising reagents for detecting the plurality of labelling agents.

117. The system of claim 116, wherein the reagents for detecting the plurality of labelling agents comprises a plurality of detectably labeled probes for binding to a reporter oligonucleotide of the plurality of labelling agents.

118. A kit, comprising:(i) a solid support;(ii) a first buffer comprising a catalyst that catalyzes de-crosslinking of molecular crosslinks;(iii) a second buffer comprising Tris and a chelating agent; and(iv) a plurality of circularizable probes configured to bind a plurality of RNAs.

119. The kit of claim 118, further comprising:(v) a plurality of labelling agents configured to bind a plurality of protein analytes and a plurality of detectably labeled probes for binding to a sequence of a reporter oligonucleotide of the plurality of labelling agents.142MF-367036897

Images