Methods and compositions for amplification reactions in situ

Abstract

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with maintaining localization of analytes or products thereof at locations of a sample. In some aspects, a method disclosed herein for amplification provides improved localization of corresponding signals for detecting analytes in a sample.

Description

PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940METHODS AND COMPOSITIONS FOR AMPLIFICATION REACTIONS IN SITUCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 715,379, filed November 1, 2024, entitled “METHODS AND COMPOSITIONS FOR IMPROVED AMPLIFICATION REACTIONS IN SITU,” and U.S. Provisional Patent Application No. 63 / 751,170 filed January 29, 2025, entitled “METHODS AND COMPOSITIONS FOR IMPROVED AMPLIFICATION REACTIONS IN SITU,” each of which is herein incorporated by reference in its entirety for all purposes.FIELD

[0002] The present disclosure generally relates to methods and compositions for accurate in situ detection of analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with maintaining localization of analytes or products thereof at locations of a sample. In some aspects, a method disclosed herein provides improved localization of corresponding signals for detecting analytes in a sample.BACKGROUND

[0003] Genomic, transcriptomic, and proteomic profiling of cells and tissue samples using microscopic imaging can resolve multiple analytes of interest at the same time, thereby providing valuable information regarding analyte abundance and localization in situ. Thus, these in situ assays are important tools, for example, for understanding the molecular basis of cell identity and diseases. In multiplex assays where multiple signals are detected simultaneously and sequentially, it is important that signals are detected accurately and localization is maintained. However, due to the length of the assay for detecting the analytes, the molecules for detection may drift or exhibit some movement in the biological sample, e.g., away from its original location. There is a need for new and improved methods for in situ assays. The present disclosure addresses these and other needs.SUMMARY

[0004] Provided herein is a method comprising adding a first reaction mixture comprising a polymerase to a cell or tissue sample comprising a primer and a circular nucleic acid, wherein the primer is exogenous to the cell or tissue sample, and wherein the circular1MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 nucleic acid comprises a hybridization region complementary to the primer; after addition of the first reaction mixture, incubating the cell or tissue sample for at least 30 minutes, wherein the circular nucleic acid is not amplified by the polymerase in the cell or tissue sample during or prior to the incubation; after the incubation, contacting the cell or tissue sample with a second reaction mixture comprising at least 10% of a crowding agent; and performing rolling circle amplification (RCA) in the cell or tissue sample contacted with the second reaction mixture, wherein the polymerase extends the primer using the circular nucleic acid as a template to generate an RCA product. In some instances, the second reaction mixture comprises at least 10% of a single crowding agent, and in some instances the second reaction mixture comprises at least 10% of a plurality of crowding agents.

[0005] In some embodiments, the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the second reaction mixture comprises a catalytic cofactor of the polymerase. In some instances, the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of at least one of four canonical bases: adenine, thymine, guanine, and cytosine; and the second reaction mixture comprises a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine, and cytosine.

[0006] In some embodiments, incubation with the first reaction mixture is performed at a temperature that impedes polymerase activity. For example, the temperature that impedes polymerase activity is at or below 4°C. In some instances, incubation with the first reaction mixture is performed at about 20°C to 25°C. In some instances, incubation with the first reaction mixture is performed for at least 60 minutes. In some instances, incubation with the first reaction mixture is performed for at least 90 minutes. In some instances, incubation with the first reaction mixture is performed for at least 120 minutes.

[0007] In some instances, incubation with the first reaction mixture is performed at between 4°C and 10°C. In some instances, incubation with the first reaction mixture is performed at 22°C. In some embodiments, RCA is performed at a temperature of greater than 25°C. In some embodiments, RCA is performed at a temperature of greater than 30°C. In some embodiments, RCA is performed at a temperature of greater than 32°C. In some embodiments, RCA is performed at a temperature of about 30°C. In some embodiments, the generated RCA product is detected at a location in the cell or tissue sample.2MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0008] Provided herein is a method comprising contacting a cell or tissue sample with a first reaction mixture, wherein: the cell or tissue sample comprises a circular nucleic acid and a primer, wherein the primer comprises a sequence complementary to a hybridization region in the circular nucleic acid, and the first reaction mixture comprises a polymerase and a non- catalytic cofactor of the polymerase; contacting the cell or tissue sample with a second reaction mixture to allow the polymerase to extend the primer using the circular nucleic acid as a template, thereby generating a rolling circle amplification (RCA) product in the cell or tissue sample, wherein the second reaction mixture comprises a catalytic cofactor of the polymerase and at least 10% of a crowding agent; and detecting the RCA product at a location in the cell or tissue sample. In some instances, the second reaction mixture comprises at least 15% of a crowding agent.

[0009] In some instances, prior to contacting the cell or tissue sample with the first reaction mixture, the cell or tissue sample is contacted with a circularizable probe and the circularizable probe is ligated to form the circular nucleic acid. In some instances, prior to contacting the cell or tissue sample with the first reaction mixture, the cell or tissue sample is contacted with a circular nucleic acid (e.g., a circular probe). In some instances, the primer is hybridized to the circular nucleic acid prior to prior to contacting the cell or tissue sample with the first reaction mixture. In some instances, the primer hybridizes to the circularizable probe prior to contacting the cell or tissue sample with the first reaction mixture.

[0010] In some instances, the first reaction mixture is free of or comprises less than 12% of a crowding agent. In some instances, the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%. In some instances, the crowding agent in the first reaction mixture is the same type of crowding agent as the crowding agent in the second reaction mixture. In some instances, the crowding agent in the first reaction mixture is a different type of crowding agent from the crowding agent in the second reaction mixture. In some instances, the second reaction mixture comprises the crowding agent at a concentration of between 10% and 20%. In some instances, the crowding agent is selected from the group consisting of polyethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate, optionally wherein the crowding agent is poly (ethylene glycol) (PEG). In some instances, the crowding agent is polyethylene glycol) (PEG). For example, the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG12000, PEG20000, and3MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940PEG35000. In some instances, the crowding agent is or comprises PEG6000. In some instances, the crowding agent is or comprises PEG20000. In some instances, the second reaction mixture comprises between about 5% and about 25% PEG, between about 5% and about 20% PEG, between about 5% and about 15% PEG, between about 5% and about 10% PEG, between about 10% and about 25% PEG, between about 10% and about 20% PEG, or between about 10% and about 15% PEG. In some instances, the second reaction mixture comprises about 10% PEG.

[0011] In some instances, the first reaction mixture is substantially free of deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs). In some instances, the second reaction mixture comprises a plurality of deoxynucleoside triphosphates (dNTPs). In some instances, the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the second reaction mixture is substantially free of the non-catalytic cofactor of the polymerase. In some instances, the non-catalytic cofactor is a di-cation cofactor. In some instances, the di-cation is Ca2+or Sr2+. In some instances, the non-catalytic cofactor stabilizes the polymerase, thereby inhibiting the polymerase activity and / or an exonuclease activity of the polymerase. In some instances, the first reaction mixture is substantially free of a catalytic cofactor of the polymerase and the second reaction mixture comprises the catalytic cofactor of the polymerase. In some instances, the catalytic cofactor is Mg2+, Co2+, and / or Mn2+.

[0012] In some instances, the first reaction mixture comprises a chelating agent. In some examples, the chelating agent comprises EDTA, EGTA, BAPTA, DTPA, or a combination thereof. In some instances, 3'^-5' exonuclease activity of the polymerase is inhibited in the first reaction mixture. In some instances, the primer comprises a 3' protective group. In some instances, the primer is 3' thiophosphate-protected, thereby protecting the primer from 3'^-5' exonuclease degradation by the polymerase while allowing priming by the polymerase.

[0013] In some embodiments, the cell or tissue sample is incubated with the first reaction mixture for at least 30 minutes. In some instances, the cell or tissue sample is incubated with the first reaction mixture for at least 60 minutes. In some instances, the cell or tissue sample is incubated with the first reaction mixture at about 30°C. In some instances, RCA is performed for no more than 60 minutes, 90 minutes, or 120 minutes.

[0014] In some embodiments, the hybridization region in the circular nucleic acid is a primer hybridization region that hybridizes to the primer, and the circular nucleic acid further4MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 comprises a target hybridization region that hybridizes to a target nucleic acid. In some instances, the target nucleic acid is an endogenous DNA or an endogenous RNA molecule in the cell or tissue sample. In some instances, the target nucleic acid is a product, optionally an amplification product of the endogenous DNA or the endogenous RNA molecule. In some instances, the target nucleic acid is a probe that directly or indirectly binds to the endogenous DNA or the endogenous RNA molecule, or a product of the probe. In some instances, the target nucleic acid comprises a genomic DNA sequence, a mtDNA sequence, an RNA sequence, and / or a cDNA sequence.

[0015] In some embodiments, one or more molecules of the polymerase that are not bound to the circular nucleic acid are removed from the cell or tissue sample prior to contacting the cell or tissue sample with the second reaction mixture. In some instances, one or more stringency washes are performed prior to contacting the cell or tissue sample with the second reaction mixture. In some instances, the cell or tissue sample is contacted with a wash solution after incubation with the first reaction mixture and prior to contacting the cell or tissue sample with the second reaction mixture. In some instances, the wash solution comprises the crowding agent. In some instances, the wash solution comprises the crowding agent at a concentration of between 0.1% and 5%. In some instances, the cell or tissue sample is incubated with the wash solution for at least 30 minutes. In some instances, the wash solution comprises dNTPs. In some instances, the concentration of dNTPs in the wash solution is lower than the concentration of dNTPs in the second reaction mixture.

[0016] In some embodiments, the polymerase and / or the primer is not attached to a surface of a solid support or to a nanopore, a nanopore membrane, or an insulating support thereof. In some instances, the polymerase or a preformed complex comprising the polymerase and the primer is diffusible in the first reaction mixture and / or when contacting with the cell or tissue sample.

[0017] In some instances, the polymerase is selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRDl polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA5MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and a variant or derivative thereof. In some instances, the polymerase is a Phi29 DNA polymerase.

[0018] In some instances, the primer is prebound to a single- stranded DNA binding domain of the Phi29 DNA polymerase in the first reaction mixture. In some instances, the primer bound to the Phi29 DNA polymerase is hybridized to the hybridization region and the Phi29 DNA polymerase is prevented from extending the primer in the first reaction mixture.

[0019] In some embodiments, the second reaction mixture comprises a deoxynucleoside triphosphate (dNTP) and / or a nucleoside triphosphate (NTP). In some aspects, the catalytic cofactor is a di-cation. In some examples, the di-cation is Ca2+. In some examples, the di-cation is Sr2+.

[0020] In some instances, the second reaction mixture is substantially free of the polymerase and / or other polymerases. In some instances, the pH of the first and second reaction mixtures is substantially the same. In some examples, the pH of the first and second reaction mixture is between pH 7.5 to pH 8.5, optionally wherein the pH is 8.

[0021] In some instances, the rolling circle amplification product is generated using a linear rolling circle amplification (RCA), a branched RCA, a dendritic RCA, or any combination thereof. In some instances, the RCA product is immobilized in the cell or tissue sample. In some instances, the RCA product is crosslinked to one or more molecules other than the RCA product in the cell or tissue sample. In some instances, the RCA product is not crosslinked to one or more other molecules in the cell or tissue sample.

[0022] In some instances, the cell or tissue sample is imaged to detect the RCA product at the location in the cell or tissue sample. In some examples, the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCA product. In some instances, a sequence of the RCA product is detected in situ in the cell or tissue sample. In some instances, a sequence of the RCA product is analyzed in situ in the cell or tissue sample. In some instances, the sequence of the RCA product is detected by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.6MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0023] In some embodiments, the sequence of the RCA product comprises a barcode sequence or complement thereof. In some instances, the circularizable probe is a padlock probe. In some instances, the circularizable probe is provided in two more or parts. In some instances, the circularizable probe comprises a barcode sequence. In some instances, the target nucleic acid is a target messenger RNA (mRNA). In some instances, the target nucleic acid is a viral DNA or a bacterial DNA. In some instances, the ligating of the circularizable probe to form the circular nucleic acid comprises performing an enzymatic ligation. In some instances, the ligating of the circularizable probe to form the circular nucleic acid comprises ligating a 5’ end to a 3’ end of the circularizable probe.

[0024] In some instances, the target nucleic acid is a reporter oligonucleotide of a labeling agent, wherein a sequence of the reporter oligonucleotide corresponds to a binding moiety and / or a non-nucleic acid target molecule bound by the labeling agent. In some instances, the non-nucleic acid target molecule bound by the labeling agent is a protein. In some embodiments, the circularizable probe comprises a padlock probe that hybridizes to the reporter oligonucleotide. In some cases, the padlock probe hybridized to the reporter oligonucleotide is ligated to form the circular nucleic acid.

[0025] In some embodiments, the cell or tissue sample comprises cells deposited on a surface. In some instances, the cell or tissue sample is a tissue sample. In some instances, the cell or tissue sample is fixed. In some instances, the cell or tissue sample is not fixed. In some instances, the cell or tissue sample is a formalin-fixed, paraffin-embedded (FFPE) sample, a frozen tissue sample, or a fresh tissue sample. In some instances, the cell or tissue sample is permeabilized. In some instances, the cell or tissue sample is processed or cleared. In some instances, the cell or tissue sample is embedded in a matrix. In some examples, the matrix is a hydrogel. In some cases, the cell or tissue sample and / or the matrix is crosslinked.

[0026] In some instances, the first reaction mixture and the second reaction mixture comprise dNTPs. In some instances, the concentration of dNTPs in the first reaction mixture is lower than the concentration of dNTPs in the second reaction mixture.

[0027] Provided herein is a method comprising contacting a cell or tissue sample with a first reaction mixture, wherein: the cell or tissue sample comprises a plurality of primers and a plurality of circular nucleic acids at a plurality of locations in the cell or tissue sample,7MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 wherein the plurality of primers are configured to hybridize to a plurality of hybridization regions in the plurality of circular nucleic acids, wherein the first reaction mixture comprises a polymerase, wherein the polymerase activity of the polymerase is inhibited; and wherein the polymerase and / or the primer binds to the plurality of circular nucleic acids; and contacting the cell or tissue sample with a second reaction mixture, allowing the polymerase to extend the plurality of primers using the plurality of circular nucleic acids as a template, thereby generating a plurality of rolling circle amplification (RCA) products in the cell or tissue sample, wherein the second reaction mixture comprises at least 10% of a crowding agent, thereby synchronizing RCA of the plurality of circular nucleic acids in the cell or tissue sample. In some instances, the second reaction mixture comprises at least 15% of a crowding agent. In some instances, the plurality of RCA products are detected at the plurality of locations in the cell or tissue sample.

[0028] In some embodiments, the first reaction mixture comprises a chelating agent. In some instances, the first reaction mixture comprises one or more deoxynucleoside triphosphates (dNTPs). In some instances, the first reaction mixture is substantially free of dNTPs. In some instances, the primer hybridization regions in two or more of the plurality of circular nucleic acids are the same in sequence. In some instances, the primer hybridization regions in two or more of the plurality of circular nucleic acids are different in sequence. In some instances, two or more primers of the plurality of primers are the same in sequence. In some instances, two or more primers of the plurality of primers are different in sequence.

[0029] In some embodiments, the polymerase is a Phi29 DNA polymerase, a Bst polymerase, a T7 RNA polymerase, or a Klenow fragment. In some instances, one or more stringency washes between contacting the cell or tissue sample with the first reaction mixture nad the second reaction mixture. In some instances, the second reaction mixture comprises deoxynucleoside triphosphates (dNTPs) and one or more catalytic cofactors of the polymerase. In some instances, the one or more catalytic cofactors comprise Mg2+, Co2+, and / or Mn2+. In some instances, the second reaction mixture does not comprise the polymerase. In some instances, the polymerase extension or RCA is performed for no more than 3 hours, no more than 2 hours, no more than 1 hour, or no more than 30 minutes. In some examples, the polymerase extension or RCA is performed for no more than 120 minutes. In some instances, RCA of the circular nucleic acids is terminated to provide a plurality of RCA products.8MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0030] In some instances, the plurality of circular nucleic acids in the cell or tissue sample comprise at least at least 1,000, at least 3,000, at least 5,000, or at least 10,000 distinguishable circular nucleic acids. In some instances, the first reaction mixture is free of a crowding agent. In some instances, the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%. In some instances, the crowding agent in the first reaction mixture is the same as the crowding agent in the second reaction mixture. In some instances, the crowding agent in the first reaction mixture is different from the crowding agent in the second reaction mixture. In some instances, the second reaction mixture comprises the crowding agent at a concentration of between 10% and 20%. In some instances, the crowding agent is selected from the group consisting of poly (ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate, optionally wherein the crowding agent is polyethylene glycol) (PEG). In some instances, the crowding agent is polyethylene glycol). In some examples, the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG12000, PEG20000, and PEG35000. In some instances, the second reaction mixture comprises a mixture of PEG (e.g., PEG6000 and PEG20000). In some instances, the second reaction mixture comprises between about 5% and about 25% PEG, between about 5% and about 20% PEG, between about 5% and about 15% PEG, between about 5% and about 10% PEG, between about 10% and about 25% PEG, between about 10% and about 20% PEG, or between about 10% and about 15% PEG. In some instances, the second reaction mixture comprises about 10% PEG.

[0031] Provided herein is a method comprising performing rolling circle amplification (RCA) of a circular nucleic acid in a cell or tissue sample using a reaction mixture comprising 10%- 15% of a crowding agent. In some instances, the cell or tissue sample comprises a polymerase and wherein the circular nucleic acid comprises a hybridization region complementary to a primer that is exogenous to the cell or tissue sample. In some instances, prior to performing RCA, contacting the cell or tissue sample with a first reaction mixture comprising the polymerase and incubating the cell or tissue sample for at least 30 minutes, wherein the circular nucleic acid is not amplified by the polymerase in the cell or tissue sample during or prior to the incubation.

[0032] In some instances, prior to performing RCA, contacting the cell or tissue sample with a circularizable probe and ligating the circularizable probe to form the circular9MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 nucleic acid. In some embodiments, the circular nucleic acid is a ligated circularizable probe, optionally, wherein the circularizable probe is a padlock probe. In some cases, the circularizable probe comprises a barcode sequence.

[0033] In some instances, the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the reaction mixture for performing RCA is a second reaction mixture comprising a catalytic cofactor of the polymerase. In some instances, the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of at least one of four canonical bases: adenine, thymine, guanine, and cytosine; and the second reaction mixture comprises a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine, and cytosine.

[0034] In some instances, prior to performing RCA, the cell or tissue sample is incubated at a temperature that impedes polymerase activity. In some examples, the temperature that impedes polymerase activity is at or below 4°C. In some instances, the incubation with the first reaction mixture is performed at about 20°C to 25°C. In some embodiments, RCA is performed at a temperature greater than 25°C, optionally at about 30°C. In some instances, the polymerase is a Phi29 DNA polymerase. In some instances, the incubation with the first reaction mixture is performed for at least 60 minutes, at least 90 minutes, or at least 120 minutes.

[0035] In some instances, a generated RCA product at a location is detected in the cell or tissue sample. In some instances, a sequence of the generated RCA product is detected in situ in the cell or tissue sample. In some embodiments, the sequence of the RCA product is detected by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some instances, the sequence of the RCA product comprises a barcode sequence or complement thereof.

[0036] In some instances, the first reaction mixture is free of or comprises less than 10% of the crowding agent. In some instances, the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%. In some instances, the crowding agent is selected from the group consisting of polyethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate, optionally wherein the crowding agent is polyethylene glycol) (PEG). In some instances, the crowding agent is poly(ethylene glycol) (PEG). In some10MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 instances, the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG 12000, PEG20000, and PEG35000.

[0037] In some instances, the non-catalytic cofactor is a di-cation cofactor. In some instances, the di-cation is Ca2+or Sr2+. In some instances, the catalytic cofactor is Mg2+, Co2+, and / or Mn2+.

[0038] In some instances, the circular nucleic acid is bound to an endogenous DNA or an endogenous RNA molecule in the cell or tissue sample. In some instances, the circular nucleic acid is bound to an endogenous messenger RNA (mRNA).

[0039] Provided herein is a system comprising a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and optionally no more than 5% crowding agent; and a second reaction mixture comprising at least 10% of a crowding agent. Provided herein is a system comprising a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and a mixture of free nucleotides lacking nucleotides of at least one of four canonical bases: adenine, thymine, guanine and cytosine; and a second reaction mixture comprising at least 10% of a crowding agent and a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine and cytosine. Provided herein is a system comprising a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase; and a second reaction mixture comprising at least 10% of a crowding agent and a catalytic cofactor of the polymerase. In some instances, the second reaction mixture comprises at least 15% of a crowding agent.

[0040] In some embodiments, the second reaction mixture comprises a plurality of deoxynucleoside triphosphates (dNTPs). In some instances, the second reaction mixture comprises a catalytic cofactor of the polymerase. In some instances, the polymerase is Phi29. In some embodiments, the system comprises a plurality of detectably labeled probes for binding to a sequence of an amplification product generated using the circular nucleic acid as a template. In some instances, reagents for sequencing are provided. In some embodiments, the system11MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 comprises a ligase for forming the circular nucleic acid. In some embodiments, the system comprises the primer. In some instances, the cell or tissue sample comprises the primer. In some instances, the first reaction mixture comprises the primer.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

[0042] FIG. 1 shows a workflow for performing rolling circle amplification (RCA) in situ including synchronizing the reaction using a first reaction mixture and a second reaction mixture.

[0043] FIGS. 2A-2D provide images of detected RCPs associated with GPX2 in human pancreas tissue sections with the indicated RCA conditions.

[0044] FIGS. 3A-3D provide images of detected RCPs associated with TFF2 in human pancreas tissue sections with the indicated RCA conditions.

[0045] FIG. 4 provides images of detected RCPs associated with glucagon (GCG) and somatostatin (SST) in a human FFPE pancreas tissue sample.

[0046] FIG. 5 provides a graph of calculated normalized median distance to the nearest neighbor used for quantifying signal mislocalization with RCA performed in the indicated conditions in various tissue samples.DETAILED DESCRIPTION

[0047] 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.

[0048] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.12MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940I. OVERVIEW

[0049] In assays involving in situ rolling circle amplification (RCA), the mislocalization of generated rolling circle amplification products can lead to inaccurate assignment of signals to locations and / or cells. In multiplex assays where multiple signals are detected simultaneously and sequentially, it is important that signals are detected accurately and localization is maintained. In some aspects, the maintaining of localization is important for assays that detect a large number of target analytes (e.g., at least 500 or at least 1,000 analytes). In some cases, due to the length of the assay for detecting the analytes or associated molecules, the molecules for detection may drift or exhibit some movement in the biological sample, e.g., away from its original location. In some aspects, addition of a crowding agent provides physical support (e.g., support in cells). However, the addition of a crowding agent may interfere with different processes of the assay during various library preparation steps (e.g., probe hybridization, ligation, extension, etc.). There is a need for new and improved methods for in situ assays. The present disclosure addresses these and other needs.

[0050] To perform amplification, a polymerase, a primer and a template nucleic acid diffuses through the sample to form a complex. For example, when a polymerase such as Phi29 is added to a sample, the enzyme diffuses from bulk solution through the sample (e.g., a cell or tissue section) to a primer hybridized to a circular template in the sample in order to start extension (e.g., an RCA reaction). In some cases, addition of a crowding agent with Phi29 can interfere with diffusion of the amplification reagents for an efficient reaction to be carried out. In some aspects, provided herein are compositions and methods for improved rolling circle amplification reaction to reduce mislocalization by using a first and second reaction mixture as described herein. For example, a first reaction mixture allows diffusion of reagents while a second reaction mixture provides a crowding agent for improving signal localization. In some aspects, use of the first reaction mixture and the second reaction mixture synchronizes the amplification reaction for a plurality of primers and template molecules located in different locations in the biological sample (e.g., a cell or tissue sample). In some embodiments, an incubation provides time for the various components of the amplification reaction to form a complex. In some aspects, provided herein are compositions and methods for generating in situ RCA products that are homogeneous in size and intensity and with preserved localization for accurate detection of corresponding analytes at locations in the biological sample. In some cases,13MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 the advantages of reducing mislocalization during RCA is applicable to a biological sample for multiplexed analyte detection. In some cases, the advantages of reducing mislocalization is useful for whole transcriptome scale assay for analyte detection in a cell or tissue sample.

[0051] In some aspects, the level of mislocalization is determined. For example, a calculated normalized median distance to the nearest neighbor metric can be used for quantifying signal mislocalization. In some aspects, the level of mislocalization is calculated based on genes with a median distance of less than 2pm to the nearest rolling circle amplification product (RCPs) of the same gene. In some aspects, performing synchronized RCA using a second reaction mixture comprising at least 10% of a crowding agent reduces the level of mislocalization as determined by a calculated normalized metric.

[0052] In some embodiments, a complex comprising a polymerase such as Phi29, a primer, and a circular nucleic acid is formed in a binding mixture (e.g., adding the first reaction mixture) that stalls or hinders the polymerase from continuous extension. In some embodiments, a complex comprising a polymerase such as Phi29, a primer, and a circular nucleic acid is formed in a binding mixture under conditions which amplification of the circular nucleic acid is not achieved. In some embodiments, a complex comprising a polymerase such as Phi29, a primer, and a circular nucleic acid is formed in a binding mixture that is substantially free of one or more catalytic cofactors (e.g., Mg2+) of the polymerase and / or dNTPs. In some aspects, the biological sample comprising a primer and a circular nucleic acid comprising a hybridization region is contacted with a first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase. In some embodiments, a plurality of complexes of RCA primers, circular templates, and polymerases (e.g., Phi29) are formed in the binding mixture at various locations in the cell or tissue sample. In some embodiments, the complexes are then contacted with reagents (e.g., in a second reaction mixture) for the amplification reaction to proceed in an amplification reaction mixture where extension of the primer occurs.

[0053] In some embodiments, after addition of the second reaction mixture to the biological sample, the polymerase is active and RCA is initiated in the amplification reaction for complexes of RCA primers, templates, and polymerases (e.g., Phi29) that were formed in the binding mixture. In some embodiments, a polymerase such as Phi29 is provided in a first reaction mixture that inhibits one or more activities of the polymerase, and then contacted with the sample. In some embodiments, RCA is not initiated until the sample is contacted with a 14MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 second reaction mixture to provide an amplification reaction mixture that allows continuous extension by the polymerase. In some embodiments, RCA is not initiated until the sample is contacted with a second reaction mixture to provide an amplification reaction mixture that lifts the inhibition on the polymerase and / or exonuclease activities of the polymerase, thereby allowing RCA in an environment with a crowding agent at a high concentration (e.g., at least 10%) to prevent mislocalization of the generated amplification products.

[0054] 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 is analyzed in situ in the sample.II. ROLLING CIRCLE AMPLIFICATION IN SITU

[0055] Provided herein is a method for performing amplification such that a circular nucleic acid and a primer comprising a sequence complementary to a hybridization region in the circular nucleic acid is contacted with a first reaction mixture under conditions wherein a polymerase is inactive or extension is impeded or hindered. In some instances, continuous extension by the polymerase does not progress in the first reaction mixture. In some instances, extension by the polymerase is stalled in the first reaction mixture. In some instances, polymerase activity is inhibited in the first reaction mixture. In some embodiments, the first reaction mixture comprises the polymerase.

[0056] Provided herein is a method for nucleic acid amplification, comprising adding a first reaction mixture comprising a polymerase to a cell or tissue sample comprising a primer and a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to the primer, and wherein the circular nucleic acid is not amplified by the polymerase in the cell or tissue sample; after addition of the first reaction mixture, incubating the cell or tissue sample for at least 30 minutes; after the incubation, contacting the cell or tissue sample with a second reaction mixture comprising at least 10% of a crowding agent, and performing rolling circle amplification (RCA) in the cell or tissue sample comprising the second reaction mixture, wherein the polymerase extends the primer using the circular nucleic acid as a template. In some embodiments, the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the second reaction mixture comprises a catalytic cofactor of the polymerase. In some embodiments, the first reaction mixture comprises a mixture of free nucleotides lacking15MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 nucleotides of at least one of four canonical bases: adenine, thymine, guanine and cytosine, and the second reaction mixture comprises a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine and cytosine. In some embodiments, the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of one or more of the four canonical bases: adenine, thymine, guanine and cytosine. In some embodiments, the cell or tissue sample is contacted with the first reaction mixture at a temperature that impedes polymerase activity. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at a temperature that impedes polymerase activity (e.g., at or below 4°C). In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at a temperature that impedes polymerase activity (e.g., at or below 4°C). In some embodiments, the incubation of the cell or tissue sample with the first reaction mixture is at about 20°C to 25°C, optionally at about 22°C. In some embodiments, the RCA product is detected at a location in the cell or tissue sample.

[0057] Provided herein is a method comprising contacting the biological sample with a first reaction mixture, wherein the biological sample comprises a circular nucleic acid and a primer comprising a sequence complementary to a hybridization region in the circular nucleic acid, wherein the first reaction mixture comprises a polymerase, and the contacting is performed under conditions wherein extension is stalled, inhibited and / or halted. Provided herein is a method for analyzing a biological sample, comprising contacting the biological sample with a first reaction mixture, wherein the biological sample comprises a circular nucleic acid and a primer comprising a sequence complementary to a hybridization region in the circular nucleic acid, the first reaction mixture comprises a polymerase and a non-catalytic cofactor of the polymerase; contacting the biological sample with a second reaction mixture comprising a catalytic co-factor of the polymerase to allow the polymerase to extend the primer using the circular nucleic acid as a template, thereby generating a rolling circle amplification product in the biological sample. In some embodiments, the second reaction mixture comprises a plurality of one more deoxynucleoside triphosphates (dNTPs) and at least 10% of a crowding agent. In some instances, the second reaction mixture comprises at least 15% of a crowding agent. In some instances, the crowding agent in the second reaction mixture comprises one or more crowding agents. In some instances, the crowding agent in the second reaction mixture comprises at least two different crowding agents. In some instances, the second reaction mixture comprises a16MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 mixture of PEG (e.g., PEG6000 and PEG20000). In some embodiments, the generated rolling circle amplification product is detected at a location in the biological sample.

[0058] In some embodiments, the circular nucleic acid is not amplified in the cell or tissue sample at or after addition of the first reaction mixture. In some embodiments, less than about 10 copies, less than about 5 copies, or less than about 2 copies of the circular nucleic acid is generated in the cell or tissue sample at or after addition of the first reaction mixture. In some embodiments, the circular nucleic acid is not amplified for more than 2 copies or for more than 5 copies with the addition of the first reaction mixture. In some embodiments, the circular nucleic acid is not amplified in the cell or tissue sample during the incubation with the first reaction mixture. In some instances, the cell or tissue sample is incubated with the first reaction mixture under conditions in which polymerase activity is stalled, inhibited and / or halted.

[0059] In some embodiments, a circular nucleic acid disclosed herein is amplified through rolling circle amplification (RCA). In some embodiments, the RCA comprises a linear RCA. In some embodiments, the RCA comprises a branched RCA. In some embodiments, the RCA comprises a dendritic RCA. In some embodiments, the RCA comprises any combination of the foregoing. In some embodiments, the circular nucleic acid is a construct formed using ligation. For example, the circular nucleic acid is formed using chemical ligation or enzymatic ligation.A. Nucleic Acid Probes

[0060] In some embodiments, the method comprises using a circular construct as template or circularizable construct hybridized to the nucleic acid of interest to generate a circular nucleic acid. In some examples, prior to contacting the biological sample with the first reaction mixture, the biological sample is contacted with a circularizable probe and the circularizable probe is ligated to form the circular nucleic acid. In some examples, prior to contacting the biological sample with the first reaction mixture, the biological sample is contacted with a primer comprising a sequence complementary to a hybridization region in the circular nucleic acid. In some aspects, the circularizable probe is ligated using a target nucleic acid (e.g., a target RNA) to form the circular nucleic acid. In some aspects, the primer hybridizes to the circularizable probe prior to the ligation. In some aspects, the primer hybridizes to the circularizable probe during the ligation. In some aspects, the primer hybridizes to the circular17MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 nucleic acid formed from the circularizable probe. In some aspects, the primer is provided with the reagents for performing the ligation of the circularizable probe. In some instances, a circularizable probe is ligated in a cell or tissue sample to form the circular nucleic acid.

[0061] Disclosed herein in some aspects are nucleic acid probes that are introduced into a cell or used to otherwise contact a biological sample such as a tissue sample. The 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. The nucleic acid probe typically contains a targeting sequence that is able to directly or indirectly bind to at least a portion of a target nucleic acid (e.g., mRNA target nucleic acid). For example, the nucleic acid probe is a circular nucleic acid or is used to generate a circular nucleic acid comprising a hybridization region complementary to a primer (e.g., for amplification). In some instances, the nucleic acid probe is a circularizable probe. In some instances, the circularizable probe is a padlock probe. In some instances, the nucleic acid probe is configured to bind to a specific target nucleic acid (e.g., an mRNA, or other nucleic acids as discussed herein). In some embodiments, the nucleic acid probes are detected using a detectable label, and / or by using detectably labeled nucleic acid probes able to bind to the nucleic acid probes or products 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 (e.g., circular nucleic acid) for a polymerase for use in an extension reaction (e.g., for rolling circle amplification).

[0062] In some embodiments, a hybridization product comprising the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules is analyzed. For example, 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.

[0063] The term “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 binding (e.g., binding comprising direct hybridization of nucleic acid sequences). The nature of the binding may vary. In some 18MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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. For example, an intermediate nucleic acid comprises a first region that binds to the first nucleic acid sequence and has a second region for binding to the second nucleic acid sequence, thereby forming a complex comprising the first and second nucleic acid sequence.

[0064] Any suitable circularizable, or indeed more generally circularizable reporter molecules, may be used to generate the RCA template (e.g., circular nucleic acid) which is used to generate the RCA product. For example, a circularizable probe is used to generate a circular nucleic acid comprising a target hybridization region that hybridizes to a target nucleic acid. In some aspects, the target nucleic acid is an endogenous DNA or an endogenous RNA molecule in the biological sample. In some instances, the target nucleic acid is a product of the endogenous DNA or the endogenous RNA molecule. In some instances, the target nucleic acid is a probe that directly or indirectly binds to the endogenous DNA or the endogenous RNA molecule, or a product of the probe. In some instances, the target nucleic acid comprises a genomic DNA sequence, a mtDNA sequence, an RNA sequence, and / or a cDNA sequence.

[0065] In some instances, the circularizable probe is provided in the form of a linear molecule having ligatable ends which may 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.

[0066] 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 19MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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 the 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 comprises 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 detection 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.

[0067] Various probes can be hybridized to an endogenous analyte and / or a labeling agent and each probe may comprise one or more barcode sequences. In some instances, a probe design is based on a padlock probe, a gapped padlock probe, a SNAIL (Splint Nucleotide Assisted Intramolecular Ligation) probe set, a PLAYR (Proximity Ligation Assay for RNA) probe set, a PLISH (Proximity Ligation in situ Hybridization) probe set, and RNA-templated ligation probes, or variations thereof.

[0068] In some aspects, a biological sample is contacted with a circularizable probe comprising a sequence complementary to a target nucleic acid. Upon hybridization to the target nucleic acid, the circularizable probe is ligated to form the circular nucleic acid. The circularizable probe and generated circular nucleic acid comprises a hybridization region20MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 complementary to a primer (e.g., for amplification). In some embodiments, the primer comprises a sequence complementary to the hybridization region. In some embodiments, the circularizable probe is provided as one nucleic acid molecule. In some embodiments, the circularizable probe is provided in two or more parts (e.g., as two or more sperate nucleic acid molecules). In some embodiments, the circularizable probe is a padlock probe. In some aspects, the 5’ and 3’ ends of the padlock probe are ligated to generate the circular nucleic acid.

[0069] In some embodiments, the target hybridization region of the circularizable probe may be positioned anywhere within the probe. For instance, the target hybridization region of a probe that binds to a target nucleic acid can be 5’ or 3’ to any barcode sequence in the probe. In some embodiments, the target hybridization region may comprise 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.

[0070] The target hybridization region of a probe (e.g., circularizable probe) may be determined with reference to a target nucleic acid (e.g., a cellular RNA or a reporter oligonucleotide of a labelling agent for a cellular analyte) that is present or suspected of being present in a sample. In some embodiments, more than one target hybridization region can be used to identify a particular analyte comprising or associated with a target nucleic acid. The more than one target hybridization region can be in the same probe or in different probes. For instance, 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 comprises target 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).

[0071] In some embodiments, a circular nucleic acid (e.g., formed from a circularizable probe) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 32 or more, 40 or more, or 50 or more barcode sequences. The barcode sequences may be positioned anywhere within the nucleic acid probe. If more than one barcode sequences are 21MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 present, the barcode sequences may be positioned next to each other, and / or interspersed with other sequences. In some embodiments, two or more of the barcode sequences may also at least partially overlap. In some embodiments, two or more of the barcode sequences in the same probe do not overlap. In some embodiments, all of the barcode sequences in the same probe are separated from one another by at least a phosphodiester bond (e.g., they may be immediately adjacent to each other but do not overlap), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides apart.

[0072] The barcode sequences, if present, may be of any length. If more than one barcode sequence is used, the barcode sequences may independently have the same or different lengths, such as at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50 nucleotides in length. In some embodiments, the barcode sequence may be no more than 120, no more than 112, no more than 104, no more than 96, no more than 88, no more than 80, no more than 72, no more than 64, no more than 56, no more than 48, no more than 40, no more than 32, no more than 24, no more than 16, or no more than 8 nucleotides in length. Combinations of any of these are also possible, e.g., the barcode sequence may be between 5 and 10 nucleotides, between 8 and 15 nucleotides, etc.

[0073] The barcode sequence may be arbitrary or random. In certain cases, the barcode sequences are chosen so as to reduce or minimize homology with other components in a sample, e.g., such that the barcode sequences do not themselves bind to or hybridize with other nucleic acids suspected of being within the cell or other sample. In some embodiments, between a particular barcode sequence and another sequence (e.g., a cellular nucleic acid sequence in a sample or other barcode sequences in probes added to the sample), the homology may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the homology may be less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases, and in some embodiments, the bases are consecutive bases.

[0074] In some embodiments, the number of distinct barcode sequences in a population of nucleic acid probes is less than the number of distinct targets (e.g., nucleic acid analytes and / or protein analytes) of the nucleic acid probes, and yet the distinct targets may still be uniquely identified from one another, e.g., by encoding a probe with a different combination of barcode sequences. However, not all possible combinations of a given set of barcode 22MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 sequences need be used. For instance, each probe may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. or more barcode sequences. In some embodiments, a population of nucleic acid probes may each contain the same number of barcode sequences, although in other cases, there may be different numbers of barcode sequences present on the various probes. In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode sequences.

[0075] As an illustrative example, a first circular nucleic acid (e.g., circularizable probe) comprises a first target-binding sequence, a first barcode sequence, and a second barcode sequence, while a second, different circular nucleic acid (e.g., circularizable probe) comprises a second target-binding sequence (that is different from the first target-binding sequence in the first circular nucleic acid), the same first barcode sequence as in the first circular nucleic acid, but a third barcode sequence instead of the second barcode sequence. Such circular nucleic acid may thereby be distinguished by determining the various barcode sequence combinations present or associated with a given circular nucleic acid at a given location in a sample.

[0076] 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. In some embodiments, the nucleic acid probes disclosed herein comprises all four canonical bases: adenine, thymine, guanine and cytosine.

[0077] In some embodiments, a nucleic acid probe disclosed herein may be labelled with 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 circularizable probe (e.g., a padlock probe) disclosed herein lacks a detectable label. While a detectable label may be incorporated into an amplification product of the primary nucleic acid probe, such as via incorporation of a modified nucleotide into an RCA product of a padlock probe, the amplification product in some embodiments is not detectably labeled.23MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0078] In some embodiments, a nucleic acid probe herein comprises a primer hybridization region for hybridizing to an amplification primer to allow for enzymatic amplification of probes. For example, the circular nucleic acid comprises a hybridization region complementary to a primer or at least a portion thereof. In some instances, a plurality of circular nucleic acids for binding to a plurality of different target nucleic acids share a common primer hybridization region (e.g., hybridize to amplification primers with the same sequence). In some instances, a plurality of primers bind to a subset of circular nucleic acids in a biological sample. In some instances, a plurality of primers bind to different circular nucleic acids that bind to a plurality of different target nucleic acids. In some instances, the circular nucleic acid comprises a hybridization region complementary to at least a portion of a nucleic acid primer.

[0079] In some instances, the primer hybridization regions in two or more of the plurality of circular nucleic acids are the same in sequence. In some instances, the primer hybridization regions in two or more of the plurality of circular nucleic acids are different in sequence. In some instances, two or more primers of a plurality of primers contacted with a cell or tissue sample are the same in sequence. In some instances, two or more primers of the plurality of primers contacted with a cell or tissue sample are different in sequence.

[0080] In some embodiments, a nucleic acid probe herein comprises a primer hybridization region, a target binding region or sequence, and a barcode sequence. In some embodiments, a nucleic acid probe herein comprises a primer hybridization region, a first and second target binding sequence, and a barcode region (e.g., comprising a first barcode sequence and a second barcode sequence). In some embodiments, the primer hybridization region and barcode sequence(s) are positioned between the first and second target binding sequences. The components of the nucleic acid probe may be arranged in any suitable order. In some embodiments, provided herein are probes, primers, and assay methods to couple target nucleic acid detection, signal amplification, and decoding of the barcodes.

[0081] In some aspects, the circular nucleic acid is a circular probe that binds directly or indirectly the to target nucleic acid. In some aspects, the circular nucleic acid is a circular probe that binds directly to the target nucleic acid. In some aspects, the circular nucleic acid is formed from a circularizable probe comprising a target hybridization region that hybridizes to a target nucleic acid. In some embodiments, a circular probe is pre-circularized prior to hybridization to a target nucleic acid. In some embodiments, a circularizable probe is24MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 circularized upon hybridization to a target nucleic acid and / or one or more other probes such as a splint. In some embodiments, a circularizable probe comprises a target recognition sequence and a sequence that does not hybridize to a target nucleic acid. In some instances, the sequence that does not hybridize to a target nucleic acid is 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 nonhybridizing to the target nucleic acid but may hybridize to one another and / or one or more other nucleic acid molecules.

[0082] In some aspects, the circular nucleic acid is formed from a padlock probe that requires gap filling to circularize upon hybridization to a template (e.g., a target nucleic acid and / or a splint). In some aspects, the circular nucleic acid is formed from a gapped padlock probe (e.g., one that require gap filling to circularize upon hybridization to a template. In some embodiments, the circular nucleic acid is formed from a probe that is ligated to itself or another probe using DNA-templated and / or RNA-templated ligation.

[0083] In some embodiments, the circular nucleic acid is formed from a DNA molecule. In some embodiments, the circular nucleic acid comprises one or more other types of nucleotides, modified nucleotides, and / or nucleotide analogues, such as one or more ribonucleotides. In some embodiments, the ligation to form the circular nucleic acid is a DNA ligation on a DNA template. In some embodiments, the ligation is a DNA ligation on an RNA template, and the probes comprise RNA-templated ligation probes.

[0084] In some embodiments, the circular nucleic acid is formed using molecular inversion probes. Like padlock probes, these are also typically linear nucleic acid molecules capable of hybridizing to a target nucleic acid molecule (such as a target analyte) and being circularized. The two ends of the molecular inversion probe may hybridize to the target nucleic acid molecule at sites which are proximate but not directly adjacent to each other, resulting in a gap between the two ends. The size of this gap may range from only a single nucleotide in some embodiments, to larger gaps of 100 to 500 nucleotides, or longer, in other embodiments. Accordingly, it is necessary to supply a polymerase and a source of nucleotides, or an additional gap-filling oligonucleotide, in order to fill the gap between the two ends of the molecular inversion probe, such that it can be circularized.25MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0085] In some embodiments, the circular nucleic acid is formed from an invader probe. Such probes are of particular utility in the detection of single nucleotide polymorphisms. The detection method of the present disclosure may, therefore, be used in the detection of a single nucleotide polymorphism, or indeed any variant base, in the target nucleic acid sequence. Probes for use in such a method may be designed such that the 3' ligatable end of the probe is complementary to and capable of hybridizing to the nucleotide in the target molecule which is of interest (the variant nucleotide), and the nucleotide at the 3' end of the 5' additional sequence at the 5' end of the probe or at the 5' end of another, different, probe part is complementary to the same said nucleotide, but is prevented from hybridizing thereto by a 3' ligatable end (e.g., it is a displaced nucleotide). Cleavage of the probe to remove the additional sequence provides a 5' ligatable end, which may be ligated to the 3' ligatable end of the probe or probe part if the 3' ligatable end is hybridized correctly to (e.g., is complementary to) the target nucleic acid molecule. Probes designed according to this principle provide a high degree of discrimination between different variants at the position of interest, as only probes in which the 3' ligatable end is complementary to the nucleotide at the position of interest may participate in a ligation reaction. In one embodiment, the probe is provided in a single part, and the 3' and 5' ligatable ends are provided by the same probe. In some embodiments, an invader probe is a padlock probe (an invader padlock or “iLock”), e.g., as described in Krzywkowski et al., Nucleic Acids Research 45, el61, 2017 and US 2020 / 0224244, which are incorporated herein by reference.

[0086] In some embodiments, the circular nucleic acid is pre-assembled from multiple components, e.g., prior to being in a complex with a target nucleic acid. In some embodiments, a circular nucleic acid disclosed herein can be assembled during and / or after contacting a target nucleic acid or a sample with multiple components. In some embodiments, a circular nucleic acid disclosed herein is assembled in situ in a sample. In some embodiments, the multiple components can be contacted with a target nucleic acid or a sample in any suitable order and any suitable combination. For instance, a first component and a second component can be contacted with a target nucleic acid, to allow binding between the components and / or binding between the first and / or second components with the target nucleic acid. Optionally a reaction involving either or both components and / or the target nucleic acid, between the components, and / or between either one or both components and the target nucleic acid can be performed, such26MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 as hybridization, ligation, primer extension and / or amplification, chemical or enzymatic cleavage, click chemistry, or any combination thereof. In some embodiments, the circularizable probe can be assembled in situ in a stepwise manner, each step with the addition of one or more components, or in a dynamic process where all components are assembled together. One or more removing steps, e.g., by washing the sample such as under stringent conditions, may be performed at any point during the assembling process to remove or destabilize undesired intermediates and / or components at that point and increase the chance of accurate probe assembly and specific target binding of the assembled probe.

[0087] In some embodiments, the circular nucleic acid used for RCA is formed in a mixture substantially free of a crowding agent. In some embodiments, the circular nucleic acid used for RCA is formed using template primer extension followed by ligation. In some embodiments, the circular nucleic acid is formed by providing an insert between ends to be ligated. In some embodiments, the circular nucleic acid is formed using a combination of any of the foregoing. In some embodiments, the ligation is a DNA-DNA templated ligation. In some embodiments, the ligation is an RNA-RNA templated ligation. Exemplary RNA-templated ligation probes and methods are described in US 2020 / 0224244 which is incorporated herein by reference in its entirety. In some embodiments, the ligation is a RNA-DNA templated ligation. In some embodiments, a splint is provided as a template for ligation. In some embodiments, the ligation to generate the circular nucleic acid is performed in a mixture substantially free of a crowding agent.

[0088] In some embodiments, the ligation involves chemical ligation (e.g., click chemistry ligation). In some embodiments, the chemical ligation involves template dependent ligation. In some embodiments, the chemical ligation involves template independent ligation. In some embodiments, the click reaction is a template-independent reaction. In some embodiments, the click reaction is a template-dependent reaction or template-directed reaction. In some embodiments, the template-dependent reaction is sensitive to base pair mismatches such that reaction rate is significantly higher for matched versus unmatched templates. In some embodiments, the click reaction is a nucleophilic addition template-dependent reaction. In some embodiments, the click reaction is a cyclopropane-tetrazine template-dependent reaction.

[0089] 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 27MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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 doublestrand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and singlestrand 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) and 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.

[0090] 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, i.e., separated by one or more intervening nucleotides or "gaps". In some embodiments, said 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. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific embodiments, the gap may be 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 may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of28MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0091] 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 containing the ligated polynucleotides prior to subsequent steps, comprising amplification and detection.

[0092] 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 Tm around 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.

[0093] 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.29MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0094] In some embodiments, a circularizable probe disclosed herein (e.g., a padlock probe) comprises a 5' flap which may be recognized by a structure-specific cleavage enzyme, e.g., an enzyme capable of recognizing the junction between single-stranded 5' overhang and a DNA duplex, and cleaving the single-stranded overhang. It will be understood that the branched three-strand structure which is the substrate for the structure- specific cleavage enzyme may be formed by 5' end of one probe part and the 3' end of another probe part when both have hybridized to the target nucleic acid molecule, as well as by the 5' and 3' ends of a one-part probe. Enzymes suitable for such cleavage include Flap endonucleases (FENS), which are a class of enzymes having endonucleolytic activity and being capable of catalyzing the hydrolytic cleavage of the phosphodiester bond at the junction of single- and double-stranded DNA. Thus, in some embodiment, cleavage of the additional sequence 5' to the first target-specific binding site is performed by a structure-specific cleavage enzyme, e.g., a Flap endonuclease. Suitable Flap endonucleases are described in US 2020 / 0224244, which is incorporated herein by reference in its entirety, and may include P. furiosus (Pfu), A. fulgidus (Afu), M. jannaschii (Mja) or M. thermoautotrophicum (Mth). In other embodiments an enzyme capable of recognizing and degrading a single-stranded oligonucleotide having a free 5' end may be used to cleave an additional sequence (5' flap) from a structure as described above. Thus, an enzyme having 5' nuclease activity may be used to cleave a 5' additional sequence. Such 5' nuclease activity may be 5' exonuclease and / or 5' endonuclease activity. A 5' nuclease enzyme is capable of recognizing a free 5' end of a single-stranded oligonucleotide and degrading said singlestranded oligonucleotide. A 5' exonuclease degrades a single-stranded oligonucleotide having a free 5' end by degrading the oligonucleotide into constituent mononucleotides from its 5' end. A 5' endonuclease activity may cleave the 5' flap sequence internally at one or more nucleotides. Further, a 5' nuclease activity may take place by the enzyme traversing the single-stranded oligonucleotide to a region of duplex once it has recognized the free 5' end, and cleaving the single-stranded region into larger constituent nucleotides (e.g., dinucleotides or trinucleotides), or cleaving the entire 5' single- stranded region. In some embodiments, the cleavage enzyme is Exonuclease VIII, or a native or recombinant DNA polymerase enzyme from Thermits aquaticus (Taq), Thermits thermophilus or Thermus flavus, or the nuclease domain therefrom.

[0095] 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, at30MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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 circular nucleic acids are amplified within a sample, e.g., a cell or tissue sample. In some instances, a plurality of circular nucleic acids for binding 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 300, at least about 500, at least about 1,000, at least about 10,000, at least about 15,000, at least about 20,000, at least about 50,000, at least about 100,000 or more different analytes (e.g., distinguishable RNAs) in a biological sample is provided.B. Compositions and Methods for Amplification

[0096] In some aspects, provided herein are a first reaction mixture that allows diffusion of reagents and a second reaction mixture for performing RCA. In some instances, the first reaction mixture added to the biological sample provides conditions which inhibit, stall or halt a polymerase from amplifying the circular nucleic acid. In some embodiments, the biological sample (e.g., cell or tissue sample) comprises a circular nucleic acid and a primer (e.g., an amplification primer). In some cases, an amplification primer is added following formation of the circular nucleic acid. In some instances, an amplification primer is added during formation of the circular nucleic acid. In some instances, an amplification primer is provided to the biological sample with reagents for ligation to form the circular nucleic acid.

[0097] In some embodiments, the biological sample is contacted with a first reaction mixture, wherein the biological sample comprises a circular nucleic acid and a primer, and the first reaction mixture comprises a polymerase. In some instances, the first reaction mixture comprises a non-catalytic cofactor of the polymerase. In some instances, the first reaction mixture comprises a non-catalytic cofactor of the polymerase. In some embodiments, the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of at least one out of four canonical bases: adenine, thymine, guanine and cytosine. In some embodiments, the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of one or more of the four canonical bases: adenine, thymine, guanine and cytosine. In some instances, the first reaction mixture is substantially free of dNTPs and / or NTPs.31MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0098] In some embodiments, the biological sample (e.g., cell or tissue sample) is incubated with the first reaction mixture. During the incubation, in some aspects, the circular nucleic acids, primers, and molecules of polymerase form complexes. In some cases, the cell or tissue samples comprise a plurality of corresponding circular nucleic acids and primers which hybridize if the sequences are complementary. In some instances, during the incubation, the biological sample comprises a primer, a circular nucleic acid, and a polymerase. In some embodiments, the circular nucleic acid is not amplified by the polymerase during or prior to the incubation with the first reaction mixture in the cell or tissue sample. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at a temperature that impedes polymerase activity (e.g., extension). In some embodiments, the biological sample is incubated with the first reaction mixture at or below 10°C. In some embodiments, the biological sample is incubated with the first reaction mixture at or below 5°C. In some embodiments, the biological sample is incubated with the first reaction mixture at or below 4°C. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at a temperature that allows hybridization (e.g., of the primer to the corresponding circular nucleic acid) and impedes polymerase activity (e.g., extension).

[0099] A primer is generally a single-stranded nucleic acid sequence having a 3’ end that, in some embodiments, is used as a substrate for a nucleic acid polymerase in a nucleic acid extension 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, in some cases, refers to a primer binding sequence. A primer extension reaction, in some embodiments, generally refers to any method where two nucleic acid sequences become linked (e.g., primer hybridized to template). Such linking, in some embodiments, is followed by nucleic acid extension (e.g., an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for32MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 extension. In some embodiments, enzymatic extension is performed by an enzyme including, but not limited to, a polymerase and / or a reverse transcriptase.

[0100] In some instances, the primer (e.g., amplification primer) is contacted with the biological sample with the first reaction mixture. In some aspects, the primer is exogenous to the biological sample. In some instances, the primer is a DNA primer. In some embodiments, RCA is not primed by the target nucleic acid. In some instances, the primer is contacted with the biological sample prior to adding the first reaction mixture to the biological sample. In some instances, the primer is contacted with the biological sample with the polymerase for performing RCA. In some instances, the primer is contacted with the biological sample with the polymerase and a non-catalytic cofactor of the polymerase. In some embodiments, the circular nucleic acid comprises a target hybridization region (e.g., for hybridizing to a nucleic acid analyte such as mRNA) and a primer hybridization region which is complementary to the primer or a portion thereof. In some instances, the primer (e.g., a portion thereof) is complementary to the target nucleic acid and the padlock probe (e.g., a SNAIL probe). In some embodiments, a washing step is performed to remove any unbound probes, primers, etc. In some embodiments, the wash is a stringency wash. Washing steps can be performed at any point during the process to remove non- specifically bound probes and / or other reagents, etc.

[0101] In some embodiments, the biological sample is incubated with the first reaction mixture for at least 30 minutes. In some embodiments, prior to RCA, the biological sample is incubated with the first reaction mixture for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 120 minutes, or at least 180 minutes. In some embodiments, the biological sample is incubated with the first reaction mixture for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 120 minutes, or at least 180 minutes. In some embodiments, prior to RCA, the biological sample is incubated with the first reaction mixture for at least 30 minutes. In some embodiments, prior to RCA, the biological sample is incubated with the first reaction mixture for at least 60 minutes. In some embodiments, prior to RCA, the biological sample is incubated with the first reaction mixture for at least 90 minutes. In some embodiments, prior to RCA, the biological sample is incubated with the first reaction mixture for at least 120 minutes. In some embodiments, prior to RCA, the biological sample is incubated with the first reaction mixture for between about 15 minutes to about 12 hours, about 15 minutes to about 10 hours, about 1533MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 minutes to about 8 hours, about 15 minutes to about 5 hours, about 15 minutes to about 2 hours, about 15 minutes to about 60 minutes, about 15 minutes to about 30 minutes, about 30 minutes to about 12 hours, about 30 minutes to about 10 hours, about 30 minutes to about 8 hours, about 30 minutes to about 5 hours, about 30 minutes to about 2 hours, about 30 minutes to about 60 minutes, about 30 minutes to about 90 minutes, about 1 hour to about 12 hours, about 1 hour to about 10 hours, about 1 hour to about 8 hours, about 1 hour to about 5 hours, about 1 hour to about 2 hours, about 90 minutes to about 12 hours, about 90 minutes to about 10 hours, about 90 minutes to about 8 hours, about 90 minutes to about 5 hours, about 90 minutes to about 2 hours, or about 2 hours to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 5 hours, about 2 hours to about 4 hours, or about 2 hours to about 3 hours.

[0102] In some embodiments, the biological sample is incubated with the first reaction mixture at or below 10°C for at least 60 minutes. In some embodiments, the biological sample is incubated with the first reaction mixture at or below 5°C for at least 60 minutes. In some embodiments, the biological sample is incubated with the first reaction mixture at or below 4°C for at least 60 minutes. In some embodiments, the biological sample is incubated with the first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase at or below 10°C for at least 60 minutes. In some embodiments, the biological sample is incubated with the first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase at or below 5°C for at least 60 minutes. In some embodiments, the biological sample is incubated with the first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase at or below 4°C for at least 60 minutes. In some instances, the first reaction mixture comprises a plurality of deoxynucleoside triphosphates (dNTP), e.g., adenine, thymine, guanine and cytosine.

[0103] In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at between about 18°C to 25°C, at between about 18°C to 22°C, at between about 18°C to 20°C, at between about 20°C to 22°C, or at between about 20°C to 25°C. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at a temperature of at least 12°C, at least 15°C, at least 18°C, at least 20°C, or at least 22°C. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at about 22°C.34MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at about 22°C for at least 30 minutes. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at about 22°C for at least 60 minutes. In some embodiments, the cell or tissue sample is incubated with the first reaction mixture at about 22°C for at least 90 minutes.

[0104] In some embodiments, the first reaction mixture stabilizes the polymerase and / or inhibit an activity of the polymerase, such as a polymerase activity and / or a nuclease activity. In some instances, the first reaction mixture comprises one or more deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs). In some instances, the first reaction mixture comprises dATP, dTTP, dCTP, and / or dGTP. Alternatively, in some instances, the first reaction mixture is substantially free of dNTPs and / or NTPs. In some embodiments, the first reaction mixture comprises a non-catalytic cofactor of the polymerase. For example, the non-catalytic cofactor of the polymerase is a dication. In some instances, the non-catalytic cofactor of the polymerase is Ca2+which can stabilize the polymerase without activating its polymerase activity and / or exonuclease activity. In some instances, the non-catalytic cofactor of the polymerase is Sr2+which can stabilize the polymerase without activating its polymerase activity and / or exonuclease activity.

[0105] In some embodiments, the first reaction mixture is substantially free of a catalytic cofactor of the polymerase. A catalytic cofactor is a non-protein chemical compound or metallic ion that is required for an enzyme’s activity as a catalyst. DNA and ribonucleic acid (RNA) polymerases in general require a divalent or trivalent metal cofactor cation to catalyze the polymerization of individual nucleotides into a polynucleotide. In some embodiments herein, the presence / absence of particular divalent cation(s) can be used to alter the kinetics of polymerases. Absent the metal cofactor in the proper oxidation state, polymerization will not occur at an appreciable rate even if all other necessary components are present. In some instances, the catalytic cofactor comprises Co2+, Mn2+, Zn2+and / or Mg2+. Suitable catalytic cofactor cations are disclosed in U.S. Patent No. 8,133,672 which is incorporated herein by reference in its entirety. In some embodiments, the catalytic cofactor is a metal provided in the forms of salts such as MgChor C0CI2. The salts form hydrates such as MgC12*(H2O)xor CoCh’nEhO (n=l, 2, 6, and 9) in aqueous solution. In some embodiments, the catalytic cofactor is magnesium. Magnesium may be present as a magnesium salt such as magnesium chloride (MgCh). Magnesium may be35MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 provided as metallic magnesium, Mg(0), and can be oxidized by electrolysis at an anode in buffered solution to generate Mg(II). Another suitable metal cofactor is cobalt. Cobalt can be provided as a cobalt complex such as a cobalt (III) complex or a cobalt (I) complex. Example cobalt complexes include trans-Dichlorobis(ethylenediamine)cobalt(III) chloride, pentaaminechlorocobalt(III) chloride, hexamine cobalt(III) chloride, trans- dichlorotetrakis(imidazole)cobalt(III) chloride or chlorotris(triphenylphosphine)cobalt(I). The cobalt complex may be reduced or oxidized to cobalt(II) chloride (C0CI2). For example, a Co(III)-complex can be reduced to a Co(II)-complex which can undergo ligand exchange with a buffered aqueous solution to form Co(II) which can then coordinate with a polymerase to activate it for polynucleotide synthesis. A ligand exchange reaction involves the substitution of one or more ligands in a complex ion with one or more different ligands.

[0106] Certain divalent or trivalent metal cofactors such as magnesium and manganese are known to interact with a polymerase to modulate the progress of the reaction. Such catalytic metal cofactors can coordinate with a polymerase and the triphosphate of a dNTP to catalyze the addition of a nucleotide to the 3’ terminal nucleotide on the end of the primer, creating a phosphodiester linkage between the nucleotide of the dNTP and the initiator and releases pyrophosphate (PPi).

[0107] In some aspects, the first reaction mixture comprises a non-catalytic cofactor of the polymerase which interacts with a polymerase, such as Phi29 or a variant or derivative thereof, to negative effect, e.g., to stabilize the enzyme and halt polymerization. Different non- catalytic co-factors can have varying effects upon the polymerization reaction depending upon the nature of the polymerization reaction, the polymerase used, the nucleotides employed, etc., and in some embodiments, the catalytic / non-catalytic effects of these cofactors are used to control initiation of polymerization such as RCA reactions. In some aspects, a catalytic cofactor for Phi29 or a variant or derivative thereof, includes Co2+, Mn2+, Zn2+or Mg2+, or any combination thereof. In some embodiments, the first reaction mixture is substantially free of Co2+, Mn2+, Zn2+and Mg2+, so as to halt the polymerization such as RCA reactions while allowing a polymerase and / or primer to diffuse in a sample and bind to circular nucleic acids, and / or complexes thereof. In some aspects, the first reaction mixture comprises strontium. In some aspects, the first reaction mixture comprises nickel. In some aspects, the first reaction mixture comprises SrCh.36MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0108] In some embodiments, the first reaction mixture comprises a chelating agent. In some embodiments, the biological sample is contacted with a chelating agent. For instance, the chelating agent can chelate a di-cation such as Mg2+from one or more prior reactions. In some embodiments, the first reaction mixture comprises EDTA, EGTA, BAPTA, DTPA, or a combination thereof. In some embodiments, one or more chelating agents in the first reaction mixture chelates catalytic metal cofactors for Phi29 or a variant or derivative thereof, such as Co2+, Mn2+, Zn2+or Mg2+, thereby sequestering these catalytic cofactors from polymerases in the reaction mixtures and / or in the sample in order to halt polymerization.

[0109] In some embodiments, the first reaction mixture comprises one or more cofactors that interact with a polymerase, but that do not promote the polymerization reaction, and in some cases act to arrest or prevent polymerization and / or inhibit one or more other activities of the polymerase, such as the 3'^-5' exonuclease activity. In some embodiments, the first reaction mixture comprises one or more non-catalytic cofactors, wherein the non-catalytic cofactor comprises metal ions, such as calcium, barium, strontium, iron, cobalt, nickel, tin, zinc, and europium. These metals can be added to the first reaction mixture or biological sample in salt form such as SrCh, Sr(OAc)2, Sr(OAc)2, C0CI2, SnCh, CaCh, or ZnSCU. A first metal cofactor that might be deemed to be catalytic under a first set of reaction conditions or relative to second metal co-factor, may be deemed to be a non-catalytic metal under another different set of reaction conditions, or with respect to a third metal co-factor. In some instances, under certain conditions, and / or relative to manganese, magnesium can operate as a non-catalytic cofactor. In some embodiments herein, a catalytic cofactor supports polymerization to a greater degree than the non-catalytic cofactor under the same reaction conditions. In some embodiments, the relative catalytic impact is a function of the reactant turnover rate of the polymerization complex, with catalytic metal cofactors promoting a turnover that is at least two times, more preferably at least 5 times, still more preferably, at least 10 times, and in some cases 20 times, 50 times or more than that of the non-catalytic metal cofactor under the same reaction conditions. In some embodiments, the presence of a non-catalytic cofactor in complex with the polymerase through binding in or around the active site, results in the inability for the synthesis reaction to proceed out of the complexed state. In particular, the presence of calcium ions can modulate both the forward progress of the polymerase reaction, as well as the reverse progress of the reaction. As a result, in the presence of calcium or other non-catalytic cofactors, the complexed nucleotide is37MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 effectively sequestered in the polymerase complex. The reaction is an unproductive nucleotide binding event, that is, it is unable to proceed forward to incorporation, or in reverse to the release of the unincorporated nucleotide to yield a free polymerase.

[0110] In some embodiments, the catalytic cofactor is a metal selected from Mg2+, Mn2+and mixtures thereof, and the non-catalytic cofactor is a metal selected from Ca2+, Zn2+, Co2+, Ni2+, EU2+, Sr2+, Ba2+, Fe2+, Eu2+and mixtures thereof. In some embodiments, the first reaction mixture comprises one or more of Ca2+, Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, and EU2+. In some embodiments, the first reaction mixture comprises Ca2+and / or Sr2+. In some embodiments, the first reaction mixture comprises one or more of Ca2+, Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, and Eu2+, and is substantially free of Mg2+and / or Mn2+. In some embodiments, the first reaction mixture comprises Ni2+and / or Sr2+and is substantially free of Mg2+and / or Mn2+.

[0111] In some embodiments, the first reaction mixture comprises one or more dNTPs. However, in some embodiments, due to the absence of one or more catalytic cofactors, the presence of one or more chelating agents for the catalytic cofactors, and / or the presence of one or more non-catalytic cofactors, the dNTPs are sequestered and the polymerase is unable to incorporate the dNTPs. In some embodiments, the first reaction mixture allows binding among a polymerase, a primer, and / or a circular nucleic acid, in order to form complexes at multiple locations in a sample that are ready to start RCA once the polymerase activity is turned on.

[0112] In some embodiments, the first reaction mixture comprises one or more polynucleotides that can function as RCA primers. In some embodiments, since Phi29 possesses a 3'^-5' exonuclease (proofreading) activity acting preferentially on single- stranded DNA or RNA, in some embodiments, the primer can be 3'-modified. In some embodiments, the primer is 3' thiophosphate-protected, which protects the polynucleotide from 3'^-5' exonuclease degradation by the polymerase while allowing priming by the polymerase. In some embodiments, the primer comprises a 3 '-tail of sufficient length in order to protect the sequence that functions as RCA primer from degradation by Phi29. In some embodiments, the 3 '-end tail can be gradually digested until the remaining part can be converted to RCA primer and is extended along the circular template via the polymerase activity of Phi29. However, in some embodiments, due to the absence of one or more catalytic cofactors, the presence of one or more chelating agents for the catalytic cofactors, and / or the presence of one or more non-catalytic38MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 cofactors, the exonuclease activity of Phi29 may be effectively inhibited such that no 3’ protective modification or 3 ’ -tail is necessary. Thus, in some embodiments, the primer has a free 3’ hydroxyl group available for nucleotide incorporation by Phi29.

[0113] In some embodiments, the first reaction mixture comprises one or more polymerases. In some embodiments, the biological sample is incubated with the first reaction mixture and one or more polymerases. In some embodiments, the polymerase comprises a modified recombinant Phi29-type polymerase. In some embodiments, the polymerase comprises a modified recombinant Phi29, B103, GA-1, PZA, Phil5, BS32, M2Y, Nf, Gl, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In some embodiments, the polymerase comprises a modified recombinant DNA polymerase having at least one amino acid substitution or combination of substitutions as compared to a wildtype Phi29 polymerase. Suitable polymerases are described in U.S. Patent Nos. 8,257,954; 8,133,672; 8,343,746; and 8,921,086, all of which are herein incorporated by reference. In some embodiments, the polymerase is not directly or indirectly immobilized to a substrate, such as a bead or planar substrate (e.g., glass slide), prior to contacting a sample, although the sample may be immobilized on a substrate. In some embodiments, the polymerase is not attached to a nanopore, a nanopore membrane or an insulating support thereof. In some embodiments, the polymerase is diffusible in the binding mixture and / or in the biological sample. In some embodiments, a polymerase and the RCA primer are diffusible in the biological sample after providing the first reaction mixture. In some instances, the primer bound to the Phi29 DNA polymerase is hybridized to the hybridization region and the Phi29 DNA polymerase is prevented from extending the primer during the incubation with the first reaction mixture.

[0114] In some embodiments, the cell or tissue sample is incubated with the polymerase in the first reaction mixture. Suitable examples of DNA polymerases that can be used include, but are not limited to: E.coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, LongAmp® Taq DNA polymerase, LongAmp® Hot Start Taq DNA polymerase, Crimson LongAmp® Taq DNA polymerase, Crimson Taq DNA polymerase, OneTaq® DNA polymerase, OneTaq® Quick-Load® DNA polymerase, Hemo KlenTaq® DNA polymerase, REDTaq® DNA polymerase, Phusion® DNA polymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA39MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase and T7 DNA polymerase enzymes.

[0115] In some instances, upon addition of a polymerase in the presence of appropriate dNTP precursors and other catalytic cofactors, the amplification primer is elongated by replication of multiple copies of the template (e.g., circular nucleic acid). The amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after formation of the circular nucleic acid by ligation (e.g., of the ends of a padlock probe) the circular nucleic acid is rolling-circle amplified to generate a RCA product containing multiple copies of the sequence(s) of the circular nucleic acid. In some embodiments, the multiple copies of the sequence(s) of the circular nucleic acid comprises at least 2 copies, at least 5 copies, at least 10 copies, at least 20 copies, at least 50 copies, at least 100 copies, or at least 500 copies of the sequence(s) of the circular nucleic acid. In some embodiments, a product generated from amplification of the sequence(s) of the circular nucleic acid comprises at least 10 copies, at least 20 copies, at least 50 copies, at least 100 copies, or at least 500 copies of the sequence(s) of the circular nucleic acid.

[0116] In some embodiments, after the incubation with the first reaction mixture, the biological sample (e.g., cell or tissue sample) is contacted with a second reaction mixture. In some embodiments, the method further comprises between the contacting with the first and second reaction mixtures, washing the biological sample. In some embodiments, the method comprises one or more stringency washes between the contacting with the first and second reaction mixtures.

[0117] In some embodiments, the second reaction mixture comprises reagents for performing RCA. For example, the biological sample is contacted with a second reaction mixture to allow the polymerase to extend the primer using the circular nucleic acid as a template, thereby generating a rolling circle amplification product in the biological sample. In some embodiments, the second reaction mixture comprises a catalytic cofactor of the polymerase. In some instances, the second reaction mixture comprises a mixture of free nucleotides comprising all four canonical bases: adenine, thymine, guanine and cytosine. In some embodiments, the second reaction mixture comprises a mixture of free nucleotides and a catalytic cofactor of the polymerase. In some embodiments, the second reaction mixture lacks additional polymerase. In some embodiments, the second reaction mixture is substantially free of polymerase.40MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0118] In some embodiments, rolling circle amplification products are generated using a polymerase selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi- PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and a variant or derivative thereof.

[0119] In some embodiments, RCA is performed after contacting the biological sample with a second reaction mixture comprising a crowding agent. In some embodiments, the second reaction mixture comprises a mixture of free nucleotides, a crowding agent and a catalytic cofactor of the polymerase. In some embodiments, the crowding agent is selected from the group comprising poly (ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate. In some embodiments, the crowding agent is polyethylene glycol) (PEG). In some embodiments, the crowding agent is PVP. A provided amount or concentration of an agent, such as a crowding agent, includes the sum amount or concentration of one or more agents with the same functional properties. For example, an amount or concentration of a crowding agent in a reagent encompasses the sum of any amount or concentration of all crowding agents in said reagents. That is, reference to “at least 10% of a crowding agent,” for example, encompasses at least 5% of a first crowding agent and at least 5% of a second crowding agent, or at least 8% of a first crowding agent and at least 2% of a second crowding agent, even if the first and second crowding agents are structurally distinct. In some embodiments, the second reaction mixture comprises at least about 10% crowding agent. For example, the percentage of the crowding agent in the second reaction mixture is a mass by volume percentage (w / v). In some instances, the percentage of the crowding agent in the second reaction mixture is a mass by volume percentage (w / v) of the total amount of two or more crowding agents. For example, the biological sample is contacted with a second reaction mixture comprising a final concertation of crowding agent that is at least 10%, at least 15%, at least 18% or at least 20% (w / v). In some embodiments, the second reaction mixture comprises at least about 15% crowding agent. In some embodiments, the second reaction mixture comprises at least about 20% crowding agent. In some41MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 embodiments, the second reaction mixture comprises no more than about 25% crowding agent. In some embodiments, the second reaction mixture comprises no more than about 20% crowding agent. In some embodiments, the second reaction mixture comprises no more than about 18% crowding agent. In some embodiments, the second reaction mixture comprises between about 10% and about 25% crowding agent. In some embodiments, the second reaction mixture comprises between about 10% and about 30% crowding agent. In some embodiments, the second reaction mixture comprises between about 10% and about 25% crowding agent. In some embodiments, the second reaction mixture comprises between about 10% and about 20% crowding agent. In some instances, the second reaction mixture comprises between about 10% and about 12.5% crowding agent.

[0120] In some instances, the crowding agent in the second reaction mixture is PVP. In some instances, the crowding agent in the second reaction mixture is PEG. In some instances, the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG12000, PEG20000, and PEG35000. In some instances, the crowding agent in the second reaction mixture comprises 15% PEG6000. In some instances, the crowding agent in the second reaction mixture comprises 15% PEG20000. In some instances, the crowding agent in the second reaction mixture is a mixture of different molecular weight polyethylene glycol. In some instances, the crowding agent in the second reaction mixture comprises PEG6000 and PEG20000. In some instances, the crowding agent in the second reaction mixture comprises at least 5% PEG20000 and at least 5% PEG6000. In some instances, the crowding agent in the second reaction mixture comprises 10% PEG20000 and 5% PEG6000. In some embodiments, the second reaction mixture comprises at least about 10% PEG. In some embodiments, the second reaction mixture comprises at least about 15% PEG. In some embodiments, the second reaction mixture comprises at least about 20% PEG. In some embodiments, the second reaction mixture comprises between about 5% and about 25% PEG, between about 5% and about 20% PEG, between about 5% and about 15% PEG, between about 5% and about 10% PEG, between about 10% and about 25% PEG, between about 10% and about 20% PEG, or between about 10% and about 15% PEG. In some instances, the second reaction mixture comprises between about 10% and about 12.5% PEG. In some instances, the second reaction mixture comprises between about 12% and about 16% PEG In some embodiments, the second reaction mixture comprises between about 10% and about 15% PEG. In some embodiments, the second reaction mixture comprises42MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 between about 10% and about 15% dextran sulfate. In some embodiments, the second reaction mixture comprises between about 10% and about 20% PEG. In some embodiments, the second reaction mixture comprises between about 12% and about 18% PEG. In some embodiments, the second reaction mixture comprises between about 10% and about 20% dextran sulfate. In some embodiments, the second reaction mixture comprises between about 10% and about 20% PVP. In some embodiments, the second reaction mixture comprises about 10% PEG. In some embodiments, the second reaction mixture comprises about 10% PVP.

[0121] In some embodiments, the first reaction mixture is substantially free of a crowding agent. In some embodiments, the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%. In some embodiments, the first reaction mixture comprises less than 12% of a crowding agent. In some embodiments, the first reaction mixture comprises less than 10% of a crowding agent. In some embodiments, the first reaction mixture comprises no more than 5% of a crowding agent. In some embodiments, the first reaction mixture comprises no more than 2% of a crowding agent. In some embodiments, the first reaction mixture comprises no more than 1% of a crowding agent. In some embodiments, the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 2%. In some instances, the crowding agent in the first reaction mixture is the same as the crowding agent in the second reaction mixture. In some instances, the crowding agent in the first reaction mixture is the same type of crowding agent as the crowding agent in the second reaction mixture (e.g., both comprise PEG, both comprise PVP, etc.). In some embodiments, the crowding agent in both the first reaction mixture and the second reaction mixture comprises PEG. In some instances, the crowding agent in the first reaction mixture is different from the crowding agent in the second reaction mixture. In some instances, the crowding agent in the first reaction mixture is a different type of crowding agent from the crowding agent in the second reaction mixture. In some instances, the crowding agent in the first reaction mixture comprises PEG and the crowding agent in the second reaction mixture does not comprise PEG. In some instances, the crowding agent in the first reaction mixture does not comprise PEG and the crowding agent in the second reaction mixture comprises PEG.

[0122] In some embodiments, the second reaction mixture comprises a deoxynucleoside triphosphate (dNTP) or derivative, variant, or analogue thereof. In some embodiments, the second reaction mixture comprises a catalytic cofactor of the polymerase. In 43MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 any of the preceding embodiments, the second reaction mixture comprises a catalytic cofactor that is a di-cation, such as Mg2+and / or Mn2+. In some embodiments, the second reaction mixture is substantially free of a non-catalytic cofactor of the polymerase, such as Ca2+, Zn2+, Co2+, Ni2+, EU2+, Sr2+, Ba2+, Fe2+, Eu2+and mixtures thereof. In some embodiments, a catalytic cofactor in the second reaction mixture can replace a non-catalytic cofactor in complex with the polymerase, the circular nucleic acid and the RCA primer, thus turning on the polymerase activity of the polymerase. In some embodiments, when the sample is contacted with a second reaction mixture comprising a catalytic cofactor (such as Mg2+and / or Mn2+), a non-catalytic cofactor (such as Ca2+, Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, and / or Eu2+) bound to Phi29 is displaced, thereby activating the 5'^-3' polymerase activity and the 3'^-5' exonuclease (proofreading) activity of Phi29.

[0123] In some embodiments, the pH of the first and second reaction mixtures can be substantially the same, e.g., about pH 8.0. In some embodiments, the pH of the first and second reaction mixtures are independently about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In any of the preceding embodiments, the pH of the first and second reaction mixtures are independently about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

[0124] In some embodiments, the first reaction mixture and the second reaction mixture both comprises dNTPs. In some embodiments, the second reaction mixture comprises dNTPs and the first reaction mixture is free of dNTPs.

[0125] In some embodiments, the second reaction mixture is substantially free of the polymerase and / or other polymerases. In some embodiments, the first reaction mixture comprises the polymerase and the second reaction mixture is substantially free of the polymerase and / or other polymerases. In some embodiments, molecules of the polymerase that are not bound to the circular nucleic acid and / or the RCA primer are removed from the biological sample. Thus, in some embodiments, substantially all of the polymerase molecules in the sample are bound to a circular nucleic acid and a RCA primer and ready to initiate RCA at the same time once the enzyme activity is turned on. In some instances, the primers hybridized to the hybridization region of the circular nucleic acids are extended by the polymerase, thereby initiating rolling circle amplification simultaneously at different circular nucleic acids in a44MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 concerted manner. In some instances, the RCA reactions are terminated at the same time to provide a plurality of rolling circle amplification products.

[0126] In some aspects, using the first and second reaction mixture allow synchronization of RCA in situ. For instance, synchronized RCA reactions (e.g., starting the reaction at the same time) of circularized probes targeting analytes at different locations in a tissue section may provide more homogeneously sized RCA products. In some aspects, the first reaction mixture allows an environment for amplification reagents and components to diffuse through the sample before amplification is initiated upon addition of the second reaction mixture to the biological sample. In some aspects, incubation with the first reaction mixture allows amplification reagents and components to diffuse through the sample and form complexes before amplification is initiated upon addition of the second reaction mixture to the biological sample. In some aspects, performing RCA in situ using the first and second reaction mixture allows generation of RCA products which remain localized throughout amplification and for detection. In some aspects, performing RCA in situ using the first and second reaction mixture allows results in at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of RCA products to remain localized throughout amplification and for detection. In some aspects, performing RCA in situ using the first and second reaction mixture allows results in at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of generated RCA products to be assigned correctly to an originating cell.

[0127] In some embodiments, the pH of the first reaction mixture and second reaction mixture is between pH 7.5 to pH 8.5. In some embodiments, the pH of the first reaction mixture and second reaction mixture is about pH 8. In some embodiments, the pH of the first reaction mixture is between pH 7.5 to pH 8.5. In some embodiments, the pH of the second reaction mixture is between pH 7.5 to pH 8.5. In some embodiments, the pH of the second reaction mixture is between pH 7.2 to pH 7.7. In some embodiments, the pH of the second reaction mixture is about pH 7.5. In some embodiments, the pH of the second reaction mixture is pH 8.3 or lower. In some embodiments, the second reaction mixture is substantially free of NaOH.

[0128] 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 45MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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. 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 35°C. In some aspects, the amplification performed with the second reaction mixture is at a temperature between at or about 25°C and at or about 35°C. In some aspects, the amplification performed with the second reaction mixture is at a temperature between at or about 32°C and at or about 35°C. In some aspects, the amplification performed with the second reaction mixture is at a temperature of about 30°C. In some aspects, the amplification performed with the second reaction mixture is at a temperature of about 35°C. In some aspects, due to the viscosity of the crowding agent in the second reaction mixture, the amplification performed with the second reaction mixture is at a temperature of at least 32°C.

[0129] In some embodiments, the amplification is performed for no more than 3 hours, no more than 2 hours, no more than 1 hour, or no more than 30 minutes. In some embodiments, the amplification is incubated with the second reaction mixture for no more than 3 hours, no more than 2 hours, no more than 1 hour, or no more than 30 minutes. In some instances, wherein the polymerase extension or RCA is performed for no more than 120 minutes. In some instances, the RCA of the circular nucleic acids is terminated to provide a plurality of RCA products. In some instances, the RCA of the circular nucleic acids is terminated to provide a plurality of RCA products after about 3 hours, 2 hours, 1 hour, or 30 minutes. In some aspects, the amplification performed with the second reaction mixture is at a temperature of about 30°C for about 90 minutes. In some aspects, the amplification performed with the second reaction mixture is at a temperature of about 35 °C for about 90 minutes.

[0130] In some aspects, provided herein is a method comprising: contacting the biological sample with a binding mixture (e.g., in a first reaction mixture that allows the primers to hybridize to the plurality of circular nucleic acids and bind to polymerases). In some embodiments, the biological sample comprises a plurality of circular nucleic acids each comprising a primer hybridization region, during incubation with the first reaction mixture, a plurality of complexes to form each comprising a polymerase, a primer, an a circular nucleic acid. In some embodiments, the primer comprises a sequence complementary to the primer hybridization region of one or more circular nucleic acids, and the polymerase activity of the polymerase is inhibited, thereby allowing the complexes to form without initiating amplification.46MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940In some cases, complexes are formed in a condition that does not allow amplification to progress. In some embodiments, the method further comprises contacting the biological sample with a second reaction mixture for RCA that provides reagents for performing amplification. In some embodiments, the amplification reaction mixture allows the polymerase to extend the primer hybridized to the primer hybridization region, thereby initiating rolling circle amplification the plurality of circular nucleic acids in the biological sample. In some instances, RCA is performed with the second reaction mixture in the biological sample to generate an amplification product.

[0131] For example, as shown in the workflow of FIG. 1, a biological sample (e.g., a tissue sample) is contacted with a circularizable probe. The circularizable probe is ligated to generate a circular nucleic acid. The biological sample is contacted with a first reaction mixture comprising polymerase. In some aspects, a complex is formed after addition of the first reaction mixture to the biological sample, where the complex comprises a polymerase such as Phi29, a primer, and the generated circular nucleic acid. In some aspects, the first reaction mixture stabilizes the complex but inhibits one or more activities of the polymerase, such as the polymerase and / or exonuclease activities. In some embodiments, the biological sample is incubated with first reaction mixture to allow diffusion of reagents and for the complexes to form. In some instances, the biological sample is contacted with a wash solution and the wash solution is removed. The biological sample is then contacted and incubated with a second reaction mixture comprising at least 10% of a crowding agent to perform RCA. In some instances, the second reaction mixture comprises at least 15% of a crowding agent. In some embodiments, the generated rolling circle amplification product is detected at a location in the biological sample.

[0132] Provided herein is a method comprising adding a first reaction mixture comprising a polymerase, a plurality of dNTPs and a non-catalytic cofactor of the polymerase to a cell or tissue sample comprising a primer and a circular nucleic acid, wherein the primer is exogenous to the cell or tissue sample, and wherein the circular nucleic acid comprises a hybridization region complementary to the primer; after addition of the first reaction mixture, incubating the cell or tissue sample for at least 60 minutes; after the incubation, removing the first reaction mixture from the cell or tissue sample; contacting the cell or tissue sample with a second reaction mixture comprising at least 15% of a crowding agent; and performing rolling47MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 circle amplification (RCA) at 30°C or higher for at least 60 minutes in the cell or tissue sample contacted with the second reaction mixture, wherein the polymerase extends the primer using the circular nucleic acid as a template to generate an RCA product. In some embodiments, removing the first reaction mixture from the cell or tissue sample comprises washing the cell or tissue sample.

[0133] Provided herein is a method comprising adding a first reaction mixture comprising a polymerase, a plurality of dNTPs and strontium to a cell or tissue sample comprising a primer and a circular nucleic acid, wherein the primer is exogenous to the cell or tissue sample, and wherein the circular nucleic acid comprises a hybridization region complementary to the primer; after addition of the first reaction mixture, incubating the cell or tissue sample for at least 60 minutes at 4°C; after the incubation, removing the first reaction mixture from the cell or tissue sample; contacting the cell or tissue sample with a second reaction mixture comprising at least 15% of a crowding agent; and performing rolling circle amplification (RCA) at 30°C or higher in the cell or tissue sample contacted with the second reaction mixture, wherein the polymerase extends the primer using the circular nucleic acid as a template to generate an RCA product. In some embodiments, removing the first reaction mixture from the cell or tissue sample comprises washing the cell or tissue sample.

[0134] Provided herein is a method comprising adding a first reaction mixture comprising a polymerase and strontium to a cell or tissue sample comprising a primer and a circular nucleic acid, wherein the primer is exogenous to the cell or tissue sample, and wherein the circular nucleic acid comprises a hybridization region complementary to the primer; after addition of the first reaction mixture, incubating the cell or tissue sample for at least 30 minutes at 20°C or higher; after the incubation, removing the first reaction mixture from the cell or tissue sample; contacting the cell or tissue sample with a second reaction mixture comprising at least 10% of a crowding agent; and performing rolling circle amplification (RCA) at 30°C or higher in the cell or tissue sample contacted with the second reaction mixture, wherein the polymerase extends the primer using the circular nucleic acid as a template to generate an RCA product. In some embodiments, removing the first reaction mixture from the cell or tissue sample comprises washing the cell or tissue sample.

[0135] In some embodiments, the wash is performed prior to contacting the biological sample (e.g., cell or tissue sample) with a second reaction mixture. For example, the 48MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 wash comprises one or more stringency washes. In some cases, the wash solution is added to the biological sample after incubating with the first reaction mixture and prior to adding the second reaction mixture. In some examples, the wash solution comprises the crowding agent and a mixture of dNTPs. In some instances, the wash solution comprises the crowding agent at a concentration of between 0.1% and 5%. In some instances, the wash solution comprises the crowding agent at a lower concentration than the second reaction mixture. In some embodiments, the wash solution comprises dNTPs. In some instances, the wash solution comprises a crowding agent and dNTPs. In some examples, the concentration of dNTPs in the wash solution is lower than the concentration of dNTPs in the second reaction mixture.

[0136] In some embodiments, the circular nucleic acids comprise probes targeting endogenous molecules in the biological sample, such as padlock probes targeting genomic DNA or cellular RNA such as mRNA. In some embodiments, the circular nucleic acids comprise products of exogenous probes targeting endogenous molecules in the biological sample.

[0137] In some embodiments, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other catalytic cofactors, a primer is elongated to produce multiple copies of the circular nucleic acid. This amplification step can utilize isothermal amplification or non-isothermal amplification. Techniques for rolling circle amplification (RCA) include linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. Exemplary polymerases for use in RCA comprise DNA polymerase such Phi29 (cp29) polymerase, Klenow fragment, Bacillus stearothermophilus 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.

[0138] In some aspects, during the amplification reaction, modified nucleotides are added to the reaction to incorporate the modified nucleotides in the amplification product (e.g., nanoball). In some instances, 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 products comprise a modified nucleotide, such as an amine-modified nucleotide. In some embodiments, the amine-modified nucleotide comprises an acrylic acid N- hydroxy succinimide moiety 49MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 modification. Examples of other amine-modified nucleotides comprise, but are not limited to, a5-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.

[0139] In some aspects, the amplification product (e.g., amplicon) are anchored to a polymer matrix. In some aspects, the amplification product (e.g., amplicon) are not anchored to a polymer matrix using functional groups. For example, the polymer matrix is a hydrogel. In some embodiments, one or more of the polynucleotide probe(s) or generated product is 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. Exemplary modification and polymer matrix that can be employed in accordance with the provided embodiments comprise those described in, for example, WO 2017 / 079406, US 2016 / 0024555, US 2018 / 0251833 and US 2017 / 0219465, 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 set or amplification product. In some examples, the scaffold can comprise oligonucleotides, polymers or chemical groups, to provide a matrix and / or support structures.

[0140] In some instances, the amplification products are immobilized within the matrix generally at the location of the nucleic acid being amplified, thereby creating a localized colony of amplicons. In some cases, the amplification products are immobilized within the matrix by steric factors. The amplification products may also be 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.

[0141] 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 50MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 within the cell thereby providing a subcellular localization distribution pattern. In some embodiments, the provided methods involve embedding the one or more polynucleotide 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 clearing, 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.

[0142] In some embodiments, the circular nucleic acid comprises a sequence associated with the target analyte, or a part 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. In some embodiments, different analytes are detected in situ in one or more cells using a RCA-based detection system, e.g., where the signal is provided by generating an RCA product from a circular RCA template which is provided or generated in the assay, and the RCA product is detected to detect the corresponding analyte. In some embodiments, the RCA product 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 RCA product 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.

[0143] Following amplification, the sequence of the RCA product or a portion thereof, is determined or otherwise analyzed, for example by using detectably labeled probes and imaging. 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. In some instances, a sequence of the RCA product is detected at a location in the biological sample. 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.51MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940C. Detection

[0144] 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.

[0145] 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.A and II.B).

[0146] 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.

[0147] 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 the primary probe or probe set, 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 more52MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 wash steps to remove unbound and / or nonspecifically bound intermediate probe molecules from the RCPs.

[0148] 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.

[0149] 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 / or hybridization-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).

[0150] 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).

[0151] In some aspects, the provided methods comprise imaging a detectably labeled probe bound directly or indirectly to the first probe or product thereof 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,53MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.

[0152] 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, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenical acetyl transferase, and urease.

[0153] 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).

[0154] 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.54MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0155] 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 as1251,35S,14C, or3H. Identifiable markers are commercially available from a variety of sources.

[0156] 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- dichlorofluorescein dyes, spectrally resolvable rhodamine dyes, 4,7- dichlororhodamine dyes, cyanine dyes, ether-substituted fluorescein dyes, energy transfer dyes, and xanthine dyes. Labeling 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. Exemplary fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer.

[0157] 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-55MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940UTP), 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.

[0158] 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.

[0159] 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).

[0160] 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 56MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0161] 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.

[0162] 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. Exemplary 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.).

[0163] In some embodiments, detection of the barcode sequences of the circular nucleic acid 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.57MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0164] 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.

[0165] 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.

[0166] 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 (a second nucleotide residue) in the template nucleotide as template. Thus, in some embodiments, cycles of introducing and removing detectably labeled nucleotides are performed.58MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0167] 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.

[0168] 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.

[0169] 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 nucleic acid 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 labeling may comprise59MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 fluorescence labeling of, e.g., the cognate nucleotide or the polymerase that participate in the ternary complex.

[0170] 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.

[0171] 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.

[0172] In some embodiments, the detecting step 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 probes or probe sets), and dehybridizing the one or more detectably labeled probes. In some embodiments, the contacting and dehybridizing steps are repeated with the one or more 60MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0173] 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).

[0174] In any of the embodiments herein, the detecting step can comprise 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 probes or probe sets or rolling circle amplification product generated using the plurality of probes or probe sets), 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 probes or probe sets or rolling circle amplification product generated using the plurality of probes or probe sets). 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., fluorophore sequences assigned to the corresponding barcode sequences or complements thereof).

[0175] 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 61MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0176] In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporations can be detected through fluorescence 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.

[0177] In some embodiments, the analysis and / or sequence determination involves washing to remove unbound polynucleotides, thereafter revealing a fluorescent product for imaging.

[0178] 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.

[0179] 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).62MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0180] 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 light, 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.

[0181] 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.63MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0182] 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 microscopy (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).

[0183] 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., RCA product 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 circular or circularizable probes hybridize to the RNA transcripts in the biological sample, and are optionally ligated and amplified by rolling circle amplification. In some embodiments, the64MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 detecting is performed at a plurality of locations to detect 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 RNA transcripts in the biological sample.III. Samples, Analytes, and Target SequencesA. Samples

[0184] 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, in 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.

[0185] 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. The65MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0186] 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.

[0187] 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, depending 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.

[0188] 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.66MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940(i) Preparation

[0189] 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.

[0190] 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 the tissue 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 pm. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 pm or more. Typically, the thickness of a tissue section is between 1-100 pm, 1-50 pm, 1-30 pm, 1-25 pm, 1-20 pm, 1-15 pm, 1-10 pm, 2-8 pm, 3-7 pm, or 4-6 pm, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analysed.

[0191] 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.

[0192] 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 67MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0193] 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., deparaffinization) 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 deparaffinization. In some embodiments, processing of the biological sample, such as de-waxing, allows the biological sample to become permeabilized.

[0194] 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.

[0195] 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 postfixing 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.

[0196] 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.68MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0197] 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.

[0198] 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- 66, 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.

[0199] 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.

[0200] 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 69MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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

[0201] 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. Biological 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.

[0202] 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.

[0203] 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.70MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0204] 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.

[0205] 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, a 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.

[0206] 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.

[0207] 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.

[0208] In some embodiments, a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers. Examples of71MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0209] 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 sample), 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.

[0210] 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.

[0211] 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.

[0212] 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.72MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0213] 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.

[0214] 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 within 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 labeling agent is added to the hydrogel before, contemporaneously with, and / or after polymerization. In some embodiments, a cell-penetrating agent is added to the hydrogel before, contemporaneously with, and / or after polymerization.

[0215] 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.

[0216] 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).73MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0217] 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 been isometrically expanded. In some embodiments, a biological sample is isometrically expanded to a size at least 2x, 2. lx, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3. lx, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4. lx, 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)

[0218] 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.

[0219] 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,74MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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).

[0220] 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.

[0221] 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.B. Analytes

[0222] A biological sample may comprise one or a plurality of analytes of interest. Methods for performing multiplexed assays to analyze two or more different analytes in a single biological sample are provided. The methods and compositions 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 is directly or indirectly detected.75MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0223] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed is 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 300, at least about 500, at least about 1,000, at least about 10,000, at least about 15,000, at least about 20,000, at least about 50,000, at least about 100,000 or more different analytes present. In some embodiments, the plurality of RNAs 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 about 500, at least about 1,000, at least about 10,000, at least about 15,000, at least about 20,000, at least about 50,000, at least about 100,000, at least 250,000, at least 500,000, or at least 1,000,000 distinguishable RNAs.

[0224] 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. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis, and / or allow access of one or more reagents (e.g., probes for analyte detection) to the analytes in the cell or cell compartment or organelle.

[0225] The analyte may include any biomolecule or chemical compound, including a macromolecule such as 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. An analyte can be any substance or entity for which a specific binding partner (e.g., an affinity binding partner) can be developed. Such a specific binding partner may be a nucleic acid probe (e.g., a padlock or other circularizable probe) and may lead directly to the generation of a RCA template (e.g., a circular nucleic acid as described in Section II). Alternatively, the specific binding partner may be coupled to a nucleic acid, which may be detected using an RCA strategy, e.g., in an assay which uses or generates a circular nucleic acid molecule which can be the RCA template.

[0226] Analytes of particular interest may include nucleic acid molecules, such as DNA (e.g., genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g., 76MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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 includes 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. The analyte may be 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 and nucleic 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.(i) Endogenous Analytes

[0227] In some embodiments, an analyte herein is endogenous to a biological sample and can include nucleic acid analytes and non-nucleic acid analytes. Methods and compositions disclosed herein can be used to analyze nucleic acid analytes (e.g., using a nucleic acid probe or probe set that directly or indirectly hybridizes to a nucleic acid analyte) and / or non-nucleic acid analytes (e.g., using a labeling agent that comprises a reporter oligonucleotide and binds directly or indirectly to a non-nucleic acid analyte) in any suitable combination.

[0228] 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 is an organelle (e.g., nuclei or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted77MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 analyte. Exemplary 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, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.

[0229] 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.

[0230] 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-stranded78MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940RNA or single-stranded RNA. In some embodiments, the RNA comprises circular RNA. In some embodiments, the RNA is a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0231] In some embodiments described herein, an analyte may be 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.

[0232] Methods and compositions 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 present in a region of the sample.(ii) Labeling Agents

[0233] In some embodiments, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, cell surface or intracellular proteins, and / or metabolites) in a sample using one or more labeling agents. In some embodiments, an analyte labeling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labeling agents can comprise a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the labeling agent. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labeling agent. In some cases, the sample contacted by the labeling agent can be further contacted with a probe (e.g., a single-stranded probe sequence), that hybridizes to a reporter oligonucleotide of the labeling agent, in order to identify the analyte associated with the labeling agent. In some embodiments, the analyte labeling agent comprises an analyte binding moiety and a labeling 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 includes 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 79MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0234] 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 labeling agents.

[0235] In the methods and systems described herein, one or more labeling agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes, cells 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.

[0236] 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 labeling 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 bispecific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a DARPin, and a protein scaffold, or any combination thereof. The labeling 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 labeling agent. For example, a labeling 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 labeling 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 a description of exemplary labeling agents, reporter80MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.

[0237] In some embodiments, an analyte binding moiety includes one or more antibodies or epitope-binding fragments thereof. The antibodies or epitope-binding fragments including the analyte binding moiety can specifically bind to a target analyte. In some embodiments, 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 labeling 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 includes a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labeling agents are the same. In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labeling agents are the different (e.g., members of the plurality of analyte labeling 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 includes multiple different species of analyte (e.g., multiple different species of polypeptides).

[0238] In other instances, e.g., to facilitate sample multiplexing, a labeling agent that is specific to a particular cell feature may have a first plurality of the labeling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labeling agent coupled to a second reporter oligonucleotide.

[0239] In some aspects, these reporter oligonucleotides may comprise nucleic acid barcode sequences that permit identification of the labeling 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, e.g., using the in situ detection techniques described herein.81MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0240] Attachment (coupling) of the reporter oligonucleotides to the labeling 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 labeling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labeling 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. Protein (e.g., antibody) and oligonucleotide biotinylation techniques are available. As a non-limiting example, in some cases click reaction chemistry is used to couple reporter oligonucleotides to labeling agents. Commercially available kits, such as those from Thunder-Link® and Abeam, and techniques common in the art may be used to couple reporter oligonucleotides to labeling agents as appropriate. In another example, a labeling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide comprising a barcode sequence that identifies the label agent. For instance, the labeling 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 labeling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labeling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be attached to the labeling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein.

[0241] In some cases, the labeling agent can comprise 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 embodiments, the label is conjugated to a labeling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labeling 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.

[0242] In some embodiments, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more 82MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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 can 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 labeling 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.IV. Systems, Compositions and Kits

[0243] In some aspects, provided herein are systems, compositions and kits comprising one or more probes (e.g., for generating the circular nucleic acids) and reagents for performing RCA as described in Section II. In some embodiments, the systems, compositions or kits comprise reagents for performing the methods provided herein, for example reagents required for one or more steps comprising hybridization, ligation, amplification, detection, sequencing, and / or sample preparation as described herein. In some aspects, the systems, compositions or kits comprise a first reaction mixture and a second reaction mixture as described herein. In some instances, a first reaction mixture comprises a polymerase.

[0244] In some embodiments, a system comprises: a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and optionally no more than 5% crowding agent; and a second reaction mixture comprising at least 10% of a crowding agent. In some embodiments, a system comprises: a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and optionally no more than 5% crowding agent; and a second reaction mixture comprising at least 10% of a crowding agent (e.g., at least 5% of a first crowding agent and at least 5% of a second crowding agent). In some embodiments, a system comprises: a cell or tissue sample comprising a circular83MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and a mixture of free nucleotides lacking at least one of four canonical bases: adenine, thymine, guanine and cytosine; and a second reaction mixture comprising at least 10% of a crowding agent and a mixture of free nucleotides comprising all four canonical bases: adenine, thymine, guanine and cytosine. In some embodiments, a system comprises a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase; and a second reaction mixture comprising at least 10% of a crowding agent and a catalytic cofactor of the polymerase. In some embodiments, the first reaction mixture is configured to prevent the polymerase from amplifying the circular nucleic acid in the cell or tissue sample. In some embodiments, In some embodiments, a system comprises: a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer; a first reaction mixture comprising a polymerase and no more than 5% crowding agent; and a second reaction mixture comprising at least 10% of a crowding agent.

[0245] In some instances, the second reaction mixture comprises at least 10% of a crowding agent. In some instances, the second reaction mixture comprises between about 10% and about 12.5% PEG. In some instances, the second reaction mixture comprises between about 12% and about 18% PEG. In some instances, the second reaction mixture comprises a mixture of dNTPs and at least 10% of a crowding agent. In some instances, the second reaction mixture comprises at least 15% of a crowding agent. For example, a first reaction mixture comprises a polymerase and a non-catalytic cofactor of the polymerase and the second reaction mixture comprises a mixture of dNTPs and at least 10% of a crowding agent. In some embodiments, the second reaction mixture comprises a catalytic cofactor of the polymerase. In some embodiments, the systems, compositions or kits further comprises a target nucleic acid. In some embodiments, any or all of the probes and / or polynucleotides are DNA molecules. In some embodiments, the target nucleic acid is a messenger RNA molecule. In some embodiments, the systems, compositions or kits further comprise a ligase, for instance for forming a circular nucleic acid from a circularizable probe (e.g., a padlock probe). In some embodiments, the ligase has DNA- splinted DNA ligase activity. In some embodiments, the ligase has RNA-splinted ligase activity.84MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940In some embodiments, the systems, compositions or kits further comprise a polymerase, for instance for performing amplification of the padlock probe, e.g., using any of the methods described in Section II.

[0246] In some embodiments, disclosed herein is a system, composition or kit for analyzing a biological sample, comprising contacting the biological sample comprising a circular nucleic acid with a first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase, and contacting the biological sample with a second reaction mixture comprising at least 10% of a crowding agent to allow the polymerase to extend the plurality of primers using the plurality of circular nucleic acids as a template, thereby generating a plurality of rolling circle amplification products in the biological sample. In some instances, the second reaction mixture comprises at least 15% of a crowding agent. In some embodiments, the biological sample is contacted by a plurality of primers. In some embodiments, the primers in the plurality of primers may be the same. Alternatively, in any of the preceding embodiments, the primers in the plurality of primers are of at least two or more different sequences. In any of the preceding embodiments, the polymerase is a Phi29 DNA polymerase and the non-catalytic cofactor of the polymerase is a di-cation, e.g., Sr2+and / or Ca2+. In any of the preceding embodiments, the polymerase can be Phi29 DNA polymerase and the catalytic cofactor of the polymerase is a dication, e.g., Mg2+, Co2+, and / or Mn2+.

[0247] The various components 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 to practice the provided methods.

[0248] In some embodiments, the system, composition or kit comprises reagents and / or consumables required for performing one or more steps of the provided methods. In some embodiments, the reagents include reagents for fixing, embedding, and / or permeabilizing the biological sample. In some embodiments, reagents such as enzymes and buffers for ligation and / or amplification, such as ligases and / or polymerases are provided. In some aspects, the system, composition or kit also comprise any of the reagents described herein, e.g., wash buffer and ligation buffer. In some embodiments, the system, composition or kit contains reagents for detection and / or sequencing, such as barcode detection probes or detectable labels. In some embodiments, the system, composition or kit optionally contains other components, for example85MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, reagents for additional assays.V. Opto-Fluidic Instruments for Analysis of Biological Samples

[0249] Provided herein is an instrument having integrated optics and fluidics modules (an “opto-fluidic instrument” or “opto-fluidic system”) for detecting target molecules (e.g., nucleic acids, etc.) in biological samples (e.g., one or more cells or a tissue sample) as described herein. In some aspects, the instrument is for generating and / or detecting amplification products generated as described in Section II at locations in the biological sample. In an opto-fluidic instrument, the fluidics module 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 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.C). In various embodiments, the captured images may be processed in real time and / or at a later time 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).

[0250] In various embodiments, the opto-fluidic instrument is configured to detect one or more target RNAs (e.g., as described in Section II) in their naturally occurring place (i.e., in situ) by detecting associated amplification products within the biological sample. In some embodiments, the opto-fluidic instrument is configured to process (e.g., perform RCA) and / or detect one or more analytes (e.g., as described in Section II) in relative spatial locations within the biological sample. For example, an opto-fluidic instrument may be 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.86MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0251] It is to be noted that, although the above discussion relates to an opto-fluidic instrument that can be used for in situ target molecule detection of amplification products, the discussion herein equally applies to any opto-fluidic instrument that employs any imaging or target molecule detection technique. That is, for example, an opto-fluidic instrument may include a fluidics module that includes fluids needed for establishing the experimental conditions required for the probing of target molecules in the sample. Further, such an opto-fluidic instrument may also include a sample module configured to receive the sample, and an optics module including an imaging system for illuminating (e.g., exciting one or more fluorescent probes within the sample) and / or imaging light signals received from the probed sample. The in- situ analysis system may also include other ancillary modules configured to facilitate the operation of the opto-fluidic instrument, such as, but not limited to, cooling systems, motion calibration systems, etc.

[0252] In some embodiments, the sample is a biological sample (e.g., a cell or tissue sample) that includes molecules such as DNA, RNA, proteins, antibodies, etc. In various embodiments, the sample is placed in the opto-fluidic instrument for analysis and detection of the molecules in the sample. In various embodiments, the opto-fluidic instrument can be a system configured to facilitate the experimental conditions conducive for the detection of the target molecules. For example, the opto-fluidic instrument can include a fluidics module, an optics module, a sample module, and an ancillary module, and these modules may be operated by a system controller to create the experimental conditions for the detection of the molecules in the sample, as well as to facilitate the imaging of the probed sample (e.g., by an imaging system of the optics module). In various embodiments, the various modules of the opto-fluidic instrument may be separate components in communication with each other, or at least some of them may be integrated together.

[0253] In various embodiments, the sample module may be configured to receive the sample into the opto-fluidic instrument. For instance, the sample module may include a sample interface module (SIM) that is configured to receive a sample device (e.g., cassette) onto which the sample can be deposited. That is, the sample may be placed in the opto-fluidic instrument by depositing the sample (e.g., the sectioned tissue) on a sample device that is then inserted into the SIM of the sample module. In some instances, the sample module may also include an X-Y stage onto which the SIM is mounted. The X-Y stage may be configured to move the SIM mounted87MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 thereon (e.g., and as such the sample device containing the sample inserted therein) in perpendicular directions along the two-dimensional (2D) plane of the opto-fluidic instrument.

[0254] The experimental conditions that are conducive for the detection of the generated amplification product in the sample may depend on the target molecule detection technique that is employed by the opto-fluidic instrument. For example, in various embodiments, the opto-fluidic instrument can be a system that is configured to detect amplification products in the sample via hybridization of probes. In such cases, the experimental conditions can include molecule hybridization conditions that result in the intensity of hybridization of the amplification product to a probe (e.g., detectably labeled probe) being significantly higher when the detectably labeled probe sequence is complementary to the amplification product than when there is a single-base mismatch. The hybridization conditions include the preparation of the sample using reagents such as washing / stripping reagents, hybridizing reagents, extension reagents etc., and such reagents may be provided by the fluidics module.

[0255] In various embodiments, the fluidics module may include one or more components that may be used for storing the reagents, as well as for transporting said reagents to and from the sample device containing the sample. For example, the fluidics module may include reservoirs configured to store the reagents, as well as a waste container configured for collecting the reagents (e.g., and other waste) after use by the opto-fluidic instrument to analyze and detect the molecules of the sample. Further, the fluidics module may also include pumps, tubes, pipettes, etc., that are configured to facilitate the transport of the reagent to the sample device (e.g., and as such the sample). For instance, the fluidics module may include pumps (“reagent pumps”) that are configured to pump washing / stripping reagents to the sample device for use in washing / stripping the sample (e.g., as well as other washing functions such as washing an objective lens of the imaging system of the optics module).

[0256] In various embodiments, the ancillary module can be a cooling system of the opto-fluidic instrument, and the cooling system may include a network of coolant-carrying tubes that are configured to transport coolants to various modules of the opto-fluidic instrument for regulating the temperatures thereof. In such cases, the fluidics module may include coolant reservoirs for storing the coolants and pumps (e.g., “coolant pumps”) for generating a pressure differential, thereby forcing the coolants to flow from the reservoirs to the various modules of the opto-fluidic instrument via the coolant-carrying tubes. In some instances, the fluidics module 88MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 may include returning coolant reservoirs that may be configured to receive and store returning coolants, e.g., heated coolants flowing back into the returning coolant reservoirs after absorbing heat discharged by the various modules of the opto-fluidic instrument. In such cases, the fluidics module may also include cooling fans that are configured to force air (e.g., cool and / or ambient air) into the returning coolant reservoirs to cool the heated coolants stored therein. In some instances, the fluidics module may also include cooling fans that are configured to force air directly into a component of the opto-fluidic instrument so as to cool said component. For example, the fluidics module may include cooling fans that are configured to direct cool or ambient air into the system controller to cool the same.

[0257] As discussed above, the opto-fluidic instrument may include an optics module which include the various optical components of the opto-fluidic instrument, such as but not limited to a camera, an illumination module (e.g., LEDs), an objective lens, and / or the like. The optics module may include a fluorescence imaging system that is configured to image the fluorescence emitted by the probes (e.g., oligonucleotides) in the sample after the probes are excited by light from the illumination module of the optics module.

[0258] In some instances, the optics module may also include an optical frame onto which the camera, the illumination module, and / or the X-Y stage of the sample module may be mounted.

[0259] In various embodiments, the system controller may be configured to control the operations of the opto-fluidic instrument (e.g., and the operations of one or more modules thereof). In some instances, the system controller may take various forms, including a processor, a single computer (or computer system), or multiple computers in communication with each other. In various embodiments, the system controller may be communicatively coupled with data storage, set of input devices, display system, or a combination thereof. In some cases, some or all of these components may be considered to be part of or otherwise integrated with the system controller, may be separate components in communication with each other, or may be integrated together. In other examples, the system controller can be, or may be in communication with, a cloud computing platform.

[0260] In various embodiments, the opto-fluidic instrument may analyze the sample and may generate the output that includes indications of the presence of the target molecules89MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940(e.g., target RNAs), the presence of which can be indicated by detecting sequences in generated amplification product in the sample. For instance, with respect to the example embodiment discussed above where the opto-fluidic instrument employs a hybridization technique for detecting generated amplification products, the opto-fluidic instrument may cause the sample to undergo successive rounds of detectably labeled probe hybridization (e.g., using two or more sets of fluorescent probes, where each set of fluorescent probes is excited by a different color channel) and be imaged to detect target molecules in the probed sample. In such cases, the output may include optical signatures (e.g., a codeword) specific to each gene, which allow the identification of the target RNAs.VI. Terminology

[0261] 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.

[0262] 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.

[0263] 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.90MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0264] 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.

[0265] 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.

[0266] 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."

[0267] 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 specifically 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.

[0268] 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 used91MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 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.EXAMPLES

[0269] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.Example 1: Synchronized rolling circle amplification (RCA) performed in the presence of a crowding agent improves signal localization

[0270] This example demonstrates the unexpected benefits of performing RCA in the presence of a crowding agent with a synchronized start to the amplification reaction (e.g., using Ca2+ synchronization).

[0271] Tissue sections of a human FFPE pancreas tissue sample were deparaffinized using xylene and a series of ethanol washes. The samples were then decrosslinked and prepared for probe hybridization. A plurality of circularizable probes (e.g., padlock probes) corresponding to a panel of 377 genes (targeted with 1365 probes) including GPX2 and TFF2 were contacted with the tissue sections. The circularizable probes were ligated using a ligation mixture comprising a ligase to generate a plurality of circular nucleic acid templates for RCA. In addition, the circularizable probes each comprise a hybridization region comprising sequences complementary to a plurality of primers for extension in the RCA reaction. The primers were added to the tissue samples simultaneously with the ligation mixture for circularization of the probes.

[0272] In the non- synchronized conditions ("non-sync, 5% PEG20K during amplification" and "non-sync, 10% PEG20K during amplification"), after ligation of the circularizable probes, the tissue samples were contacted with a reaction mixture comprising polymerase, 0.8mM dNTPs, 30 mM MgCh, and either 5% or 10% PEG (mass by volume percentage (w / v)) in a buffer and incubated at 30°C for 2 hours.92MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940

[0273] For the synchronized RCA conditions ("sync, 5% PEG20K during amplification" and "sync, 10% PEG20K during amplification"), after ligation of the circularizable probes, a first reaction mixture comprising 30mM CaCh, 2.7pM phi29 polymerase, 5% PEG20K, and 0.8mM dNTPs in a buffer was added to the tissue samples. In this first reaction mixture, phi29 polymerase was provided with dNTPs and Ca2+, a dication that is not a catalytic cofactor of the polymerase, thus halting the polymerase. The tissue samples were incubated with this first reaction mixture at 22°C (on thermocycler) for 2 hours. The mixture was removed from the tissue samples and a wash mixture comprising 5% PEG 20K and 0.8mM dNTPs in a buffer was added and incubated with the tissue samples for 1 minute. After the incubation, the wash mixture was removed from the tissue sample and a second reaction mixture comprising either 5% PEG20K or 10% PEG20K with 30mM MgCh and 0.8mM dNTPs in a buffer was added to the tissue sample. This second reaction mixture provided dNTPs and Mg2+, a dication that is a catalytic cofactor of the polymerase. This second reaction mixture did not provide additional polymerase. The tissue samples were incubated with the second reaction mixture at 30°C for 2 hours for RCA to proceed.

[0274] The generated RCA products associated with the panel of target RNAs were then detected in the tissue sample by performing sequential cycles of hybridization of detectably labeled probes and imaging the tissue sample.

[0275] Images of detected RCPs associated with two target RNAs (GPX2 and TFF2) are shown in FIGS. 2A-2D and FIGS. 3A-3D. In the non-synchronized conditions (FIGS. 2A and 2B, FIGS. 3A and 3B), adding 10% PEG reduced the number of transcripts from all genes on the gene panel that decoded with high quality (Phred quality score >= 20) ("Q20 count"). In general, Q20 count is higher in the synchronized conditions (FIGS. 2C and 2D, FIGS. 3C and 3D), as sensitivity was increased by incubating with the first reaction mixture which allows the reagents to diffuse through the tissue sample to various circular templates for RCA. Notably, in the synchronized conditions, performing RCA in 10% PEG20K in the second reaction mixture resulted in the benefit of reducing signal mislocalization compared to RCA performed in the presence of 5% PEG20K. For example, there are more diffuse signals detected in the 5% PEG20K condition than in the presence of 10% PEG20K, indicating mislocalization of signals from the cells (e.g., signals not localized to cells). Synchronization of RCA and using a second reaction mixture comprising 10% PEG showed reduced signal mislocalization (e.g., drifting of 93MF-363763353PCT / US25 / 53325 30 October 2025 (30.10.2025)202412023940 the RCA products away from the original location of the transcript). In a separate experiment with RCA synchronization performed on a human FFPE pancreas sample essentially as described above, performing RCA in 15% PEG20K in the second reaction mixture resulted in greater reduced signal mislocalization compared to performing RCA in 10% PEG20K (signals associated with two genes glucagon (GCG) and somatostatin (SST) are shown in FIG. 4). Similar benefits in reduction of signal mislocalization were observed with detected signals associated with generated RCPs of various target RNAs in human FFPE kidney, lymph node, colon, and pancreas tissue samples, and using 15mM or 30mM of SrCh instead ofCaChin the first reaction mixture. FIG. 5 shows a calculated normalized median distance to the nearest neighbor metric used for quantifying signal mislocalization (based on genes with a median distance of less than 2pm to the nearest RCPs of the same gene in at least one condition per tissue). Across various tissue types, it was observed that performing synchronized RCA in 10% PEG20K or 15% PEG20K in the second reaction mixture resulted in reduced signal mislocalization compared to non-synchronized RCA performed in the presence of 5% PEG20K.

[0276] This workflow allows for the polymerase to efficiently diffuse to its templates in the first reaction mixture and the amount of crowding agent provided in the second reaction mixture allows the added benefit during amplification of maintaining spatiality of the generated RCPs. Thus, synchronizing the RCA reaction allows the concentration of the crowding agent to be increased during RCA, resulting in reduced signal mislocalization without reducing sensitivity in analyte detection.

[0277] 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 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.94MF-363763353

Claims

202412023940CLAIMS1. A method, comprising:(a) adding a first reaction mixture comprising a polymerase to a cell or tissue sample comprising a primer and a circular nucleic acid, wherein the primer is exogenous to the cell or tissue sample, and wherein the circular nucleic acid comprises a hybridization region complementary to the primer;(b) after addition of the first reaction mixture, incubating the cell or tissue sample for at least 30 minutes, wherein the circular nucleic acid is not amplified by the polymerase in the cell or tissue sample during or prior to the incubation;(c) after the incubation, contacting the cell or tissue sample with a second reaction mixture comprising at least 10% of a crowding agent; and(d) performing rolling circle amplification (RCA) in the cell or tissue sample contacted with the second reaction mixture, wherein the polymerase extends the primer using the circular nucleic acid as a template to generate an RCA product.

2. The method of claim 1, wherein the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the second reaction mixture comprises a catalytic cofactor of the polymerase.

3. The method of claim 1, wherein: the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of at least one of four canonical bases: adenine, thymine, guanine, and cytosine; and the second reaction mixture comprises a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine, and cytosine.

4. The method of any one of claims 1 to 3, wherein the incubation in (b) is performed at a temperature that impedes polymerase activity.

5. The method of claim 4, wherein the temperature that impedes polymerase activity is at or below 4°C.

6. The method of any one of claims 1 to 3, wherein the incubation in (b) is performed at about 20°C to 25°C.95MF-3637633532024120239407. The method of claim 6, wherein the incubation in (b) is performed at 22°C.

8. The method of any one of claims 1 to 7, wherein the RCA performed in (d) is performed at a temperature greater than 25°C.

9. The method of any one of claims 1 to 8, wherein the RCA performed in (d) is performed at a temperature of about 30°C.

10. The method of any one of claims 1 to 9, wherein the incubation in (b) is performed for at least 60 minutes.

11. The method of any one of claims 1 to 10, wherein the incubation in (b) is performed for at least 90 minutes.

12. The method of any one of claims 1 to 11, wherein the incubation in (b) is performed for at least 120 minutes.

13. The method of any one of claims 1 to 12, further comprising detecting the RCA product generated in (d) at a location in the cell or tissue sample.

14. A method, comprising:(a) contacting a cell or tissue sample with a first reaction mixture, wherein: the cell or tissue sample comprises a circular nucleic acid and a primer, wherein the primer comprises a sequence complementary to a hybridization region in the circular nucleic acid, and the first reaction mixture comprises a polymerase and a non-catalytic cofactor of the polymerase;(b) contacting the cell or tissue sample with a second reaction mixture to allow the polymerase to extend the primer using the circular nucleic acid as a template, thereby generating a rolling circle amplification (RCA) product in the cell or tissue sample, wherein the second reaction mixture comprises a catalytic cofactor of the polymerase and at least 10% of a crowding agent; and(c) detecting the RCA product at a location in the cell or tissue sample.

15. The method of any one of claims 1 to 14, comprising prior to (a): contacting the cell or tissue sample with a circularizable probe and ligating the circularizable probe to form the circular nucleic acid.96MF-36376335320241202394016. The method of any one of claims 1 to 15, wherein the primer is hybridized to the circular nucleic acid prior to (a).

17. The method of claim 15, wherein the primer hybridizes to the circularizable probe prior to (a).

18. The method of any one of claims 1 to 17, wherein the first reaction mixture is free of or comprises less than 12% of a crowding agent.

19. The method of any one of claims 1 to 18, wherein the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%.

20. The method of claim 19, wherein the crowding agent in the first reaction mixture is the same type of crowding agent as the crowding agent in the second reaction mixture.

21. The method of claim 19, wherein the crowding agent in the first reaction mixture is a different type of crowding agent from the crowding agent in the second reaction mixture.

22. The method of any one of claim 1 to 21, wherein the second reaction mixture comprises the crowding agent at a concentration of between 10% and 20%.

23. The method of any one of claim 1 to 22, wherein the crowding agent is selected from the group consisting of polyethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate, optionally wherein the crowding agent is polyethylene glycol) (PEG).

24. The method of claim 23, wherein the crowding agent is poly(ethylene glycol) (PEG).

25. The method of claim 24, wherein the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG 12000, PEG20000, and PEG35000.

26. The method of claim 25, wherein the crowding agent is PEG20000.

27. The method of claim 24, wherein the second reaction mixture comprises between about 10% and about 25% PEG, between about 10% and about 20% PEG, between about 10% and about 15% PEG, or between about 10% and about 12.5% PEG.

28. The method of claim 24, wherein the second reaction mixture comprises about 10% PEG.97MF-36376335320241202394029. The method of any one of claims 1 to 28, wherein the first reaction mixture is substantially free of deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs).

30. The method of any one of claims 1 to 29, wherein the second reaction mixture comprises a plurality of deoxynucleoside triphosphates (dNTPs).

31. The method of any one of claims 1 to 30, wherein the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the second reaction mixture is substantially free of the non-catalytic cofactor of the polymerase.

32. The method of claim 31, wherein the non-catalytic cofactor is a di-cation cofactor.

33. The method of claim 32, wherein the di-cation is Ca2+or Sr2+.

34. The method of claim 31, wherein the non-catalytic cofactor stabilizes the polymerase, thereby inhibiting the polymerase activity and / or an exonuclease activity of the polymerase.

35. The method of any one of claims 1 to 34, wherein the first reaction mixture is substantially free of a catalytic cofactor of the polymerase and the second reaction mixture comprises the catalytic cofactor of the polymerase.

36. The method of claim 35, wherein the catalytic cofactor is Mg2+, Co2+, and / or Mn2+.

37. The method of any one of claims 1 to 36, wherein the first reaction mixture comprises a chelating agent.

38. The method of claim 37, wherein the chelating agent comprises EDTA, EGTA, BAPTA, DTPA, or a combination thereof.

39. The method of any one of claims 1 to 38, wherein a 3'^-5' exonuclease activity of the polymerase is inhibited in the first reaction mixture.

40. The method of any one of claims 1 to 39, wherein the primer comprises a 3' protective group.

41. The method of claim 40, wherein the primer is 3' thiophosphate-protected, thereby protecting the primer from 3'^-5' exonuclease degradation by the polymerase while allowing priming by the polymerase.98MF-36376335320241202394042. The method of any one of claims 14 to 41, wherein the cell or tissue sample is incubated with the first reaction mixture for at least 30 minutes.

43. The method of any one of claims 14 to 42, wherein the cell or tissue sample is incubated with the first reaction mixture for at least 60 minutes.

44. The method of any one of claims 14 to 43, wherein the cell or tissue sample is incubated with the first reaction mixture at about 30°C.

45. The method of any one of claims 1 to 44, wherein the RCA is performed for no more than 60 minutes, 90 minutes, or 120 minutes.

46. The method of any one of claims 1 to 45, wherein the hybridization region in the circular nucleic acid is a primer hybridization region that hybridizes to the primer, and the circular nucleic acid further comprises a target hybridization region that hybridizes to a target nucleic acid.

47. The method of claim 46, wherein the target nucleic acid is an endogenous DNA or an endogenous RNA molecule in the cell or tissue sample.

48. The method of claim 47, wherein the target nucleic acid is a product, optionally an amplification product of the endogenous DNA or the endogenous RNA molecule.

49. The method of claim 46, wherein the target nucleic acid is a probe that directly or indirectly binds to the endogenous DNA or the endogenous RNA molecule, or a product of the probe.

50. The method of claim 46, wherein the target nucleic acid comprises a genomic DNA sequence, a mtDNA sequence, an RNA sequence, and / or a cDNA sequence.

51. The method of any one of claims 1 to 50, further comprising removing one or more molecules of the polymerase that are not bound to the circular nucleic acid from the cell or tissue sample prior to contacting the cell or tissue sample with the second reaction mixture.

52. The method of any one of claims 14 to 51, further comprising performing one or more stringency washes prior to contacting the cell or tissue sample with the second reaction mixture.99MF-36376335320241202394053. The method of claim 52, wherein the cell or tissue sample is contacted with a wash solution after incubation with the first reaction mixture and prior to contacting the cell or tissue sample with the second reaction mixture.

54. The method of claim 53, wherein the wash solution comprises the crowding agent.

55. The method of claim 54, wherein the wash solution comprises the crowding agent at a concentration of between 0.1% and 5%.

56. The method of any one of claims 53 to 55, wherein the cell or tissue sample is incubated with the wash solution for at least 30 minutes.

57. The method of any one of claims 53 to 56, wherein the wash solution comprises dNTPs.

58. The method of claim 57, wherein the concentration of dNTPs in the wash solution is lower than the concentration of dNTPs in the second reaction mixture.

59. The method of any one of claims 1 to 58, wherein the polymerase and / or the primer is not attached to a surface of a solid support or to a nanopore, a nanopore membrane, or an insulating support thereof.

60. The method of any one of claims 1 to 59, wherein the polymerase or a preformed complex comprising the polymerase and the primer is diffusible in the first reaction mixture and / or when contacting with the cell or tissue sample.

61. The method of any one of claims 1 to 60, wherein the polymerase is selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRDl polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and a variant or derivative thereof.

62. The method of any one of claims 1 to 61, wherein the polymerase is a Phi29 DNA polymerase.100MF-36376335320241202394063. The method of claim 62, wherein the primer is prebound to a single-stranded DNA binding domain of the Phi29 DNA polymerase in the first reaction mixture.

64. The method of claim 63, wherein the primer bound to the Phi29 DNA polymerase is hybridized to the hybridization region and the Phi29 DNA polymerase is prevented from extending the primer in the first reaction mixture.

65. The method of any one of claims 1 to 64, wherein the second reaction mixture comprises a deoxynucleoside triphosphate (dNTP) and / or a nucleoside triphosphate (NTP).

66. The method of claim 2 or any one of claims 4 to 65, wherein the catalytic cofactor is a di-cation.

67. The method of claim 66, wherein the di-cation is Ca2+.

68. The method of claim 66, wherein the di-cation is Sr2+.

69. The method of any one of claims 1 to 68, wherein the second reaction mixture is substantially free of the polymerase and / or other polymerases.

70. The method of any one of claims 1 to 69, wherein the pH of the first and second reaction mixtures is substantially the same.

71. The method of any one of claims 1 to 70, wherein the pH of the first and second reaction mixture is between pH 7.5 to pH 8.5, optionally wherein the pH is 8.

72. The method of any one of claims 1 to 71, wherein the rolling circle amplification product is generated using a linear rolling circle amplification (RCA), a branched RCA, a dendritic RCA, or any combination thereof.

73. The method of any one of claims 1 to 72, wherein the RCA product is immobilized in the cell or tissue sample.

74. The method of any one of claims 1 to 73, wherein the RCA product is crosslinked to one or more molecules other than the RCA product in the cell or tissue sample.

75. The method of any one of claims 1 to 73, wherein the RCA product is not crosslinked to one or more other molecules in the cell or tissue sample.101MF-36376335320241202394076. The method of any one of claims 1 to 75, wherein the method comprises imaging the cell or tissue sample to detect the RCA product at the location in the cell or tissue sample.

77. The method of claim 76, wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCA product.

78. The method of any one of claims 1 to 77, wherein a sequence of the RCA product is detected in situ in the cell or tissue sample.

79. The method of claim 78, wherein the sequence of the RCA product is detected by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

80. The method of claim 78 or claim 79, wherein the sequence of the RCA product comprises a barcode sequence or complement thereof.

81. The method of any one of claims 15 to 80, wherein the circularizable probe is a padlock probe.

82. The method of any one of claims 15 to 80, wherein the circularizable probe is provided in two or more parts.

83. The method of any one of claims 15 to 82, wherein the circularizable probe comprises a barcode sequence.

84. The method of any one of claims 46 to 83, wherein the target nucleic acid is a target messenger RNA (mRNA).

85. The method of any one of claims 46 to 83, wherein the target nucleic acid is a viral DNA or a bacterial DNA.

86. The method of any one of claims 15 to 80, wherein the ligating of the circularizable probe to form the circular nucleic acid comprises performing an enzymatic ligation.

87. The method of any one of claims 15 to 80, wherein the ligating of the circularizable probe to form the circular nucleic acid comprises ligating a 5’ end to a 3’ end of the circularizable probe.102MF-36376335320241202394088. The method of any one of claims 46 to 83, wherein the target nucleic acid is a reporter oligonucleotide of a labeling agent, wherein a sequence of the reporter oligonucleotide corresponds to a binding moiety and / or a non-nucleic acid target molecule bound by the labeling agent.

89. The method of claim 88, wherein the non-nucleic acid target molecule bound by the labeling agent is a protein.

90. The method of claim 89, wherein the circularizable probe comprises a padlock probe that hybridizes to the reporter oligonucleotide.

91. The method of claim 90, further comprising ligating the padlock probe hybridized to the reporter oligonucleotide to form the circular nucleic acid.

92. The method of any one of claims 1 to 91, wherein the cell or tissue sample comprises cells deposited on a surface.

93. The method of any one of claims 1 to 92, wherein the cell or tissue sample is a tissue sample.

94. The method of any one of claims 1 to 93, wherein the cell or tissue sample is fixed.

95. The method of any one of claims 1 to 93, wherein the cell or tissue sample is not fixed.

96. The method of any one of claims 1 to 94, wherein the cell or tissue sample is a formalin- fixed, paraffin-embedded (FFPE) sample, a frozen tissue sample, or a fresh tissue sample.

97. The method of any one of claims 1 to 96, wherein the cell or tissue sample is permeabilized.

98. The method of any one of claims 1 to 97, wherein the cell or tissue sample is processed or cleared.

99. The method of any one of claims 1 to 98, wherein the cell or tissue sample is embedded in a matrix.

100. The method of claim 99, wherein the matrix is a hydrogel.

101. The method of claim 99 or claim 100, wherein the cell or tissue sample and / or the matrix is crosslinked.103MF-363763353202412023940102. The method of any one of claims 1 to 101, wherein the first reaction mixture and the second reaction mixture comprise dNTPs.

103. The method of claim 102, wherein the concentration of dNTPs in the first reaction mixture is lower than the concentration of dNTPs in the second reaction mixture.

104. A method, comprising:(a) contacting a cell or tissue sample with a first reaction mixture, wherein: the cell or tissue sample comprises a plurality of primers and a plurality of circular nucleic acids at a plurality of locations in the cell or tissue sample, wherein the plurality of primers are configured to hybridize to a plurality of hybridization regions in the plurality of circular nucleic acids, wherein the first reaction mixture comprises a polymerase, wherein the polymerase activity of the polymerase is inhibited; and wherein the polymerase and / or the primer binds to the plurality of circular nucleic acids; and(b) contacting the cell or tissue sample with a second reaction mixture, allowing the polymerase to extend the plurality of primers using the plurality of circular nucleic acids as a template, thereby generating a plurality of rolling circle amplification (RCA) products in the cell or tissue sample, wherein the second reaction mixture comprises at least 10% of a crowding agent, thereby synchronizing RCA of the plurality of circular nucleic acids in the cell or tissue sample.

105. The method of claim 104, further comprising,(c) detecting the plurality of RCA products at the plurality of locations in the cell or tissue sample.

106. The method of claim 104 or claim 105, wherein the first reaction mixture comprises a chelating agent.

107. The method of any one of claims 104 to 106, wherein the first reaction mixture comprises one or more deoxynucleoside triphosphates (dNTPs).

108. The method of any one of claims 104 to 106, wherein the first reaction mixture is substantially free of dNTPs.

109. The method of any one of claims 104 to 108, wherein the primer hybridization regions in two or more of the plurality of circular nucleic acids are the same in sequence.104MF-363763353202412023940110. The method of any one of claims 104 to 108, wherein the primer hybridization regions in two or more of the plurality of circular nucleic acids are different in sequence.

111. The method of any one of claims 104 to 110, wherein two or more primers of the plurality of primers are the same in sequence.

112. The method of any one of claims 104 to 110, wherein two or more primers of the plurality of primers are different in sequence.

113. The method of any one of claims 104 to 112, wherein the polymerase is a Phi29 DNA polymerase, a Bst polymerase, a T7 RNA polymerase, or a Klenow fragment.

114. The method of any one of claims 104 to 113, further comprising, performing one or more stringency washes between (a) and (b).

115. The method of any one of claims 104 to 114, wherein the second reaction mixture comprises deoxynucleoside triphosphates (dNTPs) and one or more catalytic cofactors of the polymerase.

116. The method of claim 115, wherein the one or more catalytic cofactors comprise Mg2+, Co2+, and / or Mn2+.

117. The method of any one of claims 104 to 116, wherein the second reaction mixture does not comprise the polymerase.

118. The method of any one of claims 1 to 117, wherein the polymerase extension or RCA is performed for no more than 3 hours, no more than 2 hours, no more than 1 hour, or no more than 30 minutes.

119. The method of claim 118, wherein the polymerase extension or RCA is performed for no more than 120 minutes.

120. The method of any one of claims 1 to 119, further comprising terminating RCA of the circular nucleic acids to provide a plurality of RCA products.

121. The method of any one of claims 104 to 120, wherein the plurality of circular nucleic acids in the cell or tissue sample comprise at least at least 1,000, at least 3,000, at least 5,000, or at least 10,000 distinguishable circular nucleic acids.105MF-363763353202412023940122. The method of any one of claims 104 to 121, wherein the first reaction mixture is free of a crowding agent.

123. The method of any one of claims 104 to 121, wherein the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%.

124. The method of any one of claims 104 to 121 or claim 123, wherein the crowding agent in the first reaction mixture is the same as the crowding agent in the second reaction mixture.

125. The method of any one of claims 104 to 121 or claim 123, wherein the crowding agent in the first reaction mixture is different from the crowding agent in the second reaction mixture.

126. The method of any one of claims 104 to 125, wherein the second reaction mixture comprises the crowding agent at a concentration of between 10% and 20%.

127. The method of any one of claims 104 to 126, wherein the crowding agent is selected from the group consisting of polyethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate, optionally wherein the crowding agent is poly (ethylene glycol) (PEG).

128. The method of claim 127, wherein the crowding agent is polyethylene glycol).

129. The method of claim 127 or claim 128, wherein the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG12000, PEG20000, and PEG35000.

130. The method of any one of claims 127 to 129, wherein the second reaction mixture comprises between about 10% and about 25% PEG, between about 10% and about 20% PEG, between about 10% and about 15% PEG, or between about 10% and about 12.5% PEG.

131. The method of any one of claims 127 to 130, wherein the second reaction mixture comprises about 10% PEG.

132. A system, comprising:(a) a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer;106MF-363763353202412023940(b) a first reaction mixture comprising a polymerase and optionally no more than 5% crowding agent; and(c) a second reaction mixture comprising at least 10% of a crowding agent.

133. A system, comprising:(a) a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer;(b) a first reaction mixture comprising a polymerase and a mixture of free nucleotides lacking nucleotides of at least one of four canonical bases: adenine, thymine, guanine and cytosine; and(c) a second reaction mixture comprising at least 10% of a crowding agent and a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine and cytosine.

134. A system, comprising:(a) a cell or tissue sample comprising a circular nucleic acid, wherein the circular nucleic acid comprises a hybridization region complementary to a primer;(b) a first reaction mixture comprising a polymerase and a non-catalytic cofactor of the polymerase; and(c) a second reaction mixture comprising at least 10% of a crowding agent and a catalytic cofactor of the polymerase.

135. The system of claim 132 or claim 134, wherein the second reaction mixture comprises a plurality of deoxynucleoside triphosphates (dNTPs).

136. The system of claim 132 or claim 133, wherein the second reaction mixture comprises a catalytic cofactor of the polymerase.

137. The system of any one of claims 132 to 136, wherein the polymerase is Phi29.

138. The system of any one of claims 132 to 137, further comprising a plurality of detectably labeled probes for binding to a sequence of an amplification product generated using the circular nucleic acid as a template.107MF-363763353202412023940139. The system of any one of claims 132 to 137, further comprising reagents for sequencing.

140. The system of any one of claims 132 to 139, further comprising a ligase for forming the circular nucleic acid.

141. The system of any one of claims 132 to 140, further comprising the primer.

142. The system of claim 141, wherein the cell or tissue sample comprises the primer.

143. The system of claim 141, wherein the first reaction mixture comprises the primer.

144. A method, comprising: performing rolling circle amplification (RCA) of a circular nucleic acid in a cell or tissue sample using a reaction mixture comprising 10% to 15% of a crowding agent.

145. The method of claim 144, wherein the cell or tissue sample comprises a polymerase and wherein the circular nucleic acid comprises a hybridization region complementary to a primer that is exogenous to the cell or tissue sample.

146. The method of claim 144 or claim 145, prior to performing RCA, contacting the cell or tissue sample with a first reaction mixture comprising the polymerase and incubating the cell or tissue sample for at least 30 minutes, wherein the circular nucleic acid is not amplified by the polymerase in the cell or tissue sample during or prior to the incubation.

147. The method of any one of claims 144 to 146, prior to performing RCA, contacting the cell or tissue sample with a circularizable probe and ligating the circularizable probe to form the circular nucleic acid.

148. The method of any one of claims 146 to 147, wherein the first reaction mixture comprises a non-catalytic cofactor of the polymerase and the reaction mixture for performing RCA is a second reaction mixture comprising a catalytic cofactor of the polymerase.

149. The method of any one of claims 146 to 147, wherein the first reaction mixture comprises a mixture of free nucleotides lacking nucleotides of at least one of four canonical bases: adenine, thymine, guanine, and cytosine; and108MF-363763353202412023940 the second reaction mixture comprises a mixture of free nucleotides comprising nucleotides of all four canonical bases: adenine, thymine, guanine, and cytosine.

150. The method of any one of claims 146 to 149, prior to performing RCA, the cell or tissue sample is incubated at a temperature that impedes polymerase activity.

151. The method of claim 150, wherein the temperature that impedes polymerase activity is at or below 4°C.

152. The method of claim 150 or claim 151, wherein the incubation is performed at about 20°C to 25°C.

153. The method of any one of claims 144 to 152, wherein RCA is performed at a temperature greater than 25°C, optionally at about 30°C.

154. The method of any one of claims 144 to 153, wherein the polymerase is a Phi29 DNA polymerase.

155. The method of any one of claims 146 to 154, wherein the incubation is performed for at least 60 minutes, at least 90 minutes, or at least 120 minutes.

156. The method of any one of claims 144 to 155, wherein the circular nucleic acid is a ligated circularizable probe, optionally, wherein the circularizable probe is a padlock probe.

157. The method of claim 156, wherein the circularizable probe comprises a barcode sequence.

158. The method of any one of claims 144 to 157, further comprising detecting a generated RCA product of the circular nucleic acid at a location in the cell or tissue sample.

159. The method of claim 158, wherein a sequence of the generated RCA product is detected in situ in the cell or tissue sample.

160. The method of claim 159, wherein the sequence of the RCA product is detected by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

161. The method of claim 159 or claim 160, wherein the sequence of the RCA product comprises a barcode sequence or complement thereof.109MF-363763353202412023940162. The method of any one of claims 144 to 161, wherein the first reaction mixture is free of or comprises less than 10% of the crowding agent.

163. The method of any one of claims 144 to 161, wherein the first reaction mixture comprises a crowding agent at a concentration of between 0.1% and 5%.

164. The method of any one of claims 144 to 163, wherein the crowding agent is selected from the group consisting of polyethylene glycol) (PEG), polyvinylpyrrolidone (PVP), glycerol, Ficoll, and dextran sulfate, optionally wherein the crowding agent is poly (ethylene glycol) (PEG).

165. The method of claim 164, wherein the crowding agent is polyethylene glycol) (PEG).

166. The method of claim 165, wherein the PEG is selected from the group consisting of PEG200, PEG6000, PEG8000, PEG 12000, PEG20000, and PEG35000.

167. The method of any one of claims 148-166, wherein the non-catalytic cofactor is a dication cofactor.

168. The method of claim 167, wherein the di-cation is Ca2+or Sr2+.

169. The method of any one of claims 148 to 168, wherein the catalytic cofactor is Mg2+, Co2+, and / or Mn2+.

170. The method of any one of claims 144 to 169, wherein the circular nucleic acid is bound to an endogenous DNA or an endogenous RNA molecule in the cell or tissue sample.

171. The method of any one of claims 144 to 169, wherein the circular nucleic acid is bound to an endogenous messenger RNA (mRNA).110MF-363763353

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