Methods and compositions for in-situ simultaneous reactions

Abstract

The present disclosure relates in some aspects to methods and compositions for accurately detecting and quantitatively being present in multiple analytes in biological samples. In some respects, provided herein are methods and compositions that solve the problem associated with the heterogeneity of analyte abundance (e.g., gene expression level) and the difference between the reactions at different positions of the sample (e.g., an amplification reaction starts earlier at one position than at another position). In some respects, compared with the method causing signal spots to be wide and uneven in size and intensity distribution, methods disclosed herein provide a tighter distribution of signal spot size and intensity in the sample.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 227,830, filed July 30, 2021, entitled “METHOD AND COMPOSITIONS FORSYNCHRONIZING REACTIONS IN SITU,” which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This disclosure generally relates to methods and compositions for in-situ detection of multiple molecules of one or more analytes in a sample. Background Technology

[0004] Genomic, transcriptomic, and proteomic analyses of cell and tissue samples using microscopic imaging can simultaneously resolve multiple analytes of interest, providing valuable in-situ information about analyte abundance and localization. Therefore, these in-situ assays are important tools, for example, for understanding the molecular basis of cell identity and developing treatments for diseases. In multiplex assays that simultaneously detect multiple signals, it is important to collect as much information as possible. However, due to the heterogeneity of analyte abundance (e.g., gene expression levels) and the differences in responses at different locations in the sample, signal “spots” in the sample can exhibit a wide and non-uniform distribution in size and intensity. Large signal spots may overlap and / or mask adjacent smaller signal spots, making the smaller spots unresolved. Additionally, some analytes may be accompanied by bright signal spots (e.g., due to high analyte abundance and / or preferential signal amplification), while others may be accompanied by signal spots that are too dark to be detected simultaneously with bright spots (e.g., within the same field of view (FOV) during microscopic observation). New and improved in-situ assay methods are needed. This disclosure addresses these and other needs. Summary of the Invention

[0005] In some embodiments, this document provides a method for analyzing a biological sample, comprising contacting the biological sample with a first reaction mixture, wherein the biological sample contains a circular nucleic acid containing a hybridization region, the first reaction mixture contains a polymerase, the circular nucleic acid or the polymerase is pre-bound to a polynucleotide containing a sequence complementary to the hybridization region, and the polymerase activity is inhibited. In some embodiments, the method further comprises contacting the biological sample with a second reaction mixture to allow the polymerase to use the circular nucleic acid as a template to extend the polynucleotide hybridizing with the hybridization region. In any of the foregoing embodiments, rolling circle amplification products of the circular nucleic acid can be generated in the biological sample, for example for in-situ analysis of the circular nucleic acid and / or one or more analytes of interest associated therewith.

[0006] In any of the foregoing embodiments, the first reaction mixture may stabilize and / or inhibit polymerase activity, such as polymerase activity and / or nuclease activity. In any of the foregoing embodiments, the first reaction mixture may contain one or more deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs). In any of the foregoing embodiments, the first reaction mixture may contain dATP, dTTP, dCTP, and / or dGTP. Alternatively, in any of the foregoing embodiments, the first reaction mixture may be substantially free of dNTPs and / or NTPs. In any of the foregoing embodiments, the first reaction mixture may contain a divalent cation that is not a cofactor of the polymerase. In some embodiments, the divalent cation is Ca2+. 2+ In any of the foregoing embodiments, the divalent cation can stabilize the polymerase. In any of the foregoing embodiments, the divalent cation can stabilize the preformed complex comprising the polymerase and the polynucleotide. In any of the foregoing embodiments, the first reaction mixture may be substantially free of polymerase cofactors. In any of the foregoing embodiments, the first reaction mixture may be substantially free of Mg. 2+ Co 2+ and / or Mn 2+ In any of the foregoing embodiments, the first reaction mixture may contain a chelating agent. For example, the chelating agent may chelate divalent cations such as Mg from one or more previously reacted reactions. 2+Therefore, chelating agents can chelate residual divalent cations in biological samples, such as tissue sections that have already been contacted with a reaction mixture containing divalent cations (e.g., a linking reaction mixture for cyclizing padlock probes to form circular nucleic acids). In any of the foregoing embodiments, the first reaction mixture may contain EDTA, EGTA, BAPTA, DTPA, or combinations thereof. In any of the foregoing embodiments, the first reaction mixture may inhibit polymerase activity and / or the exonuclease activity of the polymerase. In any of the foregoing embodiments, the 3′→5′ exonuclease activity and / or the 5′→3′ exonuclease activity of the polymerase may be inhibited in the first reaction mixture.

[0007] In any of the foregoing embodiments, the polynucleotide may contain a 3′ protecting group and / or a 5′ protecting group. In any of the foregoing embodiments, the polynucleotide may contain a free 3′ hydroxyl group that can be extended under polymerase action. In any of the foregoing embodiments, the polynucleotide may be 3′ phosphate thioester protected, thereby protecting the polynucleotide from 3′→5′ exonuclease degradation under polymerase action while allowing polymerase initiation.

[0008] In any of the foregoing embodiments, the polynucleotide may be a primer, and the primer is pre-bound to a polymerase in a first reaction mixture prior to contacting the biological sample. In any of the foregoing embodiments, the method may include removing one or more complexes comprising the polymerase and primers from the biological sample that are not bound to the circular nucleic acid before contacting the biological sample with a second reaction mixture. Accordingly, in some embodiments, this document provides 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 containing a primer hybridization region, the first reaction mixture comprising a polymerase, the circular nucleic acid or the polymerase being pre-bound to a primer containing a sequence complementary to the primer hybridization region, and the polymerase activity being inhibited; and contacting the biological sample with a second reaction mixture to allow the polymerase to use the circular nucleic acid as a template to extend primers hybridizing with the primer hybridization region, wherein rolling circle amplification products of the circular nucleic acid are generated in the biological sample.

[0009] In any of the foregoing embodiments, the polynucleotide may be pre-bound to a circular nucleic acid in the biological sample prior to contact with the first reaction mixture. In any of the foregoing embodiments, the polynucleotide may be a primer, and the primer is pre-bound to a circular nucleic acid in the biological sample prior to contact with the first reaction mixture. In any of the foregoing embodiments, the hybridization region in the circular nucleic acid may be a primer hybridization region that hybridizes with the primer, and the circular nucleic acid may also include a target hybridization region that hybridizes with the target nucleic acid. In any of the foregoing embodiments, the target nucleic acid may be a DNA or RNA molecule in the biological sample, a product of a DNA or RNA molecule, a probe directly or indirectly bound to a DNA or RNA molecule, or a probe product. In any of the foregoing embodiments, the target nucleic acid may comprise a genomic DNA sequence, an RNA sequence, and / or a cDNA sequence.

[0010] In any of the foregoing embodiments, the polynucleotide may be the target nucleic acid. In any of the foregoing embodiments, the target nucleic acid may be pre-bound to a circular nucleic acid in the biological sample prior to contact with the first reaction mixture. In any of the foregoing embodiments, the target nucleic acid may comprise a genomic DNA sequence, an RNA sequence, and / or a cDNA sequence. In any of the foregoing embodiments, the target nucleic acid may comprise a free 3' end for initiating rolling circle amplification. In any of the foregoing embodiments, the target nucleic acid may be processed (e.g., by an enzyme having 3′→5′ exonuclease activity such as Phi29) to provide a free 3′ end for initiating rolling circle amplification. In any of the foregoing embodiments, the method may further include removing one or more molecules of polymerase that are not bound to the circular nucleic acid from the biological sample prior to contacting the biological sample with the second reaction mixture.

[0011] In any of the foregoing embodiments, the polymerase may not be attached to the nanopore, nanopore membrane, or its insulating support. In any of the foregoing embodiments, the polymerase may be diffuseable in the first reaction mixture and / or in the biological sample. In any of the foregoing embodiments, the preformed complex comprising the polymerase and the polynucleotide may be diffuseable in the first reaction mixture and / or in the biological sample.

[0012] In any of the foregoing embodiments, the polymerase may be selected from 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 (exonuclease-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, the 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 variants or derivatives thereof.

[0013] In any of the foregoing embodiments, the polymerase may be Phi29 DNA polymerase or a variant or derivative thereof.

[0014] In any of the foregoing embodiments, the polynucleotide may be pre-bound to the single-stranded DNA-binding domain of the Phi29 DNA polymerase. In some embodiments, the polynucleotide is a primer pre-bound to the single-stranded DNA-binding domain of the Phi29 DNA polymerase in the first reaction mixture. In any of the foregoing embodiments, the polynucleotide bound to the Phi29 DNA polymerase may hybridize with the hybridization region and may prevent the Phi29 DNA polymerase from extending the polynucleotide until the biological sample comes into contact with the second reaction mixture.

[0015] In any of the foregoing embodiments, the method may further include a step of removing molecules of polymerase and / or polynucleotides that are not bound to the circular nucleic acid from the biological sample between contact with the first reaction mixture and contact with the second reaction mixture. In any of the foregoing embodiments, the method may further include one or more rigorous washes between the contact steps.

[0016] In any of the foregoing embodiments, the second reaction mixture may contain deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs). In any of the foregoing embodiments, the second reaction mixture may contain a cofactor for the polymerase. In any of the foregoing embodiments, the second reaction mixture may contain a divalent cation, such as Mg²⁺. 2+ Co 2+ and / or Mn 2+In any of the foregoing embodiments, the second reaction mixture may be substantially unconjugated to the polymerase and / or other polymerases. In any of the foregoing embodiments, the pH of the first reaction mixture and the second reaction mixture may be substantially the same, for example, about pH 8.5. In any of the foregoing embodiments, the pH of the first reaction mixture and the second reaction mixture may independently be 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 foregoing embodiments, the pH of the first reaction mixture and the second reaction mixture may independently be 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.

[0017] In any of the foregoing embodiments, the polynucleotide hybridized to the hybridization region can be extended by a polymerase using a circular nucleic acid as a template to generate a rolling circle amplification product. In any of the foregoing embodiments, the rolling circle amplification product can be generated using linear rolling circle amplification (RCA), branched RCA, dendritic RCA, or any combination thereof. In any of the foregoing embodiments, the rolling circle amplification product can be generated in situ. In any of the foregoing embodiments, the rolling circle amplification product can be immobilized in a biological sample. In any of the foregoing embodiments, the rolling circle amplification product can be cross-linked with one or more other molecules in the biological sample. In any of the foregoing embodiments, the method can include imaging the biological sample to detect the rolling circle amplification product. In any of the foregoing embodiments, imaging can include detecting a signal associated with a fluorescently labeled probe that binds directly or indirectly to the rolling circle amplification product.

[0018] In any of the foregoing embodiments, the signal associated with the rolling circle amplification product can be amplified in situ in the biological sample. In any of the foregoing embodiments, in-situ signal amplification can include rolling circle amplification (RCA) of probes directly or indirectly bound to the rolling circle amplification product, hybridization reaction (HCR) directly or indirectly on the rolling circle amplification product, linear oligonucleotide hybridization reaction (LO-HCR) directly or indirectly on the rolling circle amplification product, primer exchange reaction (PER) directly or indirectly on the rolling circle amplification product, branching assembly directly or indirectly on the rolling circle amplification product, hybridization of multiple detectable probes directly or indirectly on the rolling circle amplification product, or any combination thereof.

[0019] In any of the foregoing embodiments, the sequence of the rolling circle amplification product can be analyzed in situ in a biological sample. In any of the foregoing embodiments, the sequence of the rolling circle amplification product can be analyzed by sequential hybridization, sequencing-while-hybridization, sequencing-while-ligation, sequencing-while-synthesis, sequencing-while-binding, or combinations thereof. In any of the foregoing embodiments, the sequence of the rolling circle amplification product may include a barcode sequence or its complementary sequence.

[0020] In any of the foregoing embodiments, detecting rolling circle amplification products may include: contacting a biological sample with one or more detectably labeled probes that hybridize directly or indirectly with the rolling circle amplification products, and dehybridizing the one or more detectably labeled probes from the rolling circle amplification products. In some embodiments, the contacting and dehybridizing steps are repeated with the one or more detectably labeled probes and / or one or more other detectably labeled probes that hybridize directly or indirectly with the rolling circle amplification products.

[0021] In any of the foregoing embodiments, detecting rolling circle amplification products may include: contacting a biological sample with one or more intermediate probes that hybridize directly or indirectly with the rolling circle amplification products, wherein the one or more intermediate probes may be detected using one or more detectably labeled probes, and dehybridizing the one or more intermediate probes and / or the one or more detectably labeled probes from the rolling circle amplification products. In some embodiments, the contact and dehybridization steps are repeated using 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.

[0022] In any of the foregoing embodiments, the detectably labeled probe and / or intermediate probe can hybridize with the barcode sequence or its complementary sequence in the rolling circle amplification product.

[0023] In any of the foregoing embodiments, the circular nucleic acid can be formed in a biological sample by a probe or probe set of the target molecule. In any of the foregoing embodiments, the circular nucleic acid can be formed in a biological sample by a padlock probe, a SNAIL (nucleic acid-specific amplification via intramolecular linkage) probe set, a PLAYR (RNA proximity ligation assay) probe set, and a PLISH (proximity ligation in situ hybridization) probe set.

[0024] In any of the foregoing embodiments, the target molecule may be a target nucleic acid. In any of the foregoing embodiments, the target molecule may include viral DNA, bacterial DNA, or cellular DNA or RNA molecules or their products in a biological sample. In any of the foregoing embodiments, the target molecule may include a probe that binds to viral DNA, bacterial DNA, or cellular DNA or RNA molecules or their products in a biological sample. In any of the foregoing embodiments, the target molecule may include a product of a probe that binds to viral DNA, bacterial DNA, or cellular DNA or RNA molecules or their products in a biological sample. In any of the foregoing embodiments, the target molecule may include genomic DNA, mitochondrial DNA, mRNA, or cDNA, and the probe or probe set of the target molecule may include a padlock probe that hybridizes to genomic DNA, mitochondrial DNA, mRNA, or cDNA. In any of the foregoing embodiments, the method may include linking a padlock probe that hybridizes to genomic DNA, mitochondrial DNA, mRNA, or cDNA to form a circular nucleic acid.

[0025] In any of the foregoing embodiments, the target molecule may be a non-nucleic acid target molecule. In any of the foregoing embodiments, the target molecule may be a target protein. In any of the foregoing embodiments, the method may include contacting a biological sample with a labeling agent comprising (i) a binding moiety that binds directly or indirectly to a non-nucleic acid target molecule and (ii) a reporter oligonucleotide corresponding to the binding moiety and / or the non-nucleic acid target molecule. In any of the foregoing embodiments, a probe or probe set of a non-nucleic acid target molecule may comprise a padlock probe that hybridizes to a reporter oligonucleotide. In any of the foregoing embodiments, the method may include ligating a padlock probe that hybridizes to a reporter oligonucleotide to form a circular nucleic acid.

[0026] In some aspects, this document provides a method for analyzing biological samples, comprising: contacting the biological sample with a binding mixture, wherein the biological sample comprises a plurality of circular nucleic acids, each comprising a primer hybridization region, the binding mixture comprising a plurality of complexes, each comprising a polymerase bound to a primer, wherein the primers comprise sequences complementary to the primer hybridization regions of one or more circular nucleic acids, and the polymerase activity is inhibited, thereby allowing the plurality of complexes to hybridize with the plurality of circular nucleic acids. In some embodiments, the method further comprises contacting the biological sample with a primer extension reaction mixture to allow the polymerase to extend primers hybridizing with the primer hybridization regions, thereby synchronizing rolling circle amplification of the plurality of circular nucleic acids in the biological sample.

[0027] In any of the foregoing embodiments, the binding mixture may contain a chelating agent. In any of the foregoing embodiments, the binding mixture may contain one or more deoxynucleoside triphosphates (dNTPs). Alternatively, in any of the foregoing embodiments, the binding mixture may be substantially dNTP-free.

[0028] In any of the foregoing embodiments, the primers in the one or more complexes may have a 3′ hydroxyl group. In any of the foregoing embodiments, the primers in the one or more complexes may have a phosphate-thioester-protected 3′ nucleotide, thereby protecting the primer from 3′→5′ exonuclease degradation by the polymerase in the complex, while allowing initiation by the polymerase. In any of the foregoing embodiments, the hybridization regions of the primers in two or more of the plurality of circular nucleic acids may be identical in sequence. In any of the foregoing embodiments, the hybridization regions of the primers in two or more of the plurality of circular nucleic acids may be different in sequence. In any of the foregoing embodiments, the primers in two or more of the plurality of complexes may be identical in sequence. In any of the foregoing embodiments, the primers in two or more of the plurality of complexes may be different in sequence. In any of the foregoing embodiments, the polymerase may be Phi29 DNA polymerase, Bst polymerase, T7 RNA polymerase, or a Klenow fragment. In any of the foregoing embodiments, the method may further include, between contact steps, the removal of one or more complexes from the biological sample that are not bound to one or more circular nucleic acids. In any of the foregoing embodiments, the primer extension reaction mixture may contain deoxynucleoside triphosphates (dNTPs) and one or more cations. In any of the foregoing embodiments, the primer extension reaction mixture may contain Mg. 2+ Co 2+ and / or Mn 2+ In any of the foregoing embodiments, the primer extension reaction mixture may not contain polymerase.

[0029] In some aspects, this document provides a method for analyzing biological samples, comprising: contacting the biological sample with a binding mixture, wherein the biological sample contains multiple circular nucleic acids, each containing a hybridization region that hybridizes with a polynucleotide, the binding mixture containing a polymerase, and the polymerase activity of the polymerase being inhibited to allow the polymerase to bind to the multiple circular nucleic acids; and contacting the biological sample with a primer extension reaction mixture to allow the polymerase to extend the polynucleotides hybridized with the hybridization region, thereby synchronizing rolling circle amplification of the multiple circular nucleic acids in the biological sample.

[0030] In any of the foregoing embodiments, the polynucleotide may be an exogenous primer that contacts the biological sample, the hybridization region may be a primer hybridization region, and the circular nucleic acid may contain (i) an endogenous molecule in the biological sample, (ii) a product of the endogenous molecule in the biological sample, (iii) a probe that targets the endogenous molecule in the biological sample, and / or (iv) a product of an exogenous probe that targets the endogenous molecule in the biological sample.

[0031] In any of the foregoing embodiments, the polynucleotide comprises (i) an endogenous molecule in a biological sample, (ii) a product of an endogenous molecule in a biological sample, (iii) a probe targeting an endogenous molecule in a biological sample, and / or (iv) a product of an exogenous probe targeting an endogenous molecule in a biological sample.

[0032] In any of the foregoing embodiments, the method may further include terminating rolling circle amplification (RCA) of circular nucleic acids to provide a variety of rolling circle amplification products.

[0033] In any of the foregoing embodiments, the plurality of rolling circle amplification products may have an average diameter of about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1.0 μm, about 1.1 μm, about 1.2 μm, about 13 μm, about 1.4 μm, or about 1.5 μm, or any of the foregoing values. In any of the foregoing embodiments, the plurality of rolling circle amplification products may have an average diameter of less than 0.25 μm.

[0034] In any of the foregoing embodiments, the plurality of rolling circle amplification products may have an average length of about 1 kb, about 2 kb, about 5 kb, about 10 kb, about 20 kb, about 30 kb, about 40 kb, about 50 kb, about 60 kb, or about 70 kb, or any of the foregoing values. In any of the foregoing embodiments, the plurality of rolling circle amplification products may have an average length of less than 20 kb or less than 10 kb.

[0035] In any of the foregoing embodiments, the average copy number of the unit sequence complementary to the circular nucleic acid in the plurality of rolling circle amplification products may be about 10, about 50, about 100, about 500, about 1,000, about 5,000, or about 10,000 or more.

[0036] In any of the foregoing embodiments, the average copy number of the unit sequence complementary to the circular nucleic acid in the plurality of rolling circle amplification products may be less than 100 or less than 1,000.

[0037] In any of the foregoing embodiments, the average peak intensity of the plurality of rolling circle amplification products can be approximately 2 to approximately 10 times that of the rolling circle amplification products formed without rolling circle amplification of the plurality of circular nucleic acids in a concurrent biological sample.

[0038] In any of the foregoing embodiments, the distribution of the relative observed signal of the rolling circle amplification product formed in the case of rolling circle amplification of the multiple circular nucleic acids in a synchronous biological sample may be narrower than the distribution of the relative observed signal of the rolling circle amplification product formed in the case of rolling circle amplification without the multiple circular nucleic acids in a synchronous biological sample.

[0039] In any of the foregoing embodiments, polymerase extension may be performed for no more than 3 hours. In any of the foregoing embodiments, polymerase extension may be performed for no more than 2 hours. In any of the foregoing embodiments, polymerase extension may be performed for no more than 1 hour. In any of the foregoing embodiments, polymerase extension may be performed for no more than 30 minutes.

[0040] In some embodiments, this document discloses a kit for analyzing biological samples comprising: (i) a binding mixture comprising multiple complexes each containing a polymerase bound to a primer and a chelating agent, wherein the binding mixture is substantially free of deoxynucleoside triphosphates (dNTPs); and (ii) a primer extension reaction mixture comprising dNTPs and a divalent cation, wherein the primer extension reaction mixture is substantially free of the polymerase. In any of the foregoing embodiments, the primers in the multiple complexes may be the same. Alternatively, in any of the foregoing embodiments, the primers in two or more of the multiple complexes may be different. In any of the foregoing embodiments, the polymerase may be Phi29 DNA polymerase and the divalent cation may be Mg2+. 2+ Co 2+ and / or Mn 2+ .

[0041] In any of the foregoing embodiments, the biological sample may comprise cells or cellular components. In any of the foregoing embodiments, the biological sample may be a tissue sample. In any of the foregoing embodiments, the biological sample may be fixed. Alternatively, in any of the foregoing embodiments, the biological sample may not be fixed. In any of the foregoing embodiments, the biological sample may be a formalin-fixed paraffin-embedded (FFPE) sample, a frozen tissue sample, or a fresh tissue sample. In any of the foregoing embodiments, the biological sample may be permeabilized. In any of the foregoing embodiments, the biological sample may be processed. In any of the foregoing embodiments, the biological sample may be transparentized. In any of the foregoing embodiments, the biological sample may be embedded in a matrix. In any of the foregoing embodiments, the biological sample may be embedded in a hydrogel. In any of the foregoing embodiments, the biological sample may be embedded in a hydrogel and then transparentized. In any of the foregoing embodiments, the biological sample and / or the matrix may be cross-linked. In any of the foregoing embodiments, the biological sample may be fixed to a substrate such as a glass or plastic slide. Attached Figure Description

[0042] The following figures illustrate some embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0043] Figure 1A -IC illustrates a schematic diagram of an exemplary method for in-situ synchronized RCA. A circular or circularized probe that hybridizes to a target nucleic acid in the sample may contain a primer hybridization site. A polymerase (e.g., Phi29) and primer complex can be formed in an OFF buffer that inhibits the polymerase's activity. The pre-formed polymerase-primer complex can diffuse in the sample and hybridize to the primer hybridization site. Hybridization of the pre-formed complex with target nucleic acid molecules at different locations in the sample can be synchronized, while RCA does not begin until unbound complexes are removed and an ON buffer is applied to initiate RCA at different locations. Figure 1A Alternatively, primers can hybridize to circular or cyclic probes at different locations in the sample, which are then contacted with a polymerase (e.g., Phi29) in an OFF buffer that inhibits polymerase activity. The polymerase can diffuse throughout the sample and bind to the primers hybridized to the circular or cyclic probes. RCA does not begin until unbound polymerase is removed and an ON buffer is applied to initiate RCA at different locations. Figure 1BIn another example, a circular or cyclic probe hybridizes to a polynucleotide in the sample, which is then contacted with a polymerase (e.g., Phi29) in an OFF buffer that inhibits polymerase activity. RCA does not begin until unbound polymerase is removed and an ON buffer is applied to initiate RCA at different sites using the polynucleotide as an RCA primer. Figure 1C In this example, the polynucleotide can be any endogenous or exogenous nucleic acid (or its product) in the sample, such as mRNA or cDNA, and a separate RCA primer is not required.

[0044] Figure 2A-2B Compared to methods using Phi29 alone in OFF buffer (“Phi29+”), methods using primers alone in OFF buffer (“primer+”), and unsynchronized control protocols (“control”), this exemplary method demonstrates the use of a Phi29-primer complex (“complex”) in OFF buffer to synchronize RCA for detecting Gad2, highlighting the characteristic intensity in these methods. Figure 2A ) and estimated feature size ( Figure 2B The result of ).

[0045] Figures 3A-3B Compared to methods using Phi29 alone in OFF buffer (“Phi29+”), methods using primers alone in OFF buffer (“primer+”), and unsynchronized control protocols (“control”), this exemplary method demonstrates the use of a Phi29-primer complex (“complex”) in OFF buffer to synchronize RCA for detecting Slc17a7, showcasing characteristic intensity in this approach. Figure 3A ) and estimated feature size distribution ( Figure 3B The result of ).

[0046] Figures 4A-4B Results of an exemplary method for detecting Slc17a7 are shown. Figure 4A The study shows the trend of increased intensity of 60-minute and 120-minute RCA relative to the control group ("ctrl") under synchronized conditions ("synch"). Figure 4B Representative images of features detected after 120 minutes of amplification are shown, demonstrating a trend toward more uniform feature intensity in the synchronization group compared to the control group.

[0047] Figure 4C Representative images after feature detection and filtering are shown in the control and synchronous groups after 30 minutes of RCA, demonstrating a trend of improved RCA product detection at locations in brain tissue where Slc 17a7 expression is expected. Detailed Implementation

[0048] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated separately by reference. If any definition set forth herein contradicts or is contrary to a definition set forth in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein shall prevail over the definition incorporated herein by reference.

[0049] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0050] I. Overview

[0051] In assays involving in situ rolling circle amplification (RCA), the non-uniform size and intensity distribution of the RCA products can lead to a loss of sensitivity. This is because weak signals associated with some RCA products may fail to meet the detection thresholds for spot detection used in image analysis, while strong signals associated with some adjacent RCA products may cause optical congestion and prevent the detection of nearby weaker signal spots. Both weak, undetected RCA products and large, overcrowded RCA products can contribute to a loss of sensitivity. In some respects, the non-uniform size and intensity distribution may be due to the asynchronous in situ initiation of the RCA reaction at different times. In other respects, the non-uniformity may be caused by the diffusion time and rate at which one or more reagents (e.g., enzymes, primers, etc.) reach different locations in the sample (e.g., tissue sections). In some cases, when a polymerase such as phi29 is added to the RCA reaction buffer and applied to the sample, the enzyme diffuses from the bulk solution through the sample (e.g., tissue sections) to the primers and circular template in the sample to initiate the RCA reaction. Therefore, circular templates that accept functional polymerase molecules later may initiate RCA later than other circular templates that accept polymerase molecules earlier. When RCA is stopped at the same time point, circular templates that started earlier produce larger and brighter signal spots associated with rolling circle amplification products, while circular templates that started later produce smaller and weaker signal spots.

[0052] In some aspects, this document provides compositions and methods for generating rolling circle amplification (RCA) products that achieve greater uniformity in RCA product size by synchronizing the RCA start point. In other aspects, this document provides compositions and methods for in situ generation of RCA products that are more uniform in size and intensity to make spot detection more robust for image analysis. In some cases, the advantages of synchronized RCA can be applied to biological samples with highly expressed genes to be detected. In some embodiments, a polymerase such as Phi29 is used to bind single-stranded nucleic acids (e.g., ssDNA) in polynucleotides (e.g., probes or target nucleic acids such as mRNA or cDNA) via its single-stranded nucleic acid-binding domain, while its exonuclease and polymerase functions are disabled in OFF buffer, for example, substantially free of one or more cofactors (e.g., Mg). 2+ The binding buffer for RCA primers and / or dNTPs is used. In some embodiments, the complex of the RCA primer and polymerase (e.g., Phi29) is pre-formed in OFF buffer. In some embodiments, the complex is then added to a sample such as a tissue section having a pre-formed loop template (e.g., prepared by padlock probe ligation onto the target nucleic acid).

[0053] In some embodiments, after the complex hybridizes to the RCA primer site in the loop (e.g., a circular nucleic acid), the mixture is removed and replaced with an ON buffer that activates the polymerase and simultaneously initiates RCA on all loops that have accepted the polymerase-primer complex in the RCA reaction mixture. Figure 1A In some embodiments, the primers prehybridize with a circular template in the sample. In some implementations, a polymerase such as Phi29 is provided in an OFF buffer that inhibits one or more activities of the polymerase and is then contacted with the sample. The polymerase binds to the primers prehybridized with the circular template, but RCA is not initiated until the sample is contacted with an ON buffer (e.g., RCA reaction buffer) that relieves the inhibition of the polymerase and / or exonuclease activity, thereby synchronizing the RCA from the circular template at multiple sites in the sample. Figure 1B In some embodiments, the polymerase can be loaded onto a sample containing a circular nucleic acid molecule, where no separate RCA primer is provided. For example, in one embodiment, the polymerase in OFF buffer is applied to the sample and binds to the 3′ end of a polynucleotide (e.g., RNA or DNA, such as mRNA or cDNA) in the sample. In some embodiments, the polymerase loaded onto the polynucleotide in the sample can be activated with ON buffer to initiate a simultaneous RCA reaction using the polynucleotide as a primer. Figure 1C ).

[0054] In some implementations, synchronization results in more uniform RCA product size and / or brighter signal spots. In some implementations, synchronization results in fewer dark signal spots and / or more bright signal spots in the RCA products. In some implementations, when the RCA product size becomes more uniform, amplification time can be reduced, resulting in smaller but equally bright RCA products. Overall, synchronization facilitates the detection and / or resolution of more RCA products in congested spaces.

[0055] II. Samples, analytes, and target sequences

[0056] A.Sample

[0057] The samples disclosed herein can be or are derived from any biological sample. The methods and compositions disclosed herein can be used to analyze biological samples obtained from a subject using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and typically include cells and / or other biological material from the subject. In addition to the subject described above, biological samples can be obtained from prokaryotes (such as bacteria, archaea, viruses, or viroids). Biological samples can also be obtained from non-mammalian organisms (e.g., plants, insects, arthropods, nematodes, fungi, or amphibians). Biological samples can also be obtained from eukaryotes, such as tissue samples, patient-derived organoids (PDOs), or patient-derived xenografts (PDXs). Biological samples derived from an organism can contain one or more other organisms or components thereof. For example, in addition to mammalian cells and non-cellular tissue components, mammalian tissue sections can contain prions, viroids, viruses, bacteria, fungi, or components from other organisms. Subjects from whom biological samples can be obtained may be healthy or asymptomatic individuals, individuals who have or are suspected of having a disease (e.g., patients with a disease such as cancer) or are susceptible to a disease, and / or individuals who need or are suspected of needing treatment.

[0058] Biological samples can include any number of macromolecules, such as cellular macromolecules and organelles (e.g., mitochondria and the nucleus). Biological samples can be nucleic acid samples and / or protein samples. Biological samples can be carbohydrate samples or lipid samples. Biological samples can be obtained as tissue samples (such as tissue sections, biopsy samples, core needle biopsy samples, needle aspirates, or fine needle aspirates). Samples can be fluid samples, such as blood samples, urine samples, or saliva samples. Samples can be skin samples, colon samples, buccal swabs, histological samples, histopathological samples, plasma or serum samples, tumor samples, live cells, cultured cells, or clinical samples (e.g., whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions). In some embodiments, biological samples may contain cells deposited on a surface.

[0059] Cell-free biological samples may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from body samples such as blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, and tears.

[0060] Biological samples may originate from homogeneous cultures or populations of the subjects or organisms mentioned herein, or alternatively from collections of several different organisms, for example, in a community or ecosystem.

[0061] Biological samples may include one or more diseased cells. Diseased cells may have altered metabolic properties, gene expression, protein expression, and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, neurological disorders, and cancer. Cancer cells may originate from solid tumors, hematologic malignancies, cell lines, or be obtained as circulating tumor cells. Biological samples may also include fetal cells and immune cells.

[0062] Biological samples may include analytes (e.g., proteins, RNA, and / or DNA) embedded in a 3D matrix. In some embodiments, analytes (e.g., proteins, RNA, and / or DNA) or associated amplicons (e.g., rolling circle amplification products) may be embedded in the 3D matrix. In some embodiments, the 3D matrix may contain networks of natural and / or synthetic molecules linked chemically and / or enzymatically (e.g., through cross-linking). In some embodiments, the 3D matrix may contain synthetic polymers. In some embodiments, the 3D matrix comprises a hydrogel.

[0063] In some embodiments, the substrate herein can be any support insoluble in aqueous liquids, allowing for the positioning of biological samples, analytes, features, and / or reagents (e.g., probes) on said support. In some embodiments, the biological sample can be attached to the substrate. Attachment of the biological sample can be irreversible or reversible, depending on the nature of the sample and subsequent steps in the analytical method. In some embodiments, the sample can be reversibly attached to the substrate by applying a suitable polymer coating to it and bringing the sample into contact with the polymer coating. The sample can then be separated from the substrate, for example, using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers suitable for this purpose.

[0064] In some implementations, the substrate may be coated or functionalized with one or more substances to facilitate sample adhesion to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0065] Various steps can be performed to prepare or process biological samples for and / or during assays. Unless otherwise stated, the preparation or processing steps described below can generally be combined in any manner and in any order to properly prepare or process a particular sample for and / or for analysis.

[0066] (i) tissue sections

[0067] Biological samples can be obtained from a subject (e.g., via surgical biopsy, whole subject section) or grown in vitro as cell populations on a growth substrate or culture dish and prepared as tissue sections or tissue slides for analysis. The grown samples can be thin enough to be analyzed without further processing steps. Alternatively, grown samples and samples obtained via biopsy or sectioning can be prepared into thin tissue sections using mechanical cutting devices such as a vibrating blade microtome. As another alternative, in some embodiments, thin tissue sections can be prepared by applying a tactile imprint of the biological sample onto a suitable substrate material.

[0068] The thickness of a tissue section can be a fraction of the largest cross-sectional size of the cells (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections thicker than the largest cross-sectional cell size can also be used. For example, cryostat sections can be used, which can be, for example, 10-20 μm thick.

[0069] More generally, the thickness of tissue sections typically depends on the method used to prepare the sections and the physical properties of the tissue, thus sections with a wide variety of thicknesses can be prepared and used. For example, tissue section thicknesses can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 μm. Thicker sections, such as at least 70, 80, 90, or 100 μm or more, can also be used if desired or convenient. Typically, tissue section thicknesses are between 1-100 μm, 1-50 μm, 1-30 μm, 1-25 μm, 1-20 μm, 1-15 μm, 1-10 μm, 2-8 μm, 3-7 μm, or 4-6 μm, but as mentioned above, sections with thicknesses greater than or less than these ranges can also be analyzed.

[0070] Multiple slices can also be obtained from a single biological sample. For example, multiple tissue slices can be obtained from a surgical biopsy sample by serially slicing the biopsy sample using a slicing blade. Spatial information between the series of slices can be preserved in this way, and the slices can be analyzed continuously to obtain three-dimensional information about the biological sample.

[0071] (ii) Freezing

[0072] In some implementations, biological samples (e.g., tissue sections as described above) can be prepared by deep freezing at temperatures suitable for maintaining or preserving the integrity (e.g., physical properties) of the tissue structure. Frozen tissue sample sections (e.g., thin slices) can be applied to a substrate surface using any number of suitable methods. For example, tissue samples can be prepared using a cryostat (e.g., a cryostat) set at temperatures suitable for maintaining the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. Such temperatures can be, for example, below -15°C, below -20°C, or below -25°C.

[0073] (iii) Fixation and post-fixation

[0074] In some embodiments, established methods for formalin fixation and paraffin embedding (FFPE) can be used to prepare biological samples. In some embodiments, formalin fixation and paraffin embedding can be used to prepare cell suspensions and other non-tissue samples. After fixing and embedding the sample in a block of paraffin or resin, the sample can be sectioned as described above. Prior to analysis, the paraffin embedding material can be removed from the tissue sections by incubating them in a suitable solvent (e.g., xylene) followed by rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes) (e.g., dewaxing).

[0075] As an alternative to formalin fixation as described above, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, samples can be fixed by immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0076] In some embodiments, fresh frozen samples are fixed with acetone, which may include, but is not limited to, cortical tissue, mouse olfactory bulbs, human brain tumors, human post-mortem brain, and breast cancer samples. When performing acetone fixation, a pre-permeabilization step (described below) may be omitted. Alternatively, acetone fixation may be combined with a permeabilization step.

[0077] In some embodiments, the methods provided herein include one or more post-fixing (also known as postfixation) steps. In some embodiments, one or more post-fixation steps are performed after contacting the sample with the polynucleotides disclosed herein, such as one or more probes, such as circular or padlock probes. In some embodiments, one or more post-fixation steps are performed after a hybridization complex containing the probe and target has formed in the sample. In some embodiments, one or more post-fixation steps are performed prior to the ligation reactions disclosed herein, such as ligation involving the circularization of a padlock probe.

[0078] In some embodiments, one or more post-fixation steps are performed after contacting the sample with a binder or labeler (e.g., an antibody or its antigen-binding fragment) of a non-nucleic acid analyte such as a protein analyte. The labeler may comprise a nucleic acid molecule (e.g., a reporter oligonucleotide) containing a sequence corresponding to the labeler and therefore to the analyte (e.g., uniquely identified). In some embodiments, the labeler may comprise a reporter oligonucleotide containing one or more barcode sequences.

[0079] The post-fixation step can be performed using any suitable fixation reagent disclosed herein (e.g., 3% (w / v) paraformaldehyde in DEPC-PBS).

[0080] (iv) Embedding

[0081] As an alternative to the paraffin embedding described above, biological samples can be embedded in any of a variety of other embedding materials to provide a structural substrate for the sample prior to sectioning and other processing steps. In some cases, the embedding material can be removed, for example, before analyzing tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylates), epoxy resins, and agar.

[0082] In some embodiments, biological samples can be embedded in a matrix (e.g., a hydrogel matrix). Embedding samples 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 formation of the hydrogel results in its internalization within the biological sample.

[0083] In some embodiments, biological samples are immobilized in the hydrogel by crosslinking the polymeric material that forms the hydrogel. Crosslinking can be carried out chemically and / or photochemically, or alternatively, by any other hydrogel-forming method.

[0084] The composition of the hydrogel-matrix and its application to biological samples typically depend on the nature and preparation of the biological sample (e.g., sectioned, unsectioned, fixed type). As an example, when the biological sample is a tissue section, the hydrogel-matrix may include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, when the biological sample consists of cells (e.g., cultured cells or cells dissociated from a tissue sample), the cells may be incubated together with the monomer solution and the APS / TEMED solution. For cells, the hydrogel-matrix gel forms in compartments, including but not limited to devices for culturing, maintaining, or transporting cells. For example, a hydrogel-matrix with a depth range of approximately 0.1 μm to approximately 2 mm can be formed using a monomer solution added to the compartment along with APS / TEMED.

[0085] Other 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.

[0086] (v) Staining and immunohistochemistry (IHC)

[0087] To facilitate visualization, a wide variety of staining agents and staining techniques can be used to stain biological samples. For example, in some embodiments, any number of staining agents and / or immunohistochemical reagents can be used to stain the sample. One or more staining steps can be performed to prepare or process the biological sample for the assay described herein, or can be performed during and / or after the assay. In some embodiments, the sample can be contacted with one or more nucleic acid staining agents, membrane staining agents (e.g., cell membrane or nuclear membrane), cytological staining agents, or combinations thereof. In some instances, staining can be specific to proteins, phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, organelles, or cellular compartments. The sample can be contacted with one or more labeled antibodies (e.g., a first antibody specific to the analyte of interest and a second antibody labeled specifically to the first antibody). In some embodiments, one or more images of the stained sample can be used to segment the cells in the sample.

[0088] In some embodiments, lipophilic dyes are used for staining. In some instances, lipophilic carbocyanine or aminostyrene dyes or their analogues (e.g., DiI, DiO, DiR, DiD) are used. Other cell membrane staining agents may include FM and RH dyes or immunohistochemical reagents specific to cell membrane proteins. In some instances, staining agents may include, but are not limited to, acridine orange, Bismarck brown, carmine, Coomassie blue, cresol purple, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hoechst staining agent, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamine B), or safranin or their derivatives. In some embodiments, samples may be stained with hematoxylin and eosin (H&E).

[0089] Samples can be stained using hematoxylin and eosin (H&E) staining techniques, Papanicolaoustaining, Masson's trichrome staining, silver staining, Sudan staining, and / or periodic acid Schiff (PAS) staining. PAS staining is typically performed after formalin or acetone fixation. In some embodiments, samples can be stained using Romanovsky stains (including Wright's stain, Jenner's stain, Can-Grunwald stain, Leishman stain, and Giemsa stain).

[0090] In some embodiments, biological samples can be destained. Any suitable method can be used to destain or decolorize biological samples, and this typically depends on the nature of the staining agent applied to the sample. For example, in some embodiments, one or more immunofluorescent staining agents are applied to the sample via antibody conjugation. Such staining agents can be removed using techniques such as washing with reducing agents and detergents to cleave disulfide bonds, liquid salt treatment, treatment with antigen retrieval solutions, and treatment with acidic glycine buffer. Methods for multiple 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.

[0091] (vi) Isometric Expansion

[0092] In some implementations, biological samples embedded in a matrix (e.g., a hydrogel) can be isochorically expanded. Isochoric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in Chen et al., Science 347(6221):543-548, 2015, the entire contents of which are incorporated herein by reference.

[0093] Isovolescent swelling can be achieved by anchoring one or more components of a biological sample to a gel, followed by gel formation, protein hydrolysis, and swelling. In some embodiments, analytes, analyte products, and / or probes associated with analytes in the sample can be anchored to a matrix (e.g., a hydrogel). Isovolescent swelling of the biological sample can occur before or after the biological sample is immobilized on the substrate. In some embodiments, the isovolescently swelled biological sample can be removed from the substrate before contacting the substrate with the probes disclosed herein.

[0094] Typically, the steps used for isochoric expansion of biological samples can depend on the characteristics of the sample (e.g., the thickness of the tissue section, fixation, cross-linking) and / or the analyte of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to the gel).

[0095] In some embodiments, proteins in a biological sample are anchored to a swellable gel (such as a polyelectrolyte gel). Antibodies may be directed to the proteins before, after, or in combination with anchoring to the swellable gel. DNA and / or RNA in a biological sample may also be anchored to the swellable gel via suitable adapters. Examples of such adapters include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X SE) (available from Thermo Fisher, Waltham, MA), Label-IT amine (available from Mirus Bio, Madison, WI), and Label X (described, for example, in Chen et al., Nat. Methods 13:679-684, 2016, the entire contents of which are incorporated herein by reference).

[0096] Isochoric expansion of a sample can increase the spatial resolution of subsequent analyses. The increase in resolution in the spatial distribution can be determined by comparing isochorically expanded samples with those that have not been isochorically expanded.

[0097] In some embodiments, the biological sample is isochorically expanded to a size at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x of its unexpanded size. In some embodiments, the sample is isochorically expanded to at least 2x and less than 20x of its unexpanded size.

[0098] (vii) Crosslinking and decrosslinking

[0099] In some embodiments, biological samples are reversibly cross-linked before or during in-situ assay rounds. In some aspects, analytes, polynucleotides, and / or amplification products (e.g., amplicons) of analytes or probes bound thereto can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more polynucleotide probes and / or their amplification products (e.g., amplicons) can be modified to contain functional groups that can serve as anchoring sites for attaching the polynucleotide probes and / or amplification products to the polymer matrix. In some embodiments, modified probes containing oligodT can be used to bind to mRNA molecules of interest, followed by reversible cross-linking of the mRNA molecules.

[0100] In some embodiments, biological samples are immobilized within a hydrogel by crosslinking the polymeric material that forms the hydrogel. Crosslinking can be carried out chemically and / or photochemically, or alternatively, by any other hydrogel-forming method. The hydrogel may comprise a macromolecular polymer gel containing a network system. In the network system, some polymer chains may optionally be crosslinked, but crosslinking does not always occur.

[0101] In some embodiments, the hydrogel may include hydrogel subunits such as, but not limited to, acrylamide, bisacrylamide, polyacrylamide and its derivatives, poly(ethylene glycol) and its derivatives (e.g., PEG-acrylate (PEG-DA), PEG-RGD), methacrylamide gelatin (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinylpyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and combinations thereof.

[0102] In some embodiments, the hydrogel includes a hybrid material, for example, the hydrogel material includes elements of a synthetic polymer and a natural polymer. Examples of suitable hydrogels are described, for example, in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422 and U.S. Patent Application Publications Nos. 2017 / 0253918, 2018 / 0052081, and 2010 / 0055733, the entire contents of which are incorporated herein by reference.

[0103] In some embodiments, the hydrogel can form a substrate. In some embodiments, the substrate comprises 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, under, or in any other configuration with one or more second materials. In some embodiments, hydrogel formation occurs after contact with one or more second materials during substrate formation. Hydrogel formation can also occur within structures located on the substrate, such as pores, ridges, protrusions, and / or textures.

[0104] In some implementations, hydrogel formation on the substrate occurs before, simultaneously with, or after the probe is provided to the sample. For example, hydrogel formation can be performed on a substrate that already contains the probe.

[0105] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, the biological sample (e.g., a tissue section) is embedded in the hydrogel. In some embodiments, hydrogel subunits are injected into the biological sample, and the polymerization of the hydrogel is initiated by external or internal stimuli.

[0106] In embodiments of hydrogel formation within a biological sample, functionalizing chemistry may be used. In some embodiments, functionalizing chemistry includes hydrogel-tissue chemistry (HTC). Any hydrogel-tissue framework suitable for HTC (e.g., synthetic or natural) can be used to anchor biomolecules and modulate functionalization. Non-limiting examples of methods using HTC framework variants include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, hydrogel formation within the biological sample is permanent. For example, biomolecules can permanently adhere to the hydrogel, allowing for multiple rounds of interrogation. In some embodiments, hydrogel formation within the biological sample is reversible.

[0107] In some embodiments, additional reagents are added to the hydrogel subunits before, during, and / or after polymerization. These additional reagents may include, but are not limited to, oligonucleotides (e.g., probes), DNA fragmentation endonucleases, DNA fragmentation buffers, DNA polymerases, and dNTPs for amplifying nucleic acids and attaching barcodes to the amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, transposases, ligases, protease K, and DNase. Additional reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers, and switch oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, during, and / or after polymerization.

[0108] In some embodiments, the HTC reagent is added to the hydrogel before, during, and / or after polymerization. In some embodiments, a cell marker is added to the hydrogel before, during, and / or after polymerization. In some embodiments, a cell permeabilizer is added to the hydrogel before, during, and / or after polymerization.

[0109] Hydrogels embedded in biological samples can be removed using any suitable method. For example, electrophoretic tissue removal methods can be used to remove biomolecules from hydrogel-embedded samples. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixation medium, methylcellulose, or other semi-solid medium) before or after hydrogel removal.

[0110] In some embodiments, the methods disclosed herein include decrosslinking a reversibly crosslinked biological sample. Decrosslinking does not need to be complete. In some embodiments, only a subset of the crosslinked molecules in the reversibly crosslinked biological sample are decrosslinked and allowed to migrate.

[0111] (viii) Tissue permeation and treatment

[0112] In some implementations, biological samples can be permeabilized to facilitate the transfer of analytes from the sample and / or to facilitate the transfer of substances (such as probes) into the sample. If the sample is not adequately permeabilized, the amount of substances (such as probes) in the sample may be too low for sufficient analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationships of analytes within the tissue sample may be lost. Therefore, it is ideal to strike a balance between adequately permeating the tissue sample to obtain good signal intensity and still maintaining the spatial resolution of the analyte distribution within the sample.

[0113] Typically, biological samples can be permeated by exposing them to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), crosslinking agents (e.g., paraformaldehyde), and detergents (e.g., saponins, Triton X-100). TM Or Tween-20 TM The sample may contain enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample may be incubated with a cell permeabilizing agent to promote sample permeation. Other methods for sample permeation 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 sample permeation method can generally be used in conjunction with the samples described herein.

[0114] In some implementations, biological samples can be permeated by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive agents such as lysins for lysing different cell types (e.g., Gram-positive or Gram-negative bacteria, plants, yeast, mammals), such as lysozyme, colorless peptidase, lysostaphin, labiase, Rhizoctonia solani lysin (kitalase), lysozyme, and various other commercially available lysins.

[0115] Other lysis agents can be added to biological samples, either additionally or alternatively, to facilitate permeation. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions may contain ionic surfactants such as sodium dodecyl sarcosinate and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents may include, but are not limited to, organic solvents, chelating agents, detergents, surfactants, and dissociation agents.

[0116] In some implementations, biological samples can be permeated using non-chemical permeation methods. Non-chemical permeation methods include, but are not limited to, physical lysis techniques such as electroporation, mechanical permeation methods (e.g., bead tapping using homogenizers and grinding balls to mechanically disrupt the sample tissue structure), acoustic permeation (e.g., sonication), and thermal lysis techniques (e.g., heating to induce thermal permeation of the sample).

[0117] Prior to analysis, additional reagents can be added to biological samples to perform various functions. In some embodiments, DNase and RNase inactivators or inhibitors such as protease K and / or chelating agents such as EDTA can be added to the sample. For example, the methods disclosed herein may include steps to increase the binding accessibility of nucleic acids, such as a denaturation step that opens DNA in cells for probe hybridization. For example, protease K treatment can be used to release DNA bound to proteins.

[0118] (ix) Selective enrichment of RNA species

[0119] In some embodiments, when RNA is the analyte, one or more RNA analyte species of interest can be selectively enriched. For example, one or more RNA species of interest can be selected by adding one or more oligonucleotides to the sample. In some embodiments, the additional oligonucleotide is a sequence for initiating a reaction by an enzyme (e.g., polymerase). For example, one or more primer sequences that are sequence complementary to one or more target RNAs can be used to amplify the one or more target RNAs, thereby selectively enriching these RNAs.

[0120] In some implementations, oligonucleotides having sequence complementarity with the complementary strand of RNA (e.g., cDNA) can bind to cDNA. For example, biotinylated oligonucleotides having sequences complementary to one or more cDNAs of interest bind to cDNA, and biotinylated oligonucleotides can be captured using biotinylation-streptavidin affinity methods (e.g., using streptavidin beads).

[0121] Alternatively, one or more RNA species can be downselected (e.g., removed) using any of a variety of methods. For example, a probe can be applied to the sample that selectively hybridizes with ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. Additionally and alternatively, double-stranded specific nuclease (DSN) treatment can remove rRNA (see, for example, Archer et al., Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage, BMC Genomics, 15401, (2014), the entire contents of which are incorporated herein by reference). Furthermore, hydroxyapatite chromatography can remove abundant species (e.g., rRNA) (see, for example, Vandernoot, VA, cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6)373-80, (2012), the entire contents of which are incorporated herein by reference).

[0122] Biological samples may contain one or more analytes of interest. Methods are provided for performing multiple assays to analyze two or more different analytes in a single biological sample.

[0123] B. Analytes

[0124] The methods and compositions disclosed herein can be used to detect and analyze a wide variety of analytes. In some aspects, analytes may include any biological substance, structure, part, or component to be analyzed. In some aspects, the targets disclosed herein may similarly include any analyte of interest. In some instances, the target or analyte can be detected directly or indirectly.

[0125] Analytes can originate from specific cell types and / or specific subcellular regions. For example, analytes can originate from the cytosol, the nucleus, mitochondria, microsomes, and more generally, any other compartment, organelle, or part of the cell. Permeabilizers that specifically target certain cell compartments and organelles can be used to selectively release analytes from the cell for analysis and / or allow one or more reagents (e.g., probes for analyte detection) to approach the analyte within the cell or cell compartment or organelle.

[0126] Analytes can include any biomolecule or chemical compound, including macromolecules (such as proteins or peptides, lipids, or nucleic acid molecules) or small molecules (including organic or inorganic molecules). Analytes can be cells or microorganisms, including viruses or fragments or products thereof. Analytes can be any substance or entity for which specific binding couplers (e.g., affinity couplers) can be developed. Such specific binding couplers can be nucleic acid probes (for nucleic acid analytes) and can directly lead to the generation of an RCA template (e.g., padlock or other circularizable probe). Alternatively, specific binding couplers can be coupled to nucleic acids that can be detected using an RCA strategy, for example in assays using or generating circular nucleic acids that can serve as RCA templates.

[0127] 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., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.); and synthetic and / or modified nucleic acid molecules (e.g., including nucleic acid domains comprising synthetic or modified nucleotides such as LNA, PNA, morpholino, etc., or composed of such substances); protein molecules, such as peptides, polypeptides, proteins, or prions, or any molecule comprising protein or polypeptide components, or fragments thereof; or lipid or carbohydrate molecules or any molecule comprising lipid or carbohydrate components. Analytes may be single molecules or complexes containing two or more molecular subunits, such as, but not limited to, protein-DNA complexes, which may or may not be covalently bound to each other and may be the same or different. Therefore, in addition to cells or microorganisms, such complex analytes may also be protein complexes or protein-protein interactions. Thus, such complexes or interactions may be homopolymers or heteropolymers. Aggregates of molecules (e.g., proteins) can also be target analytes, such as aggregates of the same or different proteins. Analytes can also be complexes between proteins or peptides and nucleic acid molecules (such as DNA or RNA), such as interactions between proteins and nucleic acids (e.g., regulatory factors (such as transcription factors) and DNA or RNA).

[0128] (i) Endogenous analytes

[0129] In some embodiments, the analytes described herein are endogenous to the biological sample and may include both nucleic acid analytes and non-nucleic acid analytes. The methods and compositions disclosed herein may be used in any suitable combination to analyze nucleic acid analytes (e.g., using nucleic acid probes or probe sets that hybridize directly or indirectly with nucleic acid analytes) and / or non-nucleic acid analytes (e.g., using labelers that contain reporter oligonucleotides and bind directly or indirectly to non-nucleic acid analytes).

[0130] 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, amidated variants of proteins, hydroxylated variants of proteins, methylated variants of proteins, ubiquitinated variants of proteins, sulfated variants of proteins, viral capsid proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte is located inside the cell or on the cell surface, such as a transmembrane analyte or an analyte attached to the cell membrane. In some embodiments, the analyte may be an organelle (e.g., the nucleus or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Exemplary analytes include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T-cell receptors, T-cell receptors, B-cell receptors, chimeric antigen receptors, extracellular matrix proteins, and post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) of cell surface proteins, nick junctions, or adhesion junctions.

[0131] Examples of nucleic acid analytes include DNA analytes, such as single-stranded 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. DNA analytes can also be transcripts of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample.

[0132] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), including nascent RNA, pre-mRNA, primary transcribed RNA, and processed RNA (such as capped mRNA (e.g., with a 5′7-methylguanosine cap), polyadenylated mRNA (with a polyA tail at the 3′ end), and spliced ​​mRNA with one or more introns removed). Uncapped mRNA, unpolyadenylated mRNA, and unspliced ​​mRNA are also included among the analytes disclosed herein. RNA analytes can be transcripts of another nucleic acid molecule (e.g., DNA or RNA (such as viral RNA)) present in a tissue sample. Examples of non-coding RNAs (ncRNAs) that do not translate into proteins include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), extracellular RNAs (exRNAs), Cajal body-specific RNAs (scaRNAs), and long ncRNAs (such as Xist and HOTAIR). RNA can be small (e.g., less than 200 nucleotides in length) or large (e.g., more than 200 nucleotides in length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNA, piRNA, tRNA-derived small RNAs (tsRNAs), and small rDNA-derived RNAs (srRNAs). RNA can be double-stranded or single-stranded. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16S rRNA or 23S rRNA).

[0133] In some embodiments described herein, the analyte may be 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 but used in the methods disclosed herein.

[0134] In some implementations, analytes can be extracted from live cells. Processing conditions can be adjusted to ensure the biological sample remains viable during analysis and that the analyte is extracted (or released) from the viable cells of the sample. Live cell-derived analytes can be obtained only once from the sample, or they can be obtained at intervals from samples that remain continuously viable.

[0135] The methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes being 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 or in a single feature of the substrate.

[0136] In any of the embodiments described herein, the analyte comprises a target sequence. In some embodiments, the target sequence may be an endogenous sequence of the sample, a sequence generated in the sample, a sequence added to the sample, or a sequence associated with the analyte in the sample. In some embodiments, the target sequence is a single-stranded target sequence (e.g., a sequence in a rolling circle amplification product). In some embodiments, the analyte comprises one or more single-stranded target sequences. In one aspect, the first single-stranded target sequence is different from a second single-stranded target sequence. In another aspect, the first single-stranded target sequence is the same as one or more second single-stranded target sequences. In some embodiments, one or more second single-stranded target sequences are contained in an analyte (e.g., a nucleic acid) that is the same as the first single-stranded target sequence. Alternatively, one or more second single-stranded target sequences are contained in an analyte (e.g., a nucleic acid) that is different from the first single-stranded target sequence.

[0137] (ii) Markers

[0138] In some embodiments, this document provides methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, and cell surface or intracellular proteins and / or metabolites) in a sample using one or more labeling agents. In some embodiments, the analyte labeling agent may include a reagent that interacts with the analyte (e.g., an endogenous analyte in the sample). In some embodiments, the labeling agent may contain a reporter oligonucleotide indicating an analyte or a portion thereof that interacts with the labeling agent. For example, the reporter oligonucleotide may contain a barcode sequence that allows identification of the labeling agent. In some cases, the sample contacted with the labeling agent may be further contacted with a probe (e.g., a single-stranded probe sequence) that hybridizes with the reporter oligonucleotide of the labeling agent to identify an analyte associated with the labeling agent. In some embodiments, the analyte labeling agent comprises an analyte-binding moiety and a labeling agent barcode domain, the labeling agent barcode domain containing one or more barcode sequences, such as barcode sequences corresponding to the analyte-binding moiety and / or the analyte. The analyte-binding moiety barcode includes a barcode that associates with or otherwise identifies the analyte-binding moiety. In some implementations, the analyte binding moiety is identified by identifying its associated analyte binding moiety barcode, or the analyte bound to the analyte binding moiety is identified. The analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the analyte binding moiety. The analyte binding moiety barcode can generally include any of the aspects of barcodes described herein.

[0139] In some embodiments, the method includes one or more post-fixation steps following contact of the sample with one or more labeling agents.

[0140] In the methods and systems described herein, one or more labelers capable of binding to or otherwise coupling to one or more features can be used to characterize analytes, cells, and / or cell features. In some cases, cell features include cell surface features. Analytes may include, but are not limited to, proteins, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T-cell receptors, T-cell receptors, B-cell receptors, chimeric antigen receptors, nick junctions, adhesion junctions, or any combination thereof. In some cases, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation state or other post-translational modifications), nucleoproteins, nuclear membrane proteins, or any combination thereof.

[0141] In some embodiments, the analyte binding portion may include any molecule or portion capable of binding an analyte (e.g., a bioanalyte, such as a macromolecular component). The labeler may include, but is not limited to, proteins, peptides, antibodies (or epitope-binding fragments thereof), lipophilic moieties (such as cholesterol), cell surface receptor-binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T-cell adaptors, T-cell receptor adaptors, B-cell receptor adaptors, antibody prodrugs, aptamers, monoclonal antibodies, affimers, darpins, and protein scaffolds, or any combination thereof. The labeler may include (e.g., linked to) a reporter oligonucleotide that indicates the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide may contain a barcode sequence that allows identification of the labeler. For example, a labeler specific to one type of cell feature (e.g., a first cell surface feature) may have a first reporter oligonucleotide coupled thereto, while a labeler specific to a different cell feature (e.g., a second cell surface feature) may have different reporter oligonucleotides coupled thereto. For a description of exemplary labelers, reporting oligonucleotides, and methods of use, see, for example, U.S. Patent 10,550,429; U.S. Patent Publication 20190177800; and U.S. Patent Publication 20190367969, all of which are incorporated herein by reference in their entirety.

[0142] In some embodiments, the analyte-binding portion comprises one or more antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment comprising the analyte-binding portion can specifically bind to the target analyte. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, multiple analyte markers comprising multiple analyte-binding portions bind to multiple analytes present in a biological sample. In some embodiments, the multiple analytes comprise a single class of analytes (e.g., a single class of peptides). In some embodiments where the multiple analytes comprise a single class of analytes, the analyte-binding portions of the multiple analyte markers are identical. In some embodiments where the multiple analytes comprise a single class of analytes, the analyte-binding portions of the multiple analyte markers are different (e.g., members of the multiple analyte markers may have two or more classes of analyte-binding portions, each class of the two or more classes of analyte-binding portions binding to a single class of analyte, e.g., at different binding sites). In some embodiments, the multiple analytes comprise multiple different classes of analytes (e.g., multiple different classes of peptides).

[0143] In other cases, such as to facilitate sample reuse, a marker specific to a particular cell characteristic may have a first plurality of markers (e.g., an antibody or lipophilic moiety) conjugated to a first reporter oligonucleotide and a second plurality of markers conjugated to a second reporter oligonucleotide.

[0144] In some respects, these reporter oligonucleotides may contain a specific nucleic acid barcode sequence that allows for the identification of the marker to which the reporter oligonucleotide is conjugated. Selecting oligonucleotides as reporters offers the advantages of: enabling significant sequence diversity, ease of ligation to most biomolecules (e.g., antibodies), and ease of detection (e.g., using sequencing or array techniques).

[0145] Reporting oligonucleotides can be linked (conjugated) to labelers through any of a variety of direct or indirect, covalent or non-covalent associations or linkages. For example, oligonucleotides can be linked using chemical conjugation techniques (e.g., Lightning-, available from InnovaBiosciences). Antibody labeling kits can be covalently linked to a portion of a labeling agent (such as a protein, for example, an antibody or antibody fragment), and can also be linked using other non-covalent linkage mechanisms, such as using biotinylated antibodies and oligonucleotides with avidin or streptavidin linkers (or beads comprising one or more biotinylated linkers coupled to oligonucleotides). Antibody and oligonucleotide biotinylation techniques are available. See, for example, Fang et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. 15 Jan 2003; 31(2): 708-715, the entire text of which is incorporated herein by reference for all purposes. Similarly, protein and peptide biotinylation techniques have been developed and are readily available. See, for example, U.S. Patent No. 6,265,552, which is incorporated herein by reference for all purposes. Furthermore, click reaction chemistry can be used to conjugate reporter oligonucleotides to labelers. Commercially available kits (such as those from Thunderlink and Abcam) and techniques common in the art can be used, where appropriate, to conjugate reporter oligonucleotides to labelers. In another example, the labeler is indirectly (e.g., via hybridization) conjugated to the reporter oligonucleotide, which contains a barcode sequence that identifies the labeler. For example, the labeler can be directly conjugated (e.g., covalently bound) to a hybrid oligonucleotide, which contains a sequence that hybridizes with the sequence of the reporter oligonucleotide. Hybridization of the hybrid oligonucleotide with the reporter oligonucleotide conjugates the labeler to the reporter oligonucleotide. In some embodiments, such as upon application of a stimulus, the reporter oligonucleotide can be released from the labeler. For example, the reporter oligonucleotide can be linked to the labeler via an unstable bond (e.g., chemically unstable, light-instable, thermally unstable, etc.), as broadly described elsewhere herein for the release of molecules from a support. In some cases, the reporter oligonucleotides described herein may include one or more functional sequences that can be used for subsequent processing, such as adaptor sequences, unique molecular identifier (UMI) sequences, sequencer-specific flow cell ligation sequences, primers or primer-binding sequences, sequencing primers or primer-binding sequences.

[0146] In some cases, the labeling agent may comprise a reporter oligonucleotide and a tag. The tag may be a fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The tag may be conjugated directly or indirectly to the labeling agent (or reporter oligonucleotide) (or, the tag may be conjugated to a molecule that can bind to the labeling agent or reporter oligonucleotide). In some cases, the tag is conjugated to a first oligonucleotide that is sequence complementary to (e.g., hybridized to) the reporter oligonucleotide.

[0147] In some implementations, multiple different types of analytes (e.g., peptides) from a biological sample can subsequently be associated with one or more physical properties of the biological sample. For example, multiple different types of analytes can be associated with the location of the analytes within the biological sample. Such information (e.g., proteomic information when the analyte-binding moiety recognizes the peptide) can be used in conjunction with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptomic information (e.g., transcript sequences), or both). For example, cell surface proteins can be associated with one or more physical properties of the cell (e.g., cell shape, size, activity, or type). These physical properties can be characterized by cell imaging. Cells can be bound to analyte markers comprising an analyte-binding moiety that binds to a cell surface protein and an analyte-binding moiety barcode that identifies the analyte-binding moiety. Protein analysis results in a sample (e.g., a tissue sample or cells) can be associated with DNA and / or RNA analysis in the sample.

[0148] (iii) Products of endogenous analytes and / or labeling agents

[0149] In some embodiments, this document provides methods and compositions for analyzing one or more products of endogenous analytes and / or markers in biological samples. In some embodiments, the analysis involves endogenous analytes (e.g., viral or cellular DNA or RNA) or their products (e.g., hybridization products, ligation products, extension products (e.g., by DNA or RNA polymerase), replication products, transcription / reverse transcription products, and / or amplification products (such as rolling circle amplification (RCA) products)). In some embodiments, the analysis involves markers that bind directly or indirectly to the analytes in the biological sample. In some embodiments, the analysis involves products of markers that bind directly or indirectly to the analytes in the biological sample (e.g., hybridization products, ligation products, extension products (e.g., by DNA or RNA polymerase), replication products, transcription / reverse transcription products, and / or amplification products (such as rolling circle amplification (RCA) products)).

[0150] (a) hybridization

[0151] In some embodiments, the product of the endogenous analyte and / or labeler is a hybridization product comprising paired, substantially complementary or complementary nucleic acid sequences within two different molecules, one of which is the endogenous analyte or labeler (e.g., a reporter oligonucleotide linked thereto). The other molecule may be another endogenous molecule or another labeler, such as a probe. Pairing can be achieved by any method in which the nucleic acid sequences bind to substantially or completely complementary sequences through base pairing to form a hybridization complex. For hybridization purposes, two nucleic acid sequences are considered "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 each other.

[0152] Various probes and probe sets can hybridize with endogenous analytes and / or labelers, and each probe can contain one or more barcode sequences. Exemplary barcoded probes or probe sets can be based on padlock probes, notched padlock probes, SNAIL (splice nucleotide-assisted intramolecular linkers) S plint N ucleotide A ssisted I ntramolecular L igation)) probe set, PLAYR (RNA proximity assay) P roximity L igation A ssa y for R NA)) probe set and PLISH (near-neighbor in situ hybridization) P roximity L igation i n s itu H Hybridization) probe arrays. Specific probe or probe array designs can vary.

[0153] (b) Connection

[0154] In some embodiments, the product of the endogenous analyte and / or labeler is a ligation product. In some embodiments, a ligation product is formed between two or more endogenous analytes. In some embodiments, a ligation product is formed between an endogenous analyte and a labeler. In some embodiments, a ligation product is formed between two or more labelers. In some embodiments, the ligation product is an intramolecular linker of the endogenous analyte. In some embodiments, the ligation product is an intramolecular linker of the labeler, for example, a circularization of a probe or probe set that can circularize upon hybridization with a target sequence. The target sequence may be contained in an endogenous analyte (e.g., a nucleic acid, such as genomic DNA or mRNA) or its product (e.g., cDNA from cellular mRNA transcripts), or in a labeler (e.g., a reporter oligonucleotide) or its product.

[0155] In some embodiments, this document provides probes or probe sets capable of DNA templated ligation (such as from cDNA molecules). See, for example, U.S. Patent 8,551,710, the entire contents of which are incorporated herein by reference. See, for example, U.S. Patent Publication 2020 / 0224244, the entire contents of which are incorporated herein by reference. In some embodiments, the probe set is a SNAIL probe set. See, for example, U.S. Patent Publication 20190055594, the entire contents of which are incorporated herein by reference. In some embodiments, this document provides multiple proximity ligation assays. See, for example, U.S. Patent Publication 20140194311, the entire contents of which are incorporated herein by reference. In some embodiments, this document provides probes or probe sets capable of proximity ligation, such as proximity ligation assays of RNA (e.g., PLAYR) probe sets. See, for example, U.S. Patent Publication 20160108458, the entire contents of which are incorporated herein by reference. In some embodiments, circular probes can hybridize indirectly with target nucleic acids. In some embodiments, the circular construct is formed from a probe set capable of neighbor-to-neighbor ligation (e.g., a neighbor-to-neighbor in situ hybridization (PLISH) probe set). See, for example, U.S. Patent Publication 2020 / 0224243, the entire contents of which are incorporated herein by reference.

[0156] In some embodiments, the ligation involves chemical ligation. In some embodiments, the ligation involves template-dependent ligation. In some embodiments, the ligation involves template-free ligation. In some embodiments, the ligation involves enzymatic ligation.

[0157] In some embodiments, enzymatic ligation involves the use of a ligase. In some aspects, the ligases used herein include enzymes commonly used to join polynucleotides together or to join a single polynucleotide end. RNA ligases, DNA ligases, or another type of ligase can be used to join two nucleotide sequences together. Ligases include ATP-dependent double-stranded polynucleotide ligases, NAD+-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, such as any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), and EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases (such as Escherichia coli DNA ligase), Tth DNA ligase, and Thermococcus species (strain 9°N) DNA ligase (9°N). TM DNA ligase (New England Biolabs), Taq DNA ligase, Ampligase TM (Epicentre Biotechnologies) and phage ligases (such as T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase) and their mutants. 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 with DNA clamping DNA ligase activity. In some embodiments, the ligase is a ligase with RNA clamping DNA ligase activity.

[0158] In some embodiments, the ligation described herein is direct ligation. In some embodiments, the ligation described herein is indirect ligation. "Direct ligation" means that the ends of the polynucleotides hybridize adjacently to form a substrate for the ligase, thereby causing them to join together (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize not adjacently, for example, separated by one or more insert nucleotides or "gaps". In some embodiments, the ends do not join directly to each other, but occur through one or more inserts (so-called "gaps" or "gaps-filling" (oligonucleotides)) or by "filling" the "gaps" corresponding to the insert nucleotides via the 3' end of an extended probe (intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides can be "filled" by one or more "gaps" (oligonucleotides) complementary to a splint, padlock probe, or target nucleic acid. The gap can be a gap of 1 to 60 nucleotides, a gap of 1 to 40 nucleotides, or a gap of 3 to 40 nucleotides. In specific embodiments, the gap can be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides, or a gap of any integer (or integer range) of nucleotides between the indicated values. In some embodiments, the gap between the terminal regions can be filled by a gapped oligonucleotide or by extending the 3′ end of the polynucleotide. In some cases, ligation involves attaching the end of the probe to at least one gapped (oligo)nucleotide, such that the gapped (oligo)nucleotide is incorporated into the resulting polynucleotide. In some embodiments, gap filling is performed prior to ligation herein. In other embodiments, gap filling is not required for ligation herein.

[0159] In some implementations, the polynucleotide produced by polynucleotide ligation has a higher melting temperature than the unligated polynucleotide. Therefore, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotide prior to subsequent steps, including amplification and detection.

[0160] In some applications, high-fidelity ligases are used, such as thermostable DNA ligases (e.g., Taq DNA ligase). Thermostable DNA ligases are active at elevated temperatures, by operating near the melting temperature of the DNA strand (T0). m Incubation at the specified temperature allows for further differentiation. This selectively reduces the concentration of annealed mismatched substrates compared to annealed substrates with fully base-paired mating (as expected with slightly lower T values ​​around mismatches). m Therefore, high-fidelity ligation can be achieved through a combination of the inherent selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0161] In some embodiments, the ligation described herein is a proximity ligation that joins two (or more) nucleic acid sequences that are adjacent to each other, for example, by enzymatic means (e.g., ligases). In some embodiments, proximity ligation may include a “gap filling” step, which involves incorporating one or more nucleic acids across the distance between two nucleic acid molecules of interest, based on the nucleic acid sequence of the template nucleic acid molecule, using a polymerase (see, for example, U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference). Various different methods can be used for proximity ligation of nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt-end” ligation. Furthermore, single-stranded ligation can be used for proximity ligation on single-stranded nucleic acid molecules. Sticky-end proximity ligation involves hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be ligated prior to the ligation event itself. Blunt-end proximity ligation typically does not include hybridization from complementary regions of each nucleic acid molecule because both nucleic acid molecules lack single-stranded overhangs at the ligation site.

[0162] (c) Primer extension and amplification

[0163] In some embodiments, the product is a primer extension product of an analyte, a labeling agent, a circularizable probe or probe set that binds to the analyte (e.g., a padlock probe that binds to genomic DNA, mRNA, or cDNA), or a circularizable probe or probe set that binds to a labeling agent (e.g., a padlock probe that binds to one or more reporter oligonucleotides from the same or different labeling agents).

[0164] In some embodiments, primers are typically single-stranded nucleic acid sequences with a 3′ end that can be used as substrates for nucleic acid polymerases in nucleic acid extension reactions. RNA primers are formed from RNA nucleotides and used for RNA synthesis, while DNA primers are formed from DNA nucleotides and used for DNA synthesis. Primers may also contain both RNA and DNA nucleotides (e.g., in a random or designed pattern). Primers may also contain other natural or synthetic nucleotides as described herein that may have additional functions. In some instances, DNA primers can be used to initiate RNA synthesis and vice versa (e.g., RNA primers can be used to initiate DNA synthesis). The length of primers can vary. For example, primers can be from about 6 bases to about 120 bases. For example, primers can contain up to about 25 bases. In some cases, primers may be primer-binding sequences. In some embodiments, a primer extension reaction generally refers to any method of joining (e.g., hybridization) two nucleic acid sequences by overlapping their respective terminal complementary nucleic acid sequences (e.g., 3′ ends). Such joining can be followed by one or both-terminal nucleic acid extension (e.g., enzymatic extension) using another nucleic acid sequence as an extension template. Enzymatic extension can be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0165] In some embodiments, the product of the endogenous analyte and / or labeler is an amplification product of one or more polynucleotides (e.g., a circular probe, a circularizable probe, or a probe set). In some embodiments, amplification is achieved by performing rolling circle amplification (RCA). In other embodiments, primers that hybridize to the circular or circularized probe are added and used as is for amplification. In some embodiments, RCA includes linear RCA, branched RCA, dendritic RCA, or any combination thereof.

[0166] In some embodiments, amplification is performed at or between about 20°C and about 60°C. In some embodiments, amplification is performed at or between about 30°C and about 40°C. In some aspects, amplification steps (such as rolling circle amplification (RCA)) are performed at or between about 25°C and about 50°C (such as 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).

[0167] In some embodiments, after the addition of DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, primers are extended to produce multiple copies of a circular template. This amplification step can utilize isothermal or non-isothermal amplification. In some embodiments, after hybridization complex formation and amplification probe binding, the hybridization complex undergoes rolling circle amplification to produce cDNA nanospheres (e.g., amplicons) containing multiple copies of cDNA. Rolling circle amplification (RCA) techniques include linear RCA, branched RCA, dendritic RCA, or any combination thereof. (See, for example, Baner et al., Nucleic Acids Research, 26: 5073-5078, 1998; Lizardi et al., Nature Genetics 19: 226, 1998; Mohsen et al., Acc Chem Res. 2016 November 15; 49(11): 2540-2550; Schweitzer et al., Proc. Natl Acad. Sci. USA 97: 101 13-1 19, 2000; Faruqi et al., BMC Genomics 2: 4, 2000; Nallur et al., Nucl. Acids Res. 29: el 18, 2001; Dean et al., Genome Res. 1 1: 1095-1099, 2001; Schweitzer et al., Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329, and 6,368,801, all of which are incorporated herein by reference in their entirety. Exemplary polymerases for RCA include DNA polymerases, such as phi29. The polymerase may be a Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some embodiments, a DNA polymerase that has been engineered or mutated to have the desired characteristics may be used. In some embodiments, the polymerase is phi29 DNA polymerase.

[0168] In some aspects, modified nucleotides may be added to the reaction during the amplification step to incorporate the modified nucleotides into the amplification product (e.g., nanospheres). Examples of modified nucleotides include amine-modified nucleotides. In some aspects of the method, for example, they may be used to anchor or crosslink the resulting amplification product (e.g., nanospheres) to a scaffold, cellular structure, and / or other amplification products (e.g., other nanospheres). In some aspects, the amplification product contains modified nucleotides, such as amine-modified nucleotides. In some embodiments, the amine-modified nucleotides contain an N-hydroxysuccinimide acrylate moiety. Examples of other amine-modified nucleotides include, but are not limited to, 5-aminoallyl-dUTP moiety modification, 5-propynylamino-dCTP moiety modification, N6-6-aminohexyl-dATP moiety modification, or 7-deazona-7-propynylamino-dATP moiety modification.

[0169] In some aspects, polynucleotides and / or amplification products (e.g., amplicones) can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more polynucleotide probes can be modified to contain functional groups that can serve as anchoring sites for attaching the polynucleotide probes and / or amplification products to the polymer matrix. Exemplary modifications and polymer matrices that may be employed according to the provided embodiments include those described in the following documents: e.g., WO 2014 / 163886, WO 2017 / 079406, US 2016 / 0024555, US 2018 / 0251833, and US 2017 / 0219465, all of which are incorporated herein by reference in their entirety. In some instances, the scaffold also contains modifications or functional groups capable of reacting with or incorporating into modifications or functional groups of the probe set or amplification products. In some instances, the scaffold may contain oligonucleotides, polymers, or chemical groups to provide a matrix and / or support structure.

[0170] Amplification products can be immobilized within the matrix, typically at the site of nucleic acid amplification, thus creating local colonies of amplicones. Amplification products can be immobilized within the matrix by spatial factors. They can also be immobilized via covalent or non-covalent bonds. In this way, the amplification products can be considered to be attached to the matrix. By immobilizing to the matrix, such as through covalent bonds or cross-linking, the size and spatial relationships of the original amplicon are maintained. By immobilizing to the matrix, such as through covalent bonds or cross-linking, the amplification products are resistant to migration or dispersion under mechanical stress.

[0171] In some aspects, amplification products are copolymerized and / or covalently linked to the surrounding matrix, thereby preserving their spatial relationships and any inherent information. For example, if the amplification products are generated from intracellular DNA or RNA embedded in the matrix, the amplification products can also be functionalized to form covalent links with the matrix, preserving their intracellular spatial information and thus providing subcellular localization patterns. In some embodiments, the provided methods involve embedding one or more polynucleotide probe sets and / or amplification products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the described hydrogel-tissue chemistry includes covalently linking nucleic acids to in-situ synthetic hydrogels for tissue clearance, enzyme diffusion, and multi-cycle sequencing, which is not possible with existing hydrogel-tissue chemistry methods. In some embodiments, to enable the amplification products to be embedded in a tissue-hydrogel setting, amine-modified nucleotides are included in the amplification step (e.g., RCA), functionalized with N-hydroxysuccinimide acrylate to an acrylamide moiety, and copolymerized with an acrylamide monomer to form a hydrogel.

[0172] In some implementations, the RCA template may contain a target analyte or a portion thereof, wherein the target analyte is a nucleic acid, or the RCA template may be provided or generated as a substitute for or a label for the analyte. As described above, many assays can be employed to detect many different analytes, said assays using RCA-based detection systems, for example, wherein a signal is provided by generating an RCA product from a circular RCA template provided or generated in the assay, and the RCA product is detected to detect the analyte. Thus, the RCA product can be considered a reporter molecule that is detected to detect the target analyte. However, the RCA template can also be considered a reporter molecule for the target analyte; the RCA product is generated based on the RCA template and contains a complementary copy of the RCA template. The RCA template determines the signal that is detected and thus indicates the target analyte. As will be described in more detail below, the RCA template may be a probe or a portion or component of a probe, or may be generated by a probe, or may be a component of the detection assay (e.g., a reagent in the detection assay) that serves as a reporter molecule for the assay, or a portion of a reporter molecule, or a signal generation system. Therefore, the RCA template used to generate the RCA product can be a circular (e.g., circularized) reporter nucleic acid molecule, i.e., from any RCA-based assay that uses or generates a circular nucleic acid as the reporter molecule for the assay.

[0173] In some embodiments, the products herein comprise molecules or complexes generated in a series of reactions, such as hybridization, ligation, extension, replication, transcription / reverse transcription, and / or amplification (e.g., rolling circle amplification) in any suitable combination. For example, a product containing a target sequence of a probe disclosed herein may be a hybridization complex formed from cellular nucleic acids in a sample and an exogenously added nucleic acid probe. The exogenously added nucleic acid probe may contain overhangs that do not hybridize with cellular nucleic acids but hybridize with another probe. The exogenously added nucleic acid probe may optionally be ligated to a cellular nucleic acid molecule or another exogenous nucleic acid molecule. In other instances, a product containing a target sequence of a probe disclosed herein may be an RCA product of a circularizable probe or probe set that hybridizes with a cellular nucleic acid molecule (e.g., genomic DNA or mRNA) or its product (e.g., a transcript such as cDNA, or a templated ligation product of two probes). In other instances, a product containing a target sequence of a probe disclosed herein (e.g., a detectably labeled probe) may be a probe that hybridizes with an RCA product (e.g., an intermediate probe such as an L-shaped probe). The probe (e.g., an intermediate probe) may include a protruding end that does not hybridize with the RCA product but hybridizes with another probe. The probe may optionally be attached to a cellular nucleic acid molecule or another probe, such as an anchoring probe that hybridizes with the RCA product.

[0174] C. Target sequence

[0175] The target sequence of the probes disclosed herein may be included in any analyte disclosed herein, including endogenous analytes (e.g., viral or cellular nucleic acids), markers, or products of endogenous analytes and / or markers.

[0176] In some respects, one or more of the target sequences contain one or more barcodes, such as at least two, three, four, five, six, seven, eight, nine, ten, or more barcodes. Barcodes can spatially resolve molecular components found in biological samples, such as those within cell or tissue samples. Barcodes can be reversibly or irreversibly linked to an analyte or another part or structure. Barcodes can be added to fragments of samples, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), before or during sample sequencing. Barcodes can allow for the identification and / or quantification of individual sequencing reads (e.g., barcodes can be or may include a unique molecular identifier or "UMI"). In some respects, barcodes contain approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides.

[0177] In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode may contain two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences. In some embodiments, one or more barcodes may also provide a platform for targeting functions, such as oligonucleotides, oligonucleotide-antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functions, and / or enzymes for the detection and identification of polynucleotides.

[0178] In any of the foregoing embodiments, any suitable method or technique may be used to analyze (e.g., detect or sequence) barcodes (e.g., primary and / or secondary barcode sequences), including those described herein, such as target RNA sequence detection (RNA SPOT), sequential fluorescence in situ hybridization (seqFISH), single-molecule fluorescence in situ hybridization (smFISH), multiple error robust fluorescence in situ hybridization (MERFISH), in situ sequencing, hybridization-based in situ sequencing (HybISS), targeted in situ sequencing, fluorescence in situ sequencing (FISSEQ), sequencing by synthesis (SBS), ligation sequencing (SBL), hybridization sequencing (SBH), or spatial resolution transcript amplicon read mapping (STARmap). In any of the foregoing embodiments, the methods provided herein may include analyzing barcodes by sequential hybridization and detection with multiple labeled probes (e.g., detecting oligomers).

[0179] In some implementations, in barcode sequencing methods, the barcode sequence is detected to identify other molecules containing nucleic acid molecules (DNA or RNA) that are longer than the barcode sequence itself, as opposed to directly sequencing longer nucleic acid molecules. In some implementations, given an N-base sequencing read, the N-mer barcode sequence contains 4... N The complexity of barcode sequencing, and the fact that molecular identification may require much shorter sequencing reads compared to non-barcode sequencing methods such as direct sequencing. For example, a 5-nucleotide barcode sequence can identify 1024 molecular species (4... 5 =1024), and an 8-nucleotide barcode can be used to identify up to 65,536 molecular species, a number greater than the total number of different genes in the human genome. In some implementations, the detection of the barcode sequence contained in the probe or RCA product, rather than the endogenous sequence, can be a valid read for the information of each sequencing cycle. Because the barcode sequences are predetermined, they can also be designed to characterize error detection and correction mechanisms, see, for example, US Patent Publication 20190055594 and US Patent Publication. 20210164039 The entire text is quoted here.

[0180] III. Nucleic Acid Probes

[0181] In some aspects, this document discloses nucleic acid probes and / or probe sets that are introduced into cells or used to otherwise contact biological samples, such as tissue samples. Probes may comprise any of a variety of entities that can typically hybridize with nucleic acids via Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc. Nucleic acid probes typically contain a targeting sequence capable of binding directly or indirectly to at least a portion of a target nucleic acid. For example, nucleic acid probes are circular nucleic acids or are used to generate circular nucleic acids containing hybridization regions complementary to polynucleotides (e.g., amplification primers). Nucleic acid probes may be capable of binding to specific target nucleic acids (e.g., mRNA or other nucleic acids as discussed herein). In some embodiments, nucleic acid probes can be detected using detectable markers and / or by using secondary nucleic acid probes capable of binding to the nucleic acid probe. In some embodiments, nucleic acid probes (e.g., primary probes and / or secondary probes) are compatible with one or more biological and / or chemical reactions. For example, the nucleic acid probes disclosed herein can serve as templates or primers for polymerases, templates or substrates for ligases, substrates for click chemistry reactions, and / or substrates for nucleases (e.g., endonucleases or exonucleases for cleavage or digestion).

[0182] In some embodiments, more than one type of primary nucleic acid probe may be contacted with the sample, e.g., simultaneously or in any suitable order, such as in a sequential probe hybridization / dehybridization cycle. In some embodiments, the primary probe may include circular probes and / or circularizable probes (such as padlock probes). In some embodiments, more than one type of secondary nucleic acid probe may be contacted with the sample, e.g., simultaneously or in any suitable order, such as in a sequential probe hybridization / dehybridization cycle. In some embodiments, the secondary probe may include an intermediate probe that binds to the product of the primary probe targeting the analyte (e.g., an RCA product). In some embodiments, more than one type of higher-order nucleic acid probe may be contacted with the sample, e.g., simultaneously or in any suitable order, such as in a sequential probe hybridization / dehybridization cycle. In some embodiments, more than one type of detectably labeled nucleic acid probe may be contacted with the sample, e.g., simultaneously or in any suitable order, such as in a sequential probe hybridization / dehybridization cycle. In some embodiments, detectably labeled probes may include probes that bind to one or more primary probes, one or more secondary probes, one or more higher-order probes, one or more intermediate probes between primary / secondary / higher-order probes, and / or one or more detectably or non-detectably labeled probes (e.g., in the case of hybridization chain reaction (HCR), branched DNA reaction (bDNA), etc.). 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 1,000, at least 3,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 250,000, at least 500,000, or at least 1,000,000 distinguishable nucleic acid probes (e.g., primary, secondary, higher-order probes, and / or detectably labeled probes) may be contacted with the sample, for example, simultaneously or in any suitable sequential order. Between any probe contact steps disclosed herein, the method may include one or more intermediate reaction and / or processing steps, such as modification of the target nucleic acid, modification of the probe or its product (e.g., by hybridization, ligation, extension, amplification, cleavage, digestion, branching migration, primer exchange reaction, click chemistry reaction, crosslinking, attachment of a detectable label, activation of the photoreactive portion, etc.), removal of the probe or its product (e.g., cleaving a portion of the probe and / or dehybridizing the entire probe), signal modification (e.g., quenching, masking, photobleaching, signal enhancement (e.g., by FRET), signal amplification, etc.), signal removal (e.g., cleaving or permanently inactivating a detectable label), crosslinking, decrosslinking, and / or signal detection.

[0183] The target binding sequence of a probe (sometimes also referred to as the target region / sequence or recognition region / sequence) can be located anywhere within the probe. For example, the target binding sequence of a primary probe that binds to a target nucleic acid can be located at the 5' or 3' position of any barcode sequence within the primary probe. Similarly, the target binding sequence of a secondary probe (which binds to the primary probe or its complementary sequence or product) can be located at the 5' or 3' position of any barcode sequence within the secondary probe. In some embodiments, the target binding sequence may comprise a sequence substantially complementary to a portion of the target nucleic acid. In some embodiments, said portion 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.

[0184] The target binding sequence of a primary nucleic acid probe can be determined by referencing the target nucleic acid present or suspected to be present in the sample (e.g., cellular RNA or reporter oligonucleotides of markers for cellular analytes). In some embodiments, more than one target binding sequence can be used to identify a specific analyte containing or associated with a target nucleic acid. The more than one target binding sequence can be in the same probe or in different probes. For example, multiple probes can be used sequentially and / or simultaneously, which can bind to different regions of the same target nucleic acid (e.g., hybridization). In other instances, the probe can contain target binding sequences that can bind to different target nucleic acid sequences, such as various intron and / or exon sequences of the same gene (e.g., to detect splice variants), or sequences of different genes, for example, to detect products containing said different target nucleic acid sequences, such as genomic rearrangements (e.g., inversions, transposations, translocations, insertions, deletions, duplications, and / or amplifications).

[0185] After contacting the nucleic acid probe with the sample, the probe can be detected directly by determining a detectable label (if present), and / or detected by using one or more other probes that bind directly or indirectly to the probe or its product. The one or more other probes may contain a detectable label. For example, a primary nucleic acid probe can bind to a target nucleic acid in the sample, and a secondary nucleic acid probe can be introduced to bind to the amplification product of the primary nucleic acid probe, wherein the secondary nucleic acid probe or its product can then be detected using a detectably labeled probe. Higher-order probes that bind directly or indirectly to the secondary nucleic acid probe or its product can also be used, and the higher-order probe or its product can then be detected using a detectably labeled probe.

[0186] In some implementations, detection can be spatial, e.g., two-dimensional or three-dimensional. In some implementations, detection can be quantitative, e.g., determining the amount or concentration of the primary nucleic acid probe (and target nucleic acid). In some implementations, depending on the application, the primary probe, secondary probe, higher-order probe, and / or detectably labeled probe can comprise any of a variety of entities capable of hybridizing with nucleic acids, e.g., DNA, RNA, LNA, and / or PNA, etc.

[0187] Secondary nucleic acid probes (e.g., intermediate probes) may contain a recognition sequence capable of binding or hybridizing with a primary nucleic acid probe or its product, for example, at a barcode sequence or portion thereof of the primary nucleic acid probe, or at a complementary sequence or portion thereof (e.g., in the case of hybridization between the secondary probe and the RCA product of the primary probe). In some embodiments, the secondary nucleic acid probe may bind to a combination of barcode sequences (which may be consecutive or spaced apart) in the primary nucleic acid probe or its product. In some embodiments, binding is specific, or binding may cause the recognition sequence to preferentially bind or hybridize with only one of the present barcode sequences or their complementary sequences. The secondary nucleic acid probe may also contain one or more detectable markers. If more than one secondary nucleic acid probe is used, the detectable markers may be the same or different.

[0188] The recognition sequence can have any length, and multiple recognition sequences in the same or different secondary nucleic acid probes can have the same or different lengths. If more than one recognition sequence is used, the recognition sequences can independently have the same or different lengths. For example, the length of the recognition sequence can be at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 nucleotides. In some embodiments, the length of the recognition sequence can not exceed 48, 40, 32, 24, 16, 12, 10, 8, or 6 nucleotides. Any combination of these is also possible; for example, the recognition sequence can have a length between 5 and 8 nucleotides, between 6 and 12 nucleotides, or between 7 and 15 nucleotides, etc. In one embodiment, the recognition sequence has the same length as the barcode sequence or complementary sequence of the primary nucleic acid probe or its product. In some implementations, the identification sequence may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to the barcode sequence or its complementary sequence.

[0189] In some embodiments, the nucleic acid probe, such as a primary or secondary nucleic acid probe, may also contain 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 can be located anywhere within the nucleic acid probe. If more than one barcode sequence is present, the barcode sequences can be positioned adjacent to each other and / or interspersed with other sequences. In some embodiments, two or more barcode sequences may at least partially overlap. In some embodiments, two or more barcode sequences in the same probe do not overlap. In some embodiments, all barcode sequences in the same probe are separated from each other by at least one phosphodiester bond (e.g., they may be adjacent to each other but not overlapping), such as separating 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0190] If present, the barcode sequence can have any length. If more than one barcode sequence is used, the barcode sequences can 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, or at least 50 nucleotides. In some embodiments, the length of the barcode sequence can not exceed 120, 112, 104, 96, 88, 80, 72, 64, 56, 48, 40, 32, 24, 16, or 8 nucleotides. Any combination of these is also possible; for example, the barcode sequence can be between 5 and 10 nucleotides, between 8 and 15 nucleotides, etc.

[0191] The barcode sequence can be arbitrary or random. In some cases, the barcode sequence is chosen to reduce or minimize homology with other components in the sample, for example, such that the barcode sequence itself does not bind or hybridize with other nucleic acids suspected of being present in cells or other samples. In some embodiments, the homology between a particular barcode sequence and another sequence (e.g., a cellular nucleic acid sequence in the sample or other barcode sequences added to a probe in the sample) can 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 can 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.

[0192] In some implementations, the number of distinct barcode sequences in a population of nucleic acid probes is less than the number of distinct targets of the nucleic acid probes (e.g., nucleic acid analytes and / or protein analytes), but the distinct targets can still be uniquely identified from one another, for example, by encoding the probes with different combinations of barcode sequences. However, it is not necessary to use all possible combinations of a given set of barcode sequences. For example, each probe may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more barcode sequences. In some implementations, the population of nucleic acid probes may each contain the same number of barcode sequences, but in other cases, different numbers of barcode sequences may be present on various probes.

[0193] As an illustrative example, a first probe may contain a first target-binding sequence, a first barcode sequence, and a second barcode sequence, while a second, different probe may contain a second target-binding sequence (different from the first target-binding sequence in the first probe), the same first barcode sequence as the first probe, but a third barcode sequence instead of the second barcode sequence. Thus, such probes can be distinguished by identifying the various combinations of barcode sequences present at a given location in the sample or associated with a given probe.

[0194] In some embodiments, the nucleic acid probes disclosed herein can be prepared using only two or only three of the four bases, for example, by excluding all "G"s and / or all "C"s from the probe. Sequences lacking "G"s or "C"s can form very small secondary structures and, in some embodiments, can facilitate more uniform and faster hybridization.

[0195] In some embodiments, the nucleic acid probes disclosed herein may contain detectable markers such as fluorophores. In some embodiments, one or more of the multiple nucleic acid probes used in the assay may lack a detectable marker, while one or more other probes of the multiple nucleic acid probes each contain a limited pool of detectable markers selected from different detectable markers (e.g., red, green, yellow, and blue fluorophores), and the absence of a detectable marker can be used as a separate "color". Therefore, a detectable marker is not required in all cases. In some embodiments, the primary nucleic acid probes disclosed herein (e.g., padlock probes) lack a detectable marker. While a detectable marker can be incorporated into the amplification product of the primary nucleic acid probe, such as by incorporating modified nucleotides into the RCA product of the padlock probe, in some embodiments, the amplification product is not detectably labeled. In some embodiments, probes that bind to the primary nucleic acid probe or its product (e.g., secondary nucleic acid probes that bind to a barcode sequence or its complementary sequence in the primary nucleic acid probe or its product) contain a detectable marker and can be used to detect the primary nucleic acid probe or its product. In some embodiments, the secondary nucleic acid probes disclosed herein lack detectable labels, and the secondary nucleic acid probes or their products can be detected using detectably labeled probes (e.g., at barcode sequences or their complementary sequences in the secondary nucleic acid probes or their products) that bind to them. In some embodiments, signals associated with detectably labeled probes can be used to detect one or more barcode sequences in the secondary probes and / or one or more barcode sequences in the primary probes, for example, by sequential hybridization, sequencing-by-ligation, and / or sequencing-by-hybridization using detectably labeled probes. In some embodiments, barcode sequences (e.g., in the secondary probes and / or in the primary probes) are used to encode multiple analytes of interest in combination. Therefore, signals associated with detectably labeled probes at specific locations in a biological sample can be used to generate distinct signal signatures, each corresponding to an analyte in the sample, thereby identifying the analyte at that specific location, for example, for in-situ spatial analysis of the sample.

[0196] In some embodiments, the nucleic acid probes described herein contain one or more other components, such as one or more primer-binding sequences (e.g., to allow enzymatic amplification of the probe), enzyme recognition sequences (e.g., for endonuclease cleavage), etc. The components of the nucleic acid probe can be arranged in any suitable order.

[0197] In some aspects, the analyte is targeted by a primary probe, which is barcoded by incorporating one or more barcode sequences (e.g., sequences that can be detected or otherwise "read") separate from the sequence in the primary probe that directly or indirectly binds the targeted analyte. In some aspects, the primary probe is further targeted by a secondary probe, which is also barcoded by incorporating one or more barcode sequences separate from the identification sequence in the secondary probe that directly or indirectly binds the primary probe or its product. In some embodiments, the secondary probe may bind to the barcode sequence in the primary probe. In some embodiments, the secondary probe may bind to a complementary sequence of the barcode sequence in the RCA product of the primary probe. In some aspects, tertiary probes and optionally even higher-order probes can be used to target the secondary probe, for example, at the barcode sequence or its complementary sequence in the secondary probe or its product. In some embodiments, the tertiary probe and / or even higher-order probes may comprise one or more barcode sequences and / or one or more detectable tags. In some embodiments, the tertiary probe is a detectably labeled probe that hybridizes with the barcode sequence (or its complementary sequence) of the secondary probe (or its product). In some embodiments, the location and identity of one or more analytes in a sample can be determined by detecting signals associated with a detectably labeled probe in the sample. In some embodiments, the presence / absence, absolute or relative abundance, amount, level, concentration, activity, and / or relationship with another analyte of a particular analyte can be analyzed in situ within the sample.

[0198] In some implementations, this document provides probes, probe sets, and assay methods that combine target nucleic acid detection, signal amplification (e.g., hybridization of nucleic acid amplification such as RCA and / or multiple detectably labeled probes, such as in hybridization chain reaction, etc.) and barcode decoding.

[0199] In some aspects, primary probes, secondary probes, and / or higher-order probes may be selected from circular probes, circularizable probes, and linear probes. In some embodiments, circular probes may be probes that are pre-circularized before hybridization with the target nucleic acid and / or one or more other probes. In some embodiments, circularizable probes may be probes that can be circularized upon hybridization with the target nucleic acid and / or one or more other probes, such as splices. In some embodiments, linear probes may be probes containing a target recognition sequence and a sequence that does not hybridize with the target nucleic acid, such as 5' protrusions, 3' protrusions, and / or adapters or spacers (which may contain nucleic acid sequences or non-nucleic acid portions). In some embodiments, sequences (e.g., 5' protrusions, 3' protrusions, and / or adapters or spacers) do not hybridize with the target nucleic acid but may hybridize with each other and / or with one or more other probes, such as detectably labeled probes.

[0200] Specific probe designs can vary depending on the application. For example, the primary, secondary, and / or higher-order probes disclosed herein may include padlock probes that require gap filling to circularize when hybridizing with a template (e.g., a target nucleic acid and / or a probe such as a clip), gap-filled padlock probes (e.g., probes that require gap filling to circularize when hybridizing with a template), L-shaped probes (e.g., probes containing a target recognition sequence and a 5' or 3' protrusion when hybridizing with a target nucleic acid or probe), U-shaped probes (e.g., probes containing a target recognition sequence, a 5' protrusion, and a 3' protrusion when hybridizing with a target nucleic acid or probe), V-shaped probes (e.g., probes containing at least two target recognition sequences and a linker or spacer between the target recognition sequences when hybridizing with a target nucleic acid or probe), probes or probe sets for proximity ligation (such as those described in US 7,914,987 and US 8,580,504, the full text of which is incorporated herein by reference, and probes for proximity ligation assays (PLA) to simultaneously detect and quantify nucleic acid molecules and protein-protein interactions), or any suitable combination thereof. In some embodiments, the primary, secondary, and / or higher-order probes disclosed herein may include probes ligated to themselves or another probe using DNA templated and / or RNA templated ligation. In some embodiments, the primary, secondary, and / or higher-order probes disclosed herein may be DNA molecules and may contain one or more other types of nucleotides, modified nucleotides, and / or nucleotide analogs, such as one or more ribonucleotides. In some embodiments, the ligation may be a DNA ligation on a DNA template. In some embodiments, the ligation may be a DNA ligation on an RNA template, and the probe may include an RNA-templated ligation probe. In some embodiments, the primary, secondary, and / or higher-order probes disclosed herein may include padlock-like probes or probe sets. In some embodiments, the nucleic acid probes disclosed herein are SNAILs (… folder plate nuclear Glycolic acid auxiliary help point This is part of a probe suite (internal linker), such as the probe suite described in US 2019 / 0055594 or US 2021 / 0164039, the full text of which is incorporated herein by reference. In some embodiments, the nucleic acid probes disclosed herein are PLAYR ( R NA's adjacent close even catch Test This document describes a probe set, such as the probe set described in US 2016 / 0108458, the full text of which is incorporated herein by reference. In some embodiments, the nucleic acid probes disclosed herein are PLISH (… Original Bit miscellaneous In the middle adjacent close even(Continued) This refers to a probe assembly, such as the probe assembly described in US 2020 / 0224243, the entire contents of which are incorporated herein by reference. Any suitable combination of probe designs described herein may be used.

[0201] RCA templates for generating RCA products can be generated using any suitable cyclizable probe or probe set, or more generally, cyclizable reporter molecules. For example, cyclizable probes or probe sets can be used to generate circular nucleic acids containing a target hybridization region. The target hybridization region in the RCA product can contain an identifying sequence, such as the sequence of the nucleic acid analyte or probe with which the cyclizable probe or probe set hybridizes, or a barcode sequence. The cyclizable probe or reporter (RCA template) can be in the form of a linear molecule with linkable ends, which can be cyclized by directly or indirectly linking the ends together (e.g., linking them to each other or to the corresponding ends of an intermediate (“gap”) oligonucleotide or to an extended 3’ end of the cyclizable RCA template). The cyclizable template can also be provided as two or more parts, i.e., two or more molecules (e.g., oligonucleotides), which can be linked together to form a loop. When the RCA template is cyclizable, it is cyclized by linking before the RCA. Linking templates can be used to template the linking, and in the case of padlock probes and molecular inversion probes, the target analyte can provide the linking template, or it can be provided alone. The circulable RCA template (or a portion or part of the template) will contain complementarity at its respective 3' and 5' end regions with corresponding homologous complementary regions (or binding sites) in the connecting template. These complementary regions may be adjacent to or not adjacent to the places where the ends are directly connected to each other, having an intermediate "gap" sequence where indirect connection will occur.

[0202] In one embodiment, the ends of a padlock probe can be brought close together by hybridization with adjacent sequences on a target nucleic acid molecule (such as a target analyte), which acts as a linking template, allowing the ends to join together to form a circular nucleic acid molecule, thus allowing the circularized padlock probe to serve as a template for an RCA reaction. In such an example, the terminal sequences of the padlock probes hybridizing with the target nucleic acid molecule are specific to the target analyte in question and will be repeatedly replicated in the RCA product. Therefore, they can serve as marker sequences indicating the target analyte. Thus, it can be seen that the marker sequences in the RCA product can be equivalent to the sequences present in the target analyte itself. Alternatively, marker sequences (e.g., tags or barcode sequences) can be provided in the non-target complementary portion of the padlock probe. In a further embodiment, the marker sequences can be present in interstitial oligonucleotides hybridizing between the respective hybridization ends of the padlock probes, where they hybridize with non-adjacent sequences in the target molecule. Such interstitial padlock probes are similar to molecular inverted probes.

[0203] In some embodiments, molecular inverted probes can be used to generate similar circular RCA template molecules. Like padlock probes, these are typically linear nucleic acid molecules capable of hybridizing with and circularizing target nucleic acid molecules (such as target analytes). The two ends of the molecular inverted probe can hybridize with the target nucleic acid molecule at sites close to but not directly adjacent to each other, creating a gap between the two ends. In some embodiments, the size of this gap can range from a single nucleotide in some embodiments to larger gaps of 100 to 500 nucleotides or longer in others. Therefore, a polymerase and nucleotide source or additional gap-filling oligonucleotides are required to fill the gap between the two ends of the molecular inverted probe so that it can be circularized.

[0204] Similar to padlock probes, the terminal sequences of molecular inverted probes that hybridize with the target nucleic acid molecule, as well as the sequences between them, will be specific to the target analyte in question and will be repeatedly replicated in the RCA product. Therefore, they can serve as labeled sequences indicating the target analyte. Alternatively, labeled sequences (e.g., tags or barcode sequences) can be provided in the non-target complementary portion of the molecular inverted probe.

[0205] In some embodiments, the probes disclosed herein can be intrusive probes, for example, to generate circular nucleic acids such as circularized probes. Such probes are particularly useful in the detection of single nucleotide polymorphisms. Therefore, the detection methods disclosed herein can be used to detect single nucleotide polymorphisms or virtually any variant bases in a target nucleic acid sequence. Probes used in such methods can be designed such that the 3' linkable end of the probe is complementary to and hybridizes with a nucleotide in the target molecule of interest (variant nucleotide), and the nucleotide at the 3' end of a 5' appended sequence at the 5' end of the probe, or at the 5' end of another different probe portion, is complementary to the same nucleotide but is prevented from hybridizing through the 3' linkable end (e.g., which is a transposed nucleotide). Cutting the probe to remove the appended sequence will provide a 5' linkable end, which can be linked to the probe or the 3' linkable end of the probe portion if the 3' linkable end hybridizes correctly (e.g., is complementary to the target nucleic acid molecule). Probes designed according to this principle provide a high degree of differentiation between different variants at the site of interest because only probes in which the 3' linkable end is complementary to the nucleotide at the site of interest can participate in the ligation reaction. In one implementation, the probe is provided as a single part, and the 3' and 5' connectable ends are provided by the same probe. In some implementations, the intrusion probe is a padlock probe (intrusion padlock or "iLock"), as described, for example, in Krzywkowski et al., Nucleic Acids Research 45, e161, 2017 and US 2020 / 0224244, which are incorporated herein by reference.

[0206] Other types of probes that generate circular molecules detectable by RCA and contain a target analyte sequence or its complement include selector-type probes as described in US20190144940, which contain a sequence capable of guiding the cleavage of a target nucleic acid molecule (e.g., a target analyte) to release a fragment containing the target sequence from the target analyte and a sequence capable of templated circularization and ligation of the fragment. WO 2016 / 016452, the entire contents of which are incorporated herein by reference, describes probes containing a 3' sequence capable of hybridizing with a target nucleic acid molecule (e.g., a target analyte) and acting as a primer to generate a complement to the target sequence within the target nucleic acid molecule (e.g., target templated extension via a primer) and an internal sequence capable of templated circularization and ligation of the extended probe, the extended probe containing an inverse complement to the target sequence within the target analyte and a portion of the probe. In both cases, the target sequence or its complement is incorporated into a circularized molecule that acts as a template for the RCA reaction to generate an RCA product, which thus contains a tandem repeat of the target sequence. In some embodiments, the target sequence may serve as a marker sequence within or that may contain an RCA product indicating the target analyte in question. Alternatively, a marker sequence (e.g., a tag or barcode sequence) may be provided in the non-target complementary portion of the probe.

[0207] In some embodiments, the nucleic acid probes disclosed herein may be pre-assembled from multiple components, for example, before contacting the nucleic acid probe with the target nucleic acid or sample. In some embodiments, the nucleic acid probes disclosed herein may be assembled during and / or after contacting the target nucleic acid or sample with the multiple components. In some embodiments, the nucleic acid probes disclosed herein are assembled in situ in the sample. In some embodiments, the multiple components may be contacted with the target nucleic acid or sample in any suitable order and in any suitable combination. For example, a first component and a second component may be contacted with the target nucleic acid to allow binding between components and / or binding between the first and / or second components and the target nucleic acid. Optionally, reactions involving either or both of the components and / or the target nucleic acid, such as hybridization, ligation, primer extension and / or amplification, chemical or enzymatic cleavage, click chemistry, or any combination thereof, may be performed between the components and / or between any one or both of the components and the target nucleic acid. In some embodiments, a third component may be added before, during, or after the reaction. In some embodiments, a third component may be added before, during, or after contacting the sample with the first and / or second components. In some embodiments, the first, second, and third components can be contacted with the sample in any suitable combination order or simultaneously. In some embodiments, the nucleic acid probe can be assembled in situ in a stepwise manner, with one or more components added at each step, or in a dynamic process where all components are assembled together. One or more removal steps, such as washing the sample under stringent conditions, can be performed at any point during the assembly process to remove or destabilize undesirable intermediates and / or components at that point and increase the chances of accurate probe assembly and specific target binding of the assembled probe.

[0208] IV. Synchronous in situ rolling circle amplification

[0209] A. Rolling circle amplification (RCA)

[0210] In some embodiments, the probes disclosed herein are amplified via rolling circle amplification. In some embodiments, the primary probe, such as a padlock probe or a probe set including a padlock probe, contains one or more barcodes. For example, the probe or probe set used for amplification (e.g., as described in Part III) is a circular nucleic acid or a circular nucleic acid used to generate a hybridization region containing a target hybridization region and a hybridization region complementary to a polynucleotide. In some embodiments, the barcode is bound by a detection primary probe, which does not need to be fluorescent but includes a target-binding portion (e.g., for hybridization with one or more primary probes) and multiple other barcodes (e.g., secondary barcodes, relative to the primary barcode on the primary probe). In some embodiments, the barcode of the detection primary probe is targeted by a detectably labeled detection oligonucleotide, such as a fluorescently labeled oligonucleotide. In some embodiments, one or more decoding schemes are used to decode the signal, such as fluorescence, for sequence determination. Exemplary decoding schemes are described in Eng et al., “Transcriptome-scale Super-Resolved Imaging in Tissues by RNA SeqFISH+,” Nature 568(7751): 235-239 (2019); Chen et al., “Spatiallyresolved, highly multiplexed RNA profiling in single cells,” Science; 348(6233): aaa6090 (2015); US 10,457,980 B2; US 2016 / 0369329 A1; WO 2018 / 026873 A1; and US2017 / 0220733 A1, all of which are incorporated herein by reference in their entirety. In some implementations, these determinations enable simultaneous signal amplification, combined decoding, and error correction schemes.

[0211] In some embodiments, the method includes generating a circular nucleic acid using a circular or circularizable construct that hybridizes with the nucleic acid of interest. In some embodiments, the RCA includes a linear RCA. In some embodiments, the RCA includes a branched RCA. In some embodiments, the RCA includes a dendritic RCA. In some embodiments, the RCA includes any combination of the foregoing. In some embodiments, the circular nucleic acid is a construct formed using ligation. In some embodiments, a circular construct is formed by extending and then ligating using a template primer. In some embodiments, a circular construct is formed by providing an insert between the ends to be ligated. In some embodiments, a circular construct is formed using any combination 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, the entire contents of which are incorporated herein by reference. In some embodiments, the ligation is an RNA-DNA templated ligation. In some embodiments, a clip is provided as a template for ligation.

[0212] In some embodiments, the probes disclosed herein (e.g., padlock probes) may include a 5' flanking region that can be recognized by a structure-specific cleaving enzyme (e.g., an enzyme capable of recognizing the junction between a single-stranded 5' protrusion and a DNA double helix and cleaving the single-stranded protrusion). It should be understood that the branched triple-stranded structure, as a substrate for the structure-specific cleaving enzyme, can be formed by the 5' end of one probe portion and the 3' end of another probe portion (when both have hybridized with the target nucleic acid molecule) as well as by the 5' and 3' ends of a single-part probe. Enzymes suitable for such cleavage include flanking endonucleases (FENS), which are a class of enzymes with endonuclease activity capable of catalyzing the hydrolytic cleavage of the phosphodiester bond at the junction of single-stranded and double-stranded DNA. Therefore, in some embodiments, cleavage of the additional sequence at the 5' of the first target-specific binding site is performed by a structure-specific cleaving enzyme (e.g., a flanking endonuclease). Suitable flanking endonucleases are described in Ma et al., 2000, JBC 275, 24693-24700 and US 2020 / 0224244, the full text of which is incorporated herein by reference and may include P. furiosus (Pfu), A. fulgidus (Afu), M. jannaschii (Mja), or Mthermoautotrophicum (Mth). In other embodiments, an enzyme capable of recognizing and degrading single-stranded oligonucleotides having a free 5' end can be used to cleave the additional sequence (5' flanking) from the structure as described above. Thus, an enzyme with 5' nuclease activity can be used to cleave the 5' additional sequence. Such 5' nuclease activity can be 5' exonuclease activity and / or 5' endonuclease activity. 5' nucleases are capable of recognizing the free 5' end of a single-stranded oligonucleotide and degrading said single-stranded oligonucleotide. 5' exonucleases degrade single-stranded oligonucleotides having a free 5' end by degrading the oligonucleotide from its 5' end into constituent mononucleotides. 5' endonuclease activity can cleave 5' flanking sequences internally at one or more nucleotides. Once the enzyme recognizes the free 5' end, it crosses the single-stranded oligonucleotide to reach the double-stranded region and cleaves the single-stranded region into larger constituent nucleotides (e.g., dinucleotides or trinucleotides), or cleaves the entire 5' single-stranded region, thus exhibiting 5' nuclease activity, as described, for example, in Lyamichev et al., 1999. PNAS 96, 6143-6148, for Taq DNA polymerase and its 5' nuclease, the full text of which is incorporated herein by reference. Preferred enzymes with 5' nuclease activity include exonuclease VIII, or native or recombinant DNA polymerases derived from *Taq*, *Thermophilus*, or *Agrophytes*, or from their nuclease domains.

[0213] In some cases, amplification primers are added after the circular nucleic acid is formed. In other cases, amplification primers are added together with primary and / or secondary probes. In some embodiments, the circular nucleic acid contains a target hybridization region (e.g., for hybridization with a nucleic acid analyte such as mRNA) and a hybridization region complementary to a polynucleotide (e.g., the amplification primer) or a portion thereof. In some cases, the amplification primers may also be complementary to both the target nucleic acid and a 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 washing is a rigorous wash. The washing step can be performed at any point during the process to remove non-specifically bound probes, ligated probes, etc.

[0214] In some cases, with the addition of DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the amplification primers are extended by the replication of multiple copies of the template. The amplification step can utilize isothermal or non-isothermal amplification. In some embodiments, after the formation of the hybridization complex and any subsequent circularization (e.g., ligation of a padlock probe), the circular probe is circularly amplified to produce an RNA product (e.g., an amplicon) containing multiple circular copies.

[0215] Suitable examples of DNA polymerases that can be used include, but are not limited to: Escherichia coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, and VENT. TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot Start Taq DNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase DNA polymerase, Quick- DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-Fidelity DNA polymerase, Platinum Pfx DNA polymerase, AccuPrimePfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase.

[0216] In some embodiments, rolling circle amplification products are generated using polymerases selected from: 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 (exonuclease-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, the 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 variants or derivatives thereof.

[0217] Following amplification, the sequence of the amplicon (e.g., RCA product) or a portion thereof is determined or otherwise analyzed, for example, by using detectably labeled probes and imaging. Sequencing or analysis of the amplified product may include sequencing-while-hybridizing, sequencing-while-ligating, and / or fluorescence in situ sequencing, and / or where in situ hybridization includes sequential fluorescence in situ hybridization. In some cases, the sequence of the RCA product is detected using, for example, secondary and higher-order probes and detecting oligonucleotides as described herein.

[0218] B. Compositions and methods for synchronizing RCA

[0219] Synchronization of in situ RCA can offer numerous advantages. For example, simultaneous RCA reactions (e.g., simultaneous initiation of the reaction) of circularized probes of analytes targeting different locations within a tissue section can provide RCA products of more uniform size. In some aspects, synchronized RCA reactions can result in signal spots (for RCA products) with uniform signal intensity and / or brightness. In some embodiments, synchronization results in fewer dark signal spots and / or more bright signal spots in the RCA products. In some embodiments, when RCA product size becomes more uniform, amplification time can be reduced, allowing smaller RCA products with sufficient brightness to be detected. In some embodiments, when RCA product size and brightness become more uniform within the same microscope field of view, RCA reaction times can be achieved that result in fewer large signal spots (e.g., even for highly expressed genes) that overlap and / or mask neighboring smaller signal spots, thereby improving optical congestion and allowing adjacent spots to be better resolved from each other. In some embodiments, RCA reaction times can be achieved that result in very few extremely bright signal spots, allowing relatively dark spots to be detected simultaneously with bright spots. In some respects, synchronized RCA reactions can result in at least 40%, 50%, 60%, 70%, or 80% of the RCA products having a signal within 50% of the average size and / or average peak intensity at detection. In some embodiments, RCA reaction times can be achieved that allow for the detection and analysis of as many useful signal spots as possible to accurately detect and quantify (e.g., in situ) multiple analytes present in a biological sample. In some cases, by synchronizing the initiation of RCA in the biological sample, it may be easier to distinguish the RCA signal from background signals (e.g., noise).

[0220] In some aspects, this document provides a method for analyzing a biological sample, comprising: contacting the biological sample with a binding mixture (e.g., a hybridization reaction mixture that allows the multiple complexes to hybridize with the multiple circular nucleic acids). In some embodiments, the biological sample comprises multiple circular nucleic acids, each comprising a primer hybridization region, and the binding mixture comprises multiple complexes, each comprising a polymerase bound to primers, wherein the primers comprise sequences complementary to the primer hybridization regions of one or more circular nucleic acids, and the polymerase activity is inhibited, thereby allowing the multiple complexes to hybridize with the multiple circular nucleic acids. In some embodiments, the method further comprises contacting the biological sample with a primer extension reaction mixture (e.g., an amplification reaction mixture for RCA). In some embodiments, the primer extension reaction mixture allows the polymerase to extend primers hybridizing with the primer hybridization regions, thereby synchronizing rolling circle amplification of the multiple circular nucleic acids in the biological sample.

[0221] In some embodiments, this document provides a method for analyzing target nucleic acids in a biological sample, the method comprising: (a) contacting the biological sample with a circular probe or a circularizable probe or probe set that hybridizes with the target nucleic acid; (b) contacting the biological sample with a binding mixture, wherein the binding mixture comprises a polymerase pre-bound to a polynucleotide, the polynucleotide comprising a sequence complementary to a hybridization region in the circular probe or a circular probe formed from a circularizable probe or probe set, and wherein the activity of the polymerase is inhibited; and (c) contacting the biological sample with a primer extension reaction mixture to initiate polymerase activity, wherein the polynucleotide is used as a primer to generate rolling circle amplification products of the circular or circularized probe in the biological sample.

[0222] For example, such as Figure 1A As shown, a complex containing a polymerase such as Phi29 and primers can be pre-formed before contacting the complex with a buffer (OFF buffer for RCA) that will stabilize the complex but inhibit one or more activities of the polymerase, such as polymerase activity and / or exonuclease activity. In some aspects, the pre-formed complex containing the polymerase and primers can be provided as a unit to the template (e.g., circular nucleic acid in a biological sample) for extension. In some embodiments, the complex is contacted with a circular or circularized template, and RCA is then simultaneously initiated by adding an RCA reaction buffer (ON buffer for RCA) that relieves inhibition of the polymerase and / or exonuclease activity of the enzyme. In some embodiments, multiple circular nucleic acids are bound to multiple target nucleic acids at various locations in the biological sample prior to rolling circle amplification.

[0223] In some aspects, this document provides a method for analyzing biological samples, comprising: contacting the biological sample with a binding mixture, wherein the biological sample contains multiple circular nucleic acids, each containing a hybridization region that hybridizes with a polynucleotide, the binding mixture containing a polymerase, and the polymerase activity of the polymerase being inhibited to allow the polymerase to bind to the multiple circular nucleic acids; and contacting the biological sample with a primer extension reaction mixture to allow the polymerase to extend the polynucleotides hybridized with the hybridization region, thereby synchronizing rolling circle amplification of the multiple circular nucleic acids in the biological sample.

[0224] In some embodiments, a complex comprising a polymerase, a circular nucleic acid, and a polynucleotide is formed in a biological sample. For example, the polynucleotide may contain a sequence complementary to a portion of the circular nucleic acid. In some embodiments, the polynucleotide may contain exogenous primers added to the sample. For example, such as… Figure 1BAs shown, primers can hybridize to circular or circularized probes at different locations in the sample, which are then contacted with a polymerase (e.g., Phi29) in an OFF buffer that inhibits polymerase activity. The polymerase can diffuse throughout the sample and bind to the primers hybridized to the circular or circularized probes, but RCA remains blocked until unbound polymerase is removed and an ON buffer is applied to initiate RCA at different locations. In these examples, circular nucleic acids can include endogenous molecules in the biological sample, such as endogenous circular DNA or RNA molecules in cells, such as mtDNA. Alternatively, circular nucleic acids can include products of endogenous molecules in the biological sample, such as circularized molecules derived from genomic DNA, cellular RNA such as mRNA, or circularized cDNA. In other cases, circular nucleic acids can include probes targeting endogenous molecules in the biological sample, such as padlock probes targeting genomic DNA or cellular RNA such as mRNA. In still other cases, circular nucleic acids can include exogenous probes targeting endogenous molecules in the biological sample. In any of the foregoing embodiments, the circular nucleic acid can be hybridized with one or more RCA products to generate additional RCA products using the circular nucleic acid as a template.

[0225] In some embodiments, the polynucleotide may comprise an endogenous molecule (e.g., a target nucleic acid) in a biological sample, such as genomic DNA or cellular RNA such as mRNA. In some embodiments, the polynucleotide may comprise cDNA. For example, as Figure 1CAs shown, a polymerase in OFF buffer can be applied to the sample to bind to the 3′ end of a polynucleotide (e.g., RNA or DNA, such as mRNA or cDNA) in the sample, and the polymerase loaded onto the polynucleotide can be activated with ON buffer to initiate a simultaneous RCA reaction using the polynucleotide as a primer and a circular or circularized probe as a template. In some embodiments, the polynucleotide is RNA such as mRNA, the circular or circularized probe hybridizing to the RNA contains DNA, and the RNA polynucleotide is used to initiate an RCA reaction using a circular or circularized DNA template. In some embodiments, the polynucleotide may include the product of an endogenous molecule in the biological sample, such as a ligation, cleavage, or amplification product of genomic DNA or cellular RNA such as mRNA. In some embodiments, the polynucleotide may include a probe targeting an endogenous molecule in the biological sample, such as an L-shaped probe or a U-shaped probe. In these examples, the circular nucleic acid can hybridize with a polynucleotide used as a primer for the RCA, for example, similar to the RCA-smFISH (RollFISH) combinatorial method described by Wu et al., CommunBiol 1:209 (2018), the full text of which is incorporated herein by reference. In some embodiments, the polynucleotide may comprise the product of an exogenous probe targeting an endogenous molecule in a biological sample. For example, the polynucleotide itself may be an RCA product, and a circular or cyclic probe may be hybridized with this RCA product to generate another RCA product using the circular or cyclic probe as a template.

[0226] The conjugation mixture or OFF buffer can stabilize and / or inhibit polymerase activity, such as polymerase activity and / or nuclease activity. The conjugation mixture may contain one or more deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs). For example, the conjugation mixture may contain dATP, dTTP, dCTP, and / or dGTP. Alternatively, the conjugation mixture may be substantially free of dNTPs and / or NTPs. In some embodiments, the conjugation mixture may contain a divalent cation that is not a cofactor of the polymerase, such as Ca2+. 2+ It can stabilize polymerase without activating its polymerase activity and / or exonuclease activity.

[0227] In some embodiments, the binding mixture or OFF buffer or biological sample may be substantially incompatible with the polymerase cofactor. A cofactor is a non-protein chemical compound or metal ion required for the enzyme's activity as a catalyst. DNA and ribonucleic acid (RNA) polymerases typically require divalent or trivalent metal cofactor cations to catalyze the polymerization of individual nucleotides into polynucleotides. In some embodiments described herein, the presence / absence of a specific divalent cation can be used to alter the polymerase kinetics. Without a metal cofactor in the appropriate oxidation state, polymerization will not occur at a perceptible rate, even in the presence of all other necessary components. Metal cofactor cations may include Co. 2+ Mn 2+ 、Zn 2+ and / or Mg 2+ Exemplary cofactor cations are disclosed in Vashishtha et al., J Biol Chem 291(40):20869-20875, 2016; US 2021 / 0047669; US Patent Nos. 5,409,811; 8,133,672; 8,658,365; and 9,279,155, all of which are incorporated herein by reference. Metallic cofactors may be provided in the form of salts such as MgCl2 or COCl2. These salts form hydrates such as MgCl2·(H2O) in aqueous solution. x Or CoCl2·nH2O (n = 1, 2, 6, and 9). A suitable metallic cofactor is magnesium. Magnesium can be present as a magnesium salt such as magnesium chloride (MgCl2). Magnesium can be provided as metallic magnesium Mg(0) and can be oxidized to Mg(II) by electrolysis at the anode in a buffer solution. Another suitable metallic cofactor is cobalt. Cobalt can be provided as cobalt complexes such as cobalt(III) complexes or cobalt(I) complexes. Exemplary cobalt complexes include trans-dichlorobis(ethylenediamine)cobalt(III) chloride, pentaamminecobalt(III) chloride, hexaaminecobalt(III) chloride, trans-dichlorotetra(imidazolium)cobalt(III) chloride, or tris(triphenylphosphine)cobalt(I) chloride. Cobalt complexes can be reduced or oxidized to cobalt(II) chloride (CoCl2). For example, a Co(III)-complex can be reduced to a Co(II)-complex, which can then undergo ligand exchange with a buffer solution to form Co(II). Co(II) can then coordinate with a polymerase to activate it for polynucleotide synthesis. Ligand exchange reactions involve replacing one or more ligands in the complex ion with one or more different ligands.

[0228] Certain divalent or trivalent metal cofactors, such as magnesium and manganese, are known to interact with polymerases to regulate reaction progress. These catalytic metal cofactors can coordinate with the polymerase and the triphosphate of dNTPs to catalyze the addition of nucleotides to the 3' terminal nucleotide of an initiator (e.g., a primer), thereby forming a phosphodiester bond between the nucleotide of the dNTP and the initiator and releasing pyrophosphate (PPi). Other metal ions such as Ca... 2+ Metal cofactors can interact with polymerases such as Phi29 or its variants or derivatives, negatively impacting the process, for example, by stabilizing the enzyme and halting polymerization. Depending on the nature of the polymerization reaction, the polymerase used, the nucleotides employed, etc., different metal cofactors can exhibit different catalytic effects on the polymerization reaction, and in some embodiments, the catalytic / non-catalytic effects of these cofactors are used to synchronously polymerize reactions such as RCA reactions. For example, a first metal cofactor that interacts with the polymerase or polymerase-primer complex described herein under the same conditions to support the polymerization reaction at a level higher than that of a second metal cofactor is referred to as a catalytic metal ion. In some aspects, such catalytic metals support the continuous, iterative, or progressive polymerization of nucleic acids under specific polymerase reaction conditions, for example, by addition to multiple bases, while in other cases, a given type of catalytic metal cofactor may only support the addition of a single base. In some embodiments, catalytic metal cofactors, for example, targeting Phi29 or its variants or derivatives, may include Co. 2+ Mn 2+ 、Zn 2+ or Mg 2+ Or any combination thereof. In some embodiments, the binding mixtures described herein (e.g., in OFF buffer) may be substantially free of Co. 2+ Mn 2+ 、Zn 2+ and Mg 2+ This is to stop polymerization reactions such as RCA, while allowing the polymerase (or polymerase-nucleic acid complex such as polymerase-primer complex) to diffuse in the sample and bind to the circular nucleic acid, primer, and / or their complex.

[0229] In some embodiments, the conjugate mixture or OFF buffer or biological sample may contain a chelating agent. For example, the chelating agent may chelate divalent cations such as Mg from one or more previously reacted cations. 2+ Therefore, chelating agents can chelate residual divalent cations in biological samples, such as tissue sections that have been contacted with a reaction mixture containing divalent cations (e.g., a linkage reaction mixture for cyclizing padlock probes to form circular nucleic acids). In some embodiments, the binding mixture may contain EDTA, EGTA, BAPTA, DTPA, or combinations thereof. In some embodiments, one or more chelating agents in the binding mixture can chelate catalytic metal cofactors such as Co from Phi29 or its variants or derivatives.2+ Mn 2+ 、Zn 2+ or Mg 2+ This isolates these cofactors from the polymerase in the reaction mixture and / or sample in order to stop polymerization.

[0230] In some embodiments, the binding mixture or OFF buffer or biological sample may contain one or more cofactors that interact with the polymerase but do not promote the polymerization reaction, and in some cases act as a repressor or inhibitor of polymerization and / or inhibit one or more other activities of the polymerase, such as 3′→5′ exonuclease activity. In some embodiments, the binding mixture may contain one or more non-catalytic metal ions, such as calcium, barium, strontium, iron, cobalt, nickel, tin, zinc, and europium. These metals may be added to the binding mixture and / or sample in the form of salts such as Sr(OAc)2, Sr(OAc)2, CoCl2, SnCl2, CaCl2, or ZnSO4. A first metal cofactor that can be considered catalytic under a first set of reaction conditions or relative to a second metal cofactor may be considered non-catalytic under another set of different reaction conditions or relative to a third metal cofactor. For example, magnesium is known to support DNA polymerization. However, under certain conditions and / or relative to manganese, magnesium may act as a non-catalytic cofactor. In some embodiments herein, under the same reaction conditions, the catalytic cofactor supports polymerization to a greater extent than the non-catalytic metal. In some embodiments, the relative catalytic effect is a function of the reactant turnover rate of the polymerase complex, wherein, under the same reaction conditions, the catalytic metal cofactor promotes turnover at least two times, more preferably at least five times, even more preferably at least ten times, and in some cases 20 times, 50 times, or more, than the non-catalytic metal cofactor. In some embodiments, the presence of a non-catalytic metal in the polymerase complex (e.g., in a binding mixture or OFF buffer) prevents the synthesis reaction from proceeding from the complexed state by binding within or around the active site. In particular, the presence of calcium ions can modulate both the forward and reverse progression of the polymerase reaction. Thus, in the presence of calcium or other non-catalytic metals, the complexed nucleotide is effectively isolated within the polymerase complex. This reaction is a non-productive nucleotide binding event, i.e., it cannot proceed forward to incorporation or reverse to release unincorporated nucleotides to produce free polymerase.

[0231] In some implementations, the catalytic metal is selected from Mg. 2+ Mn 2+ and mixtures thereof, the non-catalytic metal being selected from Ca 2+ 、Zn 2 + Co 2+ Ni 2+ Eu2+ 、Sr 2+ 、Ba 2+ 、Fe 2+ Eu 2+ and mixtures thereof. In some embodiments, the combination mixture or OFF buffer contains Ca. 2+ 、Zn 2+ Co 2+ Ni 2+ Eu 2+ 、Sr 2+ 、Ba 2+ 、Fe 2+ and Eu 2+ One or more of the following, and substantially free of Mg. 2+ and / or Mn 2+ .

[0232] In some embodiments, the binding mixture or OFF buffer or biological sample may contain one or more dNTPs. However, in some embodiments, dNTPs are excluded and polymerases cannot incorporate dNTPs due to the absence of one or more catalytic cofactors, the presence of one or more catalytic cofactor chelators, and / or the presence of one or more non-catalytic cofactors. In some embodiments, the binding mixture allows binding between the polymerase, primers, and / or circular nucleic acids to form complexes at multiple sites in the sample, which are ready to initiate RCA as soon as polymerase activity is activated.

[0233] In some embodiments, the binding mixture or OFF buffer or biological sample may contain one or more polynucleotides that can act as RCA primers. In some embodiments, because Phi29 has 3′→5′ exonuclease (correction) activity that preferentially acts on single-stranded DNA or RNA, the polynucleotide acting as an RCA primer (e.g., an exogenously added primer or target nucleic acid in the sample) may be 3′ modified. In some embodiments, the polynucleotide may be 3′ phosphate-thioester protected, which protects the polynucleotide from degradation by 3′→5′ exonucleases under polymerase action while allowing initiation under polymerase action. In some embodiments, the polynucleotide may contain a 3′ tail of sufficient length to protect the sequence acting as an RCA primer from Phi29 degradation. In some embodiments, the 3′-terminal tail may be gradually digested until the remaining portion can be converted into an RCA primer and extended along the circular template via the polymerase activity of Phi29. However, in some embodiments, the exonuclease activity of Phi29 can be effectively inhibited in binding mixtures or OFF buffers due to the absence of one or more catalytic cofactors, the presence of one or more catalytic cofactor chelators, and / or the presence of one or more non-catalytic cofactors, thus eliminating the need for 3' protective modifications or 3'-tails. Therefore, in some embodiments, polynucleotides (e.g., primers or target nucleic acids added exogenously to the sample) can have free 3' hydroxyl groups that can be used for nucleotide incorporation via Phi29.

[0234] In some embodiments, the binding mixture or OFF buffer or biological sample may contain one or more polymerases. In some embodiments, the polymerase comprises a modified recombinant Phi29 polymerase. In some embodiments, the polymerase comprises a modified recombinant Phi29, B103, GA-1, PZA, Phi15, BS32, M2Y, Nf, G1, 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 compared to the wild-type Phi29 polymerase. Exemplary polymerases are described in U.S. Patent Nos. 8,257,954; 8,133,672; 8,343,746; 8,658,365; 8,921,086; and 9,279,155, all of which are incorporated herein by reference. In some embodiments, the polymerase is not directly or indirectly immobilized to a substrate, such as beads or a planar substrate (e.g., a glass slide), before contact with the sample, but the sample may be immobilized on the substrate. In some embodiments, the polymerase is not attached to nanopores, nanopore membranes, or their insulating supports. In some embodiments, the polymerase may diffuse within the binding mixture and / or the biological sample. In some embodiments, a pre-formed complex comprising the polymerase and RCA primers may be diffuseable within the binding mixture and / or the biological sample.

[0235] In some embodiments, the method may further include a step of removing molecules of polymerase and / or polynucleotides that are not bound to the circular nucleic acid from the biological sample between contact with the binding mixture and contact with the primer extension reaction mixture (e.g., an amplification reaction mixture). In some embodiments, the method may further include one or more rigorous washes between the contact steps.

[0236] In some embodiments, the primer extension reaction mixture may contain deoxynucleoside triphosphates (dNTPs) or their derivatives, variants, or analogs. In some embodiments, the primer extension reaction mixture may contain a catalytic cofactor of the polymerase. In any of the foregoing embodiments, the primer extension reaction mixture may contain a catalytic divalent cation, such as Mg²⁺. 2+ and / or Mn 2+ In some implementations, the primer extension reaction mixture is substantially free of non-catalytic cations, such as Ca2+. 2+ 、Zn 2 + Co 2+ Ni 2+ Eu 2+ 、Sr 2+ 、Ba 2+ 、Fe2+ Eu 2+ and mixtures thereof. In some embodiments, the catalytic cation in the primer extension reaction mixture can replace the non-catalytic cation that complexes with the polymerase bound to the circular nucleic acid or RCA primer, thereby activating the polymerase's polymerase activity. In some embodiments, when the sample is mixed with a catalytic divalent cation (such as Mg...), 2+ and / or Mn 2+ When the primer extension reaction mixture is in contact with the Phi29 primer, non-catalytic cations (such as Ca2+) bind to Phi29. 2+ 、Zn 2+ Co 2+ Ni 2 + Eu 2+ 、Sr 2+ 、Ba 2+ 、Fe 2+ and / or Eu 2+ The 5′→3′ polymerase activity and the 3′→5′ exonuclease (correction) activity of Phi29 are replaced, thereby activating the 5′→3′ polymerase activity and the 3′→5′ exonuclease (correction) activity of Phi29.

[0237] In some embodiments, the pH of the binding mixture and the primer extension reaction mixture may be substantially the same, for example, about pH 8.5. In some embodiments, the pH of the binding mixture and the primer extension reaction mixture may be 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 foregoing embodiments, the pH of the binding mixture and the primer extension reaction mixture may be 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.

[0238] In some embodiments, the primer extension reaction mixture may be substantially free of the polymerase and / or other polymerases. In some embodiments, polymerase molecules that have not bound to the circular nucleic acid and / or RCA primers are removed from the biological sample. Thus, in some embodiments, substantially all polymerase molecules in the sample are bound to the circular nucleic acid and RCA primers and are ready to initiate RCA simultaneously as soon as enzyme activity is activated. Polynucleotides that hybridize to the hybridization region of the circular nucleic acid (e.g., exogenously added primers or target nucleic acids in the sample) can be extended by the polymerase, thereby initiating rolling circle amplification simultaneously at different circular nucleic acids in a coordinated manner. The RCA reaction can be terminated simultaneously to provide multiple rolling circle amplification products.

[0239] In some embodiments, the plurality of rolling circle amplification products may have an average diameter of about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1.0 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, or about 1.5 μm, or any of the foregoing values. In some embodiments, the plurality of rolling circle amplification products may have an average diameter of less than 0.25 μm.

[0240] In some embodiments, the multiple rolling circle amplification products may have an average length of about 1 kb, about 2 kb, about 5 kb, about 10 kb, about 20 kb, about 30 kb, about 40 kb, about 50 kb, about 60 kb, or about 70 kb, or any of the foregoing values. In some embodiments, the multiple rolling circle amplification products may have an average length of less than 20 kb or less than 10 kb.

[0241] In some implementations, the mean, average, or medium copy number of the unit sequence complementary to the circular nucleic acid in the various rolling circle amplification products can be about 10, about 50, about 100, about 500, about 1,000, about 5,000, or about 10,000 or more.

[0242] In some implementations, the mean (average or medium) copy number of the unit sequence complementary to the circular nucleic acid in the various rolling circle amplification products may be less than 100 or less than 1,000.

[0243] In some implementations, the standard deviation of the diameter of the various rolling circle amplification products is smaller than the standard deviation of the diameter of the rolling circle amplification products generated using asynchronous methods.

[0244] In some embodiments, the mean (average or medium) peak intensity of the multiple rolling circle amplification products can be approximately 2 to approximately 10 times that of the mean (average or medium) peak intensity of the rolling circle amplification products formed without rolling circle amplification of the multiple circular nucleic acids in a concurrent biological sample.

[0245] In some implementations, the distribution of the relative observed signal of the rolling circle amplification product formed in the case of rolling circle amplification of the multiple circular nucleic acids in a synchronous biological sample can be narrower than the distribution of the relative observed signal of the rolling circle amplification product formed in the case of rolling circle amplification of the multiple circular nucleic acids in a synchronous biological sample.

[0246] In some embodiments, synchronization results in more uniform RCA product size and / or brighter signal spots. In some embodiments, synchronization results in fewer dark signal spots and / or more bright signal spots in the RCA product. In some embodiments, when the RCA product size becomes more uniform, amplification time can be reduced, resulting in smaller but equally bright RCA products. In some embodiments, polymerase extension can be performed for no more than 3 hours. In some embodiments, polymerase extension can be performed for no more than 2 hours. In some embodiments, polymerase extension can be performed for no more than 1 hour. In some embodiments, polymerase extension can be performed for no more than 30 minutes. In any of the embodiments herein, polymerase extension (e.g., RCA) may be performed at a temperature between about 20°C and about 40°C, for example at about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, or about 37°C for less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, less than about 60 minutes, less than about 65 minutes, less than about 70 minutes, less than about 75 minutes, less than about 80 minutes, less than about 85 minutes, less than about 90 minutes, less than about 95 minutes, less than about 100 minutes, less than about 105 minutes, less than about 110 minutes, less than about 115 minutes, or less than about 120 minutes. In some embodiments, polymerase extension can be carried out for less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, less than about 23 hours, less than about 24 hours, less than about 30 hours, less than about 35 hours, or less than about 40 hours. In one specific embodiment, polymerase extension can be carried out for between about 10 and 24 hours.

[0247] In some implementations, using the methods disclosed herein, RCA products with more uniform size (e.g., a denser size distribution) are produced within an RCA time of less than about 30 minutes. For example, Figure 4C Representative images after feature detection and filtering are shown in the control and synchronous groups after 30 minutes of RCA. The synchronous group shows a trend toward improved detection of RCA products at the expected sites of target gene expression in brain tissue. Therefore, synchronous RCA can facilitate better detection and / or resolution of more RCA products compared to asynchronous in situ RCA reactions.

[0248] V. Signal amplification, detection, and analysis

[0249] In some aspects, the provided methods involve analyzing (e.g., detecting or identifying) one or more sequences present in circular nucleic acids and / or polynucleotides and / or their products or derivatives (such as in amplified padlock probes). In some cases, analysis is performed on one or more captured images and may include processing said one or more images and / or quantifying observed signals. For example, the analysis may include processing information on one or more cell types, one or more types of biomarkers, the number or level of biomarkers, and / or the number or level of cells detected in a specific region of the sample. In some embodiments, the analysis includes detecting sequences, such as barcodes present in the sample. In some embodiments, the analysis includes quantifying puncta (e.g., if amplification products are detected). In some cases, the analysis includes determining the presence of specific cells and / or signals associated with one or more biomarkers from a specific panel. In some embodiments, the obtained information may be compared with positive and negative controls, or with a threshold of a characteristic, to determine whether the sample exhibits a certain characteristic or phenotype. In some cases, the information may include signals from cells, regions, and / or readings from multiple detectable markers. In some cases, the analysis also includes displaying information from the analytical or detection steps. In some implementations, software can be used to automate the processing, analysis, and / or display of data.

[0250] In some embodiments, the methods disclosed herein may further include one or more signal amplification components. In some embodiments, this disclosure relates to in situ detection of nucleic acid sequences using probe hybridization and generating an amplified signal associated with the probe, wherein background signal is reduced and sensitivity is increased. In some embodiments, methods including signal amplification can be used to detect RCA products generated from synchronous RCA.

[0251] Exemplary signal amplification methods include: targeted deposition of detectable reactive molecules around probe hybridization sites; targeted assembly of branched structures (e.g., bDNA or branching assays using locked nucleic acids (LNAs)); in situ growth of polyandries programmed by enzymatic rolling circle amplification (RCA) (e.g., as described in US 2019 / 0055594, which is incorporated herein by reference); hybridization chain reactions; assembly of topologically linked DNA structures using successive rounds of chemical ligation (clamp FISH); signal amplification via hairpin-mediated polyandration (e.g., as described in US 2020 / 0362398, which is incorporated herein by reference); and, for example, primer exchange reactions such as signal amplification via exchange reaction (SABER) or SABER with DNA-exchange (exchange-SABER). In some embodiments, non-enzymatic signal amplification methods may be used.

[0252] Detectable reactive molecules may include tyramine, such as for use in tyramine signal amplification (TSA) or multiple catalytic reporter deposition (CARD)-FISH. In some embodiments, the detectable reactive molecules may be released and / or cleaved from detectable tags such as fluorophores. In some embodiments, the methods disclosed herein include multiplex analysis of biological samples, comprising a sequential cycle of probe hybridization, fluorescence imaging, and signal removal, wherein signal removal comprises removing the fluorophore from the fluorophore-tagged reactive molecule (e.g., tyramine). Exemplary detectable reactive reagents and methods are described in US 6,828,109, US 2019 / 0376956, WO 2019 / 236841, WO 2020 / 102094, WO 2020 / 163397, and WO 2021 / 067475, all of which are incorporated herein by reference in their entirety.

[0253] In some implementations, signal amplification can be achieved using hybridization chain reaction (HCR). HCR is an enzyme-free nucleic acid amplification based on the hybridization-triggered chain of nucleic acid molecules. It begins with HCR monomers, which hybridize with each other to form a nicked nucleic acid polymer. This polymer is the product of the HCR reaction and is ultimately detected to indicate the presence of the target analyte. HCRs are described in detail in Dirks and Pierce, 2004, PNAS, 101(43), 15275-15278, and US 7,632,641 and US 7,721,721 (see also US 2006 / 00234261; Chemeris et al., 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al., 2010, 46, 3089-3091; Choi et al., 2010, Nat. Biotechnol. 28(11), 1208-1212; and Song et al., 2012, Analyst, 137, 1396-1401), all of which are incorporated herein by reference in full. HCR monomers typically contain hairpins or other metastable nucleic acid structures. In the simplest form of HCR, when an "initiator" nucleic acid molecule is introduced, two different types of stable hairpin monomers (here referred to as the first and second HCR monomers) undergo a hybridization chain reaction event to form a double-stranded DNA molecule with a long nick. The HCR monomer has a hairpin structure comprising a double-stranded stem region, a loop region connecting the two strands of the stem region, and a single-stranded region at one end of the double-stranded stem region. The single-stranded region exposed (and thus available for hybridization with another molecule, such as an initiator or other HCR monomers) when the monomer is in the hairpin structure can be referred to as the "foothold region" (or "input domain"). The first HCR monomer also contains a sequence complementary to the sequence in the exposed foothold region of the second HCR monomer. This complementary sequence in the first HCR monomer can be referred to as the "interaction region" (or "output domain"). Similarly, the second HCR monomer contains an interaction region (output domain), for example, a sequence complementary to the exposed foothold region (input domain) of the first HCR monomer. In the absence of an HCR initiator, these interaction regions are protected by secondary structures (e.g., they are not exposed), so the hairpin monomers are stable or kinetically trapped (also known as “metastable”) and remain as monomers (e.g., to prevent the system from rapidly equilibrating) because the first and second sets of HCR monomers cannot hybridize with each other. However, once an initiator is introduced, it is able to hybridize with and invade the exposed foothold region of the first HCR monomer, causing it to open. This exposes the interaction region of the first HCR monomer (e.g., a sequence complementary to the foothold region of the second HCR monomer), thus allowing it to hybridize with and invade the second HCR monomer at the foothold region.This hybridization and invasion then opens the second HCR monomer, exposing its interaction region (which is complementary to the foothold region of the first HCR monomer), and allows it to hybridize with and invade another first HCR monomer. The reaction continues in this manner until all HCR monomers are depleted (e.g., all HCR monomers are incorporated into the polymer chain). Ultimately, this chain reaction results in a nicked chain forming alternating units of the first and second monomer species. Therefore, the presence of an HCR initiator is required to trigger the HCR reaction by hybridizing with and invading the first HCR monomer. The first and second HCR monomers are engineered to hybridize with each other and can therefore be defined as homologous to each other. They are also homologous to a given HCR initiator sequence. HCR monomers that interact (hybridize) with each other can be described as a set of HCR monomers or an HCR monomer or hairpin system.

[0254] HCR reactions can be carried out using more than two species or types of HCR monomers. For example, a system involving three HCR monomers can be used. In such a system, each first HCR monomer may contain an interaction region that binds to the base region of a second HCR monomer; each second HCR monomer may contain an interaction region that binds to the base region of a third HCR monomer; and each third HCR monomer may contain an interaction region that binds to the base region of a first HCR monomer. The HCR polymerization reaction is then carried out as described above, except that the resulting product will be a polymer having repeating units of the first, second, and third monomers in succession. Corresponding systems with a larger number of HCR monomer groups can be readily envisioned. Branched HCR systems have also been designed and described (see, for example, WO 2020 / 123742, which is incorporated herein by reference) and can be used in the methods described herein.

[0255] In some embodiments, similar to HCR reactions using hairpin monomers, linear oligonucleotide hybridization chain reaction (LO-HCR) can also be used for signal amplification. In some embodiments, this document provides a method for detecting an analyte in a sample, comprising: (i) performing a linear oligonucleotide hybridization chain reaction (LO-HCR) wherein an initiator is contacted with a plurality of LO-HCR monomers of at least first and second species to produce a polymeric LO-HCR product hybridizing with a target nucleic acid molecule, wherein the first species comprises a first hybridization region complementary to the initiator and a second hybridization region complementary to the second species, wherein the first and second species are linear single-stranded nucleic acid molecules; wherein the initiator is provided in one or more portions and hybridizes directly or indirectly with or is contained in the target nucleic acid molecule; and (ii) detecting the polymer product, thereby detecting the analyte. In some embodiments, the first and / or second species may not contain a hairpin structure. In some embodiments, the plurality of LO-HCR monomers may not contain a metastable secondary structure. In some embodiments, the LO-HCR polymer may not contain a branched structure. In some embodiments, performing a linear oligonucleotide hybridization chain reaction includes contacting a target nucleic acid molecule with an initiator to provide an initiator for hybridization with the target nucleic acid molecule. In any embodiment herein, the target nucleic acid molecule and / or analyte may be an RCA product. Exemplary methods and compositions for LO-HCR are described in US 2021 / 0198723, the entire contents of which are incorporated herein by reference.

[0256] In some embodiments, the in situ detection of nucleic acid sequences comprises a combination of synchronous RCA and a component for branching signal amplification. In some embodiments, the component complex comprises an amplifier that hybridizes directly or indirectly (via one or more oligonucleotides) with the sequence of the RCA product. In some embodiments, the component comprises one or more amplifiers, each amplifier comprising an amplifier repeat sequence. In some aspects, the one or more amplifiers are labeled. This document describes a method of using the aforementioned component, including, for example, using the component in a multiple error robust fluorescence in situ hybridization (MERFISH) application, where branched DNA amplification is used for signal readout. In some embodiments, the amplifier repeat sequence is about 5-30 nucleotides and is repeated N times in the amplifier. In some embodiments, the amplifier repeat sequence is about 20 nucleotides and is repeated at least twice in the amplifier. In some aspects, the one or more amplifier repeat sequences are labeled. For exemplary branching signal amplification, see, for example, U.S. Patent Publication No. US20200399689A1 and Xia et al., Multiplexed Detection of RNA using MERF ISH and branched DNA amplification. Scientific Reports (2019), each of which is incorporated herein by reference in its entirety.

[0257] In some embodiments, RCA products can be detected using methods including signal amplification via primer exchange reaction (PER). In various embodiments, a primer having a domain at its 3' end binds to a catalytic hairpin and extends a new domain via a chain displacement polymerase. For example, a primer having domain 1 at its 3' end binds to a catalytic hairpin and extends a new domain 1 via a chain displacement polymerase, with repeated cycles producing polymers of repeating domain 1 sequences. In various embodiments, the chain displacement polymerase is Bst. In various embodiments, the catalytic hairpin includes a terminator that releases the chain displacement polymerase. In various embodiments, branch migration displaces the extended primer, and the extended primer can then dissociate. In various embodiments, the primer undergoes repeated cycles to form polymer primers. In various embodiments, multiple polymer primers are contacted with a sample containing RCA products generated using the methods described herein. In various embodiments, the RCA product can be contacted with multiple polymer primers and multiple labeled probes. For exemplary molecules and PER reaction components, see, for example, U.S. Patent Publication No. US20190106733, which is incorporated herein by reference.

[0258] In some embodiments, RCA products can be detected by providing detection probes, such as probes used to perform a chain reaction (e.g., HCR) to form the amplification product. In some embodiments, the analysis includes determining all or a portion of the sequence of the amplification product. In some embodiments, the analysis includes detecting sequences present in the amplification product. In some embodiments, the sequence of all or a portion of the amplification product indicates the identity of the region of interest in the target nucleic acid. In other embodiments, the provided method involves analyzing (e.g., detecting or determining) one or more sequences present in a polynucleotide probe (e.g., barcode sequences present in the protruding regions of a first and / or second probe).

[0259] In some embodiments, the method includes sequencing all or a portion of the amplification product, such as one or more barcode sequences present in the amplification product. In some embodiments, analysis and / or sequence determination includes sequencing all or a portion of the amplification product or probe and / or in situ hybridization with the amplification product or probe. In some embodiments, the sequencing step involves sequencing-while-hybridization, sequencing-while-ligation and / or fluorescence in situ sequencing, hybridization-based in situ sequencing and / or where the in situ hybridization includes sequential fluorescence in situ hybridization. In some embodiments, analysis and / or sequence determination includes detecting polymers generated by a hybridization chain reaction (HCR) reaction, for example, US 2017 / 0009278, which is incorporated herein by reference, for exemplary probes and HCR reaction components. In some embodiments, detection or determination includes hybridization of a detection oligonucleotide labeled with a fluorophore, isotope, mass tag, or combination thereof with the amplification product. In some embodiments, detection or determination includes imaging the amplification product. In some embodiments, the target nucleic acid is mRNA in a tissue sample, and detection or determination is performed when the target nucleic acid and / or amplification product are located in situ in the tissue sample.

[0260] In some aspects, the provided methods include imaging one or more portions of amplification products (e.g., amplicon) and / or polynucleotides, for example, by detecting binding to and detecting a detectable label. In some embodiments, the detection probe comprises a measurable and quantifiable detectable label. The terms "label" and "detectable label" encompass a directly or indirectly detectable portion associated with (e.g., conjugated to) a molecule to be detected, such as a detectable probe, including but not limited to fluorophores, radioisotopes, fluorescent agents, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens), etc.

[0261] The term "fluorophore" includes substances or portions thereof capable of exhibiting fluorescence within a detectable range. Specific examples of markers that may be used according to the provided embodiments include, but are not limited to, phycoerythrin, Alexa dye, luciferin, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acridinium ester, 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, renin luciferase, NADPH, β-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease.

[0262] Fluorescence detection in tissue samples is often hampered by the presence of strong background fluorescence. "Autofluorescence" is a general term used to distinguish background fluorescence (which can be generated from a variety of sources, including aldehyde fixation, extracellular matrix components, erythrocytes, lipofuscin, etc.) from the desired immunofluorescence from fluorescently labeled antibodies or probes. Tissue autofluorescence can make it difficult to distinguish signals attributed to fluorescent antibodies or probes from the general background. In some embodiments, the methods disclosed herein utilize one or more reagents to reduce tissue autofluorescence, such as autofluorescence quenchers (Sigma / EMD Millipore), TrueBlack lipofuscin autofluorescence quenchers (Biotium), MaxBlock autofluorescence reduction kits (MaxVision Biosciences), and / or very strong black dyes (e.g., Sudan Black or equivalent dark chromophores).

[0263] In some embodiments, a detectable probe containing a detectable label can be used to detect one or more polynucleotides and / or amplification products (e.g., amplicones) described herein. In some embodiments, the method involves incubating the detectable probe containing the detectable label with a sample, washing unbound detectable probes, and detecting the label, for example, by imaging.

[0264] Examples of detectable markers include, but are not limited to, various radioactive components, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, and protein-antibody binding pairs. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, and phycoerythrin.

[0265] Examples of bioluminescent labels include, but are not limited to, luciferases (e.g., bacteria, fireflies, and click beetles), luciferin, and amygdalin. Examples of enzyme systems with visually detectable signals include, but are not limited to, galactosidases, glucuronidases, phosphatases, peroxidases, and cholinesterases. Identifiable labels also include radioactive compounds such as… 125 I, 35 S. 14 C or 3 H. Identifiable markers are commercially available from a variety of sources.

[0266] Examples of fluorescent labels and nucleotides and / or polynucleotides conjugated with such fluorescent labels include, for example, those described in: Hoagland, Handbook of Fluorescent Probes and Research Chemicals, 9th edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259 (1991). In some embodiments, exemplary techniques and methods suitable for the provided embodiments include, for example, those described in US 4,757,141, US 5,151,507, and US 5,091,519, the full text of which is incorporated herein by reference. In some embodiments, one or more fluorescent dyes are used as markers for the target sequence of the label, for example, as described below: US 5,188,934 (4,7-dichlorofluorescein dye); US 5,366,860 (spectrally resolvable rhodamine dye); US 5,847,162 (4,7-dichlororhodamine dye); US 4,318,846 (ether-substituted fluorescein dye); US 5,800,996 (energy transfer dye); US 5,066,580 (xanthine dye); and US 688,648 (energy transfer dye). Quantum dots can also be used for labeling, as described in US 6,322,901, US 6,576,291, US 6,423,551, US 6,251,303, US 6,319,426, US 6,426,513, US 6,444,143, US 5,990,479, US 6,207,392, US 2002 / 0045045, and US 2003 / 0017264, all of which are incorporated herein by reference in their entirety. As used herein, the term “fluorescent label” includes a signal transduction component that conveys information through the fluorescent absorption and / or emission properties of one or more molecules. Exemplary fluorescence properties include fluorescence intensity, fluorescence lifetime, emission spectral characteristics, and energy transfer.

[0267] Examples of commercially available fluorescent nucleotide analogs that readily incorporate into nucleotide and / or polynucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-1-2-dUTP, tetramethylrhodamine-6-dUTP, and Texas Red. TM -5-dUTP, CASCADE BLUE TM -7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHOD AMINE GREEN TM -5-dUTP, OREGON GREENR TM 488-5-dUTP, Texas Red TM -12-dUTP, BODIPY TM 630 / 650-14-dUTP, BODIPY TM 650 / 665-14-dUTP, ALEXA FLUOR TM 488-5-dUTP, ALEXAFLUOR TM 532-5-dUTP, ALEXA FLUOR TM 568-5-dUTP, ALEXA FLUOR TM 594-5-dUTP, ALEXAFLUOR TM 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, Texas red TM -5-UTP, mCherry, CASCADE BLUE TM -7-UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM TR-14-UTP, RHOD AMINE GREEN TM -5-UTP, ALEXA FLUOR TM 488-5-UTP and ALEXA FLUOR TM 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.). Custom synthesis of nucleotides with other fluorophores can be found in Henegariu et al., (2000) Nature Biotechnol. 18: 345, the full text of which is incorporated herein by reference.

[0268] Other fluorophores available for post-synthesis attachment include, but are not limited to, ALEXA FLUOR. TM 350, ALEXAFLUOR TM 532. ALEXA FLUOR TM 546. ALEXA FLUOR TM 568. ALEXA FLUOR TM 594. ALEXA FLUOR TM 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Danshenyl, Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethyl Rhodamine, Texas Red (available from Molecular) Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, NJ). FRET tandem fluorophores can also be used, including but not limited to PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), and APC-Alexa dyes.

[0269] In some cases, metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or polynucleotide sequences (Lakowicz et al., (2003) Bio Techniques 34:62, the full text of which is incorporated herein by reference).

[0270] Biotin or its derivatives can also be used as a label on nucleotide and / or polynucleotide sequences and subsequently bound by a detectably labeled avidin / streptoavidin derivative (e.g., phycoerythrin-conjugated streptoavidin) or a detectably labeled avidin antibody. Digoxigenin can be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinized anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into polynucleotide sequences and subsequently conjugated with an N-hydroxysuccinimide (NHS)-derived fluorescent dye. Generally, any member of a conjugate pair can be incorporated into the detective polynucleotide, provided that the detectably labeled conjugate pair can bind to allow detection. In some embodiments, antibody refers to any class of antibody molecule or any subfraction thereof, such as Fab.

[0271] Other suitable markers for polynucleotide sequences may include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and phosphorus-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments, detection is performed using the following hapten / antibody pairs, where each antibody is derivatized with a detectable marker: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, and 5-carboxyfluorescein (FAM) / α-FAM.

[0272] In some embodiments, nucleotide and / or polynucleotide sequences may be indirectly labeled, particularly with haptens, which are then bound by a capture agent, for example, as disclosed in US 5,344,757, US 5,702,888, US 5,354,657, US 5,198,537 and US 4,849,336 and PCT Publication WO 1991 / 017160, all of which are incorporated herein by reference in their entirety. Many different hapten-capture agent pairs are available. Exemplary haptens include, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, Cy5, and digoxigenin. For biotin, the capture agent may be avidin, streptoavidin, or an antibody. Antibodies may be used as capture agents for other haptens (many dye-antibody pairs are commercially available, e.g., Molecular Probes, Eugene, Oreg.).

[0273] In some aspects, analysis and / or sequencing can be performed at room temperature to best preserve tissue morphology with low background noise and reduced errors. In some embodiments, analysis and / or sequencing includes eliminating error accumulation as sequencing proceeds.

[0274] In some implementations, analysis and / or sequence determination involves washing to remove unbound polynucleotides, followed by exposure of fluorescent products for imaging.

[0275] In some aspects, detection involves the use of detection methods such as flow cytometry, sequencing, probe binding and electrochemical detection, pH alteration, enzyme-induced catalysis bound to DNA tags, quantum entanglement, Raman spectroscopy, terahertz wave technology and / or scanning electron microscopy. In some aspects, flow cytometry is mass cytometry or fluorescence-activated flow cytometry. In some aspects, detection includes performing microscopy, scanning mass spectrometry or other imaging techniques described herein. In such aspects, detection involves measuring a signal, such as a fluorescence signal.

[0276] In some aspects, any of many different types of microscopy is used for detection (including imaging), such as confocal microscopy, two-photon microscopy, light field microscopy, whole tissue expansion microscopy, and / or CLARITY. TM - Optimized light-sheet microscopy (COLM).

[0277] In some implementations, fluorescence microscopy is used for the detection and imaging of the detection probe. In some aspects, fluorescence microscopy is an optical microscope that uses fluorescence and phosphorescence to replace or supplement reflection and absorption to study the properties of organic or inorganic substances. In fluorescence microscopy, the sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescence is then imaged through a microscope objective, where the fluorescence wavelength is typically longer than the illumination wavelength. Two filters can be used in this technique: an illumination (or excitation) filter that ensures the illumination is nearly monochromatic and at the correct wavelength, and a second emission (or barrier) filter that ensures no excitation source reaches the detector. Alternatively, both functions can be achieved with a single dichroic filter. "Fluorescence microscopy" encompasses any microscope that uses fluorescence to generate images, whether it is a simpler device like an epifluorescence microscope or a more complex design like a confocal microscope, all of which use optical sections to obtain better resolution fluorescence images.

[0278] In some implementations, confocal microscopy is used for the detection and imaging of the probe. Confocal microscopy uses point illumination and a pinhole in the optical conjugate plane in front of the detector to eliminate defocus signals. Because only fluorescence light very close to the focal plane can be detected, the optical resolution of the image, especially in the sample depth direction, is much better than that of wide-field microscopy. However, this improvement in resolution comes at the cost of reduced signal intensity—and therefore typically requires long exposure times—because much of the fluorescence light from the sample is blocked at the pinhole. Since only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning the specimen in a regular grating (e.g., a rectangular pattern of parallel scan lines). The achievable thickness of the focal plane is primarily limited by the wavelength of the light used divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. Thin optical sections make these types of microscopy particularly good for 3D imaging and surface profilometry of samples. TM - Optimized light-sheet microscopy (COLM) provides an alternative microscopy technique for rapid 3D imaging of large, clear samples. COLM interrogates large immunostained tissues, allowing for faster acquisition speeds and producing higher-quality generated data.

[0279] Other types of microscopy that can be used include bright-field microscopy, oblique illumination microscopy, dark-field microscopy, phase contrast microscopy, differential interference difference (DIC) microscopy, interferometric reflection microscopy (also known as reflection interference difference or RIC), single-plane illumination microscopy (SPIM), super-resolution 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 Microscopy (ECSTM), Electrostatic Force Microscopy (EFM), Fluid Force Microscopy (FluidFM), Force Modulation Microscopy (FMM), Feature-Guided 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, piezoelectric response force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microscopy / 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 Conductivity Microscopy (SICM), SPSM Spin Polarization Scanning Tunneling Microscopy (SPSM), SSRM Scanning Diffusion Resistance Microscopy (SSRM), SThM Scanning Thermal Microscopy (SThM), STM Scanning Tunneling Microscopy (STM), STP Scanning Tunneling Potential Method (STP), SVM Scanning Voltage Microscopy (SVM), Synchrotron X-ray Scanning Tunneling Microscopy (SXSTM), and Extensive Tissue Microscopy (exM).

[0280] In some implementations, sequencing can be performed in situ. In situ sequencing typically involves sequentially, template-dependently incorporating labeled nucleotides (e.g., fluorescently labeled mononucleotides or dinucleotides) or hybridizing labeled primers (e.g., labeled random hexamers) with a nucleic acid template such that the identity (e.g., nucleotide sequence) of the incorporated nucleotide or labeled primer extension product can be determined, and thus the nucleotide sequence of the corresponding template nucleic acid can be determined. Aspects of in situ sequencing are described, for example, in Mitra et al., (2003) Anal. Biochem. 320, 55-65 and Lee et al., (2014) Science, 343(6177), 1360-1363, the full text of which is incorporated herein by reference. In addition, examples of methods and systems used for in situ sequencing are described in US 2016 / 0024555, US 2019 / 0194709, US 10,138,509, US 10,494,662, and US 10,179,932, all of which are incorporated herein by reference in their entirety. Exemplary techniques for in situ sequencing include, but are not limited to, STARmap (described, for example, Wang et al., (2018) Science, 361(6499)5691), MERFISH (described, for example, Moffitt, (2016) Methods in Enzymology, 572, 1-49), hybridization-based in situ sequencing (HybISS) (described, for example, Gyllborg et al., Nucleic Acids Res (2020) 48(19): e112) and FISSEQ (described, for example, US 2019 / 0032121), all of which are incorporated herein by reference in their entirety.

[0281] In some implementations, sequencing can be performed via sequencing-by-synthesis (SBS). In some implementations, the sequencing primers are complementary to sequences at or near one or more barcodes. In such implementations, sequencing-by-synthesis may include reverse transcription and / or amplification to generate a template sequence from which the primer sequences can bind. Exemplary SBS methods include, for example, but not limited to, those described in US 2007 / 0166705, US 2006 / 0188901, US 7,057,026, US 2006 / 0240439, US 2006 / 0281109, US 201I / 005986, US 2005 / 0100900, US 9,217,178, US 2009 / 0118128, US 2012 / 0270305, US 2013 / 0260372 and US 2013 / 0079232, all of which are incorporated herein by reference in their entirety.

[0282] In some embodiments, sequence analysis of nucleic acids (e.g., nucleic acids such as probes or RCA products containing barcode sequences generated using circular nucleic acids as templates) can be performed via sequential hybridization (e.g., hybridization-while-sequencing and / or sequential in situ fluorescence hybridization). Sequential fluorescence hybridization can involve sequential hybridization of a detectable probe comprising an oligonucleotide and a detectable label. In some embodiments, the methods disclosed herein include sequential hybridization of the detectable probes disclosed herein, including detectably labeled probes (e.g., fluorophore-conjugated oligonucleotides) and / or probes that are not themselves detectably labeled but are capable of binding to and being detected by a detectably labeled probe (e.g., via nucleic acid hybridization). Exemplary methods of sequential fluorescence hybridization comprising detectable probes are described in US 2019 / 0161796, US 2020 / 0224244, US2022 / 0010358, US 2021 / 0340618, and WO 2021 / 138676, all of which are incorporated herein by reference. In some implementations, the methods provided herein may include analyzing identifier sequences (e.g., analyte sequences or barcode sequences) by sequential hybridization and detection with probes labeled with multiple tags (e.g., detection oligonucleotides).

[0283] In some embodiments, sequence detection includes contacting a biological sample with one or more intermediate probes that hybridize directly or indirectly with rolling circle amplification products, wherein the one or more intermediate probes can be detected using one or more detectably labeled probes, and dehybridizing the one or more intermediate probes and / or the one or more detectably labeled probes from the rolling circle amplification products. In some embodiments, the one or more intermediate probes include one or more protruding regions (e.g., 5′ and / or 3′ ends of the probe that do not hybridize with the rolling circle amplification products). A probe containing a single protruding region may be referred to as an "L-shaped probe," and a probe containing two protrusions may be referred to as a "U-shaped probe." In some cases, the protruding region includes a binding region for binding one or more detectably labeled probes. In some embodiments, detection includes contacting a biological sample with intermediate probe pools corresponding to different barcode sequences or portions thereof and with detectably labeled probe pools corresponding to different detectable labels. In some embodiments, the biological sample is contacted sequentially with different intermediate probe pools. In some cases, a common or universal detectably labeled probe pool is used in multiple sequential hybridization steps (e.g., with different intermediate probe pools).

[0284] In some embodiments, this document provides methods for in situ analysis of analytes in samples using sequential probe hybridization. In some aspects, this document provides a method for analyzing biological samples, comprising: a) generating a rolling circle amplification product (RCP) of a circular probe as described herein, the RCP comprising an identifier sequence such as a barcode sequence or an analyte sequence, wherein the identifier sequence is associated with an analyte of interest and assigned a signal code sequence; b) contacting the biological sample with a first probe (e.g., an intermediate probe such as an L-probe) and a first detectably labeled probe to generate a first complex comprising a first probe hybridized with the RCP and a first detectably labeled probe hybridized with the first probe, wherein the first probe comprises (i) an identification sequence (e.g., a target-binding sequence) complementary to the identifier sequence (e.g., a barcode sequence or an analyte sequence) and (ii) a first landing sequence (e.g., a protrusion sequence), wherein the first detectably labeled probe comprises a sequence complementary to the first landing sequence; c) detecting a first signal associated with the first detectably labeled probe, wherein the first signal corresponds to a signal code. The first signal code in the signal code sequence; d) contacting the biological sample with a second probe (e.g., an intermediate probe such as an L-probe) and a second detectably labeled probe to generate a second complex comprising a second probe hybridized with RCP and a second detectably labeled probe hybridized with the second probe, wherein the second probe comprises (i) an identification sequence (e.g., a target-binding sequence) complementary to an identifier sequence (e.g., a barcode sequence or an analyte sequence) and (ii) a second landing sequence (e.g., a protrusion sequence), wherein the second detectably labeled probe comprises a sequence complementary to the second landing sequence; and e) detecting a second signal associated with the second detectably labeled probe, wherein the second signal corresponds to a second signal code in the signal code sequence, wherein the signal code sequence containing the first signal code and the second signal code is measured at a certain location in the biological sample, thereby decoding the identifier sequence (e.g., a barcode sequence or an analyte sequence) and identifying the analyte of interest at that location in the biological sample. In some embodiments, the detectable label of the first detectably labeled probe and the detectable label of the second detectably labeled probe are the same. In some embodiments, the detectable markers of the first detectable marker probe and the second detectable marker probe are different. In some embodiments, the first signal code and the second signal code are the same. In some embodiments, the first signal code and the second signal code are different.

[0285] In some embodiments, a first probe (e.g., a first intermediate probe such as a first L-probe), a second probe (e.g., a second intermediate probe such as a second L-probe), and one or more subsequent probes (e.g., subsequent intermediate probes such as subsequent L-probes) are sequentially contacted with a biological sample in a predetermined order, the predetermined order corresponding to a signal code sequence assigned to an identifier sequence (e.g., a barcode sequence or analyte sequence), wherein each of the one or more subsequent probes comprises (i) an identification sequence complementary to the identifier sequence (e.g., a barcode sequence or analyte sequence) and (ii) a protrusion sequence complementary to a detectably labeled probe pool (e.g., a universal pool across different probe hybridization cycles). In some embodiments, the biological sample is contacted with the first probe before the second probe and one or more subsequent probes. In some embodiments, the biological sample is contacted with the second probe after the first probe and before one or more subsequent probes. In some embodiments, the biological sample is contacted with the one or more subsequent probes after the first probe. In some embodiments, the biological sample is contacted with the one or more subsequent probes after the first probe and the second probe.

[0286] In some embodiments, a first detectably labeled probe and a second detectably labeled probe are housed in a pool of detectably labeled probes. The pool of detectably labeled probes may contain at least two detectably labeled probes and may be used for multiplex analysis of two or more target analytes (e.g., target nucleic acids) in a biological sample. In some embodiments, contact b) includes contacting the biological sample with a universal pool of detectably labeled probes, and contact d) includes contacting the biological sample with a universal pool of detectably labeled probes. In some embodiments, the universal pool of detectably labeled probes used in contact b) is the same as the universal pool of detectably labeled probes used in contact d). In some embodiments, the universal pool contains detectably labeled probes, each detectably labeled probe having a detectable label corresponding to a different nucleic acid sequence for hybridization with a landing sequence (e.g., a protrusion sequence) in the probe (e.g., an intermediate probe such as an L-probe). In some embodiments, the number of different detectably labeled probes in the universal pool is four.

[0287] In some embodiments, the method contacts one or more subsequent probes with a biological sample to determine a signal code in a signal code sequence until sufficient signal codes have been determined to decode an identifier sequence (e.g., a barcode sequence or analyte sequence) to identify a target analyte (e.g., a target nucleic acid). In some embodiments, the method further includes the step of removing a first probe and / or a first detectably labeled probe from the biological sample before contacting the sample with subsequent probes and a detectably labeled probe that hybridizes with the subsequent probes. In some embodiments, the method further includes the step of removing a second probe and / or a second detectably labeled probe from the biological sample before contacting the sample with subsequent probes and a detectably labeled probe that hybridizes with the subsequent probes.

[0288] In some embodiments, the method further includes identifying multiple different target analytes present at locations (e.g., different locations) within the biological sample. In some embodiments, each different target analyte is assigned a different signal code sequence and targeted by a circular or ringable probe or probe set, the circular or ringable probe or probe set comprising complementary sequences of different barcode sequences of the multiple barcode sequences. In some embodiments, the number of different probes (e.g., L-probes with different recognition sequences binding to barcode sequences) in each probe pool is greater than the number of different detectably labeled probes in a general detectably labeled probe pool. In some embodiments, the number of different detectably labeled probes in a general pool is four. In some embodiments, the number of different probes in each probe (e.g., L-probe) pool is about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 500, about 1,000, or more. In some implementations, the number of different identification sequences (e.g., identification sequences combined with barcode sequences) of probes in each probe pool is at least about 10, such as any one of at least about 20, 30, 40, 50, 100, 200, 500, 1,000 or more.

[0289] In some implementations, sequencing can be performed using single-molecule ligation-while-sequencing. Such techniques utilize DNA ligases to incorporate oligonucleotides and identify such incorporation. Oligonucleotides typically have distinct markers associated with the identity of specific nucleotides in the sequence to which they hybridize. Aspects and characteristics involved in ligation-while-sequencing are described, for example, in Shendure et al., Science (2005), 309:1728-1732, and in US 5,599,675; US 5,750,341; US ​​6,969,488; US 6,172,218; US 6,306,597, all of which are incorporated herein by reference in their entirety.

[0290] In some embodiments, the barcode of the probe (e.g., a padlock probe or a first and / or second probe) is targeted by a detectably labeled detection oligonucleotide, such as a fluorescently labeled oligonucleotide. In some embodiments, one or more decoding schemes are used to decode the signal, such as fluorescence, for sequence determination. In any embodiment herein, any suitable method or technique can be used to analyze (e.g., detect or sequence) the barcode (e.g., primary and / or secondary barcode sequences), including those described herein, such as RNA SPOT targeting, sequential fluorescence in situ hybridization (seqFISH), single-molecule fluorescence in situ hybridization (smFISH), multiple error robust fluorescence in situ hybridization (MERFISH), hybridization-based in situ sequencing (HybISS), in situ sequencing, targeted in situ sequencing, fluorescence in situ sequencing (FISSEQ), or spatially resolved transcript amplicon readout mapping (STARmap). In some embodiments, the methods provided herein include analyzing the barcode by sequential hybridization and detection with multiple labeled probes (e.g., detection oligonucleotides). Exemplary decoding schemes are described in Eng et al., “Transcriptome-scale Super-Resolved Imaging in Tissues by RNA SeqFISH+,” Nature 568(7751): 235-239(2019); Chen et al., “Spatially resolved, highly complexed RNA profiling in single cells,” Science; 348(6233): aaa6090(2015); Gyllborg et al., Nucleic Acids Res(2020)48(19): e112; US 10,457,980B2; US 2016 / 0369329 A1; WO 2018 / 026873 A1; and US 2017 / 0220733 A1, all of which are incorporated herein by reference in their entirety. In some implementations, these determinations enable simultaneous signal amplification, combined decoding, and error correction schemes.

[0291] In some implementations, nucleic acid hybridization can be used for sequencing. These methods utilize labeled nucleic acid decoder probes that are complementary to at least a portion of the barcode sequence. Multiple decoding can be performed using pools of many different probes with distinguishable labels. Non-limiting examples of nucleic acid hybridization sequencing are described, for example, in US 8,460,865 and Gunderson et al., Genome Research 14:870-877 (2004), the full text of which is incorporated herein by reference.

[0292] In some implementations, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET), as cited 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, all of which are incorporated herein by reference in full.

[0293] VI. Reagent Kit

[0294] This document also provides kits that, for example, contain one or more probes (e.g., circular nucleic acids) and polynucleotides (e.g., any polynucleotides described in Part III) and reagents for performing the methods provided herein, such as reagents required for one or more steps including hybridization, ligation, amplification, detection, sequencing, and / or sample preparation as described herein. In some embodiments, the kit also contains a target nucleic acid, such as any target nucleic acid described in Part III. In some embodiments, any or all probes and / or polynucleotides are DNA molecules. In some embodiments, the target nucleic acid is a messenger RNA molecule. In some embodiments, the kit also contains a ligase, for example for forming a circular probe from a padlock probe. In some embodiments, the ligase has DNA clipping DNA ligase activity. In some embodiments, the ligase has RNA clipping ligase activity. In some embodiments, the kit also contains a polymerase, for example for amplifying the padlock probe using any method described in Part V. In some embodiments, the kit also contains primers for amplification.

[0295] In some embodiments, this document discloses a kit for analyzing biological samples comprising: (i) a binding mixture comprising multiple complexes of polymerases each containing primers and a chelating agent, wherein the binding mixture is substantially free of deoxynucleoside triphosphates (dNTPs); and (ii) a primer extension reaction mixture comprising dNTPs and a divalent cation, wherein the primer extension reaction mixture is substantially free of the polymerase. In any of the foregoing embodiments, the primers in the multiple complexes may be the same. Alternatively, in any of the foregoing embodiments, the primers in two or more of the multiple complexes may be different. In any of the foregoing embodiments, the polymerase may be Phi29 DNA polymerase and the divalent cation may be Mg2+. 2+ Co 2+ and / or Mn 2+ .

[0296] The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined into a single container. In some embodiments, the kit also includes instructions for carrying out the provided methods using the kit components.

[0297] In some embodiments, the kit may contain reagents and / or consumables required for performing one or more steps of the provided method. In some embodiments, the kit contains reagents for immobilizing, embedding, and / or permeabilizing biological samples. In some embodiments, the kit contains reagents, such as enzymes and buffers for ligation and / or amplification, such as ligases and / or polymerases. In some aspects, the kit may also contain any of the reagents described herein, such as wash buffers and ligation buffers. In some embodiments, the kit contains reagents for detection and / or sequencing, such as barcode detection probes or detectable markers. In some embodiments, the kit optionally contains other components, such as nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, and reagents for additional assays.

[0298] VII. Application

[0299] In some respects, the provided implementation schemes can be applied to in situ methods for analyzing nucleic acid sequences, such as in situ transcriptomics analysis or in situ sequencing, for example, nucleic acid sequences from intact tissues or samples in which spatial information has been preserved. In some respects, the implementation schemes can be applied to imaging or detection methods for multiplex nucleic acid analysis. In some respects, the provided implementation schemes can be used to identify or detect regions of interest in target nucleic acids.

[0300] In some embodiments, the region of interest contains a single nucleotide polymorphism (SNP). In some embodiments, the region of interest is a single nucleotide variant (SNV). In some embodiments, the region of interest contains a single nucleotide substitution. In some embodiments, the region of interest contains a point mutation. In some embodiments, the region of interest contains a single nucleotide insertion.

[0301] In some respects, the implementation scheme can be applied to research and / or diagnostic applications, such as for characterizing or evaluating specific cells or tissues from a subject. Applications of the provided methods can include biomedical research and clinical diagnostics. For example, in biomedical research, applications include, but are not limited to, spatial resolution gene expression analysis for biological research or drug screening. In clinical diagnostics, applications include, but are not limited to, detecting genetic markers in patient samples such as disease, immune responses, bacterial or viral DNA / RNA.

[0302] In some respects, the implementation scheme can be applied to visualize the distribution of genetically encoded markers throughout a tissue at subcellular resolution, such as chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of alleles indicating disease predisposition or good health, the likelihood of responding to treatment, or in personalized medicine or lineage.

[0303] VIII. Terminology

[0304] Specific terminology is used throughout this disclosure to explain various aspects of the described apparatus, systems, methods, and compositions.

[0305] Some illustrative embodiments of this disclosure have been described, but it will be apparent to those skilled in the art that the foregoing is merely illustrative and not restrictive, and has been presented by way of example only. Many modifications and other illustrative embodiments are within the scope of those skilled in the art and are considered to fall within the scope of this disclosure. In particular, although many of the examples presented herein relate to specific combinations of method actions or system elements, it should be understood that these actions and elements can be combined in other ways to achieve the same objective.

[0306] As used herein, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly specifies otherwise. For example, “a” or “an” means “at least one (kind)” or “one (kind) or more (kinds).”

[0307] As used herein, the term "about" refers to the typical range of error for a corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments involving that value or parameter itself.

[0308] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Therefore, the scope description should be considered as having specifically disclosed all possible subscopes and individual numerical values ​​within that scope. For example, in the case of providing a scope of values, it should be understood that every intermediate value between the upper and lower limits of that scope, as well as any other stated or intermediate value within the scope of that statement, is covered within the claimed subject matter. The upper and lower limits of these smaller scopes may be independently included in the smaller scope and also covered within the claimed subject matter, but are subject to any specific exclusion limits within the scope of that statement. Where the scope of that statement includes one or both of these limits, the scope excluding any one or both of those included limits is also included within the claimed subject matter. This applies regardless of the breadth of the scope.

[0309] The use of ordinal numbers such as "first," "second," and "third" to modify claim elements in claims does not imply any priority, order, or sequence of precedence of one claim element over another, nor does it imply a temporal order of multiple actions of a method. Rather, it serves merely as a label to distinguish one claim element with a particular name from another element with the same name (but using a different ordinal number) used to differentiate claim elements. Similarly, the use of a), b), etc., or i), ii), etc., in claims does not imply any priority, order, or sequence of steps. Likewise, the use of these terms in the specification does not imply any priority, order, or sequence of claims.

[0310] (i) Barcode

[0311] A barcode is a mark or identifier that conveys or is able to convey information (e.g., information about an analyte in a sample and / or probe). A barcode can be part of the analyte or independent of it. A barcode can be attached to the analyte. A particular barcode can be unique relative to other barcodes.

[0312] Barcodes can take many different forms. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. Barcodes can be reversibly or irreversibly linked to an analyte or another part or structure. Barcodes can be added to fragments of samples, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), before or during sample sequencing. Barcodes can allow for the identification and / or quantification of individual sequencing reads (e.g., barcodes can be or can include unique molecular identifiers or "UMIs").

[0313] Barcodes can, for example, spatially resolve molecular components present in a biological sample at single-cell resolution (e.g., a barcode can be or may include a "spatial barcode"). In some embodiments, a barcode includes both UMI and spatial barcodes. In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode may contain two or more polynucleotide sequences separated by one or more non-barcode sequences (e.g., sub-barcodes).

[0314] (ii) Nucleic acids and nucleotides

[0315] The terms “nucleic acid” and “nucleotide” are intended to be used in the art and include naturally occurring substances or their functional analogs. Particularly useful functional analogs of nucleic acids are capable of hybridizing with nucleic acids in a sequence-specific manner (e.g., hybridizing with two nucleic acids such that ligation can occur between the two hybridized nucleic acids) or can be used as templates for the replication of specific nucleotide sequences. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. Analog structures may have alternative backbone bonds, including any of a variety. Naturally occurring nucleic acids generally have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0316] Nucleic acids can contain nucleotides from a variety of analogues having these sugar moieties. Nucleic acids can contain natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G). Non-natural bases that can be included in nucleic acids or nucleotides can be utilized.

[0317] (iii) Probe and target

[0318] The term "probe" or "target," when used to refer to nucleic acids or nucleic acid sequences, is intended in the context of a method or composition to serve as a semantic identifier for the nucleic acid or sequence and does not limit the structure or function of the nucleic acid or sequence beyond what is explicitly stated.

[0319] (iv) Oligonucleotides and Polynucleotides

[0320] The terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to single-stranded nucleotide polymers of about 2 to about 500 nucleotides in length. Oligonucleotides can be prepared synthetically, enzymatically (e.g., by polymerization), or using a "split-pool" method. Oligonucleotides can include ribonucleotide monomers (e.g., oligoribonucleotides) and / or deoxyribonucleotide monomers (e.g., oligodeoxyribonucleotides). In some instances, oligonucleotides can include combinations of deoxyribonucleotide and ribonucleotide monomers (e.g., random or ordered combinations of deoxyribonucleotide and ribonucleotide monomers). For example, oligonucleotides can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400-500 nucleotides in length. Oligonucleotides may include one or more functional moieties linked to a multimeric structure (e.g., covalently or non-covalently). For example, oligonucleotides may include one or more detectable tags (e.g., radioisotopes or fluorophores).

[0321] (v) Hybridizing, Hybridize, Annealing

[0322] The terms “hybridizing” and “annealing” are used interchangeably in this disclosure and refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any method in which nucleic acid sequences bind to substantially or completely complementary sequences through base pairing to form a hybridization complex. For hybridization purposes, 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 each other.

[0323] (vi) Primers

[0324] A primer is a single-stranded nucleic acid sequence having a 3' end, which can be used as a substrate for nucleic acid polymerases in nucleic acid extension reactions. RNA primers are formed from RNA nucleotides and are used for RNA synthesis, while DNA primers are formed from DNA nucleotides and are used for DNA synthesis. Primers may also contain both RNA and DNA nucleotides (e.g., in a random or designed pattern). Primers may also contain other natural or synthetic nucleotides as described herein that may have additional functions. In some instances, DNA primers can be used to initiate RNA synthesis and vice versa (e.g., RNA primers can be used to initiate DNA synthesis). The length of primers can vary. For example, primers can be from about 6 bases to about 120 bases. For example, primers can contain up to about 25 bases. In some cases, primers may be primer-binding sequences.

[0325] (vii) Primer extension

[0326] Two nucleic acid sequences can be joined (e.g., hybridization) by overlapping their respective complementary terminal nucleic acid sequences (e.g., 3′ ends). Such ligation can be followed by one or both ends of nucleic acid extension using another nucleic acid sequence as an extension template (e.g., enzymatic extension). Enzymatic extension can be performed using enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0327] (viii) Nucleic acid extension

[0328] Nucleic acid extension typically involves the template-dependent incorporation of one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs or derivatives thereof) into a molecule (such as, but not limited to, nucleic acid sequences) such that consecutive nucleic acids are incorporated by an enzyme (such as a polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. For example, primers hybridizing with complementary nucleic acid sequences can be used to synthesize new nucleic acid molecules by using complementary nucleic acid sequences as templates for nucleic acid synthesis. Similarly, the 3' polyadenylated tail of mRNA transcripts hybridized with poly(dT) sequences (e.g., capture domains) can be used as templates for the single-stranded synthesis of the corresponding cDNA molecule.

[0329] (ix)PCR amplification

[0330] “PCR amplification” refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for performing PCR are described, for example, in U.S. Patents 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture comprises the genetic material to be amplified, an enzyme, one or more primers for primer extension reactions, and reagents for the reaction. Oligonucleotide primers are of sufficient length to provide hybridization with complementary genetic material under annealing conditions. The length of primers typically depends on the length of the amplified domain, but is generally at least 4, 5, 6, 8, 9, 10 base pairs (bp), at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 bp, and can be as long as 40 bp or longer. Primer lengths are typically in the range of 18 to 50 bp. Genetic material can be contacted with a single primer or a set of two primers (forward and reverse primers), depending on whether primer extension, linear or exponential amplification of the genetic material is required.

[0331] In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In some embodiments, the DNA polymerase is derived from bacteria; for example, the DNA polymerase is a bacterial DNA polymerase. For instance, the DNA polymerase can be derived from bacteria of the genus *Escherichia*, *Bacillus*, *Thermophilus*, or *Pomacea*.

[0332] Suitable examples of DNA polymerases that can be used include, but are not limited to: Escherichia coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, and VENT. TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot Start Taq DNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase DNA polymerase, Quick- DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-Fidelity DNA polymerase, Platinum Pfx DNA polymerase, AccuPrimePfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase.

[0333] The term "DNA polymerase" includes not only naturally occurring enzymes but also all modified derivatives thereof, as well as derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase may have been modified to remove 5′–3′ exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that may be used include, but are not limited to, mutants that retain at least some of the functions (e.g., DNA polymerase activity) of the wild-type sequence. Mutations can affect the distribution of enzyme activity under different reaction conditions (e.g., temperature, template concentration, primer concentration, etc.), for example, increasing or decreasing the polymerization rate. Mutations or sequence modifications may also affect exonuclease activity and / or the thermostability of the enzyme.

[0334] In some implementations, PCR amplification may include reactions such as, but not limited to, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, isothermal amplification, and / or loop-mediated amplification.

[0335] In some embodiments, PCR amplification uses a single primer complementary to the 3′ tag of the target DNA fragment. In some embodiments, PCR amplification uses a first primer and a second primer, wherein at least the 3′ terminal portion of the first primer is complementary to at least a portion of the 3′ tag of the target nucleic acid fragment, and wherein at least the 3′ terminal portion of the second primer displays the sequence of at least a portion of the 5′ tag of the target nucleic acid fragment. In some embodiments, the 5′ terminal portion of the first primer is not complementary to the 3′ tag of the target nucleic acid fragment, and the 5′ terminal portion of the second primer does not display the sequence of at least a portion of the 5′ tag of the target nucleic acid fragment. In some embodiments, the first primer contains a first universal sequence and / or the second primer contains a second universal sequence.

[0336] In some implementations (e.g., when PCR amplifies captured DNA), DNA ligases can be used to ligate the PCR amplification product with additional sequences. DNA ligase activity can be provided by one or more different DNA ligases. In some implementations, the DNA ligase is derived from bacteria; for example, the DNA ligase is a bacterial DNA ligase. In some implementations, the DNA ligase is derived from viruses (e.g., bacteriophages). For example, the DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Thermococcus (strain 9oN) DNA ligase (9oN™ DNA ligase, available from New England Biolabs, Ipswich, MA), and Ampligase. TM (Available from Epicentre Biotechnologies, Madison, WI). Derivatives of these derivatives (e.g., sequence-modified derivatives) and / or mutants may also be used.

[0337] In some implementations, genetic material is amplified via reverse transcription polymerase chain reaction (RT-PCR). The required reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which include, but are not limited to: M-MLV, MuLV, AMV, HIV, and ArrayScript. TM MultiScribe TM ThermoScript TM and Reverse transcriptases include enzymes I, II, III, and IV. The term "reverse transcriptase" encompasses not only naturally occurring enzymes but also all their derivatives with such modifications, as well as derivatives of naturally occurring reverse transcriptases.

[0338] Furthermore, reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases (including mutants that retain at least some functional activity (e.g., reverse transcriptase activity) of the wild-type sequence. Reverse transcriptases can be provided as part of a composition comprising other components, such as stabilizing components that enhance or improve reverse transcriptase activity, such as RNase inhibitors or DNA-dependent DNA synthesis inhibitors, such as actinomycin D. Many sequence-modified derivatives or mutants of reverse transcriptases (e.g., M-MLV), and compositions comprising unmodified and modified enzymes, are commercially available, for example, ArrayScript. TM MultiScribe TM ThermoScript TM ,as well as Enzymes I, II, III, and IV.

[0339] Some reverse transcriptases (such as avian myeloblastic leukemia virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can synthesize complementary DNA strands using both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates. Therefore, in some embodiments, the reverse transcription reaction can use enzymes (reverse transcriptases) capable of using both RNA and ssDNA as templates for the extension reaction, such as AMV or MMLV reverse transcriptases.

[0340] In some implementations, techniques well known in the art, such as, but not limited to, “TAQMAN”, are used. TM "or Or in the capillary (“ On a capillary tube, RNA and / or DNA are quantified by real-time PCR (also known as quantitative PCR or qPCR). In some embodiments, the quantification of genetic material is determined by optical absorbance and real-time PCR. In some embodiments, the quantification of genetic material is determined by digital PCR. In some embodiments, the analyzed gene can be compared with a reference nucleic acid extract (DNA and RNA) corresponding to expression (mRNA) and quantity (DNA) to compare the expression levels of the target nucleic acid.

[0341] (x) antibody

[0342] An antibody is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a Y-shaped molecular structure. Naturally occurring antibodies, called immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can also be produced synthetically. For example, recombinant antibodies, which are monoclonal antibodies, can be synthesized using synthetic genes. This involves recovering the antibody gene from a source cell, amplifying it into a suitable vector, and introducing the vector into a host to allow the host to express the recombinant antibody. Generally, recombinant antibodies can be cloned from antibody-producing animals of any species using appropriate oligonucleotide primers and / or hybridization probes. Recombinant technologies can be used to generate antibodies and antibody fragments, including in non-endogenous species.

[0343] Synthetic antibodies can be derived from non-immunoglobulin sources. For example, antibodies can be produced from nucleic acids (e.g., aptamers) and from non-immunoglobulin protein scaffolds (such as peptide aptamers), where hypervariable loops are inserted to form antigen-binding sites. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue penetration.

[0344] Antibodies may also include affimer proteins, which are affinity agents typically with a molecular weight of about 12-14 kDa. Affimer proteins typically bind to targets (e.g., target proteins) with both high affinity and high specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive proteins. In some embodiments, the affimer protein is derived from a cysteine ​​protease inhibitor and includes a peptide ring and a variable N-terminal sequence that provides a binding site.

[0345] Antibodies can also refer to “epitope-binding fragments” or “antibody fragments,” as used herein, and generally refer to a portion of a complete antibody capable of binding to the same epitope as the complete antibody, although the extent may not be identical. While many types of epitope-binding fragments are possible, epitope-binding fragments typically contain at least one pair of heavy chain variable regions and light chain variable regions (VH and VL, respectively) bound together (e.g., via disulfide bonds) to retain the antigen-binding site, and do not contain all or part of the Fc region. Epitope-binding fragments of antibodies can be obtained from a given antibody by any suitable technique (e.g., recombinant DNA technology, or enzymatic or chemical cleavage of the complete antibody) and can generally be screened for specificity in the same manner as screening for the complete antibody. In some embodiments, epitope-binding fragments include F(ab')2 fragments, Fab' fragments, Fab fragments, Fd fragments, or Fv fragments. In some implementations, the term "antibody" includes antibody-derived peptides such as single-chain variable fragments (scFv), dimer or other multimer scFv, heavy chain antibodies, single-domain antibodies, or other peptides that contain a sufficient portion of an antibody (e.g., one or more complementarity-determining regions (CDRs)) to confer specific antigen-binding ability to the peptide.

[0346] (xi) Markings, detectable markings, and optical markings

[0347] The terms “detectable label,” “optical label,” and “label” are used interchangeably herein to refer to a directly or indirectly detectable portion associated with (e.g., conjugated to) a molecule to be detected, which has, for example, a probe or analyte for in situ assay. A detectable label can be directly detectable itself (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, indirectly detectable, for example, by catalyzing a chemical change in a substrate compound or composition that is directly detectable. Detectable labels can be suitable for small-scale assays and / or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.

[0348] Detectable markers can be detected qualitatively (e.g., optically or spectroscopically) or quantitatively. Qualitative detection typically includes detection methods in which the presence or occurrence of a detectable marker is confirmed, while quantifiable detection typically includes detection methods with quantifiable (e.g., numerically reportable) values ​​such as intensity, duration, polarization, and / or other properties. In some embodiments, the detectable marker is combined with a feature or a probe associated with the feature. For example, the feature of a detectably labeled marker may include a fluorescent, colorimetric, or chemiluminescent marker attached to a bead (see, for example, Rajeswali et al., J. Microbiol Methods 139: 22-28, 2017 and Forcucci et al., J. Biomed Opt. 10: 105010, 2015, the entire contents of each of these documents are incorporated herein by reference).

[0349] In some implementations, multiple detectable tags can be bound to the feature to be detected, a probe, or a composition. For example, detectable tags can be incorporated during nucleic acid polymerization or amplification (e.g., -Tagged nucleotides, such as -dCTP). Any suitable detectable label can be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be derived from the group including: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), Alexa. 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546. Alexa 555, Alexa 568, Alexa 594. Alexa 633, Alexa 647. Alexa 660, Alexa 680, Alexa 700, Alexa 750, Allophycocyanin (APC), AMCA / AMCA-X, 7-Aminoactinomycin D (7-AAD), 7-Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Folgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBOTM -1 / BO-PRO TM -1, BOBO TM -3 / BO-PRO TM -3、 FL, TMR, TR-X, 530 / 550 558 / 568 564 / 570 581 / 591 630 / 650-X, 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson TM Calcium Green-1 TM Calcium Orange TM , White, 5-Carboxyfluorescein (5-FAM), 5-Carboxynaphthylfluorescein, 6-Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade CascadeYellow TM CCF2 (GeneBlaze) TM CFP (cyan fluorescent protein), CFP / YFP FRET, chromopycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC methyl , Cychrome (PE-Cy5), dansamide, dansamide, dansamide chloride, DAPI, dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4ANEPPS, Di-8ANEPPS, DM-NERF (4.5-6.5pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, -97 alcohol, eosin, erythrosine, ethidium bromide, ethidium homodimer-1 (EthD-1), europium(III) chloride, 5-FAM (5-carboxyfluorescein), fast blue, fluorescein-dT phosphorous amide, FITC, Fluo-3, Fluo-4 Fluoro-Gold TM (High pH), Fluoro-Gold TM(Low pH), Fluoro-Jade 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red TM (High Calcium), Fura Red TM / Fluo-3、GeneBLAzer TM (CCF2), GFP Red Shifted (rsGFP), GFP Wild-type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342&33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5-IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indo-dicarbonyl cyanine, Indo-tricarbonyl cyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751(+DNA), LDS 751(+RNA), LOLO TM -1 / LO-PRO TM -1. Fluorescent Yellow, LysoSensor TM Blue (pH 5), LysoSensor TM Green (pH 5), LysoSensor TM Yellow / Blue (pH 4.2) green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM Marina 4-Methylumbelliferone, sclerotinib, green, orange, Red, NBD (amine), Nile Red, Oregon 488, Oregon 500, Oregon 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (polydinophyll chlorophyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1, POPO TM -3 / PO-PRO TM-3. Propidium iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantum Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein (DsRed), Halogen, RH 414, Rhod-2, Rhodamine B, Rhodamine Green TM Rhodamine Red TM Rhodamine phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS -1 (high pH) -2、 -1 (high pH) -1 (low pH), Sodium Green TM , #1 #2 11. 13. 17. 45. blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC) / -X、 -X (NHS ester), thiazolinone, thiazolinone orange -1 / TO- -1、 -3 / TO- -3、TO- -5. Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (Yellow Fluorescent Protein) -1 / YO- -1、 -3 / YO- -3, 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5' 700, 5' 800, 5' 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye 640 (NHS ester) and Dy 750 (NHS ester).

[0350] As mentioned above, in some embodiments, a luminescent or chemiluminescent moiety can be detected or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. Given a suitable substrate (e.g., an oxidizing agent plus a chemiluminescent compound), these protein moieties can catalyze a chemiluminescent reaction. Many families of compounds are known to provide chemiluminescence under a variety of conditions. Non-limiting examples of families of chemiluminescent compounds include 5-amino-6,7,8-trimethoxy-2,3-dihydro-1,4-phthalazine dione luminol and dimethylamino[ca]benzo[a] analogues. These compounds can emit light in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of families of chemiluminescent compounds include, for example, 2,4,5-triphenylimidazolium, p-dimethylamino and -methoxy substituents, oxalates such as oxaloyl active ester, p-nitrophenyl, N-alkyl acridine ester, luciferin, lusterin, or acridine ester. In some implementations, the detectable marker is or includes a metal-based or mass-based marker. For example, small clusters of metal ions, metals, or semiconductors can serve as mass codes. In some instances, the metal can be selected from groups 3-15 of the periodic table, such as Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, Bi, or combinations thereof.

[0351] Example

[0352] The following examples are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0353] Example 1: Synchronous in situ rolling circle amplification (RCA)

[0354] This embodiment discloses an exemplary method for improving in-situ target detection and image analysis. Certain known methods for in-situ signal amplification using RCA produce amplified products with non-uniform intensity and size, where weak and / or large / small RCA products may fail to reach the detection threshold. In contrast, non-uniformly strong and / or non-uniformly large RCA products can lead to optical overcrowding, especially when they are close together. In both cases, sensitivity is reduced, thereby degrading the quality of image analysis. The method in this embodiment can promote the generation of uniform amplified products, thus providing a solution to the previously unmet need for improved target analysis.

[0355] Improved target detection and image analysis can be achieved by synchronizing the initiation of in situ RCA, thereby providing RCA products of uniform intensity and size generated in a coordinated reaction at multiple locations in the sample. Synchronization can be achieved by conjugating primers with a polymerase (e.g., Phi29) before adding them to a sample containing a circular template (e.g., a circular nucleic acid containing a hybridization region with the primers), as demonstrated in the following experiments.

[0356] Glutamate decarboxylase 2 (Gad2)

[0357] Fresh / frozen mouse brain samples were prepared for in situ analysis of glutamate decarboxylase 2 (Gad2) gene expression. A padlock probe for Gad2 was added to the sample, and hybridization was initiated. After probe hybridization, the sample was washed to remove unbound probes. For padlock probe ligation, T4 RNA ligase 2 (T4 Rnl2) and an RNase inhibitor were added to the sample, and the sample was incubated. Experimental and control groups were set up as shown in Table 1 below.

[0358] Table 1

[0359]

[0360] In the "complex" group, a Phi29-primer complex is pre-formed by mixing the two together in a binding buffer. The EDTA in the binding buffer ("OFF buffer") chelates Mg in the sample. 2+ The Mg 2+ It may exist as a residue from one or more earlier reactions, such as when using Mg-containing... 2+ The padlock connection of the buffer solution. Due to Mg 2+It is a cofactor of Phi29, so binding buffer can inhibit polymerase activity. The reaction mixture containing the pre-formed Phi29-primer complex was then added to the tissue sample and incubated at 37°C for 50 minutes. This incubation allows the Phi29-primer complex to diffuse through the sample and “find” circularized padlock probes that hybridize with the target nucleic acid of interest, such as Gad2 mRNA molecules in this example. Unbound complex was removed by washing three times. The remaining Phi29-primer complex in the sample was then bound with a buffer containing Mg... 2+ The tissue was contacted with a reaction buffer (pH 8.5) containing dNTPs and Tris-HCl. Inhibition of Phi29 polymerase activity was relieved, and the tissue was subjected to RCA at 37°C for 3 hours, followed by tissue washing with TE buffer.

[0361] For comparison, similar experiments were performed using Phi29 alone (“Phi29+”) or primers alone (“Primer+”) in binding buffer (Table 1). In the “Primer+” group, Phi29 was provided in the amplification reaction buffer (“Reaction Mixture” in Table 1), while the primers were provided in the binding buffer. In the “Phi29+” group, the primers were provided in the ligation reaction mixture (“Lie Mixture” in Table 1), while Phi29 was provided in the binding buffer. Note that in the “Complex” group, Phi29 and primers were pre-complexed in the binding buffer; no primers were included in the ligation mixture, and no Phi29 was included in the amplification reaction buffer. In the “Control” group, the primers were provided in the ligation mixture, Phi29 was provided in the amplification reaction buffer, and the samples were incubated with the amplification reaction buffer (“Reaction Mixture”) and then incubated with TE buffer for 3 hours before stopping. Sample processing, probe hybridization, ligation, and RCA in both experimental and control groups were performed essentially as described above. All experiments were performed in duplicate.

[0362] Prior to imaging, tissue samples were treated with Gold quench-resistant mounting medium to optimize the optical path and extend sample integrity. Imaging was performed using an Orca Fusion camera at 40x magnification, with each section having a 4×4 field of view (FOV).

[0363] Figure 2A-2B The characteristic intensity is shown between experiments using the Phi29-primer complex in OFF buffer, experiments using Phi29 alone in OFF buffer, experiments using the primer alone in OFF buffer, and the control group. Figure 2A ) and estimated feature size ( Figure 2B The result of ). Figure 2A The study shows a trend where the feature intensity detected in the "complex" group is greater than that detected in other groups. Figure 2BThe characteristic area (μm) of the "complex" group compared to other groups is shown. 2 The distribution tends to be narrower. Therefore, a trend of larger spot sizes and narrower spot size distribution was observed in the "complex" group using simultaneous RCA reactions. The control group showed a higher number of RCA products per pixel area, which may be due to the longer incubation time of Phi29 in the sample resulting in more RCA products.

[0364] In summary, these results indicate that polymerase-primer complexation prior to sample contact can influence characteristic properties by enhancing RCA product strength and reducing inhomogeneity in characteristic size distribution.

[0365] Vesicle glutamate transporter 1 (Slc17a7)

[0366] To further evaluate the impact of simultaneous amplification on characteristic properties, another set of experiments was performed using vesicle glutamate transporter 1 (Slc17a7) as the target of interest, essentially as described above for Gad2. In the Slc17a7 experiments, the Phi29-primer complex in OFF buffer was incubated with the tissue sample at 37°C for 120 minutes. Imaging was performed essentially as described above.

[0367] Figures 3A-3B The characteristic intensity and apparent size between the experimental group and the control group are shown separately. Figure 3A The trend of increasing characteristic intensity in the "complex" group relative to other groups is shown. Figure 3B The characteristic area (μm) of the "complex" group compared to the control group is shown. 2 The distribution tends to be narrower. In summary, these results are consistent with those observed for Gad2. Applying a pre-formed Phi29-primer complex to synchronize RCA appears to result in increased spot intensity and a tighter size distribution of the RCA products detected in the sample.

[0368] Time-course experiments were conducted to investigate the effectiveness of simultaneous amplification using Slc17a7 as a target by conjugating polymerase (Phi29) with primers. RCA was run for 60 minutes and 120 minutes to test the effect on characterization by increasing amplification time.

[0369] Figure 4A The study showed a trend of increased intensity of 60-minute and 120-minute RCA compared to the control group under synchronous conditions. These results are consistent with observations of synchronous amplification in the Gad2 and Slc17a7 studies described herein. Figure 4B Representative images of features detected in the control and synchronization groups 120 minutes after amplification are shown. Compared with the control group, the synchronization group showed a tendency towards more uniform feature intensity.

[0370] Figure 4C Representative images after feature detection and filtering are shown in the control and synchronous groups after 30 minutes of RCA. The synchronous group shows a trend of better detection of RCA products at locations in brain tissue where Slc17a7 expression is expected.

[0371] In summary, these results indicate that synchronizing RCA by conjugating polymerase with primers prior to sample application enhances feature intensity and reduces size inhomogeneity. Compared to the unsynchronized control group, RCA products generated using synchronized RCA exhibit improved feature properties, facilitating detection and image analysis, and ultimately leading to more accurate in-situ analyte detection.

[0372] This disclosure is not intended to limit its scope to the specific embodiments disclosed, which are provided, for example, to illustrate various aspects of the disclosure. Various modifications to the described compositions and methods will become apparent from the description and teaching herein. Such changes may be practiced without departing from the true scope and spirit of this disclosure, and such changes are intended to fall within the scope of this disclosure.

Claims

1. A method for analyzing biological samples, the method comprising: (a) Contacting the biological sample with the first reaction mixture, wherein: The biological sample may comprise cells or tissue samples, and the biological sample may contain circular nucleic acids, which may contain hybridization regions. The first reaction mixture contains polymerase. The first reaction mixture contains a chelating agent, contains no catalytic cofactor of the polymerase, and / or contains one or more non-catalytic cofactors of the polymerase, wherein the catalytic cofactors contain Mg. 2+ Co 2+ and Mn 2+ The one or more non-catalytic cofactors comprise calcium and / or strontium, and the chelating agent comprises EDTA, EGTA, BAPTA, DPTA, or a combination thereof. The circular nucleic acid or the polymerase is pre-bound to a polynucleotide containing a sequence complementary to the hybridization region, and The polymerase activity of the polymerase is inhibited; and (b) Contact the biological sample with the second reaction mixture to allow the polymerase to use the circular nucleic acid as a template to extend the polynucleotide that hybridizes with the hybridization region. The circular nucleic acid is generated in the biological sample through rolling circle amplification.

2. The method according to claim 1, wherein the first reaction mixture stabilizes the polymerase.

3. The method according to claim 1, wherein the first reaction mixture is substantially free of deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs).

4. The method of claim 1, wherein the first reaction mixture comprises a divalent cation that is not a cofactor of the polymerase.

5. The method of claim 4, wherein the divalent cation stabilizes the polymerase and / or a pre-formed complex comprising the polymerase and the polynucleotide, thereby inhibiting the polymerase activity and / or exonuclease activity of the polymerase.

6. The method of claim 1, wherein the first reaction mixture is substantially free of any catalytic cofactor of the polymerase, wherein the catalytic cofactor comprises Mg 2+ Co 2+ and Mn 2+ .

7. The method of claim 6, wherein the first reaction mixture comprises a chelating agent.

8. The method according to claim 1, wherein the 3'→5' exonuclease activity of the polymerase is inhibited in the first reaction mixture.

9. The method of claim 1, wherein the polynucleotide comprises a 3' protecting group.

10. The method of claim 9, wherein the polynucleotide is 3'-thiophosphate protected, thereby protecting the polynucleotide from 3'→5' exonuclease degradation by the polymerase while allowing initiation by the polymerase.

11. The method of claim 1, wherein the polynucleotide is a primer, and the primer is pre-bound to the polymerase in the first reaction mixture prior to contacting the biological sample in step (a).

12. The method of claim 11, further comprising removing from the biological sample one or more complexes comprising the polymerase and the primer that are not bound to the circular nucleic acid before contacting the biological sample with the second reaction mixture.

13. The method of claim 1, wherein the polynucleotide is pre-bound to the circular nucleic acid in the biological sample prior to contacting the first reaction mixture in step (a).

14. The method of claim 13, wherein the polynucleotide is a primer, and the primer is pre-bound to the circular nucleic acid in the biological sample prior to contacting the first reaction mixture in step (a).

15. The method of claim 14, wherein the hybridization region in the circular nucleic acid is a primer hybridization region that hybridizes with the primer, and the circular nucleic acid further comprises a target hybridization region that hybridizes with the target nucleic acid.

16. The method of claim 15, wherein the target nucleic acid is a DNA or RNA molecule in the biological sample, a product of the DNA or RNA molecule, a probe that binds directly or indirectly to the DNA or RNA molecule, or a product of the probe.

17. The method of claim 13, wherein the polynucleotide is a target nucleic acid, and the target nucleic acid is pre-bound to the circular nucleic acid in the biological sample before and / or during contact with the first reaction mixture in step (a).

18. The method of claim 17, wherein the target nucleic acid comprises a genomic DNA sequence, an mtDNA sequence, an RNA sequence, and / or a cDNA sequence.

19. The method of claim 13, further comprising removing one or more molecules of the polymerase that are not bound to the circular nucleic acid from the biological sample before contacting the biological sample with the second reaction mixture.

20. The method of claim 1, wherein the polymerase is not attached to the nanopore, nanopore membrane or its insulating support.

21. The method of claim 1, wherein the polymerase or the preformed complex comprising the polymerase and the polynucleotide is diffusible in the first reaction mixture and / or in contact with the biological sample.

22. The method according to claim 1, wherein the polymerase is selected from 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 (exonuclease-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, the 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, and T3 RNA polymerase.

23. The method according to claim 1, wherein the polymerase is Phi29 DNA polymerase.

24. The method of claim 23, wherein the polynucleotide is pre-bound to the single-stranded DNA-binding domain of the Phi29 DNA polymerase in the first reaction mixture.

25. The method of claim 24, wherein in step (a), the polynucleotide bound to the Phi29 DNA polymerase hybridizes with the hybridization region, and the Phi29 DNA polymerase is prevented from elongating the polynucleotide.

26. The method of claim 1, further comprising, between the contacts in steps (a) and (b), a step of removing molecules of the polymerase and / or the polynucleotide from the biological sample that are not bound to the circular nucleic acid.

27. The method of claim 1, wherein the method comprises one or more rigorous washes between the contacts in step (a) and step (b).

28. The method of claim 1, wherein the second reaction mixture comprises deoxynucleoside triphosphates (dNTPs) and / or nucleoside triphosphates (NTPs).

29. The method of claim 1, wherein the second reaction mixture comprises a catalytic cofactor of the polymerase.

30. The method of claim 1, wherein the second reaction mixture is substantially free of the polymerase and / or other polymerases.

31. The method according to claim 1, wherein the first reaction mixture and the second reaction mixture have substantially the same pH.

32. The method of claim 1, wherein the polynucleotide hybridized to the hybridization region is extended by the polymerase using the circular nucleic acid as a template to generate the rolling circle amplification product.

33. The method of claim 1, wherein the rolling circle amplification product is generated using linear rolling circle amplification (RCA), branched RCA, dendritic RCA, or any combination thereof.

34. The method according to claim 1, wherein the rolling ring amplification product is generated in situ.

35. The method of claim 1, wherein the rolling circle amplification product is immobilized in the biological sample.

36. The method of claim 1, wherein the rolling circle amplification product is crosslinked with one or more other molecules in the biological sample.

37. The method of claim 1, wherein the method includes imaging the biological sample to detect the rolling circle amplification product.

38. The method of claim 37, wherein the imaging includes detecting a signal associated with a fluorescently labeled probe that is directly or indirectly bound to the rolling circle amplification product.

39. The method of claim 1, wherein the sequence of the rolling circle amplification product is analyzed in situ in the biological sample.

40. The method of claim 39, wherein the sequence of the rolling circle amplification product is analyzed by sequential hybridization, sequencing-while-hybridization, sequencing-while-ligation, sequencing-while-synthesis, sequencing-while-binding, or a combination thereof.

41. The method of claim 39, wherein the sequence of the rolling circle amplification product comprises a barcode sequence or its complementary sequence.

42. The method of claim 1, wherein the circular nucleic acid is formed in the biological sample by a probe or probe set of the target molecule, wherein the probe or probe set is selected from padlock probes, SNAIL (nucleic acid-specific amplification via intramolecular ligation) probe set, PLAYR (RNA proximity assay) probe set, and PLISH (proximity ligation in situ hybridization) probe set.

43. The method of claim 42, wherein the target molecule is a target nucleic acid.

44. The method of claim 42, wherein the target molecule is viral DNA, bacterial DNA, or cellular DNA or RNA molecule or its product in the biological sample.

45. The method of claim 42, wherein the target molecule is genomic DNA, mitochondrial DNA, mRNA or cDNA, and the probe or probe set of the target molecule comprises a padlock probe that hybridizes to the genomic DNA, mitochondrial DNA, mRNA or cDNA.

46. ​​The method of claim 45, wherein the method comprises ligating the padlock probe, which hybridizes with the genomic DNA, mitochondrial DNA, mRNA, or cDNA, to form the circular nucleic acid.

47. The method of claim 42, wherein the target molecule is a non-nucleic acid target molecule.

48. The method of claim 47, the method comprising contacting the biological sample with a labeling agent, the labeling agent comprising (i) a binding moiety that binds directly or indirectly to the non-nucleic acid target molecule and (ii) a reporter oligonucleotide corresponding to the binding moiety and / or the non-nucleic acid target molecule.

49. The method of claim 48, wherein the probe or probe set of the non-nucleic acid target molecule comprises a padlock probe that hybridizes with the reporter oligonucleotide.

50. The method of claim 49, the method comprising connecting the padlock probe that hybridizes with the reporter oligonucleotide to form the circular nucleic acid.

51. The method of claim 1, wherein the biological sample comprises cells or cellular components.

52. The method according to claim 1, wherein the biological sample is a tissue sample.

53. The method of claim 1, wherein the biological sample is immobilized.

54. The method of claim 1, wherein the biological sample is not immobilized.

55. The method according to claim 1, wherein the biological sample is a formalin-fixed paraffin-embedded (FFPE) sample, a frozen tissue sample, or a fresh tissue sample.

56. The method of claim 1, wherein the biological sample is permeabilized.

57. The method of claim 1, wherein the biological sample is processed or transparentized.

58. The method of claim 1, wherein the biological sample is embedded in a matrix.

59. The method of claim 58, wherein the matrix is ​​a hydrogel.

60. The method of claim 58, wherein the biological sample and / or the matrix is ​​cross-linked.

61. The method according to any one of claims 1-60, wherein the signal associated with the rolling circle amplification product is amplified in situ in the biological sample.

62. The method of claim 61, wherein in situ signal amplification comprises rolling circle amplification (RCA) of probes directly or indirectly bound to the rolling circle amplification product, hybridization chain reaction (HCR) directly or indirectly on the rolling circle amplification product, linear oligonucleotide hybridization chain reaction (LO-HCR) directly or indirectly on the rolling circle amplification product, primer exchange reaction (PER) directly or indirectly on the rolling circle amplification product, branching structure assembly directly or indirectly on the rolling circle amplification product, hybridization of multiple detectable probes directly or indirectly on the rolling circle amplification product, or any combination thereof.

63. A method for analyzing biological samples, the method comprising: (a) Contacting the biological sample with the binding mixture, wherein: The biological sample may comprise cells or tissue samples, and may contain multiple circular nucleic acids, each containing a primer hybridization region. The binding mixture comprises multiple complexes, each containing a polymerase that binds to a primer, wherein the primer contains a sequence complementary to the primer hybridization region of one or more circular nucleic acids. The binding mixture comprises a chelating agent, contains no catalytic cofactor of the polymerase, and / or comprises one or more non-catalytic cofactors of the polymerase, wherein the catalytic cofactor comprises Mg 2+ Co 2+ and Mn 2+ The one or more non-catalytic cofactors comprise calcium and / or strontium, and the chelating agent comprises EDTA, EGTA, BAPTA, DPTA, or a combination thereof. The polymerase activity of the polymerase is inhibited. This allows the various complexes to hybridize with the various circular nucleic acids; and (b) Contact the biological sample with the primer extension reaction mixture to allow the polymerase to extend the primers that hybridize with the primer hybridization region. This allows for the simultaneous rolling circle amplification of the various circular nucleic acids in the biological sample.

64. The method of claim 63, wherein the binding mixture comprises a chelating agent.

65. The method of claim 63, wherein in one or more of the complexes, the primer has a 3' hydroxyl group.

66. The method of claim 63, wherein in one or more of the complexes, the primer is 3'-thiophosphate protected, thereby protecting the primer from 3'→5' exonuclease degradation by the polymerase in the complex, while allowing initiation by the polymerase.

67. The method of claim 63, wherein the primer hybridization regions of two or more of the plurality of circular nucleic acids are identical in sequence, and / or the primer hybridization regions of two or more of the plurality of circular nucleic acids are different in sequence.

68. The method of claim 63, wherein the primers in two or more of the plurality of complexes are identical in sequence, and / or the primers in two or more of the plurality of complexes are different in sequence.

69. The method of claim 63, wherein the polymerase is a Phi29 DNA polymerase, Bst polymerase, T7 RNA polymerase, or a Klenow fragment.

70. The method of claim 63, wherein the method further comprises, between the contacts in step (a) and step (b), removing one or more complexes from the biological sample that are not bound to the one or more circular nucleic acids.

71. The method of claim 63, wherein the primer extension reaction mixture comprises deoxynucleoside triphosphates (dNTPs) and one or more cations.

72. The method of claim 71, wherein the one or more cations comprise Mg 2+ Co 2+ and / or Mn 2+ .

73. The method of claim 63, wherein the primer extension reaction mixture does not contain the polymerase.

74. A method for analyzing biological samples, the method comprising: (a) Contacting the biological sample with the binding mixture, wherein: The biological sample comprises cells or tissue samples, and the biological sample contains multiple circular nucleic acids, each containing a hybridization region that hybridizes with a polynucleotide. The conjugated mixture contains a polymerase. The binding mixture comprises a chelating agent, contains no catalytic cofactor of the polymerase, and / or comprises one or more non-catalytic cofactors of the polymerase, wherein the catalytic cofactor comprises Mg 2+ Co 2+ and Mn 2+ The one or more non-catalytic cofactors comprise calcium and / or strontium, and the chelating agent comprises EDTA, EGTA, BAPTA, DPTA, or a combination thereof. The polymerase activity of the polymerase is inhibited. This allows the polymerase to bind to the various circular nucleic acids; and (b) Contact the biological sample with the primer extension reaction mixture to allow the polymerase to extend the polynucleotide that has hybridized with the hybridization region. This allows for the simultaneous rolling circle amplification (RCA) of the various circular nucleic acids in the biological sample.

75. The method of claim 74, wherein the polynucleotide is an exogenous primer in contact with the biological sample, the hybridization region is a primer hybridization region, and the circular nucleic acid comprises (i) an endogenous molecule in the biological sample, (ii) a product of the endogenous molecule in the biological sample, (iii) a probe targeting the endogenous molecule in the biological sample, and / or (iv) a product of an exogenous probe targeting the endogenous molecule in the biological sample.

76. The method of claim 74, wherein the polynucleotide comprises (i) an endogenous molecule in the biological sample, (ii) a product of the endogenous molecule in the biological sample, (iii) a probe targeting the endogenous molecule in the biological sample, and / or (iv) a product of an exogenous probe targeting the endogenous molecule in the biological sample.

77. The method according to any one of claims 63-76, further comprising terminating the RCA of the circular nucleic acid to provide a plurality of RCA products.

78. The method of claim 77, wherein the plurality of RCA products have an average diameter of 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, or 1.5 μm, or any of the foregoing values.

79. The method of claim 78, wherein the plurality of RCA products have an average diameter of less than 0.25 μm.

80. The method of claim 77, wherein the plurality of RCA products have an average length between 1kb, 2kb, 5kb, 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, or 70kb or any of the foregoing values.

81. The method of claim 77, wherein the plurality of RCA products have an average length of less than 20 kb or less than 10 kb.

82. The method of claim 77, wherein the average copy number of the unit sequence complementary to the circular nucleic acid in the plurality of RCA products is 10, 50, 100, 500, 1,000, 5,000, or 10,000 or more.

83. The method of claim 77, wherein the average copy number of the unit sequence complementary to the circular nucleic acid in the plurality of RCA products is less than 100 or less than 1,000.

84. The method of claim 77, wherein the standard deviation of the diameter of the plurality of RCA products is less than the standard deviation of the diameter of the RCA products produced using an asynchronous method.

85. The method of claim 77, wherein the average peak intensity of the plurality of RCA products is 2 to 10 times greater than the average peak intensity of the RCA products formed without synchronizing the RCAs of the plurality of circular nucleic acids in the biological sample.

86. The method of claim 77, wherein the distribution of the relative observed signal of the RCA product formed when the RCA of the multiple circular nucleic acids in the biological sample is synchronized is narrower than the distribution of the relative observed signal of the RCA product formed when the RCA of the multiple circular nucleic acids in the biological sample is not synchronized.

87. The method of claim 77, wherein the polymerase extension is performed for no more than 3 hours, no more than 2 hours, no more than 1 hour, or no more than 30 minutes.

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