Simultaneous imaging of nucleic acids and proteins in a sample

Abstract

The present disclosure relates to simultaneous imaging of nucleic acids and proteins in a sample. In particular, the present disclosure provides compositions, methods, systems, and kits for imaging at least one target protein and at least one target nucleic acid in a single sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 421,390, filed November 1, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Field The present disclosure relates to the simultaneous imaging of nucleic acids and proteins in a sample. [Background technology]

[0003] background Analysis of the abundance and distribution of nucleic acids and proteins is useful for understanding complex biological systems. Traditional strategies for detecting and characterizing nucleic acids and proteins, such as in situ hybridization, Western blot analysis, and immunofluorescence detection, have been useful for identifying target nucleic acids and proteins involved in the growth and development of whole organisms and specifically investigating the cause and progression of a wide variety of diseases. However, these strategies do not allow for the simultaneous detection of nucleic acids and proteins in a single sample. For example, the fixation and permeabilization techniques typically required for nucleic acid detection can hinder accurate protein detection in the same sample. Integrating multimodal analysis not only has the potential to enhance the discovery and explanation of potential correlations between the abundance and distribution of nucleic acids, such as mRNA, and the abundance and distribution of target proteins, but can also facilitate the identification of cellular phenotypes. Given the advantages associated with multimodal analysis, the art needs additional methods that enable the simultaneous imaging of nucleic acids and proteins. Summary of the Invention

[0004] overview The present disclosure provides a method for imaging target proteins and target nucleic acids in a sample. In certain embodiments, the method includes: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, where the protein-binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample to generate an amplicon by performing an amplification process; and (e)(i) contacting the sample with a first labeled detection probe comprising a sequence complementary to the oligonucleotide coupled to the protein-binding reagent. (ii) contacting the sample with a first bridging oligonucleotide comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein-binding reagent and contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the first bridging oligonucleotide; (f) imaging the first labeled detection probe to detect the target protein; (g) (i) contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the amplicon; or (ii) contacting the sample with a second bridging oligonucleotide comprising a sequence complementary to the sequence of the amplicon and contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0005] The present disclosure further provides a method for imaging a target protein and a target nucleic acid in a sample, the method comprising: (a) binding a protein-binding reagent coupled to an oligonucleotide to a target protein in the sample; (b) hybridizing a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, to the oligonucleotide coupled to the protein-binding reagent; (c) amplifying the target nucleic acid in the sample by performing an amplification process to generate an amplicon; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide coupled to the protein-binding reagent or hybridizes to a first bridging oligonucleotide hybridized to the oligonucleotide coupled to the protein-binding reagent; and (e) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the amplicon or hybridizes to a second oligonucleotide hybridized to the amplicon.

[0006] In certain embodiments, the amplification process is a rolling circle amplification process. In certain embodiments, the rolling circle amplification process includes (a) contacting a sample with (i) a padlock probe comprising two nucleotide sequences complementary to a target nucleic acid and (ii) a ligase to generate a circular DNA template, and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0007] In certain embodiments, rolling circle amplification is primed using a blocking oligonucleotide, eg, an exonuclease-blocking oligonucleotide, that is complementary to the oligonucleotide coupled to the protein-binding reagent.

[0008] In certain embodiments, the protein binding reagent is an antibody or an antigen-binding fragment thereof.

[0009] In certain embodiments, the target nucleic acid comprises RNA.

[0010] In certain embodiments, the target nucleic acid is (i) an oligonucleotide coupled to a protein-binding reagent and / or (ii) a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, complementary to the oligonucleotide coupled to the protein-binding reagent. In certain embodiments, the target nucleic acid is an oligonucleotide coupled to a protein-binding reagent. In certain embodiments, the target nucleic acid is a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, complementary to the oligonucleotide coupled to the protein-binding reagent.

[0011] In certain embodiments, the nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent is located at the 5'-end of the blocking oligonucleotide, e.g., the exonuclease-blocking oligonucleotide. In certain embodiments, the oligonucleotide coupled to the protein-binding reagent comprises a barcode sequence, and the blocking oligonucleotide, e.g., the exonuclease-blocking oligonucleotide, does not bind to the barcode sequence. In certain embodiments, the blocking oligonucleotide comprises one or more modified nucleotides. In certain embodiments, the one or more modified nucleotides are located at the 3'-end of the blocking oligonucleotide, e.g., the exonuclease-blocking oligonucleotide. In certain embodiments, the one or more modified nucleotides comprise a nucleotide having a phosphorothioate bond.

[0012] In certain embodiments, providing a sample includes (a) treating the sample with a fixative, (b) dehydrating the sample, and / or (c) permeabilizing the sample. In certain embodiments, the sample is post-fixed before amplifying the target nucleic acid. In certain embodiments, the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid. In certain embodiments, the sample is treated with NHS-acetate before amplifying the target nucleic acid.

[0013] In certain embodiments, at least 10 target proteins are imaged in the sample. In certain embodiments, at least 10 target nucleic acids are imaged in the sample.

[0014] In certain embodiments, the sample is a tissue sample.

[0015] The present disclosure further provides kits for carrying out the methods disclosed herein. In certain embodiments, the kits include at least one container containing a blocking oligonucleotide. [Brief explanation of the drawings]

[0016] [Figure 1] 1 provides a schematic diagram of an exemplary method according to the present disclosure. [Figure 2] 1 provides a schematic diagram of an exemplary method according to the present disclosure, the method including cyclic imaging. [Figure 3] Images of samples stained for both protein and mRNA using exemplary methods according to the present disclosure are provided. The samples were stained for CD97B protein and Cd79a RNA to label B cells, and for F4 / 80 to label macrophages. [Figure 4] 1 provides images showing degradation of the 3' end of an oligonucleotide coupled to an antibody by Phi29 and protection of the 3' end of the oligonucleotide using a blocking oligonucleotide (eg, an exonuclease-blocking oligonucleotide). [Figure 5] provides images of a sample stained for both protein and mRNA over multiple imaging cycles using an exemplary method according to the present disclosure. The sample was stained and imaged for CD22 protein and mRNA, then for CD4 protein and mRNA, and finally for F4 / 80 protein and mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description The present disclosure relates to compositions and methods for simultaneous imaging of nucleic acids and proteins in a single sample. For example, the disclosed methods include staining one or more proteins, followed by staining one or more nucleic acids, such as mRNA, in a single sample, followed by imaging of both the proteins and the nucleic acids.

[0018] For clarity, and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following sections: I. Definition; II. Compositions and methods for simultaneous imaging of nucleic acids and proteins; III. SYSTEMS AND KITS; AND IV. Exemplary Embodiments.

[0019] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which the subject matter of this disclosure belongs.The following references provide those skilled in the art with the general definitions of many terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings described below unless otherwise specified.

[0020] As used herein, in the claims and / or specification, when used in conjunction with the term "comprising," the use of the words "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0021] The term "about" or "approximately" means within an acceptable error range for a particular value determined by a person skilled in the art, which depends to some extent on the method of measurement or determination, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations depending on the technical field. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, this term can mean within one order of magnitude of a value, preferably within 5 times, and more preferably within 2 times.

[0022] The term "amplification process" generally refers to any process in which a portion of a nucleic acid is copied or replicated into at least one additional nucleic acid molecule.

[0023] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0024] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0025] As used herein, the terms "comprise(s) / include(s)," "having / has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0026] The term "coupled" can refer to connecting or binding two or more components by an interaction, bond, link, force, or connection to hold the two or more components together. In certain embodiments, the term "coupled" encompasses direct or indirect bonding, for example, where a first component is directly bonded to a second component, or where one or more intermediate molecules are disposed between the first and second components. Exemplary bonds include covalent bonds, ionic bonds, van der Waals interactions, and other bonds identifiable by one of skill in the art.

[0027] As used herein, the term "detect" or "detection" refers to the determination of the existence and / or presence of a target, e.g., a protein target or a nucleic acid target, in a limited portion of space, including, but not limited to, a sample. As used herein, the term "detect" or "detection" can include the determination of the chemical and / or biological properties of a target, including, but not limited to, its ability to interact with, particularly bind to, other compounds, its ability to activate another compound, and additional properties identifiable by one of skill in the art upon reading this disclosure. Detection can be quantitative or qualitative. Detection is "quantitative" when it refers to, relates to, or includes the measurement of the quantity or amount of a target or signal (also called quantification), including, but not limited to, any analysis designed to determine the amount or proportion of a target or signal. Detection is "qualitative" when it refers to, relates to, or includes the identification of the quality or type of a target or signal in terms of its relative abundance relative to another target or signal that is not quantified.

[0028] As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently join to form a stable double-stranded polynucleotide.

[0029] As used herein, the term "imaging" refers to microscopy. In certain embodiments, microscopy includes immunofluorescence microscopy.

[0030] As used herein, the term "individual" or "subject" refers to a vertebrate or invertebrate, e.g., a human or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, pets, etc. Non-limiting examples of non-human animal subjects include rodents, e.g., mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes, and monkeys. In certain embodiments, the individual or subject is a human.

[0031] As used herein, "label" refers to an agent that allows for direct or indirect detection. Labels include, but are not limited to, fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels. Non-limiting examples of labels are green fluorescent protein ("GFP"), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005), incorporated herein by reference). 32 P, 14 C. 125 I, 3 H and 131 Examples of suitable fluorescent substances include rare earth chelates or lucifer yellow and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial fluorescent protein enzymes (U.S. Pat. No. 4,737,456), fluorescein, 2,3-dihydrophthalazine diketone, and enzymes that generate a detectable signal, such as horseradish peroxidase (HRP), alkaline phosphatase enzymes, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD), and heterocyclic oxidases such as uricase and xanthine oxidase.

[0032] As used herein, the term "ligation" refers to the formation of a covalent bond or linkage between the ends of two or more nucleic acids.

[0033] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding, for example, variant antibodies that contain naturally occurring mutations or that may arise during the production of a monoclonal antibody preparation (such variants are generally present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the presently disclosed subject matter can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0034] The terms "nucleic acid" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by their base sequence, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. The term nucleic acid encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), e.g., messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acids described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases, including derivatized sugar or phosphate backbone bonds or chemically modified residues.

[0035] The term "plurality" refers to a number greater than one. In certain embodiments, the term "plurality of proteins" refers to several proteins greater than one. For example, without limitation, a plurality of proteins includes at least two proteins. In certain embodiments, the term "plurality of nucleic acids" refers to a number greater than one nucleic acid. For example, without limitation, a plurality of nucleic acids includes at least two nucleic acids.

[0036] The term "reverse transcription process" refers to the process of producing a complementary strand of DNA using an enzyme called reverse transcriptase.

[0037] As used herein, the term "sample" refers to any sample containing one or more individual cells. In certain embodiments, a "sample" refers to a sample of biological material obtained from a subject, e.g., a tissue biopsy or tissue sample. In certain embodiments, a sample can be obtained from a tissue, e.g., a tissue sample. Non-limiting examples of tissues include eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchus, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testis, ovary, uterus, fallopian tube, thymus, pituitary gland, thyroid gland, adrenal gland, or parathyroid gland tissue. In certain embodiments, a sample is obtained from a subject. In certain embodiments, the subject may be a human, a non-human primate, such as an ape or monkey, a livestock animal, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, a cat, a sheep, a pig, a goat, a cow, or a horse. In certain embodiments, the subject is a human. In certain embodiments, the sample may be obtained from preserved tissue, such as a fixed tissue, a frozen tissue, or a fresh tissue, such as a tissue sample. In certain embodiments, the sample that can be analyzed using the methods of the present disclosure comprises at least two or more cells. For example, and without limitation, a sample may contain about 10 or more cells, about 100 or more cells, about 1,000 or more cells, about 5,000 or more cells, about 10,000 or more cells, about 20,000 or more cells, about 30,000 or more cells, about 40,000 or more cells, about 50,000 or more cells, about 100,000 or more cells, about 150,000 or more cells, about 200,000 or more cells, about 300,000 or more cells, about 400,000 or more cells, or 500,000 or more cells.

[0038] As used herein, the term "simultaneous" is not limited to two actions occurring simultaneously in time, but rather includes two actions occurring on a single sample, for example, staining a sample for the presence of one or more target protein(s) and subsequently staining the same sample for the presence of one or more target nucleic acid(s) results in "simultaneous" staining of the sample for both the target nucleic acid(s) and the target protein(s).

[0039] As used herein, the term "specifically binds" refers to preferential binding to a target molecule, e.g., a protein or nucleic acid, compared to other molecules, e.g., proteins or nucleic acids, in a sample.

[0040] II. Compositions and Methods for Simultaneous Imaging of Nucleic Acids and Proteins The present disclosure relates to compositions and methods for simultaneous imaging of nucleic acids and proteins in a sample. The disclosed methods can be used for a variety of applications. For example, the disclosure provides a method for determining the spatial distribution of one or more proteins and one or more nucleic acids in a single sample. The disclosed methods also enable visualization of both target nucleic acids (e.g., target DNA or target RNA) and target proteins in the same sample. In certain embodiments, the sample used in the disclosed methods can contain multiple target nucleic acids, multiple target proteins, or both multiple target nucleic acids and target proteins. For example, but not limited to, the disclosed methods enable visualization of at least two or more target proteins (e.g., at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, or at least ten or more target proteins) and at least two or more target nucleic acids (e.g., at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, or at least ten or more target nucleic acids) in a single sample.

[0041] In certain embodiments, the compositions and methods of the present disclosure can also be used to determine specific characteristics of cells present in a sample. In certain embodiments, one or more target proteins can be present in cells in a sample that are different from one or more target nucleic acids. In certain embodiments, each target protein of a plurality of target proteins can be present in different cells within a sample, and / or each target nucleic acid of a plurality of target nucleic acids can be present in different cells within a sample. For example, but not limited to, the compositions and methods of the present disclosure can be used to identify different cell types present in a sample.

[0042] In certain embodiments, the disclosed compositions and methods enable multimodal analysis over time, facilitating the detection of changes in cellular characteristics and / or the presence of different cell types in samples taken at independent time points. For example, without limitation, the disclosed methods may include obtaining samples (e.g., of tissue) at different time points and visualizing one or more target nucleic acids (e.g., one or more target DNAs or RNAs) and one or more target proteins in each sample to identify changes in cellular characteristics of the tissue over time. In certain embodiments, the disclosed methods may include obtaining samples (e.g., of tissue) at different time points and visualizing one or more target nucleic acids (e.g., one or more target DNAs or RNAs) and one or more target proteins in each sample to identify changes in the types of cells present in the tissue over time.

[0043] In certain embodiments, the compositions and methods of the present disclosure can be used for diagnostic purposes. For example, but not limited to, the compositions and methods of the present disclosure can be used to determine the presence and / or absence of protein and / or nucleic acid variants associated with a disease, e.g., to diagnose a subject with a disease. In certain embodiments, the compositions and methods of the present disclosure can be used in pathological dissection of tissue, e.g., to identify a disease. Furthermore, the compositions and methods of the present disclosure enable multimodal analysis over time to facilitate diagnostic evaluation.

[0044] 1 provides a flowchart of an exemplary method of the present disclosure. In certain embodiments, the method of the present disclosure can include providing a sample, detecting one or more target proteins in the sample, detecting one or more target nucleic acids in the sample, and imaging the one or more target proteins and one or more target nucleic acids in the sample. In certain embodiments, providing the sample includes preparing the sample.

[0045] A. Sample Preparation As shown in FIG. 1, the methods of the present disclosure include preparing a sample for imaging of one or more target proteins and one or more target nucleic acids in the sample.

[0046] In certain embodiments, the sample to be analyzed can be prepared prior to detection of one or more target proteins and one or more target nucleic acids. In certain embodiments, such sample preparation can include a fixation process, a permeabilization process, a dehydration process, a rehydration process, a post-fixation process, and / or a nuclease inhibition process. In certain embodiments, the sample preparation includes a fixation process. In certain embodiments, the sample preparation includes a fixation process and a dehydration process. In certain embodiments, the sample preparation can further include a permeabilization process. In certain embodiments, the sample preparation further includes a rehydration process. In certain embodiments, the sample preparation includes a fixation process, a dehydration process, and a rehydration process. In certain embodiments, the sample preparation includes a fixation process, a dehydration process, a permeabilization process, and a rehydration process. In certain embodiments, the sample preparation further includes a post-fixation process. In certain embodiments, the sample preparation includes a fixation process, a dehydration process, a permeabilization process, a rehydration process, and a post-fixation process. In certain embodiments, the sample preparation further includes a nuclease inhibition process. In certain embodiments, sample preparation includes a fixation process, a dehydration process, a permeabilization process, a rehydration process, a post-fixation process, and a nuclease inhibition process.

[0047] In certain embodiments, the fixation process involves contacting the sample with a fixative. Non-limiting examples of fixatives include aldehydes (e.g., formaldehyde, paraformaldehyde, and glutaraldehyde), imidoesters, N-hydroxysuccinimide (NHS) esters (e.g., bis-NHS esters), alcohols (e.g., methanol and ethanol), acetone, and acetic acid. In certain embodiments, the fixative is formaldehyde. In certain embodiments, the fixative comprises two or more fixatives. For example, the fixative can comprise formaldehyde and glutaraldehyde. In certain embodiments, the sample is fixed at a final fixative concentration of about 0.1% to about 10%, about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, about 3% to about 6%, or about 3% to about 5%. In certain embodiments, the sample is fixed at a final fixative concentration of about 3% to about 6%. In certain embodiments, the sample is fixed at a final fixative concentration of about 3% to about 5%. In certain embodiments, the sample is fixed at a final fixative concentration of about 4%. In certain embodiments, the sample is fixed at a final formaldehyde concentration of about 0.1% to about 10%, about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, about 3% to about 6%, or about 3% to about 5%, e.g., about 4%. In certain embodiments, the sample can be contacted with the fixative for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, or about 5 minutes or less. In certain embodiments, the sample can be contacted with the fixative for about 5 minutes to about 1 hour, e.g., about 5 minutes to about 30 minutes. In certain embodiments, the sample can be contacted with the fixative for about 5 minutes to about 1 hour. In certain embodiments, the sample can be contacted with the fixative for about 5 minutes to about 30 minutes. In certain embodiments, the sample can be contacted with the fixative at a temperature ranging from about 0°C to 50°C, e.g., room temperature (RT). In certain embodiments, the method can include a post-fixation process. For example, the sample can be fixed after contacting the sample with the protein-binding reagent but before detecting the target nucleic acid in the sample. The fixatives disclosed herein can be used to post-fix the sample.In certain embodiments, samples can be post-fixed at a final fixative concentration of about 3% to about 6%, e.g., about 4%. In certain embodiments, the post-fixative comprises two or more fixatives, such as formaldehyde (e.g., at a concentration of about 3% to about 6%, e.g., about 4%) and glutaraldehyde (e.g., at a concentration of about 0.5% to about 2%, e.g., about 1%).

[0048] In certain embodiments, sample preparation can include a dehydration process. In certain embodiments, the dehydration process results in a reduction in the amount of water in the sample. In certain embodiments, such dehydration is achieved by contacting the sample with an alcohol, e.g., a series of alcohols. In certain embodiments, dehydration includes contacting the sample with solutions of increasing alcohol content. For example, without limitation, the dehydration process can include contacting the sample with an ethanol series, where the sample is contacted with ethanol solutions of increasing concentrations. In certain embodiments, the sample can be contacted with each concentration of alcohol (e.g., ethanol) in the series (e.g., ethanol series) for about 0.5 minutes to about 1 hour, e.g., about 1 minute. In certain embodiments, the ethanol series includes 70%, 75%, 80%, 85%, 90%, 95%, and / or 100% ethanol, e.g., 70%, 85%, and / or 100% ethanol. In certain embodiments, the ethanol series includes 70%, 85%, and 100% ethanol. In certain embodiments, the dehydration process includes contacting the sample with 70% ethanol, followed by contacting the sample with 85% ethanol, and then contacting the sample with 100% ethanol. In certain embodiments, the sample can be contacted with, for example, an alcohol series at a temperature ranging from about 0°C to 50°C, e.g., room temperature (RT). In certain embodiments, the sample can be contacted with different concentrations of alcohol, e.g., ethanol, for about 0.5 minutes to about 1 hour, e.g., about 0.5 minutes to about 10 minutes, e.g., about 1 minute.

[0049] In certain embodiments, the sample can be permeabilized before contacting the sample with a protein-binding reagent used to detect the target protein. For example, but not limited to, the sample can be fixed and then permeabilized before contacting the sample with the protein-binding reagent. Techniques for permeabilizing cells are known in the art, and one of ordinary skill in the art would be able to evaluate the suitability of a particular technique for use in connection with the methods of the present disclosure. Non-limiting examples of reagents for permeabilizing cells include detergents (e.g., saponin, Tween-20, and Triton X-100) and fixatives (e.g., acetone, methanol, and ethanol). For example, but not limited to, the sample can be permeabilized with an alcohol, such as methanol, and / or a detergent, such as Triton X-100. In certain embodiments, the sample is permeabilized with a detergent. In certain embodiments, the sample is permeabilized with a fixative. In certain embodiments, the permeabilization reagent can be used at a concentration of about 0.1% to about 10%, e.g., about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%. In certain embodiments, the permeabilization reagent (e.g., detergent) can be used at a concentration of about 0.1% to about 1.0%, e.g., 0.5%. In certain embodiments, permeabilization can be performed by contacting the fixed sample with 0.5% Triton X-100. In certain embodiments, the sample can be contacted with the permeabilization reagent for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, or about 5 minutes or less. In certain embodiments, the sample can be contacted with the permeabilization reagent, e.g., Triton X-100, for about 20 minutes. In certain embodiments, the sample can be contacted with the permeabilization reagent, e.g., Triton X-100, at a temperature ranging from about 0°C to 50°C, e.g., room temperature (RT).

[0050] B. Protein staining As shown in Figure 1, the methods of the present disclosure can include staining and imaging one or more target proteins in a sample. For example, without limitation, the methods of the present disclosure can include staining and imaging one target protein, two or more target proteins, three or more target proteins, four or more target proteins, five or more target proteins, six or more target proteins, seven or more target proteins, eight or more target proteins, nine or more target proteins, or ten or more target proteins. In certain embodiments, the methods of the present disclosure can include staining and imaging at least five target proteins, at least ten target proteins, at least fifteen target proteins, at least twenty target proteins, at least thirty-five target proteins, at least forty-five target proteins, at least forty-five target proteins, at least fifty target proteins, at least fifty-five target proteins, at least sixty-five target proteins, at least sixty-five target proteins, at least seventy-five target proteins, at least seventy-five target proteins, at least eighty-five target proteins, at least eighty-five target proteins, at least ninety-five target proteins, at least ninety-five target proteins, at least ninety-five target proteins, at least one hundred target proteins, at least one hundred five target proteins, at least one hundred ten ... At least 115 target proteins, at least 120 target proteins, at least 125 target proteins, at least 130 target proteins, at least 135 target proteins, at least 140 target proteins, at least 145 target proteins, at least 150 target proteins, at least 155 target proteins, at least 160 target proteins, at least 165 target proteins, at least 170 target proteins, at least 175 target proteins, at least 180 target proteins, at least 185 target proteins, at least 190 target proteins, at least 195 target proteins, or at least 200 target proteins are imaged in a single sample using the methods of the present disclosure. In certain embodiments, about 15 to about 100 target proteins are stained and imaged in a single sample using the methods of the present disclosure.In certain embodiments, about 15 to about 30 target proteins are stained and imaged in a single sample using the methods of the present disclosure. In certain embodiments, one or more target proteins may be present in the same cell within the sample. Alternatively, one or more target proteins may be present in different cells (e.g., different cell types) within the sample (e.g., as shown in Figures 3, 4, and 5).

[0051] In certain embodiments, proteins that can be stained and imaged using the methods of the present disclosure include any proteins present in or on the surface of a cell.For example, but not limited to, the target protein can be an intracellular protein, an extracellular protein, or a transmembrane protein.In certain embodiments, the target protein is a mutant form of a protein or a wild-type form of a protein.In certain embodiments, the target protein is an exogenous protein, for example, a protein that is exogenously expressed in a sample.In certain embodiments, the target protein is an endogenous protein, for example, a protein that is endogenously expressed in a sample.In certain embodiments, the target protein is a post-translationally modified form of a protein.

[0052] In certain embodiments, staining and imaging one or more protein targets in a sample may include contacting the sample with a reagent that binds to the target protein in the sample (also referred to herein as a "protein binding reagent"). In certain embodiments, the protein binding reagent specifically binds to the target protein, e.g., a reagent that specifically binds to the target protein in cells in the sample. In certain embodiments, the reagent that binds to the target protein allows for imaging of the target protein. In certain embodiments, the reagent that binds to the target protein allows for quantitative analysis of the target protein. Non-limiting examples of protein binding reagents include antibodies (or antigen-binding fragments thereof), aptamers, affimers, peptides, and small molecules.

[0053] In certain embodiments, the protein binding reagent is an antibody (or antigen-binding fragment thereof) specific for the target protein. In certain embodiments, the affinity between the antibody (or antigen-binding fragment thereof) and the target protein is such that the affinity is such that the dissociation constant (K) is ≦1 M, ≦100 mM, ≦10 mM, ≦1 mM, ≦100 μM, ≦10 μM, ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM. d In certain embodiments, the antibody specific for the target protein is characterized by a -3 Less than or equal to 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 K of M d In certain embodiments, the antibody may be an antibody fragment as described herein. For example, but not limited to, the antibody may be a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, diabody, or single domain antibody. In certain embodiments, the antibody may be a humanized antibody or a chimeric antibody.

[0054] In certain embodiments, the protein-binding reagent is coupled to an oligonucleotide. In certain embodiments, the antibody is coupled to an oligonucleotide, also referred to herein as an "antibody-oligonucleotide conjugate." Exemplary antibody-oligonucleotide conjugates are shown and used in Figures 1, 2, 4, and 5. In certain embodiments, the oligonucleotide coupled to the protein-binding reagent (e.g., an antibody or antigen-binding fragment thereof) can be about 5 to about 200 nucleotides in length, e.g., about 5 to about 150 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 50 nucleotides in length, about 10 to about 150 nucleotides in length, about 20 to about 100 nucleotides in length, or about 10 to about 100 nucleotides in length. In certain embodiments, the oligonucleotide index sequence coupled to the antibody can be about 5 to about 50 nucleotides in length, e.g., about 5 to about 45 nucleotides, about 5 to about 40 nucleotides, about 5 to about 35 nucleotides, about 5 to about 30 nucleotides, about 5 to about 25 nucleotides, about 5 to about 20 nucleotides, about 5 to about 15 nucleotides, about 5 to about 10 nucleotides, about 10 to about 50 nucleotides, about 15 to about 50 nucleotides, about 20 to about 50 nucleotides, about 25 to about 50 nucleotides, about 30 to about 50 nucleotides, about 35 to about 50 nucleotides, about 40 to about 50 nucleotides, about 10 to about 40 nucleotides, or about 10 to about 30 nucleotides in length. TM(Trademark) antibody (BioLegend, San Diego, CA). In certain embodiments, the use of antibody-oligonucleotide conjugates allows for protein imaging by binding a detection probe to the oligonucleotide conjugated to the antibody and / or a bridging oligonucleotide that binds to the oligonucleotide conjugated to the antibody. In certain embodiments, the use of antibody-oligonucleotide conjugates allows for protein imaging by amplifying the oligonucleotide conjugated to the antibody, followed by binding a detection probe to the amplicon and / or detecting a bridging oligonucleotide that binds to the resulting amplicon. Alternatively, the use of antibody-oligonucleotide conjugates allows for protein imaging by amplifying a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that binds to the oligonucleotide conjugated to the antibody, followed by binding a detection probe to the amplicon and / or detecting a bridging oligonucleotide that binds to the resulting amplicon.

[0055] In certain embodiments, the oligonucleotide conjugated to the protein-binding reagent can include a barcode, e.g., as shown in FIG. 1. In certain embodiments, the barcode is a unique nucleotide sequence that can be used to identify the antibody coupled to the barcode. In certain embodiments, the barcode is about 10 to about 50 nucleotides in length, e.g., about 10 to about 30 nucleotides in length. In certain embodiments, the barcode is about 10 to about 20 nucleotides in length. In certain embodiments, the barcode is about 15 nucleotides in length. In certain embodiments, a detection probe can be bound to the barcode, e.g., the detection probe can comprise a sequence at least partially complementary to the barcode. Alternatively or additionally, the detection probe can be bound to a bridging oligonucleotide that is bound to the barcode, e.g., the detection probe can comprise a sequence at least partially complementary to a bridging oligonucleotide that is at least partially complementary to the barcode, as shown in FIG. 1.

[0056] In certain embodiments, the oligonucleotide conjugated to the protein-binding reagent can further comprise a primer sequence, for example, as shown in FIG. 1. In certain embodiments, the primer sequence can be used for amplification of the oligonucleotide. For example, but not by way of limitation, a primer sequence present in the oligonucleotide coupled to the protein-binding reagent can be used to amplify the oligonucleotide during an amplification reaction used to amplify a target nucleic acid in a sample. In certain embodiments, the primer sequence is about 10 to about 50 nucleotides in length, e.g., about 10 to about 30 nucleotides in length. In certain embodiments, the barcode is about 20 nucleotides in length.

[0057] In certain embodiments, multiple protein binding reagents for binding multiple different target proteins can be used in the present disclosure.For example, but not limited to, each protein binding reagent, such as each antibody-oligonucleotide conjugate, specifically binds to a single target protein.In certain embodiments, two or more protein binding reagents, three or more protein binding reagents, four or more protein binding reagents, five or more protein binding reagents, six or more protein binding reagents, seven or more protein binding reagents, eight or more protein binding reagents, nine or more protein binding reagents, or ten or more protein binding reagents can be used in the present disclosure, and each protein binding reagent specifically binds to a single target protein.In certain embodiments, the number of protein binding reagents is at least 5, at least 10, at least 15, at least 20, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 120, at least 140, at least 160, at least 180, at least 190, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 420, at least 430, at least 440, at least 450, at least 460, at least 470, at least 480, at least 490, at least 500, at least 510, at least 520, at least 530, at least 540, at least 550, at least 560, at least 570, at least 580, at least 590, at least 600, at least 610, at least 620, at least 630, at least 640, at least 650, at least 700, at least 750, at least 800, at least 850 At least 15 protein binding reagents, at least 120 protein binding reagents, at least 125 protein binding reagents, at least 130 protein binding reagents, at least 135 protein binding reagents, at least 140 protein binding reagents, at least 145 protein binding reagents, at least 150 protein binding reagents, at least 155 protein binding reagents, at least 160 protein binding reagents, at least 165 protein binding reagents, at least 170 protein binding reagents, at least 175 protein binding reagents, at least 180 protein binding reagents, at least 185 protein binding reagents, at least 190 protein binding reagents, at least 195 protein binding reagents, or at least 200 protein binding reagents can be used to detect target proteins in a single sample by the methods of the present disclosure. In certain embodiments, about 15 to about 100 protein binding reagents can be used to detect target proteins in a single sample by the methods of the present disclosure. In certain embodiments, about 15 to about 30 protein binding reagents can be used to detect target proteins in a single sample by the methods of the present disclosure.The use of multiple protein binding reagents, such as antibody-oligonucleotide conjugates, can allow for the imaging of multiple proteins in a single sample and also allow for analysis of the spatial location of each protein relative to one another.

[0058] In certain embodiments, the sample can be contacted with a protein-binding reagent, such as an antibody specific to the target protein, for a time and under conditions that support specific binding of the protein-binding reagent to the target protein. In certain embodiments, the sample can be contacted with a protein-binding reagent, such as an antibody specific to the target protein, for about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less, about 20 hours or less, about 19 hours or less, about 18 hours or less, about 17 hours or less, about 16 hours or less, about 15 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less, about 11 hours or less, about 10 hours or less, about 9 hours or less, about 8 hours or less, about 7 hours or less, about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, or about 60 minutes or less. In certain embodiments, the sample can be contacted with a protein-binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 48 hours, e.g., about 1 to about 42 hours, about 1 to about 40 hours, about 1 to about 38 hours, about 1 to about 36 hours, about 1 to about 34 hours, about 1 to about 32 hours, about 1 to about 30 hours, about 1 to about 28 hours, about 1 to about 26 hours, about 1 to about 24 hours, about 1 to about 22 hours, about 1 to about 20 hours, about 1 to about 18 hours, about 1 to about 16 hours, about 1 to about 14 hours, about 1 to about 12 hours, about 1 to about 10 hours, about 1 to about 8 hours, about 1 to about 6 hours, about 1 to about 4 hours, or about 1 to about 2 hours. In certain embodiments, the sample can be contacted with a protein-binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 24 hours. In certain embodiments, the sample can be contacted with the protein binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 20 hours. In certain embodiments, the sample can be contacted with the protein binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 16 hours. In certain embodiments, the sample can be contacted with the protein binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 12 hours. In certain embodiments, the sample can be contacted with the protein binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 8 hours. In certain embodiments, the sample can be contacted with the protein binding reagent, e.g., an antibody specific for the target protein, for about 1 to about 3 hours.In certain embodiments, the sample can be contacted with a protein binding reagent, e.g., an antibody specific for the target protein, at a temperature ranging from about 0° C. to 50° C., e.g., room temperature (RT). In certain embodiments, the sample can be contacted with a protein binding reagent, e.g., an antibody specific for the target protein, at a temperature ranging from about 0° C. to 10° C., e.g., 4° C.

[0059] In certain embodiments, contacting the protein-binding reagent with the sample may include a blocking step to reduce background noise. In certain embodiments, the blocking step includes contacting the sample with one or more oligonucleotides, e.g., blocking oligonucleotides, that are complementary to one or more nucleotide sequences, e.g., two or more, three or more, four or more, or five or more nucleotide sequences, present in the oligonucleotides conjugated to the antibodies. In certain embodiments, the blocking oligonucleotides may be complementary to conserved sequences present in the oligonucleotides coupled to the protein-binding reagents, e.g., conserved sequences present in each of the oligonucleotides conjugated to the multiple protein-binding reagents used in the disclosed methods. In certain embodiments, the blocking oligonucleotides are about 10 to about 100 nucleotides in length, e.g., about 10 to about 50 nucleotides in length. In certain embodiments, the blocking step can be performed before incubating the protein-binding reagent with the sample. Alternatively, the blocking step can be performed simultaneously with incubating the protein-binding reagent with the sample. In certain embodiments, the blocking step can be performed after incubating the protein-binding reagent with the sample. In certain embodiments, the blocking step is carried out for about 5 minutes to 24 hours, for example, about 15 minutes to about 24 hours, about 15 minutes to about 20 hours, about 15 minutes to about 16 hours, about 15 minutes to about 12 hours, about 15 minutes to about 8 hours, about 15 minutes to about 4 hours, about 15 minutes to about 1 hour, about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 3 hours to about 24 hours, about 4 hours to about 24 hours, about 5 hours to about 24 hours, The blocking step can be carried out for 4 hours, about 6 hours to about 24 hours, about 7 hours to about 24 hours, about 8 hours to about 24 hours, about 9 hours to about 24 hours, about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 14 hours to about 24 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, about 20 hours to about 24 hours, about 22 hours to about 24 hours, about 2 hours to about 12 hours, or about 2 hours to about 6 hours. In certain embodiments, the blocking step can be carried out for about 1 to about 24 hours. In certain embodiments, the blocking step can be carried out for about 1 to about 20 hours.In certain embodiments, the blocking step can be carried out for about 1 to about 16 hours. In certain embodiments, the blocking step can be carried out for about 1 to about 12 hours. In certain embodiments, the blocking step can be carried out for about 1 to about 8 hours. In certain embodiments, the blocking step can be carried out for about 1 to about 3 hours. In certain embodiments, the blocking step can be carried out at a temperature ranging from about 0°C to 25°C, for example, 4°C or room temperature.

[0060] In certain embodiments, samples analyzed by the methods of the present disclosure can be crosslinked. For example, without limitation, samples can be crosslinked prior to detecting nucleic acids in the sample. Crosslinking of a sample can be achieved by exposing cells to any crosslinking agent. In certain embodiments, crosslinking can be performed using a fixative such as those disclosed herein. In certain embodiments, crosslinking can be performed by exposing the sample to formaldehyde. In certain embodiments, crosslinking can be performed by exposing the sample to glutaraldehyde. In certain embodiments, crosslinking can be performed by exposing the sample to a solution containing formaldehyde and glutaraldehyde. In certain embodiments, crosslinking can be performed by exposing the sample to a solution containing about 1% to about 10%, e.g., about 4%, paraformaldehyde and about 0.1% to about 5%, e.g., about 1%, glutaraldehyde. In certain embodiments, crosslinking is performed as a post-fixation step as described herein. In certain embodiments, crosslinking is performed to maintain binding of target proteins to protein-binding reagents during sample processing steps required to prepare the sample for nucleic acid detection. For example, but not by way of limitation, HCl, which is used to permeabilize cells for nucleic acid detection, abolishes the binding of target proteins to protein-binding reagents; cross-linking prior to HCl treatment prevents this from happening, thereby allowing for the simultaneous detection of proteins and nucleic acids in a single sample.

[0061] C. Nucleic acid staining In certain embodiments, the disclosed methods can further include staining and imaging one or more nucleic acids in a sample. For example, without limitation, the disclosed methods can further include staining and / or imaging one or more nucleic acids in a sample before or after staining and / or imaging one or more proteins in a sample. In certain embodiments, the disclosed methods can further include staining one or more nucleic acids in a sample before or after staining one or more proteins in a sample. In certain embodiments, the disclosed methods can further include imaging one or more nucleic acids in a sample before or after imaging one or more proteins in a sample. In certain embodiments, the disclosed methods can further include staining one or more nucleic acids in a sample after staining one or more proteins in a sample, as described in Example 1. In certain embodiments, the disclosed methods can further include imaging one or more nucleic acids in a sample after imaging one or more proteins in a sample, as described in Example 1.

[0062] In certain embodiments, the disclosed methods further comprise staining and imaging one target nucleic acid, two or more target nucleic acids, three or more target nucleic acids, four or more target nucleic acids, five or more target nucleic acids, six or more target nucleic acids, seven or more target nucleic acids, eight or more target nucleic acids, nine or more target nucleic acids, or ten or more target nucleic acids. In certain embodiments, at least five target nucleic acids, at least 10 target nucleic acids, at least 15 target nucleic acids, at least 20 target nucleic acids, at least 35 target nucleic acids, at least 40 target nucleic acids, at least 45 target nucleic acids, at least 50 target nucleic acids, at least 55 target nucleic acids, at least 60 target nucleic acids, at least 65 target nucleic acids, at least 70 target nucleic acids, at least 75 target nucleic acids, at least 80 target nucleic acids, at least 85 target nucleic acids, at least 90 target nucleic acids, at least 95 target nucleic acids, or at least 100 target nucleic acids are stained and imaged in a single sample using the disclosed methods. In certain embodiments, at least about 100 target nucleic acids, at least about 200 target nucleic acids, at least about 300 target nucleic acids, at least about 400 target nucleic acids, at least about 500 target nucleic acids, at least about 600 target nucleic acids, at least about 700 target nucleic acids, at least about 800 target nucleic acids, at least about 900 target nucleic acids, at least about 1,000 target nucleic acids, at least about 1,500 target nucleic acids, at least about 2,000 target nucleic acids, at least about 2,500 target nucleic acids, at least about 3,000 target nucleic acids, at least about 3,500 target nucleic acids, at least about 4,000 target nucleic acids, at least about 4,500 target nucleic acids, or at least about 5,000 target nucleic acids are stained and imaged in a single sample using the methods of the present disclosure. In certain embodiments, about 100 target nucleic acids are stained and imaged in a single sample using the methods of the present disclosure.

[0063] In certain embodiments, the target nucleic acid can be any nucleic acid molecule (e.g., a DNA molecule or an RNA molecule) present in the sample to be analyzed. In certain embodiments, the target nucleic acid is mRNA. In certain embodiments, the target nucleic acid is a non-coding RNA, such as tRNA, rRNA, or microRNA (miRNA). In certain embodiments, the target nucleic acid is a genomic DNA molecule. In certain embodiments, the target nucleic acid is genomic DNA. In certain embodiments, the target nucleic acid is an exogenous nucleic acid, such as a viral nucleic acid. In certain embodiments, the target nucleic acid is a mutant of the target nucleic acid. In certain embodiments, the target nucleic acid is an engineered barcode RNA. In certain embodiments, the target nucleic acid is a guide RNA that can be used, for example, in gene editing technology. In certain embodiments, the target nucleic acid is a guide RNA for use with a CRISPR enzyme.

[0064] In certain embodiments, the target nucleic acid is an oligonucleotide coupled to a protein-binding reagent and / or a blocking oligonucleotide complementary to the oligonucleotide coupled to the protein-binding reagent, such as an exonuclease-blocking oligonucleotide. For example, but not limited to, the target nucleic acid is an oligonucleotide coupled to a protein-binding reagent used in a method for detecting a target protein, such as an oligonucleotide of an antibody-oligonucleotide conjugate. In certain embodiments, the target nucleic acid is a blocking oligonucleotide complementary to the oligonucleotide coupled to the protein-binding reagent, such as an exonuclease-blocking oligonucleotide. In certain embodiments, the target nucleic acid is a blocking oligonucleotide complementary to the oligonucleotide of an antibody-oligonucleotide conjugate, such as an exonuclease-blocking oligonucleotide.

[0065] The target nucleic acid that is stained and imaged using the disclosed method can have various lengths.In certain embodiments, the target nucleic acid is about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 60 or more, about 80 or more, about 100 or more, about 150 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more nucleotides in length.In certain embodiments, the target nucleic acid comprises 10 or more consecutive nucleotides of a known sequence. For example, but not by way of limitation, a target nucleic acid can comprise about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 60 or more, about 80 or more, about 100 or more, about 150 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more consecutive nucleotides of a known sequence. In certain embodiments, the target nucleic acid comprises 10 or more consecutive nucleotides of an unknown sequence.

[0066] In certain embodiments, staining and imaging of nucleic acids in a sample involves performing an amplification process to amplify the target nucleic acid. Suitable nucleic acid amplification methods known to those skilled in the art can be evaluated to identify a suitable strategy for amplifying the target nucleic acid. Non-limiting examples of such amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, rolling circle amplification (RCA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR), nicking endonuclease-assisted nanoparticle activation (NENNA), and ligase chain reaction (LCR). Fakruddin et al., J. Pharm. Bioallied. Sci. 5(4):245-252 (2013) and Yan et al., Mol. BioSyst. 10:970-1003 (2014) disclose additional amplification methods for use in the present disclosure, the contents of each of which are herein incorporated by reference in their entirety. In certain embodiments, the amplification process is RCA.

[0067] In certain embodiments, the amplification process includes an amplification reaction using a polymerase with exonuclease activity, such as a polymerase with 3' to 5' exonuclease activity. In certain embodiments, the amplification reaction includes an amplification reaction comprising a polymerase with strand displacement activity. Non-limiting examples of polymerases with strand displacement and / or exonuclease activity include Bst polymerase, DNA polymerase ε (Polε), DNA polymerase δ (Polδ), and Phi29 polymerase, as well as derivatives thereof. In certain embodiments, a derivative of Phi29 polymerase includes a Phi29 polymerase that includes one or more modifications, e.g., amino acid mutations, compared to wild-type Phi29 polymerase. In certain embodiments, the Phi29 polymerase is EquiPhi29™ (Thermo Scientific). In certain embodiments, the amplification process includes an amplification reaction comprising Phi29 polymerase.

[0068] In certain embodiments, performing an amplification process involves contacting a sample with reagents necessary for the amplification process and carrying out the process under conditions suitable for amplifying the target nucleic acid. Non-limiting examples of such reagents include polymerases, reverse transcriptases, nucleoside triphosphates or NTP analogs, primers, probes, primers, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers, or labels. In certain embodiments, additional reagents can include RNAse inhibitors to protect the integrity of RNA in the sample, for example, by inhibiting the activity of RNase A, B, and / or C.

[0069] In certain embodiments, nucleic acid staining and imaging in a sample can use padlock probes, i.e., linear probes that can be converted into circular DNA molecules by ligation upon hybridization to a target nucleic acid, such as a target mRNA. In certain embodiments, ligation is performed using a ligase that ligates single-stranded DNA, such as Splint® ligase. Further disclosure regarding the use of padlock oligonucleotides is provided in Sountoulidis et al., PLoS Biology 18(11):e3000675 (2020), the contents of which are incorporated herein by reference.

[0070] In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur in a hybridization buffer for a period of about 1 hour to about 24 hours, e.g., about 2 hours to about 20 hours, about 2 hours to about 16 hours, or about 2 hours to about 12 hours. In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur in a hybridization buffer for a period of about 2 hours to about 20 hours. In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur at a temperature ranging from about 20°C to about 60°C, e.g., about 40°C or about 45°C. In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur at a temperature ranging from about 30°C or about 50°C. In certain embodiments, the concentration of the oligonucleotide in the hybridization buffer is about 1 nM to about 1,000 nM, e.g., about 1 nM to about 900 nM, about 1 nM to about 800 nM, about 1 nM to about 700 nM, about 1 nM to about 600 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 200 nM, about 1 nM to about 100 nM, or about 1 nM to about 50 nM. In certain embodiments, the concentration of the oligonucleotide in the hybridization buffer is about 1 nM to about 50 nM. In certain embodiments, the hybridization buffer may contain formamide, e.g., about 10% to about 30% formamide.

[0071] In certain embodiments, ligation using a ligase, such as Splint® ligase, can be performed after hybridization of the padlock oligonucleotide with the target nucleic acid. In certain embodiments, ligation can be performed for about 1 hour to about 24 hours, e.g., about 1 hour to about 20 hours, about 1 hour to about 16 hours, about 1 hour to about 12 hours, about 5 hours to about 20 hours, or about 5 hours to about 16 hours. In certain embodiments, ligation can be performed for about 1 hour to about 24 hours. In certain embodiments, ligation can be performed for about 1 hour to about 20 hours. In certain embodiments, ligation can be performed for about 1 hour to about 16 hours. In certain embodiments, ligation can be performed for about 1 hour to about 12 hours. In certain embodiments, ligation can be performed at a temperature ranging from about 10°C to 60°C, e.g., 25°C, 30°C, or 40°C. In certain embodiments, ligation can be performed at a temperature ranging from about 20°C to 50°C.

[0072] In certain embodiments, the present disclosure can use multiple oligonucleotides, e.g., padlock oligonucleotides, to detect multiple different target nucleic acids. For example, but not limited to, each oligonucleotide, e.g., padlock oligonucleotide, specifically binds to a single target nucleic acid. For example, but not limited to, two or more padlock oligonucleotides, three or more padlock oligonucleotides, four or more padlock oligonucleotides, five or more padlock oligonucleotides, six or more padlock oligonucleotides, seven or more padlock oligonucleotides, eight or more padlock oligonucleotides, nine or more padlock oligonucleotides, or ten or more padlock oligonucleotides can be used in the present disclosure, where each padlock oligonucleotide specifically binds to a single target nucleic acid. In certain embodiments, the disclosed methods can include contacting a sample with two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more padlock probes, where each padlock oligonucleotide specifically binds to a single target nucleic acid. In certain embodiments, one or more of the padlock oligonucleotides specifically binds to an oligonucleotide coupled to a protein-binding reagent used to detect a target protein in the sample. In certain embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more padlock probes can each bind to a different oligonucleotide of a protein binding reagent, e.g., a plurality of protein binding reagents.

[0073] Alternatively and / or additionally, other techniques for detecting target nucleic acids can be used in the present disclosure. In certain embodiments, other techniques that can be used in the present disclosure for detecting target nucleic acids include techniques that generate templates, such as circular DNA templates, that can be amplified using a polymerase with strand displacement activity, such as Phi29 polymerase or its derivatives. In certain embodiments, other techniques that can be used in the present disclosure for detecting target nucleic acids include techniques that use amplification with a polymerase with strand displacement activity, such as Phi29 polymerase-based amplification. For example, but not limited to, other techniques that can be used in the present disclosure for detecting target nucleic acids include SNAIL, RCP-FISH, DARTFISH, and OPS. In certain embodiments, SNAIL involves the use of two oligonucleotides, one of which binds to the target nucleic acid and contains a ligation junction, and the second of which binds to the target nucleic acid and the ligation junction of the first oligonucleotide, followed by ligation to generate a circular DNA molecule. In certain embodiments, DARTFISH involves the generation of cDNA complementary to the target nucleic acid, followed by the use of a padlock probe that binds to the cDNA and ligation to generate a circular DNA molecule.

[0074] In certain embodiments, the resulting circularized single-stranded DNA molecules can then be amplified in an RCA process using a polymerase with strand displacement activity, such as Phi29 polymerase or its derivatives. This RCA process generates single-stranded DNA molecules containing multiple tandem repeats of the original target nucleic acid sequence, referred to herein as "RCA amplicons." In certain embodiments, the RCA process can be carried out for at least about 1 hour, at least about 5 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours. In certain embodiments, the RCA process can be carried out for at least about 24 hours. In certain embodiments, the RCA process can be carried out for at least about 20 hours. In certain embodiments, the RCA process can be carried out for at least about 16 hours. In certain embodiments, the RCA process can be carried out for at least about 12 hours. In certain embodiments, the RCA process can be carried out for at least about 24 hours. In certain embodiments, the RCA process can be carried out for about 1 to about 24 hours, about 1 to about 22 hours, about 1 to about 20 hours, about 1 to about 18 hours, about 1 to about 16 hours, about 1 to about 14 hours, about 1 to about 12 hours, about 1 to about 10 hours, about 1 to about 8 hours, about 1 to about 6 hours, about 1 to about 4 hours, or about 1 to about 2 hours. In certain embodiments, the RCA process can be carried out for about 8 to about 24 hours. In certain embodiments, the RCA process can be carried out for about 12 to about 24 hours. In certain embodiments, the RCA process can be carried out at a temperature ranging from about 0°C to 50°C, for example, about 30°C.

[0075] In certain embodiments, a sample can be treated with a reagent that prevents the degradation of oligonucleotides conjugated to a protein-binding reagent by preventing the exonuclease activity of a polymerase used in an amplification process (e.g., an RCA process). For example, but not limited to, a sample can be treated with a reagent that prevents the degradation of oligonucleotides conjugated to a protein-binding reagent by preventing the exonuclease activity, 3' to 5' exonuclease activity, of a polymerase (e.g., a polymerase with strand displacement activity, such as Phi29 polymerase or its derivatives) used in an amplification process (e.g., an RCA process). For example, but not limited to, a sample can be treated with a reagent that prevents the degradation of oligonucleotides of an antibody-oligonucleotide conjugate (e.g., the 3' end of an oligonucleotide of an antibody-oligonucleotide conjugate) by preventing the 3' to 5' exonuclease activity of Phi29 polymerase or its derivatives used in an amplification process (e.g., an RCA process).

[0076] In certain embodiments, a blocking oligonucleotide, also referred to herein as a "Phi29 blocking oligonucleotide" or "exonuclease blocking oligonucleotide," that binds to an oligonucleotide conjugated to a protein-binding reagent can be used to prevent degradation of the oligonucleotide conjugated to the protein-binding reagent by a polymerase with strand displacement activity, such as Phi29 polymerase or a derivative thereof. In certain embodiments, a sample is contacted with the exonuclease blocking oligonucleotide before performing an amplification process. In certain embodiments, a sample is contacted with the exonuclease blocking oligonucleotide before contacting the sample with a polymerase with strand displacement activity. In certain embodiments, a sample is contacted with the exonuclease blocking oligonucleotide after binding of the protein-binding reagent but before contacting the sample with a polymerase with strand displacement activity and / or 3' to 5' exonuclease activity. In certain embodiments, a sample is contacted with the exonuclease blocking oligonucleotide before contacting the sample with Phi29 polymerase.

[0077] In certain embodiments, the exonuclease blocking oligonucleotide has the structure shown in Figure 4. In certain embodiments, the exonuclease blocking oligonucleotide comprises a sequence complementary to an oligonucleotide conjugated to a protein-binding reagent. For example, without limitation, the exonuclease blocking oligonucleotide may comprise a sequence complementary to a region adjacent to a barcode sequence present in the oligonucleotide conjugated to the protein-binding reagent, as shown in Figure 4. In certain embodiments, this region is located 3' from the barcode sequence of the oligonucleotide conjugated to the protein-binding reagent.

[0078] In certain embodiments, the exonuclease blocking oligonucleotide does not bind to the barcode sequence of the oligonucleotide, e.g., as shown in Figure 4. In certain embodiments, the exonuclease blocking oligonucleotide does not comprise a nucleotide sequence that is complementary to the barcode sequence of the oligonucleotide. In certain embodiments, the exonuclease blocking oligonucleotide does not bind to the primer sequence of the oligonucleotide, e.g., as shown in Figure 4. In certain embodiments, the exonuclease blocking oligonucleotide does not comprise a nucleotide sequence that is complementary to the primer sequence of the oligonucleotide. In certain embodiments, the exonuclease blocking oligonucleotide comprises a nucleotide sequence at its 3' end that does not bind to (e.g., is not complementary to) the sequence of the oligonucleotide, e.g., as shown in Figure 4.

[0079] In certain embodiments, the exonuclease blocking oligonucleotide has a 5' to 3' structure that includes (i) a nucleotide sequence that is complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a first nucleotide sequence, e.g., a complementary region or domain), and (ii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a second nucleotide sequence, e.g., a 3' flap region or domain). In certain embodiments, the exonuclease blocking oligonucleotide has a 5' to 3' structure that includes (i) a nucleotide sequence that includes an extension template (e.g., a first nucleotide sequence, e.g., a 5' extension template region or domain), (ii) a nucleotide sequence that is complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a second nucleotide sequence, e.g., a complementary region or domain), and (iii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region or domain), as shown, for example, in FIG. In certain embodiments, the nucleotide sequence comprising the extension template (e.g., the first nucleotide sequence) is not complementary to the oligonucleotide conjugated to the protein-binding reagent. In certain embodiments, the nucleotide sequence of the oligonucleotide coupled to the protein-binding reagent that is bound by the exonuclease-blocking oligonucleotide can be conserved among each oligonucleotide coupled to the multiple protein-binding reagents used in the disclosed methods.

[0080] In certain embodiments, the exonuclease blocking oligonucleotide can further comprise an extension template at its 5' end. In certain embodiments, the extension template at the 5' end of the exonuclease blocking oligonucleotide can prevent the exonuclease activity of a polymerase, for example, a polymerase with strand displacement activity, such as Phi29 polymerase. In certain embodiments, the extension template at the 5' end of the exonuclease blocking oligonucleotide can prevent the 3' to 5' exonuclease activity of a polymerase on a single-stranded nucleic acid by generating a double-stranded nucleic acid. In certain embodiments, the extension template at the 5' end of the exonuclease blocking oligonucleotide can prevent the exonuclease activity of a polymerase, for example, a polymerase with strand displacement activity, such as Phi29 polymerase, on the oligonucleotide of the protein binding reagent-oligonucleotide conjugate. In certain embodiments, the extension template at the 5' end of the exonuclease-blocking oligonucleotide can prevent the exonuclease activity of Phi29 polymerase on the oligonucleotide of the protein binding reagent-oligonucleotide conjugate.

[0081] In certain embodiments, the exonuclease blocking oligonucleotide is about 5 to about 200 nucleotides in length, e.g., about 5 to about 150 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 50 nucleotides in length, about 10 to about 150 nucleotides in length, about 20 to about 100 nucleotides in length, or about 10 to about 100 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 10 to about 100 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 20 to about 90 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 30 to about 90 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 40 to about 90 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 50 to about 90 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 60 to about 90 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 20 to about 80 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 30 to about 80 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 40 to about 80 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 50 to about 80 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 60 to about 80 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 45 to about 75 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 30 to about 60 nucleotides in length.In certain embodiments, the exonuclease blocking oligonucleotide can be about 5 to about 50 nucleotides in length, e.g., about 5 to about 45 nucleotides, about 5 to about 40 nucleotides, about 5 to about 35 nucleotides, about 5 to about 30 nucleotides, about 5 to about 25 nucleotides, about 5 to about 20 nucleotides, about 5 to about 15 nucleotides, about 5 to about 10 nucleotides, about 10 to about 50 nucleotides, about 15 to about 50 nucleotides, about 20 to about 50 nucleotides, about 25 to about 50 nucleotides, about 30 to about 50 nucleotides, about 35 to about 50 nucleotides, about 40 to about 50 nucleotides, about 10 to about 40 nucleotides, or about 10 to about 30 nucleotides in length. In certain embodiments, the exonuclease blocking oligonucleotide is about 30 to about 50 nucleotides in length.

[0082] In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., that is complementary to the oligonucleotide of the protein binding reagent) is about 5 to about 100 nucleotides in length (e.g., if the nucleotides are contiguous). For example, and without limitation, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) can be about 5 to about 95 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 85 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 75 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 65 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 55 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 100 nucleotides in length, about 15 about 100 nucleotides in length, about 20 to about 100 nucleotides in length, about 25 to about 100 nucleotides in length, about 30 to about 100 nucleotides in length, about 35 to about 100 nucleotides in length, about 40 to about 100 nucleotides in length, about 45 to about 100 nucleotides in length, about 50 to about 100 nucleotides in length, about 55 to about 100 nucleotides in length, about 60 to about 100 nucleotides in length, about 65 to about 100 nucleotides in length, about 70 to about 100 nucleotides in length, The length is about 75 to about 100 nucleotides, about 80 to about 100 nucleotides, about 85 to about 100 nucleotides, about 90 to about 100 nucleotides, about 95 to about 100 nucleotides, about 30 to about 90 nucleotides, about 30 to about 80 nucleotides, about 30 to about 70 nucleotides, about 30 to about 60 nucleotides, about 30 to about 50 nucleotides, about 10 to about 40 nucleotides, or about 10 to about 30 nucleotides.In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) is about 5 to about 50 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) is about 5 to about 40 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) is about 5 to about 30 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) is about 10 to about 50 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that binds to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) is about 10 to about 40 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the oligonucleotide of the protein binding reagent (e.g., complementary to the oligonucleotide of the protein binding reagent) is about 20 to about 30 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the oligonucleotide of the protein binding reagent is about 50 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the oligonucleotide of the protein binding reagent is about 60 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the oligonucleotide of the protein binding reagent is about 70 to about 100 nucleotides in length.In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is about 80 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is about 90 to about 100 nucleotides in length.

[0083] In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is at least about 90% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is at least about 95% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is at least about 97% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is at least about 98% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is at least about 99% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide bound to the protein binding reagent oligonucleotide is 100% complementary to the nucleotide sequence of the protein binding reagent oligonucleotide.

[0084] In certain embodiments, about 10% to about 90% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. For example, but not limited to, about 15% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 35% to about 90%, about 40% to about 45%, about 50% to about 90%, about 55% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 80% to about 90%, about 85% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 80%, or about 60% to about 70% is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 50% to about 90% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 60% to about 90% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 70% to about 90% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 80% to about 90% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 20% to about 60% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent.In certain embodiments, about 10% to about 50% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 20% to about 60% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 30% to about 60% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 40% to about 60% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 50% to about 60% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 20% to about 50% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 30% to about 50% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 40% to about 50% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent. In certain embodiments, about 30% to about 40% of the nucleotide sequence of the exonuclease blocking oligonucleotide is complementary to the oligonucleotide of the protein binding reagent.

[0085] In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising the extension template is from about 5 to about 100 nucleotides in length (eg, if the nucleotides are contiguous). For example, but not limited to, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising the extension template may be about 5 to about 95 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 85 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 75 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 65 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 55 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 100 nucleotides in length, about 15 to about 100 nucleotides in length, about 20 to about 100 nucleotides in length, The length of the amino acid sequence is about 25 to about 100 nucleotides, about 30 to about 100 nucleotides, about 35 to about 100 nucleotides, about 40 to about 100 nucleotides, about 45 to about 100 nucleotides, about 50 to about 100 nucleotides, about 55 to about 100 nucleotides, about 60 to about 100 nucleotides, about 65 to about 100 nucleotides, about 70 to about 100 nucleotides, about 75 to about 100 nucleotides, about 80 to about 100 nucleotides, about 85 to about 100 nucleotides, about 90 to about 100 nucleotides, about 95 to about 100 nucleotides, about 30 to about 90 nucleotides, about 30 to about 80 nucleotides, about 30 to about 70 nucleotides, about 30 to about 60 nucleotides, about 30 to about 50 nucleotides, about 10 to about 40 nucleotides, or about 10 to about 30 nucleotides. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising the extension template is about 50 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising the extension template is about 60 to about 100 nucleotides in length.In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 70 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 80 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 90 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 20 to about 60 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 20 to about 50 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 20 to about 40 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 20 to about 30 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 10 to about 40 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 10 to about 30 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 10 to about 20 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide comprising an extension template is about 20 to about 30 nucleotides in length.

[0086] In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent (e.g., not complementary to the oligonucleotide of the protein binding reagent) is about 5 to about 100 nucleotides in length (e.g., if the nucleotides are contiguous). In certain embodiments, the nucleotide sequence of the 3' end of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent (e.g., not complementary to the oligonucleotide of the protein binding reagent) is about 5 to about 100 nucleotides in length (e.g., if the nucleotides are contiguous).For example, but not limited to, the nucleotide sequence of an exonuclease blocking oligonucleotide that does not bind to an oligonucleotide of a protein binding reagent (e.g., that is not complementary to an oligonucleotide of a protein binding reagent) can be about 5 to about 95 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 85 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 75 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 65 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 55 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 2 ... 5 to about 100 nucleotides in length, about 20 to about 100 nucleotides in length, about 25 to about 100 nucleotides in length, about 30 to about 100 nucleotides in length, about 35 to about 100 nucleotides in length, about 40 to about 100 nucleotides in length, about 45 to about 100 nucleotides in length, about 50 to about 100 nucleotides in length, about 55 to about 100 nucleotides in length, about 60 to about 100 nucleotides in length, about 65 to about 100 nucleotides in length, about 70 to about 100 nucleotides in length, The length of the exonuclease-blocking oligonucleotide is about 75 to about 100 nucleotides, about 80 to about 100 nucleotides, about 85 to about 100 nucleotides, about 90 to about 100 nucleotides, about 95 to about 100 nucleotides, about 30 to about 90 nucleotides, about 30 to about 80 nucleotides, about 30 to about 70 nucleotides, about 30 to about 60 nucleotides, about 30 to about 50 nucleotides, about 10 to about 40 nucleotides, or about 10 to about 30 nucleotides. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is about 50 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is about 60 to about 100 nucleotides in length.In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent is about 70 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent is about 80 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent is about 90 to about 100 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent is about 20 to about 80 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent is about 20 to about 60 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the oligonucleotide of the protein binding reagent is about 20 to about 50 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide is about 25 to about 45 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide is about 5 to about 40 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide (e.g., at the 3' end) is about 5 to about 35 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide (e.g., at the 3' end) is about 5 to about 30 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide (e.g., at the 3' end) is about 5 to about 25 nucleotides in length.In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide (e.g., at the 3' end) is about 5 to about 20 nucleotides in length. In certain embodiments, the nucleotide sequence of the exonuclease blocking oligonucleotide that does not bind to the protein binding reagent oligonucleotide (e.g., at the 3' end) is about 10 to about 20 nucleotides in length.

[0087] In certain embodiments, the exonuclease blocking oligonucleotide can comprise one or more modified nucleotides. In certain embodiments, the exonuclease blocking oligonucleotide comprises at least 5 or more, at least 6 or more, at least 7 or more, at least 8 or more, at least 9 or more, at least 10 or more, at least 11 or more, at least 12 or more, at least 13 or more, at least 14 or more, at least 15 or more, at least 16 or more, at least 17 or more, at least 18 or more, at least 19 or more, or at least 20 or more modified nucleotides. In certain embodiments, the exonuclease blocking oligonucleotide comprises from about 5 to about 20 modified nucleotides. In certain embodiments, the exonuclease blocking oligonucleotide comprises from about 5 to about 15 modified nucleotides. In certain embodiments, the exonuclease blocking oligonucleotide comprises from about 10 to about 15 modified nucleotides. In certain embodiments, the modified nucleotides are nucleotides that are resistant to exonuclease cleavage. In certain embodiments, the modified nucleotide comprises a modified nucleotide base having a derivatized sugar or phosphate backbone linkage or a chemically modified residue. In certain embodiments, the modified nucleotide is a nucleotide having a phosphate backbone modification. In certain embodiments, the modified nucleotide is a nucleotide having a phosphorothioate linkage at its 3' end. In certain embodiments, the modified nucleotide is a 2',3'-dideoxynucleoside-alpha-thiol nucleotide, such as 2',3'-dideoxyadenosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxycytidine-5'-O-(1-thiotriphosphate), 2',3'-dideoxyguanosine-5'-O-(1-thiotriphosphate), and / or 2',3'-dideoxythymidine-5'-O-(1-thiotriphosphate).In certain embodiments, the exonuclease blocking oligonucleotide comprises at least 5 or more, at least 6 or more, at least 7 or more, at least 8 or more, at least 9 or more, at least 10 or more, at least 11 or more, at least 12 or more, at least 13 or more, at least 14 or more, at least 15 or more, at least 16 or more, at least 17 or more, at least 18 or more, at least 19 or more, or at least 20 or more nucleotides having phosphorothioate linkages at the 3' end of the exonuclease blocking oligonucleotide that do not bind to (e.g., are not complementary to) the barcode sequence of the oligonucleotide. In certain embodiments, the exonuclease blocking oligonucleotide comprises at least 10 or more modified nucleotides, e.g., nucleotides having a phosphate backbone modification, e.g., nucleotides having phosphorothioate linkages, at the 3' end. In certain embodiments, the modified nucleotides, e.g., nucleotides having phosphorothioate linkages, are contiguous. The use of phosphorothioate bonds can prevent the DNA barcode from becoming double-stranded (e.g., by contacting it with an exonuclease-blocking oligonucleotide). In certain embodiments, the sample is contacted with an exonuclease-blocking oligonucleotide before performing the amplification process.

[0088] In certain embodiments, the sample may be contacted with the exonuclease blocking oligonucleotide for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, or about 30 minutes or less, hi certain embodiments, the sample may be contacted with the exonuclease blocking oligonucleotide for about 30 minutes.

[0089] In certain embodiments, one or more of the padlock oligonucleotides can hybridize to a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) complementary to an oligonucleotide coupled to a protein-binding reagent. In certain embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more padlock oligonucleotides can each be bound to a separate blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide). In certain embodiments, each different blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) hybridizes to an oligonucleotide coupled to a protein-binding reagent.

[0090] In certain embodiments, one or more of the plurality of padlock oligonucleotides can hybridize to a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) that is complementary to the oligonucleotide coupled to the protein binding reagent, and a second padlock oligonucleotide of the plurality of padlock oligonucleotides binds to a target nucleic acid, e.g., RNA or genomic nucleic acid of the sample.

[0091] In certain embodiments, samples processed according to the methods disclosed herein can be permeabilized a second time, e.g., before performing the amplification process and after contacting the sample with a protein-binding reagent. For example, such samples can be permeabilized a second time using a permeabilization reagent disclosed herein. In certain embodiments, permeabilization is performed using an acid. For example, but not limited to, permeabilization can be performed using HCl, e.g., about 0.01 N to about 10 N HCl or about 0.01 N to about 1.0 N HCl. In certain embodiments, permeabilization can be performed using a solution containing an acid, e.g., HCl, and a peptidase. In certain embodiments, the peptidase can be pepsin. In certain embodiments, the peptidase, e.g., pepsin, can be present in the solution at a concentration of about 0.1 mg / ml to about 10 mg / ml or about 0.1 mg / ml to about 5 mg / ml. In certain embodiments, permeabilization can be performed using a solution containing about 0.01 N to about 1 N HCl and about 0.1 mg / ml to about 5 mg / ml of a peptidase, such as pepsin. In certain embodiments, the sample can be contacted with HCl for about 1 to about 10 minutes.

[0092] In certain embodiments, the sample processed according to the method disclosed herein is treated with NHS-acetate before the amplification process for nucleic acid detection.For example, but not limited to, the sample can be treated with NHS-acetate after post-fixation and HCl permeabilization of the sample.In certain embodiments, the sample can be contacted with NHS-acetate for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, or about 30 minutes or less.In certain embodiments, the sample can be contacted with NHS-acetate for about 30 minutes.

[0093] D. Imaging Strategies As described above, the disclosed methods include imaging target proteins and target nucleic acids in a sample. In certain embodiments, protein-binding reagents and / or oligonucleotides coupled to amplicons generated by the amplification process facilitate imaging of the target proteins and nucleic acids.

[0094] In certain embodiments, detection probes can be used to image oligonucleotides bound to protein-binding reagents and / or amplicons utilized in the methods disclosed herein. A "detection probe" refers to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of a target sequence amplified during RCA or a portion of an oligonucleotide coupled to a protein-binding reagent) under appropriate hybridization conditions. In certain embodiments, a detection probe can comprise or consist of about 10 to about 50 nucleotides, e.g., about 15 to about 30 nucleotides. In certain embodiments, a detection probe for use in the present disclosure comprises a sequence that specifically hybridizes to an amplicon, e.g., an RCA amplicon. In certain embodiments, a detection probe for use in the present disclosure comprises a sequence that specifically hybridizes to a DNA barcode sequence of an oligonucleotide coupled to a protein-binding reagent, e.g., an antibody. In certain embodiments, a detection probe for use in the present disclosure comprises a sequence that specifically hybridizes to a detection bridging oligonucleotide.

[0095] In certain embodiments, a detection bridge oligonucleotide for use in protein imaging is an oligonucleotide that hybridizes to a nucleotide sequence (e.g., a DNA barcode sequence) of an oligonucleotide coupled to a protein-binding reagent, e.g., an antibody. In certain embodiments, the detection bridge oligonucleotide can comprise a sequence complementary to the DNA barcode and additional nucleotides present in the oligonucleotide coupled to the protein-binding reagent, e.g., an antibody, as shown in FIG. 1 . For example, without limitation, the detection bridge oligonucleotide can comprise a sequence complementary to the DNA barcode and at least five additional nucleotides present in the oligonucleotide coupled to the protein-binding reagent, e.g., an antibody. In certain embodiments, the detection bridge oligonucleotide comprises a nucleotide sequence having a length of about 5 to about 100 nucleotides (e.g., when the nucleotides are contiguous) that is complementary to the oligonucleotide coupled to the protein-binding reagent, e.g., an antibody. In certain embodiments, where the method involves amplification of an exonuclease-blocking oligonucleotide and / or amplification of an oligonucleotide coupled to a protein-binding reagent, the detection bridge oligonucleotide can comprise a sequence complementary to the nucleotide sequence of the resulting amplicon. In certain embodiments, the detection bridge oligonucleotide comprises a nucleotide sequence having a length of about 5 to about 100 nucleotides (e.g., when the nucleotides are contiguous) that is complementary to the nucleotide sequence of the resulting amplicon.

[0096] In certain embodiments, a detection bridge oligonucleotide for use in nucleic acid imaging is an oligonucleotide that hybridizes to a sequence present in an amplicon generated during an amplification reaction, for example, as shown in Figure 1. In certain embodiments, the padlock oligonucleotide comprises such a sequence that is amplified during the amplification process, e.g., RCA. In certain embodiments, the amplicon comprises one or more sequences, two or more sequences, three or more sequences, four or more sequences, or five or more sequences that can hybridize to the detection bridge oligonucleotide.

[0097] In certain embodiments, the detection bridging oligonucleotide comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nucleotide sequences capable of hybridizing to the detection probe. In certain embodiments, the detection bridging oligonucleotide comprises five or more nucleotide sequences capable of hybridizing to the detection probe. In certain embodiments, the detection bridging oligonucleotide comprises ten or more nucleotide sequences capable of hybridizing to the detection probe. In certain embodiments, the detection bridging oligonucleotide comprises from about one to about eight nucleotide sequences capable of hybridizing to the detection probe. In certain embodiments, one or more nucleotide sequences capable of hybridizing to the detection probe are identical.

[0098] In certain embodiments, the detection bridge oligonucleotide is about 5 to about 200 nucleotides in length, e.g., about 5 to about 150 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 50 nucleotides in length, about 10 to about 150 nucleotides in length, about 20 to about 100 nucleotides in length, or about 10 to about 100 nucleotides in length. In certain embodiments, the detection bridge oligonucleotide is about 25 to about 100 nucleotides in length or about 25 to about 120 nucleotides in length. In certain embodiments, the detection bridge oligonucleotide comprises one or more nucleotide sequences capable of hybridizing to a detection probe. In certain embodiments, the detection bridge oligonucleotide comprises about 1 to about 8 nucleotide sequences capable of hybridizing to a detection probe. In certain embodiments, the detection bridge oligonucleotide comprises about 1 to about 10 nucleotide sequences capable of hybridizing to a detection probe.

[0099] In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence complementary to the nucleotide sequence of the amplicon and having a length of about 5 to about 100 nucleotides (e.g., when contiguous nucleotides). In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence complementary to the nucleotide sequence of the amplicon and having a length of about 5 to about 50 nucleotides (e.g., when contiguous nucleotides). In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence complementary to the nucleotide sequence of the amplicon and having a length of about 10 to about 40 nucleotides (e.g., when contiguous nucleotides). In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence complementary to the nucleotide sequence of the amplicon and having a length of about 10 to about 30 nucleotides (e.g., when contiguous nucleotides). In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence complementary to the nucleotide sequence of the amplicon and having a length of about 10 to about 20 nucleotides (e.g., when contiguous nucleotides).

[0100] In certain embodiments, a detection bridge oligonucleotide for use in detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of about 5 to about 40 nucleotides (e.g., when contiguous nucleotides) that is complementary to the nucleotide sequence of an amplicon obtained from amplification of the target nucleic acid. In certain embodiments, a detection bridge oligonucleotide for use in detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of about 5 to about 35 nucleotides (e.g., when contiguous nucleotides) that is complementary to the nucleotide sequence of an amplicon obtained from amplification of the target nucleic acid. In certain embodiments, a detection bridge oligonucleotide for use in detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of about 5 to about 30 nucleotides (e.g., when contiguous nucleotides) that is complementary to the nucleotide sequence of an amplicon obtained from amplification of the target nucleic acid. In certain embodiments, a detection bridge oligonucleotide for use in detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of about 5 to about 25 nucleotides (e.g., when contiguous nucleotides) that is complementary to the nucleotide sequence of an amplicon obtained from amplification of the target nucleic acid. In certain embodiments, a detection bridge oligonucleotide for use in detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of about 5 to about 20 nucleotides (e.g., when the nucleotides are contiguous) that is complementary to the nucleotide sequence of an amplicon resulting from amplification of the target nucleic acid.

[0101] In certain embodiments, a detection bridging oligonucleotide for use in detecting an oligonucleotide coupled to a protein-binding reagent comprises a nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent and having a length of about 5 to about 50 nucleotides (e.g., if the nucleotides are contiguous). In certain embodiments, a detection bridging oligonucleotide for use in detecting an oligonucleotide coupled to a protein-binding reagent comprises a nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent and having a length of about 5 to about 40 nucleotides (e.g., if the nucleotides are contiguous). In certain embodiments, a detection bridging oligonucleotide for use in detecting an oligonucleotide coupled to a protein-binding reagent comprises a nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent and having a length of about 5 to about 35 nucleotides (e.g., if the nucleotides are contiguous). In certain embodiments, a detection bridging oligonucleotide for use in detecting an oligonucleotide coupled to a protein-binding reagent comprises a nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent and having a length of about 5 to about 30 nucleotides (e.g., if the nucleotides are contiguous). In certain embodiments, a detection bridging oligonucleotide for use in detecting an oligonucleotide coupled to a protein-binding reagent comprises a nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent and having a length of about 10 to about 30 nucleotides (e.g., if the nucleotides are contiguous). In certain embodiments, a detection bridging oligonucleotide for use in detecting an oligonucleotide coupled to a protein-binding reagent comprises a nucleotide sequence complementary to the oligonucleotide coupled to the protein-binding reagent and having a length of about 20 to about 30 nucleotides (e.g., if the nucleotides are contiguous).

[0102] In certain embodiments, the detection probe is conjugated to a detectable label to facilitate imaging. Non-limiting examples of detectable labels include fluorescent labels (e.g., fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliproteins and R-phycoerythrin, and quantum dots (e.g., zinc sulfide-capped cadmium selenide)), chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels. In certain embodiments, the detection probe is fluorescently labeled. In certain embodiments, the detection probe is covalently bound to a fluorescent label at its 5' or 3' end.

[0103] In certain embodiments, the detection bridge oligonucleotide can comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more detectable labels as described herein, which can eliminate the need for a detection probe.

[0104] In certain embodiments in which multiple target nucleic acids are imaged in a single sample, the specific detection probes, e.g., the detection probes for specific target nucleic acids, can each be labeled with a different label, e.g., a fluorophore, thereby allowing for simultaneous imaging of multiple target nucleic acids. Similarly, in certain embodiments in which multiple target nucleic acids are imaged in a single sample, the specific detection probes, e.g., the detection probes for specific target nucleic acids, can each be labeled with a different label, e.g., a fluorophore, thereby allowing for simultaneous imaging of multiple target nucleic acids.

[0105] In certain embodiments, periodic imaging is carried out to visualize labeled target proteins and target nucleic acids in a single sample.In certain embodiments, periodic imaging includes the periodic addition and removal of labeled detection probes.In certain embodiments, the removal of labeled detection probes is achieved by thermal denaturation.Alternatively or additionally, the removal of labeled detection probes is achieved by using detection probes with disulfide-conjugated dyes, thereby allowing disulfide cleavage to remove the label from the detection probes.

[0106] In certain embodiments, periodic imaging of all protein targets is performed first, followed by periodic imaging of all nucleic acid targets. Alternatively, periodic imaging of all nucleic acid targets is performed first, followed by periodic imaging of all protein targets. In certain embodiments, imaging of a first target protein is performed, followed by imaging of a first target nucleic acid, and so on until all protein and nucleic acid targets have been imaged.

[0107] In certain embodiments, periodic imaging can be used to detect target proteins in samples.For example, but not limited to, periodic imaging can be carried out by contacting the sample with a detection probe, such as a fluorescently labeled detection probe, that is specific to the barcode sequence of the oligonucleotide that is conjugated to the antibody that binds to the target sequence.In certain embodiments, the detection probe is then imaged and subsequently removed.In certain embodiments, the detection probe is removed by a chaotropic solvent.In certain embodiments, one or more new detection probes are bound and then imaged.In certain embodiments, this periodic imaging process is repeated until all the antibodies that bind to target proteins in the sample are imaged.

[0108] In certain embodiments, periodic imaging of target nucleic acid can be performed.For example, but not limited to, periodic imaging can be performed by contacting the sample with a detection probe, such as a fluorescently labeled detection probe, specific to the amplicon, such as the RCA amplicon, generated during the amplification of target nucleic acid.In certain embodiments, the detection probe is then imaged, and then removed, for example, by a chaotropic solvent and / or thermal process.In certain embodiments, one or more new detection probes are bound and then imaged.In certain embodiments, this periodic imaging process is repeated until all target nucleic acids in the sample are imaged.

[0109] Additional disclosure regarding periodic imaging is provided in Black et al., Nature Protocols 16:3802-3835 (2021) and Kennedy-Darling et al., Eur. K. Immunol. 51(5):1262-1277 (2021), the contents of each of which are incorporated herein by reference in their entirety.

[0110] In certain embodiments, a method for imaging target proteins and target nucleic acids in a sample includes: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to the target protein in the sample, where the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (d) amplifying the target nucleic acid in the sample to generate an amplicon by performing an amplification process; (e) contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the amplicon; and (h) imaging the second labeled detection probe to detect the target nucleic acid. In certain embodiments, the target nucleic acid is a blocking oligonucleotide (eg, an exonuclease-blocking oligonucleotide) and / or the target nucleic acid is an oligonucleotide coupled to a protein-binding reagent.

[0111] In certain embodiments, a method for imaging target proteins and target nucleic acids in a sample includes: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, where the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (d) performing an amplification process to amplify the target nucleic acid in the sample to generate an amplicon; (e) contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the amplicon; (f) imaging the first labeled detection probe to detect the target nucleic acid; (g) contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; and (h) imaging the second labeled detection probe to detect the target protein. In certain embodiments, the target nucleic acid is a blocking oligonucleotide and / or the target nucleic acid is an oligonucleotide coupled to a protein binding reagent.

[0112] In certain embodiments, a method for imaging target proteins and target nucleic acids in a sample includes: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, where the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; and (d) performing an amplification process to detect the blocking oligonucleotide and / or protein binding agent in the sample. The method includes (a) amplifying an oligonucleotide coupled to the protein-binding reagent to produce a first amplicon; (b) performing an amplification process to amplify a target nucleic acid in the sample to produce a second amplicon; (c) contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein-binding reagent and / or contacting the sample with a first labeled detection probe comprising a sequence complementary to the first amplicon; (d) imaging the first labeled detection probe to detect the target protein; (e) contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the second amplicon; and (f) imaging the second labeled detection probe to detect the target nucleic acid. In certain embodiments, the generation of the first amplicon and the second amplicon occurs simultaneously during a single amplification process. In certain embodiments, the generation of the first amplicon occurs during a first amplification process, and the generation of the second amplicon occurs during a second amplification process, which are performed separately.

[0113] In certain embodiments, a method for imaging target proteins and target nucleic acids in a sample includes: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, where the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide, e.g., an exonuclease blocking oligonucleotide, comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; and (d) contacting the blocking oligonucleotide and / or oligonucleotide coupled to the protein binding reagent. (e) amplifying the target nucleic acid in the sample by performing an amplification process to generate a first amplicon; (f) contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the second amplicon; (g) imaging the first labeled detection probe to detect the target nucleic acid; (h) contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent and / or contacting the sample with a second labeled detection probe comprising a sequence complementary to the first amplicon; and (i) imaging the second labeled detection probe to detect the target protein. In certain embodiments, the generation of the first amplicon and the second amplicon occurs simultaneously during a single amplification process. In certain embodiments, the generation of the first amplicon occurs during a first amplification process and the generation of the second amplicon occurs during a second amplification process, which are performed separately.

[0114] In certain embodiments, a method for imaging a target protein and a target nucleic acid in a sample includes: (a) allowing a protein-binding reagent coupled to an oligonucleotide to bind to the target protein in the sample; (b) hybridizing a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, to the oligonucleotide coupled to the protein-binding reagent; (c) performing an amplification process to amplify the target nucleic acid in the sample to generate an amplicon; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide coupled to the protein-binding reagent; and (e) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the amplicon. In certain embodiments, the target nucleic acid is the blocking oligonucleotide and / or the target nucleic acid is the oligonucleotide coupled to the protein-binding reagent.

[0115] In certain embodiments, a method for imaging a target protein and a target nucleic acid in a sample includes: (a) allowing a protein-binding reagent coupled to an oligonucleotide to bind to a target protein in the sample; (b) hybridizing a blocking oligonucleotide to the oligonucleotide coupled to the protein-binding reagent; (c) performing an amplification process to amplify the target nucleic acid in the sample to generate an amplicon; (d) imaging the target nucleic acid by detecting a first labeled detection probe that hybridizes to the amplicon; and (e) imaging the target protein by detecting a second labeled detection probe that hybridizes to the oligonucleotide coupled to the protein-binding reagent. In certain embodiments, the target nucleic acid is the blocking oligonucleotide, and / or the target nucleic acid is the oligonucleotide coupled to the protein-binding reagent.

[0116] In certain embodiments, a method for imaging a target protein and a target nucleic acid in a sample includes: (a) allowing a protein-binding reagent bound to an oligonucleotide to bind to the target protein in the sample; (b) hybridizing a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, to the oligonucleotide coupled to the protein-binding reagent; (c) performing an amplification process to amplify the blocking oligonucleotide and / or the oligonucleotide coupled to the protein-binding reagent in the sample to generate a first amplicon; (d) performing an amplification process to amplify the target nucleic acid in the sample to generate a second amplicon; (e) imaging the target protein by detecting a first labeled detection probe hybridized to the oligonucleotide coupled to the protein-binding reagent and / or hybridized to the first amplicon; and (f) imaging the target nucleic acid by detecting a second labeled detection probe hybridized to the second amplicon. In certain embodiments, the generation of the first amplicon and the second amplicon occurs simultaneously during a single amplification process. In certain embodiments, the generation of the first amplicon occurs during a first amplification process and the second amplicon occurs during a second amplification process, which are performed separately.

[0117] In certain embodiments, a method for imaging target proteins and target nucleic acids in a sample includes: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, wherein the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide, e.g., an exonuclease blocking oligonucleotide, comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; and (d) performing an amplification process to amplify and image the target nucleic acid in the sample. (e) contacting the sample with a first bridging oligonucleotide comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein-binding reagent, and contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the first bridging oligonucleotide; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second bridging oligonucleotide comprising a sequence complementary to the sequence of the amplicon, and contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0118] In certain embodiments, a method for imaging target proteins and target nucleic acids in a sample includes: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to the target protein in the sample, where the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; and (d) performing an amplification process to detect target proteins and target nucleic acids in the sample. (e) contacting the sample with a first bridging oligonucleotide comprising a sequence complementary to that of the amplicon, and contacting the sample with a first labeled detection probe comprising the sequence of the amplicon; (f) imaging the first labeled detection probe to detect the target nucleic acid; (g) contacting the sample with a second bridging oligonucleotide comprising a sequence complementary to that of the oligonucleotide coupled to the protein binding reagent, and contacting the sample with a second labeled detection probe comprising a sequence complementary to that of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target protein.

[0119] III. Systems and Kits The present disclosure provides systems and kits for carrying out the methods of the present disclosure. For example, without limitation, the present disclosure provides systems and kits that include materials for carrying out methods of imaging nucleic acids and proteins in a sample.

[0120] In certain embodiments, the disclosed system or kit includes a container containing one or more protein binding reagents. In certain embodiments, the disclosed system or kit can further include a container containing one or more blocking oligonucleotides. In certain embodiments, the disclosed system or kit can further include one or more detection probes, such as fluorescently labeled detection probes. Non-limiting examples of suitable containers include bottles, test tubes, vials, and microtiter plates. The container can be formed from a variety of materials, such as glass or plastic.

[0121] In certain embodiments, the system or kit further comprises a package insert providing instructions for use of the components provided in the system or kit. For example, a system or kit of the present disclosure can include a package insert providing instructions for performing a method for imaging one or more target proteins and one or more target nucleic acids in a single sample.

[0122] In certain embodiments, the disclosed systems or kits may further include reagents for performing an amplification reaction, such as an RCA reaction, which may include one or more of a polymerase, a reverse transcriptase, a nucleoside triphosphate or NTP analog, a primer, a cofactor, a ligation reaction reagent, an endonuclease, a lysis reagent, a dye, a marker, an RNase inhibitor, and a label.

[0123] In certain embodiments, the system or kit may include other materials desirable from a commercial and user perspective, including other buffers and diluents. In certain embodiments, the system or kit may include materials or reagents for permeabilizing, fixing, and / or crosslinking cells or nuclei. In certain embodiments, the system or kit of the present disclosure may include a fixative, such as formaldehyde. In certain embodiments, the system or kit may include NHS-acetate. In certain embodiments, the system or kit may include HCl.

[0124] In certain embodiments, the components of the system or kit are provided in predetermined ratios, with the relative amounts of the various reagents appropriately varied to obtain the desired sensitivity and throughput of the disclosed methods.

[0125] IV. Illustrative Embodiments A. The present disclosure provides a method for imaging target proteins and target nucleic acids in a sample, the method including: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, where the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (d) amplifying the target nucleic acid in the sample to produce an amplicon by performing an amplification process; (e) contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the amplicon; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0126] A1. The method according to A, wherein the amplification process is a rolling circle amplification process.

[0127] A2. The method of A1, wherein the rolling circle amplification process includes: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0128] A3. The method according to any one of A to A2, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

[0129] A4. The method according to any one of A to A3, wherein the target nucleic acid comprises RNA.

[0130] A5. A method according to any one of A-A4, wherein providing a sample comprises one or more of the following: (a) treating the sample with a fixative; (b) dehydrating the sample; (c) permeabilizing the sample.

[0131] A6. The method of any one of A-A5, wherein at least 10 target proteins are imaged in the sample.

[0132] A7. The method of any one of A-A6, wherein at least 10 target nucleic acids are imaged in the sample.

[0133] A8. The method according to any one of A to A7, wherein the sample is post-fixed before amplifying the target nucleic acid.

[0134] A9. The method according to any one of A to A8, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

[0135] A10. The method according to any one of A to A9, wherein the sample is treated with NHS-acetate before amplifying the target nucleic acid.

[0136] A11. The method according to any one of A to A10, wherein the sample is a tissue sample.

[0137] A12. The method according to any one of A to A11, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein-binding reagent is located at the 5' end of the blocking oligonucleotide.

[0138] A13. The method according to any one of A-A12, wherein the oligonucleotide coupled to a protein-binding reagent comprises a barcode sequence, and the blocking oligonucleotide does not bind to the barcode sequence.

[0139] A14. The method according to any one of A to A13, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

[0140] A15. The method of A14, wherein the one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide, eg, within the 3' flap region.

[0141] A16. The method of any one of A-A13, wherein the blocking oligonucleotide comprises at least about 10 modified nucleotides.

[0142] A17. The method of A16, wherein the at least about 10 modified nucleotides are located at the 3' end of the blocking oligonucleotide, eg, within the 3' flap region.

[0143] A18. The method of any one of A14 to A17, wherein the one or more modified nucleotides and / or the at least 10 modified nucleotides comprise nucleotides having a phosphate backbone modification, such as a phosphorothioate linkage.

[0144] A19. The method according to any one of A to A18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein-binding reagent is about 50 to about 100 nucleotides in length.

[0145] A20. The method according to any one of A-A18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein binding reagent is from about 10 to about 40 nucleotides in length.

[0146] A21. The method according to any one of A to A18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to the protein-binding reagent is about 20 to about 30 nucleotides in length.

[0147] A22. The method according to A19, A20 or A22, wherein said about 50 to about 100 nucleotides, said about 10 to about 40 nucleotides and / or said about 20 to about 30 nucleotides are consecutive.

[0148] A23. The method according to any one of A to A22, wherein the blocking oligonucleotide comprises a 5' extension template.

[0149] A24. The method according to A23, wherein the 5' extension template is not complementary to the oligonucleotide coupled to the protein binding reagent.

[0150] A25. The method according to A23 or A24, wherein the 5' extension template is about 10 to about 30 nucleotides in length.

[0151] A26. The method according to any one of A to A25, wherein the blocking oligonucleotide comprises a 3' flap region.

[0152] A27. The method according to A26, wherein the 3' flap region is not complementary to an oligonucleotide coupled to a protein binding reagent.

[0153] A28. The method according to A26 or A27, wherein the 3' flap region is about 5 to about 15 nucleotides in length.

[0154] A29. The method of any one of A-A28, wherein the blocking oligonucleotide comprises a 5' to 3' structure comprising: (i) a nucleotide sequence comprising an extension template (e.g., a first nucleotide sequence), (ii) a nucleotide sequence complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a second nucleotide sequence), and (iii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region).

[0155] B. The present disclosure further provides a method for imaging a target protein and a target nucleic acid in a sample, the method comprising: (a) binding a protein-binding reagent coupled to an oligonucleotide to a target protein in the sample; (b) hybridizing a blocking oligonucleotide to the oligonucleotide coupled to the protein-binding reagent; (c) performing an amplification process to amplify the target nucleic acid in the sample to generate an amplicon; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide coupled to the protein-binding reagent; and (e) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the amplicon.

[0156] B1. The method according to B, wherein the amplification process is a rolling circle amplification process.

[0157] B2. The method of B1, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template comprising the target nucleic acid; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0158] B3. The method of any one of B to B2, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

[0159] B4. The method according to any one of B to B3, wherein the target nucleic acid comprises RNA.

[0160] B5. The method of any one of B-B4, wherein at least 10 target proteins are imaged in the sample.

[0161] B6. The method of any one of B-B5, wherein at least 10 target nucleic acids are imaged in the sample.

[0162] B7. The method according to any one of B to B6, wherein the sample is post-fixed before amplifying the target nucleic acid.

[0163] B8. The method according to any one of B to B7, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

[0164] B9. The method according to any one of B to B8, wherein the sample is treated with NHS-acetate before amplifying the target nucleic acid.

[0165] B10. The method according to any one of B to B9, wherein the sample is a tissue sample.

[0166] B11. The method of any one of B-B10, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein-binding reagent is located at the 5' end of the blocking oligonucleotide.

[0167] B12. The method of any one of B-B11, wherein the oligonucleotide coupled to a protein-binding reagent comprises a barcode sequence, and the blocking oligonucleotide does not bind to the barcode sequence.

[0168] B13. The method of any one of B-B12, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

[0169] B14. The method of any one of B-B13, wherein the one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide, eg, within a 3' flap region.

[0170] B15. The method of any one of B-B12, wherein the blocking oligonucleotide comprises at least about 10 modified nucleotides.

[0171] B16. The method of B15, wherein the at least about 10 modified nucleotides are located at the 3' end of the blocking oligonucleotide, eg, within a 3' flap region.

[0172] B17. The method of any one of B13 to B116, wherein said one or more modified nucleotides and / or said at least 10 modified nucleotides comprise nucleotides having a phosphate backbone modification, such as a phosphorothioate linkage.

[0173] B18. The method of any one of B to B17, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to the protein-binding reagent is about 50 to about 100 nucleotides in length.

[0174] B19. The method of any one of B-B17, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to the protein-binding reagent is about 10 to about 40 nucleotides in length.

[0175] B20. The method of any one of B-B17, wherein the nucleotide sequence of said blocking oligonucleotide complementary to the oligonucleotide coupled to the protein binding reagent is about 20 to about 30 nucleotides in length.

[0176] B21. The method according to B18, B19 or B20, wherein said about 50 to about 100 nucleotides, said about 10 to about 40 nucleotides and / or said about 20 to about 30 nucleotides are consecutive.

[0177] B22. The method of any one of B to B21, wherein the blocking oligonucleotide comprises a 5' extension template.

[0178] B23. The method of B22, wherein said 5' extension template is not complementary to an oligonucleotide coupled to a protein binding reagent.

[0179] B24. The method of B22 or B23, wherein the 5' extension template is about 10 to about 30 nucleotides in length.

[0180] B25. The method of any one of B to B24, wherein the blocking oligonucleotide comprises a 3' flap region.

[0181] B26. The method of B25, wherein the 3' flap region is not complementary to an oligonucleotide coupled to a protein binding reagent.

[0182] B27. The method according to B25 or B26, wherein the 3' flap region is about 5 to about 15 nucleotides in length.

[0183] B28. The method of any one of B-B27, wherein the blocking oligonucleotide comprises a 5' to 3' structure comprising: (i) a nucleotide sequence comprising an extension template (e.g., a first nucleotide sequence), (ii) a nucleotide sequence complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a second nucleotide sequence), and (iii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region).

[0184] C. The present disclosure provides a method for imaging target proteins and target nucleic acids in a sample, comprising: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, wherein the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide, e.g., an exonuclease-blocking oligonucleotide, comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; and (d) performing an amplification process to amplify the target nucleic acid in the sample to form amplicons. (e) contacting the sample with a first bridging oligonucleotide comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein-binding reagent, and contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the first bridging oligonucleotide; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second bridging oligonucleotide comprising a sequence complementary to the sequence of the amplicon, and contacting the sample with a second labeled detection probe comprising a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0185] C1. The method of C, wherein the amplification process is a rolling circle amplification process.

[0186] C2. The method of C1, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0187] C3. The method of any one of C to C2, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

[0188] C4. The method of any one of C to C3, wherein the target nucleic acid comprises RNA.

[0189] C5. The method of any one of C-C4, wherein providing a sample comprises one or more of the following: (a) treating the sample with a fixative; (b) dehydrating the sample; (c) permeabilizing the sample.

[0190] C6. The method of any one of C-C5, wherein at least 10 target proteins are imaged in the sample.

[0191] C7. The method of any one of C-C6, wherein at least 10 target nucleic acids are imaged in the sample.

[0192] C8. The method according to any one of C to C7, wherein the sample is post-fixed before amplifying the target nucleic acid.

[0193] C9. The method according to any one of C to C8, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

[0194] C10. The method according to any one of C to C9, wherein the sample is treated with NHS-acetate before amplifying the target nucleic acid.

[0195] C11. The method according to any one of C to C10, wherein the sample is a tissue sample.

[0196] C12. The method of any one of C to C11, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein binding reagent is located at the 5' end of the blocking oligonucleotide.

[0197] C13. The method of any one of C to C12, wherein the oligonucleotide coupled to a protein binding reagent comprises a barcode sequence, and the blocking oligonucleotide does not bind to the barcode sequence.

[0198] C14. The method of any one of C to C13, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

[0199] C15. The method of any one of C-C14, wherein the one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide, eg, within a 3' flap region.

[0200] C16. The method of any one of C-C13, wherein the blocking oligonucleotide comprises at least about 10 modified nucleotides.

[0201] C17. The method of C16, wherein the at least about 10 modified nucleotides are located at the 3' end of the blocking oligonucleotide, eg, within the 3' flap region.

[0202] C18. The method of any one of C14 to C17, wherein the one or more modified nucleotides and / or the at least 10 modified nucleotides comprise nucleotides having a phosphate backbone modification, such as a phosphorothioate linkage.

[0203] C19. The method of any one of C to C18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein-binding reagent is about 50 to about 100 nucleotides in length.

[0204] C20. The method of any one of C to C18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein binding reagent is about 10 to about 40 nucleotides in length.

[0205] C21. The method of any one of C to C18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide coupled to a protein binding reagent is about 20 to about 30 nucleotides in length.

[0206] C22. The method according to C19, C20 or C21, wherein the sequence is about 50 to about 100 consecutive nucleotides, about 10 to about 40 consecutive nucleotides, and / or about 20 to about 30 consecutive nucleotides.

[0207] C23. The method of any one of C-C22, wherein the blocking oligonucleotide comprises a 5' extension template.

[0208] C24. The method of C23, wherein the 5' extension template is not complementary to the oligonucleotide coupled to the protein binding reagent.

[0209] C25. The method of C23 or C24, wherein the 5' extension template is about 10 to about 30 nucleotides in length.

[0210] C26. The method of any one of C-C25, wherein the blocking oligonucleotide comprises a 3' flap region.

[0211] C27. The method of C26, wherein said 3' flap region is not complementary to an oligonucleotide coupled to a protein binding reagent.

[0212] C28. The method of C26 or C27, wherein the 3' flap region is about 5 to about 15 nucleotides in length.

[0213] C29. The method of any one of C-C28, wherein the blocking oligonucleotide comprises a 5' to 3' structure comprising: (i) a nucleotide sequence comprising an extension template (e.g., a first nucleotide sequence), (ii) a nucleotide sequence complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a second nucleotide sequence), and (iii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region).

[0214] D. The present disclosure further provides a kit or system for carrying out any one of methods A-C29.

[0215] D1. The kit or system of D, comprising at least one container containing said blocking oligonucleotide. [Example]

[0216] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter and not by way of limitation.

[0217] Example 1: Simultaneous imaging of RNA and proteins in a sample This example describes a method for detecting RNA and protein in a single biological sample, as shown in FIG.

[0218] Sample preparation: This method begins by preparing a sample, e.g., a portion of a sample, for protein and nucleic acid detection. If the sample is frozen, it can be thawed from -80°C for 5 minutes in a covered chamber. Sample preparation begins by fixing the sample in 4% formaldehyde in 1x PBS for 5 minutes to 1 hour. The sample is then dehydrated in an ethanol series: 70%, 85%, and 100% for 1 minute each. The sample is then dried in a sealed container at room temperature for 5 minutes or until dry. The sample is then mounted on a flow cell and washed twice with PBS-Tween (PBST).

[0219] Antibody staining: The samples were then stained for protein using a protein-specific antibody coupled to an oligonucleotide containing a barcode sequence. The samples were then permeabilized with PBS + TritonX (0.5%) + 1:80 RiboLock for 20 minutes. The samples were then blocked for 15 minutes to overnight at 4°C or room temperature and stained with one or more Total-Seq antibodies for 1.5 hours to overnight at 4°C or room temperature. The blocking solution further contained oligonucleotides complementary to conserved sequences in the oligonucleotides conjugated to the antibodies. Making RNA accessible in many tissue types requires treatment with hydrochloric acid (HCl) and often proteinases (see below), and such treatments have been found to make the detection of RNA and protein in a single sample by imaging difficult. Notably, HCl treatment to permeabilize samples for RNA detection abolished antibody binding. Therefore, to preserve antibody binding during RNA staining, the samples were subsequently washed twice with PBST, post-fixed for 5 minutes to 1 hour in 4% PFA or 4% PFA and 1% glutaraldehyde, and washed three times with PBST. This post-fixation treatment preserved the antibody while facilitating RNA hybridization even in HCl-denatured tissue.

[0220] mRNA detection: After protein staining, samples were stained for mRNA using the following protocol. Samples stained for antibodies as described above were permeabilized in 0.1 N HCl for 3 minutes. The 0.1 N HCl-containing solution can also contain 1 mg / ml pepsin. Because treatment with HCl before antibody staining was found to significantly reduce and modify the antibody staining pattern, HCl permeabilization was performed after antibody staining. We also found that RNA staining in tissues was suboptimal after the pretreatment steps performed for protein and RNA staining. To facilitate improved staining, samples were treated with NHS-acetate before RNA hybridization but after HCl permeabilization. Specifically, samples were washed five times with PBST and treated with NHS-acetate for 30 minutes.

[0221] The samples were then blocked in hybridization blocking buffer and Phi29 blocking oligonucleotide for 30 minutes and washed twice in PBST. The Phi29 blocking oligonucleotide can have the structure shown in Figure 4. Phi29 blocking oligonucleotides were used to protect antibody-conjugated oligonucleotides from Phi29 3'→5' exonuclease activity, which can rapidly degrade the single-stranded 3' end of the oligonucleotide, as shown in the right panel of Figure 4. Phi29 blocking oligonucleotides have two features: (1) a long 5' extension template, which was found to prevent Phi29 exonuclease activity on the antibody-oligonucleotide conjugate, and (2) a 3' flap with at least 10 phosphorothioate bonds, which was found to prevent Phi29 from double-stranding the DNA barcode and maintain the hybridization readout of the DNA barcode. The 3' flap of the Phi29 blocking oligonucleotide can be approximately 10-20 nucleotides in length with at least 10 phosphorothioate bonds. The 5' extension template of the Phi29 blocking oligonucleotide can have a length of about 15 to 25 nucleotides, such as about 22 nucleotides. The length of the nucleotide sequence present in the Phi29 blocking oligonucleotide that is complementary to the oligonucleotide of the antibody-oligonucleotide conjugate can be about 20 to 30 nucleotides. As shown in Figure 4, the use of Phi29 blocking oligonucleotides significantly improved the sensitivity of protein detection.

[0222] The samples were then hybridized in 20% formamide hybridization buffer and 10 nM padlock oligonucleotide at 45°C for 16-18 hours. The samples were washed three times with PBST. Ligation was performed overnight at room temperature using SPLINTR ligase, followed by three washes with PBST. Rolling circle amplification (RCA) was performed overnight at 30°C. The samples were washed twice with PBST and post-fixed for 15 minutes in 4% PFA. During RCA, the polymerase activity of Phi29 polymerase extends the 3' end of the antibody-conjugated oligonucleotide to generate a complementary sequence to the 5' extension template of the Phi29 blocking oligonucleotide, as shown in Figure 4.

[0223] Periodic imaging: Imaging of the target protein and target nucleic acid was performed by cyclic imaging, as shown in FIGS.

[0224] For protein detection, a protein detection bridge oligonucleotide and an associated readout oligonucleotide were hybridized to an antibody-coupled oligonucleotide as shown in Figure 1. The protein detection bridge oligonucleotide binds to the barcode sequence on the antibody-oligonucleotide conjugate and five nucleotides of a conserved sequence on the oligonucleotide to maximize stability. The protein detection bridge oligonucleotide has a nucleotide sequence approximately 28 nucleotides long that is complementary to the oligonucleotide on the antibody-oligonucleotide conjugate. The protein detection bridge oligonucleotide contains a repeat of the nucleotide sequence that binds to a fluorescently labeled probe to amplify the detected fluorescent signal. The sample was then washed and imaged. This cycle was repeated until all proteins of interest were imaged. Heat denaturation was performed to remove the probe and protein detection bridge oligonucleotide before the next imaging cycle. Alternatively, we used a probe with a disulfide-conjugated dye, which cleaves the disulfide and allows for removal of the fluorescent signal from the probe.

[0225] For mRNA detection, an mRNA detection bridge oligonucleotide and an associated readout oligonucleotide were hybridized to the mRNA of interest. The mRNA detection bridge oligonucleotide contained a nucleotide sequence complementary to the amplicon generated during RCA (e.g., the sequence present in the padlock oligonucleotide) and also contained repeats of a nucleotide sequence that binds to a fluorescently labeled probe to amplify the detected fluorescent signal. The mRNA detection bridge oligonucleotide had a nucleotide sequence complementary to the amplicon, approximately 12 nucleotides in length. The sample was then washed and imaged. This cycle was repeated until all of the mRNA of interest was imaged. As described above for protein detection, probes with heat-denatured or disulfide-conjugated dyes were used to enable cyclic imaging.

[0226] As shown in Figures 3 and 5, this method allows for the simultaneous detection of multiple proteins and mRNAs in a single sample.

[0227] While the subject matter disclosed herein and its advantages have been described in detail, it is to be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods, and steps described in the specification. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0228] Throughout this application, various patents, patent applications, publications, product descriptions, and protocols are cited, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. A method for imaging target proteins and target nucleic acids in a sample, comprising: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to a target protein in the sample, wherein the protein binding reagent is coupled to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide comprising a nucleotide sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (d) amplifying target nucleic acids in said sample by performing an amplification process to generate amplicons; (e) contacting the sample with a first labeled detection probe comprising a sequence complementary to the sequence of the oligonucleotide coupled to the protein binding reagent; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second, labeled detection probe comprising a sequence complementary to a sequence of the amplicon; (h) imaging the second labeled detection probe to detect the target nucleic acid.

2. 2. The method of claim 1, wherein the amplification process is a rolling circle amplification process.

3. the rolling circle amplification process (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template; (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

4. The method of any one of claims 1 to 3, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

5. The method of any one of claims 1 to 4, wherein the target nucleic acid comprises RNA.

6. The method of any one of claims 1 to 4, wherein the target nucleic acid is the oligonucleotide coupled to the protein binding reagent, the blocking oligonucleotide, or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the nucleotide sequence complementary to the oligonucleotide coupled to a protein-binding reagent is located at the 5' end of the blocking oligonucleotide.

8. 8. The method of any one of claims 1 to 7, wherein the oligonucleotide coupled to a protein-binding reagent comprises a barcode sequence, and the blocking oligonucleotide does not bind to the barcode sequence.

9. The method of any one of claims 1 to 8, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

10. 10. The method of claim 9, wherein the one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide.

11. 11. The method of claim 9 or 10, wherein the one or more modified nucleotides comprise a nucleotide having a phosphorothioate linkage.

12. The method of any one of claims 1 to 12, wherein the 3' end of the blocking oligonucleotide comprises about 1 to about 10 nucleotides having phosphorothioate linkages.

13. Providing a sample may include: (a) treating the sample with a fixative; (b) dehydrating the sample; and (c) permeabilizing the sample; The method of any one of claims 1 to 12, comprising one or more of:

14. The method of any one of claims 1 to 13, wherein at least 10 target proteins are imaged in the sample.

15. The method of any one of claims 1 to 14, wherein at least 10 target nucleic acids are imaged in the sample.

16. The method of any one of claims 1 to 15, wherein the sample is post-fixed before amplifying the target nucleic acid.

17. The method according to any one of claims 1 to 16, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

18. The method of any one of claims 1 to 17, wherein the sample is treated with NHS-acetate before amplifying the target nucleic acid.

19. The method of any one of claims 1 to 18, wherein the sample is a tissue sample.

20. 1. A method for imaging target proteins and target nucleic acids in a sample, comprising: (a) allowing a protein binding reagent coupled to an oligonucleotide to bind to a target protein in the sample; (b) hybridizing a blocking oligonucleotide to the oligonucleotide coupled to the protein binding reagent; (c) amplifying target nucleic acids in said sample by performing an amplification process to generate amplicons; (d) imaging the target protein by detecting a first labeled detection probe hybridized to the oligonucleotide coupled to the protein binding reagent; (e) imaging the target nucleic acid by detecting a second labeled detection probe that is hybridized to the amplicon.

21. 21. The method of claim 20, wherein the amplification process is a rolling circle amplification process.

22. the rolling circle amplification process (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template comprising the target nucleic acid; (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

23. The method of any one of claims 20 to 22, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

24. 24. The method of any one of claims 20 to 23, wherein the target nucleic acid comprises RNA.

25. The method of any one of claims 20 to 23, wherein the target nucleic acid is the oligonucleotide coupled to the protein binding reagent, the blocking oligonucleotide, or a combination thereof.

26. The method of any one of claims 20 to 25, wherein the nucleotide sequence complementary to the oligonucleotide coupled to a protein-binding reagent is located at the 5' end of the blocking oligonucleotide.

27. 27. The method of any one of claims 20 to 26, wherein the oligonucleotide coupled to a protein binding reagent comprises a barcode sequence, and the blocking oligonucleotide does not bind to the barcode sequence.

28. 28. The method of any one of claims 20 to 27, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

29. 29. The method of claim 28, wherein the one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide.

30. 30. The method of claim 28 or 29, wherein the one or more modified nucleotides comprise a nucleotide having a phosphorothioate linkage.

31. The method of any one of claims 20 to 30, wherein at least 10 target proteins are imaged in the sample.

32. The method of any one of claims 20 to 31, wherein at least 10 target nucleic acids are imaged in the sample.

33. The method of any one of claims 20 to 32, wherein the sample is post-fixed before amplifying the target nucleic acid.

34. The method of any one of claims 20 to 33, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

35. The method of any one of claims 20 to 34, wherein the sample is treated with NHS-acetate before amplifying the target nucleic acid.

36. The method of any one of claims 20 to 35, wherein the sample is a tissue sample.

37. A kit for carrying out the method according to any one of claims 1 to 36.

38. 38. The kit of claim 37, comprising at least one container containing the blocking oligonucleotide.

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