In situ epitranscriptome profiling

Abstract

The present disclosure provides methods, compositions, and systems for profiling epitranscriptomic RNA modifications in cells. The present disclosure also provides methods for profiling the interaction between one or more RNAs of interest in cells and RNA binding proteins (for example, proteins that introduce epitranscriptomic modifications of RNAs of interest). The present disclosure also provides methods for diagnosing a disease or disorder in a subject based on the profile of epitranscriptomic RNA modifications in cells, including cells inside intact tissue, or the profile of interactions between RNA binding proteins and RNAs. The present disclosure also provides methods for screening or testing candidate agents that can modulate the epitranscriptomic modifications of one or more RNAs or the interactions between one or more RNAs and RNA binding proteins. The present disclosure also provides methods for treating a disease or disorder in a subject in need of such treatment. The present disclosure also describes pairs of probes and sets of probes that include oligonucleotide moieties that can be useful for carrying out the methods described herein. Additionally, the present disclosure provides kits that include any of the probes described herein.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. USSN63 / 231,585, filed August 10, 2021, which is incorporated herein by reference. [Background technology]

[0002] 2. Background of the Invention Cellular RNA contains a rich chemical repertoire of enzyme-catalyzed modifications (i.e., the epitranscriptome) (Roundtree, IA et al., Dynamic RNA modifications in gene expression regulation. Cell 169, 1187 (2017)). Genetic analysis of model organisms has revealed that RNA-modifying enzymes are essential for higher eukaryotes. Furthermore, RNA modification pathways have been identified to regulate cell fate, organ development and human diseases (e.g., cancer) (Jonkhout, N. et al., The RNA modification landscape in human disease. RNA 23, 1754 (2017); Li, X. et al., Epitranscriptome sequencing technologies: decoding RNA modifications. Nature Methods 14, 23 (2017)). However, our understanding of how these epitranscriptome modifications affect cellular functions in complex biological systems remains limited. This is because traditional analysis of RNA modifications relies on mixtures of millions of cells using bulk RNA sequencing or mass spectrometry (Li, X. et al., Epitranscriptome sequencing technologies: decoding RNA modifications. Nature Methods 14, 23 (2017)). However, recent studies with single-cell sequencing approaches have revealed that multicellular organisms consist of diverse cell types, and even apparently homogeneous cell populations have variable single-cell states. Furthermore, the same type of RNA modification may have opposite regulatory effects depending on the cell type and physiological context. Although single-cell RNA sequencing techniques have been transformative for transcriptome analysis, the lack of single-cell and subcellular resolution in epitranscriptome studies has obscured the heterogeneity of RNA modification patterns in biological tissues and impaired the ability to analyze how RNA modifications control gene expression across different cell types.Therefore, there is a need for a system to profile epitranscriptomic RNA modifications with single-cell and / or subcellular resolution. Summary of the Invention

[0003] Summary of the Invention To address limitations associated with previously developed single-cell RNA sequencing methods, platforms for three-dimensional (3D) in situ sequencing of RNA modifications, as well as platforms for profiling interactions between RNA and proteins that anchor such RNA modifications, have been developed (Figures 1, 10A, 12A, 13A, and 13C). These platforms can be utilized to dissect gene regulatory mechanisms mediated by RNA modifications at single-cell or subcellular resolution in intact living tissues. Thus, epitranscriptomic RNA modifications (e.g., N-terminal domains) along with interactions between RNA-binding proteins (e.g., enzymes that anchor epitranscriptomic RNA modifications) and RNA can be analyzed. 6 -Methyladenosine (m 6 A)) can be profiled. The methods, probes, compositions and systems disclosed herein are broadly applicable to profiling various epitranscriptomic RNA modifications or interactions between RNA and RNA-binding proteins in various tissues. These methods and systems can also be useful for studying how epitranscriptomic RNA modifications or RNA-binding proteins in various cell types interact to define cellular states and regulate, for example, brain function (Widagdo, J. et al., The Journal of Biological Chemistry, 2011, 113:131-132, 2011). 6A-epitranscriptomic signature in neurobiology: from neurodevelopment to brain plasticity. Journal of Neurochemistry 147, 137 (2018)). The methods, compositions, and systems described herein may also be useful for establishing new principles of post-transcriptional gene regulatory mechanisms at single-cell or subcellular resolution (spatial resolution of approximately 150-400 nm, depending on both the size of the DNA amplicon and the optical limits) in complex biological systems, as well as discovering the role of RNA chemical fingerprints (epitranscriptomic RNA modifications) in health and disease. The methods, compositions, and systems described herein may also be used on cells present inside intact tissues (e.g., tissue samples provided by or from human or non-human subjects, such as biopsies).

[0004] Thus, in one aspect, the present disclosure provides methods, compositions and systems for profiling epitranscriptome RNA modification in a cell or a number of cells (see, for example, Figures 1, 10A, and 12A).In the methods and systems disclosed herein, cells can be contacted with one or more pairs or sets of probes, which are further described herein and can be used to amplify (for example, by rolling circle amplification) the epitranscriptome modified RNA of interest, to produce one or more concatenated amplicons.The one or more concatenated amplicons can then be embedded in a polymer matrix and sequenced (for example, by SEDAL sequencing (sequencing by dynamic annealing and ligation error reduction) as further described herein) to determine the identity of the transcripts and their positions within the polymer matrix. The location of the modified transcript of interest can be used to profile the RNA of interest, including at least one epitranscriptome modification, and to obtain spatiotemporal information to improve understanding of how epitranscriptome modifications, subcellular location, and timing affect cellular function in health and disease. The method and system can be useful, for example, to compare epitranscriptome RNA modifications in cells (or multiple cells) from diseased and healthy tissue samples; or, for example, to compare epitranscriptome RNA modifications in cells treated with an agent (e.g., a therapeutic agent or potential therapeutic agent such as a small molecule, protein, peptide, nucleic acid, lipid, or carbohydrate) and untreated cells, or in diseased and healthy cells.

[0005] In some embodiments, the present disclosure provides a method for profiling epitranscriptomic RNA modifications in a cell, comprising: a) contacting a cell with one or more sets of probes, where each set of probes includes a first probe (i.e., a "padlock probe"), a second probe (i.e., a "sprint probe"), and a third probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein), an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell. The present invention provides a method comprising:

[0006] In some embodiments, the present disclosure provides a method for profiling epitranscriptomic RNA modifications in a cell, comprising: a) contacting a cell with one or more pairs of probes, where each probe pair comprises a first probe (i.e., a "padlock probe") and a second probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) contacting a second probe, the second probe comprising a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell. In certain embodiments where only two probes are used in the methods herein, no splint probe is used.

[0007] Thus, these methods can be used to determine the location of the RNA of interest modified with one or more epitranscriptome modifications of interest in a cell or cell population (for example, in intact tissue), or in a cell organelle.In some embodiments, the methods described herein are used to simultaneously profile more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 RNAs of interest.In some embodiments, epitranscriptome RNA modifications are N 6 -Methyladenosine (m 6 A), N 1 -Methyladenosine (m 1 A), pseudouridine, N 6 ,2'-O-Dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -Acetylcytidine (ac 4 C), 2'-O-methylated (Nm), or 5-methylcytosine (m 5 C).

[0008] In another aspect, the present disclosure provides methods, probes, compositions and systems for profiling the interaction between RNA-binding proteins and RNA in a cell or a number of cells, or in a subcellular location, such as one or more specific organelles (see, for example, Figures 13A and 13C). In the methods and systems disclosed herein, cells can be contacted with one or more pairs or sets of probes as further described herein, and can be used to amplify (e.g., by rolling circle amplification) the RNA of interest bound by the RNA-binding protein to produce one or more concatenated amplicons. The one or more concatenated amplicons can then be embedded in a polymer matrix and sequenced (e.g., by SEDAL sequencing (sequencing by dynamic annealing and ligation error reduction) as further described herein) to determine the identity of the transcripts and their location within the polymer matrix. The location of the modified transcript of interest can be used to profile the RNA of interest bound by at least one RNA binding protein, and spatiotemporal information can be obtained to improve understanding of how the interaction between the RNA binding protein and the RNA, the subcellular location of such interaction, and the timing of such interaction affect the function of cells in health and disease. The methods and systems can be useful, for example, to compare the interaction between the RNA binding protein and the RNA in cells (or multiple cells) from diseased and healthy tissue samples; or, for example, to compare the interaction between the RNA binding protein and the RNA in cells treated with an agent (e.g., a therapeutic agent or a potential therapeutic agent) and untreated cells, or in diseased and healthy cells.

[0009] In some embodiments, the present disclosure provides a method for profiling interactions between an RNA binding protein and one or more RNAs of interest in a cell, comprising: a) contacting a cell with one or more sets of probes, where each set of probes includes a first probe (i.e., a "padlock probe"), a second probe (i.e., a "sprint probe"), and a third probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein), an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each RNA of interest bound by the RNA-binding protein in the cell. The present invention provides a method comprising:

[0010] Thus, these methods can be used to determine the location of an RNA of interest bound by one or more RNA-binding proteins (e.g., enzymes that install epitranscriptomic RNA modifications) within a cell or population of cells (e.g., in intact tissues), or within organelles of a cell.

[0011] The methods, compositions, and systems described herein can be useful for studying epitranscriptome RNA modifications and interactions between RNA binding proteins and RNA in tissues (e.g., developing tissues, normal tissues, diseased tissues, treated tissues), for diagnosing and treating various diseases, for research purposes, and for drug discovery. Thus, in one aspect, the present disclosure provides a method for diagnosing a disease or disorder in a subject. For example, the method for profiling epitranscriptome RNA modifications or interactions between RNA binding proteins and RNA described herein can be performed on a cell or a number of cells taken from a subject (e.g., a subject that is thought to have or is at risk of having a disease or disorder, or a subject that is healthy or thought to be healthy). The expression of the various epitranscriptome modified RNAs of interest or the RNA of interest bound by the RNA binding proteins in the cells can then be compared with the expression of the same modified RNA of interest in non-disease cells or cells from a non-disease tissue sample (e.g., a cell from a healthy individual, or a number of cells from a population of healthy individuals). Any difference in the epitranscriptome RNA modification profile of a cell or the interaction profile between RNA binding proteins and RNA in a cell (including a single RNA or multiple RNAs of interest, for example, specific disease signatures) compared to one or more non-disease cells can indicate that the subject has a disease or disorder. The epitranscriptome RNA modification and / or the interaction between RNA binding proteins and RNA in one or more non-disease cells (for example, normal cells) can be profiled in parallel with the expression in disease cells as a control experiment. The epitranscriptome RNA modification and / or the interaction between RNA binding proteins and RNA in one or more non-disease cells (for example, normal cells) can also be profiled in advance, and the profile of the disease cells can be compared to this reference data for non-disease cells.

[0012] In another aspect, the present disclosure provides a method for screening agents that can modulate the epitranscriptome modification of one or more RNAs of interest or the interaction between RNA binding protein and one or more RNAs of interest.For example, the method for profiling epitranscriptome RNA modification or the interaction between RNA binding protein and RNA described herein can be carried out in cells in the presence of one or more candidate agents.The expression of various epitranscriptome modified RNAs of interest in cells and / or the interaction profile between RNA binding protein and RNA in cells (for example, normal cells or diseased cells) can then be compared with the expression of the same modified RNA of interest or RNA bound by RNA binding protein in cells that have not been exposed to one or more candidate agents.The difference in either epitranscriptome RNA modification profile or the interaction profile between RNA binding protein and RNA compared to cells that have not been exposed to the candidate agent can indicate that the epitranscriptome modification of one or more RNAs of interest is modulated by the candidate agent. In some embodiments, a particular signature known to be associated with the treatment of a disease (e.g., a number of epitranscriptome-modified RNAs of interest, or interactions between RNA-binding proteins and a number of RNAs of interest) can be used to identify agents that can modulate epitranscriptome RNA modifications and / or interactions between RNA-binding proteins and RNAs in a desired manner, thus treating the disease.The methods and systems described herein can also be used to identify drugs with particular side effects, for example, by looking for specific epitranscriptome RNA modification signatures or RNA-binding protein-RNA interaction signatures when one or more cells are treated with a candidate agent or known drug (or a combination of a number of candidate agents and / or known drugs, such as provided in a screening library of compounds).The methods or systems described herein may also be used to identify research reagents or chemical probes that may be useful for studying the basic biology of epitranscriptome modifications or interactions between RNA-binding proteins and RNA.

[0013] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject. For example, the method and system for profiling epitranscriptome RNA modification or interaction between RNA binding protein and RNA described herein can be performed on cells from a sample taken from a subject (e.g., a subject who is thought to have or is at risk of having a disease or disorder). The epitranscriptome RNA modification profile or interaction profile of one or more RNAs of interest of the cells with RNA binding protein can then be compared with the epitranscriptome RNA modification profile or interaction profile of one or more RNAs of interest with RNA binding protein in cells from a non-disease tissue sample. If any difference in the epitranscriptome RNA modification profile or the interaction profile between RNA binding protein and RNA compared to non-disease cells is observed, a treatment for the disease or disorder (e.g., pharmaceutical agents, surgery, radiation therapy, surgery, physical therapy, lifestyle changes, etc.) can then be administered to the subject. The epitranscriptome RNA modification and / or interaction between RNA binding protein and RNA in one or more non-disease cells can be profiled together with the epitranscriptome RNA modification in disease cells as a control experiment. Epitranscriptomic RNA modifications and / or interactions between RNA binding proteins and RNA in one or more non-diseased cells (e.g., normal cells) may also be previously profiled, and the epitranscriptomic RNA modifications and / or interactions between RNA binding proteins and RNA in the diseased cells may be compared to this reference data for the non-diseased cells.

[0014] In another aspect, the disclosure provides a pair of probes comprising a first probe (also referred to herein as a "padlock" probe) and a second probe (also referred to herein as a "primer" probe), wherein: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) The second probe comprises a portion that recognizes the epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe.

[0015] In another aspect, the disclosure provides a set of probes comprising a first probe (i.e., a "padlock" probe), a second probe (i.e., a "sprint" probe), and a third probe (i.e., a "primer" probe), wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) The third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe.

[0016] In another aspect, the disclosure provides a set of probes comprising a first probe (i.e., a "padlock" probe), a second probe (i.e., a "sprint" probe), and a third probe (i.e., a "primer" probe), wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) The third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe.

[0017] In another aspect, the present disclosure provides a kit (for example, a kit that includes any of the probe pairs or probe sets disclosed herein).In some embodiments, the kit includes a number of probe pairs or probe sets as described herein, each of which can be used to identify specific epitranscriptome-modified RNA of interest or the interaction between specific RNA-binding protein of interest and RNA.In certain embodiments, the kit includes more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 probe pairs.In certain embodiments, the kit includes more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 probe sets. The kits described herein may also include any other reagents or components useful for carrying out the methods described herein, including, but not limited to, cells, enzymes such as ligases and / or polymerases, amine-modified nucleotides, RNA-modified binding agents (e.g., primary antibodies, secondary antibodies, proteins, peptides, aptamers, small molecules, etc.), buffers, reagents (including dyes, stains, buffers, and more), and monomers for making polymer matrices (e.g., polyacrylamide matrices).

[0018] In another aspect, the present disclosure provides a system for profiling epitranscriptomic RNA modifications in a cell. In some embodiments, such a system comprises: a) a cell (e.g., an isolated cell or a cell present within an intact tissue); b) one or more probe pairs comprising a first probe (i.e., a "padlock probe") and a second probe (i.e., a "primer probe"), wherein: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) one or more pairs of probes, wherein the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope; and d) Computer Includes.

[0019] In some aspects, the present disclosure provides a system, comprising: a) Cell; b) a set of one or more probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) a third probe, a set of one or more probes, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; c) a microscope; and d) Computer The present invention provides a system including:

[0020] In some aspects, the present disclosure provides a system, comprising: a) Cell; b) a set of one or more probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) a third probe, a set of one or more probes, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; c) a microscope; and d) Computer The present invention provides a system including:

[0021] Any of the probes (ie, probe pairs and probe sets) described herein may be used in the systems contemplated by this disclosure.

[0022] It should be understood that the foregoing concepts, and additional concepts described below, may be arranged in any suitable combination, as the disclosure is not limited in this respect. Moreover, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.

[0023] The following drawings form a part of this specification and are included to further demonstrate certain specific aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0024] [Figure 1-1]Figure 1 provides an overview of the method for profiling m6A modified RNA in situ. An anti-m6A secondary antibody is conjugated to a polymerizable DNA primer that anneals to a padlock probe present on the same RNA. A functionalized cDNA amplicon is then generated from the co-localized DNA primer and padlock probe. The chemically functionalized cDNA amplicon is then covalently linked to a polyacrylamide matrix to allow optical clearing of tissue and processing of biomolecules. [Figure 1-2] Figure 1 provides an overview of the method for profiling m6A modified RNA in situ. An anti-m6A secondary antibody is conjugated to a polymerizable DNA primer that anneals to a padlock probe present on the same RNA. A functionalized cDNA amplicon is then generated from the co-localized DNA primer and padlock probe. The chemically functionalized cDNA amplicon is then covalently linked to a polyacrylamide matrix to allow optical clearing of tissue and processing of biomolecules.

[0025] [Figure 2A-2B] Figures 2A-2D show examples of single cell in situ profiles of m6A RNA modification. Figure 2A shows detection of m6A modified β-actin RNA. Figures 2B-2C provide several negative controls, namely no primary antibody (Figure 2B), secondary antibody incubation without DNA conjugate (Figure 2C), and no secondary antibody (Figure 2D). [Fig. 2C-2D] Figures 2A-2D show examples of single cell in situ profiles of m6A RNA modification. Figure 2A shows detection of m6A modified β-actin RNA. Figures 2B-2C provide several negative controls, namely no primary antibody (Figure 2B), secondary antibody incubation without DNA conjugate (Figure 2C), and no secondary antibody (Figure 2D).

[0026] [Figure 3A]Figures 3A-3D are images showing how RNA modification-mediated gene regulation in living tissues can be resolved by enabling single-cell in situ epitranscriptome profiling. Figure 3A shows various chemical modifications to eukaryotic messenger RNA. The collection of all modification sites is called the epitranscriptome. [Figure 3B] Figure 3B provides a schematic illustrating the single-cell heterogeneity of epitranscriptome states across different cell types, conditions, and subcellular locations. These issues have not been addressed by previously developed bulk transcriptome or epitranscriptome analysis methods. [Figure 3C] Figure 3C shows three-dimensional (3D) in situ sequencing of RNA and RNA modifications. DNA-conjugated modification-specific binding agents are used with RNA sequence-specific DNA probes that hybridize to cellular mRNAs to selectively visualize modified RNA in intact tissues. Each RNA sequence-specific probe contains a barcode that encodes the identity of a gene that is read out by image-based in situ sequencing. Such highly multiplexed single-cell quantification of RNA with respect to chemical modification status in 3D allows single-cell discovery of cell types and epitranscriptomic cell states. [Figure 3D] Figure 3D shows the decomposition of RNA modification-mediated gene regulation in tissues, as exemplified by four possible changes in RNA modifications during brain activity. The shifts in epitranscriptome patterns, together with gene expression changes of RNA modification pathways, provide information about possible gene regulatory schemes in different cells and brain regions. In situ epitranscriptome sequencing can also be combined with precise gene perturbations to fully analyze gene regulatory mechanisms.

[0027] [Figure 4] FIG. 4 provides a schematic illustrating the issues regarding RNA modifications that cannot be analyzed by bulk epitranscriptome sequencing.

[0028] [Diagram 5] FIG. 5 shows examples of mutagenic and non-mutagenic epitranscriptome RNA modifications.

[0029] [Figure 6] Figure 6 provides a schematic showing the role of m6A in brain function. The m6A modification is reversible and is recognized by m6A-binding proteins that regulate multiple stages of the mRNA life cycle. m6A-protein complexes have been found to be abundant in synapses. Alterations in the m6A pathway affect multiple physiological functions and are associated with a range of psychiatric disorders.

[0030] [Figure 7] Figure 7 provides a schematic of the 3D-m6A-seq method. After brain tissue is prepared, DNA-conjugated m6A-specific binders hybridize to cellular mRNA in intact tissue along with RNA sequence-specific DNA probes. Only m6A-modified sites are enzymatically replicated as cDNA amplicons, whereas unmodified RNA is not amplified. Each RNA sequence-specific probe contains a barcode that encodes the identity of the gene and m6A site, which is read out by in situ sequencing (SEDAL).

[0031] [Figure 8]Figure 8 provides a schematic of four exemplary biological investigations that can be performed using single-cell in situ sequencing of RNA modifications: (1) using 3D-m6A-seq mapping of cortex to determine whether the m6A methylome is cell type and region specific; (2) using 3D-m6A-seq to test global stimulation of neuronal cultures (KCl depolarization) or mouse visual cortex (dark / light conditioning) to determine whether activity-regulated genes (ARGs) are differentially expressed in different cell types and whether m6A states covary with ARGs; (3) m6A dynamics during specific experiences (as an example, acute restraint stress) to determine whether m6A has circuit selectivity and variable responses in different brain regions. and (4) integrated analysis of single-cell m6A patterns and gene expression levels of m6A pathway proteins (e.g., methyltransferases, demethylases, and binding proteins) to determine which factors shape epitranscriptomic patterns and have regulated gene expression in various cell types, and whether m6A-dependent gene regulation during neural stimulation is universal for all neurons or is more active (or more silent) in defined neuronal cell types of specific neural circuits compared to the average activity of the brain.

[0032] [Figure 9A] Figures 9A-D show the background and applications of spatial epitranscriptomics. Figure 9A shows that m6A and its associated RNA-binding proteins (RBPs) regulate various important pathways. [Figure 9B-9C] 9B and 9C show a knowledge gap in epitranscriptomics that the methods provided herein are applicable to elucidating. [Figure 9D] FIG. 9D provides a schematic of in situ single-cell epitranscriptomics.

[0033] [Figure 10A]10A to 10E show the design and principle of m6AMap v1. Fig. 10A shows the workflow of m6AMap v1. [Figure 10B] FIG. 10B shows the conjugation strategy used to synthesize PAPG-oligos. [Fig. 10C-10D] Figure 10C demonstrates detection of actin beta (ACTB) site 1217 m6A in HeLa cells via antibody-PAPG-oligo detection, showing 20-30 fold signal enrichment over the negative control. Figure 10D demonstrates detection of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) site 2601 m6A in HeLa cells via antibody-independent biotinylated YTH-streptavidin-oligo detection, showing no enrichment over the negative control. [Figure 10E] FIG. 10E demonstrates detection of MALAT1 site 1248 m6A in mouse hippocampus via antibody-PAPG-oligo detection, showing approximately 15-fold signal enrichment compared to the negative control.

[0034] [Figure 11A-11B] Figures 11A-11B show the displacement of a secondary antibody against PAPG. Figure 11A shows the labeling chemistry used by the SiteClick antibody labeling kit (subsequent conjugation with alkyne-oligo can be performed using click chemistry). Figure 11B demonstrates the detection of ACTB site 1217m6A in HeLa cells via antibody-PAPG-oligo detection and antibody-antibody-oligo detection. The antibody-antibody-oligo detection scheme showed much lower signal enrichment than the IgG control group.

[0035] [Figure 12A-1] Figures 12A-12D show the design and principle of m6Amap v2. Figure 12A shows the workflow of m6Amap v2, which achieves the simultaneous detection of m6A-methylated RNAs and their unmethylated counterparts. [Figure 12A-2]Figures 12A-12D show the design and principle of m6Amap v2. Figure 12A shows the workflow of m6Amap v2, which achieves the simultaneous detection of m6A-methylated RNAs and their unmethylated counterparts. [Fig. 12B-12D] Figure 12B demonstrates the "winner takes all" behavior of probes targeting the same RNA: when two STARmap probe pairs (see PCT Publication No. WO 2019 / 199579, incorporated herein by reference) target the same ACTB mRNA in HeLa cells, only one of them is amplified in each ACTB mRNA molecule. Figure 12C demonstrates detection of MALAT1 site 2601 m6A in HeLa cells via m6A map v2, showing higher m6A stoichiometry in M ​​and early G1 phases. Figure 12D demonstrates detection of ACTB site 1217 m6A in HeLa cells via m6A map v2, showing higher m6A stoichiometry in non-dividing cells.

[0036] [Figure 13A] Figures 13A-13C show the principle and experimental results of the RBP map. Figure 13A shows the workflow of single RBP-RNA interaction mapping. [Figure 13B] FIG. 13B shows that HeLa cells transfected with 3xFLAG-YTH(WT / mut)-T2A-mCherry were tested for YTH binding of ACTB via the workflow shown in FIG. 13A. [Figure 13C] Figure 13C shows the workflow for multiplexed RBP-RNA mapping. Each antibody is conjugated to a unique DNA primer, and a corresponding gap-filling probe is annealed to it. First, a three-part probe is hybridized to the mRNA. Then, a mixture of different antibodies targeting different RBPs is added to the sample, followed by ligation and RCA. The barcode information of the probes can be read out by in situ sequencing.

[0037] [Figure 14]Figure 14 provides representative images of the first round of sequencing in the m6A map 100 gene data collection experiment. Ch01 and ch04 correspond to the STARmap amplicons, and ch02 and ch03 correspond to the m6A amplicons.

[0038] [Figure 15A-15B] Figures 15A-15H show the quality control and statistical summary of the m6A map v1 100 gene dataset. Well labeling: A1: STARmap; A2-anti-m6A: m6A map v1 with Abcam ab151230; A3-anti-m6A: m6A map v1 with SYSY 202003; B3-anti-m6A: m6A map v1 with Invitrogen RM362; B2-anti-m6A: negative control of m6A map v1 using CST 2729S normal rabbit IgG. Figure 15A shows the average number of reads per cell for each gene. Figure 15B shows the signal to noise ratio (calculated using the ratio of anti-m6A signal to IgG) in each well. [Fig. 15C-15D] Figure 15C shows the detected genes and reads per cell in each well, and Figure 15D shows the correlation of the estimated relative m6A stoichiometry between wells A2 and B3. [Fig. 15E-15F] Figure 15E shows the correlation between estimated m6A stoichiometry and STARmap reads, and Figure 15F shows the relative m6A stoichiometry of each gene measured in each well. [Fig. 15G-15H] Figure 15G provides a comparison of estimated m6A stoichiometry with m6A map v1 versus reported m6A stoichiometry for representative loci. Figure 15H provides a scatter plot of the data shown in Figure 15G.

[0039] [Figure 16A] Figures 16A-16C show subcellular and cell cycle analyses of the 100 gene dataset of m6A map v1. Figure 16A shows the effect of m6A deposition on RNA subcellular localization (x-axis: log2 fold change in nuclear percentage of m6A deposited relative to non-m6A deposited portions of a given RNA; y-axis: log10 of p-values). [Figure 16B] FIG. 16B shows that the cell cycle was determined using FUCCI fluorescence intensity. [Figure 16C] FIG. 16C shows representative genes with cell cycle-dependent m6A variation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: 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 Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0041] The terms "administer," "administering," and "administration" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a treatment or therapeutic agent, or a composition of a treatment or therapeutic agent, into or onto a subject.

[0042] The term "amplicon" as used herein refers to a nucleic acid (e.g., RNA) that is the product of an amplification reaction (i.e., the generation of one or more copies of a genetic fragment or target sequence) or a replication reaction. An amplicon can be formed artificially, for example, using PCR or other polymerization reactions. The term "concatenated amplicon" refers to multiple amplicons that are joined together to form a single nucleic acid molecule. A concatenated amplicon can be formed, for example, by rolling circle amplification (RCA), in which a circular oligonucleotide is amplified to produce multiple linear copies of the oligonucleotide as a single nucleic acid molecule that includes multiple concatenated amplicons.

[0043] "Antibody" refers to a glycoprotein that belongs to the immunoglobulin superfamily. The terms antibody and immunoglobulin are used interchangeably. With some exceptions, mammalian antibodies are typically made of a basic structural unit with two large heavy chains and two small light chains, respectively. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped together into different isotypes based on which heavy chains they possess. Five different antibody isotypes (IgG, IgA, IgE, IgD, and IgM) are known in mammals, which play different roles and help direct the appropriate immune response against each different type of foreign body encountered. The term "antibody" as used herein also encompasses antibody fragments, nanobodies, and single chain antibodies, as well as antibody variants. The term "antibody variant" may also be used to encompass antibody fragments. In some embodiments, the antibody or antibody variant is administered as a treatment for a disease or disorder (e.g., one associated with an altered profile of epitranscriptome RNA modifications in cells removed from a subject). In some embodiments, the antibody is conjugated to an oligonucleotide probe as described herein. In certain embodiments, the antibody is conjugated to an oligonucleotide probe that detects epitranscriptome RNA modifications (e.g., the antibody is an anti-mRNA probe).6 A antibody, anti-m 1 A antibody, anti-pseudouridine antibody, anti-m 6 Am antibody, anti-m 7 G antibody, anti-ac 4 C antibody, anti-Nm antibody or anti-m 5 C antibody).

[0044] "Cells" as used herein may be present in a population of cells (e.g., in a tissue, sample, biopsy, organ, or organoid). In some embodiments, the population of cells is composed of multiple different cell types. Cells for use in the disclosed methods and systems can be present in an organism, a single cell type derived from an organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, cells from subjects, and the like. Virtually any cell type and size can be adapted to the methods and systems described herein. In some embodiments, the cells are mammalian cells (e.g., complex cell populations such as naturally occurring tissues). In some embodiments, the cells are of human origin. In certain embodiments, the cells are retrieved from a subject (e.g., a human) through a medical procedure such as a biopsy. Alternatively, the cells can be a cultured population (e.g., a culture derived from a complex population, or a culture derived from a single cell type in which the cells have differentiated into multiple lineages). The cells can also be provided in situ in a tissue sample.

[0045] Cell types contemplated for use in the disclosed methods and systems include, but are not limited to, stem and progenitor cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc.), endothelial cells, muscle cells, cardiomyocytes, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells, hematopoietic cells, lymphocytes such as T cells (e.g., Thl T cells, Th2 T cells, ThO T cells, cytotoxic T cells) and B cells (e.g., pre-B cells), monocytes, dendritic cells, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, immune cells, neurons, hepatocytes, and cells involved in specific organs (e.g., thymus, endocrine glands, pancreas, brain, neurons, glia, astrocytes, dendrocytes, and genetically modified cells thereof). The cells can also be different types of transformed or neoplastic cells (e.g., cancers of different cellular origins, lymphomas of different cell types, etc.) or any kind of cancerous cell (e.g., from any of the cancers disclosed herein). Cells of different origins (e.g., ectoderm, mesoderm, and endoderm) are also contemplated for use in the disclosed methods and systems. In some embodiments, the cells are microglia, astrocytes, oligodendrocytes, excitatory neurons, or inhibitory neurons. In certain embodiments, the cells are HeLa cells. In some embodiments, cells of multiple cell types are present in the same sample.

[0046] The term "complementary" is used herein to refer to two oligonucleotide sequences (e.g., DNA or RNA) that contain bases that hydrogen bond with each other. The degree of complementarity between two oligonucleotide sequences can vary from complete complementarity to no complementarity (e.g., 100% complementarity, 99% complementarity, 98% complementarity, 97% complementarity, 96% complementarity, 95% complementarity, 90% complementarity, 85% complementarity, 80% complementarity, or less than 80% complementarity). For example, two oligonucleotide sequences can be only partially complementary to each other (e.g., in the probes described herein, where only a portion of the probe is complementary to another probe or RNA of interest). In some embodiments, a sequence is complementary to only a portion of another sequence. In some embodiments, a sequence is complementary to another sequence under a certain condition (e.g., a certain salt concentration, pH, etc.).

[0047] The terms "epitranscriptome modification", "epitranscriptome RNA modification", and "post-transcriptional modification" are used interchangeably throughout this disclosure. Epitranscriptome modification includes any biochemical modification of RNA in a cell. Such modifications can be chemical modifications, including, for example, methylation of nucleotides at various locations. Chemical epitranscriptome modifications include N 6 -Methyladenosine (m 6 A), N 1 -Methyladenosine (m 1 A), pseudouridine, N 6 ,2'-O-Dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -Acetylcytidine (ac 4 C), 2'-O-methylated (Nm), and 5-methylcytosine (m 5 In certain embodiments, the epitranscriptome modification includes, but is not limited to, N 6 -Methyladenosine (m 6A). Other chemical epitranscriptomic RNA modifications include adenosine to inosine mutations and queuosine (i.e., queuosine substitutes another nucleotide in RNA). Various types of cellular RNA can be epitranscriptomic modified, including but not limited to ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). Other epitranscriptomic modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, EM et al. Nature. 541, 339-346 (2017).

[0048] The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") of DNA and RNA, and mean any chain of two or more nucleotides. Polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, and single-stranded or double-stranded. Oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability of the molecule, its hybridization parameters, etc. An "aptamer" is a type of oligonucleotide molecule that binds to a specific target molecule (e.g., epitranscriptome modification).

[0049] A "protein," "peptide," or "polypeptide" includes a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein is at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. A protein may contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain) and / or amino acid analogs known in the art may alternatively be used. Also, one or more amino acids in a protein may be modified by the addition of a chemical entity, such as, for example, a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or a complex of multiple molecules. A protein may be a fragment or peptide of a naturally occurring protein. A protein may be naturally occurring, recombinant, synthetic, or any combination thereof. The protein may also be a therapeutic protein administered as a treatment for a disease or disorder (e.g., one associated with a change in the profile of epitranscriptomic RNA modifications in cells removed from a subject). In certain embodiments, the protein is an antibody or antibody variant (including an antibody fragment). In some embodiments, the protein binds to an epitranscriptomic RNA modification (e.g., m 6 A-specific YTH domain protein).

[0050] "RNA binding protein" refers to any protein that can bind to RNA or epitranscriptome modification of RNA. For example, RNA binding protein can be a protein that introduces epitranscriptome modification to RNA. RNA binding protein can also be a protein that recognizes and binds epitranscriptome modification of RNA. In certain embodiments, RNA binding protein comprises YTH family protein (e.g., YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2), IGF2BP family protein (e.g., IGF2BP1, IGF2BP2, or IGF2BP3), or FMR1. Additional RNA binding proteins that can be profiled using the methods provided herein are also described in Wang, X. et al., N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell 161, 1388-1399, doi:10.1016 / j.cell.2015.05.014(2015); Wang, X. et al., N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117-120, doi:10.1038 / nature12730(2014); Shi, H. et al., YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res 27, 315-328, doi:10.1038 / cr.2017.15(2017); Xiao, W. et al.,Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.Mol Cell 61,507-519,doi:10.1016 / j.molcel.2016.01.012(2016);Roundtree,IAet al.,YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs.Elife 6,doi:10.7554 / eLife.31311(2017);Hsu,PJet al.,Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis.Cell Res 27,1115-1127,doi:10.1038 / cr.2017.99(2017);Huang,H.et al.,Recognition of RNA N(6)-methyladenosine by IGF2BP enhances proteins mRNA stability and translation.Nat Cell Biol 20,285-295,doi:10.1038 / s41556-018-0045-z(2018); and Edens,BMet al.,FMRP Modulates Neural Differentiation through m(6)A-Dependent mRNA Nuclear Export.Cell Rep 28, 845-854 e845, doi:10.1016 / j.celrep.2019.06.072(2019), but are not limited to those disclosed therein.

[0051] "Transcript" or "RNA transcript" is the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. If the RNA transcript is a complementary copy of the DNA sequence, it is referred to as the primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as mature RNA. "Messenger RNA (mRNA)" refers to the RNA that does not contain introns and can be translated into a polypeptide by the cell.

[0052] The term "sample" or "biological sample" refers to any sample, including tissue samples (such as tissue sections, surgical biopsies, and needle biopsies of tissues); cell samples; or cell fractions, fragments, or organelles (such as obtained by lysing cells and centrifuging or otherwise separating their components). Other examples of biological samples include, but are not limited to, blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules derived from a first biological sample. In some embodiments, the biological sample is a surgical biopsy taken from a subject, such as a biopsy of any of the tissues described herein. In certain embodiments, the biological sample is a tumor biopsy. In some embodiments, the sample is brain tissue. In some embodiments, the tissue is cardiac tissue. In some embodiments, the sample is epithelial tissue, connective tissue, muscle tissue, or nerve tissue. In some embodiments, the sample is tissue from the central nervous system (e.g., the brain). In some embodiments, the cells used in the methods described herein are derived from such samples or biological samples.

[0053] The "subject" to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, or an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly person)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus or rhesus monkey) or a mouse). The term "patient" refers to a subject in need of treatment for a disease. In some embodiments, the subject is a human. In some embodiments, the patient is a human. A human can be male or female at any stage of development. Subjects or patients "in need" of treatment for a disease or disorder include, but are not limited to, those that exhibit any risk factor or symptom of a disease or disorder. In some embodiments, the subject is a non-human experimental animal (e.g., a mouse, a rat, a dog, or a non-human primate).

[0054] The term "therapeutic agent" as used herein refers to any agent that can be used to treat a disease or disorder, or to reduce or alleviate symptoms of a disease or disorder. In some embodiments, the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate. In some embodiments, the therapeutic agent is a known drug and / or an FDA-approved drug. In certain embodiments, the protein is an antibody. In certain embodiments, the protein is an antibody variant. In certain embodiments, the protein is a receptor, or a fragment or variant thereof. In certain embodiments, the protein is a cytokine. In certain embodiments, the nucleic acid is an mRNA, an antisense RNA, a miRNA, an siRNA, an RNA aptamer, a double-stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).

[0055] A "therapeutically effective amount" of a treatment or therapeutic agent is an amount sufficient to provide a therapeutic benefit in treating a condition or to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount of a treatment or therapeutic agent refers to an amount of a treatment that, alone or in combination with other treatments, provides a therapeutic benefit in treating a condition. The term "therapeutically effective amount" can encompass an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of symptoms, and / or enhances the therapeutic effectiveness of another therapeutic agent.

[0056] As used herein, a "tissue" is a group of cells from the same origin and their extracellular matrix. Together, the cells perform a specific function. The association of multiple tissue types together forms an organ. The cells can be of different cell types. In some embodiments, the tissue is an epithelial tissue. Epithelial tissue is formed by cells that cover organ surfaces (e.g., the skin, airways, soft organs, the surface of the reproductive tract, and the inside of the digestive tract). Epithelial tissue performs protective functions and is also involved in secretion, excretion, and absorption. Examples of epithelial tissue include, but are not limited to, simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified epithelium, columnar epithelium, and glandular epithelium. In some embodiments, the tissue is a connective tissue. Connective tissue is a fibrous tissue composed of cells separated by non-living materials (e.g., extracellular matrix). Connective tissue provides shape to organs and holds them in place. Connective tissue includes fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Examples of connective tissue include, but are not limited to, blood, bone, tendons, ligaments, fat, and loose connective tissue. In some embodiments, the tissue is muscle tissue. Muscle tissue is an active contractile tissue formed from muscle cells. Muscle tissue functions to generate force and cause movement. Muscle tissue includes smooth muscle (e.g., as found inside organs), skeletal muscle (e.g., typically attached to bone), and cardiac muscle (e.g., as found in the heart, which contracts to pump blood throughout an organism). In some embodiments, the tissue is nervous tissue. Nervous tissue includes cells that comprise the central and peripheral nervous systems. Nervous tissue forms the brain, spinal cord, cranial nerves, and spinal nerves (e.g., motor neurons). In certain embodiments, the tissue is brain tissue.

[0057] The terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset or inhibiting the progression of the diseases described herein. In some embodiments, treatment can be administered after one or more signs or symptoms of disease have developed or been observed (e.g., prophylactically or following suspicion or risk of disease). In other embodiments, treatment can be administered in the absence of signs or symptoms of disease. For example, treatment can be administered to susceptible subjects prior to the onset of symptoms (e.g., in light of a history of symptoms in the subject or family members of the subject). Treatment can also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment can be administered after using the methods disclosed herein and observing changes in the profile of epitranscriptome RNA modifications in cells or tissues compared to healthy cells or tissues.

[0058] Detailed Description of Specific Embodiments The aspects described herein are not limited to specific embodiments, systems, compositions, methods, or configurations, which, as such, can, of course, vary. The terminology used herein is for the purpose of describing specific aspects only and is not intended to be limiting, unless specifically defined herein.

[0059] The present disclosure provides methods, compositions and systems for profiling epitranscriptomic RNA modifications in cells or a number of cells (e.g., cells present inside intact tissue or isolated cells) at single cell and subcellular resolution. The present disclosure also provides methods for profiling the interaction between one or more RNAs of interest in a cell and an RNA binding protein (e.g., a protein that introduces an epitranscriptomic modification of an RNA of interest). The present disclosure also provides methods for diagnosing a disease or disorder in a subject based on a profile of epitranscriptomic RNA modifications in a cell, including cells inside intact tissue, or a profile of the interaction between an RNA binding protein and an RNA. The present disclosure also provides methods for treating a disease or disorder in a subject in need of such treatment. The present disclosure also provides methods for screening or testing candidate agents that can modulate epitranscriptomic modifications of one or more RNAs or the interaction between one or more RNAs and an RNA binding protein. The present disclosure also provides kits that include any of the probes described herein, as well as probe pairs and sets that can be useful for carrying out the methods described herein.

[0060] Methods for profiling epitranscriptomic RNA modifications or interactions between RNA and RNA-binding proteins in cells - Patents.com In one aspect, the present disclosure provides a method for profiling epitranscriptome RNA modification in a cell (or in a large number of cells, for example, in intact tissue).In the method disclosed herein, a cell can be contacted with one or more pairs or sets of probes, which can be used to identify and locate the RNA of interest that comprises at least one epitranscriptome modification to produce one or more concatenated amplicons, as described further herein.The one or more concatenated amplicons can then be embedded in a polymer matrix and sequenced to determine the identity of the transcripts and their positions within the polymer matrix (for example, by SEDAL sequencing, as described further herein).The positions of the modified transcripts of interest can be used to profile the epitranscriptome modified RNA in the cell.

[0061] In some embodiments, the present disclosure provides a method for profiling epitranscriptomic RNA modifications in a cell, comprising: a) contacting a cell with one or more sets of probes, where each set of probes includes a first probe (i.e., a "padlock probe"), a second probe (i.e., a "sprint probe"), and a third probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein), an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell. The present invention provides a method comprising:

[0062] In some embodiments, the present disclosure provides a method for profiling epitranscriptomic RNA modifications in a cell (or in a large number of cells, e.g., in an intact tissue), comprising: a) contacting a cell with one or more pairs of probes, where each probe pair comprises a first probe (i.e., a "padlock probe") and a second probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) contacting a second probe, the second probe comprising a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell. The present invention provides a method comprising:

[0063] Any epitranscriptome RNA modification profile is contemplated by the present disclosure. In some embodiments, the epitranscriptome RNA modification is 6 -Methyladenosine (m 6 A), N 1 -Methyladenosine (m 1 A), pseudouridine, N 6 ,2'-O-Dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -Acetylcytidine (ac 4 C), 2'-O-methylated (Nm), or 5-methylcytosine (m 5C). Other epitranscriptome modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, EM et al., Nature. 541, 339-346 (2017). In some embodiments, the epitranscriptome RNA modification is an adenosine to inosine modification. In some embodiments, the epitranscriptome modification is a queuosine (i.e., queuosine substitutes another nucleotide in the RNA), polyadenylation, intron splicing, or histone mRNA processing. In certain embodiments, the epitranscriptome RNA modification is an N 6 -Methyladenosine (m 6 A) A single epitranscriptome modification can be profiled in a cell using the methods disclosed herein, or multiple different epitranscriptome modifications (e.g., 2, 3, 4, 5, or more) can be profiled simultaneously in a cell.

[0064] In some embodiments, the method for profiling epitranscriptome RNA modifications disclosed herein contemplates the use of a set of probes, including a first probe, a second probe, and a third probe. Such methods utilize what is referred to herein as a "three-probe strategy." The second probe of the set of probes (also referred to herein as a "sprint probe") comprises a portion that recognizes an epitranscriptome RNA modification (i.e., a post-transcriptional modification present in a particular RNA of interest). In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises a peptide. In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptome modification via a mechanism that comprises a biotin-streptavidin interaction. The portion of the probe that recognizes the epitranscriptome RNA modification can be a protein (e.g., an antibody or an antibody variant, or any protein that can otherwise bind to a specific epitranscriptome modification). In certain embodiments, the protein is PAPG.In some embodiments, the part of the second probe that recognizes epitranscriptome RNA modification comprises an agent that binds to antibody or antibody variant.For example, the second probe can comprise a secondary antibody and can be bound to the epitranscriptome RNA modification using a primary antibody.The secondary antibody of the second probe then recognizes the primary antibody that binds to the epitranscriptome RNA modification.For example, see Figure 1.In another example, the second probe can comprise a protein PAPG that can bind to the antibody that binds to the epitranscriptome RNA modification.When the portion of the second probe that recognizes the epitranscriptome RNA modification is a protein, the method may optionally further comprise contacting the cell with an antibody that recognizes the epitranscriptome RNA modification, where the antibody that recognizes the epitranscriptome RNA modification is recognized and bound by the protein of the second probe (e.g., PAPG). In certain embodiments, the portion of the second probe that recognizes the epitranscriptome RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant. When the portion of the second probe that recognizes the epitranscriptome RNA modification is a secondary antibody, the method may optionally further comprise contacting the cell with a primary antibody that recognizes the epitranscriptome RNA modification and is recognized by the secondary antibody of the second probe. The cell may be contacted with the primary antibody before or after being contacted with one or more pairs of probes. In certain embodiments, the cell is contacted with the primary antibody before being contacted with one or more pairs of probes. In some embodiments, the primary antibody is an anti-m. 6 A antibody, anti-m 1 A antibody, anti-pseudouridine antibody, anti-m 6 Am antibody, anti-m 7 G antibody, anti-ac 4 C antibody, anti-Nm antibody or anti-m 5 Instead of the primary antibody, the present disclosure also contemplates the use of any agent capable of directly binding the epitranscriptome RNA modification in the probe described herein. In some embodiments, the portion of the second probe that directly binds the epitranscriptome RNA modification comprises a protein. In certain embodiments, the protein is m 6 A specific YTH domain protein. In some embodiments, the portion of the second probe that directly binds the epitranscriptome RNA modification comprises an antibody, or an antibody variant.

[0065] In some embodiments, the second probe of the set of probes further comprises a polymerization blocker. The polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the method described herein. In some embodiments, the polymerization blocker is at the 3' end of the second oligonucleotide probe. The polymerization blocker can be, for example, any chemical moiety that prevents the polymerase from using the second oligonucleotide probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue that comprises a blocked 3' hydroxyl group (e.g., an oxygen protecting group at the 3' hydroxyl group). In some embodiments, the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.

[0066] In addition to the portion that recognizes the epitranscriptome RNA modification, the second probe also includes a portion that is complementary to a portion of the first probe. In the set of probes provided herein, the portions of the first and second probes that are complementary to each other can be the same in each set of probes. In some embodiments, the portions of the first and second probes that are complementary to each other are unique in each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0067] In some embodiments, each portion of the second probe is connected by an optional linker. In some embodiments, the optional linker is a nucleotide linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In some embodiments, the second probe of the set of probes used in the method described herein has the structure: 5'-[part that recognizes epitranscriptome RNA modification]-[part complementary to the first probe]-3' Including, Here, ]-[ includes any linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second probe. In some embodiments, the portion recognizing the epitranscriptomic RNA modification and the oligonucleotide portion of the probe are attached to each other via click chemistry (see, e.g., FIG. 10B).

[0068] The first probe (also referred to herein as a "padlock" probe) of the set of probes used in the methods described herein includes an oligonucleotide portion that is complementary to the second probe. In some embodiments, the portion of the first probe that is complementary to the portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' and 3' ends of the first probe.

[0069] The first probe of the set of probes used in the methods disclosed herein also comprises an oligonucleotide portion complementary to the RNA of interest. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22 or about 19-21 nucleotides in length. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.

[0070] The first probe of the set of probes used in the methods disclosed herein also contains an oligonucleotide barcode sequence composed of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length. The barcode of the oligonucleotide probe described herein may contain a gene-specific sequence used to identify an RNA of interest (i.e., an RNA modified with at least one specific epitranscriptomic modification).

[0071] Any order of arrangement of the parts of the first oligonucleotide probe is contemplated by the present disclosure.In some embodiments, the part of the first probe is connected to another part by an optional linker.In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.In some embodiments, the first probe has the structure: 5'-[portion complementary to the second probe]-[oligonucleotide barcode sequence]-[portion complementary to the RNA of interest]-[portion complementary to the third probe]-[portion complementary to the second probe]-3' Including, Here, ]-[ includes any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe. In some embodiments, any of the oligonucleotide portions of the probes that make up the set of probes provided herein comprises DNA.

[0072] The third probe (also referred to as "primer probe") of the set of probes used in the methods provided herein comprises a portion complementary to the RNA of interest and a portion complementary to the first probe of the set of probes. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5 to 15, 6 to 14, 7 to 13, 8 to 12, or 9 to 11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0073] In certain embodiments, the third probe of the set of probes has the structure: 5'-[part complementary to the RNA of interest]-[part complementary to the first probe]-[barcode sequence]-3' where ]-[ comprises any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0074] In some embodiments, the method of profiling epitranscriptome RNA modifications disclosed herein contemplates the use of a pair of probes comprising a first probe and a second probe. Such methods utilize what is referred to herein as a "two-probe strategy." The second probe of the pair of probes (also referred to herein as a "primer-probe") comprises a portion that recognizes an epitranscriptome RNA modification (i.e., a post-transcriptional modification present in a particular RNA of interest). In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises a peptide. In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptome modification via a mechanism that comprises a biotin-streptavidin interaction. The portion of the probe that recognizes the epitranscriptome RNA modification can be a protein (e.g., an antibody or antibody variant, or any protein that can otherwise bind to a specific epitranscriptome modification). In some embodiments, the portion of the second probe that recognizes the epitranscriptome RNA modification comprises an antibody, or an agent that binds to an antibody variant. For example, the second probe can comprise a secondary antibody and can be bound to the epitranscriptome RNA modification using a primary antibody. The secondary antibody of the second probe then recognizes the primary antibody bound to the epitranscriptome RNA modification. See, for example, FIG. 1. In certain embodiments, the portion of the second probe that recognizes the epitranscriptome RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant. If the portion of the second probe that recognizes the epitranscriptome RNA modification is a secondary antibody, the method may optionally further comprise contacting the cell with a primary antibody that recognizes the epitranscriptome RNA modification and is recognized by the secondary antibody of the second probe. The cell may be contacted with the primary antibody before or after being contacted with one or more pairs of probes.In certain embodiments, the cells are contacted with a primary antibody prior to being contacted with one or more pairs of probes. In some embodiments, the primary antibody is anti-m. 6 A antibody, anti-m 1 A antibody, anti-pseudouridine antibody, anti-m 6 Am antibody, anti-m 7 G antibody, anti-ac 4 C antibody, anti-Nm antibody or anti-m 5 The second probe is a C antibody. Instead of an antibody, the present disclosure also contemplates the use of any agent capable of binding the epitranscriptome RNA modification in the probe described herein. In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises a protein. In certain embodiments, the protein is a PAPG. In some embodiments, when the portion of the second probe that recognizes the epitranscriptome RNA modification comprises a protein, the method further comprises contacting the cell with an antibody that recognizes the epitranscriptome RNA modification, where the antibody that recognizes the epitranscriptome RNA modification is recognized and bound by the PAPG. In certain embodiments, the protein is a m that can directly bind to the epitranscriptome RNA modification. 6 A specific YTH domain protein. In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification binds directly to the modification and comprises an antibody or antibody variant.

[0075] In addition to the portion that recognizes the epitranscriptome RNA modification, the second probe of the pair of probes also contains a portion that is complementary to a portion of the first probe. In the pairs of probes provided herein, the portions of the first and second probes that are complementary to each other can be the same in each set of probes. In some embodiments, the portions of the first and second probes that are complementary to each other are unique in each pair of probes. In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0076] In some embodiments, each portion of the second probe of the pair of probes is connected by an optional linker. In some embodiments, the optional linker is a nucleotide linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In some embodiments, the second probe used in the method described herein has the structure: 5'-[part that recognizes epitranscriptome RNA modification]-[part complementary to the first probe]-3' Including, Here, ]-[ includes any linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second probe.

[0077] The first probe (also referred to herein as the "padlock" probe) of the pair of probes used in the methods described herein includes an oligonucleotide portion that is complementary to the second probe. In some embodiments, the portion of the first probe that is complementary to the portion of the second probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the second probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' and 3' ends of the first probe.

[0078] The first probe of the pair of probes used in the methods disclosed herein also comprises an oligonucleotide portion complementary to the RNA of interest. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.

[0079] The first probe of the pair of probes used in the methods disclosed herein also contains an oligonucleotide barcode sequence composed of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length. The barcode of the oligonucleotide probe described herein may contain a gene-specific sequence used to identify the RNA of interest (i.e., the RNA modified with at least one specific epitranscriptomic modification).

[0080] Any order of arrangement of the first oligonucleotide probe portion of the pair of probes is contemplated by the present disclosure.In some embodiments, the first probe portion is connected to another portion by an optional linker.In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.In some embodiments, the first probe has the structure: 5'-[part complementary to second probe]-[part complementary to RNA of interest]-[barcode sequence]-[part complementary to second probe]-3'; 5'-[part complementary to second probe]-[barcode sequence]-[part complementary to RNA of interest]-[part complementary to second probe]-3'; 5'-[part complementary to second probe]-[part complementary to RNA of interest]-[barcode sequence]-3'; 5'-[part complementary to second probe]-[barcode sequence]-[part complementary to RNA of interest]-3'; 5'-[part complementary to the RNA of interest]-[barcode sequence]-[part complementary to the second probe]-3'; or 5'-[barcode sequence]-[part complementary to the RNA of interest]-[part complementary to the second probe]-3' Including, Here, each instance of ]-[ includes an optional linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe. In some embodiments, either the oligonucleotide portion of the first probe and / or the second probe comprises DNA.

[0081] In some embodiments, the disclosure provides a method for profiling interactions between an RNA binding protein and one or more RNAs of interest in a cell, the method comprising: a) contacting a cell with one or more sets of probes, where each set of probes comprises a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each RNA of interest bound by the RNA-binding protein in the cell. The present invention provides a method comprising:

[0082] The interaction between any RNA binding protein and one or more RNAs of interest can be profiled using the method provided herein.In some embodiments, RNA binding protein is the protein that introduces epitranscriptome modification to RNA.In certain embodiments, RNA binding protein comprises YTH family protein (for example, YTHDF1, YTHDF2, YTHDF3, YTHDC1 or YTHDC2), IGF2BP family protein (for example, IGF2BP1, IGF2BP2 or IGF2BP3) or FMR1. Additional RNA binding proteins that can be profiled using the methods provided herein are also described in Wang, X. et al., N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell 161, 1388-1399, doi:10.1016 / j.cell.2015.05.014(2015); Wang, X. et al., N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117-120, doi:10.1038 / nature12730(2014); Shi, H. et al., YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res 27, 315-328, doi:10.1038 / cr.2017.15(2017); Xiao, W. et al.,Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.Mol Cell 61,507-519,doi:10.1016 / j.molcel.2016.01.012(2016);Roundtree,IAet al.,YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs.Elife 6,doi:10.7554 / eLife.31311(2017);Hsu,PJet al.,Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis.Cell Res 27,1115-1127,doi:10.1038 / cr.2017.99(2017);Huang,H.et al.,Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.Nat Cell Biol 20,285-295,doi:10.1038 / s41556-018-0045-z(2018); and Edens, BMet al., FMRP Modulates Neural Differentiation through m(6)A-Dependent mRNA Nuclear Export.Cell Rep 28,845-854 e845, doi:10.1016 / j.celrep.2019.06.072(2019), but are not limited to those disclosed therein.

[0083] In some embodiments, the method for profiling the interaction between an RNA-binding protein and an RNA of interest disclosed herein contemplates the use of a set of probes comprising a first probe, a second probe and a third probe. The second probe (also referred to herein as a "sprint probe") of the set of probes comprises a portion that recognizes an RNA-binding protein (e.g., an enzyme that introduces epitranscriptomic modifications to RNA). In some embodiments, the portion of the second probe that binds to the RNA-binding protein comprises a peptide. In some embodiments, the portion of the second probe that binds to the RNA-binding protein comprises an aptamer. In some embodiments, the portion of the second probe that binds to the RNA-binding protein comprises a small molecule. In certain embodiments, the second probe binds to the RNA-binding protein via a mechanism that comprises a biotin-streptavidin interaction. The portion of the probe that recognizes the RNA-binding protein can be a protein (e.g., an antibody or antibody variant, or any protein that can otherwise bind to a specific RNA-binding protein). In some embodiments, the portion of the second probe that recognizes the RNA-binding protein comprises an agent that binds to an antibody, or an antibody variant. For example, the second probe may comprise a secondary antibody, and the primary antibody may be used to bind to the RNA-binding protein. The secondary antibody of the second probe then recognizes the primary antibody bound to the RNA-binding protein. See, for example, FIG. 13A and FIG. 13C. In certain embodiments, the portion of the second probe that recognizes the RNA-binding protein comprises an antibody (e.g., a secondary antibody), or an antibody variant. If the portion of the second probe that recognizes the RNA-binding protein is a secondary antibody, the method may optionally further comprise contacting the cell with a primary antibody that recognizes the RNA-binding protein and is recognized by the secondary antibody of the second probe. The cell may be contacted with the primary antibody before or after being contacted with one or more pairs of probes. In certain embodiments, the cell is contacted with the primary antibody before being contacted with one or more pairs of probes.Instead of primary antibody, the present disclosure also contemplates the use of any agent that can recognize and directly bind to the RNA binding protein of the probe described herein.In some embodiments, the part of the second probe that directly binds to RNA binding protein comprises protein.In some embodiments, the part of the second probe that directly binds to RNA binding protein comprises antibody, or antibody variant.

[0084] In some embodiments, the second probe of the set of probes further comprises a polymerization blocker. The polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the method described herein. In some embodiments, the polymerization blocker is at the 3' end of the second oligonucleotide probe. The polymerization blocker can be, for example, any chemical moiety that prevents the polymerase from using the second oligonucleotide probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue that comprises a blocked 3' hydroxyl group (e.g., an oxygen protecting group at the 3' hydroxyl group). In some embodiments, the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.

[0085] In addition to the portion that recognizes the RNA-binding protein, the second probe also includes a portion that is complementary to a portion of the first probe. In the set of probes provided herein, the portions of the first and second probes that are complementary to each other can be the same in each set of probes. In some embodiments, the portions of the first and second probes that are complementary to each other are unique in each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0086] In some embodiments, each portion of the second probe is connected by an optional linker. In some embodiments, the optional linker is a nucleotide linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In some embodiments, the second probe of the set of probes used in the method described herein has the structure: 5'-[RNA-binding protein recognition portion]-[portion complementary to the first probe]-3' Including, Here, ]-[ includes any linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second probe.

[0087] The first probe (also referred to herein as a "padlock" probe) of the set of probes used in the methods described herein includes an oligonucleotide portion that is complementary to the second probe. In some embodiments, the portion of the first probe that is complementary to the portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' and 3' ends of the first probe.

[0088] The first probe of the set of probes used in the method for profiling the interaction between an RNA-binding protein and an RNA disclosed herein also comprises an oligonucleotide portion complementary to the RNA of interest. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22 or about 19-21 nucleotides in length. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.

[0089] The first probe of the set of probes used in the methods disclosed herein also contains an oligonucleotide barcode sequence composed of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length. The barcode of the oligonucleotide probe described herein may contain a gene-specific sequence used to identify the RNA of interest (i.e., the RNA bound by at least one RNA-binding protein).

[0090] Any order of arrangement of the parts of the first oligonucleotide probe is contemplated by the present disclosure.In some embodiments, the part of the first probe is connected to another part by an optional linker.In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.In some embodiments, the first probe has the structure: 5'-[portion complementary to the second probe]-[oligonucleotide barcode sequence]-[portion complementary to the RNA of interest]-[portion complementary to the third probe]-[portion complementary to the second probe]-3' where ]-[ includes any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe. In some embodiments, any of the oligonucleotide portions of the probes that make up the set of probes provided herein comprises DNA.

[0091] The third probe (also referred to as "primer probe") of the set of probes used in the method for profiling the interaction between an RNA-binding protein and an RNA provided herein comprises a portion complementary to the RNA of interest and a portion complementary to the first probe of the set of probes. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5 to 15, 6 to 14, 7 to 13, 8 to 12, or 9 to 11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0092] In certain embodiments, the third probe of the set of probes has the structure: 5'-[part complementary to the RNA of interest]-[part complementary to the first probe]-[barcode sequence]-3' where ]-[ comprises any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0093] The use of any type of cell in the methods disclosed herein is contemplated by the present disclosure (e.g., any of the cell types described herein). In some embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. The present disclosure also contemplates carrying out the method for profiling epitranscriptome RNA modification or the interaction between RNA binding proteins and RNA described herein in multiple cells simultaneously. In some embodiments, the method is carried out on multiple cells of the same cell type. In some embodiments, the method is carried out on multiple cells, including cells of different cell types. In some embodiments, epitranscriptome RNA modification or the interaction between RNA binding proteins and RNA is profiled simultaneously in more than 10 cells, more than 20 cells, more than 50 cells, more than 100 cells, more than 200 cells, more than 300 cells, more than 400 cells, more than 500 cells, or more than 1000 cells. The cell types in which epitranscriptome RNA modifications or interactions between RNA-binding proteins and RNA can be profiled using the methods disclosed herein include, but are not limited to, stem cells, progenitor cells, neural cells, astrocytes, dendritic cells, endothelial cells, microglia, oligodendrocytes, myocytes, cardiomyocytes, mesenchymal cells, epithelial cells, immune cells, hepatocytes, smooth and skeletal muscle cells, hematopoietic cells, lymphocytes, monocytes, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, and neurons. In certain embodiments, the cell or cells are present within an intact tissue (e.g., any of the tissue types described herein). In certain embodiments, the intact tissue is a fixed tissue sample. In some embodiments, the intact tissue comprises multiple cell types. In some embodiments, the tissue is epithelial tissue, connective tissue, muscle tissue, or nerve tissue. In certain embodiments, the tissue is heart tissue, lymph node tissue, liver tissue, muscle tissue, bone tissue, eye tissue, brain tissue, or ear tissue.

[0094] In the methods described herein, the RNA of interest that is profiled for epitranscriptome modification or interaction with RNA binding protein can be the transcript expressed from the genomic DNA of a cell.In some embodiments, the RNA of interest is messenger RNA (mRNA), transfer RNA (tRNA), and / or ribosomal RNA (rRNA).In some embodiments, the RNA of interest comprises a transcript that has not yet been processed (e.g., pre-mRNA).The methods described herein can be used to profile one epitranscriptome-modified RNA or RNA that is bound by RNA binding protein in a cell at a time, or multiple epitranscriptome-modified RNAs or RNA that is bound by RNA binding protein simultaneously. In some embodiments, epitranscriptomic RNA modifications or interactions between RNA binding proteins and RNAs in a cell or multiple cells are profiled simultaneously for more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 RNAs.

[0095] The polymer matrix is ​​used in the methods described herein to facilitate sequencing and imaging of epitranscriptome modified RNA of interest or RNA of interest bound by RNA binding protein in cells after rolling circle amplification. The use of various polymer matrices is contemplated by the present disclosure, and any polymer matrix in which one or more concatenated amplicons can be embedded is suitable for use in the methods described herein. In some embodiments, the polymer matrix is ​​a hydrogel (i.e., a network of crosslinked polymers that are hydrophilic). In some embodiments, the hydrogel is a polyvinyl alcohol hydrogel, a polyethylene glycol hydrogel, a polyacrylate hydrogel, or a polyacrylamide hydrogel. In certain embodiments, the hydrogel is a polyacrylamide hydrogel. Such hydrogels can be prepared, for example, by incubating a sample in a buffer containing acrylamide and bisacrylamide, removing the buffer, and incubating the sample in a polymerization mixture (including, for example, ammonium persulfate and tetramethylethylenediamine). Such reagents can also be provided in a kit, for example, a kit for performing any of the methods described herein, or any of the kits described herein.

[0096] In some embodiments, the step of performing rolling circle amplification to amplify the circular oligonucleotide to produce one or more concatenated amplicons further comprises providing a nucleotide modified with a reactive chemical group (e.g., an amine-modified nucleotide, such as 5-(3-aminoallyl)-dUTP). In some embodiments, the nucleotide modified with a reactive chemical group comprises about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the nucleotides used in the amplification reaction. For example, the step of performing rolling circle amplification to amplify the circular oligonucleotide to produce one or more concatenated amplicons may further comprise providing an amine-modified nucleotide, such as 5-(3-aminoallyl)-dUTP. During the amplification process, the amine-modified nucleotide is incorporated into one or more concatenated amplicons as they are generated. The resulting amplicons are functionalized with primary amines, which can be further reacted with another compatible chemical moiety (e.g., N-hydroxysuccinimide) to facilitate embedding the concatenated amplicons in a polymer matrix. In some embodiments, embedding one or more concatenated amplicons in a polymer matrix comprises reacting the amine-modified nucleotides of one or more concatenated amplicons with acrylic acid N-hydroxysuccinimide ester, and copolymerizing one or more concatenated amplicons and the polymer matrix.

[0097] The methods disclosed herein also include sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix. In some embodiments, the sequencing step includes performing "sequencing by error reduction by dynamic annealing and ligation" (SEDAL sequencing). SEDAL sequencing is further described in X. et al., Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 2018, 361, 380, and International Patent Application Publication WO 2019 / 199579, published October 17, 2019, each of which is incorporated herein by reference. Briefly, an oligonucleotide probe containing a detectable label (i.e., any label that can be used to visualize the location of additional oligonucleotide probes, for example by imaging) is provided to a cell. In certain embodiments, the detectable label is fluorescent (e.g., a fluorophore). The additional oligonucleotide probe is complementary to the oligonucleotide barcode sequence of the first probe, and is thus linked to the identity of the RNA of interest, and can be used to identify the location of the RNA of interest inside the cell (or within an organelle, if the method is performed with subcellular resolution, e.g., using stains to identify the location of individual organelles).

[0098] The additional oligonucleotide probes used in the methods described herein (e.g., when used in SEDAL sequencing) can be read out using any suitable imaging technique known in the art. For example, in embodiments where the additional oligonucleotide probe comprises a fluorophore, the fluorophore can be read out using imaging to identify the RNA of interest. As discussed above, the additional oligonucleotide probe comprises a sequence complementary to the barcode sequence of the first oligonucleotide probe used to detect the specific RNA of interest. By imaging the location of the additional oligonucleotide probe comprising the fluorophore, the location of the specific RNA of interest within the sample can be determined. In some embodiments, the imaging step comprises fluorescent imaging. In certain embodiments, the imaging step comprises confocal microscopy. In certain embodiments, the imaging step comprises epifluorescence microscopy. In certain embodiments, two rounds of imaging are performed. In certain embodiments, three rounds of imaging are performed. In certain embodiments, four rounds of imaging are performed. In certain embodiments, five or more rounds of imaging are performed.

[0099] In some embodiments, the method for profiling epitranscriptome RNA modification or the interaction between RNA binding protein and RNA described herein can be combined with a method for profiling additional molecules inside a cell.For example, the expression of additional RNA (including RNA that is not epitranscriptome modified and RNA that is not bound by RNA binding protein) can be profiled using a probe that does not contain a moiety that recognizes epitranscriptome RNA modification or RNA binding protein, together with epitranscriptome modified RNA or RNA that is bound by one or more RNA binding proteins.Methods for profiling other types of molecules (e.g., DNA, protein, carbohydrate, or lipid) can also be combined with the methods described herein.In certain embodiments, the additional molecule to be profiled is unmodified RNA.In some embodiments, profiling unmodified RNA includes: a) contacting a cell with one or more pairs of probes, where each pair of probes comprises a first probe and a second probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, and an oligonucleotide portion complementary to an unmodified RNA of interest; and ii) contacting a second probe, the second probe comprising a portion complementary to the unmodified RNA of interest and a portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each unmodified RNA of interest in the cell. Includes.

[0100] In some embodiments, the methods provided herein further comprise determining the cell type of the profiled cell by comparing the epitranscriptomic RNA modification profile (or the profile of interaction between RNA and RNA binding protein) of the cell with reference data comprising epitranscriptomic RNA modification profiles (or the profile of interaction between RNA and RNA binding protein) of various cell types. In some embodiments, the method further comprises overexpressing or knocking out one or more genes in the cell to determine whether the one or more genes are involved in the epitranscriptomic modification of the RNA of interest. In some embodiments, the method further comprises repeating steps (a)-(e) at multiple time points to profile the epitranscriptomic RNA modification or the interaction between RNA binding protein and RNA in the cell over time. In some embodiments, the method further comprises examining how the epitranscriptomic modified RNA or the interaction between RNA and RNA binding protein in the cell or multiple cells is affected by an immune response within an intact tissue or how the profile is affected due to proximity to a tumor.

[0101] In some embodiments, any of the methods described herein further include resolving the location of the modified RNA of interest with subcellular resolution (i.e., inside a specific organelle inside the cell; with a spatial resolution of about 150-400 nm, depending on both the size of the DNA amplicon and the optical limits). This can be accomplished by organelle staining procedures well known in the art. For example, various organelles such as the endoplasmic reticulum (ER), cytoskeleton, and mitochondria can be stained. In some embodiments, agents used to stain various organelles of the cell include small molecule dyes, antibodies, and / or protein dyes.

[0102] Methods for diagnosing a disease or disorder in a subject In another aspect, the present disclosure provides a method for diagnosing disease or disorder in a subject.For example, the method of profiling epitranscriptome RNA modification or interaction between RNA binding protein and RNA described herein can be performed on a cell or a number of cells (e.g., in intact tissue) taken from a subject (e.g., a subject that is thought to have or is at risk of having a disease or disorder, or a subject that is healthy or thought to be healthy).The expression of various RNAs of interest in the cell can then be compared with the expression of the same RNAs of interest in non-disease cells or cells from a non-disease tissue sample (e.g., a cell from a healthy individual, or a number of cells from a population of healthy individuals).Any difference in the epitranscriptome RNA modification profile or interaction profile between RNA binding protein and RNA (of a single RNA or of multiple RNAs of interest, e.g., including specific disease signatures) of a cell compared to one or more non-disease cells can indicate that the subject has a disease or disorder.The epitranscriptome RNA modification or interaction between RNA binding protein and RNA in one or more non-disease cells can be profiled in parallel with the expression in disease cells as a control experiment. Epitranscriptomic RNA modifications or interactions between RNA binding proteins and RNA in one or more non-diseased cells can also be profiled in advance and expression in the diseased cells can be compared to this reference data for non-diseased cells.

[0103] In some embodiments, a method for diagnosing a disease or disorder in a subject comprises: a) contacting a cell removed from the subject with one or more sets of probes, where each set of probes comprises a first probe, a second probe, and a third probe, where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell; Including, Here, a difference in the epitranscriptomic RNA modification profile of the cell compared to one or more non-diseased cells is indicative that the subject has a disease or disorder.

[0104] In some embodiments, a method for diagnosing a disease or disorder in a subject comprises: a) contacting a cell removed from a subject (or in a plurality of cells, e.g., in an intact tissue) with one or more probe pairs, where each probe pair comprises a first probe (i.e., a "padlock probe"), and a second probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) contacting a second probe, the second probe comprising a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell; Including, Here, a difference in the epitranscriptomic RNA modification profile of the cell compared to one or more non-diseased cells is indicative that the subject has a disease or disorder.

[0105] In some embodiments, a method for diagnosing a disease or disorder in a subject comprises: a) contacting a cell removed from the subject with one or more sets of probes, where each set of probes comprises a first probe, a second probe, and a third probe, where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each RNA of interest bound to the RNA-binding protein in the cell; Including, Here, a difference in the profile of interactions between RNA and RNA binding proteins in the cell compared to one or more non-diseased cells is indicative that the subject has a disease or disorder.

[0106] In some embodiments, epitranscriptome RNA modification in one or more non-disease cells, or the interaction between RNA and RNA binding protein in one or more non-disease cells, is profiled simultaneously with cells taken from the subject using the method disclosed herein as a control experiment. In some embodiments, the profile of epitranscriptome RNA modification in one or more non-disease cells, or the profile of the interaction between RNA and RNA binding protein in one or more non-disease cells, which is compared with the expression in disease cells, includes reference data from when the method was performed on one or more non-disease cells before. Any epitranscriptome RNA modification profile for the diagnosis of disease or disorder in a subject is contemplated by the present disclosure. In some embodiments, epitranscriptome RNA modification is N 6 -Methyladenosine (m 6 A), N 1 -Methyladenosine (m 1 A), pseudouridine, N 6 ,2'-O-Dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -Acetylcytidine (ac 4 C), 2'-O-methylated (Nm), or 5-methylcytosine (m 5 C). Other epitranscriptome modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, EM et al., Nature. 541, 339-346 (2017). In some embodiments, the epitranscriptome RNA modification is an adenosine to inosine modification. In some embodiments, the epitranscriptome modification is a queuosine (i.e., queuosine substitutes another nucleotide in the RNA), polyadenylation, intron splicing, or histone mRNA processing. In certain embodiments, the epitranscriptome RNA modification is an N 6 -Methyladenosine (m6 A). A single epitranscriptome modification can be profiled in cells to diagnose disease or disorder in a subject using the methods disclosed herein, or multiple different epitranscriptome modifications (for example, 2, 3, 4, 5, or more) can be profiled simultaneously in cells. The interaction profile between RNA and any RNA-binding protein is also contemplated by the present disclosure. In some embodiments, the RNA-binding protein is an enzyme that introduces epitranscriptome modification to RNA. In certain embodiments, the RNA-binding protein is a YTH family protein (for example, YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2), an IGF2BP family protein (for example, IGF2BP1, IGF2BP2, or IGF2BP3), or FMR1. The interaction between an RNA and a single RNA binding protein can be profiled in a cell to diagnose a disease or disorder in a subject using the methods disclosed herein, or the interaction with multiple different RNA binding proteins (e.g., 2, 3, 4, 5, or more) can be profiled simultaneously in a cell.

[0107] The diagnosis of any disease or disorder is contemplated by the method described herein.In some embodiments, disease or disorder is genetic disease, proliferation disease, inflammatory disease, autoimmune disease, liver disease, spleen disease, lung disease, blood disease, neurological disease, psychiatric disease, gastrointestinal (GI) tract disease, genitourinary disease, infectious disease, musculoskeletal disease, endocrine disease, metabolic disorder, immune disorder, central nervous system (CNS) disorder or cardiovascular disease.

[0108] In some embodiments, the cells are present in a tissue (e.g., epithelial tissue, connective tissue, muscle tissue, or nerve tissue). In some embodiments, the tissue is a tissue sample from a subject. In some embodiments, the subject is a non-human experimental animal (e.g., a mouse). In some embodiments, the subject is a livestock animal. In some embodiments, the subject is a human. In some embodiments, the tissue sample comprises a fixed tissue sample. In certain embodiments, the tissue sample is a biopsy (e.g., a bone, bone marrow, breast, gastrointestinal tract, lung, liver, pancreas, prostate, brain, nerve, kidney, endometrial, cervical, lymph node, muscle, or skin biopsy). In certain embodiments, the biopsy is a tumor biopsy. In certain embodiments, the tissue is brain tissue. In certain embodiments, the tissue is from the central nervous system.

[0109] Methods for Treating a Disease or Disorder in a Subject In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject. For example, the method for profiling epitranscriptomic RNA modification or interaction between RNA binding protein and RNA described herein can be carried out in cells (or in a number of cells, for example, in intact tissue) from a sample taken from a subject (for example, a subject who is thought to have or is at risk of having a disease or disorder). The epitranscriptomic modification profile of one or more RNAs or the interaction profile between RNA binding protein and one or more RNAs in the cell can then be compared with the epitranscriptomic modification profile of the same RNA of interest and the interaction profile of RNA binding protein in cells from a non-disease tissue sample. If any difference is observed in the epitranscriptomic RNA modification profile or the interaction profile between RNA binding protein and one or more RNAs in the cell compared to non-disease cells, a treatment for the disease or disorder can then be administered to the subject. The epitranscriptomic RNA modification and / or the interaction between RNA binding protein and RNA in one or more non-disease cells can be profiled together with the epitranscriptomic RNA modification in disease cells as a control experiment. Epitranscriptomic RNA modifications and / or interactions between RNA-binding proteins and RNA in one or more non-diseased cells may also be previously profiled, and the profile of epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNA in the diseased cells may be compared to this reference data for the non-diseased cells.

[0110] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject, comprising: a) contacting a cell removed from the subject with one or more sets of probes, where each set of probes comprises a first probe, a second probe, and a third probe, where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell; and f) administering to the subject a treatment for the disease or disorder if a difference is observed in the profile of epitranscriptomic RNA modifications in the cell compared to one or more non-diseased cells. The present invention provides a method comprising:

[0111] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject, comprising: a) contacting a cell removed from a subject (or in a plurality of cells, e.g., in an intact tissue) with one or more probe pairs, where each probe pair comprises a first probe (i.e., a "padlock probe"), and a second probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) contacting a second probe, the second probe comprising a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell; and f) administering to the subject a treatment for the disease or disorder if a difference is observed in the profile of epitranscriptomic RNA modifications in the cell compared to one or more non-diseased cells. The present invention provides a method comprising:

[0112] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject, comprising: a) contacting a cell removed from the subject with one or more sets of probes, where each set of probes comprises a first probe, a second probe, and a third probe, where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each RNA of interest bound by the RNA-binding protein in the cell; and f) administering to the subject a treatment for the disease or disorder if a difference is observed in the profile of interactions between RNA and RNA binding proteins in the cell compared to one or more non-diseased cells. The present invention provides a method comprising:

[0113] In some embodiments, the epitranscriptome RNA modification in one or more non-disease cells, or the interaction between RNA and RNA-binding protein in one or more non-disease cells, is profiled simultaneously using the method disclosed herein as a control experiment.In some embodiments, the epitranscriptome RNA modification data in one or more non-disease cells, or the interaction profile between RNA and RNA-binding protein in one or more non-disease cells, which is compared with the profile of disease cells, comprises reference data from the time when the method was previously performed on non-disease cells.

[0114] Any suitable treatment for the disease or disorder may be administered to the subject. In some embodiments, the treatment includes administering a therapeutic agent. In some embodiments, the treatment includes surgery. In some embodiments, the treatment includes imaging. In some embodiments, the treatment includes performing an additional diagnostic method. In some embodiments, the treatment includes radiation therapy. In some embodiments, the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate. In some embodiments, the therapeutic agent is a known drug and / or an FDA-approved drug. In certain embodiments, the protein is an antibody. In certain embodiments, the protein is an antibody variant. In certain embodiments, the protein is a receptor, or a fragment or variant thereof. In certain embodiments, the protein is a cytokine. In certain embodiments, the nucleic acid is an mRNA, an antisense RNA, a miRNA, an siRNA, an RNA aptamer, a double-stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).

[0115] The treatment of any disease or disorder is contemplated by the method described herein.In some embodiments, the disease or disorder is genetic disease, proliferation disease, inflammatory disease, autoimmune disease, liver disease, spleen disease, lung disease, blood disease, neurological disease, gastrointestinal (GI) tract disease, genitourinary disease, infectious disease, musculoskeletal disease, endocrine disease, metabolic disorder, immune disorder, central nervous system (CNS) disorder, neurological disorder, eye disease or cardiovascular disease.

[0116] In some embodiments, the subject is a human. In some embodiments, the sample comprises a biological sample. In some embodiments, the sample comprises a tissue sample. In certain embodiments, the tissue sample is a biopsy (e.g., bone, bone marrow, breast, gastrointestinal tract, lung, liver, pancreas, prostate, brain, nerve, kidney, endometrium, cervix, lymph node, muscle, or skin biopsy). In certain embodiments, the biopsy is a tumor biopsy. In certain embodiments, the biopsy is a solid tumor biopsy. In some embodiments, the tissue sample is a brain tissue sample. In certain embodiments, the tissue sample is a central nervous system tissue sample. In certain embodiments, the epitranscriptome profile of the biological sample informs a prognosis that guides therapy, including but not limited to diabetes, psychiatric disorders, liver disease, kidney disease, blood disorders, endocrine or exocrine disorders, cardiac disorders, cancer treatments such as chemotherapy, targeted therapy, immunotherapy (e.g., checkpoint inhibition, CAR-T, cancer vaccines, etc.), metabolic disorders, or immune and autoimmune disorders, and other conditions.

[0117] Methods for screening agents capable of modulating epitranscriptomic modifications of one or more RNAs - Patents.com In another aspect, the present disclosure provides a method for screening agents that can modulate the epitranscriptome modification of one or more RNAs of interest or the interaction between RNA binding protein and one or more RNAs of interest.For example, the method for profiling epitranscriptome RNA modification or the interaction between RNA binding protein and RNA described herein can be carried out in cells (or in a number of cells, for example, in intact tissue) in the presence of one or more candidate agents.Then, the expression of the RNA of interest that is bound by various epitranscriptome modified RNAs of interest or RNA binding protein in cells (for example, normal cells or diseased cells) can be compared with the expression of the same RNA of interest in cells that have not been exposed to one or more candidate agents.The difference in either the profile of epitranscriptome RNA modification or the profile of interaction between RNA binding protein and RNA compared to cells that have not been exposed to the candidate agent can indicate that the epitranscriptome modification of one or more RNAs of interest or the interaction between one or more RNA binding protein of interest is modulated by the candidate agent. In some embodiments, a particular signature known to be associated with disease treatment (for example, the epitranscriptome modification of multiple RNAs of interest, or the interaction between RNA binding proteins and multiple RNAs of interest) can be used to identify candidate agents that can modulate epitranscriptome RNA modification in a desired manner.The methods described herein can also be used to identify drugs that have particular side effects, for example, by looking for specific epitranscriptome RNA modification signatures or the signatures of interactions between RNA binding proteins and RNAs of interest when one or more cells are treated with a candidate agent or a known drug.

[0118] In some embodiments, the present disclosure provides a method for screening an agent capable of modulating epitranscriptomic modifications of one or more RNAs, comprising: a) contacting a cell that is being or has been treated with a candidate agent with one or more sets of probes, where each set of probes includes a first probe, a second probe, and a third probe, where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell; Including, Here, a method is provided in which a difference in the epitranscriptomic RNA modification profile of a cell in the presence of a candidate agent compared to the absence of the candidate agent indicates that the candidate agent modulates epitranscriptomic RNA modification.

[0119] In some embodiments, the present disclosure provides a method for screening an agent capable of modulating epitranscriptomic modifications of one or more RNAs, comprising: a) contacting a cell (or a plurality of cells, e.g., in an intact tissue) that is being or has been treated with a candidate agent (or a combination of a plurality of candidate agents and / or known drugs, e.g., as provided in a screening library of compounds) with one or more probe pairs, where each probe pair comprises a first probe (i.e., a "padlock probe") and a second probe (i.e., a "primer probe"), where: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) contacting a second probe, the second probe comprising a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to generate a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to generate one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons or portions thereof embedded in the polymer matrix to determine the identity and location of each epitranscriptome-modified RNA of interest in the cell; Including, Here, a method is provided in which a difference in the profile of epitranscriptomic RNA modifications in a cell in the presence of a candidate agent compared to the absence of the candidate agent indicates that the candidate agent modulates epitranscriptomic RNA modifications.

[0120] In some embodiments, the disclosure provides a method for screening an agent capable of modulating an interaction between an RNA and an RNA binding protein, the method comprising: a) contacting a cell removed from the subject with one or more sets of probes, where each set of probes comprises a first probe, a second probe, and a third probe, where: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) contacting a third probe, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; b) ligating together the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the third probe as a primer to produce one or more concatenated amplicons; d) embedding one or more concatenated amplicons in a polymer matrix; and e) sequencing the concatenated amplicons, or portions thereof, embedded in the polymer matrix to determine the identity and location of each RNA of interest bound by the RNA-binding protein in the cell; Including, Here, a difference in the profile of the interaction between the RNA and the RNA binding protein in the presence of the candidate agent compared to the absence of the candidate agent provides a method indicating that the candidate agent modulates the interaction between the RNA and the RNA binding protein.

[0121] In some embodiments, the candidate agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate. In some embodiments, the candidate agent comprises a known drug or an FDA-approved drug. In certain embodiments, the protein is an antibody. In certain embodiments, the protein is an antibody fragment or an antibody variant. In certain embodiments, the protein is a receptor. In certain embodiments, the protein is a cytokine. In certain embodiments, the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double-stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO). In some embodiments, a large number of candidate agents are provided as a screening library. Any candidate agent can be screened using the methods described herein. In particular, any candidate agent that is believed to be capable of modulating the epitranscriptome modification of one or more RNAs of interest or the interaction of one or more RNAs of interest with an RNA-binding protein can be screened using the methods described herein. In some embodiments, the epitranscriptome modification of one or more RNAs of interest and / or the modulation of the interaction of one or more RNAs of interest with an RNA-binding protein by a candidate agent is associated with reducing, alleviating, or eliminating symptoms of a disease or disorder, or preventing the onset or progression of a disease or disorder. In some embodiments, the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a blood disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease.

[0122] probe The present disclosure also provides a pair of probes for use in the methods and systems for profiling epitranscriptome RNA modifications described herein. In one aspect, the present disclosure provides a pair of probes comprising a first probe (also referred to herein as a "padlock" probe) and a second probe (also referred to herein as a "primer" probe), wherein: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) The second probe comprises a portion that recognizes the epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe.

[0123] All of the probes described herein may optionally have spacers or linkers of various nucleotide lengths between each of the listed components, or the components of the oligonucleotide probe may be joined directly to each other (i.e., by phosphodiester bonds). All of the probes described herein may contain standard nucleotides, or some of the standard nucleotides may be replaced with any modified nucleotide known in the art.

[0124] The second probe of the pair of probes (also referred to herein as "primer probe") comprises a portion that recognizes an epitranscriptomic RNA modification (i.e., a post-transcriptional modification present in a particular RNA of interest). In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification comprises a peptide. In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptomic RNA modification via a mechanism that comprises a biotin-streptavidin interaction. The portion of the probe that recognizes the epitranscriptomic RNA modification can be a protein (e.g., an antibody or antibody variant, or any protein that can otherwise bind to a specific epitranscriptomic modification). In some embodiments, the protein is a PAPG. In certain embodiments, the PAPG recognizes and binds to an antibody that recognizes the epitranscriptomic RNA modification. In some embodiments, the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an agent that binds to an antibody, or an antibody variant. For example, the second probe may comprise a secondary antibody and may be bound to the epitranscriptome RNA modification using the primary antibody. The secondary antibody of the second probe then recognizes the primary antibody bound to the epitranscriptome RNA modification. See, for example, FIG. 1. In certain embodiments, the portion of the second probe that recognizes the epitranscriptome RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant. When the portion of the second probe that recognizes the epitranscriptome RNA modification is a secondary antibody, the secondary antibody may bind to the primary antibody that recognizes the epitranscriptome RNA modification. In some embodiments, the primary antibody is an anti-m 6 A antibody, anti-m 1 A antibody, anti-pseudouridine antibody, anti-m 6 Am antibody, anti-m 7 G antibody, anti-ac 4C antibody, anti-Nm antibody or anti-m 5 C antibody. Other epitranscriptome modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, EM et al., Nature. 541, 339-346 (2017), each of which is incorporated herein by reference. Instead of an antibody, the present disclosure also contemplates the use of any agent capable of binding the epitranscriptome RNA modification in the probes described herein. In some embodiments, the portion of the second probe that binds to the epitranscriptome RNA modification comprises a protein. In certain embodiments, the protein is m 6 A-specific YTH domain protein or a portion thereof. In some embodiments, the portion of the second probe that binds the epitranscriptome RNA modification comprises an antibody, or an antibody variant.

[0125] In addition to the portion that recognizes the epitranscriptome RNA modification, the second probe also includes a portion that is complementary to a portion of the first probe. In the probe pairs provided herein, the portions of the first and second probes that are complementary to each other can be the same in each set of probes. In some embodiments, the portions of the first and second probes that are complementary to each other are unique in each pair of probes. In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0126] In some embodiments, each portion of the second probe is connected by an optional linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the linker is a non-nucleotide linker (e.g., amino acids, chemical linkers, polymers, etc.). In some embodiments, the second probe of the probe pair has the structure: 5'-[part that recognizes epitranscriptome RNA modification]-[part complementary to the first probe]-3' Including, Here, ]-[ includes any linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second probe.

[0127] The first probe (also referred to herein as a "padlock" probe) of the probe pairs provided herein includes an oligonucleotide portion that is complementary to the second probe. In some embodiments, the portion of the first probe that is complementary to the portion of the second probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the second probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' and 3' ends of the first probe.

[0128] The first probe of the pair of probes provided herein also comprises an oligonucleotide portion complementary to the RNA of interest. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22 or about 19-21 nucleotides in length. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.

[0129] The first probe of the pair of probes disclosed herein also comprises an oligonucleotide barcode sequence composed of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length. The barcode of the oligonucleotide probe described herein may comprise a gene-specific sequence used to identify the RNA of interest (i.e., the RNA modified with at least one epitranscriptomic modification).

[0130] Any order of arrangement of the portions of the first oligonucleotide probe is contemplated by the present disclosure. In some embodiments, each portion of the first probe is connected by an optional linker. In some embodiments, the optional linker is a nucleotide linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the optional linker includes other types of linkages other than nucleotides (e.g., chemical linkers, peptide linkers, etc.). In some embodiments, the first probe has the structure: 5'-[part complementary to second probe]-[part complementary to RNA of interest]-[barcode sequence]-[part complementary to second probe]-3'; 5'-[part complementary to second probe]-[barcode sequence]-[part complementary to RNA of interest]-[part complementary to second probe]-3'; 5'-[part complementary to second probe]-[part complementary to RNA of interest]-[barcode sequence]-3'; 5'-[part complementary to second probe]-[barcode sequence]-[part complementary to RNA of interest]-3'; 5'-[part complementary to the RNA of interest]-[barcode sequence]-[part complementary to the second probe]-3'; or 5'-[barcode sequence]-[part complementary to the RNA of interest]-[part complementary to the second probe]-3' Including, Here, each instance of ]-[ includes an optional linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe. In some embodiments, either the oligonucleotide portion of the first probe and / or the second probe comprises DNA.

[0131] The present disclosure also provides a set of probes for use in the methods and systems for profiling epitranscriptome RNA modifications described herein.In one aspect, the present disclosure provides a pair of probes, including a first probe (also referred to herein as a "padlock" probe), a second probe (also referred to herein as a "sprint" probe), and a third probe (also referred to herein as a "primer" probe), wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) The third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe.

[0132] All of the probes in the set of probes described herein may optionally have spacers or linkers of various nucleotide lengths between each of the listed components, or the components of the oligonucleotide probe may be directly joined to each other (i.e., by phosphodiester bonds). All of the probes described herein may contain standard nucleotides, or some of the standard nucleotides may be replaced with any modified nucleotide known in the art.

[0133] The second probe of the set of probes (also referred to herein as "sprint probe") comprises a portion that recognizes an epitranscriptomic RNA modification (i.e., a post-transcriptional modification present in a particular RNA of interest). In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification comprises a peptide. In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds to the epitranscriptomic RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptomic RNA modification via a mechanism that comprises a biotin-streptavidin interaction. The portion of the probe that recognizes the epitranscriptomic RNA modification can be a protein (e.g., an antibody or antibody variant, or any protein that can otherwise bind to a specific epitranscriptomic modification). In certain embodiments, the protein is a PAPG. In some embodiments, the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an antibody, or an agent that binds to an antibody variant. For example, the second probe may comprise a secondary antibody and may be bound to the epitranscriptome RNA modification using a primary antibody. The secondary antibody of the second probe then recognizes the primary antibody bound to the epitranscriptome RNA modification. See, for example, FIG. 1. In another example, the second probe may comprise a protein PAPG that can bind to the antibody that binds to the epitranscriptome RNA modification. In some embodiments, the PAPG recognizes and binds to the antibody, and the antibody recognizes and binds to the epitranscriptome RNA modification. In certain embodiments, the portion of the second probe that recognizes the epitranscriptome RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant. In some embodiments, the secondary antibody recognizes and binds to the primary antibody that recognizes and binds to the epitranscriptome RNA. The cell may be contacted with the primary antibody before or after being contacted with one or more pairs of probes.In certain embodiments, the cells are contacted with a primary antibody prior to being contacted with one or more pairs of probes. In some embodiments, the primary antibody is anti-m. 6 A antibody, anti-m 1 A antibody, anti-pseudouridine antibody, anti-m 6 Am antibody, anti-m 7 G antibody, anti-ac 4 C antibody, anti-Nm antibody or anti-m 5 Instead of the primary antibody, the present disclosure also contemplates the use of any agent capable of directly binding the epitranscriptome RNA modification in the probe described herein. In some embodiments, the portion of the second probe that directly binds the epitranscriptome RNA modification comprises a protein. In certain embodiments, the protein is m 6 A specific YTH domain protein. In some embodiments, the portion of the second probe that directly binds the epitranscriptome RNA modification comprises an antibody, or an antibody variant.

[0134] In some embodiments, the second probe of the set of probes further comprises a polymerization blocker. The polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the method described herein. In some embodiments, the polymerization blocker is at the 3' end of the second oligonucleotide probe. The polymerization blocker can be, for example, any chemical moiety that prevents the polymerase from using the second oligonucleotide probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue that comprises a blocked 3' hydroxyl group (e.g., an oxygen protecting group at the 3' hydroxyl group). In some embodiments, the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.

[0135] In addition to the portion that recognizes the epitranscriptome RNA modification, the second probe also includes a portion that is complementary to a portion of the first probe. In the set of probes provided herein, the portions of the first and second probes that are complementary to each other can be the same in each set of probes. In some embodiments, the portions of the first and second probes that are complementary to each other are unique in each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0136] In some embodiments, each portion of the second probe is connected by an optional linker. In some embodiments, the optional linker is a nucleotide linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In some embodiments, the second probe of the set of probes described herein has the structure: 5'-[part that recognizes epitranscriptome RNA modification]-[part complementary to the first probe]-3' Including, Here, ]-[ includes any linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second probe.

[0137] The first probe (also referred to herein as a "padlock" probe) of the set of probes described herein includes an oligonucleotide portion that is complementary to the second probe. In some embodiments, the portion of the first probe that is complementary to the portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' and 3' ends of the first probe.

[0138] The first probe of the set of probes disclosed herein also comprises an oligonucleotide portion complementary to the RNA of interest. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22 or about 19-21 nucleotides in length. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.

[0139] The first probe of the set of probes disclosed herein also comprises an oligonucleotide barcode sequence composed of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length. The barcode of the oligonucleotide probe described herein may comprise a gene-specific sequence used to identify an RNA of interest (i.e., an RNA modified with at least one particular epitranscriptomic modification).

[0140] Any order of arrangement of the parts of the first oligonucleotide probe is contemplated by the present disclosure.In some embodiments, the part of the first probe is connected to another part by an optional linker.In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.In some embodiments, the first probe has the structure: 5'-[portion complementary to the second probe]-[oligonucleotide barcode sequence]-[portion complementary to the RNA of interest]-[portion complementary to the third probe]-[portion complementary to the second probe]-3' Including, Here, ]-[ includes any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe. In some embodiments, any of the oligonucleotide portions of the probes that make up the set of probes provided herein comprises DNA.

[0141] The third probe (also referred to as "primer probe") of the set of probes provided herein comprises a portion complementary to the RNA of interest and a portion complementary to the first probe of the set of probes. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5 to 15, 6 to 14, 7 to 13, 8 to 12, or 9 to 11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0142] In certain embodiments, the third probe of the set of probes has the structure:

[0143] 5'-[part complementary to the RNA of interest]-[part complementary to the first probe]-3' where ]-[ comprises any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0144] The present disclosure also provides a set of probes for use in the methods and systems for profiling interactions between RNA-binding proteins and RNA described herein.In one aspect, the present disclosure provides a pair of probes, including a first probe (also referred to herein as a "padlock" probe), a second probe (also referred to herein as a "sprint" probe), and a third probe (also referred to herein as a "primer" probe), wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) The third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe.

[0145] The second probe (also referred to herein as "sprint probe") of the set of probes for profiling the interaction between an RNA-binding protein and an RNA comprises a portion that recognizes an RNA-binding protein (e.g., an enzyme that introduces an epitranscriptomic modification to an RNA). In some embodiments, the portion of the second probe that binds to the RNA-binding protein comprises a peptide. In some embodiments, the portion of the second probe that binds to the RNA-binding protein comprises an aptamer. In some embodiments, the portion of the second probe that binds to the RNA-binding protein comprises a small molecule. In certain embodiments, the second probe binds to the RNA-binding protein through a mechanism that comprises a biotin-streptavidin interaction. The portion of the probe that recognizes the RNA-binding protein can be a protein (e.g., an antibody or antibody variant, or any protein that can otherwise bind to a specific RNA-binding protein). In some embodiments, the portion of the second probe that recognizes the RNA-binding protein comprises an agent that binds to an antibody, or an antibody variant. For example, the second probe can comprise a secondary antibody, and a primary antibody can be used to bind to the RNA-binding protein. The secondary antibody of the second probe then recognizes the primary antibody bound to the RNA-binding protein. See, for example, Figure 13A and Figure 13C. In certain embodiments, the portion of the second probe that recognizes the RNA binding protein comprises an antibody (e.g., a secondary antibody), or an antibody variant. In some embodiments, the secondary antibody recognizes and binds to the primary antibody that recognizes the RNA binding protein. The cells can be contacted with the primary antibody before or after being contacted with one or more pairs of probes. In certain embodiments, the cells are contacted with the primary antibody before being contacted with one or more pairs of probes. Instead of a primary antibody, the present disclosure also contemplates the use of any agent that can recognize and directly bind to the RNA binding protein of the probe described herein. In some embodiments, the portion of the second probe that directly binds to the RNA binding protein comprises a protein.In some embodiments, the portion of the second probe that directly binds the RNA binding protein comprises an antibody, or an antibody variant.

[0146] In some embodiments, the second probe of the set of probes further comprises a polymerization blocker. The polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the method described herein. In some embodiments, the polymerization blocker is at the 3' end of the second oligonucleotide probe. The polymerization blocker can be, for example, any chemical moiety that prevents the polymerase from using the second oligonucleotide probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue that comprises a blocked 3' hydroxyl group (e.g., an oxygen protecting group at the 3' hydroxyl group). In some embodiments, the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.

[0147] In addition to the portion that recognizes the RNA-binding protein, the second probe also includes a portion that is complementary to a portion of the first probe. In the set of probes provided herein, the portions of the first and second probes that are complementary to each other can be the same in each set of probes. In some embodiments, the portions of the first and second probes that are complementary to each other are unique in each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0148] In some embodiments, each portion of the second probe is connected by an optional linker. In some embodiments, the optional linker is a nucleotide linker. In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the second probe of the set of probes has the structure: 5'-[RNA-binding protein recognition portion]-[portion complementary to the first probe]-3' Including, Here, ]-[ includes any linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second probe.

[0149] A first probe (also referred to herein as a "padlock" probe) of a set of probes used in the methods for profiling interactions between an RNA-binding protein and an RNA described herein includes an oligonucleotide portion that is complementary to a second probe. In some embodiments, the portion of the first probe that is complementary to the portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' and 3' ends of the first probe.

[0150] The first probe of the set of probes used in the method for profiling the interaction between an RNA-binding protein and an RNA disclosed herein also comprises an oligonucleotide portion complementary to the RNA of interest. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22 or about 19-21 nucleotides in length. In some embodiments, the oligonucleotide portion of the first probe complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.

[0151] The first probe of the set of probes disclosed herein also comprises an oligonucleotide barcode sequence composed of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length. The barcode of the oligonucleotide probe described herein may comprise a gene-specific sequence used to identify the RNA of interest (i.e., the RNA bound by at least one RNA-binding protein).

[0152] Any order of arrangement of the parts of the first oligonucleotide probe is contemplated by the present disclosure.In some embodiments, the part of the first probe is connected to another part by an optional linker.In certain embodiments, the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.In some embodiments, the first probe has the structure: 5'-[portion complementary to the second probe]-[oligonucleotide barcode sequence]-[portion complementary to the RNA of interest]-[portion complementary to the third probe]-[portion complementary to the second probe]-3' Including, Here, ]-[ includes any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe. In some embodiments, any of the oligonucleotide portions of the probes that make up the set of probes provided herein comprises DNA.

[0153] The third probe (also referred to as "primer probe") of the set of probes used in the method for profiling the interaction between an RNA-binding protein and an RNA provided herein comprises a portion complementary to the RNA of interest and a portion complementary to the first probe of the set of probes. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5 to 15, 6 to 14, 7 to 13, 8 to 12, or 9 to 11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0154] In certain embodiments, the third probe of the set of probes has the structure: 5'-[part complementary to the RNA of interest]-[part complementary to the first probe]-[barcode sequence]-3' where ]-[ comprises any nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0155] All of the probes described herein may optionally have spacers or linkers of various nucleotide lengths between each of the listed components, or the components of the oligonucleotide probe may be joined directly to each other (i.e., by phosphodiester bonds). All of the probes described herein may contain standard nucleotides, or some of the standard nucleotides may be replaced with any modified nucleotide known in the art.

[0156] In some embodiments, the present disclosure provides a plurality of probes, comprising a number of probe pairs or probe sets, as described herein.In certain embodiments, each probe pair or probe set in the plurality of probes comprises an oligonucleotide portion that is complementary to different RNAs of interest.In some embodiments, the plurality of probes comprises more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 probe pairs or probe sets.

[0157] In some aspects, the present disclosure provides a library that comprises any of the multiple sets of probes described herein.In some aspects, the present disclosure provides a library that comprises any of the multiple pairs of probes provided herein.In some aspects, the present disclosure provides a library that comprises a multiple first probe from a set or pair of probes provided herein, a multiple second probe from a set or pair of probes provided herein, or a multiple third probe from a set of probes provided herein.

[0158] kit The present disclosure also provides a kit. In one aspect, the kit provided may include one or more probes as described herein. In some embodiments, the kit includes any of the probe pairs or probe sets described herein, or multiple probe pairs or probe sets. In certain embodiments, the kit includes more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 probe pairs or probe sets. In some embodiments, the kit may further include a container (e.g., a vial, an ampoule, a bottle, and / or a dispenser package, or other suitable container). The kit may also include cells for performing control experiments. In some embodiments, the kit may further comprise other reagents for carrying out the methods disclosed herein (e.g., enzymes such as ligases or polymerases, amine-modified nucleotides as described herein, primary antibodies, secondary antibodies, buffers, and / or reagents and monomers for making polymer matrices (e.g., polyacrylamide matrices)). In some embodiments, the kit is useful for profiling epitranscriptome RNA modifications in cells. In some embodiments, the kit is further useful for profiling unmodified RNA along with epitranscriptome modified RNA (i.e., the kit also includes a pair of probes for profiling an unmodified RNA of interest). In some embodiments, the kit is useful for profiling interactions between RNA binding proteins and RNA in cells. In some embodiments, the kit is useful for diagnosing a disease in a subject. In some embodiments, the kit is useful for screening agents capable of modulating epitranscriptome modifications of one or more RNAs. In some embodiments, the kit is useful for diagnosing a disease or disorder in a subject.In some embodiments, the kits are useful for treating a disease or disorder in a subject. In certain embodiments, the kits described herein further comprise instructions for using the kit.

[0159] system In one aspect, the present disclosure provides a system for profiling epitranscriptomic RNA modifications in a cell. In some embodiments, such a system comprises: a) in a cell (or in a large number of cells, e.g., in an intact tissue); b) one or more probe pairs comprising a first probe (i.e., a "padlock probe") and a second probe (i.e., a "primer probe"), wherein: i) the first probe comprises an oligonucleotide portion complementary to the second probe, an oligonucleotide portion complementary to the RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, e.g., by SEDAL sequencing as discussed herein); and ii) one or more pairs of probes, wherein the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope; and d) Computer Includes.

[0160] In some aspects, the present disclosure provides a system, comprising: a) Cell; b) a set of one or more probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptome RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) a third probe, a set of one or more probes, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; c) a microscope; and d) Computer The present invention provides a system including:

[0161] In some aspects, the present disclosure provides a system, comprising: a) Cell; b) a set of one or more probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to an RNA of interest, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) a third probe, a set of one or more probes, the third probe comprising an oligonucleotide portion complementary to the RNA of interest and an oligonucleotide portion complementary to a portion of the first probe; c) a microscope; and d) Computer The present invention provides a system including:

[0162] Any of the probes (i.e., pairs of probes) described herein may be used in the systems contemplated by the present disclosure. In some embodiments, the epitranscriptome RNA modification is 6 -Methyladenosine (m 6 A), N 1 -Methyladenosine (m 1 A), pseudouridine, N 6,2'-O-Dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -Acetylcytidine (ac 4 C), 2'-O-methylated (Nm), or 5-methylcytosine (m 5 In certain embodiments, the epitranscriptome RNA modification is N 6 -Methyladenosine (m 6 A).

[0163] In some embodiments, the system further comprises software running on the computer. In some embodiments, the system may further comprise other reagents (e.g., enzymes such as ligases or polymerases, amine-modified nucleotides as described herein, primary antibodies, secondary antibodies, buffers, and / or reagents and monomers for making polymer matrices (e.g., polyacrylamide matrices)).

[0164] example Example 1: Single-cell profiling of the epitranscriptome in situ A series of single-cell multi-omics techniques have been developed that cover a wide range of individual information from chromosome structure to cell surface protein display. The methodology for single-cell profiling of epitranscriptome RNA modifications described herein is unique in the field of single-cell multi-omics techniques due to its remarkable versatility and ability to be adapted to various applications. A variety of chemical and biotechnological tools, including RNA-hydrogel cross-linking, metabolic labeling, DNA barcoding and in situ sequencing, are combined in the method described herein to detect epitranscriptome RNA modifications in a high-throughput manner. The method described herein is useful for understanding RNA modifications and for improving understanding of human diseases associated with epitranscriptome modifications.

[0165] Chemical modifications of the transcriptome play important roles in regulating RNA activity, processing (e.g., of pre-mRNA), stability, transport and translation. These chemical modifications of cellular RNAs are diverse and ubiquitous, providing an additional layer of complexity in regulating gene expression. Although epitranscriptomics has been studied in second generation sequencing, strategies to study the native cellular environment as well as the spatial arrangement of modified RNAs in a multiplexed and high-throughput manner are needed to advance our understanding of RNA modifications. The methods described herein address these two issues through the use of probes in which an RNA modification-binding moiety (e.g., an RNA modification-binding antibody) is conjugated to an oligonucleotide primer. Thus, the identity of RNAs with specific epitranscriptomic modifications can be sequentially decoded and identified using the oligonucleotide barcode sequences present in the probes (Figure 1).

[0166] The utility of this method is demonstrated by the addition of one of the most abundant and common RNA modifications, N6-methyladenosine (m 6 A) Demonstrated by detection of key epitranscriptomic markers in mRNA processing, translation, and degradation. 6 A-modified β-actin transcripts were detected in the cells (Figures 2A-2D). 6 It is a common and abundant mRNA in cells modified by A. The raw images show that a signal corresponding to β-actin mRNA can be seen (Figure 2A) compared to several negative controls (Figure 2B-2D).

[0167] Example 2: Single-cell in situ analysis of RNA modifications in intact tissues Recent evidence indicates that RNA modifications are not only subtle structural modifications but also active gene regulators 3 In particular, mRNA modifications (e.g., N 6-methyladenosine) affects nearly every step in the life cycle of an RNA transcript from birth to death, including splicing, nuclear export, storage, cellular localization, translation, and decay. 3 However, it remains unclear why mRNA modifications are non-stoichiometric. For a given gene, many modification sites are not 100% marked, and some RNA demodification enzymes dynamically adjust the state of modifications. New methods are needed to determine what role these contrasting RNA modification states play in mRNA behavior, which is severely perturbed by bulk sampling. It is also unclear how mRNA modifications affect the subcellular location of mRNAs inside cells. Given the non-stoichiometric nature of mRNA modifications, there are at least two plausible hypotheses: that within a single cell, mRNAs with different modification states have different spatiotemporal properties to precisely control the location and duration of protein production; or that the apparent non-stoichiometry is a result of mixing different cell states and cell types, as all previous measurements were performed in millions of cells. Finally, it is also unclear how the effects of mRNA modifications differ between different cell types and states. Results of bulk epitranscriptome sequencing have revealed that different organs have distinct epitranscript signatures that vary across different developmental and disease stages. 3 Moreover, given the diversity of cell types within any given organ, single-cell epitranscriptome sequencing is required to reveal cell-type specific modification patterns. Analyzing this diversity will allow a deeper understanding of epitranscriptomic pathways.

[0168] Current bulk epitranscriptome sequencing methods have many limitations, including that they require millions of cells as input material. This results in loss of single molecule stoichiometry and single cell sensitivity. Furthermore, in applications where spatial relationships are essential, such as developmental pathways and neural tissue, dissociated tissue samples are insufficient. 5Currently, imaging methods for RNA modifications can only target one gene at a time or detect all modification sites in total without distinguishing by gene identity. It is noteworthy that many modified RNAs are regulated as large groups of genes. Therefore, for meaningful data analysis, at least several hundred modified genes need to be measured simultaneously (ideally across the transcriptome).

[0169] Overall, the methods described herein represent a transformative toolbox for single-cell epitranscriptome profiling with spatial resolution. Such a system has broad applications in diverse biological processes. Given that RNA modifications are shared among all eukaryotic cells and RNA viruses that replicate inside the cell nucleus, RNA modifications have been actively studied in cancer, immunology, RNA virology, and more. Furthermore, the spatial distribution of these modifications is likely to be important in fields encompassing neuroscience, stem cell differentiation, and developmental biology. The methods described herein also have the potential to provide an integrated transcriptome and epitranscriptome spatial cellular atlas of the brain that cannot be charted by existing approaches and new scientific knowledge of how RNA modifications regulate cellular function across various cell types and how single-cell events collectively affect the function of tissues. 6 In addition to A, there are other types of mRNA modifications that can be approached using similar strategies (e.g., 1-methyladenosine, pseudouridine, etc.). Additionally, tRNAs have a large number of diverse modifications. 3 Increasing evidence indicates that they can also affect protein translation in response to various signals, stresses, and human diseases. 3、5

[0170] N 6 -Methyladenosine (m 6A) is the most common internal modification present in the messenger RNAs of all higher eukaryotes. This modification is non-stoichiometric and its deletion is lethal in vertebrates. 6 A methyltransferase complex METTL3 / 14 ("Writer") is involved; its presence affects development, fertility, and nutritional metabolism. 6 It is further dynamically regulated by A demethylase (ALKBH5 / FTO; “Eraser”). 3 m 6 The A family of A-binding proteins, YTHDF and YTHDC, are 6 It specifically recognizes A-modified mRNA and regulates mRNA splicing, transport, translation, and degradation. 12、13 Together, m 6 A regulatory protein confers gene expression with fast response and controllable protein production essential during stem cell differentiation and animal development 3、14 .

[0171] Rodent bulk m 6 A sequencing is 6 We revealed that A is common in the whole-brain transcriptome, modifying thousands of coding and hundreds of noncoding genes (Figure 6). 15、16 m in animals 6 Research on the A pathway is 6 These results suggest that A modulates neuronal functions including cortical formation, dopaminergic signaling in the mouse midbrain, flight and locomotion behavior in flies, neurogenesis in adult mice, and axon regeneration in mice. 6 Upregulation of A has been observed to occur with brain maturation, behavioral experience, and memory formation, and is associated with m 6 This suggests a link between A accumulation and brain activity. 6 In addition to normal physiological processes, m 6 A methyltransferase and demethylase 6 A abundance and genetic variants are associated with attention-deficit / hyperactivity disorder (ADHD), major depressive disorder (MDD), addiction, epilepsy, and neurodegeneration17 .

[0172] However, m 6 Despite these early discoveries of links between A and brain function, there are still huge knowledge gaps between the associations and mechanisms. Single-cell RNA sequencing has revealed hundreds of molecularly defined cell types. 18 But bulk m 6 A-seq is a method to detect m 6 Masking potential diversity in the epitranscriptome. For example, 6 The A demethylase FTO regulates reward behavior via a meso-striatal-prefrontal dopamine signaling reward circuit 19 , m 6 These results suggest that A-dependent gene regulation may be biased toward deeper and more specific neural circuits in defined neuronal cell types. Furthermore, many brain-related studies have demonstrated that A-dependent gene regulation in cell cultures 6 A methodology that directly recognizes and utilizes molecular mechanisms discovered in A to explain mouse phenotypes. 6 This can be problematic because the A pathway involves a large number of enzymes and binding proteins, different combinations of which can lead to dramatically different functional outcomes. Moreover, such in vitro experiments remove much of the context-dependence of these cellular behaviors. Therefore, instead of using in vitro cell culture models, it is best to perform in vitro cell culture models to model the A pathway in the animal model of interest. 6 It is highly desirable to directly study the epitranscriptome to explain behavioral phenotypes. 6 It is clear that the lack of A-seq is an important limitation, and that the methods described herein address these issues.

[0173] 3D-m 6 The method for A-seq is 6 The following were developed by utilizing A-specific binding agents, proximity amplification, and in situ RNA sequencing (Figure 7): (1) m 6 A-Binding agent (anti-m 6A antibody or biochemically purified m 6 A-specific YTH domain proteins 12、13 ) is conjugated to a DNA primer and used to stain the sample of interest; (2) a DNA padlock probe is then used to detect the m 6 Hybridize to RNA sequences adjacent to the A site. Bulk m 6 Previously obtained m from A-seq 6 The A site informs the design of these probes, and the library contains thousands of m 6 (3) the padlock probe is circularized by an RNA-templated DNA ligase (e.g., SplintR); (4) the padlock probe is then circularized by a RNA-templated DNA ligase (e.g., SplintR); 6 Circularized DNA probes close to the A site are amplified by rolling circle amplification (RCA, thousands of tandem repeats), whereas those close to the unmethylated site lack the m required for circularization. 6 (5) Each padlock probe contains a 5-base or longer barcode to encode gene identity (>1000 genes) and a m-seq sequence within the transcript. 6 A site identity (five rounds of sequencing allow for up to 20 sites per transcript; >99% m 6 (5) A-modified genes contain a separate one-base barcode for encoding gene identity and m 6 A-site identity is read out by 6 rounds of combinatorial SEDAL sequencing and 5 rounds of sequential SEDAL sequencing, respectively 11 Such a 3D-m 6 A-seq results provide gene identities, 3D coordinates, and m 6 A is a collection of single RNA molecules resolved by the number / location of A peaks. Such data can be directly visualized and analyzed to reveal the spatial distribution of gene expression with subcellular resolution (spatial resolution of approximately 150-400 nm, depending on both the size of the DNA amplicon and the optical limits). 11After cell body staining (e.g., Nissl staining) and cell segmentation, RNA can be attributed to each cell. The data can then be used for simultaneous cell type classification, assessment of single cell variation in gene expression, and m 6 A methylation status can be analyzed.

[0174] Previous studies have provided evidence that: (1) activity-regulated genes (ARGs) and synaptic RNAs are involved in 6 A-modified; (2) m 6 Disruption of the A pathway impairs neuroplasticity and long-term memory formation; and (3) once animals engage in defined types of behaviors (fear, reward, stress, etc.), they experience an overall memory deficit. 6 Based on the change in the abundance of A, m 6 The method disclosed herein establishes a strong link between m A and neural activity during global neural stimulation with subcellular resolution. 6 It is useful to further analyze the spatiotemporal patterns of A. 6 A-seq can be applied to study two of the most established biological systems for molecular-level investigation of neuronal activity: potassium chloride (KCl) depolarization of primary neuronal cell cultures and dark / light conditioning in mice (Figure 8). 11 Samples were collected at a series of time points before and after stimulation to determine the overall neuronal response to stimulation. 6 Tracking the spatiotemporal dynamics and single-cell diversity of the epitranscriptome and m in immediate early genes (IEGs) versus late response genes (LRGs) 6 A RNA distribution can be assessed.

[0175] Single cell dissociation 6 After obtaining the A pattern, the gene regulatory mechanisms that shape such a pattern can be further identified. Two approaches to address this issue can be used: (1) determining whether a protein is expressed and m 6 To generate hypotheses related to changes in patterns 6 Expression levels of A-associated proteins (methyltransferases, demethylases, and binding proteins) along with single-cell m 6A-pattern integrative analysis; (2) cell-type-specific gene perturbation (knockdown or overexpression) experiments to determine which factors may affect epitranscriptomic gene expression regulation in different cell types.

[0176] Existing bulk epitranscriptome sequencing methods require millions of cells as input material and lack single-molecule stoichiometry, single-cell sensitivity, and spatial resolution. Current imaging methods of RNA modifications can only target one gene at a time or detect all modification sites in total without distinguishing gene identity. 3D in situ sequencing of RNA modifications enables simultaneous and highly multiplexed mapping of the epitranscriptome with unprecedented resolution and precision: single-molecule stoichiometry with 3D coordinates of thousands of genes in intact living tissues.

[0177] Example 3: Three-dimensional in situ profiling of single-cell epitranscriptomics The transcriptome serves as a pivotal mechanism that transmits information from the upstream genome to the downstream proteome and regulates numerous cellular processes. In recent years, single-cell RNA sequencing and spatial transcriptome techniques have successfully mapped the transcriptomes of all kinds of biological samples, revealing complex cell-to-cell heterogeneity in various systems. However, additional information beyond the RNA sequence is embedded in the transcriptome. This additional information, called the epitranscriptome, i.e., the N-terminal domain, which is important in regulating almost all stages of the mRNA life cycle, ranging from mRNA synthesis, translocation, translation, and degradation. 6 -Methyladenosine (m 6 A) and their associated RNA-binding proteins (RBPs). Knowing the spatially resolved single-cell profile of the epitranscriptome is important to more fully understand how mRNAs are regulated in different cell types and conditions. Furthermore, 6 The intracellular distribution and pattern of A6 Little is known about how A is related to its regulatory role. This disclosure provides a novel method to characterize the mechanism of action of A in vivo and in vivo signaling. 6 The in situ m is a state-of-the-art technology platform for three-dimensional (3D) in situ profiling of A. 6 A mapping (m 6 This method describes a method for the A-map. 6 It is used to generate imaging-based single-cell descriptions at a subcellular level, aiming to provide a fresh perspective on the spatial distribution of A. From a fundamental research perspective, this method can be generalized to aid in the understanding of other RNA modifications, their associated RBPs, and their interactions. 6 The application of A-map also reduces the abnormal m 6 Facilitate identification of causes and understanding of development of diseases and disorders associated with A levels.

[0178] To better understand different cell states and types, various single-cell and spatial transcriptome profiling methods have been developed to map heterogeneity at the transcriptome level, but these tools only describe the cell state rather than revealing the mechanisms underlying heterogeneous transcriptomes. Therefore, the epigenome and epitranscriptome need to be interrogated at the single-cell level with spatial resolution, as they encompass additional information carried by nucleic acids that regulate all stages of the mRNA life cycle.

[0179] Among all epitranscriptome modifications, m 6 A is the most abundant and perhaps most important modification in mRNAs, and has been reported to regulate mRNA translation efficiency, stability, translocation and splicing (Figure 9A). 6 A has also been implicated in many biological processes at the tissue level, including learning and memory, cancer progression, and neurodegeneration. Thus, different cells may be involved in heterogeneous m 6A methylome and differentially regulate their transcriptomes 6 There is a high possibility that A will be used.

[0180] Nevertheless, the lack of single-cell resolution in epitranscriptome studies remains a challenge in living tissues. 6 It hides the heterogeneity of the A pattern and m 6 This impairs our ability to analyze how A regulates gene expression across different cell types. Thus far, only a few studies have shown that mRNA expression is modulated in different cell types and conditions. 6 A and its intracellular distribution pattern 6 Little is known about how A is related to its regulatory role (Figure 9B). For example, cell type / state-specific m 6 What exactly is the A methylome, and the same cell type has m 6 Whether the granules can be divided into subtypes based on the A methylome, m 6 A-mRNA and non-mRNA 6 Whether and how A-mRNAs exhibit different localization at the subcellular and tissue levels and how heterogeneous mRNAs 6 Whether A affects different dynamics of the RNA life cycle in different cell types, and many other such questions remain unanswered (Figure 9C).

[0181] Previously, m 6 Although various tools have been developed to qualitatively and quantitatively detect A modifications, none of them simultaneously provide high throughput and single-cell, single-base, and spatial resolution. The present disclosure overcomes the above-mentioned limitations of previous methods and provides a method to detect mA modifications with subcellular resolution. 6 A novel method for decomposing the A methylome, m 6 A-Map(m 6 Single-cell in situ mapping of A is described (Figure 9D).

[0182] Method design and development in HeLa cells (m 6 A Map v1) First, two sets of triplicate probes, each set containing a primer and a 5' phosphorylated unique barcoded padlock probe, were added to each putative m 6 The mRNA region adjacent to the A site was hybridized in situ. 6 To achieve A detection, m 6 The recognition of A was coupled with the recruitment of an oligonucleotide probe (the third probe of the set of probes). PAPG, a chimeric protein that specifically recognizes the Fc region of an antibody, is conjugated to the oligo (Figure 10B). The oligo serves as a splint probe for in situ proximity ligation of the padlock probe. In this way, m 6 Only sites with A circularize their corresponding padlock probes. Furthermore, to overcome autofluorescence and light scattering, the circularized padlock probes were amplified using in situ rolling circle amplification (RCA). To preserve the location of the amplicons and stabilize the cellular structure during multiple rounds of sequencing and stripping, amino-allyl-dUTP was spiked into the RCA reaction so that the amplification products (amplicons) were functionalized with primary amine groups, which were further acryloylated by methacrylic acid N-hydroxysuccinimide ester (MA-NHS) and embedded in a polyacrylamide hydrogel network. Finally, the barcodes of the padlock probes were sequenced in situ, achieving multiplexing of up to several thousand genes (Figure 10A). Single m in both cell cultures and tissues was identified using a 3D-PCR technique. 6 Detection of A sites (ACTB and MALAT1) was achieved by anti-m 6 This is demonstrated by a 15- to 30-fold signal enrichment in group A (Figures 10C and 10E).

[0183] In the above workflow, the PAPG can be replaced with a secondary antibody. The secondary antibody can be conjugated to an oligo, for example, using the SiteClick antibody labeling kit (FIG. 11A). 6A-map v1 was tested with a rabbit secondary antibody (Figure 11B). An antibody-independent biotinylated-YTH-streptavidin-oligo detection scheme was also tested (Figure 10D).

[0184] The method can also be optimized in a number of ways. For example: (1) The optimal location of the probe is determined by the m of interest. 6 (2) rRNA m 6 m from A site 6 To reduce the A signal, 6 An rRNA blocking probe that can hybridize to rRNA sequences containing A sites can be added to the hybridization mixture; and (3) the mRNA can be immobilized in a hydrogel prior to post-hybridization proteinase treatment to allow better antibody diffusion into the sample (as an example, EDC, which preferentially reacts with RNA, can be used to attach a polymerizable handle to the RNA).

[0185] Design and development of the method in Hela cells (m 6 A Map v2) m 6 A map v1 is m 6 Although only A-modified RNA can be detected, and not unmethylated RNA, it would be highly beneficial to simultaneously detect both methylated and unmethylated RNA, given the following considerations: (1) classification of cell status or cell type using marker gene expression; (2) detection of higher m from specific genes; 6 A signal indicates higher expression levels or higher m 6 m to distinguish whether it is due to the A fraction 6 (3) estimation of methylation stoichiometry; (4) comparison of differential localization of methylated and unmethylated RNAs; and (5) integration with other in situ sequencing data. To address this issue, an updated method 6 A-Map v2 was devised. The second pre-hybridization step was m 6The first RCA is followed by a second hybridization with a STARmap probe, which is the same as A-map v1. 6 A allows the detection of RNA, followed by ligation and a second RCA (Figure 12A). If the RNA already has a DNA amplicon attached, the second RCA cannot occur because the first DNA amplicon occupies most of the available space (Figure 12B). This allows the detection of RNA, followed by ligation and a second RCA (Figure 12B). 6 A and non-m 6 Both A and B RNA can be quantified separately, 6 The proportion of A RNA in a single cell culture can be calculated. 6 m at A site (ACTB and MALAT1) 6 Quantification of the A fraction was demonstrated (Figures 12C and 12D). 6 The A fraction is actually different, m 6 A RNA and non-m 6 A RNA showed different subcellular localization.

[0186] Method design and development in Hela cells (RBP-RNA mapping) m 6 Apart from A, RBP-RNA interactions are another important aspect of the epitranscriptome, as most mRNA modifications achieve their functions through differential binding of various RBPs. Although numerous single-cell RBP-RNA interactome mapping methods have been developed, these methods are either tailored to specifically detect ribosome-mRNA interactions or require transfection of exogenous gene constructs. Moreover, they cannot preserve the cellular spatial location and subcellular location of RBP-RNA interactions.

[0187] In addition, m 6A can have opposing functions depending on the context, and the main reason is that it can be bound by different leader RBPs that trigger completely different downstream pathways. A multiplexed detection method would be ideal for interpretation. Therefore, an in situ multiplexed RBP-RNA mapping technique was developed (Figure 13A), which involves transferring the information of oligo-conjugated antibodies to padlock probes, followed by in situ sequencing of the transferred barcode information. The feasibility of single RBP-RNA mapping has already been demonstrated (Figure 13B), and the next step is to multiplex this method (Figure 13C). Such a method is useful to determine how the interaction of epitranscriptome with RBP-RNA contributes to the regulation of cell state / type-specific transcriptome and tissue function.

[0188] 100 gene detection in HeLa cells as a proof-of-concept study m 6 A map with many m 6 To further demonstrate that A sites can be simultaneously detected in a high-throughput manner, we performed fluorescent ubiquitination-based cell cycle index (FUCCI) 100-gene detection in HeLa cells. Spatial transcriptome profiles (STARmap) and m 6 A map v1 was performed simultaneously. Antibodies from different sources were tested to select the one with the highest detection efficiency. Nuclei were stained with DAPI, while cell bodies were stained with Flamingo and endoplasmic reticulum (ER) was stained with Concanavalin A, which allowed visualization of probes against three moderately expressed housekeeping genes (HMBS, MRPL19, and PGK1). Consistent anti-mRNA binding to IgG controls was observed. 6 A signal enrichment was observed (Figure 14), further validating the signal-to-noise ratio (SNR) achieved with method optimization.

[0189] m 6 Findings from the A-map v1 100 gene dataset m 6A-map v1 in situ sequencing and FUCCI imaging data were preprocessed by deconvolution, spot finding, read assignment, cellular and subcellular segmentation, alignment, filtering, and normalization. 6 The number of reads in A-map is smaller than that in STARmap (Figures 15A and 15C). 6 The A antibody achieved the best signal-to-noise ratio (SNR), reaching an SNR of 25 for most genes (Figure 15B). 6 Measured m between A antibodies 6 A Stoichiometric correlation (m 6 The m of different genes (A map reads / STARmap reads) were well correlated, verifying the robustness of the method (Figure 15D). 6 A stoichiometry has a slight negative correlation with their expression levels, m 6 A primarily promoted mRNA decay (Fig. 15E). 6 A can be used to semi-quantitatively estimate stoichiometry and, for several representative genes, shows a positive correlation with literature-reported stoichiometries (Figures 15F and 15H).

[0190] m 6 To benchmark and gain more biological insight into Abiology, 6 A vs. non-m 6 The subcellular distribution pattern of A mRNA was analyzed. 6 It was found that mRNAs with A deposits tend to localize more readily to the cytoplasm, facilitating nuclear export. 6 The biological function of A was confirmed (Figure 16A). 6 Genes strongly affected by A deposition include many genes related to translation, such as EEF2, SRP19, and MRPL20, 6 These results indicate that YTHDC1-targeted mRNAs may be important for efficient production of translation-associated proteins. 6 It may show a higher dependence on A.

[0191] Cell cycle analysis was also performed on the dataset. FUCCI fluorescence intensity was quantified for each cell to determine the cell cycle phase of the different cells (Figure 16B). 6 A stoichiometry was calculated for each gene in different cell cycle phases. Many genes exhibited cell cycle-dependent m 6 SMAD3, a gene related to transcriptional regulation, was found to exhibit higher mA fluctuations in G1 and S. 6 A stoichiometry, but lower in G2M, which is consistent with literature results (Fei, Q. et al., YTHDF2 promotes mitotic entry and is regulated by cell cycle mediators. PLoS Biol 18, e3000664 (2020)). Other genes, including SON, have higher mA stoichiometry in S and G1 but lower mA stoichiometry in G2M. 6 A stoichiometry, and some, such as MALAT1, have higher m in G2M and G1 but lower m in S 6 It was also found to have an A stoichiometry.

[0192] Consideration In summary, hundreds or thousands of microns of single cells with subcellular resolution 6 A novel strategy, m, that can be used to measure A loci and semi-quantitatively estimate the relative stoichiometry of each locus 6 A map was developed. 6 The A-Map reveals whether different epitranscriptome states affect the subcellular localization of RNAs, how the epitranscriptome exhibits cell-to-cell heterogeneity, and how the 6 The method described herein can be used to discover entirely new biological insights related to the epitranscriptome, such as how A levels fluctuate in different cell cycle phases. 6 A versatile tool for exploring biology.

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[0216] Incorporation by Reference This application refers to various issued patents, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. Details of one or more aspects of the invention are set forth herein. Other features, objects, and advantages of the invention will become apparent from the detailed description, figures, examples, and claims.

[0217] Equivalents and Scope Articles such as "a," "an," and "the" may mean one or more, unless the contrary is indicated or otherwise clear from the context. An embodiment or description including "or" between one or more members of a group satisfies whether one, more than one, or all of the members of the group are present in, employed in, or otherwise related to a given product or process, unless the contrary is indicated or otherwise clear from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise related to a given product or process. The invention includes embodiments in which more than one, or all of the members of the group are present in, employed in, or otherwise related to a given product or process.

[0218] Furthermore, the disclosure covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as, for example, a list in Markush group format, each subgroup of elements is also disclosed, and any element(s) can be removed from the group. In general, when an invention, or an aspect of an invention, is referred to as comprising certain elements and / or features, it should be understood that a particular embodiment or aspect of the disclosure consists of, or consists essentially of, such elements and / or features. For purposes of brevity, those embodiments have not been specifically recited in haec verba herein. It should also be noted that the terms "comprising" and "containing" are intended to be open and permit the inclusion of additional elements or steps. Where ranges are given, the endpoints are included. Additionally, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any specific value, or subrange within the ranges set forth in different aspects of the invention, down to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0219] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. In addition, any particular aspect of the invention that falls within the prior art may be expressly excluded from any one or more of the aspects. Because such aspects would be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not expressly stated herein. Any particular aspect of the invention may be excluded from any aspect for any reason, whether or not related to the existence of prior art.

[0220] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather as set forth in the accompanying embodiments. Those skilled in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

1. 1. A method for identifying modifications of ribonucleic acid (RNA) molecules in a cell, comprising: a) contacting a cell with a set of one or more probes, wherein the set of one or more probes comprises a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion complementary to the RNA molecule, and an oligonucleotide portion complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes a modification of the RNA molecule and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion complementary to the RNA molecule and an oligonucleotide portion complementary to a portion of the first probe; b) ligating the 5' and 3' ends of the first probe to produce a circular oligonucleotide; c) amplifying the circular oligonucleotide using the third probe as a primer to produce one or more amplicons; d) sequencing one or more amplicons or portions thereof to identify modifications of the RNA molecule; The method comprising:

2. The method of claim 1, wherein step c) comprises rolling circle amplification.

3. The method of claim 1, further comprising embedding one or more amplicons in a polymer matrix prior to step d).

4. The modification is N 6 -methyladenosine (m 6 The method according to claim 1, wherein A).

5. The modification is N 1 -methyladenosine (m 1 A) Modification, pseudouridine modification, N 6 ,2'-O-dimethyladenosine (m 6 Am) modified, 7-methylguanosine (m 7 G) Modification, N 4 -Acetylcytidine (ac 4 C) modified, 2'-O-methylated (Nm) modified, or 5-methylcytosine (m 5 C) modification.

6. A method according to any one of claims 1 to 5, wherein the portion of the second probe that recognizes the modification comprises an antibody or an antibody variant.

7. The method of claim 6, wherein the antibody is a secondary antibody.

8. 8. The method of claim 7, further comprising contacting the cells with a primary antibody that recognizes the modification and that is recognized by the secondary antibody of the second probe.

9. A method according to any one of claims 1 to 5, wherein the portion of the second probe that recognises the modification comprises an agent that binds to an antibody or antibody variant.

10. The method of claim 1, wherein the portion of the second probe that recognizes the modification comprises a protein.

11. The method of claim 10, wherein the protein comprises PAPG.

12. 12. The method of claim 11, further comprising contacting the cell with an antibody that recognizes the modification, wherein the antibody that recognizes the modification is recognized and bound by the PAPG.

13. A method according to any one of claims 1 to 5, wherein the portion of the second probe that recognises the modification comprises an agent that binds directly to the modification.

14. The method of claim 13, wherein the agent that directly binds to the modification comprises a protein.

15. Protein, m 6 The method of claim 14, wherein the YTH domain protein is an A-specific YTH domain protein.

16. A method described in any one of claims 1 to 5, further comprising simultaneously analyzing more than 10 RNA molecules.

17. The method of any one of claims 1 to 5, wherein the second probe further comprises a polymerization blocker.

18. 18. The method of claim 17, wherein the polymerization blocker is located at the 3' end of the second probe.

19. 18. The method of claim 17, wherein the polymerization blocker comprises an inverted nucleic acid residue.

20. 20. The method of claim 19, wherein the inverted nucleic acid residue is an inverted thymine residue.

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