Single-cell profiling of RNA translational state

Abstract

The present disclosure provides a method and system for profiling RNA translated in cells.The present disclosure also provides a method for diagnosing disease or disorder in a subject based on the profile of RNA translated in cells, including cells in intact tissue.The present disclosure also provides a method for screening or testing candidate agents that can modulate the translation of one or more RNAs.The present disclosure also provides a method for treating disease or disorder in a subject that requires the treatment of disease or disorder.The present disclosure also describes a pair of probes and a set of probes that comprise oligonucleotide moieties, which can be useful for carrying out the method described herein.In addition, the present disclosure provides a kit that includes 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 / 216,315, filed June 29, 2021, which is incorporated herein by reference. [Background technology]

[0002] 2. Background of the Invention Transcriptional profiles in single cells do not always correlate with proteomic profiles from the same cells. This suggests that there are various mechanisms at the post-transcriptional level to regulate protein production and degradation, some of which have yet to be characterized. Thus, mRNA levels are an imperfect proxy for estimating protein production and may be biased in defining the state of the cell. Additional methods for directly quantifying the proteome of single cells can provide a more functionally relevant basis for interpreting cell type and cell type-specific responses to the environment. Accurate determination of protein production in cells would also be useful in the study of many physiological conditions, or the treatment of various diseases, as well as drug development. Thus, additional and better systems for accurately quantifying protein production in single cells are needed. Summary of the Invention

[0003] Summary of the Invention Methods, compositions, kits, and systems for in situ ribosome profiling (i.e., profiling of an actively translated RNA of interest) are described herein. Such systems represent an essential technology to bridge the gap between the transcriptome and proteome of a cell by quantifying / profiling actively translated mRNA. A proximity ligation-based strategy is described herein to achieve specific and high-throughput characterization of the translation state of RNA in situ as an estimate of the proteome of a single cell. The system utilizes a ribosome-recognizing agent conjugated to an oligonucleotide probe that recognizes the sequence of the corresponding probe annealed to the ribosome-bound mRNA molecule (Figure 1). Briefly, the system utilizes a probe or set of probes that contain a unique molecular barcode to identify a target sequence within the mRNA of interest. The ribosome is then bound by an oligonucleotide sequence portion of a primer that is complementary to a portion of the ribosome, or by a ribosome-specific primary antibody bound to a secondary antibody conjugated to a DNA primer. An oligonucleotide sequence complementary to a portion of the ribosome, or a DNA primer conjugated to a secondary antibody (and thus bound to the ribosome) is ligated to the probe. Using the probe as a primer, the mRNA of interest is amplified by rolling circle amplification to produce a measurable sequence signal. Additionally, the antibody can be detected through antibody staining to provide spatial information. By labeling the ribosome and positioning the mRNA of interest, precise spatial information of the actively translated mRNA can be extracted by means of highly selective RNA-ribosome colocalization, which is an important readout for understanding the spatiotemporal patterns of translation in both health and disease. The interaction between ribosomes and mRNA can be probed to determine the translation of the mRNA of interest.

[0004] Thus, in one aspect, the present disclosure provides a method for profiling RNA being translated in a cell (see, for example, FIG. 1, FIG. 3, and FIG. 6). In the method disclosed herein, a cell can be contacted with one or more pairs or sets of probes, which can be further described herein and used to amplify (e.g., by rolling circle amplification) RNA being actively translated by ribosomes, to generate one or more concatenated amplicons. One or more concatenated amplicons can then be embedded in a polymer matrix and sequenced (e.g., by SEDAL sequencing (Error-reduction by Dynamic Annealing and Ligation) sequencing, as further described herein) to determine the identity of the transcripts and their location within the polymer matrix. Using the location of the transcripts of interest, RNA being translated in a cell can be profiled, and spatiotemporal information can be obtained to improve the field's understanding of how translation location and timing affect cellular function in health and disease. The methods may be useful, for example, for comparing RNA translation in cells (or multiple cells) from diseased and healthy tissue samples; or, for example, for comparing RNA translation in cells treated with an agent and untreated cells.

[0005] Importantly, the method described herein can include two or three probes for profiling mRNA molecules during translation.Although each method is useful for profiling mRNA molecules during translation, it has been demonstrated that the three-probe method produces a better signal-to-noise ratio.

[0006] In some embodiments, the present disclosure provides a method for producing a method for the treatment of a disease comprising the steps of: a) contacting a cell with one or more pairs of probes, each pair of probes comprising a first probe and a second probe; 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; contacting the cells with one or more pairs of probes; 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest in the cell. The present invention provides a two-probe method for profiling RNA being translated in a cell, comprising:

[0007] In some embodiments, the present disclosure provides a method for producing a method for the treatment of a disease comprising the steps of: a) contacting a cell with one or more sets of probes, each set of probes comprising a first probe, a second probe, and a third probe; i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; contacting the cells with one or more sets of probes; 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 third 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest in the cell. The present invention provides a three-probe method for profiling RNA being translated in a cell, comprising:

[0008] The methods, compositions, and systems described herein can be useful for studying RNA translation in tissues (e.g., developing tissues, normal tissues, diseased 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 (e.g., cancer) in a subject. For example, the method of profiling translated RNA described herein can be performed on a cell or a number of cells taken from a subject (e.g., a subject believed to have or at risk of having a disease or disorder, or a subject that is or is believed to be healthy). The expression of various RNAs of interest in the cells can then be compared to 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 RNA translation profile of a cell (including a single RNA or multiple RNAs of interest, e.g., a specific disease signature) compared to one or more non-disease cells may indicate that the subject has a disease or disorder. RNA translation in one or more non-disease cells (e.g., normal cells) may be profiled with expression in disease cells as a control experiment. RNA translation in one or more non-disease cells (e.g., normal cells) may also be profiled in advance, and expression in disease cells may be compared to this reference data for non-disease cells.

[0009] In another aspect, the present disclosure provides a method for screening agents capable of modulating the translation of one or more RNAs of interest. For example, the method of profiling translated RNAs described herein can be performed in cells in the presence of one or more candidate agents. The expression of various RNAs of interest that are translated in cells (e.g., normal or diseased cells) can then be compared to the expression of the same RNAs of interest in cells that have not been exposed to one or more candidate agents. Any difference in the RNA translation profile compared to the translation in cells that have not been exposed to the candidate agent(s) can indicate that the translation (or potentially the transcription) of one or more RNAs of interest is modulated by the candidate agent(s). In some embodiments, a particular signature (e.g., of multiple RNAs of interest that are translated) known to be associated with the treatment of a disease can be used to identify other agents that can modulate translation in a desired manner and thus treat the disease. The methods described herein can also be used to identify drugs that have certain specific side effects, for example, by searching for specific RNA translation signatures when one or more cells are treated with a candidate agent or known drug.The methods described herein can also be used to identify research reagents or chemical probes that can be useful for studying the basic biology of cellular translation and protein production.

[0010] In another aspect, the present disclosure provides a method for treating a disease or disorder (e.g., cancer) in a subject. For example, the method for profiling translated RNA described herein can be performed in cells from a sample taken from a subject (e.g., a subject who is thought to have a disease or disorder or is at risk of having a disease or disorder). The profile of one or more RNAs translated in the cells can then be compared to the translation state of the same RNA of interest in cells from a non-disease tissue sample. If any difference is observed in the RNA translation profile compared to non-disease cells, a treatment for the disease or disorder can then be administered to the subject. The RNA translation in one or more non-disease cells can be profiled along with the RNA translation in disease cells as a control experiment. The RNA translation in one or more non-disease cells (e.g., normal cells) can also be profiled in advance, and the translation in disease cells can be compared to this reference data for non-disease cells.

[0011] 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), 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; and ii) The second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe.

[0012] In another aspect, the present disclosure provides a set of oligonucleotide probes comprising a first probe (also referred to herein as a "padlock" probe), a second probe (also referred to herein as a "sprint probe" or "blocked probe"), and a third probe (also referred to herein as a "primer probe"); i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe.

[0013] In another aspect, the disclosure provides kits, including, for example, kits including any of the pairs or sets of probes disclosed herein. In some embodiments, the kits include multiple pairs or sets of probes as described herein, each of which can be used to identify a unique RNA of interest that is being translated. The kits described herein can 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, primary antibodies, secondary antibodies, buffers, reagents (including dyes, stains, etc.), and monomers for making polymer matrices (including, for example, polyacrylamide matrices).

[0014] In another aspect, the present disclosure provides a system for profiling RNA translation in a cell. In some embodiments, such a system includes: a) Cell; b) one or more pairs of probes comprising a first probe and a second probe, 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; one or more pairs of probes comprising a first probe and a second probe; c) a microscope; and d) Computer. In some embodiments, such a system includes: a) Cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome, 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; one or more sets of probes including a first probe, a second probe, and a third probe; c) a microscope; and d) Computer.

[0015] Any of the probes (ie, probe pairs or probe sets) described herein may be used in the systems contemplated by this disclosure.

[0016] 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. [Brief description of the drawings]

[0017] 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.

[0018] [Figure 1] Figure 1 is a schematic diagram of a method for characterizing / profiling RNA translation in situ. A DNA-conjugated secondary antibody is used to recognize padlock probes that are in close proximity and belong to the same RNA, allowing their amplification. Each padlock probe contains a unique barcode that encodes the identity of the RNA site. The barcode information is amplified by rolling circle amplification and decoded by SEDAL in situ sequencing.

[0019] [Figure 2A-2B] Figures 2A-D are images of single cell profiling of RNA translation in situ. Figure 2A shows detection of actively transcribed beta-actin (ACTB) RNA compared to negative controls in Figure 2B (no primary antibody), Figure 2C (secondary antibody without conjugated DNA), and Figure 2D (no secondary antibody). [Fig. 2C-2D] Figures 2A-D are images of single cell profiling of RNA translation in situ. Figure 2A shows detection of actively transcribed beta-actin (ACTB) RNA compared to negative controls in Figure 2B (no primary antibody), Figure 2C (secondary antibody without conjugated DNA), and Figure 2D (no secondary antibody).

[0020] [Diagram 3] Figure 3 is a schematic diagram of the in situ ribosome profiling method using ribosome antibodies and three-part DNA probes. A DNA-conjugated secondary antibody is used to recognize and allow ligation of padlock probes that are in close proximity and belong to the same RNA. Each padlock probe contains a unique barcode that codes for the identity of the RNA site. The barcode sequence is amplified by rolling circle amplification to produce an optimally measurable signal. The method is compatible with immunofluorescence staining to label intracellular organelles, such as the endoplasmic reticulum and mitochondria.

[0021] [Figure 4A-4B] Figure 4A-4C are images of in situ ribosome profiling based on ribosomal protein antibodies using anti-40S ribosomal protein S3 (RPS3) antibody. Figure 4A shows the in situ ribosome profiling signal for ACTB. Figure 4B shows the ribosome profiling signal of a negative control using IgG instead of anti-RPS3 antibody. Figure 4C shows the in situ ribosome profiling signal for non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). [Figure 4C] Figure 4A-4C are images of in situ ribosome profiling based on ribosomal protein antibodies using anti-40S ribosomal protein S3 (RPS3) antibody. Figure 4A shows the in situ ribosome profiling signal for ACTB. Figure 4B shows the ribosome profiling signal of a negative control using IgG instead of anti-RPS3 antibody. Figure 4C shows the in situ ribosome profiling signal for non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).

[0022] [Figure 5A-5B]Figure 5A-Figure 5C are images of in situ ribosome profiling based on ribosomal protein antibodies using anti-60S ribosomal protein L4 (RPL4) antibody. Figure 5A shows the in situ ribosome profiling signal for ACTB. Figure 5B shows the ribosome profiling signal of the negative control using IgG instead of anti-RPL4 antibody. [Figure 5C] Figures 5A-5C are images of ribosomal protein antibody-based in situ ribosome profiling using anti-60S ribosomal protein L4 (RPL4) antibody. Figure 5C shows the in situ ribosome profiling signal for the non-coding RNA MALAT1.

[0023] [Figure 6] Figure 6 is a schematic diagram of the in situ ribosome profiling method using a three-part DNA probe hybridizing to rRNA and mRNA. The rRNA hybridization probe is used to recognize and allow ligation of padlock probes that are in close proximity and belong to the same RNA. Each padlock probe contains a unique barcode that codes for the identity of the RNA site. The barcode information is amplified by rolling circle amplification and decoded by SEDAL in situ sequencing. The method is compatible with immunofluorescence staining to label intracellular organelles (e.g., endoplasmic reticulum and mitochondria).

[0024] [Figure 7A-7B] Figure 7A-7C are images of in situ ribosome profiling based on rRNA hybridized probes. Figure 7A shows the in situ ribosome profiling signal for ACTB. Figure 7B shows the ribosome profiling signal of a negative control using a probe that does not contain a moiety that hybridizes to rRNA in the ribosome. [Figure 7C]Figures 7A-7C are images of in situ ribosome profiling based on rRNA-hybridized probes. Figure 7C shows the in situ ribosome profiling signal for the non-coding RNA MALAT1.

[0025] [Figure 8] FIG. 8 shows detailed designs of exemplary probes useful in the RNA profiling methods described herein.

[0026] [Figure 9A] Figure 9A-9J show RIBOmap for in situ profiling of mRNA translation at subcellular resolution. Figure 9A provides a schematic of RIBOmap. After the sample (cell line or tissue sample) is prepared, a barcoded padlock probe and primer pair is hybridized to the targeted intracellular RNA, and the splint probe is hybridized to the ribosome via 18S rRNA. The splint probe is used as a template for proximity ligation to circularize the padlock probe. The splint probe is designed with a 3' inverted dT modification to block its extension during rolling circle amplification RCA. The intact padlock probe can then be amplified to create amine-modified DNA amplicons in situ. These DNA amplicons are then copolymerized into hydrogels via tissue-hydrogel chemistry for in situ mapping. The gene-specific identifier sequences (labeled "barcodes") of the cDNA amplicons can then be read out by circular in situ sequencing with error reduction by dynamic annealing and ligation (SEDAL). [Figure 9B-9C]Figures 9A-9J show RIBOmap for in situ profiling of mRNA translation at subcellular resolution. Figure 9B shows an example of the tri-probe strategy. (Left) Fluorescence image of a tri-probe sample shows RIBOmap signal of ACTB mRNA in HeLa cells. (Center) Fluorescence image of a negative control sample without primers or splint probe shows minimal DNA amplicon signal. (Right) Fluorescence image of a control sample using splint probes without rRNA hybridization segment shows no DNA amplicon signal. Scale bar, 10 μm. Figure 9C shows quantification of DNA amplicon signal intensity normalized to DAPI signal in Figure 9B. Error bars, standard deviation. n=3 images per condition. Student's t-test, **P<0.01. [Fig. 9D-9E] Figure 9A-9J show RIBOmap for in situ profiling of mRNA translation at subcellular resolution. Figure 9D provides a schematic representation of the validation of RIBOmap signals by targeting mRNA (ACTB) and non-coding RNAs (MALAT1 and vtRNA1-1). Figure 9E provides fluorescent images showing the detection of RNA by STARmap and the detection of translation of RNA by RIBOmap. Scale bar, 10 μm. [Fig. 9F-9G] Figures 9A-9J show RIBOmap for in situ profiling of mRNA translation at subcellular resolution. Figure 9F shows quantification of DNA amplicon signal intensity normalized to the DAPI signal shown in Figure 9E. Error bars, standard deviation. n=3 images per condition. Student's t-test, ****P<0.0001. Figure 9G provides a schematic of translational regulation by harringtonine. [Fig. 9H-9I]Figure 9A-9J show RIBOmap for in situ profiling of mRNA translation at subcellular resolution. Figure 9H shows three pairs of SNAIL probes targeting different sites of ACTB mRNA. Probe pair 1 is near the start codon, while probe pair 2 and probe pair 3 are 115 nt and 405 nt downstream of the start codon in the CDS region, respectively. Figure 9I provides fluorescent images showing the RIBOmap signals of three sets of probes targeting different regions of ACTB mRNA in HeLa cells before and after harringtonine treatment. Scale bar, 10 μm. [Figure 9J] Figures 9A-J show RIBOmap for in situ profiling of mRNA translation at subcellular resolution. Figure 9J shows quantification of RIBOmap signal intensity normalized to the DAPI signal shown in Figure 9I. Error bars, standard deviation. n=3 images per condition. Student's t-test, **P<0.01.

[0027] [Figure 10A] Figure 10A-10K show that RIBOmap simultaneously measures the intracellular translation of 981 genes in human HeLa cells. Figure 10A provides a schematic of RIBOmap detection in HeLa / FUCCI cells to measure localized mRNA translation. RIBOmap reads out ribosome-bound mRNA signals, and STARmap detects targeted RNA. Organelle staining was combined in the procedure to visualize the nucleus, ER, and cell morphology. [Figure 10B-10C]Figure 10A-10K show that RIBOmap simultaneously measures intracellular translation of 981 genes in human HeLa cells. Figure 10B provides representative images showing simultaneous mapping of FUCCI fluorescent signals, cDNA amplicons, and organelle staining signals in HeLa cells. Left, FUCCI fluorescent imaging results of cells for RIBOmap (top) and STARmap (bottom). Fluorescent signals indicate cell cycle stages of HeLa cells. Center, representative images showing maximum intensity projections (MAX) of the first sequencing cycle for 981 genes with zoomed-in views of representative cells, and single frame views of representative cells over 6 sequencing cycles. Right: zoomed-in views of organelle staining signals of representative cells. Alexa Fluor 594 conjugate staining of concanavalin A (ConA) of the ER, Flamingo fluorescent staining of cell morphology, and DAPI staining of nuclei are indicated by the legends provided. FIG. 10C shows diffusion map embeddings of cell cycle stage clusters determined using single-cell expression profiles of 41 cell cycle marker genes for STARmap (top left) and RIBOmap (top right) measurements, along with the corresponding single-cell protein fluorescence profiles of monomeric Kusabira-Orange 2 (mKO2)-hCDT1 and monomeric Azami-Green (mAG)-hGEN FUCCI cell cycle markers (STARmap, bottom left; RIBOmap, bottom right). [Figure 10D] Figure 10A-10K show that RIBOmap simultaneously measures the intracellular translation of 981 genes in human HeLa cells. Figure 10D shows a single-cell translatome covariation matrix showing the pairwise Pearson correlation coefficients of cell-to-cell variation across the 981 measured genes, together with their average expression levels. Five modules of strongly correlated genes are indicated by gray boxes in the matrix, two of which (modules 3 and 5) are expanded on the right. Genes involved in translation complexes and identified as cell cycle markers are annotated in the expanded matrix. [Figure 10E]Figure 10A-10K show that RIBOmap simultaneously measures the intracellular translation of 981 genes in human HeLa cells. Figure 10E shows a matrix of pairwise colocalization p-values ​​describing the degree to which reads of two genes tend to colocalize within a 3um radius sphere of the same cell in the RIBOmap results (left) and the STARmap results (right), along with the expression levels of these genes. The STARmap matrix uses the same order of genes as the RIBOmap matrix. Modules of five strongly correlated genes are indicated by grey boxes in the matrix. [Figure 10F] Figure 10A-K show that RIBOmap simultaneously measures the intracellular translation of 981 genes in human HeLa cells. Figure 10F shows a bar plot visualizing the most significantly enriched GO terms (up to 3) in each of the five gene modules. [Figure 10G] Figures 10A-K show that RIBOmap simultaneously measures the intracellular translation of 981 genes in human HeLa cells. Figure 10G shows an expanded matrix of gene module 3 and gene module 4. [Fig. 10H-10J] Figure 10A-10K show that RIBOmap simultaneously measures intracellular translation of 981 genes in human HeLa cells. Figure 10H shows the spatial distribution of RIBOmap signals of module 3 and module 4 genes overlaid on ER images. Images are gene dots and MAX of ER signals. Figure 10I shows quantification of ER localization percentages of module 3 genes, module 4 genes, and all detected genes. Wilcoxon signed rank test, ****P<0.0001. Figure 10J provides a diagram showing the calculation of the spatial parameter distance ratio (DR). [Figure 10K]Figures 10A-K show that RIBOmap simultaneously measures intracellular translation of 981 genes in human HeLa cells. Figure 10K provides a violin plot of ribosome-bound RNA DR and RNA DR values ​​for m6A and non-m6A genes. Wilcoxon signed rank test, ****P<0.0001.

[0028] [Figure 11A] Figure 11A-N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11A provides a diagram of targeted mouse coronal hemibrain regions (marked with boxes) for RIBOmap. [Figure 11B] Figure 11A-11N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11B provides representative images showing measurement of localized translation of 5,413 genes by RIBOmap in mouse coronal hemibrain slices. (Left) Maximum intensity projection of the first sequencing round of 5,413 genes, showing all five channels simultaneously. Box, zoomed-in area. (Center) Zoomed-in view of three cells showing the MAX view of the first sequencing round. (Right) Zoomed-in view of three cells showing spatial arrangement of amplicons in a single z-frame across nine sequencing rounds. [Figure 11C] Figures 11A-N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11C provides RIBOmap images of three cell type marker genes compared to Allen Brain ISH images showing the expression patterns of the corresponding genes. [Figure 11D]Figure 11A-11N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11D shows visualization of Uniform Manifold Approximation and Projection (UMAP) plots of translation profiles of 62,753 cells recovered from mouse coronal hemibrain. Using Leiden clustering, 57 cell types were identified. [Figure 11E] Figures 11A-11N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11E provides a representative spatial cell type atlas. (Left) Schematic highlighting different brain regions in an imaged coronal mouse brain slice. (Center) Representative spatial cell type atlas in an imaged coronal hemibrain region using the same code as in Figure 11D. Scale bar, 500 μm. (Right) Zoomed-in sections of the hippocampus and CA1 region showing different cell types and gaps. Scale bar, 200 μm. [Fig. 11F-11G] Figures 11A-N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11F provides a scheme of a hippocampal slice showing the cell bodies and processes of hippocampal neurons. Figure 11G shows a section of the CA1 region showing the cell body leads and neuronal and glial process leads. Scale bar, 50 μm. [Fig. 11H-11I] Figure 11A-11N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11H shows the projection read percentages of individual genes in the RIBOmap measurement of 5,413 genes, with genes ranked based on their projection read percentages. Nine example genes are labeled in the inset. Figure 11I shows the top 10 significantly enriched GO terms for projection-enriched translation genes. [Fig. 11J-11K]Figure 11A-11N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figure 11J shows the top 10 significantly enriched GO terms for soma-enriched translation genes. Figure 11K-11L show spatial translation maps of example process-enriched translation genes (Figure 11K) and soma-enriched translation genes (Figure 11L) in the hippocampus region, showing soma and process leads. [Figure 11L] Figures 11A-11N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figures 11K-11L show spatial translation maps of example process-enriched (Figure 11K) and soma-enriched (Figure 11L) translated genes in the hippocampal region, showing soma and process leads. [Fig. 11M-11N] Figures 11A-N show spatially resolved single-cell translatome profiling of 5,413 genes in mouse brain. Figures 11M-N show spatial translation maps of example glial marker genes of translation genes enriched in processes (M) and in somas (N) in the hippocampal region, showing soma and process leads.

[0029] [Figures 12A-12C]Figure 12A-Figure 12F show an example of an antibody-based RIBOmap strategy. Figure 12A provides a synthetic scheme for the preparation of splint probe-conjugated protein A / G. Figure 12B shows the RIBOmap signal based on anti-ribosomal protein S3 (RPS3) antibody of ACTB mRNA in HeLa cells. The RPS3 antibody binds to the RPS3 protein of the small unit of the ribosome and takes the protein A / G-conjugated splint probe to the translating mRNA. The padlock probe targeted to the translating mRNA can then be ligated and amplified to generate an amine-modified DNA amplicon in situ. Figure 12C shows the RIBOmap signal based on anti-ribosomal protein L4 (RPL4) antibody of ACTB mRNA in HeLa cells. [Fig. 12D-12E] Figures 12A-F show an example of an antibody-based RIBOmap strategy. Figure 12D shows an IgG control antibody-based RIBOmap signal of ACTB mRNA in HeLa cells. Figure 12E shows quantification of antibody-assisted RIBOmap signal intensity normalized to DAPI in Figures 12B-D. Error bars, standard deviation. n=3 images per condition. Student's t-test, **P<0.01, ***P<0.001. [Figure 12F] Figures 12A-F show examples of antibody-based RIBOmap strategies. Figure 12F shows a quantitative comparison of the signal-to-noise ratios of rRNA probe-based and antibody-based RIBOmap strategies. Error bars, standard deviation. n=3 images per condition. Student's t-test, **P<0.01.

[0030] [Figure 13A] Figures 13A-13B show transcriptomic and translatomic detailed analysis of cell cycle-related variations: Figure 13A shows the expression of cell cycle marker genes at cell cycle stage cluster embeddings. [Figure 13B]Figures 13A-13B show transcriptomic and translatomic detailed analysis of cell cycle-associated variations. Figure 13B shows the translation of cell cycle marker genes at cell cycle stage cluster embeddings.

[0031] [Figure 14A-14B] Figure 14A-Figure 14F show the covariation analysis of single-cell translatome. Figure 14A shows the top three significantly enriched GO terms in each of the five modules identified in the single-cell translatome covariation matrix. Figure 14B-Figure 14D show the expanded module 1 (Figure 14B), module 2 (Figure 14C), and module 4 (Figure 14D) identified in the single-cell translatome covariation matrix. [Fig. 14C-14D] Figures 14A-F show covariation analysis of the single-cell translatome. Figures 14B-D show the expanded module 1 (Figure 14B), module 2 (Figure 14C), and module 4 (Figure 14D) identified in the single-cell translatome covariation matrix. [Fig. 14E-14F] Figure 14A-Figure 14F show covariation analysis of single cell translatome. Figure 14E provides a zoom-in view showing the correlation between gene module 3 (G2M marker genes) and gene module 5 (G1S marker genes). Figure 14F provides a zoom-in view showing the correlation between cell cycle marker gene modules and translation machinery gene modules.

[0032] [Figure 15A] Figures 15A-F show protein-protein interaction analysis and spatial distribution of co-localized gene modules. Figure 15A shows the String protein network analysis of genes in module 3 (left) and module 4 (right). [Figure 15B]Figure 15A-F show protein-protein interaction analysis and spatial distribution of colocalized gene modules. Figure 15B shows expanded module 1 (left), module 2 (top right), and module 5 (bottom right) in the subcellular colocalization matrix of ribosome-bound RNA. [Figure 15C] Figures 15A-F show protein-protein interaction analysis and spatial distribution of colocalized gene modules. Figure 15C shows String protein network analysis of genes in module 1 (top), module 2 (middle), and module 5 (bottom). [Fig. 15D-15E] Figure 15A-F show protein-protein interaction analysis and spatial distribution of colocalized gene modules. Figure 15D shows spatial distribution of RIBOmap signals of module 1, module 2, and module 5 genes overlaid on ER images. Figure 15E shows quantification of ER localization percentage of module 1 genes, module 2 genes, module 5 genes, and all detected genes. Wilcoxon signed rank test, ****P<0.0001. [Figure 15F] Figures 15A-F show protein-protein interaction analysis and spatial distribution of colocalized gene modules. Figure 15F shows colocalization pairwise correlation p-values ​​of the hierarchical clustering matrix of the covariation analysis.

[0033] [Figure 16A] Figures 16A-C show cross-validation of RIBOmap measurements using in situ hybridization and immunofluorescence: Figure 16A provides RIBOmap images of six example cell marker genes compared with Allen Brain ISH images showing the expression patterns of the corresponding genes. [Figure 16B]Figures 16A-C show cross-validation of RIBOmap measurements using in situ hybridization and immunofluorescence. Figures 16B-C show translational profiles derived from RIBOmap, in situ hybridization (ISH) data from the Allen Brain Atlas, and antibody-based immunofluorescence (IF) profiling from the HPA Brain Atlas for Sst (Figure 16B) and Nefl (Figure 16C). [Figure 16C] Figures 16A-C show cross-validation of RIBOmap measurements using in situ hybridization and immunofluorescence. Figures 16B-C show translational profiles derived from RIBOmap, in situ hybridization (ISH) data from the Allen Brain Atlas, and antibody-based immunofluorescence (IF) profiling from the HPA Brain Atlas for Sst (Figure 16B) and Nefl (Figure 16C).

[0034] [Figure 17A] Figure 17A-B show the quality control and cell type marker expression levels of RIBOmap mouse brain data. Figure 17A provides histograms showing the number of transcripts and genes in each cell after logarithmic transformation of the RIBOmap mouse brain dataset. The vertical lines represent the filtering threshold estimated by median absolute deviation (MAD). [Figure 17B] Figures 17A-B show quality control and cell type marker expression levels for RIBOmap mouse brain data. Figure 17B provides a UMAP showing the expression levels of canonical markers for the major cell types in mouse brain in the RIBOmap dataset.

[0035] [Figure 18A] Figures 18A-D show cell type clustering based on single cell translation profiling. Figure 18A shows a hierarchical classification of cell types, showing levels 1-3 of cell clustering. [Fig. 18B-18C]Figure 18A-D show cell type clustering based on single cell translation profiling. Figure 18B provides a visualization of the uniform manifold approximation and projection (UMAP) plot of level 1 clustering to classify cells into neurons and glial cells. Figure 18C provides a visualization of the UMAP plot of level 2 clustering to classify cells into excitatory neurons, inhibitory neurons, astrocytes, oligodendrocytes, oligodendrocyte progenitors, microglia, and vascular cells. [Figure 18D] Figure 18A-D show cell type clustering based on single cell translation profiling. Figure 18D provides gene expression heatmaps of representative markers aligned with cell types identified in level 2 clustering. Expression of each gene is z-scored across all genes in each cell.

[0036] [Figure 19A] Figure 19A-C provide gene expression and spatial maps of excitatory neurons. Figure 19A provides a gene expression heat map of representative markers aligned with excitatory neuron subtypes. Expression of each gene is z-scored across all genes in each cell. [Figure 19B] Figures 19A-C provide gene expression and spatial maps of excitatory neurons, and Figure 19B shows a visualization of UMAP plots of 21 excitatory neuron subtypes. [Figure 19C] Figures 19A-C provide gene expression and spatial maps of excitatory neurons. Figure 19C provides spatial cell maps of excitatory neuron subtypes in imaged coronal hemibrain regions with a zoomed-in view showing spatial cell maps in cortical and hippocampal regions.

[0037] [Figure 20A]Figure 20A-C provide gene expression and spatial maps of inhibitory neurons. Figure 20A provides a gene expression heat map of representative markers aligned with inhibitory neuron subtypes. The expression of each gene is z-scored across all genes in each cell. [Figure 20B] Figures 20A-C provide gene expression and spatial maps of inhibitory neurons. Figure 20B shows a visualization of the UMAP plots of 18 inhibitory neuron subtypes. [Figure 20C] Figures 20A-C provide gene expression and spatial maps of inhibitory neurons. Figure 20C shows spatial cell maps of inhibitory neuron subtypes in imaged coronal hemibrain regions with a zoomed-in view showing spatial cell maps in cortical and striatal regions.

[0038] [Figure 21A] Figure 21A-C provide gene expression and spatial maps of glial cells. Figure 21A provides a gene expression heat map of representative markers aligned with glial cell subtypes. Expression of each gene is z-scored across all genes in each cell. [Figure 21B] Figures 21A-C provide gene expression and spatial maps of glial cells, and Figure 21B shows a visualization of UMAP plots of 18 glial cell subtypes. [Figure 21C] Figures 21A-C provide gene expression and spatial maps of glial cells. Figure 21C provides spatial cell maps of glial cell subtypes in imaged coronal hemibrain regions with a zoomed-in view showing spatial cell maps in the cortical and hippocampal regions.

[0039] [Figure 22A]Figures 22A-D provide spatial maps of examples of translation genes enriched in processes. Figures 22A-B show translation spatial maps of additional examples of translation genes enriched in processes (Figure 22A) and in somas (Figure 22B) in the hippocampal region. Soma and process leads are shown. [Figure 22B] Figures 22A-D provide spatial maps of examples of translation genes enriched in processes. Figures 22A-B show translation spatial maps of additional examples of translation genes enriched in processes (Figure 22A) and in somas (Figure 22B) in the hippocampal region. Soma and process leads are shown. [Figure 22C] Figures 22A-D provide spatial maps of examples of translation genes enriched in processes. Figures 22C-D provide translation spatial maps of examples of additional glial cell marker genes enriched in processes (Figure 22C) and in somas (Figure 22D) in the hippocampal region. Soma and process leads are shown. [Figure 22C] Figures 22A-D provide spatial maps of examples of translation genes enriched in processes. Figures 22C-D provide translation spatial maps of examples of additional glial cell marker genes enriched in processes (Figure 22C) and in somas (Figure 22D) in the hippocampal region. Soma and process leads are shown.

[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," or "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 generate 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 each. 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 and nanobodies, as well as variants of antibodies and variants of antibody fragments and nanobodies. In some embodiments, the antibody is administered as a treatment for a disease or disorder, e.g., one associated with an altered profile of RNA being translated in cells removed from the subject. In some embodiments, the antibody is conjugated to an oligonucleotide probe as described herein. In certain embodiments, the antibody binds to a ribosome, e.g., the antibody is an anti-40S ribosomal protein S3 (RPS3) antibody or an anti-60S ribosomal protein L4 (RPL4) antibody.

[0044] The term "cancer" (including cancers that may be studied, characterized, diagnosed, and / or treated using the methods described herein) refers to a class of diseases characterized by the development of abnormal cells that have the ability to grow uncontrollably and invade and destroy normal body tissues. See, for example, Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Cancer is an example of a proliferative disease. Exemplary cancers include acoustic neuroma; adenocarcinoma; adrenal cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma); appendix cancer; benign monoclonal gammopathy; bile duct cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, breast cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchial cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma; connective tissue cancer; epithelial carcinoma; ependymoma; endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); well-known hypereosinophilia; gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, hematopoietic cancers (e.g., leukemias such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML) and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL);Hodgkin's lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin's lymphoma (NHL) (e.g., B-cell NHL, such as diffuse large cell lymphoma (DLCL)) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, Lymphomas, such as lymphoplasmacytic lymphoma (i.e. Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma; and T-cell NHLs, such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphomas (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sézary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy leukemia / lymphoma; mixed leukemia / lymphoma of one or more of the above; and multiple myeloma (MM); heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharyngeal carcinoma; inflammatory myofibroblastic tumor; immune cell amyloidosis; kidney cancer (e.g., nephroblastoma, also known as Willems tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, etc.) cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); cancer of muscle; myelodysplastic syndromes (MDS); mesothelioma; myeloproliferative disorders (MPDs) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (AMM), also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or 2, schwannomatosis);Neuroendocrine cancers (e.g., gastrointestinal pancreatic neuroendocrine tumors (GEP-NETs), carcinoid tumors); osteosarcomas (e.g., bone cancer); ovarian cancers (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancers (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), pancreatic islet tumors); penile cancers (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumors (PNT); plasmacytoma; paraneoplastic syndromes; intraepithelial neoplasia; prostate cancers (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancers (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small intestine bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovium; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva);

[0045] "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 methods of the present disclosure may 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 a subject, and the like. Virtually any cell type and size can be accommodated in 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 may 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 may also be provided in situ in a tissue sample.

[0046] Cell types contemplated for use in the methods of the present disclosure 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 associated with specific organs (e.g., thymus, endocrine glands, pancreas, brain, neurons, glia, astrocytes, dendrocytes, and genetically modified cells thereof). The cells may 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 methods of the present disclosure. In some embodiments, the cells are microglia, astrocytes, oligodendrocytes, excitatory neurons, or inhibitory neurons. In some embodiments, cells of multiple cell types are present within the same sample.

[0047] 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. For example, two oligonucleotide sequences can be only partially complementary to each other (e.g., in the probes described herein, only a portion of the probe is complementary to another probe or RNA of interest). Two oligonucleotide sequences can be, for example, 70% or more complementary to each other, 75% or more complementary to each other, 80% or more complementary to each other, 85% or more complementary to each other, 90% or more complementary to each other, 95% or more complementary to each other, 96% or more complementary to each other, 97% or more complementary to each other, 98% or more complementary to each other, 99% or more complementary to each other, or 100% complementary to each other.

[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, and the like.

[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. The proteins of the invention preferably 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 of these. The protein may also be a therapeutic protein administered as a treatment for a disease or disorder (e.g., one that is associated with a change in the profile of RNA being translated in cells removed from a subject). In certain embodiments, the protein is an antibody.

[0050] A "transcript" or "RNA transcript" is a product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. If the RNA transcript is a complementary copy of a DNA sequence, it is referred to as a primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of a primary transcript, and is referred to as a mature RNA. "Messenger RNA (mRNA)" refers to an RNA that is free of introns and can be translated into a polypeptide by a cell. "cRNA" refers to a complementary RNA transcribed from a recombinant cDNA template. "cDNA" refers to a DNA that is complementary to and derived from an mRNA template.

[0051] 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 (such as, for example, cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); 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 (such as, for example, 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 removed from a subject, such as a biopsy of any of the tissues described herein. In certain embodiments, the biological sample is a tumor biopsy (e.g., from a subject diagnosed with, suspected of, or believed to have cancer). In some embodiments, a tumor biopsy captures primary tumor cells and tissue for direct study. In some embodiments, a liquid biopsy can be used with tumor cells captured and / or sorted via conventional methods (e.g., FACS, affinity capture, etc.) from blood or other bodily fluids (CNS fluid, lymph, etc.). In some embodiments, the sample is brain tissue. In some embodiments, the tissue is cardiac tissue. In some embodiments, the tissue is muscle tissue.

[0052] The term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Typically, a small molecule is an organic compound (e.g., that contains carbon). A small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, and heterocycles). In certain embodiments, the molecular weight of a small molecule is about 1,000 g / mol or less, about 900 g / mol or less, about 800 g / mol or less, about 700 g / mol or less, about 600 g / mol or less, about 500 g / mol or less, about 400 g / mol or less, about 300 g / mol or less, about 200 g / mol or less, or about 100 g / mol or less. In certain embodiments, the molecular weight of the small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges are also possible (e.g., at least about 200 g / mol and up to about 500 g / mol). In certain embodiments, the small molecule is a therapeutically active agent, such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration). The small molecule may also be complexed with one or more metal atoms and / or metal ions. Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, and more preferably humans. In certain embodiments, the small molecule is a drug. Preferably, although not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental or regulatory agency.

[0053] A "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 who exhibit any risk factors or symptoms 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] 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.

[0055] 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, such as the skin, airways, soft organs, the surface of the reproductive tract, and the inside of the digestive tract. Epithelial tissues perform protective functions and are also involved in secretion, excretion, and absorption. Examples of epithelial tissues 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 that consists of cells separated by non-living substances, such as the extracellular matrix. Connective tissues provide shape to organs and hold them in place. Connective tissues include fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Examples of connective tissues 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. In certain embodiments, the tissue is placental tissue. In some embodiments, the tissue is cardiac tissue.

[0056] The term "translatome" refers to all open reading frames (i.e., the DNA sequences between the start and stop codons) in a particular cell or organism. In some embodiments, the methods, probes, and kits provided herein are useful for studying and / or profiling the translatome.

[0057] The terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset or inhibiting the progression of a disease described herein, e.g., cancer. In some embodiments, treatment may be administered after one or more signs or symptoms of a disease have developed or been observed (e.g., prophylactically (as may be further described herein) or following suspicion or risk of a disease). In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms in the subject or a family member of the subject). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. In some embodiments, treatment may be administered after observing changes in the profile of RNA expressed in a cell or tissue compared to a healthy cell or tissue using the methods disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] DETAILED DESCRIPTION OF CERTAIN SPECIFIC EMBODIMENTS The aspects described herein are not limited to specific embodiments, systems, compositions, methods, or configurations, which, as such, can, of course, vary, and 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 RNA translated in cells.The present disclosure also provides a method for diagnosing a disease or disorder of a subject based on the profile of RNA translated in cells, including cells in intact tissue.The present disclosure also provides a method for screening or testing a candidate agent capable of modulating the translation of one or more RNAs.The present disclosure also provides a method for treating a disease or disorder in a subject in need of such treatment.The present disclosure also describes a kit that includes any of the oligonucleotide probes described herein, as well as pairs and sets of oligonucleotide probes that can be useful for carrying out the methods described herein.

[0060] Methods for profiling translated RNA in cells In one aspect, the present disclosure provides a method for profiling RNA that is translated in cells. In the method disclosed herein, cells can be contacted with one or more pairs or sets of probes, which can be further described herein and used to amplify RNA that is actively translated by ribosomes, to generate one or more concatenated amplicons. One or more concatenated amplicons can then be embedded in a polymer matrix and sequenced to determine the identity of transcripts and their positions within the polymer matrix (for example, by SEDAL sequencing, as further described herein). Using the positions of transcripts of interest, RNA that is translated in cells can be profiled. The method provided herein has several advantages over previously disclosed methods and systems, including but not limited to the ability to profile RNA translation in samples without disturbing the spatial information of intracellular structure, cell morphology, and / or tissue composition in samples.

[0061] In some embodiments, the present disclosure provides a method for producing a method for the treatment of a disease comprising the steps of: a) contacting a cell with one or more pairs of probes, each pair of probes comprising a first probe and a second probe; 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; contacting the cells with one or more pairs of probes; 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest in the cell. The present invention provides a method for profiling RNA being translated in a cell, comprising:

[0062] The method disclosed herein contemplates the use of a pair of probes, including a first probe and a second probe. The second probe (also referred to herein as a "primer probe") includes a portion that recognizes a ribosome (i.e., a ribosome that binds to and actively translates an RNA of interest). The portion of the probe that recognizes a ribosome can be a protein, a peptide, a nucleic acid, or a small molecule. In some embodiments, the portion of the second probe that recognizes a ribosome is an agent that binds to an antibody, or an antibody variant or fragment. In certain embodiments, the portion of the second probe that recognizes a ribosome includes an antibody (e.g., a secondary antibody), or an antibody variant or fragment. If the portion of the second probe that recognizes a ribosome is a secondary antibody, the method may optionally further include contacting the cell with a primary antibody that recognizes a ribosome and is recognized by the secondary antibody of the second probe. The primary antibody may recognize any portion of the ribosome, for example, any protein of the ribosome, nucleic acid (e.g., rRNA), or a combination thereof. In some embodiments, the primary antibody is an anti-40S ribosomal protein S3 (RPS3) antibody (e.g., an anti-RPS3 monoclonal antibody). In some embodiments, the antibody is an anti-60S ribosomal protein L4 (RPL4) antibody (e.g., an anti-RPL4 polyclonal antibody). Instead of an antibody, the present disclosure also contemplates the use of any agent capable of recognizing the ribosome of the probe described herein. In some embodiments, the portion of the second probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the ribosomal RNA (rRNA) in the ribosome. In some embodiments, the portion of the second probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the 40S small ribosomal subunit (e.g., including 18S rRNA) or a portion of the 60S large ribosomal subunit (e.g., including 5S rRNA, 28S rRNA, and 5.8S rRNA).In some embodiments, the portion of the second probe that recognizes ribosome comprises the oligonucleotide that is complementary to the portion of 18S rRNA.In certain embodiments, the oligonucleotide that is complementary to the portion of rRNA is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides long.In certain embodiments, the oligonucleotide that is complementary to the portion of rRNA is about 25 nucleotides long.

[0063] In addition to the portion that recognizes ribosomes, the second probe also comprises a portion that is complementary to the portion of the first probe.In some embodiments, the portion of the second probe that is complementary to the portion of the first probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long.The present disclosure contemplates any arrangement of the portion of the second probe.In some embodiments, the second probe used in the method described herein has the structure: 5'-[ribosome-recognizing portion]-[portion complementary to the first probe]-3' or 5'-[part complementary to the first probe]-[part recognizing ribosome]-3' Includes; Here, ]-[ includes any linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the second oligonucleotide probe.

[0064] The first probe (also referred to herein as a "padlock" probe) used in the methods described herein includes an oligonucleotide barcode sequence consisting of a unique sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 3 to about 20, about 5 to about 15, about 6 to about 14, about 7 to about 13, about 8 to about 12, or about 9 to about 11 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. The barcodes of the oligonucleotide probes described herein can include gene-specific sequences that are used to identify the RNA of interest that is being translated (i.e., each barcode sequence is associated with a unique gene or transcript). The use of barcodes for probes similar to those described herein is further described, for example, in International Patent Application Publication WO 2019 / 199579, published October 17, 2019, and Wang et al., Science 2018, 361, 380, both of which are incorporated by reference in their entireties.

[0065] The first probe also comprises an oligonucleotide portion that is complementary to the second probe and an oligonucleotide portion that is complementary to the RNA of interest. In some embodiments, the portion of the first probe that is complementary to the RNA of interest is 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, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 or more nucleotides in length. Any order of arrangement of the portions of the first oligonucleotide probe is contemplated by the present disclosure. In some embodiments, the first probe has the structure: 5'-[part complementary to the second probe]-[part complementary to the RNA of interest]-[barcode sequence]-3' or 5'-[part complementary to the RNA of interest]-[part complementary to the second probe]-[barcode sequence]-3' or 5'-[barcode sequence]-[part complementary to the RNA of interest]-[part complementary to the second probe]-3' or 5'-[barcode sequence]-[part complementary to the second probe]-[part complementary to the RNA of interest]-3' or 5'-[part complementary to the RNA of interest]-[barcode sequence]-[part complementary to the second probe]-3' or 5'-[part complementary to the second probe]-[barcode sequence]-[part complementary to the RNA of interest]-3' Including, Here, ]-[ includes an optional linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0066] In some embodiments, the present disclosure provides a method for producing a method for the treatment of a disease comprising the steps of: a) contacting a cell with one or more sets of probes, each set of probes comprising a first probe, a second probe, and a third probe; i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; contacting the cells with one or more sets of probes; 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 third 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest in the cell. The present invention provides a method for profiling RNA being translated in a cell, comprising:

[0067] The method disclosed herein contemplates the use of a set of probes, comprising a first probe, a second probe and a third probe.The addition of the third probe (also referred to herein as "primer probe") can result in improved specificity and / or reduced off-target amplification compared to the embodiment of the method in which the third probe is not used.

[0068] As described herein, the second probe (also referred to herein as a "sprint probe" or "blocked probe") comprises a moiety that recognizes a ribosome (i.e., a ribosome that is bound to and actively translating the RNA of interest). In some embodiments, the second probe further comprises a polymerization blocker. The addition of the polymerization blocker prevents the second probe from being used as a primer in the amplification of step (c) and ensures that the third probe is used as a primer during the amplification. The polymerization blocker of the second probe can be any moiety that can prevent the use of the second probe as a primer in the amplification of step (c) of the methods described herein. In some embodiments, the polymerization blocker is at the 3' end of the second probe. The polymerization blocker can be, for example, any chemical moiety that prevents the polymerase from using the second 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 some chemical moiety in place of the 3' hydroxyl group that prevents the addition of additional nucleotides. 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.

[0069] In addition to the portion that recognizes the ribosome, the second probe also includes a portion that is complementary to a portion of the first probe. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the portion of the second probe that recognizes the ribosome and the portion of the second probe that is complementary to the first probe are joined by a polyA nucleotide linker. In some embodiments, the polyA nucleotide linker is 20-80, 30-70, or 40-60 nucleotides in length (e.g., about 50 nucleotides in length).

[0070] The present disclosure contemplates any arrangement of the portion of the second probe. In some embodiments, the second probe used in the methods described herein has the structure: 5'-[ribosome-recognizing portion]-[portion complementary to the first probe]-3' or 5'-[part complementary to the first probe]-[part recognizing ribosome]-3' Includes; Here, ]-[ includes any linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct link between two portions of the second oligonucleotide probe. In some embodiments, the second probe has the structure: 5'-[ribosome-recognizing portion]-[portion complementary to the first probe]-[polymerization blocker]-3' Including, Here, ]-[ includes any nucleotide linker.

[0071] The first probe (also referred to herein as a "padlock" probe) used in the methods described herein includes a first oligonucleotide barcode sequence and a second oligonucleotide barcode sequence that are composed of a unique sequence of nucleotides. In some embodiments, the first and second oligonucleotide barcode sequences of the first probe comprise the same nucleotide sequence. The presence of the second barcode sequence of the first oligonucleotide probe may improve the specificity of the detection of the RNA of interest in the methods described herein (i.e., compared to when the method is performed using a first oligonucleotide probe that does not include the second barcode sequence). The use of an additional oligonucleotide barcode sequence of the first oligonucleotide probe may also play a role in reducing non-specific amplification of the RNA of interest in the methods described herein. This is achieved because the oligonucleotide barcode sequence of the third probe used in the methods described herein is complementary to the first oligonucleotide barcode sequence of the first probe, adding an additional layer of specificity that is required before amplification can occur using the third probe as a primer. In some embodiments, the portion of the first probe that is complementary to the third 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 first probe that is complementary to the third probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0072] The first probe also includes an oligonucleotide portion complementary to the second probe and a portion complementary to the RNA of interest. In some embodiments, the portion of the first probe complementary to the second probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the first probe complementary to the second probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe complementary to the second probe is split between the 5' and 3' ends of the first probe. In some embodiments, the portion of the first 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 first probe that is complementary to the RNA of interest is 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, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 or more nucleotides in length.

[0073] In some embodiments, the first probe has the structure: 5'-[part complementary to the second probe]-[first barcode sequence]-[part complementary to a part of the third probe]-[part complementary to the RNA of interest]-[second barcode sequence]-3' Including, Here, ]-[ includes an optional linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0074] The third probe (also referred to herein as a "primer" probe) used in the methods disclosed herein includes a barcode sequence consisting of a unique sequence of nucleotides. In some embodiments, the barcode sequence of the third probe is about 3 to about 20, about 5 to about 15, about 6 to about 14, about 7 to about 13, about 8 to about 12, or about 9 to about 11 nucleotides in length. In some embodiments, the barcode sequence of the third probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In certain embodiments, the barcode sequence of the third probe is 10 nucleotides in length.

[0075] The third probe also includes a portion complementary to the RNA of interest and a portion complementary to a portion of the first probe. In some embodiments, the first and third probes are complementary to and bind to different portions of the RNA of interest. 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 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, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the third probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 or more nucleotides in length. In some embodiments, the portion of the third probe that is complementary to 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 the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0076] In some embodiments, the third probe has the structure: 5'-[part complementary to the RNA of interest]-[part complementary to the first probe]-[barcode sequence]-3' Including, Here, ]-[ includes an optional linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct link between the two portions of the third probe. All arrangements of portions of the third probe are contemplated by the present disclosure.

[0077] 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 target cell is a mammalian cell. In certain embodiments, the cell is a human cell. The present disclosure also contemplates performing the methods described herein on a large number of cells simultaneously (e.g., more than 100 cells, more than 200 cells, more than 300 cells, more than 400 cells, more than 500 cells, more than 1000 cells, more than 10,000 cells, more than 20,000 cells, more than 30,000 cells, more than 40,000 cells, or more than 50,000 cells simultaneously). In some embodiments, the method is performed on a large number of cells of the same cell type. In some embodiments, the method is performed on a large number of cells, including cells of different cell types. Using the methods disclosed herein, cell types in which translated RNA can be profiled include, but are not limited to, stem cells, progenitor cells, neural cells, astrocytes, dendritic cells, endothelial cells, microglia, oligodendrocytes, muscle cells, 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, one or more cells are present in 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 certain embodiments, the tissue is cardiac tissue, lymph node tissue, liver tissue, muscle tissue, bone tissue, eye tissue, or ear tissue. In certain embodiments, the tissue is brain tissue.

[0078] The RNA of interest whose gene expression is profiled in the methods described herein can be a transcript expressed from the genomic DNA of a cell.In some embodiments, the RNA of interest is mRNA.The methods described herein can be used to simultaneously profile one RNA that is translated in a cell at a time, or multiple RNAs of interest.In some embodiments, the RNA translation in a cell or multiple cells is simultaneously profiled 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, more than 3000 RNAs, more than 4000 RNAs, or more than 5000 RNAs.

[0079] The polymer matrix is ​​used in the methods described herein to facilitate sequencing and imaging of RNA of interest that is translated 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 sodium polyacrylate hydrogel, an acrylate polymer 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).

[0080] In some embodiments, performing rolling circle amplification to amplify the circular oligonucleotide to generate one or more concatenated amplicons further comprises providing a nucleotide modified with a reactive chemical group, for example, 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, performing rolling circle amplification to amplify the circular oligonucleotide to generate 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 an acrylic acid N-hydroxysuccinimide ester and copolymerizing the one or more concatenated amplicons and the polymer matrix.

[0081] The methods disclosed herein also include a step of sequencing the concatenated amplicons embedded in the polymer matrix. In some embodiments, the step of sequencing includes performing "sequencing by error reduction by dynamic annealing and ligation" (SEDAL sequencing). In some embodiments, SEDAL is performed two, three, four, five, or more than five times for a particular sample. 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 within the cell.

[0082] The additional oligonucleotide probes used in the methods described herein (e.g., when used in SEDAL sequencing) may 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 may 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 unique RNA of interest. By imaging the location of the additional oligonucleotide probe comprising the fluorophore, the location of the unique 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.

[0083] In some embodiments, the method of profiling RNA translation described herein can be combined with a method of profiling additional molecules in a cell. For example, the expression of non-translating RNA can be profiled together with RNA translation using a probe that does not contain a moiety that recognizes ribosomes. RNA in other subcellular locations that are not actively translated can also be profiled together with RNA translation. Protein expression profiling (e.g., using traditional proteomic methods) can also be combined with the methods described herein, as well as transcriptome, lipid, and / or small molecule profiling. In some embodiments, any of the methods provided herein further comprises overexpressing or knocking out one or more genes in a cell to determine whether one or more genes are involved in regulating the translation of the RNA of interest.

[0084] Any of the methods described herein can also be used to determine the cell type and / or cell state of one or more cells.In some embodiments, any of the methods provided herein further comprises determining the cell type of the profiled cell, or the cell type of the profiled multiplicity of cells, by comparing the RNA translation profile of one or more cells with reference data comprising the RNA translation profiles of cells of various cell types.In some embodiments, any of the methods provided herein further comprises determining the cell state of the profiled cell, or the cell state of the profiled multiplicity of cells, by comparing the RNA translation profile of one or more cells with reference data comprising the RNA translation profiles of cells of various cell states.

[0085] Methods for diagnosing a disease or disorder in a subject In another aspect, the present disclosure provides a method for diagnosing a disease or disorder in a subject. For example, the method of profiling translated RNA described herein can be performed on a cell or a number of cells taken from a subject (e.g., a subject who is thought to have a disease or disorder or is at risk of having a disease or disorder, or a subject who is healthy or thought to be healthy). The expression of various RNAs of interest in the cell can then be compared to 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 RNA translation profile of a cell (of a single RNA or of multiple RNAs of interest, including, e.g., a specific disease signature) compared to one or more non-disease cells can indicate that the subject has a disease or disorder. The RNA translation in one or more non-disease cells can be profiled along with the expression in a diseased cell as a control experiment. The RNA translation in one or more non-disease cells can also be profiled in advance, and the expression in the diseased cell can be compared to this reference data for non-disease cells.

[0086] In some embodiments, a method for diagnosing a disease or disorder in a subject comprises the steps of: a) contacting a cell removed from the subject with one or more pairs of probes, each pair of probes comprising a first probe and a second probe; 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; contacting cells removed from the subject with one or more pairs of probes; 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest to profile the RNA being translated in the cell; Including, Here, a difference in the RNA translation profile of the cell relative to one or more non-diseased cells is indicative that the subject has a disease or disorder.

[0087] In some embodiments, a method for diagnosing a disease or disorder in a subject comprises the steps of: a) contacting a cell removed from the subject with one or more sets of probes, each set of probes comprising a first probe, a second probe, and a third probe; i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; contacting cells removed from the subject with one or more sets of probes; 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 third 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest in the cell. Including, Here, a difference in the RNA translation profile of the cell relative to one or more non-diseased cells is indicative that the subject has a disease or disorder.

[0088] In some embodiments, the RNA translated in one or more non-disease cells is profiled simultaneously with cells removed from the subject as a control experiment using the methods disclosed herein. In some embodiments, the profile of the RNA translated in one or more non-disease cells that is compared to the expression in the disease cells includes reference data from when the method was previously performed on one or more non-disease cells.

[0089] The diagnosis 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, 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.In certain embodiments, the disease is cancer.

[0090] In some embodiments, the cells are present in a tissue, such as epithelial, connective, muscle, or nervous tissue. In some embodiments, the tissue is a tissue sample from a subject. In some embodiments, the subject is a non-human experimental animal, such as a mouse, rat, dog, pig, or non-human primate. In some embodiments, the subject is a farm 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, such as 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.

[0091] Methods for screening agents capable of modulating the translation of one or more RNAs - Patents.com In another aspect, the present disclosure provides a method for screening agents capable of modulating the translation of one or more RNAs of interest. For example, the methods of profiling translated RNAs described herein can be performed in cells in the presence of one or more candidate agents. The expression of various RNAs of interest that are translated in cells (e.g., normal or diseased cells) can then be compared to the expression of the same RNAs of interest in cells that have not been exposed to one or more candidate agents. Any difference in the RNA translation profile compared to the translation in cells that have not been exposed to the candidate agent(s) can indicate that the translation of one or more RNAs of interest is modulated by the candidate agent(s). In some embodiments, a particular signature (e.g., of multiple RNAs of interest that are translated) known to be associated with the treatment of a disease can be used to identify a candidate agent capable of modulating translation in a desired manner. The methods described herein can also be used to identify drugs that have certain unique side effects, for example, by looking for specific RNA translation signatures when one or more cells are treated with a candidate agent or a known drug.

[0092] In some embodiments, the disclosure provides a method for screening for an agent capable of modulating translation of one or more RNAs, the method comprising the steps of: a) contacting a cell that is being treated or has been treated with a candidate agent with one or more pairs of probes, each pair of probes comprising a first probe and a second probe; 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; contacting cells that are being treated, or have been treated, with a candidate agent with one or more pairs of probes; 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest to profile the RNA being translated in the cell; Including, Here, a difference in the profile of RNA being translated in the presence of the candidate agent compared to the absence of the candidate agent indicates that the candidate agent modulates the translation of one or more RNAs.

[0093] In some embodiments, the disclosure provides a method for screening for an agent capable of modulating translation of one or more RNAs, the method comprising the steps of: a) contacting a cell that is being treated or has been treated with a candidate agent with one or more sets of probes, each set of probes comprising a first probe, a second probe, and a third probe; i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; contacting cells that are being treated, or have been treated, with a candidate agent with one or more sets of probes; 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 third 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest bound to ribosomes in the cell; Including, Here, a difference in the profile of RNA being translated in the presence of the candidate agent compared to the absence of the candidate agent indicates that the candidate agent modulates the translation of one or more RNAs.

[0094] 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). Any candidate agent can be screened using the methods described herein. In particular, any candidate agent that may be capable of modulating the translation of one or more RNAs can be screened using the methods described herein. In some embodiments, the modulation of the translation of one or more RNAs of interest by the 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 modulated by the candidate agent 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. In certain embodiments, the disease or disorder modulated by the candidate agent is cancer.

[0095] Methods of Treating a Disease or Disorder in a Subject In another aspect, the present disclosure provides a method for treating disease or disorder in a subject.For example, the method of profiling translated RNA described herein can be carried out in cells from a sample taken from a subject (e.g., a subject who is thought to have a disease or disorder or is at risk of having a disease or disorder).The profile of one or more RNAs translated in the cell can then be compared with the translation state of the same RNA of interest in cells from a non-disease tissue sample.Then, if any difference is observed in the RNA translation profile compared to non-disease cells, a treatment for disease or disorder can be administered to the subject.The RNA translation in one or more non-disease cells can be profiled together with the RNA translation in disease cells as a control experiment.The RNA translation in one or more non-disease cells can also be profiled in advance, and the translation in disease cells can be compared to this reference data for non-disease cells.

[0096] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject, the method comprising the steps of: a) contacting a cell that is being treated or has been treated with a candidate agent with one or more pairs of probes, each pair of probes comprising a first probe and a second probe; 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; contacting cells that are being treated, or have been treated, with a candidate agent with one or more pairs of probes; 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest to profile the RNA being translated in the cell; Including, Here, a difference in the profile of RNA being translated in the presence of the candidate agent compared to the absence of the candidate agent indicates that the candidate agent modulates the translation of one or more RNAs.

[0097] In some embodiments, the disclosure provides a method for treating a disease or disorder in a subject, the method comprising the steps of: a) contacting a cell that is being treated or has been treated with a candidate agent with one or more sets of probes, each set of probes comprising a first probe, a second probe, and a third probe; i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; contacting cells that are being treated, or have been treated, with a candidate agent with one or more sets of probes; 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 third 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 embedded in the polymer matrix to determine the identity and location of each RNA of interest bound to ribosomes in the cell; Including, Here, a difference in the profile of RNA being translated in the presence of the candidate agent compared to the absence of the candidate agent indicates that the candidate agent modulates the translation of one or more RNAs.

[0098] In some embodiments, RNA translation in one or more non-diseased cells is profiled simultaneously as a control experiment using the methods disclosed herein. In some embodiments, the RNA translation data in one or more non-diseased cells that is compared to the translation in the diseased cells includes reference data from a time point when the method was previously performed on the non-diseased cells.

[0099] 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 fragment or 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).

[0100] 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, ophthalmic disease or cardiovascular disease.In certain embodiments, the disease is cancer.

[0101] 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., 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 biopsy is a biopsy of a solid tumor. In some embodiments, the tissue sample is a brain tissue sample. In certain embodiments, the tissue sample is a central nervous system tissue sample.

[0102] probe The present disclosure also provides pairs and sets of probes for use in the methods and systems 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), 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; and ii) The second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe.

[0103] 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.

[0104] The pair of probes described herein includes a first probe and a second probe. The second probe includes a portion that recognizes a ribosome (i.e., a ribosome that is bound to and actively translating an RNA of interest). In some embodiments, the portion of the second probe that recognizes the ribosome is an agent that binds to an antibody, or an antibody variant or fragment. In certain embodiments, the portion of the second probe that recognizes the ribosome includes an antibody (e.g., a secondary antibody), or an antibody variant or fragment. When the portion of the second probe that recognizes the ribosome is a secondary antibody, the secondary antibody can bind to the primary antibody that recognizes the ribosome. In some embodiments, the primary antibody is an anti-40S ribosomal protein S3 (RPS3) antibody. In some embodiments, the primary antibody is an anti-60S ribosomal protein L4 (RPL4) antibody. Instead of an antibody, the present disclosure also contemplates the use of any agent that can recognize the ribosome of the probe described herein. In some embodiments, the portion of the second probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the ribosomal RNA (rRNA) in the ribosome. In some embodiments, the portion of the second probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the 40S small ribosomal subunit (including, for example, the 18S rRNA) or a portion of the 60S large ribosomal subunit (including, for example, the 5S rRNA, the 28S rRNA, and the 5.8S rRNA). In some embodiments, the portion of the second probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the 18S rRNA. In certain embodiments, the oligonucleotide that is complementary to a portion of the rRNA is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. In certain embodiments, the oligonucleotide complementary to a portion of the rRNA is about 25 nucleotides in length.

[0105] The first probe (also referred to herein as the "padlock" probe) of the probe pairs described herein includes an oligonucleotide barcode sequence consisting of a unique sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequence of the first probe is about 3 to about 20, about 5 to about 15, about 6 to about 14, about 7 to about 13, about 8 to about 12, or about 9 to about 11 nucleotides in length. In some embodiments, the oligonucleotide barcode sequence of the first probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. The barcodes of the oligonucleotide probes described herein may include gene-specific sequences used to identify an RNA of interest (i.e., an RNA being translated by a ribosome).

[0106] The first probe also includes a portion that is complementary to the second probe and a portion that is complementary to the RNA of interest. Any order of arrangement of the portions of the first oligonucleotide probe is contemplated by the present disclosure. In some embodiments, the first probe has the structure: 5'-[part complementary to the second probe]-[part complementary to the RNA of interest]-[barcode sequence]-3' Including, 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.

[0107] In addition to the portion that recognizes the ribosome, the second probe also includes a portion that is complementary to the portion of the first probe. The present disclosure contemplates any arrangement of the portion of the second probe. In some embodiments, the second probe of the pair of probes described herein has the structure: 5'-[ribosome-recognizing portion]-[portion complementary to the first probe]-3' Including, Here, ]-[ includes any linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct link between two portions of the second oligonucleotide probe.

[0108] In another aspect, the present disclosure provides a set of oligonucleotide probes comprising a first probe (also referred to herein as a "padlock" probe), a second probe (also referred to herein as a "sprint probe" or "blocked probe"), and a third probe (also referred to herein as a "primer probe"); i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe.

[0109] In comparison to the pairs of probes described herein, the set of probes contemplated by the present disclosure includes a third probe. The addition of a third probe may result in improved specificity and / or reduced off-target amplification compared to embodiments in which a third probe is not used. As described herein, the second probe includes a moiety that recognizes a ribosome (i.e., a ribosome that is bound to and actively translating the RNA of interest). In some embodiments, the second probe further includes a polymerization blocker. The addition of the polymerization blocker prevents the second probe from being used as a primer in the amplification of step (c) and ensures that the third probe is used as a primer during amplification. The polymerization blocker of the second probe can be any moiety that can prevent the use of the second probe as a primer in the amplification of step (c) of the method described herein. In some embodiments, the polymerization blocker is at the 3' end of the second probe. The polymerization blocker can be, for example, any chemical moiety that prevents the polymerase from using the second probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue that includes a blocked 3' hydroxyl group (e.g., an oxygen protecting group at the 3' hydroxyl group). In some embodiments, the polymerization blocker includes a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker includes some chemical moiety in place of the 3' hydroxyl group that prevents the addition of additional nucleotides. In some embodiments, the polymerization blocker includes 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.

[0110] In addition to the portion that recognizes the ribosome, the second probe also includes a portion that is complementary to a portion of the first probe. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the portion of the second probe that recognizes the ribosome and the portion of the second probe that is complementary to the first probe are joined by a polyA nucleotide linker. In some embodiments, the polyA nucleotide linker is 20-80, 30-70, or 40-60 nucleotides in length (e.g., about 50 nucleotides in length).

[0111] The present disclosure contemplates any arrangement of portions of the second probe. In some embodiments, the second probe has the structure: 5'-[ribosome-recognizing portion]-[portion complementary to the first probe]-3' Including, Here, ]-[ includes any nucleotide linker. In some embodiments, ]-[ represents a direct link between the two portions of the second oligonucleotide probe. In some embodiments, the second probe has the structure: 5'-[ribosome-recognizing portion]-[portion complementary to the first probe]-[polymerization blocker]-3' Including, Here, ]-[ includes an optional linker (eg, a nucleotide linker).

[0112] The first probe of the set of probes described herein (also referred to herein as a "padlock" probe) comprises a first oligonucleotide barcode sequence and a second oligonucleotide barcode sequence, which are composed of a unique sequence of nucleotides. In some embodiments, the first and second oligonucleotide barcode sequences of the first probe comprise the same nucleotide sequence. The second barcode sequence of the first oligonucleotide probe may improve the specificity of the detection of the RNA of interest in the methods described herein (i.e., compared to when the method is performed using a first probe that does not include the second barcode sequence). The use of an additional oligonucleotide barcode sequence of the first oligonucleotide probe may also serve to reduce non-specific amplification of the RNA of interest being translated by ribosomes. This is achieved by the fact that the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe, adding an additional layer of the desired specificity before amplification can occur using the third probe as a primer. In some embodiments, the portion of the first probe that is complementary to the portion of the third 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 first probe that is complementary to the portion of the third probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0113] The first probe also includes an oligonucleotide portion complementary to the second probe and a portion complementary to the RNA of interest. In some embodiments, the portion of the first probe complementary to the portion of the second probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the first probe complementary to the portion of the second probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe complementary to the second probe is split between the 5' and 3' ends of the first probe. In some embodiments, the portion of the first 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 first probe that is 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, or about 30 nucleotides in length. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides in length. 5'-[part complementary to the second probe]-[first barcode sequence]-[part complementary to a part of the third probe]-[part complementary to the RNA of interest]-[second barcode sequence]-3' Including, Here, ]-[ includes an optional linker, such as a nucleotide linker. In some embodiments, ]-[ represents a direct linkage (i.e., a phosphodiester bond) between the two portions of the first probe.

[0114] The third probe (also referred to herein as a "primer" probe) of the set of probes disclosed herein comprises a barcode sequence consisting of a unique sequence of nucleotides. In some embodiments, the barcode sequence of the third probe is about 3 to about 20, about 5 to about 15, about 6 to about 14, about 7 to about 13, about 8 to about 12, or about 9 to about 11 nucleotides in length. In some embodiments, the barcode sequence of the third probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In certain embodiments, the barcode sequence of the third probe is 10 nucleotides in length.

[0115] The third probe also includes a portion complementary to the RNA of interest and a portion complementary to a portion of the first probe. In some embodiments, the first and third probes are complementary to and bind to different portions of the RNA of interest. 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 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, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In some embodiments, the third probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 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.

[0116] In some embodiments, the third probe has the structure: 5'-[part complementary to the RNA of interest]-[part complementary to the first probe]-[barcode sequence]-3' Including, Here, ]-[ includes any nucleotide linker. In some embodiments, ]-[ represents a direct link between the two portions of the third probe. All arrangements of the portions of the third probe are contemplated by the present disclosure.

[0117] In some embodiments, the present disclosure provides a plurality of probes, comprising multiple pairs of probes as described herein.In some embodiments, the present disclosure provides a plurality of probes, comprising multiple sets of probes as described herein.In certain embodiments, each pair or set of probes in the plurality of probes comprises an oligonucleotide portion that is complementary to a different RNA of interest.

[0118] kit The present disclosure also provides kits. In one aspect, the kits provided may include one or more probes as described herein. In some embodiments, the kits include any of the pairs of probes or any of the multiple pairs of probes described herein. In some embodiments, the kits include any of the sets of probes or any of the multiple sets of probes described herein. In some embodiments, the kits may further include a container (e.g., a vial, an ampoule, a bottle, and / or a dispenser package, or other suitable container). The kits may also include cells for performing control experiments. In some embodiments, the kits may further include other reagents for performing 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 kits are useful for profiling gene and protein expression in cells. In some embodiments, the kits are useful for diagnosing disease in a subject. In some embodiments, the kits are useful for screening agents capable of modulating RNA translation. In some embodiments, the kits are 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.

[0119] system In one aspect, the present disclosure provides a system for profiling RNA being translated in a cell. In some embodiments, such a system includes: a) Cell; b) one or more pairs of probes comprising a first probe and a second probe, 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; and ii) the second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe; one or more pairs of probes comprising a first probe and a second probe; c) a microscope; and d) Computer. In some embodiments, such a system includes: a) Cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, i) the first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to an RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprises a portion that recognizes the ribosome, 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 of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe; one or more sets of probes including a first probe, a second probe, and a third probe; c) a microscope; and d) Computer.

[0120] Any of the probes (i.e., pairs of probes or sets of probes) described herein may be used in the systems contemplated by the present disclosure. In some embodiments, the microscope is a confocal microscope. In some embodiments, the system further comprises a CPU. In some embodiments, the system further comprises computer storage and / or memory, or a storage device. In some embodiments, the system further comprises a camera. In some embodiments, the system further comprises a CCD. In some embodiments, the system further comprises software for performing microscopy and / or image analysis. In some embodiments, the system further comprises a ligase. In some embodiments, the system further comprises a polymerase. In some embodiments, the system further comprises amine-modified nucleotides. In some embodiments, the system further comprises reagents for making a polymer matrix, e.g., a polyacrylamide matrix. The cells in the systems of the present disclosure may be any of the cell types disclosed herein. In some embodiments, the system comprises a multiplicity of cells. In some embodiments, the cells are of different cell types. In certain embodiments, the cells are present in a tissue. In some embodiments, the tissue is provided by the subject or is a tissue sample from the subject. In certain embodiments, the subject is a human.

[0121] example Example 1: Single-cell profiling of RNA translational state in situ In situ ribosome profiling was used to visualize active translation of beta-actin (ACTB) mRNA (Figure 2A-D). Oligonucleotide-conjugated ribosome antibodies converted ribosome binding to amplifiable primers that could be easily amplified and detected by fluorescent methods. Pools of padlock probes were designed to convert segments of targeted sequences into unique barcodes. Combinatorial and sequential SEDAL sequencing rounds were then performed to read out the barcodes and decipher the identity of the mRNA and the number of ribosomes on that mRNA.

[0122] To further improve the detection specificity of in situ ribosome profiling, a three-part probe strategy was developed and utilized (Figure 3). Anti-ribosome antibody-conjugated DNA probes are blocked at the 3' end and thus can only be used for ligation of padlock probes. The intact padlock probes can then be amplified and DNA amplicons can be created using primer probes hybridizing to adjacent sites. In this way, only ribosome-bound RNA creates cDNA amplicons, while ribosome-unbound mRNA cannot be ligated and amplified. The identity of each gene is encoded in the padlock and primer probes and read out by SEDAL in situ sequencing. To obtain subcellular localization information, organelle staining was incorporated into the in situ ribosome profiling procedure (Figure 3). Overall, using this improved strategy, highly specific single-cell translational states in situ with subcellular localization information can be obtained.

[0123] Eukaryotic ribosomes consist of a small 40S subunit and a large 60S subunit, which together contain a total of approximately 80 structurally distinct proteins. Antibodies targeting both the small subunit protein RPS3 (ribosomal protein S3) and the large subunit protein RPL4 (ribosomal protein L4) were utilized to test a three-probe in situ ribosome profiling method. Both anti-RPS3 and anti-RPL4 antibodies were shown to work with this method (Figures 4A-C and 5A-C). A strong in situ ribosome profiling signal was observed for ACTB using anti-RPS3 or anti-RPL4 antibodies (Figures 4A and 5A), while a negative control sample using immunoglobulin G (IgG) as the antibody showed a weak signal (Figures 4B and 5B). Noncoding metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) RNA was also tested, and little signal was observed for MALAT1 using either anti-RPS3 or anti-RPL4 antibodies (Figure 4C and Figure 5C), supporting the hypothesis that this approach specifically detects translating mRNA and not noncoding RNA.

[0124] The antibody-based in situ ribosome profiling method described here can accurately detect ribosome-bound mRNA; however, the procedure relies on the specificity of anti-ribosome antibodies and requires an extra step of antibody-DNA conjugation. Because ribosomes consist of ribosomal RNA (rRNA) and ribosomal proteins, a procedure was developed that utilizes rRNA-hybridized probes for padlock probe ligation to avoid the antibody binding step (Figure 6). The procedure utilizes the three-probe strategy described herein and is also compatible with the two-probe strategy described herein. Similar to the antibody-based approach, the rRNA-hybridized probes are blocked at the 3' end and cannot be used as primers for rolling circle amplification. A probe hybridizing to 18S rRNA was used to test the in situ ribosome profiling method. This procedure was found to give the best signal-to-noise contrast compared to the previously described approach (Figures 7A-C). A strong in situ ribosome profiling signal for ACTB was observed close to the plasma membrane (Figure 7A), consistent with localized translation of ACTB RNA. Negative control samples utilizing a probe without the hybridization portion of rRNA or targeting the noncoding MALAT1 gene both showed minimal signal (Figures 9B and 9C).

[0125] Example 2: Spatially resolved single-cell translatomics at subcellular resolution Precise spatial control of mRNA translation is an essential part of post-transcriptional and translational gene regulation of cell physiology. However, systematically studying localized mRNA translation at the transcriptome scale in single cells remains a major challenge. Thus, the development of RIBOmap is described herein. RIBOmap is a three-dimensional (3D) in situ profiling method to detect mRNA translation of thousands of genes simultaneously at subcellular resolution in intact cells and tissues. Using RIBOmap, 981 genes were mapped in human HeLa cells, revealing cell cycle-dependent and subcellular localized translation. Furthermore, a single-cell spatial translatome of 5,413 genes in adult mouse brain tissue was created with a spatial cellular atlas of 62,753 cells. Localized translation in neuronal and glial cells was also detected. Taken together, RIBOmap presents the first spatially resolved single-cell translatomics technology, facilitating the understanding of localized protein synthesis in the context of subcellular architecture, cell state, and tissue anatomy. Overall, RIBOmap enables single-cell in situ profiling of mRNA translation in 3D for thousands of genes simultaneously in intact cells and tissues.

[0126] Cellular proteins are the end products of gene expression to carry out cellular functions and shape cell types and states, and measuring genome-wide translation patterns at single-cell and spatial resolution is a paramount goal that can transform our understanding of translational regulation in heterogeneous cell types and states. While large-scale single-cell or spatially resolved proteome profiling remains challenging (1), it has been common practice to use mRNA levels to infer the abundance of corresponding proteins in single cells. However, gene expression is regulated at both transcriptional and translational levels, and many studies have concluded that the correlation between mRNA and protein levels is low (2-5). Moreover, many genes undergo localized translation at unique subcellular locations (6). Therefore, methods for scalable single-cell and spatially resolved profiling of protein synthesis are needed for a comprehensive understanding of translational control.

[0127] Existing bulk and single-cell ribosome profiling methods have enabled the analysis of protein translation at the transcriptome (7-12). However, these methods cannot preserve spatial information of subcellular structures, cell morphology, or tissue organization, limiting their ability to chart protein synthesis in space. In contrast, imaging-based methods that can track mRNA translation in their physical coordinates (13-19) are limited to one gene at a time. Thus, highly multiplexed spatial ribosome profiling in single cells at subcellular resolution remains challenging to achieve. To fill this gap, a novel 3D in situ mapping method (RIBOmap) is described herein to highly multiplex and chart protein and gene expression at subcellular resolution. Protein synthesis of 981 genes was profiled in intact cells with RIBOmap, and a computational pipeline was developed to analyze the subcellular patterning of local translation. RIBOmap was also applied to profile 5,413 genes in intact mouse brain tissue at subcellular resolution, revealing spatial patterns of protein synthesis across cell types and tissue regions.

[0128] RIBOmap is built on a targeted sequencing strategy that uses a unique design of tri-probes to selectively detect and amplify ribosome-bound mRNAs, followed by hydrogel-tissue embedding and highly multiplexed in situ sequencing readout (20). In particular, the RIBOmap tri-probe set encompasses (1) a splint DNA probe that hybridizes to ribosomal RNA (rRNA) and acts as a splint to circularize nearby padlock probes (see below); (2) a padlock probe that targets a specific mRNA species of interest and encodes a gene-unique identifier; and (3) a primer that targets adjacent sites of the padlock probe of the same mRNA and acts as a primer to enable amplification of the circularized padlock probe by rolling circle amplification (RCA) to generate DNA nanoballs (amplicons). Importantly, the gene-specific hybridization pair of primer-padlock probes excludes false-positive signals arising from nonspecific hybridization of single probes (20) (Figure 9A). Taken together, the RNA signal is amplified into a DNA amplicon only if all three probes are present in close proximity (Figure 9B and Figure 9C). The DNA amplicon is then embedded in situ into a polyacrylamide hydrogel matrix using tissue-hydrogel conjugation chemistry (20). The gene-unique barcode of the DNA amplicon is then decoded by in situ sequencing with error reduction by dynamic annealing and ligation (SEDAL, Figure 9A) (20).

[0129] An alternative strategy to the RIBOmap workflow was also designed utilizing a primary antibody targeting a ribosomal protein, which is then detected by splint-conjugated protein A / G for tri-probe amplification (Figure 12A). Specifically, this strategy was evaluated using anti-RPS3 (small 40S subunit ribosomal protein 3) and anti-RPL4 (large 80S subunit ribosomal protein 4) antibodies (Figure 12B-Figure 12E). The signal-to-noise ratio (SNR) of the antibody-based strategy ranged from 13.6 to 16.7. The SNR of the rRNA-targeting strategy was 93 ± 17 (Figure 12F).

[0130] Next, the specificity of RIBOmap triprobe amplification for ribosome-bound mRNA was evaluated. First, detection specificity was tested using non-translating long non-coding RNAs (nuclear-localized MALAT1 RNA and cytoplasmic-localized vtRNA1-1 RNA) as negative controls. RIBOmap and previously reported STARmap (20) imaging were performed on ACTB and negative control RNA in HeLa cells to detect ribosome-bound RNA and total RNA, respectively (Figure 9D). It was found that RIBOmap only detected ACTB mRNA, while STARmap detected both mRNA and non-coding RNA (Figure 9E and Figure 9F). Next, the subtranscript specificity of RIBOmap in detecting ribosome loading sites was verified. Here, harringtonine, a translation inhibitor, was used, which can trap translation-initiating ribosomes at the start codon and displace elongating ribosomes from the transcript (Figure 9G). Three tri-probe sets were designed to target subtranscript regions at different distances (-16, 115, and 405 nt) from the start codon (Figure 1H). After 5 min of harringtonine treatment (2 μg / ml final concentration), ACTB mRNA, when detected by probes targeting 115 or 405 nt downstream of the start codon, showed a significant decrease in signal intensity in RIBOmap (p = 7.5 × 10, respectively). -5 , 4.7×10 -5, Student's t test), whereas the signal generated by a probe targeting -16 nt from the start codon showed a slight decrease (Figure 9I and Figure 9J). Taken together, it was demonstrated that RIBOmap can specifically detect ribosome-bound mRNA.

[0131] After benchmarking the SNR and specificity of RIBOmap, cell cycle-dependent and localized mRNA translation in single cells was profiled at subcellular resolution by performing a highly multiplexed 981-gene RIBOmap experiment in HeLa cells (Figure 10A). The 981 genes represent a curated list composed of cell cycle gene markers, genes with diverse subcellular patterns, and genes of variable RNA stability (21-24). A multimodal RIBOmap experiment was designed that further incorporated cell cycle stage and subcellular organelle information: (1) the cell cycle phase of each cell was captured by fluorescent ubiquitination-based cell cycle indicator (FUCCI) (25, 26); (2) ribosome-bound mRNAs (981 genes) were sequenced in situ after FUCCI imaging (20); (3) finally, nuclei, endoplasmic reticulum, and cell shape were stained and imaged (Figure 2B). All three imaging modalities were registered by a shared channel of nuclear (DAPI) staining. To compare spatial translatome vs. spatial transcriptome, paired total RNA mapping of the same 981 genes was performed using STARmap (with identical RNA hybridization sequences) on the same batch of HeLa FUCCI cells (Figure 10A). In total, after quality control filtering, 1,813 cells were sequenced by RIBOmap (1,500 ribosome-bound RNA reads per cell on average) and 1,757 cells were sequenced by STARmap (2,349 RNA reads per cell on average).

[0132] First, it was assessed whether RIBOmap could dissect cell cycle-dependent mRNA translation. To this end, single-cell translatome profiles generated by RIBOmap and single-cell transcriptome profiles generated by STARmap were subjected to single-cell trajectory analysis (21). In both RIBOmap and STARmap samples, single-cell profiles of known cell cycle gene markers (21) were used to embed single cells into diffusion maps with pseudotime values ​​to delineate cell cycle progression (Figure 10C and Figures 13A and 13B). For both datasets, RNA-defined G1, S, and G2 / M cell cycle phases were consistent with the expected cell cycle-dependent patterns of FUCCI protein fluorescence (Figure 10C), demonstrating the ability of RIBOmap in detecting cell states.

[0133] After confirming the accuracy of RIBOmap in depicting cell cycle progression, co-regulated gene modules were identified by covariation analysis. Based on single-cell translatome expression, pairwise correlations of all gene pairs were calculated, and hierarchical clustering was used to identify genes with high correlation coefficients across single cells. Notably, five gene modules with substantial intra-module correlations and enrichment of distinct functional pathways were found (modules 1–5, Figure 10D and Figure 14A–D). Notably, genes in modules 3 and 5 were enriched with G2 / M and G1 / S cell cycle marker genes, respectively (Figure 10E). These two gene modules are also negatively correlated (Figure 14E). This result provided single-cell level evidence supporting previous findings that these cell cycle marker genes are co-regulated during cell cycle progression due to the synchronized execution of their gene functions (27). Moreover, module 2 contains genes encoding protein translation machinery such as ribosomal proteins (RPS3, RPS28, and RPL37) and translation factors (EEF2, EIF3B, and ETF1) (Figure 14C). These genes show a positive correlation with module 5 (G1 / S marker genes) and a negative correlation with module 3 (G2 / M marker genes) (Figure 14F). This leads to the conclusion that protein translation machinery is more actively produced in the G1 / S phase to supply protein production for the physical expansion and duplication of intracellular organelles.

[0134] Leveraging the high spatial resolution of the dataset, we investigated whether RIBOmap could detect subcellular patterns of translation. To identify translating genes that showed a tendency to colocalize (exhibit spatial proximity) in the subcellular space, nearest-neighbor profiles were created for each RNA in RIBOmap, and the significance of gene colocalization was assessed by comparing against a randomized control. Subsequent hierarchical clustering of the gene colocalization matrix identified five gene modules with highly correlated subcellular spatial organization and distinct functional enrichment (modules 1–5, Figures S1E–S1G and S15A–S15C). Among them, modules 2, 4, and 5 were functionally enriched with membrane proteins and the secretory pathway (Figures S1F–S1G and S15A–S15B) and physically colocalized with ER staining (Figures S1H–S10I and S14D–S14E), suggesting that these are ER-translated genes. Meanwhile, genes in modules 1 and 3 are enriched in large protein complexes of the mitotic spindle (e.g., CDK1, NUSAP1, KIF23, SPAG5, TACC3, FAM83D) and translation machinery (e.g., RPS3, RPS28, RPL36A, EEF2, DHX9), respectively (Figure 10F and Figure 10G and Figure 15A and Figure 15C). Such observations point to the fact that subunits of protein complexes may be generated in spatial proximity for efficient assembly. Finally, we investigated whether co-regulated genes tend to be co-localized for translation. To this goal, the co-localization p-values ​​from the subcellular co-localization analysis were plotted in a single-cell covariation gene matrix. The five gene modules with high single-cell covariation correlation were also found to show strong subcellular co-localization (Figure 16F). These results lead to the conclusion that functionally related gene groups can be co-regulated by co-localized translation in cells.

[0135] Second, a crucial posttranscriptional RNA modification (28), N 6 -Methyladenosine modification (m 6It was determined whether RNAs with mA (A) have distinct spatial translation patterns. To this end, the translation distribution of each gene was quantified with the distance ratio (DR) metric, which estimates the relative position of each amplicon between the nuclear and cytoplasmic membranes (Figure 10J). In particular, 6 The DR value of the A gene is non-m 6 The DR value was significantly lower than that of the A gene (Fig. 10K), and m 6 This difference indicates that the A gene is physically translated close to the nuclear membrane. 6 It was hypothesized that this could be explained by the shorter lifetime and, thus, shorter transport time of A-modified RNAs. (23) Taken together, we demonstrated the power of RIBOmap for the integrated analysis of spatial translatomics at the subcellular level, providing an unprecedented approach for future studies of RNA processing and translation.

[0136] To further demonstrate the spatial profiling power of RIBOmap in analyzing tissue samples, this approach was applied to mouse brain slices to reveal in situ single-cell translatome profiles. Here, a targeted gene list of 5,413 genes, curated from single-cell sequencing studies of previously published mouse cell atlases (29–35), was mapped. Nine rounds of in situ sequencing revealed a single-cell translatome profile in the mouse left hemisphere (27.6 mm), which contains a large number of brain regions. 2 , 62,753 cells) were imaged with a voxel size of 90 nm × 90 nm × 300 nm (Fig. 1A). As expected, minimal RIBOmap signal was observed in the nucleus, in contrast to a dense signal in the cytosol, where protein translation occurs (Fig. S11B).

[0137] First, tissue RIBOmap data were benchmarked by comparing spatial translation patterns of well-known cell type marker genes (e.g., Slc17a7, Gad2, and Pcp4) with in situ hybridization (ISH) images of corresponding genes from the Allen Brain database (36), and the comparison showed concordant spatial patterns (Figures S11C and S16A). Notably, it was observed that RIBOmap reads of unique genes correlated better with protein signals than RNA signals (ISH) in unique brain regions, such as Sst in CA3 and Nefl in CA1 in the hippocampus (Figures S16B and S16C). This result is consistent with previous reports that ribosome profiling results correlated better with protein abundance than RNA-seq data (7, 11, 12). Next, all 62,753 cells were subjected to brain cell type identification (Figure S17). Here, a hierarchical clustering strategy was employed (20, 31): Level 1 clustering classified cells into neurons and glial cells (Figure 18A and Figure 18B); Level 2 clustering identified excitatory neurons, inhibitory neurons, astrocytes, oligodendrocytes, oligodendrocyte progenitors, microglia, and vascular cells (Figure 18A, Figure 18C, and Figure 18D); Level 3 clustering identified 57 distinct subtypes (Figure 11D and Figures 19-21). Based on the cell typing results, a spatial cell map was produced from the imaged hemi-brain region (Figure 11E). The spatial distribution of these translatomic defined cell types is largely as expected. For example, the cortex is dominated by layer-specific excitatory neurons (layer 2 / 3: Cplx2 + , layer 4 / 5:Dkk3 + , layer 6:Nr4a2 + , layer 6: Pcp4 + ), and the corpus callosum is made up of oligodendrocytes (Mbp + , Mal + , Cnp + , Plp1 + ), and the striatum is composed of medium spiny neurons (Tac1 + , Adora2a + , Ppp1r1b+ , Penk + , Rasd2 + ), and the thalamus is composed of excitatory neurons (Prkcd + , Synpo2 + ) and the hypothalamus contains peptidergic neurons (Hap1 + , Tac1 + , Dlk + ) were enriched in the pyramidal layer of CA1-3 and the granular layer of DG. Region-specific excitatory neurons were also observed in the pyramidal layer of CA1-3 and the granular layer of DG, respectively. Thus, the potential of RIBOmap to identify diverse brain cell types based on single-cell translatomes was demonstrated as an alternative strategy to generate spatial tissue atlases.

[0138] Subcellular localized mRNA translation is essential for both neuronal (37-39) and glial cell function (40-42). Notably, the subcellular resolution of RIBOmap enabled the investigation of localized translation in the cell bodies and processes of neuronal and glial cells. To this goal, RIBOmap reads were separated into cell body reads (i.e., inside the cell body regions identified by ClusterMap (43)) and process reads (i.e., the remaining reads) (Figure ​(Figure11F​and​Figure11G). Next, the top 10% genes with the highest process-to-cell body ratio were defined as translated genes enriched in processes, and the top 10% genes with the lowest process-to-cell body ratio were defined as translated genes enriched in cell bodies (Figure ​(Figure11H). 11H). Gene Ontology (GO) analysis showed that the translated genes enriched in processes were related to translation, synapses, and postsynaptic thickening (Figure ​(Figure11I). In contrast, the translation genes enriched in the cell body are associated with the plasma membrane, extracellular matrix, and endoplasmic reticulum (Figure S1J). This result further validated the ability of RIBOmap in detecting subcellularly localized translation in tissue samples, as membrane and secretory proteins are expected to be translated in the ER in the cell body region. Notably, exceptionally high abundance of process-enriched translation signals, such as postsynaptic density proteins (e.g., Shank1, Dlg4, and Grik5), translation machinery proteins (e.g., Eef2, Eef1a, Rpl3, and Rps5), motor proteins (e.g., Kif5a and Kif1a), and calcium sensor and signaling proteins (e.g., Calm1 and Camk2n1), was observed in hippocampal neuropil (Figure S1K and Figure S2A). In comparison, translation genes enriched in the cell body, such as App, which encodes a transmembrane precursor protein and is associated with Alzheimer's disease, and Rtn4, known to be associated with the endoplasmic reticulum, showed sparse RIBOmap signals in hippocampal neuropil (Figure 11L and Figure 22B). In addition to these exemplary genes translated in neurons, localized translation of glial cell marker genes was also observed (Figure 11M and Figure 11N and Figure 22C and Figure 22D).For oligodendrocytes, the marker genes Mbp and Plekhb1 were identified as translation genes enriched in processes, while Mal and Cnp were translation genes enriched in the cell body (Figure 11M and 11N and Figure 22C and 22D). Astrocytes have many processes, and their marker genes were enriched in the translation category enriched in processes, such as Gfap, Ttyh1, Apoe, and Clu (Figure 11M and 22C). For vascular cells, their marker genes were enriched in the translation gene group enriched in the cell body, such as Ptgds, Itm2a, and Bsg (Figure 22C). Overall, RIBOmap was demonstrated to be a powerful tool to study localized translation in processes of both neuronal and glial cells in mouse brain tissue.

[0139] With the development of a novel spatial omic method, RIBOmap, spatial mRNA translation can be mapped at single-cell and subcellular levels. It is demonstrated herein that RIBOmap probes the translation of 981 genes during the cell cycle and reveals distinct subcellular distribution patterns of translating RNA in human cells, enabling a critical step toward a more comprehensive understanding of mRNA regulation and protein synthesis. To illustrate this, the present disclosure demonstrates spatial profiling of translatome states in situ in intact tissue samples with high 3D spatial resolution, and creates translatome-defined spatial cell maps in mouse brain tissue. Moreover, compared to Ribo-STAMP and TRAP methods, RIBOmap does not rely on gene manipulation.

[0140] material and method cell culture Human HeLa cells (CCL-2) were obtained from ATCC and cultured in DMEM supplemented with 10% FBS at 37°C and 5% CO. Harringtonine treatment was performed by adding drug to a final concentration of 2 μg / ml and incubating at 37°C for 5 min.

[0141] HeLa FUCCI cells were generated using lentiviral particles as previously described (26). Briefly, HEK 293T cells were transfected with pBOB-EF1-FastFUCCI-Puro (obtained from Addgene, #86849) and packaging plasmids (psPAX2 and VSVG). 48 hours after transfection, supernatants were harvested from the culture medium containing the virus. The supernatants were filtered through 0.45 μm PES filters, and the viral supernatants were used to transduce HeLa cells. Following puromycin selection, stable cell lines were grown.

[0142] mouse All animal procedures followed animal care guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute of MIT and Harvard under animal protocol #0255-08-19. Animal experiments were performed in compliance with IACUC policies and NIH guidelines. C57BL / 6 (male, 16 weeks old) mice used for RIBOmap in this study are purchased from The Jackson Laboratory (JAX).

[0143] Chemicals and enzymes Chemicals and enzymes are listed as name (vendor, catalog number): DMEM (Gibco, 11995), Harringtonine (Abcam, ab141941). Lipofectamine MessengerMAX transfection reagent (Thermo Scientific, LMRNA003). Tissue-Tek OCT compound (SAKURA, 4583). Glass bottom 24-well plates (Cellvis, P24-1.5HN). Glass bottom 12-well and 24-well plates (MatTek, P12G-1.5-14-F). 3-(trimethoxysilyl)propyl methacrylate (Sigma, M6514). Poly-D-lysine (Sigma-Aldrich, A-003-M). 16% PFA (Electron Microscope Sciences, 15710-S). Methanol (Sigma-Aldrich, 34860-1L-R). PBS (Gibco, 10010-023). Tween-20, 10% solution (Calbiochem, 655206). Yeast tRNA (Thermo Scientific, AM7119). SUPERase·In RNase Inhibitor (Thermo Scientific, AM2696). 20×SSC (Sigma-Aldrich, S6639). Formamide (Calbiochem, 655206). Ribonucleoside vanadyl complex (New England Biolabs, S1402S). T4 DNA ligase, 5 Weiss U / μL (Thermo Scientific, EL0012). Phi29 DNA polymerase (Thermo Scientific, EP0094). 10 mM dNTP mix (Thermo Scientific, 18427089). UltraPure BSA (Thermo Scientific, AM2618). 5-(3-aminoallyl)-dUTP (Thermo Scientific, AM8439). Methacrylic acid NHS ester, 98% (Sigma-Aldrich, 730300). DMSO, anhydrous (Molecular Probes, D12345). Acrylamide solution, 40% (Bio-Rad, 161-0140). Bis solution, 2% (Bio-Rad, 161-0142).Ammonium persulfate (Sigma-Aldrich, A3678). N,N,N',N'-Tetramethylethylenediamine (Sigma-Aldrich, T9281). OminPur SDS, 20% (Calbiochem, 7991). Proteinase K solution, RNA grade (Thermo Scientific, 25530049). Antarctic phosphatase (New England Biolabs, M0289L). DAPI (Molecular Probes, D1306). 10% Triton X-100 (Sigma-Aldrich, 93443). Concanavalin A, Alexa Fluor 594 conjugate (Thermo Scientific, C11253). Flamingo fluorescent protein gel stain (Bio-Rad, 1610490). DL-Dithiothreitol (Sigma-Aldrich, D9779). 0.5ml 30-kDa Amicon Ultra Filters (Millipore, UFC503024). Ribosomal protein L4 polyclonal antibody (Thermo Scientific, 11302-1-AP). RPS3 monoclonal antibody (Thermo Scientific, 66046-1-IG). Micro Bio-Spin™ P-6 gel columns, Tris buffer (Bio-Rad, 7326221). Pierce™ SM(PEG)2, No-Weigh™ format (Thermo Scientific, A35397). Zeba™ spin desalting columns, 7K MWCO, 0.5mL (Thermo Scientific, 89882).

[0144] Design of RIBOmap and STARmap probes RIBOmap was performed using a tri-probe strategy containing a splint probe and a SNAIL probe. The splint probe consisted of three parts: a 25-nt sequence at the 5' end that hybridizes to 18S ribosomal RNA (rRNA), a 50-nt deoxyadenosine (dA) sequence in the middle, and a 12-nt splint-padlock annealing sequence at the 3' end. The design of the 25-nt sequence that hybridizes to 18S rRNA was applied as previously described (44). The probes are targeted to regions of 18S rRNA that are relatively unstructured and available for chemical modification. In total, five unique probes were designed. The 3' end of the splint probe is blocked by a 3' inverted dT modification and cannot be used as a primer for RCA amplification. The splint probes were synthesized by Integrated DNA Technologies (IDT).

[0145] The design of SNAIL probes was applied as previously described (20). Briefly, Picky 2.2 was used to design the hybridization sequence of each SNAIL probe pair with the length set at 40–46 nt, and then the resulting sequences were split into halves 20–25 nt and separated by 0–2 nt to best match the melting temperature (Tm) between the two halves. For the 981 gene experiment in HeLa cells, 5 or 6 pairs of SNAIL probes were designed for each gene. For the 5,433 gene experiment in mouse brain, 4 pairs of SNAIL probes were designed for each gene. SNAIL probes were synthesized and pooled by IDT.

[0146] RIBOmap and STARmap sample preparation for cell culture HeLa FUCCI cells were cultured in 24-well plates, washed with PBS and fixed with 300 μl of 1.6% PFA in PBS buffer for 15 min at room temperature before sample collection. After fixation, cells were permeabilized with 450 μl of cold methanol and incubated at −20° C. for 1 h. HeLa FUCCI cell samples were then removed from −20° C. to room temperature for 5 min and then quenched with 200 μl of quenching solution (1 mg / ml yeast tRNA, 0.1 U / μl SUPERase In RNase inhibitor, 100 mM glycine, 0.1% Tween-20 in PBS) for 10 min at room temperature. After quenching, samples were incubated with 200 μl of 1× hybridization buffer (2× SSC, 10% formamide, 20 mM ribonucleoside vanadyl complex, 0.1 mg / ml yeast tRNA, 0.5% SUPERase In, 0.1% Tween-20, pooled SNAIL probes at 1 nM per oligo and splint probes for RIBOmap samples at 1 μM per probe) for 12 h with parafilm wrapping and shaking in a humidified oven at 40°C. Samples were washed twice with 300 μl PBSTR (0.1 U / μl SUPERase In RNase inhibitor in PBS, 0.1% Tween-20) and once with 300 μl high salt wash buffer (4× SSC in PBSTR) for 20 min each wash at 37°C. Finally, samples were rinsed once with 300 μl of PBSTR at room temperature.

[0147] The samples were then incubated with 200 μl of ligation mixture (0.25 U / μl T4 DNA ligase, 0.5 mg / ml BSA, and 0.4 U / μl SUPERase·In RNase inhibitor in 1× T4 DNA ligase buffer) for 2 h at room temperature with gentle shaking. After the ligation reaction, the samples were washed twice with 300 μl PBSTR and then incubated with 200 μl of rolling circle amplification (RCA) mixture (0.5 U / μl Phi29 DNA polymerase, 250 μM dNTPs, 20 μM 5-(3-aminoallyl)-dUTP, 0.5 mg / ml BSA, and 0.4 U / μl SUPERase·In RNase inhibitor in 1× Phi29 buffer) for 30 min at 4°C and 2 h at 30°C with gentle shaking. The samples were then washed twice with PBST (0.1% Tween-20 in PBS). Imaging was then performed to simultaneously record the FUCCI fluorescence signals of the RIBOmap and STARmap samples. The samples were then treated with 200 μl of modification mixture (25 mM methylacrylic acid NHS ester in 100 mM sodium bicarbonate buffer) for 1 h at room temperature, then rinsed once with PBST. The samples were incubated with 150 μl of monomer buffer (4% acrylamide, 0.2% bisacrylamide in 2×SSC) for 15 min at room temperature. The buffer was then aspirated, and 25 μL of polymerization mixture (0.2% ammonium persulfate, 0.2% tetramethylethylenediamine dissolved in monomer buffer) was added to the center of the sample, which was immediately covered with a Gel Slick-coated coverslip. The polymerization reaction was performed in a N2 box at room temperature for 1 h, then washed twice with PBST for 5 min each.

[0148] The tissue-gel hybrids were then digested with 200 μl of proteinase K mix (0.2 mg / ml proteinase K, 1% SDS, 100 mM NaCl, and 50 mM Tris) for 1 h at 37° C., followed by three washes with PBST for 5 min each. Samples were then treated with 200 μl of dephosphorylation mix (0.25 U / μL Antarctic phosphatase, 0.5 mg / mL BSA in 1× Antarctic phosphatase buffer) for 1 h at 37° C., followed by three washes with PBST for 5 min each.

[0149] For SEDAL sequencing, each sequencing cycle started with treating the sample with 200 μl of stripping buffer (60% formamide, 0.1% Triton X-100 in H2O) twice for 10 min each at room temperature, followed by washing with PBST three times for 5 min each. The sample was then incubated with 200 μl of sequencing mix (0.1875 U / μl T4 DNA ligase, 0.5 mg / ml BSA, 10 μM leading probe, and 5 μM fluorescent oligo in 1× T4 DNA ligase buffer) for at least 3 h at room temperature. The sample was washed with 300 μl of washing and imaging buffer (10% formamide in 2× SSC buffer) three times for 10 min each, and then immersed in the washing and imaging buffer for imaging. Images were acquired using a Leica TCS SP8 confocal microscope with a 40x oil immersion objective (NA 1.3) and a voxel size of 94.64nm x 94.64nm x 350nm. DAPI signals were imaged in the first round of sequencing. Six cycles of imaging were performed to detect 981 genes.

[0150] After SEDAL sequencing, samples were treated with 200 μl stripping buffer three times for 10 min each at room temperature. Samples were then washed three times with PBST for 5 min each, incubated with 200 μl ER staining mixture (0.05 mg / ml concanavalin A diluted in 100 mM sodium bicarbonate buffer), and then washed twice with PBST for 5 min each. Samples were then incubated with 200 μl Flamingo staining mixture (1× Flamingo fluorescent gel stain in washing and imaging buffer) overnight at room temperature, and then washed three times with 300 μl PBST.

[0151] RIBOmap sample preparation for mouse tissues Mice used in this study were anesthetized with isoflurane and then rapidly decapitated. Mouse brain tissue was collected and placed in Tissue-Tek OCT compound. The brain tissue in OCT was then frozen in liquid nitrogen and kept at -80°C. For sectioning of mouse brain tissue, the brain tissue was transferred to a cryostat (Leica CM1950) and cut into 20 μm coronal sections at -20°C. The slices were transferred and mounted on glass-bottom 12-well plates pretreated with 3-(trimethoxysilyl)propyl methacrylate and poly-D-lysine. Brain slices were fixed with 4% PFA in PBS for 15 min at room temperature, then permeabilized with cold methanol and placed at -80°C for 1 h. The experimental procedure for mouse brain tissue is almost the same as that for HeLa cells, except that all reaction volumes were doubled when brain tissue was prepared in 12-well plates. No fluorescent protein imaging procedure was performed and no organelle staining procedure was involved. Images were acquired using a Leica TCS SP8 confocal microscope with a 63x oil immersion objective (NA 1.3) and a voxel size of 90.14 nm × 90.14 nm × 300 nm. DAPI signals were imaged during the first round of sequencing, with nine cycles of imaging performed to detect 5,413 genes.

[0152] Antibody-Based RIBOmap Strategy for Cell Cultures Protein A / G conjugated with splint probe was first prepared. Briefly, 5'-thiol modified splint probe (IDT) was activated with DL-dithiothreitol (DTT) for 2 h at room temperature and purified by Micro Bio-Spin™ P-6 gel column to remove excess DTT. Protein A / G was reacted with PEGylated SMCC crosslinker [SM(PEG)2] for 2 h at 4°C and then purified by Zeba desalting column to remove excess crosslinker. The activated splint probe was incubated with Protein A / G (molar ratio of Protein A / G to splint probe of about 1:11) overnight at 4°C. The final conjugated Protein A / G was washed five times by using 0.5 ml of 30-kDa Amicon Ultra Filter to remove unreacted DNA oligonucleotides.

[0153] The fixation step was the same as for the rRNA probe-based RIBOmap strategy. For the hybridization step, only the SNAIL probe was added, not the splint probe. Other reagents for this step were the same as for the rRNA probe-based RIBOmap strategy. After hybridization, ribosomal protein (RPS3 or RPL4) antibody incubation was performed. The cell samples were blocked with blocking solution (5 mg / ml BSA in PBSTR) for 30 min at room temperature, then stained with primary antibodies (RPS3 or RPL4, IgG for control samples) diluted 1:100 in blocking solution for 1 h at 4°C. The samples were washed three times for 5 min each at room temperature with PBSTR. The samples were then incubated with splint probe-conjugated protein A / G for 1 h at room temperature, and washed three times for 5 min each at room temperature with PBSTR. The ligation and RCA steps were the same as for the rRNA probe-based RIBOmap strategy. The samples were then incubated with 50 nM fluorescent oligos complementary to the DNA amplicons and DAPI in PBST. The DNA amplicon signals were detected by imaging.

[0154] Preprocessing of RIBOmap and STARmap imaging. Image deconvolution was achieved using Huygens Essential version 21.04 (Scientific Volume Imaging, The Netherlands, svi.nl) using the CMLE algorithm with an SNR of 10 and 10 iterations. Similar image registration, spot calling, and barcode filtering with fine-tuning were applied as previously reported ( 20 ).

[0155] RIBOmap / STARmap cell culture sample data processing 3D cell segmentation: Image segmentation was achieved with a strategy combining 2D reference segmentation generated by CellProfiler v4.1.3, and 3D stained images were processed with a customized MATLAB script. First, an overlay image combining amplicon channel and Flamingo fluorescent gel stained images was created to represent cell boundaries. Maximum projections of both 3D DAPI stained images and overlay images were input into a customized pipeline created with CellProfiler v4.1.3. A median filter was used to remove high frequency noise on the image, and the IdentifyPrimayObjects function was used to detect cell nuclei. Then, based on the identified nuclei, cell boundaries were contoured by applying the IdentifySecondaryObjects function to the filtered 2D overlay image. Both the nuclei and cell segmentation masks in the output were used as reference segmentations in the following steps to create 3D segmentations.

[0156] For 3D segmentation, images targeting different cellular compartments (DAPI staining of nuclei, overlaid images of cell boundaries, and Concanavalin A staining of ER) were first processed by a median filter and binarized with a manually curated threshold. All connected components (objects) with less than 200 voxels were removed from the binary image. The image was then expanded with a disk structure element with a radius equal to 10. Then, 3D segmentation masks targeting each cellular region were created by an element-wise multiplication process between the binary image and the 2D reference cell segmentation from the previous step. Nuclear regions were removed from the 3D cell segmentation mask to create the cytoplasmic segmentation. Finally, a gene expression matrix per cell in each cellular compartment was created by quantifying the filtered amplicons overlapping each labeled region in 3D.

[0157] Quantification of protein signals in FUCCI cell lines: Both mAG and mKO2 fluorescent images were aligned with the sequencing images via image registration according to previous reports. The signal level of each protein was quantified as the integrated intensity of voxels overlapping with the 3D nuclear segmentation mask.

[0158] Single-cell quality control: To ensure high-quality libraries, single-cell profiles measured by both the RIBOmap and STARmap methods were filtered based on the following metrics and thresholds, respectively. First, the desired range (0.5 × 10 6 ~2×10 6 Cells with physical volumes defined by 3D segmentation outside the 3D voxel size were excluded. Sample-specific thresholds for the number of transcripts per cell were then used to remove outliers in each dataset (RIBOmap lower limit (LB): 300, upper limit (UB): 3500, STARmap LB: 500, UB: 6000). Additionally, transcript density was defined as the ratio between the number of transcripts and the physical volume of each cell, which was used as a threshold metric (LB: 0.00055) to further exclude cells. Genes were filtered based on the percentage of cells that expressed them (LB: 10%) and their maximum count in cells (RIBOmap LB: 2; STARmap LB: 4). The difference in filtering criteria for RIBOmap and STARmap arose from different expectations for each technique. After filtering, 1,813 cells for the RIBOmap dataset and 1,757 cells for STARmap of 897 genes entered the subsequent analysis.

[0159] Covariation analysis: To identify covarying gene modules, expression matrices were first normalized to the same total number of transcripts per cell, and then Pearson correlation coefficients across each gene pair were calculated to measure similarity of cell-to-cell variation. Hierarchical clustering using ward linkage was performed on RIBOmap with n_cluster set to 10.

[0160] Colocalization analysis: To reveal colocalized read clusters, permutation analysis was performed. Given one read in a cell, the gene identities of neighboring reads (within a radius of 3 μm) were recorded. By screening all cells, counts of each neighboring gene pair identified as observed counts were obtained. Then, 1000 permutation trials were performed to randomly shuffle the gene labels of RNA reads without perturbing their subcellular locations. For each permutation round, counts of each neighboring gene pair were collected to create a permutation distribution. Then, one-sided p-values ​​of each gene pair were inferred by comparing the observed counts with the permutation distribution. The pairwise p-values ​​were further used to construct a heatmap (Figure 10E). The rows and columns in the heatmap of the RIBOmap samples were ordered by hierarchical clustering. The heatmap of the STARmap samples used the same order as RIBOmap to detect gene modules using differences in spatial distribution. To quantitatively compare endoplasmic reticulum (ER) localization levels between gene modules, the percentage of reads localized to the ER for genes within a target gene module at the single-cell level was calculated. The same percentage for all genes was used as a comparison baseline. Wilcoxon signed-rank tests were performed to test for statistical significance.

[0161] Cell cycle and pseudotime: To construct pseudotime trajectories representing cell cycle progression, cell cycle phases (G1, G1S, G2M) were first identified using the expression profile of cells restricted to cycle marker genes (score_genes_cell_cycle function in Scanpy). STARmap cells were then embedded in a diffusion map constructed by the expression of the same set of genes. After transforming this diffusion map into a polar coordinate system, a "root" cell representing the start of the cell cycle was identified and a pseudotime value was assigned to each cell by its angle relative to the "root" cell on the polar coordinates. To assign pseudotime values ​​to RIBOmap cells on the same trajectory, cells from the two datasets were registered to the same embedding defined by the fluorescence intensity of mAG and mKO2. The pseudotime value of each RIBOmap cell was defined as the average value of its nearest neighbors (k=3) in STARmap.

[0162] DR calculation: To quantitatively analyze the localization of RNA reads within the cytoplasm, a distance ratio (DR) was calculated for each cytoplasmic read. The DR value of a read was defined as the shortest distance of the read to the nuclear boundary (d1), as determined by nuclear segmentation, normalized by the sum of this distance and the shortest distance of the read to the cell membrane (d2), as determined by cell segmentation. Scipy's Euclidean distance transform function (distance_transform_edit) was used to calculate the shortest distance between each read and the target surface defined by the 3D segmentation.

[0163] Data processing of RIBOmap brain tissue samples Cell segmentation using ClusterMap: An RNA amplicon-based segmentation method (ClusterMap (43)) was used to achieve single-cell signal quantification. A basic processing pipeline involving background signal rejection, DAPI signal sampling, and density-based segmentation was applied to each field of view (FOV). Specifically, 10% of the RNA signals were identified as locally low-density signals and excluded for single-cell level quantification. To further improve segmentation accuracy, additional signal spots were sampled from the coupled DAPI stained images with the parameter dapi_grid_interval set equal to 5. During segmentation in 3D, the parameter cell_num_threshold was set to 0.02 based on a qualitative test compared to the DAPI staining. Cells identified by the algorithm as having less than 5 transcripts or not overlapping with the coupled DAPI staining were excluded. The ClusterMap signal assignments of all FOVs were merged to create a cell-gene matrix.

[0164] Quality control and pre-processing: After signal quantification at the single cell level, the number of transcripts and genes per cell was used to filter out low-quality cells. In detail, the median absolute deviation (MAD) was used to estimate the threshold of reads per cell filtering, as shown by the following formula: Lower limit = Median(reads per cell)-4*MAD Upper limit = median (reads per cell) + 4*MAD

[0165] Other standard filtering strategies were also applied, such as: 1. A minimum of 10 expressed genes is required for a cell to pass the filter; and 2. A minimum of 10 expressed cells are required for a gene to pass filtering.

[0166] After filtering, transcriptome profiles of 62,753 cells and 5,413 genes were obtained. To ensure a high-quality gene library, genes with a maximum count less than 3 were further filtered out across cells, resulting in 3,995 genes that were used in downstream analyses such as cell type classification. The transcriptome profiles were then normalized by the median number of transcripts per cell by the pp.normalize_total function (Scanpy v1.8.2). A logarithmic transformation was then performed on the data using the pp.log1p function. Finally, the data matrix was scaled to unit variance by the pp.scale function and used in downstream analyses such as dimensionality reduction and unsupervised clustering.

[0167] Cell type classification: A hierarchical clustering strategy was applied to generate a three-level cell type annotation for the RIBOmap mouse brain dataset. First, a scaled matrix restricted to 3,995 highly abundant genes was used to perform a principal component analysis (PCA). The function tl.pca was used to calculate the top 30 principal components, all of which were used to construct a k-nearest neighbor (k-NN) graph, in which cells were connected with their neighbors in a high-dimensional space based on the similarity of their transcriptome profiles. The Leiden community detection algorithm was then applied to the kNN graph to detect cell clusters. Based on the expression profiles of canonical markers of common cell types in the brain (i.e., Slc17a7, Gad1, Gad2, Plp1, Slc1a3, etc.), each cluster was classified as either neuronal or glial cells as their first level annotation.

[0168] To assign more detailed annotations, the same analysis described previously was applied to each of the populations (neurons, glia) under the first level of annotation. The elbow method was used to identify significant principal components after PCA. By plotting the variance ratio of each principal component (pl.pca_variance_ratio function), the top 10 and 15 principal components with the highest values ​​were selected to perform the following analysis for neurons and glial cells, respectively. For neurons, they were further divided into excitatory and inhibitory neurons based on their expression profiles of Slc17a7, Gad1, and Gad2. For glial cells, numerous subtypes were identified for some of the major glial cells (i.e., for astrocytes, Astro1, Astro2, and Astro3). In this case, two levels of annotation were given to each cluster; the second level of annotation indicated its major glial cell type (i.e., astrocytes, oligodendrocytes, vascular cells, etc.), and the third level of annotation (unique identifiers) represented subtypes within the major population.

[0169] To identify neuronal subtypes, the same unsupervised clustering was applied to both excitatory and inhibitory neuronal populations separately. Specifically, 15 and 10 principal components were used to construct kNN graphs of excitatory and inhibitory neurons, respectively. Most of the neuronal clusters were annotated based on their spatial representation in the brain sections (i.e., anatomical regions such as L2 / 3, L4, CA, DG, TH, etc.), and some of the inhibitory neuronal clusters were annotated based on their genetic markers (i.e., Sst and Npy) that code for distinct neuropeptides.

[0170] Identification of region-enriched translated genes: RNA amplicons excluded by ClusterMap in single-cell quantification were treated as RNA outside the soma region of the cell. RNA counts for each gene in both the soma and process regions were calculated and normalized to a percentage based on the total RNA count for each gene. The top 10% genes with the highest RNA percentage in each region were annotated as region-enriched translated genes, and the spatial distribution of their RNA was visualized on brain sections.

[0171] Gene Ontology (GO) enrichment analysis The DAVID database (david.ncifcrf.gov) (45, 46) was used for GO enrichment analysis. Benjamini and Hochberg false discovery rate (FDR) was performed to adjust the p-value in multiple hypergeometric tests. Results from biological process (BP) and cellular component (CC) (FDR < 0.05) were selected as enriched GO terms. The top 3 most significantly enriched GO terms were used for covariation analysis and gene colocalization investigation, while the top 10 enriched terms were extracted in the translation genes enriched in the cell body and the translation genes enriched in the processes.

[0172] References 1.JAAlfaro,P.Bohlander,M.Dai,M.Filius,CJHoward,XFvan Kooten,S.Ohayon,A.Pomorski,S.Schmid,A.Aksimentiev,EVAnslyn,G.Bedran,J.Cao,M.Chinappi,E.Coyau d,J.Dekker,G.Dittmar,N.Drachman,R.Eelkema,D.Goodlett,S.Hentz,U.Kalathia,NLKelleher,RTKelly,Z .Kelman,ShKim,B.Kuster,D.Rodriguez-Larrea,S.Lindsay,G.Maglia,EMMarcotte,JPMarino,J.Masselon,M.Mayer,P.Samaras,K.Sarthak,L.Sepiashvili,D.Stein,M.Wanunu,M.Wilhelm,P.Yin,A.Joller,J. emerging landscape of single-molecule protein sequencing technologies.Nat.Methods.18,604-617(2021).

[0173] 2.T.Maier,M.Guell,L.Serrano,Correlation of mRNA and protein in complex biological samples.FEBS Lett.583,3966-3973(2009).

[0174] 3.R.de Sousa Abreu,LOPenalva,EMMarcotte,C.Vogel,Global signatures of protein and mRNA expression levels.Mol.Biosyst.5,1512-1526(2009).

[0175] 4.C.Vogel,E.M.Marcotte,Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.Nat.Rev.Genet.13,227-232(2012).

[0176] 5.B.Schwanhausser,D.Busse,N.Li,G.Dittmar,J.Schuchhardt,J.Wolf,W.Chen,M.Selbach,Global quantification of mammalian gene expression control.Nature.473,337-342(2011).

[0177] 6.S.Das,M.Vera,V.Gandin,R.H.Singer,E.Tutucci,Intracellular mRNA transport and localized translation.Nat.Rev.Mol.Cell Biol.22,483-504(2021).

[0178] 7.N.T.Ingolia,S.Ghaemmaghami,J.R.S.Newman,J.S.Weissman,Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.Science.324,218-223(2009).

[0179] 8.C.H.Jan,C.C.Williams,J.S.Weissman,Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science(2014),doi:10.1126 / science.1257521.

[0180] 9.C.C.Williams,C.H.Jan,J.S.Weissman,Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling.Science.346,748-751(2014).

[0181] 10.M.VanInsberghe,J.van den Berg,A.Andersson-Rolf,H.Clevers,A.van Oudenaarden,Single-cell Ribo-seq reveals cell cycle-dependent translational pausing.Nature.597,561-565(2021).

[0182] 11.N.T.Ingolia,Ribosome Footprint Profiling of Translation throughout the Genome.Cell.165,22-33(2016).

[0183] 12.Y.Liu,A.Beyer,R.Aebersold,On the Dependency of Cellular Protein Levels on mRNA Abundance.Cell.165,535-550(2016).

[0184] 13.J.M.Halstead,T.Lionnet,J.H.Wilbertz,F.Wippich,A.Ephrussi,R.H.Singer,J.A.Chao,Translation.An RNA biosensor for imaging the first round of translation from single cells to living animals.Science.347,1367-1671(2015).

[0185] 14.B.Wu,C.Eliscovich,Y.J.Yoon,R.H.Singer,Translation dynamics of single mRNAs in live cells and neurons.Science.352,1430-1435(2016).

[0186] 15.T.Morisaki,K.Lyon,K.F.DeLuca,J.G.DeLuca,B.P.English,Z.Zhang,L.D.Lavis,J.B.Grimm,S.Viswanathan,L.L.Looger,T.Lionnet,T.J.Stasevich,Real-time quantification of single RNA translation dynamics in living cells.Science.352,1425-1429(2016).

[0187] 16.X.Yan,T.A.Hoek,R.D.Vale,M.E.Tanenbaum,Dynamics of Translation of Single mRNA Molecules In Vivo.Cell.165,976-989(2016).

[0188] 17.S.Boersma,D.Khuperkar,B.M.P.Verhagen,S.Sonneveld,J.B.Grimm,L.D.Lavis,M.E.Tanenbaum,Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding.Cell.178,458-472.e19(2019).

[0189] 18.Z.B.Katz,B.P.English,T.Lionnet,Y.J.Yoon,N.Monnier,B.Ovryn,M.Bathe,R.H.Singer,Mapping translation「hot-spots」in live cells by tracking single molecules of mRNA and ribosomes.Elife.5,e10415(2016).

[0190] 19.S.T.Dieck,L.Kochen,C.Hanus,M.Heumuller,I.Bartnik,B.Nassim-Assir,K.Merk,T.Mosler,S.Garg,S.Bunse,D.A.Tirrell,E.M.Schuman,Direct visualization of newly synthesized target proteins in situ.Nature Methods.12(2015),pp.411-414.

[0191] 20.X.Wang,W.E.Allen,M.A.Wright,E.L.Sylwestrak,N.Samusik,S.Vesuna,K.Evans,C.Liu,C.Ramakrishnan,J.Liu,G.P.Nolan,F.-A.Bava,K.Deisseroth,Three-dimensional intact-tissue sequencing of single-cell transcriptional states.Science.361(2018),doi:10.1126 / science.aat5691.

[0192] 21.I.Tirosh,B.Izar,SMParade,MHWadsworth 2nd, D. Treacy, JJ Trombetta, A. Rotem, C. Rodman, C. Lian, G. Murphy, M. Fallahi-Sichani, K. Dutton-Regester, J.-R. Lin, O. Cohen, P.Shah,D.Lu,ASGenshaft,TKHughes,CGKZiegler,SWKazer,AGaillard,KEKolb,A-C Villani,CMJohannessen,AYAndreev,EMVan Allen,M.Bertagnolli,PKSorger,RJSullivan,KTFlaherty,DTFrederick,JJane-Valbuena,CHYoon,ORozenblatt-Rosen,AKShalek,ARegev,LAGarraway,Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.Science.352,189–196(2016).

[0193] 22.FMFazal,S.Han,KRParker,P.Kaewsapsak,J.Xu,ANBoettiger,HYChang,AYTing,Atlas of Subcellular RNA Localization Revealed by APEX-Seq.Cell.178,473-490.e26(2019).

[0194] 23. X. Wang, Z. Lu, A. Gomez, GCHon, Y. Yue, D. Han, Y. Fu, M. Parisen, Q. Dai, G. Jia, B. Ren, T. Pan, C. He, N.S 6 -methyladenosine-dependent regulation of messenger RNA stability.Nature.505,117–120(2014).

[0195] 24.H.Shi,X.Wang,Z.Lu,B.S.Zhao,H.Ma,P.J.Hsu,C.Liu,C.He,YTHDF3 facilitates translation and decay of N 6 -methyladenosine-modified RNA.Cell Res.27,315-328(2017).

[0196] 25.A.Sakaue-Sawano,H.Kurokawa,T.Morimura,A.Hanyu,H.Hama,H.Osawa,S.Kashiwagi,K.Fukami,T.Miyata,H.Miyoshi,T.Imamura,M.Ogawa,H.Masai,A.Miyawaki,Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.Cell.132,487-498(2008).

[0197] 26.S.-B.Koh,P.Mascalchi,E.Rodriguez,Y.Lin,D.I.Jodrell,F.M.Richards,S.K.Lyons,Quantitative FastFUCCI assay defines cell cycle dynamics at single-cell level.Journal of Cell Science.130(2),512-520(2016)doi:10.1242 / jcs.195164.

[0198] 27.D.Mahdessian,A.J.Cesnik,C.Gnann,F.Danielsson,L.Stenstrom,M.Arif,C.Zhang,T.Le,F.Johansson,R.Shutten,A.Backstrom,U.Axelsson,P.Thul,N.H.Cho,O.Carja,M.Uhlen,A.Mardinoglu,C.Stadler,C.Lindskog,B.Ayoglu,M.D.Leonetti,F.Ponten,D.P.Sullivan,E.Lundberg,Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.Nature.590,649-654(2021).

[0199] 28.B.S.Zhao,I.A.Roundtree,C.He,Post-transcriptional gene regulation by mRNA modifications.Nat.Rev.Mol.Cell Biol.18,31-42(2017).

[0200] 29.A.Saunders,E.Z.Macosko,A.Wysoker,M.Goldman,F.M.Krienen,H.de Rivera,E.Bien,M.Baum,L.Bortolin,S.Wang,A.Goeva,J.Nemesh,N.Kamitaki,S.Brumbaugh,D.Kulp,S.A.McCarroll,Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain.Cell.174,1015-1030.e16(2018).

[0201] 30.Z.Yao,CTJvan Velthoven,TNNguyen,J.Goldy,AESedeno-Cortes,F.Baftizadeh,D.Bertagnolli,T.Casper,M.Chiang,K.Crichton,S.-L.Ding,O.Fong,E.Garren,A.Glandon,NWGouwens,J.Gray,MLTGHawry,DJD. .Hirschstein,M.Kroll,K.Lathia,J.Lee,B.Levy,D.McMillen,S.Mock,T.Pham,Q.Ren,J.Rimorin,N.Sha povalova,J.Sulc,SMSunkin,M.Tieu,A.Torkelson,H.Tung,K.Ward,N.Dee,KASmith,B.Tasic,H.Zeng,A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation.Cell.184(12),3222-3241(2021),doi:10.1016 / j.cell.2021.04.021.

[0202] 31.A.Zeisel,H.Hochgerner,P.Lonnerberg,A.Johnsson,F.Memic,J.van der Zwan,M.Haring,E.Braun,LEBorm,G.La Manno,S.Codeluppi,A.Furlan,K.Lee,N.Skene,KDHarris,J.Hjerling-Leffler,E.Arenas,P.Ernfors,U.Marklund,S.Linnarsson,Molecular Architecture of the Mouse Nervous System.Cell.174,999-122(2012).

[0203] 32.Tabula Muris Consortium,Overall coordination,Logistical coordination,Organ collection and processing,Library preparation and sequencing,Computational data analysis,Cell type annotation,Writing group,Supplemental text writing group,Principal investigators,Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.Nature.562,367-372(2018).

[0204] 33.Tabula Muris Consortium,A single-cell transcriptomic atlas characterizes ageing tissues in the mouse.Nature.583,590-595(2020).

[0205] 34.X.Han,R.Wang,Y.Zhou,L.Fei,H.Sun,S.Lai,A.Saadatpour,Z.Zhou,H.Chen,F.Ye,D.Huang,Y.Xu,W.Huang,M.Jiang,X.Jiang,J.Mao,Y.Chen,C.Lu,J.Xie,Q.Fang,Y.Wang,R.Yue,T.Li,H.Huang,S.H.Orkin,G.-C.Yuan,M.Chen,G.Guo,Mapping the Mouse Cell Atlas by Microwell-Seq.Cell.173,1307(2018).

[0206] 35.J.Cao,M.Spielmann,X.Qiu,X.Huang,DMIbrahim,AJHill,F.Zhang,S.Mundlos,L.Christiansen,FJSteemers,C.Trapnell,J.Shendure,The single-cell transcriptional landscape of mammalian organogenesis.Nature.566,496-502(2019).

[0207] 36.ESLein,MJHawrylycz,N.Ao,M.Ayres,A.Bensinger,A.Bernard,AFBoe,MSBoguski,KSBrockway,EJByrnes,L.Chen,L.Chen, T.-M.Chen,MCChin,J.Chong,BECrook,A.Czaplinska,CNDang,S.Datta,NRDee,ALDesaki,T.Desta,E.Diep,TADolbeare,MJDone lan,H.-W.Dong,JGDougherty,BJDuncan,AJEbbert,G.Eichele,LKEstin,C.Faber,BAFacer,R.Fields,SRFischer,TPFliss,C.F rensley,SNGates,KJGlattfelder,KRHalverson,MRHart,JGHohmann,MPHowell,DPJeung,RAJohnson,PTKarr,R.Kawal,JMKidne y,RHKnapik,CLKuan,JHLake,ARLaramee,KDLarsen,C.Lau,TALemon,AJLiang,Y.Liu,LTLuong,J.Michaels,JJMorgan,RJMorga n,MTMortrud,NFMosqueda,LLNg,R.Ng,GJOrta,CCOverly,THPak,SEParry,SDPathak,OCPearson,RBPuchalski,ZLRiley,HRRock ett,SARowland,JJRoyall,MJRuiz,NRSarno,K.Schaffnit,NVShapovalova,T.Sivisay,CRSlaughterbeck,SCSmith,KASmith,BI Smith,AJSodt,NNStewart,K.-R.Stumpf,SMSunkin,M.Sutram,A.Tam,CDTeemer,C.Thaller,CLThompson,LRVarnam,A.Wisel,RMWhitlock,P.E.Wohnoutka,C.K.Wolkey,V.Y.Wong,M.Wood,M.B.Yaylaoglu,R.C.Young,B.L.Youngstrom,X.F.Yuan,B.Zhang,T.A.Zwingman,A.R.Jones,Genome-wide atlas of gene expression in the adult mouse brain.Nature.445,168-176(2007).

[0208] 37.M.S.Fernandopulle,J.Lippincott-Schwartz,M.E.Ward,RNA transport and local translation in neurodevelopmental and neurodegenerative disease.Nat.Neurosci.24,622-632(2021).

[0209] 38.A.Biever,C.Glock,G.Tushev,E.Ciirdaeva,T.Dalmay,J.D.Langer,E.M.Schuman,Monosomes actively translate synaptic mRNAs in neuronal processes.Science.367(2020),doi:10.1126 / science.aay4991.

[0210] 39.C.Glock,A.Biever,G.Tushev,B.Nassim-Assir,A.Kao,I.Bartnik,S.Tom Dieck,E.M.Schuman,The translatome of neuronal cell bodies,dendrites,and axons.Proc.Natl.Acad.Sci.U.S.A.118(2021),doi:10.1073 / pnas.2113929118.

[0211] 40.L.M.Meservey,V.V.Topkar,M.-M.Fu,mRNA Transport and Local Translation in Glia.Trends Cell Biol.31,419-423(2021).

[0212] 41.K.Sakers,A.M.Lake,R.Khazanchi,R.Ouwenga,M.J.Vasek,A.Dani,J.D.Dougherty,Astrocytes locally translate transcripts in their peripheral processes.Proc.Natl.Acad.Sci.U.S.A.114,E3830-E3838(2017).

[0213] 42.C.Muller,N.M.Bauer,I.Schafer,R.White,Making myelin basic protein-from mRNA transport to localized translation.Frontiers in Cellular Neuroscience.7(2013)doi:10.3389 / fncel.2013.00169.

[0214] 43.Y.He,X.Tang,J.Huang,J.Ren,H.Zhou,K.Chen,A.Liu,H.Shi,Z.Lin,Q.Li,A.Aditham,J.Ounadjela,E.I.Grody,J.Shu,J.Liu,X.Wang,ClusterMap for multi-scale clustering analysis of spatial gene expression.Nat.Commun.12,5909(2021).

[0215] 44.KSBurke,KAAntilla,DATirrell,A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome-mRNA Interactions in Single Cells.ACS Cent.Sci.3,425-433(2017).

[0216] 45.DWHuang,BTSherman,RALempicki,Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat.Protoc.4,44-57(2009).

[0217] 46. ​​DW Huang, BT Sherman, RALempicki, Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1-13 (2008).

[0218] 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 embodiments 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.

[0219] Equivalents and Scope Articles such as "a," "an," and "the" can mean one or more than one, 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, used 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, used 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, used in, or otherwise related to a given product or process.

[0220] 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 of the disclosure or an 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 embodiments of the invention, down to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0221] 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 embodiment of the invention that falls within the prior art may be expressly excluded from any one or more of the embodiments. Because such embodiments 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 embodiment of the invention may be excluded from any embodiment for any reason, whether or not related to the existence of prior art.

[0222] 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. A method for profiling RNA being translated in a cell, the method comprising: a) contacting the cell with one or more sets of probes, each set of probes comprising a first probe, a second probe, and a third probe, i) the first probe comprising an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to the RNA of interest, and a second oligonucleotide barcode sequence; ii) the second probe comprising a portion that recognizes a ribosome and an oligonucleotide portion complementary to a portion of the first probe; and iii) the third probe comprising an oligonucleotide portion complementary to the RNA of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe, contacting the cell with one or more sets of probes; b) ligating the 5' and 3' ends of the first probe together to generate a circular oligonucleotide; c) performing rolling circle amplification and amplifying the circular oligonucleotide using the third probe as a primer to generate one or more concatenated amplicons; d) embedding the 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 RNA of interest in the cell The method comprising.

2. The portion of the second probe that recognizes a ribosome is 1) an agent that binds to an antibody, or an antibody variant or fragment; 2) an antibody, or an antibody variant or fragment; or 3) an oligonucleotide complementary to a portion of the rRNA within the ribosome The method according to claim 1, comprising.

3. The portion of the second probe that recognizes a ribosome comprises an antibody, wherein the antibody is a secondary antibody, and The method further comprises contacting the cells with a primary antibody that can recognize ribosomes and be bound by a secondary antibody of a second probe. The method according to claim 2.

4. The method according to claim 3, wherein the primary antibody is an anti-40S ribosomal protein S3 (RPS3) antibody or an anti-60S ribosomal protein L4 (RPL4) antibody.

5. The method according to claim 2, wherein the portion of the second probe that recognizes ribosomes comprises an oligonucleotide complementary to a portion of 18S rRNA.

6. The method according to claim 1, wherein the second probe further comprises a polymerization blocker.

7. (i) The first probe has the structure: 5'-[portion complementary to the second probe]-[first barcode sequence -[portion complementary to the third probe]-[portion complementary to the RNA of interest]-[second barcode sequence]-3' and comprises; (ii) The second probe has the structure: 5'-[portion that recognizes ribosomes]-[portion complementary to the first probe]-3' and comprises; and / or (iii) The third probe has the structure: 5'-[portion complementary to the RNA of interest -[portion complementary to the first probe]-[barcode sequence]-3' and comprises, The method according to claim 1.

8. The method according to claim 1, wherein the portion of the first probe that is complementary to the portion of the second probe is split between the 5' and 3' ends of the first probe.

9. The method according to any one of claims 1 to 8, wherein the translated RNA is profiled simultaneously in a large number of cells.

10. The method according to claim 9, wherein the cells comprise a plurality of cell types.

11. The method according to any one of claims 1 to 8, wherein the cells are present within intact tissue.

12. The method according to any one of claims 1 to 8, wherein the translation of more than 1000 RNAs is profiled simultaneously.

13. The method according to any one of claims 1 to 8, wherein the second oligonucleotide barcode sequence of the first probe is a gene-specific sequence used to identify the RNA of interest.

14. The method according to any one of claims 1 to 8, wherein the sequencing step comprises performing sequencing by error reduction by dynamic annealing and ligation (SEDAL).

15. The method according to any one of claims 1 to 8, wherein the step of performing rolling circle amplification further comprises providing an amine-modified nucleotide, and the amine-modified nucleotide is incorporated into one or more concatenated amplicons.

16. The method according to claim 15, wherein the step of embedding one or more concatenated amplicons in a polymer matrix comprises reacting the amine-modified nucleotides of the one or more amplicons with N-hydroxysuccinimide ester of acrylic acid, and copolymerizing the one or more concatenated amplicons and the polymer matrix.

17. The method according to any one of claims 1 to 8, wherein the method does not disrupt the spatial information of intracellular structures, cell morphology, and / or tissue constitution.

18. A set of probes comprising a first probe, a second probe, and a third probe, i) The first probe comprises an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to the RNA of interest, and a second oligonucleotide barcode sequence; ii) The second probe comprises a portion that recognizes ribosomes and an oligonucleotide portion complementary to a portion of the first probe; and iii) The third probe comprises an oligonucleotide portion complementary to the RNA of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, and the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe, Said set of probes.

19. A plurality of probes comprising a plurality of sets of the probes according to claim 18, wherein each set of probes comprises an oligonucleotide portion complementary to a different RNA of interest, said plurality of probes.

20. A system for profiling RNA being translated in a cell, comprising a) a cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, i) The first probe includes an oligonucleotide portion complementary to the second probe, a first oligonucleotide barcode sequence, an oligonucleotide portion complementary to a portion of the third probe, an oligonucleotide portion complementary to the RNA of interest, and a second oligonucleotide barcode sequence; ii) The second probe includes a portion that recognizes ribosomes and an oligonucleotide portion complementary to a portion of the first probe; and iii) The third probe includes an oligonucleotide portion complementary to the RNA of interest, an oligonucleotide portion complementary to a portion of the first probe, and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the third probe is complementary to the first oligonucleotide barcode sequence of the first probe, One or more sets of probes including the first probe, the second probe, and the third probe; c) A microscope; and d) A computer A system comprising the same.

Images