Spatial visualization of adenosine-to-inosine editing in cells

Abstract

Endonuclease V Immunostaining Assay (EndoVIA) is provided herein as the first approach for quantifying and visualizing the landscape of Adenosine-to-Inosine (A-to-I) edited RNAs in situ. EndoVIA provides rapid quantification of overall inosine abundance and allows cell-to-cell comparison of A-to-I editing levels without the need for RNA sequencing. EndoVIA contributes valuable new insights into the dynamic interplay between A-to-I editing and subcellular localization that are undetectable with currently available approaches.

Description

SPATIAL VISUALIZATION OF ADENOSINE-TO-INOSINE EDITING IN CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 661,727 filed on June 19, 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under GM144075 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The present disclosure generally relates to characterization of Adenosine-to- Inosine (A-to-I) editing in cells.BACKGROUND

[0004] Adenosine-to-Inosine (A-to-I) editing is one of the most widespread post- transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity.BRIEF DESCRIPTION OF THE DISCLOSURE

[0005] Among the various aspects of the present disclosure is the provision of a workflow process for A-to-I editing visualization and characterization, including for drug target identification.

[0006] [Independent claims will be added here once finalized ]

[0007] Other objects and features will be in part apparent and in part pointed out hereinafter.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Those of skill in the art will understand that the drawings described herein are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0009] FIG. 1 is a schematics showing A-to-I editing impacts essential biological functions. In FIG. 1(a), ADAR enzy mes catalyze adenosine-to-inosine RNA editing, recoding edited sites to be read as guanosine to cellular machinery. In FIG. 1(b) Loss of A-to-I editing in endogenous RNAs triggers the innate immune response, synonymous to viral infections. In FIG. 1 (c) Presence of extensively edited 3’ UTR in mRNA results in nuclear retention to paraspeckles.

[0010] FIG 2 shows Detection of inosine-containing RNA in cells using Endo VIA. in relation to the current state of the art detecting inosine via sequencing (a) RNA is isolated from cells and then sequenced to identify A-G transitions between RNA sequencing reads (purple) and a reference genome (blue), thus mapping A-to-I edited sites, (b) EndoVIA detects cell-to- cell variation in A-to-I editing and localization of edited transcripts at the nanoscopic level, (c) EndoVIA workflow; cells are fixed, treated with glyoxal (blue pentagons) to denature RNA secondary structure, prepped, and stained using EndoV and a series of antibodies to detect A- to-I edited sites in situ.

[0011] FIG. 3 shows binding curves of anti-inosine antibodies. Quantification of the MBL anti-inosine antibody and the Diagenode anti-inosine antibody binding affinity towards ssRNA I (red) and ssRNA A (black) using MST. Values represent mean with standard deviation, and id denotes mean with 95% confidence interval. Data are representative of three independent experiments; n=3 individual trials.

[0012] FIG. 4 shows fluorescence in situ hybridization (FISH) of tRNA using different fixatives. HEK293T cells were fixed with either formaldehyde or methanol and stained for tRNA (green) and cell nuclei (blue). Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 200 pm.

[0013] FIG. 5 shows detection of tRNA in formaldehyde-fixed and methanol -fixed cells, (a) HEK293T and G-402 cells were fixed with either formaldehyde or methanol and stained for tRNA (green) and cell nuclei (blue), (b) Close-up view of images in (a) with nuclear puncta identified using white triangles. Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, (a) 50 pm and (b) 25 pm.

[0014] FIG. 6 shows quantification of residual tRNA in methanol-fixed cells. tRNA fluorescence in Figure 5 was quantified for HEK293T (black) and G-402 (gray) cells fixed with formaldehyde or methanol. Data are representative of three independent experiments; n 3 wells from a 96-well plate. Data are shown as mean ± s.d. in arbitrary units (A.U.). Statistical significance was determined by unpaired / -test; **P < 0.01, ***P < 0.001.

[0015] FIG. 7 shows a summary of EndoVIA workflow. Cultured cells are fixed and permeabilized with 100% ice cold methanol follow ed by w ashing to remove tRNA. Fixed cells are then treated with a 12% glyoxal solution (blue hexagons) to denature RNA secondary structure. Following subsequent permeabilization and blocking steps, cells are incubated with a 1 :50 EndoV calcium-containing solution to bind inosine-containing transcripts. Following EndoV treatment, cells are incubated with a 1 : 1000 anti-MBP antibody calcium-containing solution to bind the MBP tag fused to EndoV. Finally, cells are stained with a secondary antibody conjugated to Alexa Fluor 647 that is specific for the anti-MBP antibody. Cells are next imaged using fluorescence microscopy and analyzed.

[0016] FIG. 8 shows detection of Nupl53 and (3-actin using modified immunofluorescence w orkflow; (a) HEK293T cells w ere fixed and stained for Nupl53 (red) or P-actin (green) and cell nuclei (blue). Images were taken using widefield microscopy, (b) HEK293T cells were prepared as previously described in (b) and imaged using confocal microscopy. Data are representative of three independent experiments; n=3 wells from a 96- well plate. Scale bar, (a) 200 pm and (b) 50 pm.

[0017] FIG. 9 show s non-specific binding of MBP of the EndoV -MBP fusion protein. HEK293T cells were fixed and stained for edited RNA (red) and cell nuclei (blue) using EndoV-MBP fusion protein (+ EndoV), MBP alone (+ MBP), or neither (-). Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 200 pm.

[0018] FIG. 10 shows optimization of glyoxal concentration using GAPDH fluorescence in situ hybridization (FISH). HEK293T cells were fixed and treated with increasing amounts of glyoxal (3%, 6%, 12%, 30%) and stained for GAPDH mRNA using FISH probes (red) and quantified for mean corrected total cellular fluorescence (CTCF). Data are representative of three independent experiments; n=50 cells. Scale bar, 50 pm. Data are shown as CTCF per cell in arbitrary units (A.U.). Statistical significance was determined by one-way ANOVA; not significant (ns), ***P < 0.001, ****p < 0.0001.

[0019] FIG. 11 shows cellular morphology of cells treated with varying glyoxal concentrations using (3-actin. HEK293T cells were fixed and treated with increasing amounts of glyoxal (3%, 12%, 30%) and stained for p-actin (green) and cell nuclei (blue). Data are representative of three independent experiments. Scale bar, 200 pm.

[0020] FIG. 12 shows optimization of EndoV concentration. HEK293T cells were fixed and stained for edited RNA (red) with increasing amounts of EndoV or MBP and quantified for mean corrected total cellular fluorescence (CTCF). Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 200 pm. Data are shown as mean ± s.d. in arbitrary units (A.U.). Statistical significance was determined by oneway ANOVA; not significant (ns), *P < 0.05, **P < 0.01 , ***P < 0.001.

[0021] FIG. 13 shows antibody negative control for confocal imaging. HEK293T cells were fixed and stained using EndoVIA with (+) or without (-) EndoV, primary, and secondary antibody. Data are representative of three independent experiments; n=3 coverslips. Scale bar, 10 pm.

[0022] FIG. 14 shows EndoV stained cells treated with EDTA. HEK293T cells were fixed and stained for edited RNA, treated with EDTA, and quantified for mean corrected total cellular fluorescence (CTCF). Data are representative of three independent experiments; n=3 wells from a 96-well plate. Data are shown as mean ± s.d. in arbitrary units (A. U.). Statistical significance was determined by unpaired / -test; *P < 0.05.

[0023] FIG. 15 shows immunostaining AD ARI. WT HEK293T cells and AD ARI KO cells were fixed and stained for AD ARI. Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar. 200 pm.

[0024] FIG. 16 shows staining edited RNA in cells with varying levels of A-to-I editing, (a) Fixed WT and ADAR1 KO HEK293T cells were immunostained using the optimized EndoVIA workflow for edited RNA (red) and cell nuclei (blue), (b) Quantification of mean corrected total cellular fluorescence (CTCF) in (a). Data in (a) and (b) are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 200 pm. Data are shown as mean ± s.d. in arbitrary units (A.U.). Statistical significance was determined by unpaired / -test; **P < 0.01.

[0025] FIG. 17 shows AEI Values of WT and AD ARI KO HEK293T cells. Total RNA was isolated, purified, and sequenced. Resulting datasets were then trimmed, aligned, and sorted to calculate the AEI values.

[0026] FIG. 18 shows detection of an increase in A-to-I editing in HEK293T cells, (a) HEK293T cells were transfected with increasing amounts of GFP tagged ADAR1 pl 10 plasmid (0-200 ng, green), fixed, and stained for edited RNA (red) using EndoVIA. (b) Quantification of mean corrected total cellular fluorescence (CTCF) of edited RNA in ADAR- GFP positive (+) and negative (-) cells in (a). Data in (a) and (b) are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 200 pm. Data are shown as mean ± s.d. in arbitrary units (A.U.). Statistical significance was determined by unpaired t- test; **** / > < 0.0001.

[0027] FIG. 19 shows detection of an increase in A-to-I editing in HEK293T cells. HEK293T cells were transfected with increasing amounts of GFP tagged ADAR1 pl 50 plasmid (0-200 ng), fixed, and stained for edited RNA (red) using EndoVIA. Quantification of mean corrected total cellular fluorescence (CTCF) of edited RNA was performed in ADAR- GFP positive (+) and negative (-) cells. Data are representative of three independent experiments; n=3 wells from a 96-well plate. Data are shown as mean ± s.d. in arbitrary units (A.U.). Statistical significance was determined by unpaired / -test; ****P < 0.0001.

[0028] FIG. 20 show s quantification of A-to-I editing in HEK293T cells transfected with coilin-GFP. HEK293T cells were transfected with increasing amounts of coilin-GFP plasmid (0-500 ng), fixed, and stained for edited RNA (red) using EndoVIA. Coilin-GFP and edited RNA fluorescence were quantified for mean corrected total cellular fluorescence (CTCF). Data are representative of three independent experiments; n=3 w ells from a 96-well plate. Data are shown as mean ± s.d. in arbitrary units (A.U.).

[0029] FIG. 21 shows identification of hyper- and hypo-editing in cancerous breast and kidney cells, (a) MCF10A cells (non-malignant) and ZR-75-1 cells (malignant) and (b) HEK293T cells (non-malignant) and G-402 cells (malignant) w ere fixed and stained for edited RNA (red) and cell nuclei (blue) using EndoVIA. (c) Quantification of mean corrected total cellular fluorescence (CTCF) in (a), (d) Quantification of mean corrected total cellular fluorescence (CTCF) in (b). Data in (a-d) are representative of three independent experiments; n=9 wells from a 96-well plate. Scale bar, 200 pm. Data are shown as mean ± s.d. in arbitrary units (A.U.). Statistical significance was determined by unpaired / -test; ***P < 0.001, ****P < 0.0001.

[0030] FIG. 22 shows quantification of mRNA in non-malignant and malignant cell lines. The mRNA from (a) breast cell lines MCF10A and ZR-75-1 and (b) kidney cell lines HEK293T and G-402 were isolated and quantified. Data are representative of threeindependent experiments; n=3 wells from a 6-well plate. Data are shown as mean ± s.d. Statistical significance was determined by unpaired / -test; not significant (ns).

[0031] FIG. 23 shows cellular heterogeneity in non-malignant and malignant cell lines. The kernel density estimation was determined for (a) HEK293T (green) and G-402 (red) cells and (b) MCF10A (green) and ZR-75-1 (red) cells using the CTCF values of each individual cell. Data are representative of three independent experiments; n=9 wells from a 96-well plate.

[0032] FIG. 24 shows immunostaining dsRNA and edited RNA in healthy and diseased cells. Fixed HEK293T and G-402 cells were immunostained for dsRNA (J2. KI) or edited RNA (EndoVIA). Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 50 pm.

[0033] FIG. 25 shows super-resolution microscopy of edited RNA using EndoVIA. TIRF (dark images) and TIRF-dSTORM images (light images, whole cell and close-up view of boxed regions respectively) from (a) HEK293T cells and (b), adhesion site of HEK293T cells and (c) G-402 cells labeled with the described immunostaining workflow. Data in (a-c) are representative of three independent experiments; n=3 coverslips. Scale bar, 10 pm.

[0034] FIG. 26 shows antibody negative control for dSTORM in TIRF illumination imaging. HEK293T cells were fixed and stained using EndoVIA with (+) or without (-) EndoV, primary, and secondary antibody. Data are representative of three independent experiments; n=3 coverslips. Scale bar, 10 pm.

[0035] FIG. 27 shows immunostaining AD ARI in HEK293T and G-402 cells. Fixed HEK293T and G-402 cells were immunostained for AD ARI. Data are representative of three independent experiments; n=3 wells from a 96-well plate. Scale bar, 50 pm.DETAILED DESCRIPTION OF THE DISCLOSURE

[0036] State-of-the-art A-to-I editing approaches mainly rely on in vitro characterization of extracted and pooled RNA. which ultimately erases subcellular localization and cell-to-cell variation, both of which are arguably critical to fully understanding the impact of editing on disease. An approach for detecting and quantifying edited RNA in situ remains an unmet challenge.

[0037] To address this need, we have repurposed Endonuclease V (EndoV), a magnesium dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces Endonuclease V Immunostaining Assay (EndoVIA), a workflow that provides spatial visualization of editedtranscripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

[0038] As disclosed herein, EndoVIA is the first approach for quantifying and visualizing the landscape of A-to-I edited RNAs in situ. EndoVIA provides rapid quantification of overall inosine abundance and allows cell-to-cell comparison of A-to-I editing levels without the need for RNA sequencing. EndoVIA contributes valuable new- insights into the dynamic interplay between A-to-I editing and subcellular localization that are undetectable with currently available approaches.

[0039] Among the objects of the disclosure are methods of detecting ribonucleic acid (RNA) containing one or more inosine bases in cells comprising: fixing cells to retain large RNAs and not retain small RNAs to form fixed cells; denaturing the secondary structure of the large RNAs to form denatured RNA; incubating the denatured large RNAs with a calcium- containing solution of endonuclease V (EndoV) to form a complex between EndoV and RNA; tagging EndoV with a fluorophore; imaging the cells using fluorescence microscopy to detect the RNA containing one or more inosine bases.

[0040] The methods of detecting RNA containing one or more inosine bases described herein can have the tagging EndoV with a fluorophore comprise using EndoV fused with an affinity tag (EndoV-affinity tag) and conjugating a fluorophore to the affinity tag.

[0041] Also, the methods of detecting RNA containing one or more inosine bases can have the tagging EndoV with a fluorophore comprise using EndoV fused with an affinity tag (EndoV-affinity tag) and forming a complex between EndoV-affinity tag and RNA; incubating the complex of EndoV-affinity tag and RNA with an antibody for the affinity tag conjugated to the fluorophore to form a complex of EndoV-affinity tag, antibody to the affinity tag, and RNA.

[0042] The methods of detecting RNA containing one or more inosine bases can have the tagging endo V with a fluorophore comprise using EndoV fused with an affinity tag (EndoV-affinity tag) and forming a complex between EndoV-affinity tag and RNA; incubating the complex of EndoV-affinity tag and RNA with an antibody for the affinity tag to form a complex of EndoV-affinity tag, antibody to the affinity tag, and RNA; and incubating the complex of EndoV-affinity tag, antibody to the affinity tag. and RNA with a secondary antibody specific for the antibody to the affinity tag and conjugated to a fluorophore.

[0043] The methods of detecting RNA containing one or more inosine bases in cells disclosed herein can have the cells be fixed using methanol, glyoxal, formaldehyde, or paraformaldehyde. Preferably, the cells are fixed with methanol.

[0044] The methanol used as the cell fixing agent can be 80%, 90%, or 100% methanol. Preferably, 100% methanol is used. Additionally, preferably ice cold 100% methanol is used as the fixing agent.

[0045] The cells can be fixed by incubating the cells with ice cold 100% methanol at from about 0°C to about 10°C, from about 0°C to about 5°C, from about 1°C to about 5°C, from about 2°C to about 5°C, from about 3°C to about 5°C. or about 4°C.

[0046] The methods of detecting RNA containing one or more inosine bases in cells can have large RNAs have at least 100 nucleotides. The large RNAs can have from 100 nucleotides to 10,000 nucleotides, from 100 nucleotides to 5000 nucleotides, from 100 nucleotides to 4000 nucleotides, from 100 nucleotides to 3000 nucleotides, from 100 nucleotides to 2000 nucleotides or from 100 nucleotides to 1000 nucleotides.

[0047] The methods of detecting RNA containing one or more inosine bases in cells can have small RNAs have less than 100 nucleotides. The small RNAs can have from 50 to 100 nucleotides, from 60 to 100 nucleotides, from 70 to 100 nucleotides, or from 70 to 95 nucleotides.

[0048] The methods of detecting RNA containing one or more inosine bases in cells can have denaturing the secondary7structure of the large RNAs comprise treating the cells with a glyoxal solution, a urea solution, a guanidinium solution, or a methylglyoxal solution. Preferably, the cells are denatured with a glyoxal solution.

[0049] The glyoxal solution can contain from about 4% to about 15% glyoxal, from about 4% to about 12% glyoxal, from about 8% to about 12% glyoxal, from about 10% to about 12% glyoxal or about 12% glyoxal.

[0050] The cells were incubated with the glyoxal solution for about 50 to about 70 minutes at a temperature of about 40°C to about 60°C, about 45°C to about 55°C, or 50°C.

[0051] The methods of detecting RNA containing one or more inosine bases in cells can have the secondary' structure of a RNA double strand be denatured to a RNA single strand.

[0052] Further, the methods of detecting RNA containing one or more inosine bases in cells can have the calcium-containing solution of EndoV not significantly cleave inosine- containing RNAs.

[0053] The methods of detecting RNA containing one or more inosine bases in cells can have the affinity tag be maltose-binding protein (MBP), a chitin binding protein (CBP), Strep-tag, glutathione-S-transferase (GST), poly(His), FLAG octapeptide, human influenza hemagglutinin (HA) tag, or cognate 13-amino acid peptide (SpyTag).

[0054] The affinity tags can also be those that are well known in the field. Generally, the above-referenced affinity’ tags are commercially available from ThermoFischer or MilliporeSigma.

[0055] Also, the methods of detecting RNA containing one or more inosine bases in cells can have the antibody to the affinity tag be anti-MBP, anti-CBD, anti-Strep tag, anti-GST, anti-His-tag, anti-FLAG, anti-HA, or Streptococcus pyogenes surface protein (SpyCatcher).

[0056] In some cases, the system of affinity tag and antibody to the affinity tag are sold in a kit. For example, the SpyTag / SpyCatcher system is commercially available from Bio-Rad.

[0057] The methods of detecting RNA containing one or more inosine bases in cells can have the secondary antibody specific for the anti-affinity tag antibody comprises an antibody specific for anti-MBP, anti-CBD, anti-Strep tag, anti-GST, anti-His-tag, anti-FLAG, anti-HA, or SpyTag.

[0058] In many cases, the secondary antibody to the anti-affinity tag antibodies are developed and are also commercially available.

[0059] For the methods of detecting RNA containing one or more inosine bases in cells can have the fluorophore comprise a fluorescent dye that absorbs light at a particular wavelength and emits light of longer wavelength (i.e.. it fluoresces).

[0060] The methods of detecting RNA containing one or more inosine bases in cells can further comprise permeabilizing the fixed cells by contacting with detergent. The detergent can be a surfactant including nonionic surfactants like octyl phenol ethoxylate having on average from 8 to 20 ethylene oxide units per ethoxylate group. When there are an average of 9.5 ethylene oxide groups in the octyl phenol ethoxylate, the surfactant is commercially available as Triton™ X-100. Additionally, polyoxyethylene sorbitan monolaurates having from 20 to 80 repeat units of polyethylene glycol can be used as the detergent. In some cases, polyoxyethylene sorbitan monolaurates having 20 repeat units of polyethylene glycol is used.

[0061] The methods of detecting RNA containing one or more inosine bases in cells can further comprise incubating the fixed cells with a protein blocking agent.

[0062] The protein blocking agent can comprise bovine serum albumin, normal serum, milk powder, gelatin, a protein solution, or a combination thereof. Preferably, bovine serum albumin is used.

[0063] The bovine serum albumin can have a concentration of from about 2% to about 4% or about 3% in the total solution.

[0064] Also, the methods of detecting RNA containing one or more inosine bases in cells can have the endo V-alTinity tag be EndoV fused with MBP that is a recombinant Escherichia coli EndoV -MBP.

[0065] The methods of detecting RNA containing one or more inosine bases in cells can have the abundance of RNA containing one or more inosine bases in a cell be quantified.

[0066] Additionally, the methods of detecting RNA containing one or more inosine bases in cells can have the location of the RNA containing one or more inosine bases in the cell is detected.

[0067] Further, methods of detecting RNA containing one or more inosine bases in cells can have the subcellular localization of Adenosine-to-Inosine (A-to-I) editing in RNA characterized.

[0068] Another aspect of the disclosure is a method of determining a disease or condition in a subject in need thereof comprising performing the methods of detecting RNA containing one or more inosine bases in cells described herein on a cell sample from the subject; identifying an abundance of RNA containing one or more inosine bases in the cell sample and comparing the abundance to the abundance in healthy cells; and determining a suitable treatment for the disease or condition based on the difference in the abundance of RNA containing one or more inosine bases in the cell sample.

[0069] A further aspect of the disclosure is a method of determining a disease or disorder treatment for a subject in need thereof, the method comprising performing the methods of detecting RNA containing one or more inosine bases in cells disclosed herein on a cell sample from the subject; identifying the subcellular localization of the RNA containing one or more inosine bases in the cell and comparing to the subcellular localization of the RNA containing one or more inosine bases in healthy cells; and determining a suitable treatment for the disease or condition based on the difference in the location of RNA containing one or more inosine bases in the cell sample.

[0070] The methods of determining a disease or disorder treatment described herein can have the disease or disorder be a disease or disorder wherein misregulation of A-to-I editing is implicated. In some instances, the disease or disorder can be cancer.

[0071] A kit for carrying out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein comprising a calcium-containing solution of endonuclease V (EndoV); a fluorophore; and instructions for conjugating the EndoV with the fluorophore, fixing cells, denaturing cells and imaging the cells using fluorescence microscopy.

[0072] The kit for carrying out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, wherein the EndoV is fused with an affinity tag (EndoV- affnity tag).

[0073] The kit for earn ing out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, wherein the affinity' tag is conjugated with the fluorophore.

[0074] The kit for carrying out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, further comprising an antibody to the affinity tag.

[0075] The kit for carry ing out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, wherein the antibody to the affinity tag is conjugated to the fluorophore.

[0076] The kit for carrying out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, further comprising a secondary' antibody specific for the antibody to the affinity tag and being conjugated to the fluorophore.

[0077] The kit for carrying out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, further comprising a cell fixing agent.

[0078] The kit for carrying out the methods of detecting RNA containing one or more inosine bases in cells disclosed herein, further comprising a denaturing agent.

[0079] Yet another aspect of the disclosure are methods of characterizing the subcellular localization of Adenosine-to-Inosine (A-to-I) editing in RNA, the method comprising: performing an Endonuclease V Immunostaining Assay (EndoVIA) on a cell sample comprising edited RNA; and providing a spatial visualization of A-to-I editing in the cell sample.

[0080] The EndoVIA method is described herein above as methods of detecting RNA containing one or more inosine bases in cells.

[0081] Additional objects of the disclosure are methods of detecting ribonucleic acid (RNA) containing one or more inosine bases in cells comprising: fixing cells to retain large RNAs and not retain small RNAs to form fixed cells; denaturing the secondary structure of the large RNAs to form denatured RNA; incubating the denatured large RNAs with a calcium- containing solution of endonuclease V fused with a maltose-binding protein (EndoV-MBP) to form a complex between EndoV-MBP and RNA; incubating the complex of EndoV-MBP and RNA with an anti-MBP antibody to form a complex of EndoV-MBP, anti-MBP, and RNA; incubating the complex of EndoV-MBP, anti-MBP. and RNA with a secondary antibody specific for the anti-MBP antibody conjugated to a fluorescent dye; and imaging the cells using fluorescence microscopy to detect the RNA containing one or more inosine bases.

[0082] In accordance with an aspect of the present disclosure, a method of characterizing a level of Adenosine-to-Inosine (A-to-I) in cell RNA is provided. The method comprises: performing an Endonuclease V Immunostaining Assay (Endo VIA) on a cell sample comprising edited RNA; and providing a spatial visualization of A-to-I editing in the cell sample.

[0083] In accordance with another aspect of the present disclosure, a method of quantifying overall inosine abundance in a cell is provided. The method comprises: performing an Endonuclease V Immunostaining Assay (EndoVlA) on a cell sample; and measunng an amount of inosine based on an anti-inosine antibody comprising EndoV.

[0084] In accordance with a further aspect of the present disclosure, a method of determining a disease or disorder treatment for a subject in need thereof is provided. The method comprises: performing an Endonuclease V Immunostaining Assay (EndoVlA) on a unhealthy cell sample from the subject; identifying a hyper-editing unhealthy cell line or a hypo-editing unhealthy cell line based on an Adenosine-to-Inosine (A-to-I) level from the EndoVlA; and determining a suitable disease or disorder treatment based on the identified hyper- or hypo-editing unhealthy cell line.

[0085] The methods of determining a disease or disorder treatment described herein can have the disease or disorder be a disease or disorder wherein misregulation of A-to-I editing is implicated. In some instances, the disease or disorder can be cancer.

[0086] Adenosine-to-Inosine (A-to-I) editing is one of the most widespread RNA modifications in metazoans and is catalyzed by adenosine deaminases acting on RNA (ADARs).1Among the three ADAR genes encoded in vertebrates (ADAR ADAR2, and ADAR3), AD ARI is ubiquitously expressed and is responsible for the majority of editing incells.2’3AD ARI is expressed in a pl 10 isoform that primarily resides in the nucleus, in addition to an IFN-inducible pl 50 isoform that localizes to the cytoplasm. Adenosine deamination results in a change of hydrogen bonding such that the resulting inosine base pairs with cytidine, effectively recoding the site to be recognized as guanosine by cellular machinery (Figure la).2Therefore, editing that occurs in protein-coding regions of messenger RNA (mRNA) can lead to multiple protein isoforms and altered function.2While A-to-I editing is most abundant in non-coding, repetitive regions of mRNA and impacts transcript stability and localization,2-4non-coding RNAs such as small-interfering RNAs (siRNAs) and microRNAs (miRNAs) are also edited, affecting global gene expression and cellular functionality.5’6Collectively, A-to-I editing has proven essential for stem cell differentiation, embryogenesis, brain development, and cellular immunity.2’7’8

[0087] AD ARI is believed to play a critical role in regulating the activation of innate cellular immunity by targeting double-stranded RNA (dsRNA) made from long, inverted Alu repetitive elements that are located within introns and untranslated regions.9These embedded Alu repeats make up 10% of the human genome and create -300 base pair long RNAs when transcribed that look very similar to foreign, viral RNA. ADAR prevents the activation of the cytosolic innate immune system by editing these endogenous dsRNAs and marking them as ■‘self (Figure lb). Given the significant role A-to-I editing plays in maintaining cellular function, it’s unsurprising that dysregulation has been linked to multiple neurological disorders, autoimmune disorders, and cancers.6 10 11A-to-I editing levels can display hyper- or hypo-editing depending on the specific cancer type.12’13Notably, knockout of AD ARI has been shown to be lethal in cell and animal models, and AD ARI inhibition shows significant promise as a therapeutic strategy for treating cancer.9’14 20Moreover, it has been observed that in melanoma, an A-to-I editing deficiency directly contributes to the melanoma metastatic phenotype.21Collectively, these findings along with others highlight a clear link between A- to-I editing and the disease state, while speaking to the intricate relationship between editing and cancer that has yet to be fully elucidated. Leveraging this connection toward diagnostics and therapeutics will require greater understanding of the relationship between editing and disease progression, which has yet to be achieved with currently available methods.

[0088] The significance of A-to-I editing extends beyond sheer quantity; its influence on RNA localization is equally pivotal. Extensive A-to-I editing of inverted Alu repeats located within the 3’ UTR of mRNAs plays an essential role in nuclear retention22 25and the presence or absence of an edited 3’ UTR has been demonstrated to drastically alter RNA subcellularlocalization (Figure 1c). One example of this change in RNA localization was observed by Chen et al. with the human gene, Nicolin 1. Nicolin 1 is expressed in multiple isoforms, but strikingly the only isoform that remains within the nucleus features an extensively edited, inverted Alu repeat in its 3’ UTR.24Beyond Alu repeats, the 3’ UTRs of RNAs also contain cA-regulatory elements, often referred to as “zipcodes,’’ that influence the localization of rnRNA.26’27These sequences serve as encoded cellular addresses that RNA-binding proteins (RBPs) interact with to facilitate trafficking.28Considering that A-to-I editing primarily occurs within these non-coding regions, mRNA localization is subject to change based on the extent of editing. These examples underscore the profound connection between A-to-I editing and RNA localization. However, these findings also represent the extent of our current knowledge of the A-to-I editing landscape due to the lack of tools capable of mapping inosine within cells.

[0089] The most widespread technique used to characterize A-to-I editing is RNA sequencing (RNA-seq), in which RNA is extracted from cells, pooled, and subjected to high- throughput sequencing. Resulting sequences are compared to a reference genome and edited sites are identified from A-G transitions.5While it remains the gold standard for identifying edited transcripts, unfortunately RNA sequencing along with other common methods requires RNA extraction that ultimately erases subcellular localization (Figure 2a). Extraction also precludes the opportunity to compare cell-to-cell variation in situ, which is a hallmark of tumor heterogeneity.

[0090] Traditional cellular RNA imaging techniques, such as fluorescence in situ hybridization (FISH) and genetically encodable tags that bind fluorescent proteins or dyes, have revolutionized the way RNA is visualized in cells.29 31However, these approaches alone cannot detect editing status and lack the sensitivity required to discern single nucleotide modifications. As a result, specialized methods have been developed to image the localization of RNA modifications. Some of the first examples are FISH-inspired methods that leverage toehold probes to detect single nucleotide variants (SNVs) in RNA, including the first demonstration of visualizing A-to-I editing status of specific transcripts using inosine fluorescence in situ hybridization (inoFISH) thus providing insights into the link between editing patterns and localization.3233Approaches have also been designed to detect cytidine- to-uridine (C-to-U) editing of RNAs of interest in cells using forced intercalation probes (FIT) probes.34,35A generalizable method for targeting polyadenylated RNAs coined click-encoded rolling FISH (ClickerFISH) was also developed and allowed the visualization of higher-order structures at the subcellular level with cell-to-cell variation.36Most recently, deaminationadjacent to RNA modification targets FISH (DART-FISH) was developed to enable in situ detection of individual m6A-modified and unmodified transcripts and determined that m6A alone is not sufficient to localize mRNA to stress granules.37Together, these select examples highlight the importance of novel techniques to reveal the impact RNA modifications play intracellularly. Despite these advances that target specific transcripts, the need remains for a generalizable approach for quantifying and mapping the localization of inosine-containing RNAs across the transcriptome.33Antibodies could prove advantageous towards this goal, yet efforts to generate an anti-inosine antibody have fallen short, with the resulting affinity reagents lacking the affinity and reproducibility needed for rigorous analytical methods (Figure 3).38Taken together, a method for detecting and quantifying A-to-I edited RNA in situ remains an unmet challenge.

[0091] To address this need, we report herein Endonuclease V Immunostaining Assay (EndoVIA) which enables visualization of inosine-containing transcripts in cells using Endonuclease V (EndoV, Figure 2b). EndoV is a magnesium-dependent ribonuclease that cleaves inosine-containing nucleic acid substrates; however, its activity can be controlled by replacing its natural magnesium cofactor with calcium such that EndoV binds inosine with high specificity without initiating cleavage.39 42Our group has leveraged this binding event to ultimately repurpose EndoV as an "anti-inosine antibody” for enriching and quantifying edited RNA in vitro, which has in turn led to improved mapping of editing sites via RNA-seq as well as an ELISA inspired microplate-based assay for quantifying global editing levels.43,44While our group’s previous efforts have leveraged EndoV to accurately quantify A-to-I editing, these methods still require RNA extraction and pooling of RNA from a population of cells. These steps effectively erase information regarding the spatial distribution of edited RNA in situ as well as variation in editing signatures between cells. Seeking to overcome the limitations of these in vitro approaches, we recognized that the inosine binding capabilities of EndoV could be harnessed for detecting A-to-I edited transcripts in situ to retain RNA localization and cellular heterogeneity by recapitulating the principles of immunofluorescence (Figure 2c). After optimization of our immunostaining protocol, we validated our EndoVIA workflow using cells with vary ing levels of A-to-I editing. We then demonstrated that our workflow could be used to detect both elevated levels of A-to-I editing in a hyper-editing cancerous cell line and reduced editing levels in a hypo-editing cancerous cell line. Finally, we used total internal reflection fluorescence (TIRF) illumination coupled with super-resolution microscopy to detect edited RNA at the single-molecule level. Excitingly, we observe distinct subcellularlocalization paterns for edited RNA in both healthy and diseased cell line models. Together, these results demonstrate that EndoVIA can provide spatial visualization of A-to-I editing, enable quantification of total cellular inosine content at the single-cell level, and be used to map subcellular localization of edited transcripts. This in turn provides a powerful tool for acquiring previously unatainable insights into the localization of A-to-I editing and its role in cellular processes and disease progression.Development of EndoVIA for in situ Imaging of A-to-I Edited Transcripts

[0092] Seeking to advance our use of EndoV as an “anti -inosine antibody” and develop a method for detecting edited RNA in situ, we turned to immunofluorescence for inspiration. Immunofluorescence is a technique used to image cellular components by directly or indirectly labeling targets of interest with fluorophore-tagged antibodies.45The immunofluorescence staining process involves cellular fixation, permeabilization, blocking, and staining with a primary antibody specific for the target, followed by a secondary' antibody conjugated to a fluorophore.45Using this basic protocol for immunostaining as a scaffold, we integrated essential steps necessary for imaging inosine-containing RNA with EndoV and optimized this workflow in a simple model system, human embryonic kidney 293T (HEK293T) cells.

[0093] Our first goal was to strategically fix cells in a way that maximizes staining specificity7by presenting RNA integrity' and eliminating cellular components that could lead to potential off-target binding. Although widespread and effective, formaldehyde crosslinks not only large RNAs such as ribosomal RNA (rRNA) and messenger RNA (mRNA), but also small RNAs including transfer RNA (tRNA) that are edited within the anticodon loop by adenosine deaminases acting on tRNA ( AD ATs).46,47While essential for cell viability, tRNA editing is unrelated to ADAR-mediated A-to-I editing and tRNA is at least 100 times more abundant than mRNA in mammalian cells.48Therefore, a significant concern was that fixation of tRNAs could mask our ability to visualize ADAR-edited targets. To circumvent this issue, we chose to fix cells using methanol (MeOH), another commonly used fixative reagent that retains large RNAs and allows small RNAs, like tRNA, to be washed away.49’50To confirm that tRNA is indeed removed upon MeOH fixation, we performed tRNA FISH and observed that the presence of tRNA dramatically decreased after fixation and washing (Figure 4). In comparison to formaldehyde-fixed cells, methanol fixation decreased the presence of tRNA by -72% in two different cell lines (Figure 5, Figure 6).

[0094] In addition to fixation method, we also explored additional workflow steps to achieve optimal EndoV binding of the target edited RNAs. Our group has shown that the commercially available, recombinant Escherichia coli EndoV binds edited single-stranded RNA (ssRNA) with a higher affinity than edited dsRNA.43ADAR primarily targets RNA within double stranded regions, and thus an RNA denaturation step is required to maximize EndoV binding to ADARl-edited sites.1We were inspired by our previous use of glyoxal to denature RNA secondary structure while preserving EndoV binding capability7in vitro, and hypothesized that we could recapitulate this approach in cells.43Glyoxal is a reversible, chemical denaturant that forms bis-hemiaminal adducts with guanine, adenine, and cytosine nucleobases, thus disrupting Watson-Crick-Franklin base pairing.43’44’51Importantly, glyoxal is unreactive tow ards inosine as inosine differs from guanine by the lack of an exocyclic amine that is critical for glyoxal adduct formation. Following methanol fixation and glyoxal treatment, cells were permeabilized with detergent and blocked with bovine serum albumin (BSA) to prevent nonspecific binding. To achieve staining, cells were then incubated sequentially with EndoV, primary antibody, fluorophore-labeled secondary7antibody, all of which were diluted in a calcium-containing blocking buffer to support EndoV binding (Figure 7).Optimization of Endo VIA

[0095] Our workflow as described is representative of typical immunofluorescence experiments, with some modifications to support use of EndoV as an “anti-inosine antibody.” To confirm that our modified workflow was suitable for immunofluorescence and that the modifications made to enable use of EndoV would not alter cellular morphology or interfere with imaging, we first sought to detect and image well-known and characterized cellular components using commercially available antibodies. We chose to stain |3-actin. a highly abundant structure in the cytoskeleton, and nucleoporin 153 (Nupl53), a less abundant protein located in nuclear pore complexes.52-53-54-55Images for both proteins were representative of previous reports, exhibiting sufficient signal and minimal background (Figure 8a and 8b).

[0096] Given that EndoV is fused to MBP, we considered the possibility that MBP could potentially engage in non-specific binding and give rise to unwanted background signal. To assess nonspecific binding of the fusion protein, we stained HEK293T cells initially with a 1 : 1000 dilution EndoV -MBP fusion or Escherichia coli MBP using our previously outlined staining workflow and imaged using widefield microscopy (Figure 9). Encouragingly,fluorescence was only observed for cells treated with EndoV-MBP, and cells treated with MBP only were comparable to negative controls. These results suggest the observed fluorescence is derived from the binding of EndoV to edited RNAs.

[0097] Detecting the maximum number of edited sites requires the complete denaturation of RNA secondary structure in order to grant EndoV access to these regions. To find the optimal glyoxal concentration for achieving this outcome, we stained GAPDH mRNA using FISH in HEK293T cells at varying concentrations of glyoxal (Figure 10). As RNA is increasingly glyoxylated, FISH probes are no longer able to hybridize to their targets, resulting in a decrease in fluorescence. We also stained (3-actin in parallel under each glyoxal concentration to monitor cellular morphology (Figure 1 1). Taken together, we determined that 12% glyoxal was sufficient to denature RNA secondary structure without impacting cellular morphology.

[0098] Immunofluorescence protocols often require antibody optimization to minimize background and achieve maximum signal-to-noise ratio and sensitivity. To meet this need, we systematically screened EndoV concentrations to identify conditions where EndoV would stain the maximum number of targets while avoiding nonspecific binding of MBP. HEK293T cells were cultured and stained with increasing amounts of EndoV or MBP ranging from 1:4000 to 1:25. along with the appropriate controls (Figure 12). After quantifying the fluorescence, we determined that 1 :50 EndoV was sufficient to achieve saturation while mitigating nonspecific binding of MBP. To confirm antibody specificity, we also stained HEK293T cells with the same workflow but omitting EndoV and observed no detectable fluorescence from control wells treated with only primary and secondary antibodies (Figure 13). For further rigor, we also hypothesized that removing calcium, which is essential for EndoV binding, would greatly reduce the fluorescence of inosine-containing RNA. To test this, we treated cells with EDTA following EndoVIA and found that fluorescence significantly decreased, further supporting that the signal observed could be attributed to EndoV binding to edited RNA (Figure 14).Validation of EndoVIA

[0099] Having optimized our immunofluorescence protocol, we next sought to validate the ability of EndoVIA to detect biologically relevant differences in inosine by staining cells having varying levels of A-to-I editing. We predicted that if our workflow was targeting edited RNA as expected, then staining cells with little to no editing would lead to less fluorescence signal than staining of wildtype cells. To test our hypothesis, we chose to stain a HEK293Tcell line with the ADAR1 gene knocked out via CRISPR-Cas9 genome editing.9We first immunostained AD ARI in both the WT and KO cell lines and confirmed that indeed AD ARI was absent from the KO cell line (Figure 15). Using our optimized workflow, we then immunostained inosine-containing RNA in both wildtype and AD ARI KO HEK293T cells and quantified the fluorescence (Figure 16a and 16b). Image analysis revealed that WT HEK293T cells show ~2-fold greater fluorescence than ADAR1 KO cells, strongly suggesting that edited RNA is responsible for the staining.

[0100] While we w ere encouraged by this significant difference, we were not entirely surprised by the remaining fluorescence from the AD ARI KO cells. Other enzymes that are large contributors of cellular inosine, such as ADAR2 and AD AT, are still present and may contribute to editing. We turned to RNA-seq and the Alu editing index (AEI) to compare the WT HEK293T and AD ARI KO cells. The AEI was developed by Roth and coworkers and is a power computational tool that maps editing in Alu repeats across large datasets generated from RNA sequencing and has become widely accepted as the gold standard for quantifying ADAR editing activity.56Briefly, the AEI value is generated from RNA-seq data by' identifying sites that are called as guanine in RNA but adenosine in DNA and then calculating the ratio of these edited sites to the total coverage of adenosines. We determined that the AEI values for the WT HEK293T and AD ARI KO cells were 1.4 and 0.2. respectively (Figure 17). These results suggest that there is some remaining edited tRNA or other unwanted species giving rise to background signal. While methanol fixation removes the majority' of tRNA, it’s important to note that residual tRNA likely contributes to some of the observed fluorescence (Figure 6). Although the observed background signal was not ideal, we recognized that this could be especially noticeable in these very low-editing cells whereas it might not be as problematic in other cell lines and thus we carried forward. Encouragingly, our results described below indicate that this background signal appears to be nominal in the context of cells having more typical editing levels.

[0101] We were curious to explore whether we could detect an increase in A-to-I editing as well. Immortalized cell lines have low editing activity7in comparison to human primary7cells. Specifically, HEK293T cells have low' editing levels, making them an ideal cell line to stimulate ADARf upregulation.57To explore this hypothesis, we transfected HEK293T cells with increasing amounts of a GFP-tagged AD ARI pl 10 overexpression vector (pCMV) plasmid and stained accordingly (Figure 18a). We quantified the fluorescence of edited RNA in ADAR-GFP positive cells and determined that across all plasmid concentrations, cellsexpressing ADAR1-GFP displayed significantly higher immunostaining signal in comparison to cells that did not express ADAR1-GFP (Figure 18b). We observed the same trend in GFP- tagged ADAR1 pl50 transfected cells as well (Figure 19). To confirm that changes in fluorescence were due to ADAR transfection, we also transfected HEK293T cells with increasing amounts of coilin-GFP, another nuclear protein. We found that there was no change in edited RNA fluorescence (Figure 20).Detecting A-to-1 Editing in Cancer

[0102] Dysregulated A-to-I editing has been linked to various cancer types, and while many cancer types present a hyper-editing signature, some also display hypo-editing.58Most notably, hyper-editing is evident in thyroid, head, neck, lung, and breast cancers whereas hypoediting is observed in metastatic melanoma, invasive breast cancer, and renal cancer.20-59 61As a result, A-to-I editing is rapidly emerging as not only a biomarker for diagnosing and studying cancers, but also as a potential therapeutic target. We recognized that EndoVIA could prove to be particularly powerful in this context, as it could enable rapid quantification of editing levels across multiple samples and with the ability to observe cell-to-cell heterogeneity. Additionally, we envisioned that the ability to discriminate between inosine levels in healthy vs cancerous cells could serve as a foundation for the development of phenotypic screening assays for ADAR inhibition.

[0103] Fortuitously, Schaffer et al. recognized the importance of having proper cellular models that reflect the A-to-I editing levels of a given tissue condition or disease signature and using the AEI computational tool, they determined the AEI values for more than 1,000 human cell line types.62This provided a wealth of validated cell lines for us to choose from in exploring the ability of EndoVIA to detect cancer-related changes in inosine levels. Drawing inspiration from the Cell Line A-to-I Editing Catalogue, we first chose to test our workflow against ZR- 75-1 and MCF10A cell lines. ZR-75-1 cells are a malignant breast cell line that displays hyperediting signatures with an AEI value of 2.1 whereas the MCF10A breast cell line as a non- malignant control that has an AEI value of 1.4 (Figure 21a). Both cell lines were subjected to our EndoVIA workflow, and we were very excited to see that the ZR-75-1 cell line did indeed show higher fluorescence than the MCF10A cells (Figure 21 c). Even more encouraging, the ratio between the immunofluorescence signal for the tw o cell lines w as comparable to that of the reported AEI values. This demonstrates that EndoVIA is able to detect a cancer-relatedincrease in RNA editing without the need for sequencing, which dramatically increases potential throughput.

[0104] Having established the ability of EndoVIA to detect cancer-related hyperediting. we were also curious to explore whether we could detect cancer-related hypo-editing. Kidney cancers such as kidney chromophobe (KICH) and kidney renal papillary cell carcinoma (KIRP) in particular have been identified to display hypo-editing.63Therefore, we selected the G-402 kidney cell line, characterized with renal leiomyoblastoma and an AEI value of 0.8. We also stained HEK293T cells that have an AEI value of 1.1 in parallel as a non-malignant counterpart (Figure 21b). As anticipated, we were able to detect a decrease in fluorescence in the G-402 cell line (Figure 21 d). We also confirmed that there were comparable amounts of mRNA across each pair of healthy and diseased cell lines, strongly suggesting that differences in fluorescence can be attributed to changes in A-to-I editing (Figure 22).

[0105] Having demonstrated that EndoVIA can detect global changes in inosine levels between healthy and cancerous cells, we sought to also capture nuanced shifts in cell-to-cell variation, as cellular heterogeneity is a hallmark of cancer. Using the CTCF values of individual cells across all four cell lines, we employed kernel density estimation to unveil the distribution of fluorescence within each cell population.64There was a notable difference in the distribution patterns that emerged between healthy and diseased cells. Particularly striking was the comparison between HEK2393T and G-402 cells, where we observed a distinct migration of the density peak. In HEK293T cells, a higher density of cells exhibited elevated levels of A- to-I editing, while in G-402 cells the density' shifted toward lower CTCF values, signaling an increase in cells displaying reduced levels of editing (Figure 23a). A similar trend was also observed in MCF10A and ZR-75-1 cells, where higher A-to-I editing levels in ZR-75-1 cells displayed a shift in cell density7towards higher CTCF values (Figure 23b). These findings aligned with our expectations, reinforcing that EndoVIA is capable of not only detecting widespread changes in A-to-I editing but also uncovering nuances in cellular heterogeneity.

[0106] EndoVIA serves as the first method capable of detecting inosine-containing transcripts in situ, thus we were curious about its performance against traditional approaches for characterizing A-to-I editing in cells, namely dsRNA antibodies. Given that dsRNA is the primary7target of ADAR, the J2 and KI antibodies have been routinely employed for detecting and enriching ADAR substrates. To put these antibodies to the test, we chose to stain dsRNA with J2 and KI in HEK293T and G-402 cells and compare to EndoVIA that was completed in parallel (Figure 24). The localization patterns for all three treatments varied across bothHEK293T and G-402 cells. Notably, the dsRNA antibodies exhibited a surprising absence of fluorescence in the nucleus, despite this being ADAR’s primary' residence for RNA editing. KI. specifically in HEK293T cells, showed localization patterns most comparable to EndoV, demonstrating increased fluorescence near the cell membrane. These observable differences across treatments underscore the unknown binding preferences of J2 and KI in the context of A-to-I editing status. In comparison, EndoVIA offers a higher degree of confidence in staining edited RNA as it directly detects inosine, providing a higher level of detail and reliable staining approach.Detecting Subcellular Localization of Inosine-Containing Transcripts

[0107] In addition to the ability to rapidly detect global inosine levels in cell samples, we envisioned that EndoVIA might also enable imaging of the subcellular localization of edited RNAs. The majority7of A-to-I editing occurs within the 3’ UTRs of rnRNA which heavily influences RNA localization. The loss of an edited 3’ UTR has been shown to completely alter the destination of a given RNA.22 25Despite this evident link between A-to-I editing and RNA localization, many unanswered questions remain due to the lack of available methods. As an example of this, the images of HEK293T cells presented in the figures above show observable disparities in fluorescence signal, with signal seeming to be concentrated near the cellular membrane. In order to more rigorously study these localization patterns and achieve greater resolution, we hypothesized that TIRF illumination coupled with super-resolution microscopy would yield high-quality7, nanometric spatial insight from EndoVIA. Thus, we performed the immunostaining on both HEK293T and G-402 cell lines using the EndoVIA protocol.

[0108] Imaging of HEK293T cells using dSTORM in TIRF illumination enabled us to observe single EndoV binding events (presumably from individual editing sites) with large populations near the membrane (Figure 25a) and at the adhesion site (Figure 25b). Interestingly, while we observe edited RNAs in both subcellular locations, the edited RNA is concentrated at the cellular membrane in contrast to displaying a more even distribution throughout the adhesion sight. Interestingly, previous reports have determined that cellular adhesion and motility is partly regulated through RNA localization and translation at focal adhesions.65’66Encouragingly, when cells were stained omitting EndoV and imaged using dSTORM in TIRF illumination, there was no detectable fluorescence and thus consistent with previous experiments (Figure 26). Together, these findings offer new potential insights into thedownstream functions of editing, as well as mark the first demonstration of a method that provides spatial distribution of A-to-I editing across the cell at the single-molecule level.

[0109] dSTORM TIRF imaging of cancerous G-402 cells revealed similar localization of edited RNAs at the membrane, though less pronounced than in the HEK293T cells. G-402 cells also appear to display more even distribution of edited transcripts throughout the cytoplasm. Interestingly, upon closer inspection, super-resolution imaging of the G-402 cells reveals some clustering in the cytoplasm that may represent subcellular structures for organizing edited RNAs that are not present in HEK293T cells and not visible using standard TIRF or confocal imaging (Figure 25c). Despite these differences in the localization of edited RNA, ADAR1 localization in both HEK293T cells and G-402 cells remains in the nucleus, hinting at potential underlying mechanisms dictating the localization of edited transcripts that have yet to be discovered (Figure 27). These results not only underscore the power of EndoVIA to lead to new biological discoveries and knowledge, but also establish its compatibility with advanced imaging modalities.

[0110] These results represent the first example of transcriptome-wide visualization of edited RNAs with nanoscale resolution in the cellular environment. We do note that some residual tRNA can be present after the fixation steps and may lead to background signal. However, tRNA FISH in the HEK293T cells reveals that that this remaining tRNA is primarily sequestered to nuclear foci (Figure 5), and we do not observe a strong corresponding EndoV signal in our single-molecule EndoVIA experiments. This suggests that the interference of residual tRNA is minimal, though out of an abundance of caution we do recommend that tRNA FISH be included as a control experiment when using EndoVIA to map subcellular localization of edited RNAs. Together, these experiments open up a wide range of potential explorations that will reveal new links between A-to-I editing and RNA localization and in turn provide novel insights into the role of this important process in development and disease.[OHl] A-to-I editing is the most widespread post-transcriptional modification and is essential for multiple biological processes. One way that editing impacts cellular pathways is thought to be through directing the localization of RNAs to subcellular compartments by editing within the 3 ’ UTR of mRNAs. Although there are a few demonstrations that nuclear retention of specific mRNAs is a result of A-to-I editing, the broader impact of editing on RNA localization is underexplored due to a lack of methods for imaging inosine-containing RNAs in the cellular context. In parallel, dysregulation of A-to-I editing is intricately linked to neurological disorders, autoimmune diseases, and multiple cancers, highlighting its critical rolein disease pathogenesis. However, the tremendous potential of editing to serve as a biomarker or therapeutic target is limited by the lack of methods for observing editing directly in situ. Specifically, the current gold standard technique of high-throughput RNA sequencing requires RNA extraction and pooling of RNA from multiple cells in a sample. This essentially erases information relating to cell-to-cell variation of global inosine levels and precludes studying the specific subcellular localization of edited RNAs. Moreover, RNA-seq remains limited in throughput, making it poorly suited for high-throughput drug screening campaigns.

[0112] Herein, we introduce EndoVIA as the first approach for imaging and quantifying the full breadth of inosine-containing transcripts in cells. Key to the development of this technique is our repurposing of EndoV to act as an “anti-inosine antibody,” which in turn enables us to develop a protocol analogous to immunofluorescence staining that is aimed at detecting edited RNAs in situ. We have validated our approach using cells having varying editing levels and demonstrate the ability to detect biologically relevant differences in inosine between healthy and cancerous cell lines. We also show that EndoVIA is compatible with super-resolution microscopy techniques to image edited RNAs at nanoscale resolution and provide previously unattainable insight into the subcellular localization and organization of edited transcripts.

[0113] We envision that the availability of EndoVIA will open up numerous avenues for studying RNA editing and harnessing this important process for diagnostics and therapeutics. For example, efforts are underway in our laboratory to further elaborate and refine EndoVIA to enable high-throughput phenotypic screening for the discovery of novel small molecules to regulate A-to-I editing. Additionally, the ability to image the subcellular localization of edited RNAs can be directed toward probing a wide range of biological questions regarding how this localization is impacted by cell type, disease state, and external stimuli. Thus, this first-in-class approach for imaging A-to-I editing at the cellular level is expected to not only advance research in our own laboratory but also empower other groups studying A-to-I editing and in turn advance both the basic science and translational potential of this important biological process.

[0114] For the EndoVIA work flow generally, the cells were seeded as described below in the example section. Cells were then fixed in 100% ice cold methanol and incubated at from 2°C to 5°C or 4°C for 15-25 or 20 minutes. The methanol was removed, and wells were washed twice with IX PBS. Cells were next rehydrated with IX PBS by incubating for 1 hour at room temperature with gentle shaking. A 10% to 14% or 12% glyoxal denaturing solution wasprepared by combining the following in order: 1.974 mL nuclease-free water, 0.796 mL of 100% ethanol, 1.200 mL of glyoxal, and 0.030 mL acetic acid. These volumes were scaled up or down as needed for the experiment. The IX PBS was removed and the glyoxal denaturing solution was placed on cells and incubated at 45°C to 55°C or 50°C for 50 minutes to 70 minutes or 60 minutes.

[0115] Cells were then incubated with a 0.05% to 0.15% or 0.1% Triton X-100 in IX PBS permeabilization solution at 20°C to 25°C or room temperature for 10 minutes followed by two washes in IX PBS. Cells were then incubated in a blocking solution containing 2% to 4% or 3% bovine serum albumin, 0.05% to 0. 15% or 0. 1% Triton X-100. and 0.05% to 0. 15% or 0.1% Tween 20 in IX PBS for 50 minutes to 70 minutes or 60 minutes at 20°C to 25°C or room temperature. Wells were then washed twice with IX PBS. A calcium containing blocking buffer was prepared as follows: 2% to 4% or 3% bovine serum albumin and 0.8 nM to 1.2nM or 1 mM calcium chloride in IX PBS.

[0116] Cells were incubated with 1 :20 to 1 :80 or 1:50 Endonuclease V (New England BioLabs) in the calcium containing blocking buffer, or the calcium containing blocking buffer alone as a negative control, at 20°C to 25°C or room temperature for 50 minutes to 70 minutes or 60 minutes. A calcium containing wash buffer with 0.8 mM to 1.2mM or 1 mM calcium chloride. 15 mM to 25 mM or 20 mM tris hydrochloride, and 100 mM to 200 mM or 150 mM sodium chloride w as made and used for the remainder of the washing steps. Cells were then washed three times, incubating for 5 minutes each time.

[0117] The commercially available EndoV is fused to a maltose-binding protein (MBP) tag that is commonly used for purification purposes. Thus, an anti-MBP primary antibody that binds MBP of the EndoV-MBP fusion protein was used. Cells were then incubated for 50 minutes to 70 minutes or 60 minutes at20°C to 25 °C or room temperature with a 1:800 to 1: 1200 or 1 :1000 anti-MBP antibody (New England BioLabs) solution diluted in calcium containing blocking buffer. Cells were washed three times, with 5-minute incubation periods. Finally, a staining solution containing 1 : 800 to 1: 1200 or 1 : 1000 goat anti -mouse Alexa Flour 647 and 1:800 to 1 : 1200 or 1 : 1000 Floechst nuclear dye in calcium containing blocking buffer was incubated in the wells for 1 hour at room temperature in the dark. Cells w ere then washed three times for 5 minutes each time.

[0118] Cells were then imaged. Because EndoV is used in a manner analogous to an antibody for inosine, optimizing concentration was thus imperative for developing a robust fluorescence-based assay that accurately detects edited substrates. The EndoV concentrationused for the EndoVIA workflow was determined by following the previously described protocol using a range of EndoV and MBP (Novus Biologicals) concentrations (1:4000-1:25). As a negative control, a set of cells were not treated with EndoV fusion or MBP but were still incubated with primary and secondary antibody. The calcium dependence of EndoVIA was confirmed by treating wells with 5 mM EDTA and incubating for for 50 minutes to 70 minutes or 60 minutes at 20°C to 25°C or room temperature following EndoVIA and then imaged. All immunostaining incubations were completed with 200 mL volumes and solutions were prepared fresh for each experiment. EndoVIA was performed under nuclease-free conditions.MOLECULAR ENGINEERING

[0119] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0120] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0121] The terms "heterologous DNA sequence", "exogenous DNA segment", or "heterologous nucleic acid”, “transgene”, “exogenous polynucleotide” as used herein, each refers to a sequence that originates from a source foreign (e g., non-native) to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinanly found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0122] Sequences described herein can also be the reverse, the complement, or the reverse complement of the nucleotide sequences described herein. The RNA goes in the reverse direction compared to the DNA, but its base pairs still match (e.g., G to C). The reverse complementary' RNA for a positive strand DNA sequence will be identical to the correspondingnegative strand DNA sequence. Reverse complement converts a DNA sequence into its reverse, complement, or reverse-complement counterpart.

[0123] Complementarity is aproperty shared between two nucleic acid sequences (e.g., RNA, DNA), such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. Two bases are complementary if they form Watson-Crick base pairs.

[0124] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in. for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleicacid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0125] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology7for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coh is used as the host for protein production, but other cell types may also be used.

[0126] In molecular biology , an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:(i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes. An operon is a cluster of genes that are transcribed together to give a single messenger RNA (mRNA) molecule, which therefore encodes multiple proteins.(ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0127] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0128] For a gene to be expressed, its DNA sequence (or polynucleotide sequence) must be copied (in a process known as transcription) to make a smaller, mobile moleculecalled messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.

[0129] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0130] A “promoter"’ is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary' nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0131] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0132] A ribosomal skipping sequence (e.g., 2A sequence such as furin-GSG-T2A) can be used in a construct to prevent covalently linking translated amino acid sequences.

[0133] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and usingconstructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory7Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN- 10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory7Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzy mology7167, 747-754).

[0134] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0135] "Operably -linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory7sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0136] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, vims, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule w here one or more nucleic acid molecule has been operably linked.

[0137] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatorynucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory’ nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0138] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0139] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using self-replicating primers, paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0140] "Wild-type" refers to a virus or organism found in nature without any known mutation.

[0141] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0142] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the twosequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity7. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST. BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity7= X / Y I OO. where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%. about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%. about 99%, or about 100%.

[0143] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.

[0144] “Point mutation" refers to when a single base pair is altered. A point mutation or substitution is a genetic mutation where a single nucleotide base is changed, inserted, or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product — consequences that are moderately predictable based upon the specifics of the mutation. These consequences can range from no effect (e.g., synonymous mutations) to deleterious effects (e.g.. frameshift mutations), with regard to protein production, composition, and function. Point mutations can have one of three effects. First, the base substitution can be a silent mutation where the altered codon corresponds to thesame amino acid. Second, the base substitution can be a missense mutation where the altered codon corresponds to a different amino acid. Or third, the base substitution can be a nonsense mutation where the altered codon corresponds to a stop signal. Silent mutations result in a new codon (a triplet nucleotide sequence in RNA) that codes for the same amino acid as the wild type codon in that position. In some silent mutations the codon codes for a different amino acid that happens to have the same properties as the amino acid produced by the wild type codon. Missense mutations involve substitutions that result in functionally different amino acids; these can lead to alteration or loss of protein function. Nonsense mutations, which are a severe type of base substitution, result in a stop codon in a position where there was not one before, which causes the premature termination of protein synthesis and can result in a complete loss of function in the finished protein.

[0145] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by He, Leu by He, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine. Selenocysteine. Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0146] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If aparticular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm= 81.5 °C + 16.6(logio[Na+]) + 0.41(fraction G / C content) - 0.63(% formamide) - (600 / 1). Furthermore, the Tmof a DNA:DNA hybrid is decreased by 1-1.5°C for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0147] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory’ Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed.. Current Protocols. ISBN- 10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory' Manual, 3d ed., Cold Spring Harbor Laboratory' Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile- mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

[0148] Exemplary nucleic acids that may be introduced to a host cell include, for example. DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0149] Host strains developed according to the approaches described herein can be evaluated by a number of means know n in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukary otic Expression Systems, Wiley-VCH. ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis. ISBN-10: 0954523253).

[0150] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g.. small interfering RNAs (siRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review ofPhysiology 67, 147-173, describing RNAi; Dykxhoom and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g.. Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.GENOME EDITING

[0151] As described herein, signals can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing.

[0152] As described herein, activity, signals, expression, or function can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing (e.g., upregulate, downregulate, overexpress, underexpress, express (e.g., transgenic expression), knock in, knock out, knockdown).

[0153] Processes for genome editing are well known; see e g., Aldi 2018 Nature Communications 9(1911). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0154] For example, genome editing can comprise CRISPR / Cas9, CRISPR-Cpfl, TALEN, or ZNFs. Adequate blockage by genome editing can result in protection from various diseases.

[0155] As an example, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N)2oNGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example,using CRISPR / Cas systems could be useful tools for therapeutic applications to target cells by the removal or addition of signals (e.g., activate (e.g., CRISPRa), upregulate, overexpress, downregulate).

[0156] For example, the methods as described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.GENE THERAPY AND GENOME EDITING

[0157] Gene therapies can include inserting a functional gene with a viral vector. Gene therapies are rapidly advancing.

[0158] There has recently been an improved landscape for gene therapies. For example, in the first quarter of 2019, there were 372 ongoing gene therapy clinical trials (Alliance for Regenerative Medicine, 5 / 9 / 19).

[0159] Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof.Gene therapy strategies.

[0160] Gene therapy can allow for the constant delivery of the enzyme directly to target organs and eliminates the need for weekly infusions. Also, correction of a few cells could lead to the enzyme being secreted into the circulation and taken up by their neighboring cells (crosscorrection), resulting in widespread correction of the biochemical defects. As such, the number of cells that must be modified with a gene transfer vector is relatively low.

[0161] Genetic modification can be performed either ex vivo or in vivo. The ex vivo strategy is based on the modification of cells in culture and transplantation of the modified cell into a patient. Cells that are most commonly considered therapeutic targets for monogenic diseases are stem cells. Advances in the collection and isolation of these cells from a variety of sources have promoted autologous gene therapy as a viable option.

[0162] The use of endonucleases for targeted genome editing can solve the limitations presented by the usual gene therapy protocols. These enzy mes are custom molecular scissors, allowing cutting DNA into well-defined, perfectly specified pieces, in virtually all cell types. Moreover, they can be delivered to the cells by plasmids that transiently express the nucleases, or by transcribed RNA, avoiding the use of viruses.FORMULATION

[0163] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety'. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0164] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0165] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc., may also be used.

[0166] The term “pharmaceutically acceptable excipient,’7as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro. Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0167] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0168] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0169] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-releasepreparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled- release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g.. change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0170] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.THERAPEUTIC METHODS

[0171] Also provided are processes of detecting, detecting and treating, or detecting and reversing autoimmune disorders, epilepsy, autism, schizophrenia, Alzheimer’s disease, and / or cancer in a subject in need thereof. Based on the detection, a suitable therapeutic agent (or agents) can be selected for administration to the subject.

[0172] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing autoimmune disorders, epilepsy, autism, schizophrenia. Alzheimer’s disease, and / or cancer. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subj ect.

[0173] Generally, a safe and effective amount of a suitable therapeutic agent (or agents) is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a suitable therapeutic agent (or agents) described herein can substantially inhibit, slow' the progress of, orlimit the development of autoimmune disorders, epilepsy, autism, schizophrenia, Alzheimer’s disease, and / or cancer.

[0174] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0175] When used in the treatments described herein, a therapeutically effective amount of a suitable therapeutic agent (or agents) can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to produce a desired functional outcome.

[0176] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0177] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0178] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453;Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw- Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0179] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes reversing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g.. causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.

[0180] Administration of a suitable therapeutic agent (or agents) can occur as a single event or over a time course of treatment. For example, a suitable therapeutic agent (or agents) can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0181] Treatment in accord with the methods described herein can be performed prior to or before, concurrent with, or after conventional treatment modalities for autoimmune disorders, epilepsy, autism, schizophrenia. Alzheimer's disease, and / or cancer.A suitable therapeutic agent (or agents) can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.For example, a suitable therapeutic agent (or agents) can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a suitable therapeutic agent (or agents), an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a suitable therapeutic agent (or agents), an antibiotic, an anti-inflammatory, or another agent. A suitable therapeutic agent (or agents) can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a suitable therapeutic agent (or agents)can be administered before or after administration of an antibiotic, an antiinfl ammatory, or another agent.

[0182] Active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal, such as the model systems shown in the examples and drawings.

[0183] An effective dose range of a therapeutic can be extrapolated from effective doses determined in animal studies for a variety of different animals. In general, a human equivalent dose (HED) in mg / kg can be calculated in accordance with the following formula (see e.g., Reagan-Shaw et al., FASEB J, 22(3):659-661, 2008, which is incorporated herein by reference):HED (mg / kg) = Animal dose (mg / kg) * (Animal Km / Human Km)

[0184] Use of the Kmfactors in conversion results in more accurate HED values, which are based on body surface area (BSA) rather than only on body mass. Km values for humans and various animals are well known. For example, the Kmfor an average 60 kg human (with a BSA of 1.6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Kmof 25. Km for some relevant animal models are also well known, including: mice Kmof 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Km of 5 (given a weight of 0.08 kg and BSA of 0.02); rat Km of 6 (given a weight of 0. 15 kg and BSA of 0.025) and monkey Km of 12 (given a weight of 3 kg and BSA of 0.24).

[0185] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are peculiar to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of thepatient, the route of administration, the intended goal of treatment, and the potency, stability, and toxicity of the particular therapeutic formulation.

[0186] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the ty pe of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dosage may be adjusted by the individual physician in the event of any complication.

[0187] In some embodiments, the suitable therapeutic agent (or agents) may be administered in an amount from about 1 mg / kg to about 100 mg / kg. or about 1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg, or about 1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg, or about 3 mg / kg. In some embodiments, a suitable therapeutic agent (or agentsjsuch as a compound described herein may be administered in a range of about 1 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 200 mg / kg. or about 50 mg / kg to about 100 mg / kg, or about 75 mg / kg to about 100 mg / kg, or about 100 mg / kg.

[0188] The effective amount may be less than 1 mg / kg / day, less than 500 mg / kg / day, less than 250 mg / kg / day, less than 100 mg / kg / day, less than 50 mg / kg / day, less than 25 mg / kg / day or less than 10 mg / kg / day. It may alternatively be in the range of 1 mg / kg / day to 200 mg / kg / day.

[0189] In other non-limiting examples, a dose may also comprise from about 1 micro- gram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / bodyweight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above.CELL THERAPY

[0190] Cells generated according to the methods described herein can be used in cell therapy. Cell therapy (also called cellular therapy, cell transplantation, or cytotherapy) can be a therapy in which viable cells are injected, grafted, or implanted into a patient in order to effectuate a medicinal effect or therapeutic benefit. For example, transplanting T-cells capable of fighting cancer cells via cell-mediated immunity can be used in the course of immunotherapy, grafting stem cells can be used to regenerate diseased tissues, or transplanting beta cells can be used to treat diabetes.

[0191] Stem cell and cell transplantation has gained significant interest by researchers as a potential new therapeutic strategy for a wide range of diseases, in particular for degenerative and immunogenic pathologies.

[0192] Allogeneic cell therapy or allogenic transplantation uses donor cells from a different subject than the recipient of the cells. A benefit of an allogeneic strategy is that unmatched allogenic cell therapies can form the basis of "off the shelf products.

[0193] Autologous cell therapy or autologous transplantation uses cells that are derived from the subject’s own tissues. It could also involve the isolation of matured cells from diseased tissues, to be later re-implanted at the same or neighboring tissues. A benefit of an autologous strategy is that there is limited concern for immunogenic responses or transplant rejection.

[0194] Xenogeneic cell therapies or xenotransplantation uses cells from another species. For example, pig derived cells can be transplanted into humans. Xenogeneic cell therapies can involve human cell transplantation into experimental animal models for assessment of efficacy and safety or enable xenogeneic strategies to humans as well.ADMINISTR TION

[0195] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0196] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular,intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0197] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g.. up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g.. 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0198] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled penod of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0199] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Deliver}’, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent deliver}- can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow- colocalized deposition w ith other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to highinitial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.SCREENING

[0200] Also provided are screening methods.

[0201] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g.. less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals.

[0202] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0203] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database. UCSF. with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com: and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0204] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0205] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like"’. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0206] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0207] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8 A to about 15 A.KITS

[0208] Also provided are kits. Such kits can include an agent or composition described herein and. in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to components and / or reagents described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack.Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0209] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0210] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0211] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0212] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology. 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods inEnzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis. ISBN-10: 0954523253).

[0213] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherw ise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0214] Unless otherwise indicated, an “antibody” is a peptide or protein that interacts with or binds an antigen (e.g., peptide, protein, polysaccharide, lipid, or nucleic acid). Typically, there are antigen-antibody pairs that are commercially available. Also, an antibody can interact with or bind an affinity tag in order to bind or form a complex between the peptide or protein attached to the affinity tag and the antibody.

[0215] Unless otherwise indicated an “affinity tag” or a “protein tag” is a peptide sequence that is conjugated or otherwise attached to another agent, such as a protein, peptide. Affinity tags include the agents described herein.

[0216] Unless otherwise specified, the term “complex” means that two or more compounds (e.g.. peptides, proteins, or other molecules) are associated with each other either through a covalent bond, hydrogen bonding, or van der Waal forces.

[0217] Unless otherwise specified, the term “conjugate” or “conjugated” means that the group is associated with the other through a covalent bond or hydrogen bonding.

[0218] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numencal parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported asprecisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0219] In some embodiments, the terms “a’' and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0220] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0221] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherw ise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0222] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in. or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletionoccurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0223] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0224] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0225] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Materials and MethodsMaterials

[0226] Methanol, 4% paraformaldehyde, ethanol, 40% glyoxal, acetic acid, Triton X- 100, Tween 20, tris hydrochloride, calcium chloride, sodium chloride, cholera toxin, TE buffer, and EDTA were purchased from Sigma Aldrich. Nuclease-free water, Hoechst 33342. and 6- well plates were purchased from Thermo Fisher Scientific.RNA Oligoribonucleotides

[0227] All oligoribonucleotides used in this study were custom designed and purchased from Integrated DNA Technologies (IDT). Complete sequences can be found in Table SI.Table SI. Synthetic oligoribonucleotides used in this study. The following synthetic oligoribonucleotides are labeled with Cy5 and were used for MST analysis.Cell Culture and Transfection

[0229] The G-402 cell line (ATCC) was cultured in McCoy’s 5a Medium Modified (ATCC) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). The HEK293T cell line (ATCC) and the HEK293T ADAR1 KO cell line (gifted by Dr. Charles Rice) was cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The MCF10A cell line (ATCC) was cultured in MEBM Basal Medium (Lonza) supplemented with the included additives, excluding GA-1000. and 100 ng mL1cholera toxin. The ZR-75-1 cell line (ATCC) was cultured in RPM1-1640 Medium (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cell lines were cultured at 37°C in a humidified incubator with 5% CO2. For FISH and immunofluorescence experiments, black 96-well plates (Cellvis) were coated in poly-D-lysine (Gibco) following the manufacturer’s protocol. Cells were seeded at a density of 10,000 cells per well followed by a 48-hour incubation at 37°C in a humidified incubator with 5% CO2. For transfection, cells were seeded as previously described and 24 hours post seeding (-70% confluent), cells were transfected with increasing amounts of ADAR1-GFP plasmid (pl 10 and pl50, Addgene) or coilin-GFP plasmid (Addgene) using Opti-MEM Reduced Serum Medium (Gibco) and lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. Cells were then incubated for 48 hours at 37°C in a humidified incubator with 5% CO2 before completing subsequent immunostaining. tRNA and GAPDH Fluorescence In Situ Hybridization (FISH)

[0230] For tRNA FISH, cells were seeded as previously described. Wells were then fixed with ice cold 100% methanol or 4% paraformaldehyde. Methanol treated wells were incubated at 4°C for 20 minutes and paraformaldehyde treated wells were incubated at room temperature for 15 minutes. Fixative reagents were then removed, and wells were washed with IX PBS (Invitrogen) for 1 hour at room temperature. Cells were permeabilized in 0.1% Triton X-100 in IX PBS for 10 minutes. The Stellaris RNA FISH hybridization buffer, Wash BufferA, and Wash Buffer B were prepared according to the manufacturer’s instructions (Stellaris). The IX PBS was then replaced with the prepared Stellaris RNA FISH Wash Buffer A and incubated at room temperature for 5 minutes. tRNA FISH probes (IDT, Table S2) were dissolved in TE buffer (10 mM tris hydrochloride. 1 mM EDTA, pH 8.0) at a concentration of 100 mM and then diluted in the prepared hybridization buffer to a concentration of 250 nM. Wash Buffer A was then replaced with the diluted probes, the plate was sealed to prevent evaporation, and incubated in the dark at 37°C overnight. The probes were removed and replaced with Wash Buffer A and incubated in the dark at 37°C for 30 minutes. Wash Buffer A was removed and replaced with Wash Buffer B and incubated at room temperature for 5 minutes. Wash Buffer B was exchanged for IX PBS and cells were imaged. For GAPDH FISH, cells w ere seeded as previously described. Wells w ere then fixed with ice cold 100% methanol at 4°C for 20 minutes. The methanol was replaced with IX PBS and washed at room temperature for 1 hour. Wells were treated with either 3%, 6%, 12%. or 30% glyoxal solution and incubated at 50°C for 1 hour. The glyoxal solutions were removed and wells were washed twice with IX PBS. Cells were then permeabilized in 0.1% Triton X-100 in IX PBS for 10 minutes. GAPDH FISH was completed by using Human GAPDH FISH Probes (Stellaris) and the previously described protocol for tRNA FISH. Cells were then imaged. FISH was completed under nuclease-free conditions.Table S2. tRNA Fluorescence in situ hybridization (FISH) probes. The following locked nucleic acid (LNA) FISH probes were used to detect tRNA Ser AGA. LNA bases are denoted with (+) following the nucleobase and probes are labeled with Alexa Fluor 488 (3AlexF88N).

[0231] Immunofluorescence

[0232] Nupl53 and 0-actin w ere immunstained to ensure that the EndoVIA workflow has the dynamic range needed to capture the signal associated with edited RNA. These control proteins were immunostained in in the following manner. Cells were cultured and seeded as previously described. Cells were then fixed in 100% ice cold methanol and incubated at 4°C for 20 minutes. The methanol was removed, and wells were washed twice with IX PBS. Cellswere next rehydrated with IX PBS by incubating for 1 hour at room temperature with gentle shaking. A 4% glyoxal denaturing solution from Fu and Zhuang was prepared by combining the following in order: 1.974 mL nuclease-free water, 0.796 mL of 100% ethanol, 1.200 mL of glyoxal, and 0.030 mL acetic acid and incubated at 37°C for 1 hour.1Wells were then washed twice with IX PBS. The IX PBS was replaced with a 0.1% Triton X-100 in IX PBS permeabilization solution and incubated at room temperature for 10 minutes followed by two washes in IX PBS. Cells were then incubated in a blocking solution containing 3% bovine serum albumin (Gibco), 0. 1% Triton X-100, and 0. 1% Tween 20 in IX PBS for 1 hour at room temperature. Wells were washed twice in IX PBS and then incubated in 1 : 100 anti-Nupl53 antibody (Abeam) or 1 : 1000 anti-P-actin antibody (Invitrogen) in blocking buffer for 1 hour at room temperature. The primary antibody solutions were removed, and wells were washed three times in IX PBS. Finally, a staining solution comprised of 1: 1000 Hoechst nuclear dye and 1: 1000 goat anti-rabbit Alexa Fluor 647 (Invitrogen. Nupl53) or 1: 1000 goat anti-rabbit Alexa Fluor 488 (Invitrogen, -actin) diluted in blocking buffer were added and incubated for 1 hour at room temperature in the dark. Wells were then washed in IX PBS three times and imaged. As negative controls, cells were also stained by omitting the primary or secondary antibodies to assess nonspecific binding and autofluorescence. To assess the cellular morphology’ under varying glyoxal conditions, P-actin was stained in the following manner. Cells were cultured and seeded as previously described. Cells were then immunostained as previously described, with the exception that multiple glyoxal concentrations were tested (4%, 12%, 30%). AD ARI and dsRNA were stained in the following manner. Cells were cultured and seeded as previously described. Cells were then fixed in 4% paraformaldehyde at room temperature for 15 minutes and then washed, permeabilized, and blocked as previously described for Nupl53 and P-actin. After washing with IX PBS, cells were incubated in primary antibody solutions consisting of 1 :25 anti-dsRNA J2 antibody (Sigma Aldrich), 1 :25 anti-dsRNA KI antibody (Nordic MUbio), or 1:400 anti-ADARl antibody (Atlas Antibodies) in blocking buffer and incubated for 1 hour at room temperature. The primary antibody solutions were removed, and wells were washed three times in IX PBS. Finally, a staining solution comprised of 1 :1000 Hoechst nuclear dye and 1:500 goat anti-mouse Alexa Fluor 647 (Invitrogen, J2 and KI) or 1 : 1000 goat anti-rabbit Alexa Fluor 647 (Invitrogen, AD ARI) diluted in blocking buffer were added and incubated for 1 hour at room temperature in the dark. Wells were then washed in IX PBS three times and imaged. All immunostaining incubations were completed with 200 mL volumes and solutionswere prepared fresh for each experiment. Immunofluorescence was completed under nuclease- free conditions.EndoVIA

[0233] Cells were seeded as previously described. Cells were then fixed in 100% ice cold methanol and incubated at 4°C for 20 minutes. The methanol was removed, and wells were washed twice with IX PBS. Cells were next rehydrated with IX PBS by incubating for 1 hour at room temperature with gentle shaking. A 12% glyoxal denaturing solution inspired by Fu and Zhuang was prepared by combining the following in order: 1.974 mL nuclease-free water, 0.796 mL of 100% ethanol, 1.200 mL of glyoxal, and 0.030 mL acetic acid.1These volumes were scaled up or down as needed for the experiment. The IX PBS was removed and the glyoxal denaturing solution was placed on cells and incubated at 50°C for 1 hour. Cells were then incubated with a 0.1% Triton X-100 in IX PBS permeabilization solution at room temperature for 10 minutes followed by two washes in IX PBS. Cells were then incubated in a blocking solution containing 3% bovine serum albumin, 0.1% Triton X-100, and 0.1% Tween 20 in IX PBS for 1 hour at room temperature. Wells were then washed twice with IX PBS. A calcium containing blocking buffer was prepared as follows: 3% bovine serum albumin and 1 mM calcium chloride in IX PBS. Cells were incubated with 1 :50 Endonuclease V (New England BioLabs) in the calcium containing blocking buffer, or the calcium containing blocking buffer alone as a negative control, at room temperature for 1 hour. A calcium containing wash buffer with 1 mM calcium chloride, 20 mM tris hydrochloride, and 150 mM sodium chloride was made and used for the remainder of the washing steps. Cells were then washed three times, incubating for 5 minutes each time. The commercially available EndoV is fused to a maltose-binding protein (MBP) tag that is commonly used for purification purposes. Thus, we chose to use an anti-MBP primary antibody that binds MBP of the EndoV -MBP fusion protein. Cells were then incubated for 1 hour at room temperature with a 1 : 1000 anti- MBP antibody (New England BioLabs) solution diluted in calcium containing blocking buffer. Cells were washed three times, with 5-minute incubation periods. Finally, a staining solution containing 1 : 1000 goat anti-mouse Alexa Flour 647 and 1 : 1000 Hoechst nuclear dye in calcium containing blocking buffer was incubated in the wells for 1 hour at room temperature in the dark. Cells were then washed three times for 5 minutes each time. Cells were then imaged. Because EndoV is used in a manner analogous to an antibody for inosine, optimizing concentration was thus imperative for developing a robust fluorescence-based assay thataccurately detects edited substrates. The EndoV concentration used for the EndoVIA workflow was determined by following the previously described protocol using a range of EndoV and MBP (Novus Biologicals) concentrations (1:4000-1 :25). As a negative control, a set of cells were not treated with EndoV fusion or MBP but were still incubated with primary’ and secondary antibody. The calcium dependence of EndoVIA was confirmed by treating wells with 5 mM EDTA and incubating for 1 hour at room temperature following EndoVIA and then imaged. All immunostaining incubations were completed w ith 200 mL volumes and solutions were prepared fresh for each experiment. EndoVIA was performed under nuclease-free conditions. mRNA Quantification

[0234] Cells were seeded in 6-well plates at a density of 300,000 cells per well and incubated at 37°C in a humidified incubator with 5% CO2 for 24 hours before harvesting for RNA. Cells were lysed and mRNA was isolated and purified using the Magnetic mRNA Isolation Kit (New England BioLabs). Resulting purified mRNA was quantified using Nanodrop.Microscopy and Image Analysis

[0235] Stained cells were imaged using a Nikon Spinning Disk for widefield microscopy with a 20x air objective and confocal microscopy with a 60x oil objective. Laser excitation at 405 nm was used to image Hoechst 33342; excitation at 640 nm w as used to image Alexa Fluor 647; excitation at 488 nm was used to image ADAR-GFP; excitation at 560 nm was used to image Quasar570 labeled GAPDH FISH probes. Gain and exposure settings for each laser were optimized to achieve sufficient fluorescence and minimize oversaturation. The resulting images were analyzed to determine the fluorescence of each cell using FIJI. The area and integrated density were measured for each cell and then the Corrected Total Cellular Fluorescence was calculated as follows: CTCF = integrated density — cell area X mean background fluorescence).Alu Editing Index (AE1)

[0236] Cells were seeded in 6-well plates at a density’ of 300,000 cells per well and incubated at 37°C in a humidified incubator with 5% CO2 for 48 hours. Cells were lysed and total RNA was isolated and purified using the Monarch Total RNA Miniprep Kit (New EnglandBioLabs). This purified RNA was then used to prepare sequencing libraries with the Tru-Seq Stranded with RiboZero Gold (Human / Mouse / Rat) Kit. Standard 8-bp i5 and i7 Illumina index barcode and adapters were added to each library . Libraries were sequence using a NovaSeq X Plus 300 cycles (Illumina) to produce paired end 150-bp reads (approximately 30M reads per sample). Raw FASTQ files were trimmed using Trimmomaticl with the parameter HEADCROP:3 to remove the first 3 bp.2FASTQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) was then used to check read quality (PHRED33) after data trimming. Reads were next aligned to the human reference genome hg38 using STAR 2.5.23 with the additional parameter outFilterMatchNminOverLread 0.95 to detect A-to-I editing.3The resulting .bam files were sorted, and duplicates were removed using Samtools 1.35.4Finally, the RNA editing indexer package was used with the default settings to calculate the AEI for each sample.5(1) Ye Fu, X. Z. m6A-Binding YTHDF Proteins Promote Stress Granule Formation. Physiol. Behav. 2016, 176 (3). 139-148. https: / / doi.org / 10. 1038 / s41589-020-0524-y.m.(2) Bolger, A. M.; Lohse, M.; Usadel, B. Trimmomatic: A Flexible Trimmer for IlluminaSequence Data. Bioinformatics 2014. 30 (15), 2114-2120. https : / / doi. org / 10.1093 / bioinformatics / btu 170.(3) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T. R. STAR: Ultrafast Universal RNA-Seq Aligner. Bioinformatics 2013, 29 (1), 15-21. https: / / doi.org / 10.1093 / bioinformatics / bts635.(4) Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G; Abecasis, G.; Durbin, R. The Sequence Alignment / Map Format and SAMtools. Bioinformatics 2009, 25 (16), 2078-2079. https: / / doi.org / 10. 1093 / bioinformatics / btp352.(5) Roth, S. H ; Levanon, E. Y.; Eisenberg, E. Genome-Wide Quantification of ADAR Adenosine-to-Inosine RNA Editing Activity. Nat. Methods 2019, 16 (11), 1131-1138. https : / / doi. org / 10. 1038 / s41592-019-0610-9.Kernel Density Estimation

[0237] This analysis utilized Python with the pandas, seaborn, and matplotlib libraries to create Kernel Density Estimation (KDE) plots from the individual CTCF values of each cell. The CTCF values underwent preprocessing and were loaded into a pandas DataFrame. KDE plots were then generated using the seaborn library’s kdeplot () function. The python code was written and executed using Visual Studio Code. dSTORM super-resolution Setup and Imaging

[0238] dSTORM images were acquired on a home-built setup based on a Nikon Ti2 Eclipse microscope body operated with Micro-Manager 2.0. Excitation was performed by a 180mW 638nm laser (Cobolt 06-MLD, Coherent) to which the beam was circularly polarized (ThorLabs), expanded (GBE10-A . ThorLabs) and focused at the back focal plane of the objective (SR HP TIRF, 100X, 1.49NA, Nikon) by an achromatic lens (f=300mm, AC254- 300- A, ThorLabs) mounted on a motorized translating stage ( KMTS25E, ThorLabs) to achieve TIRF illumination. A filter cube (TRF89901v2, Chroma) containing excitation and emission filters ensure spectral filtering of both light paths. Emission was detected by a sCMOS camera (Orca Fusion, Hamamatsu) with 2x2 binning, resulting in effective pixel size of 130 nm. Prior to imaging, cells were treated with dSTORM imaging buffer (GLOX) consisting of 0.56 mg.mL-1 Glucose Oxidase (Sigma), 34 pg.mL-1 Catalase (Sigma), 50 mM Tris (pH 8.00), 10 mM NaCl, 10% glucose (w / v) and 143 mM P-mercaptothanol. For imaging, a stack of 20,000 images were taken per field at maximum laser power. The first 1,000 images were discarded and the remaining stacks were analyzed by ThunderSTORM imageJ plugin. Microscope’s Perfect Focus System (PFS, Nikon) compensate for Z drift, while lOOnm fiducial marker (TetraSpeck, Invitrogen) were added post-fixation to compensate for XY drift.Glyoxal Treatment

[0239] 200 pmol of RNA A or RNA I (Table SI) was added to 14.5 pL of 40% glyoxal, 50 pL of DMSO, and nuclease-free water to a final volume of 100 pL. Samples were reacted for 1 hour at 50 °C and ethanol precipitated.Microscale Thermophoresis (MST)

[0240] For EndoV binding studies, varying amounts of EndoV (New England BioLabs) diluted in Diluent C (New England BioLabs) were combined with 0.4 pmol of the respective glyoxalated RNA A or RNA I targets in a final volume of 20 pL and incubated at room temperature for 30 minutes. Samples were then loaded into standard glass capillaries and MST was performed using a Nanotemper Monolith NT. l 15 Instrument. All measurements were analyzed using the Nano-Red filter with medium MST-Power and 100% excitation-power. Data were fitted using GraphPad Prism 8 to determine KA values. For the anti-inosine antibody binding studies, varying amounts of each antibody were diluted in 50% glycerol 50% IX PBS (MBL) or IX PBS (Diagenode) and prepared and analyzed as previously described for EndoV.Statistics

[0241] A minimum of three biological replicates were completed for each experiment. Statistical analyses were completed using GraphPad Prism 10 and all values and error bars indicate the mean ± s.d. unless otherwise noted. For comparison of two independent groups, an unpaired / -test was performed. For multiple comparisons, one-way ANOVA was performed.References(1) Nishikura, K. Functions and Regulation of RNA Editing by ADAR Deaminases. Annu. Rev. Biochem. 2010, 79. 321-349. https: / / doi.org / 10.1146 / annurev-biochem-060208- 105251.(2) Nishikura, K. A-to-I Editing of Coding and Non-Coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 2016, 77 (2), 83-96. https: / / doi.Org / 10.1038 / nrm.2015.4.(3) Kobayashi, Y.; Kawamura, F. Molecular Cloning of cDNA for Double-Stranded RNA Adenosine Deaminase, a Candidate Enzyme for Nuclear RNA Editing. Biotechnol. Read. Mass 1992, 22 (November), 123-141. https: / / doi.org / 10.1016 / b978-0-12-821471- 8.00011-8.(4) Wang, I. X.; So, E.; Devlin, J. L.; Zhao, Y.; Wu, M.; Cheung, V. G. ADAR Regulates RNA Editing, Transcript Stability, and Gene Expression. Cell Rep. 2013, 5 (3), 849-860. https : / / doi. org / 10. 1016 / j . celrep.2013. 10.002.(5) Yang, Y.; Okada, S.; Sakurai, M. Adenosine-to-Inosine RNA Editing in NeurologicalDevelopment and Disease. RNA Biol. 2021, 18 (7), 999-1013. https: / / doi.org / ! 0. 1080 / 15476286.2020. 1867797.(6) Verghese. Redirection of Silencing Targets by Adenosine-to-Inosine Editing of miRNAs. Bone 2011, 23 (1), 1-7. https: / / doi.org / 10.1126 / science.1138050.Redirection.(7) Motorin, Y.; Helm, M. Methods for RNA Modification Mapping Using Deep Sequencing:Established and New Emerging Technologies. Genes 2019, 10 (1). https: / / doi.org / 10.3390 / genesl0010035.(8) Pinto, Y.; Levanon, E. Y. Computational Approaches for Detection and Quantification ofA-to-I RNA-Editing. Methods 2019, 156 (August 2018), 25-31. https : / / doi. org / 10. 1016 / j .ymeth.2018.1 1.011.(9) Chung, H.; Calis, J. J. A.; Wu, X.; Sun, T.; Yu, Y.; Sarbanes, S. L.; Dao Thi, V. L.; Shilvock. A. R.; Hoffmann. H. H.; Rosenberg, B. R.; Rice, C. M. Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown. Cell 2018, 172 (4), 811-824. e!4. https: / / doi.Org / 10.1016 / j.cell.2017.12.038.(10) Knutson, S. D.; Ayele, T. M.; Heemstra, J. M. Chemical Labeling and Affinity' Capture of Inosine-Containing RNAs Using Acrylamidofluorescein. Bioconjug. Chem. 2018. 29 (9), 2899-2903. https: / / doi.org / 10.1021 / acs.bioconjchem.8b00541.(11) Li, Y.; Gohl, M.; Ke, K.; Vanderwal. C. D.; Spitale, R. C. Identification of Adenosine-to- Inosine RNA Editing with Acrylonitrile Reagents. Org. Lett. 2019, 27 (19), 7948-7951. https : / / doi. org / 10. 1021 / acs. orglett.9b02929.(12) Paz, N.; Levanon, E. Y.; Amariglio, N.; Heimberger, A. B.; Ram, Z.; Constantini, S.; Barbash, Z. S.; Adamsky, K; Safran. M.; Hirschberg, A.; Krupsky, M.; Ben-Dov. L; Cazacu, S.; Mikkelsen, T.; Brodie, C.; Eisenberg, E.; Rechavi, G. Altered Adenosine-to- Inosine RNA Editing in Human Cancer. Genome Res. 2007, 77 (11), 1586-1595. https: / / doi.org / 10.1101 / gr.6493107.(13) Frezza, V.; Chellini, L.; Del Verme, A.; Paronetto, M. P. RNA Editing in Cancer Progression. Cancers 2023, 15 (21), 5277. htps: / / doi.org / 10.3390 / cancersl5215277.(14) Higuchi, M.; Maas, S.; Single, F. N.; Hartner, J.; Rozov, A.; Bumashev, N.; Feldmeyer, D.; Sprengel, R.; Seeburg, P. H. Point Mutation in an AMPA Receptor Gene Rescues Lethality in Mice Deficient in the RNA-Editing Enzyme ADAR2. Nature 2000, 406 (6791), 78-81. htps: / / doi.org / 10.1038 / 35017558.(15) Wang, Q.; Miyakoda, M.; Yang, W.; Khillan, J.; Stachura, D. L.; Weiss, M. J.; Nishikura,K. Stress-Induced Apoptosis Associated with Null Mutation of ADAR1 RNA Editing Deaminase Gene *. J. Biol. Chem. 2004, 279 (6). 4952-4961. htps : / / doi. org / 10. 1074 / j be. M310162200.(16) Hartner, J. C.; Schmitwolf, C.; Kispert, A.; Muller. A. M.; Higuchi, M.; Seeburg, P. H.Liver Disintegration in the Mouse Embryo Caused by Deficiency in the RNA-Editing Enzyme ADAR1 *. J. Biol. Chem. 2004, 279 (6), 4894-4902. htps: / / doi. org / 10. 1074 / jbc. M311347200.(17) Hartner, J. C.; Walkley, C. R.; Lu, J.; Orkin, S. H. ADAR1 Is Essential for the Maintenance of Hematopoiesis and Suppression of Interferon Signaling. Nat. Immunol. 2009, 10 (1), 109-115. htps: / / doi.org / 10.1038 / ni.1680.(18) Sakurai, M.; Shiromoto, Y.; Ota, H.; Song, C.; Kossenkov, A. V.; Wickramasinghe, J.; Showe, L. C.; Skordalakes, E.; Tang. H. Y.; Speicher, D. W.; Nishikura, K. AD ARI Controls Apoptosis of Stressed Cells by Inhibiting Staufenl -Mediated mRNA Decay. Nat. Struct. Mol. Biol. 2017, 24 (6), 534-543. htps: / / doi.org / 10.1038 / nsmb.3403.(19) Fritzell, K.; Xu, L.-D.; Otrocka, M.; Andreasson, C.; Ohman, M. Sensitive ADAR Editing Reporter in Cancer Cells Enables High-Throughput Screening of Small Molecule Libraries. Nucleic Acids Res. 2019, 47 (4), e22. htps: / / doi.org / 10.1093 / nar / gkyl228.(20) Christofi, T.; Zaravinos, A. RNA Editing in the Forefront of Epitranscriptomics and Human Health. J. Transl. Med. 2019, 77 (1). htps: / / doi.Org / 10. l 186 / sl2967-019-2071-4.(21) Shoshan, E ; Mobley, A. K.; Braeuer, R. R.; Kamiya, T.; Huang, L.; Vasquez, M. E.; Salameh, A.; Lee, H. J.: Kim, S. J.; Ivan, C.; Velazquez-Torres, G.; Nip, K. M.; Zhu, K.; Brooks, D.; Jones, S. J. M.; Birol, I.; Mosqueda, M.; Wen, Y.; Eterovic, A. K.; Sood, A. K.; Hwu, P.; Gershenwald, J. E.; Gordon Robertson, A.; Calin, G. A.; Markel, G ; Fidler, I. J.; Bar-Eli, M. Reduced Adenosine-to-Inosine miR-455-5p Editing Promotes Melanoma Growth and Metastasis. Nat. Cell Biol. 2015, 17 (3), 311-321. htps: / / doi.org / 10.1038 / ncb3110.(22) Zhang, Z.; Carmichael, G. G. The Fate of dsRNA in the Nucleus: A P54(Nrb)-Containing Complex Mediates the Nuclear Retention of Promiscuously A-to-I Edited RNAs. Cell 2001, 106 (4), 465-475. htps: / / doi.org / 10. 1016 / s0092-8674(01)00466-4.(23) Prasanth, K. V.; Prasanth, S. G.; Xuan, Z.; Hearn, S.; Freier, S. M.; Bennet, C. F.; Zhang, M. Q.; Spector, D. L. Regulating Gene Expression through RNA Nuclear Retention. Cell 2005, 123 (2), 249-263. htps: / / doi.Org / 10.1016 / j.cell.2005.08.033.(24) Chen, L.-L.; DeCerbo, J. N.; Carmichael, G. G. Alu Element-Mediated Gene Silencing. EMBO J. 2008, 27 (12), 1694-1705. htps: / / doi.org / 10.1038 / emboj.2008.94.(25) Chen. L.-L.; Carmichael, G. G. Altered Nuclear Retention of mRNAs Containing Inverted Repeats in Human Embry onic Stem Cells: Functional Role of a Nuclear Noncoding RNA. Mol. Cell 2009. 35 (4), 467-478. htps: / / doi.org / 10. 1016 / j.molcel.2009.06.027.(26) Mendonsa, S.; von Kiigelgen, N.; Dantsuji, S.; Ron, M.; Breimann, L.; Baranovskii, A.;Lodige. I.; Kirchner. M.; Fischer, M.; Zema. N.; Bujanic. L.; Mertins, P.; Ulitsky, I.; Chekulaeva, M. Massively Parallel Identification of mRNA Localization Elements in Primary Cortical Neurons. Nat. Neurosci. 2023, 26 (3). 394-405. htps : / / doi. org / 10. 1038 / s41593 -022-01243 -x.(27) Kislauskis, E. H.; Singer, R. H. Determinants of mRNA Localization. Curr. Opin. Cell Biol. 1992, 4 (6), 975-978. https: / / doi.org / 10. 1016 / 0955-0674(92)90128-y.(28) Martin, K. C.; Ephrussi, A. mRNA Localization: Gene Expression in the Spatial Dimension. Cell 2009, 736 (4), 719-730. https: / / doi.Org / 10.1016 / j.cell.2009.01.044.(29) Bertrand, E.; Chartrand, P.; Schaefer, M.; Shenoy, S. M.; Singer, R. H.; Long, R. M. Localization of ASH1 mRNA Particles in Living Yeast. Mol. Cell 1998, 2 (4), 437-445. https: / / doi.org / 10. 1016 / sl097-2765(00)80143-4.(30) Rudkin, G. T.; Stollar, B. D. High Resolution Detection of DNA-RNA Hybrids in Situ byIndirect Immunofluorescence. Nature 1977, 265 (5593), 472-473. https: / / doi.org / 10.1038 / 265472a0.(31) Neubacher. S.; Hennig. S. RNA Structure and Cellular Applications of Fluorescent Light- Up Aptamers. Angew. Chem. Int. Ed Engl. 2019, 58 (5), 1266-1279. https: / / doi.org / 10.1002 / anie.201806482.(32) Levesque, M. J.; Ginart, P.; Wei, Y.; Raj, A. Visualizing SNVs to Quantify Allele- Specific Expression in Single Cells. Nat. Methods 2013, 10 (9), 865-867. https: / / doi.org / 10.1038 / nmeth.2589.(33) Meilis, I. A.; Gupte, R.; Raj, A.; Rouhanifard, S. H. Visualizing Adenosine-to-Inosine RNAediting in Single Mammalian Cells. Nat. Methods 2017, 14 (8), 801-804. https : / / doi. org / 10.1038 / nMeth.4332.(34) Fang, G; Chamiolo, J.; Kankowski, S.; Hbvelmann, F.; Friedrich, D.; Lower, A.; Meier, J. C.; Seitz, O. A Bright FIT-PNA Hybridization Probe for the Hybridization State Specific Analysis of a C — > U RNA Edit via FRET in a Binary System. Chem. Sci. 2018, 9 (21), 4794-4800. https: / / doi.org / 10.1039 / C8SC00457A.(35) Knoll, A.; Kankowski, S.; Schbllkopf, S.; Meier, J. C.; Seitz, O. Chemo-Biological mRNA Imaging with Single Nucleotide Specificity. Chem. Commun. 2019, 55 (98), 14817-14820. https: / / doi.org / 10.1039 / C9CC06989E.(36) Chen, F.; Bai, M.; Cao. X.; Zhao, Y.; Xue. J.; Zhao, Y. Click-Encoded Rolling FISH for Visualizing Single-Cell RNA Polyadenylation and Structures. Nucleic Acids Res. 2019, 47 (22), el45. https: / / doi.org / 10.1093 / nar / gkz852.(37) Sheehan, C. J.; Marayati, B. F.; Bhatia, J.; Meyer, K. D. In Situ Visualization of m6A Sites in Cellular mRNAs. Nucleic Acids Res. 2023, 51 (20), elOl. https : / / doi. org / 10.1093 / nar / gkad787.(38) Inouye, H.; Fuchs, S.; Sela. M.; Littauer, U. Z. Detection of Inosine Containing Transfer Ribonucleic Acid Species by Affinity Chromatography on Columns of Anti Inosine Antibodies. J. Biol. Chem. 1973, 248 (23), 8125-8129. https: / / doi.org / 10.1016 / s0021- 9258(19)43202-x.(39) Vik, E. S.; Sameen Nawaz, M.; Strom Andersen, P.; Fladeby, C.; Bjoras, M.; Dalhus, B.; Alseth, I. Endonuclease v Cleaves at Inosines in RNA. Nat. Commun. 2013, 4. https : / / doi. org / 10.1038 / ncomms3271.(40) Wu, J.; Samara, N. L.; Kuraoka, I.; Yang, W. Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase. Mol. Cell 2019, 76 (1), 44-56. e3. https : / / doi. org / 10. 1016 / j .molcel.2019.06.046.(41) Yao, M.; Hatahet, Z.; Melamede, R. J.; Kow, Y. W. Purification and Characterization of a Novel Deoxyinosine-Specific Enzyme, Deoxyinosine 3’ Endonuclease, from Escherichia Coll. J. Biol. Chem. 1994, 269 (23), 16260-16268. https: / / doi.org / 10. 1016 / s0021-9258(17)34002-4.(42) Dalhus, B.; Arvai, A. S.; Rosnes, I.; Olsen, 0. E.; Backe, P. H.; Alseth, L; Gao, H.; Cao, W.; Tainer. J. A.; Bjoras, M. Structures of Endonuclease V with DNA Reveal Initiation of Deaminated Adenine Repair. Nat. Struct. Mol. Biol. 2009, 16 (2), 138-143. https: / / doi.org / 10.1038 / nsmb.1538.(43) Knutson, S. D.; Arthur, R. A.; Johnston, H. R.; Heemstra, J. M. Selective Enrichment of A-to-I Edited Transcripts from Cellular RNA Using Endonuclease v. ACS Appl. Mater. Interfaces 2020. https: / / doi.org / 10.1021 / jacs.9bl3406.(44) Knutson, S. D.; Arthur, R. A.; Johnston, H. R.; Heemstra, J. M. Direct Immunodetection of Global A-to-I RNA Editing Activity with a Chemiluminescent Bioassay. Angew. Chem. - Int. Ed. 2021, 60 (31), 17009-17017. https: / / doi.org / 10.1002 / anie.202W2762.(45) Im, ; Mareninov, S.; Diaz, M. F. P.; Yong, W. H. An Introduction to Performing Immunofluorescence Staining. Methods Mol. Biol. 2019, 1897 (310), 299-311. https: / / doi.org / 10.1007 / 978-l-4939-8935-5_26.(46) Sarkar, S.; Hopper, A. K. tRNA Nuclear Export in Saccharomyces Cerevisiae: In SituHybridization Analysis. Mol. Biol. Cell 1998. 9 (11), 3041-3055. https: / / doi.Org / 10.1091 / mbc.9. 11.3041.(47) Tones, A. G.; Pineyro. D.; Filonava, L.; Stracker. T. H.; Batlie, E.; Ribas De Pouplana, L. A-to-I Editing on tRNAs: Biochemical, Biological and Evolutionary Implications. FEBS Lett. 2014, 588 (23), 4279-4286. https: / / doi.Org / 10.1016 / j.febslet.2014.09.025.(48) Palazzo, A. F.; Lee, E. S. Non-Coding RNA: What Is Functional and What Is Junk? Front. Genet. 2015, 6.(49) Hobro, A. J.; Smith, N. I. An Evaluation of Fixation Methods: Spatial and Compositional Cellular Changes Observed by Raman Imaging. Vib. Spectrosc. 2017, 97, 31-45. https: / / doi.Org / 10.1016 / j.vibspec.2016.10.012.(50) Ye Fu, X. Z. m6A-Binding YTHDF Proteins Promote Stress Granule Formation. Physiol. Behav. 2016, 776 (3), 139-148. https: / / doi.org / 10.1038 / s41589-020-0524-y.rn.(51) Richter. K. N.; Revelo, N. H.; Seitz, K. J.; Helm, M. S.; Sarkar, D.; Saleeb, R. S.; D’Este,E.; Eberle, J.; Wagner, E.; Vogl, C.; Lazaro, D. F.; Richter, F.; Coy-Vergara, J.; Coceano, G.; Boyden, E. S.; Duncan, R. R.; Hell, S. W.; Lauterbach, M. A.; Lehnart, S. E.; Moser, T.; Outeiro, T. F.; Rehling, P.; Schwappach, B.; Testa, L; Zapiec, B.; Rizzoli, S. O. Glyoxal as an Alternative Fixative to Formaldehyde in Immunostaining and Superresolution Microscopy. EMBO J. 2018, 37 (1), 139-159. https: / / doi.org / 10.15252 / embj.201695709.(52) Bunnell, T. M.; Burbach, B. J.; Shimizu, Y .; Ervasti, J. M. P-Actin Specifically Controls Cell Growth, Migration, and the G-Actin Pool. Mol. Biol. Cell 2011, 22 (21), 4047-4058. https: / / doi.org / 10.1091 / mbc.El 1-06-0582.(53) Moroianu, J.; Blobel, G.; Radu, A. RanGTP-Mediated Nuclear Export of Karyopherin a Involves Its Interaction with the Nucleoporin Nupl53. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (18), 9699-9704. https: / / doi.org / 10.1073 / pnas.94.18.9699.(54) Chen, L.; Kashina, A. Quantification of Intracellular N-Terminal P-Actin Arginylation. Sci. Rep. 2019, 9 (1), 1-9. https: / / doi.org / 10.1038 / s41598-019-52848-5.(55) Kadota, S.; Ou, J.; Shi, Y.; Lee, J. T.; Sun, J.; Yildirim, E. Nucleoporin 153 Links Nuclear Pore Complex to Chromatin Architecture by Mediating CTCF and Cohesin Binding. Nat. Commun. 2020, 77 (1). https: / / doi.org / 10.1038 / s41467-020-16394-3.(56) Roth, S. H.; Levanon, E. Y.; Eisenberg, E. Genome-Wide Quantification of ADAR Adenosine-to-lnosine RNA Editing Activity. Nat. Methods 2019, 16 (11), 1131-1138. https: / / doi.org / 10.1038 / s41592-019-0610-9.'(57) Knutson, S. D.; Arthur. R. A.; Johnston, H. R.; Heemstra, J. M. Direct Immunodetection of Global A-to-I RNA Editing Activity with a Chemiluminescent Bioassay. Angew. Chem. 2021, 133 (31), 17146-17154. https: / / doi.org / 10.1002 / ange.202102762.(58) Paz-Yaacov, N.; Bazak, L.; Buchumenski, L; Porath, H. T.; Danan-Gotthold, M.; Knisbacher. B. A.; Eisenberg, E.; Levanon. E. Y. Elevated RNA Editing Activ ity Is a Major Contributor to Transcriptomic Diversity in Tumors. Cell Rep. 2015, 13 (2), 267- 276. https: / / doi.Org / 10.1016 / j.celrep.2015.08.080.(59) Yuan, J.; Xu, L.; Bao, H.-J.; Wang, J.; Zhao, Y.; Chen, S. Biological Roles of A-to-I Editing: Implications in Innate Immunity, Cell Death, and Cancer Immunotherapy. J. Exp. Clin. Cancer Res. 2023, 42 (1), 149. https: / / doi.org / 10.1 186 / sl3046-023-02727-9.(60) Gumireddy. K.; Li, A.; Kossenkov, A. V.; Sakurai, M.; Yan, J.; Li, Y .; Xu, H.; Wang, J.; Zhang, P. J.; Zhang, L.; Showe, L. C.; Nishikura, K; Huang, Q. The mRNA-Edited Form of GABRA3 Suppresses GABRA3-Mediated Akt Activation and Breast Cancer Metastasis. Nat. Commun. 2016, 7, 10715. https: / / doi.org / 10.1038 / ncommsl0715.(61) Hu, X.; Wan, S.; Ou, Y.; Zhou, B.; Zhu, J.; Yi, X.; Guan, Y.; Jia, W.; Liu, X.; Wang, Q.; Qi, Y.; Yuan. Q.; Huang. W.; Liao. W.: Wang. Y.; Zhang, Q.; Xiao, H.; Chen. X.; Huang, J. RNA Over-Editing of BLCAP Contributes to Hepatocarcinogenesis Identified by Whole-Genome and Transcriptome Sequencing. Cancer Lett. 2015, 357 (2), 510-519. https: / / doi.Org / 10.1016 / j.canlet.2014.12.006.(62) Schaffer, A. A.; Kopel, E.; Hendel, A.; Picardi. E.; Levanon, E. Y.i Eisenberg, E. The Cell Line A-to-I RNA Editing Catalogue. Nucleic Acids Res. 2020, 48 (11), 5849-5858. https: / / doi. org / 10.1093 / nar / gkaa305.(63) Han, L.; Diao, L.; Yu, S.; Xu, X.; Li, J.; Zhang, R.; Yang, Y .; Wemer, H. M. J.; Eterovic, A. K.; Yuan, Y.; Li, J.; Nair. N.; Minelli, R.; Tsang. Y. H.; Cheung, L. W. T.; Jeong. K. J.; Roszik, J.; Ju, Z.; Woodman, S. E.; Lu, Y.; Scott, K. L.; Li, J. B.; Mills, G. B.; Liang, H. The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers. Cancer Cell 2015, 28 (4), 515-528. https: / / doi.Org / 10.1016 / j.ccell.2015.08.013.(64) Hasenauer, J.; Waldherr, S.; Doszczak, M.; Scheurich, P.; Radde. N.; Allgower. F. Analysis of Heterogeneous Cell Populations: A Density-Based Modeling and Identification Framework. J. Process Control 2011, 21 (10), 1417-1425. https : / / doi. org / 10. 1016 / j .j procont.2011.06.020.(65) Katz, Z. B.; Wells. A. L.; Park, H. Y.; Wu, B.; Shenoy, S. M.; Singer, R. H. (3-Actin mRNA Compartmentalization Enhances Focal Adhesion Stability and Directs Cell Migration. Genes Dev. 2012, 25 (17), 1885. https: / / doi.org / 10.1101 / gad.190413.112.(66) Katz, Z. B.; English, B. P.; Lionnet, T.; Yoon, Y. J.; Monnier, N.; Ovryn, B.; Bathe, M.; Singer, R. H. Mapping Translation "hot-Spots" in Live Cells by Tracking Single Molecules of mRNA and Ribosomes. eLife 5, el 0415. https: / / doi.org / 10.7554 / eLife.10415.

Claims

CLAIMS1. A method of detecting ribonucleic acid (RNA) containing one or more inosine bases in cells comprising: fixing cells to retain large RNAs and not retain small RNAs to form fixed cells; denaturing the secondary' structure of the large RNAs to form denatured RNA; incubating the denatured large RNAs with a calcium-containing solution of endonuclease V (EndoV) to form a complex between EndoV and RNA; tagging EndoV with a fluorophore; imaging the cells using fluorescence microscopy to detect the RNA containing one or more inosine bases.

2. The method of claim 1, wherein tagging EndoV with a fluorophore comprises using EndoV fused with an affinity' tag (EndoV -affinity tag) and conjugating a fluorophore to the affinity' tag.

3. The method of claim 1, wherein tagging EndoV with a fluorophore comprises using EndoV fused with an affinity' tag (EndoV -affinity tag) and forming a complex between EndoV -affinity' tag and RNA; incubating the complex of EndoV -affinity' tag and RNA with an antibody for the affinity tag conjugated to the fluorophore to form a complex of EndoV - affinity tag, antibody to the affinity tag, and RNA.

4. The method of claim 1, wherein tagging endo V with a fluorophore comprises using EndoV fused with an affinity' tag (EndoV -affinity tag) and forming a complex between EndoV -affinity tag and RNA; incubating the complex of EndoV -affinity tag and RNA with an antibody' for the affinity tag to form a complex of EndoV-affinity tag, antibody to the affinity' tag, and RNA; and incubating the complex of EndoV-affinity' tag, antibody to the affinity' tag, and RNA with a secondary' antibody specific for the antibody to the affinity' tag and conjugated to a fluorophore.

5. The method of any one of claims 1 to 4, wherein cells are fixed using methanol, glyoxal, formaldehyde, or paraformaldehyde.

6. The method of any one of claims 1 to 5, wherein large RNAs have at least 100 nucleotides.

7. The method of any one of claims 1 to 6, wherein small RNAs have less than 100 nucleotides.

8. The method of any one of claims 1 to 7, wherein denaturing the secondary structure of the large RNAs comprises treating the cells with a glyoxal solution, a urea solution, a guanidinium solution, or a methylglyoxal solution.

9. The method of any one of claims 1 to 8, wherein the secondary structure of a RNA double strand is denatured to a RNA single strand.

10. The method of any one of claims 1 to 9, wherein the calcium-containing solution of EndoV does not significantly cleave inosine-containing RNAs.

11. The method of any one of claims 2 to 10, wherein the affinity tag is maltose-binding protein (MBP). a chitin binding protein (CBP), Strep-tag. glutathione-S-transferase (GST). poly(His), FLAG octapeptide, human influenza hemagglutinin (HA) tag, or cognate 13- amino acid peptide (SpyTag).

12. The method of any one of claims 3 to 11, wherein the antibody to the affinity tag is anti-MBP, anti-CBD, anti-Strep tag, anti-GST, anti-His-tag, anti-FLAG, anti-HA, or Streptococcus pyogenes surface protein (Spy Catcher).

13. The method of any one of claims 4 to 12, wherein the secondary antibody specific for the anti-affinity tag antibody comprises an antibody specific for anti-MBP, anti-CBD, anti- Strep tag, anti-GST, anti-His-tag, anti-FLAG, anti-HA, or SpyCatcher.

14. The method of any one of claims 1 to 13, wherein the fluorophore comprises a fluorescent dye that absorbs light at a particular wavelength and emits light of longer wavelength.

15. The method of any one of claims 1 to 14, further comprising permeabilizing the fixed cells by contacting with detergent.

16. The method of any one of claims 1 to 15, further comprising incubating the fixed cells with a protein blocking agent.

17. The method of claim 16, wherein the protein blocking agent comprises bovine serum albumin, normal serum, milk powder, gelatin, a protein solution, or a combination thereof.

18. The method of any one of claims 2 to 17, wherein the endo V-affinity tag is EndoV fused with MBP and is a recombinant Escherichia coli EndoV -MBP.

19. The method of any one of claims 1 to 18, wherein the abundance of RNA containing one or more inosine bases in a cell is quantified.

20. The method of any one of claims 1 to 19, wherein the subcellular localization of Adenosine-to-Inosine (A-to-I) editing in RNA characterized.

21. A method of determining a disease or condition in a subject in need thereof comprising performing the method of any one of claims 1 to 20 on a cell sample from the subject; identifying an abundance of RNA containing one or more inosine bases in the cell sample and comparing the abundance to the abundance in healthy cells; and determining a suitable treatment for the disease or condition based on the difference in the abundance of RNA containing one or more inosine bases in the cell sample.

22. A method of determining a disease or condition treatment for a subject in need thereof, the method comprising performing the method of any one of claims 1 to 20 on a cell sample from the subject; identifying the location of the RNA containing one or more inosine bases in the cell and comparing to the location in healthy cells: and determining a suitable treatment for the disease or condition based on the difference in the location of RNA containing one or more inosine bases in the cell sample.

23. A kit for earn ing out the method of any one of claims 1 to 20, comprising a calcium-containing solution of endonuclease V (EndoV); a fluorophore; and instructions for conjugating the EndoV with the fluorophore, fixing cells, denaturing cells and imaging the cells using fluorescence microscopy.

24. The kit of claim 23, wherein the EndoV is fused with an affinity tag (EndoV -affinity tag).

25. The kit of claim 24, wherein the affinity tag is conjugated with the fluorophore.

26. The kit of claim 23, further comprising an antibody to the affinity tag.

27. The kit of claim 26, wherein the antibody to the affinity tag is conjugated to the fluorophore.

28. The kit of claim 26, further comprising a secondary antibody specific for the antibody to the affinity' tag and being conjugated to the fluorophore.

29. The kit of any one of claims 23 to 28, further comprising a cell fixing agent.

30. The kit of any one of claims 23 to 29, further comprising a denaturing agent.

31. A method of characterizing the subcellular localization of Adenosine-to-Inosine (A- to-I) editing in RNA. the method comprising: performing an Endonuclease V Immunostainmg Assay (EndoVIA) on a cell sample comprising edited RNA; and providing a spatial visualization of A-to-I editing in the cell sample.

32. A method of quantifying overall inosine abundance in a cell, the method comprising: performing an Endonuclease V Immunostaining Assay (EndoVIA) on a cell sample; andmeasuring an amount of inosine based on an anti-inosine antibody comprising EndoV.

33. A method of determining a cancer treatment for a subject in need thereof, the method comprising: performing an Endonuclease V Immunostaining Assay (EndoVIA) on a cancerous cell sample from the subject; identifying a hyper-editing cancerous cell line or a hypo-editing cancerous cell line based on an Adenosine-to-Inosine (A-to-I) level from the EndoVIA; and determining a suitable cancer treatment based on the identified hyper- or hypo-editing cancerous cell line.

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