Selaginella. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte  Howeveor (PUF) . Other vor (PUF) . Althougor (PUF) ), these or (PUF) , 26. In or (PUF) . The rolor (PUF) , 27. Theor (PUF) . In addior (PUF) , 30. Addor (PUF) —at leastor (PUF) , which hor (PUF) . These pbacteria  and in vbacteria  in the aaginella , 24. Thi2.2.19 PPR array, the hydrophobic residues in the first helix of the S motif were replaced by hydrophilic residues. All the sequences used for the design of the PPR proteins can be found in Four designed synthetic S (dsnS) motifs  were used in this study . The des2.2https://www.genscript.com/). The TRX-9P-S2 gene comprised two gBlocks (https://www.idtdna.com) and assembled together by Gibson assembly into pETM20 , 0.05 U/µl Phusion polymerase (New England BioLabs) and 8 U/µl Taq DNA ligase (New England Bioabs)). The reaction was transformed into E. coli DH5α competent cells following 30-min incubation at 50°C. The genes encoding the GFP-9S proteins were made in two steps. Initially, we prepared a pETM11 plasmid  and clone them into a pET-GFP plasmid that already contained Motifs 5–9. The ligation was transformed directly into Rosetta 2(DE3) cells and used for picking colonies into 96 deep well plates for screening of binding. We sequenced a library of 644 clones to identify the fifth/last combination in the fourth PPR motif in each of the randomized constructs. This represents about 1.5 times the potential diversity of the library.To partially randomize nucleotide recognition by Motif 4, a motif with fifth/last codons Asn/Asp was amplified from the pGEM plasmid with a set of four forward primers (in the ratio 12:6:1:1) containing nucleotides NDT, VMA, ATG or TGG at the fifth codon and a set of four reverse primers in the same ratio with the complementary partially degenerate codons at the last codon position of the motif . The choAll PCR amplifications were done with PrimeSTAR HS ExTaq DNA polymerase (Takara Bio) according to the manufacturer’s recommendations .2.3600 0.4–0.8 at 37°C, transferred to 16°C and, once the culture had time to cool down, 0.1 mM IPTG was added and the bacteria continued growing at 16°C overnight. After spinning down the bacteria the next day, the pellet was either stored at −80°C or immediately resuspended in lysis buffer  and lysed by sonication. Examples of protein expression for the constructs used in this work are shown in The proteins were made by transforming the plasmids into Rosetta 2(DE3) cells (Novagen) that were grown overnight at 37°C. This preculture was used to inoculate 0.5 l of a fresh LB media at a ratio of 1:50. The culture was grown to OD2.42PO4 pH 7.5, 100 mM KF) to 0.1 mg/ml immediately before CD wavelength scan analysis. CD measurements were performed in triplicate using a JASCO J-810 spectropolarimeter with quartz cuvette of 1 mm path length, 100 millidegree sensitivity, 1 nm data pitch, 100 nm/min scanning speed, 2 s response time, 4 nm bandwidth and three accumulations between 185 and 260 nm at room temperature (24°C).Purified proteins were concentrated to 10 mg/ml using 10 kDa M.W.C.O. centrifugal filter (Amicon) in Tris pH 8, 200 mM KCl, 5% glycerol buffer. Proteins were then diluted 100 times in circular dichroism (CD) buffer  were made up in the same buffer to 200 mM. Protein samples were placed in the sample cell (cell volume = 200 µl) and titrated with RNA. Titrations were performed at 25°C with a stirring speed of 1000 rpm. RNA was injected 18 times from a computer-controlled syringe at a volume of 2 µl over 4 s for each injection, with a spacing of 150 s between injections. Only 0.5 µl was injected for the first injection and ignored in analysis to minimize potential errors from preparation. Experimental data were fitted to a theoretical titration curve using the Origin software .A Microcal iTC200 from GE Healthcare was used to perform isothermal titration calorimetry (ITC) analysis. Proteins were diluted to 20 mM into Tris pH 8, 200 mM KCl, 5% glycerol buffer. RNA target ligands featuring the 2.6ds were estimated from the binding curves taking into account the probe concentration  was added to a final concentration of 5 nM. Readings were performed on a CLARIOstar Plus microplate reader (BMG Labtech) 532 ± 25 nm/595 ± 35 nm fitted with a polarization membrane. Kntration .2.7ds were estimated from the binding curves taking into account the probe concentration (Cy5-labeled RNA target (Cy5-rpoA) was added at a final concentration of 20 nM to serial dilutions of the protein made in binding buffer . Following 30 min incubation, we measured binding at 20% LED and 40% laser power using a Monolith NT.115 MicroScale Thermophoresis apparatus (NanoTemper Technologies GmbH). The measurements were repeated three times. Kntration .2.8ds were estimated from the binding curves taking into account the probe concentration  in a polypropylene PCR plate. After binding, the beads were washed with binding buffer  and resuspended in binding buffer containing 2 nM fluorescently labeled RNA probe. All the washes were done by fixing the beads with a magnet and aspirating the liquid with a pipette. The reaction volume was normally 20 µl. The beads were incubated in the dark between 30 min and an hour and then cooled down on ice for approximately 15 min. The next steps were all done on ice. As quickly as possible, the supernatant of the reaction was removed by a multi-channel pipette, and the pellet was washed in 100-µl ice-cold binding buffer and resuspended in 8 M urea to elute the bound RNA. The eluent was transferred into a fresh PCR plate alongside serial dilutions of the original RNA solution and imaged by a Typhoon scanner  to detect fluorescein (473/>510 nm), Cy5 (635/>665 nm) and IRDye® 800CW (785/814–826 nm). The image was quantified by ImageQuant software . Kntration .Labeled RNA sequences used in this study: Cy5-rpoA = Cy5-AUUACACGUGA; Cy5-rpoA-G = Cy5-AUUAGACGUGA; IR-rpoA-A = IRDye® 800CW-AUUAAACGUGA; IR-rpoA-U = IRDye® 800CW-AUUAUACGUGA; Cy5-clpP = Cy5-CAGCAACAGAAGCCCAAGCUCAUGGA.2.9ds were estimated from the binding curves taking into account the probe concentration  and scaling the values between 0 and 1. For comparison with the data from .Rosetta 2(DE3) colonies containing the GFP-9S library were picked into 96 deep well plates and grown overnight in 0.5 ml LB at 37°C. This preculture was used to inoculate a fresh 0.5-ml LB following the same protocol as growing culture for protein purification. After the overnight induction at 16°C, the plates were spun and the pellet remaining in the plate was frozen at −80°C (to facilitate cell lysis upon thawing). The cells were resuspended in lysis buffer supplemented with 1 unit of DNaseI (ThermoFisher), 50 mg/ml lysozyme and 0.05% Triton X-100. The resuspended cells were incubated 1 h at 37°C shaking and then spun. The supernatant was used for further analysis. The protein content was quantified by measuring GFP fluorescence from 100 µl lysate and comparing to a standard curve made of dilutions of recombinant GFP purified by nickel affinity column similar to the PPR protein purification and gel filtration (Amersham S200 column using 50 mM Tris and 100 mM NaCl buffer). Unless otherwise specified, 100 µl lysate was used in the screens using the same protocol as for the other RNA-binding measurements. Kntration . Estimatata from , their r2.10rpoA editing site of CLB19 was cloned into the 3ʹ untranslated region (UTR) of the TRX-9S-DYW transcript by amplifying the entire TRX-9S-DYW plasmid using PrimeSTAR HS ExTaq DNA polymerase (Takara Bio) with primers RpoA_GA.For and RpoA_GA.Rev. The resulting PCR fragment was DpnI-digested, gel-purified and circularized using Gibson assembly before transforming into E. coli DH5α. The sequence encoding for the amino acids 73–193 of Arabidopsis thaliana MORF2 protein was amplified from Arabidopsis cDNA using PrimeSTAR HS DNA polymerase (Takara Bio) with primers MORF2_73.For and MORF2_193.Rev  cells. Five-milliliter starter cultures of Rosetta 2(DE3) were inoculated from single colonies and grown overnight at 37°C at 200 rpm. Two hundred fifty microliter of each overnight culture was inoculated into an Erlenmeyer flask with 25 ml of LB supplemented with the appropriate antibiotics and grown at 37°C 200 rpm until OD600 reached 0.4. The cultures were cooled at 4°C for 15 min and then placed at 16°C 180 rpm for a further 15 min. The media was supplemented with 0.4 mM ZnSO4 and expression of the PPR and MORF2 proteins was induced by adding IPTG to a final concentration of 0.4 mM. The cultures were grown at 16°C 180 rpm for 20 h before harvesting 1 ml of each culture by centrifugation at 4000 rpm for 2 min. The supernatant was discarded after which the pellets were flash frozen in liquid nitrogen and stored at −80°C until RNA extraction. RNA was extracted from the E. coli pellets using the Zymo Direct-Zol RNA miniprep kit (Zymo Research) following the manufacturer’s protocol.The TRX-9S-DYW and petM11-MORF2 plasmids were sequence-verified and transformed into clumpify  from the bbmap package (https://sourceforge.net/projects/bbmap/), then trimmed of adapters with bbduk (parameters: ktrim = r k = 23 mink = 11 hdist = 1 tpe tbo ftm = 5) and mapped to the E. coli BL21 genome (accession CP010816) and the relevant pETM11 and pETM20 constructs with bbmap (parameters: ambiguous = random mappedonly = t). Strand-specific nucleotide counts were obtained using in-house code . Nucleotide count data was analyzed statistically with a Fisher exact test as implemented in the Python scipy.stats package and the p-values were corrected for multiple testing using statsmodels.stats.multitest.multipletests with the Simes-Hochberg procedure. Odds ratios were calculated after adding a pseudocount of 0.5 to all observations to avoid division by zero.Total RNA extracted as described above was DNase digested using Turbo DNase (Ambion) and quantified using Qubit® . 400 ng of the DNase-digested total RNA was depleted of ribosomal RNA  and used3.3.1Selaginella moellendorffii genome, where such S-type arrays are common. In these natural PPR proteins, the motifs at the N and C termini of PPR arrays differ slightly in characteristic ways from those toward the center of the arrays. Whether these differences are functionally significant has never been tested. Therefore, from these alignments, we derived four designed synthetic S (dsnS) motifs for different positions in the protein  that imitate the subtle differences between these motifs in native S-type PPR proteins  of the aligned motifs came from the proteins . In natuArabidopsis thaliana chloroplast rpoA transcript. This sequence is part of the binding site for the RNA editing factor CLB19 that has been extensively studied in our lab   , Table 2s [MST]) , Table 2s [MST]) , Table 23.3Beyond the most common amino-acid combinations at the fifth/last positions in natural PPR proteins, there are many more combinations that occur rarely and which might bind to the four RNA nucleotides with varying affinity and specificity. A few studies have attempted to infer the binding preferences of the various amino-acid-acid combinations from a statistical analysis of the known native PPR proteins and their RNA targets , 46–49. rpoA RNA target. All nine motifs were based on the dsnSc consensus. Eight of the motifs were held constant, whilst the fourth motif was a different amino-acid combination in each protein . A library was prepared by randomizing the fifth and last codons of the fourth motif  to four RNA targets differing only in the nucleotide aligned to the fourth motif . We founth motif  using prth motif . The PPRds. To be able to assess the full range of Kds accurately, we performed the screen three times with five different protein concentrations  are also relatively rare in natural S-type proteins  is similar to the rpoA target site (UUACACGUGCAAAAUC), making it a likely off-target. However, the amount of editing is extremely low (14 edited reads out of 10 303), and the identical sequence in tufA is edited even less (1 edited read out of 10 419).In plant organelles, S-type PPR motifs are predominantly found in RNA editing factors. This prompted us to design a synthetic RNA editing factor based on an S-type PPR motif array. To test for the ability to edit RNA, we expressed the  E. coli . The tar E. coli . RNA edi E. coli . The exp E. coli . The speactor Tu . This ed4.E. coli, is relatively soluble and is predicted to fold into a typical PPR structure composed of tandem helix-turn-helix repeats , i.e. 5–12 nM for complexes with the rpoA target. The apparent Kd measured by ITC was considerably higher (48–68 nM). Whereas the pulldown assay, fluorescence anisotropy and MST are all end-point assays measuring the ‘true’ Kd at equilibrium (if the protein/RNA mix has reached equilibrium before the assay), ITC measures the transient enthalpy change when the RNA and protein are mixed. In the case of a complex, slow interaction (likely to be the case for PPR-RNA binding) ITC may reveal the thermodynamics for an initial fast phase of the binding, likely leading to a less stable complex (and thus a higher Kd) than the final bound state.In this paper, we present a novel designed PPR protein chassis that we developed based on the alignment of native S-type PPR motifs in a similar approach to that taken previously for P-type proteins . The cha repeats . We have repeats . Many diThe affinity of PPR proteins for their RNA targets is determined by the identity of two amino acids at the fifth and last position of each motif . We wereex-vivo applications and suggests that it should function in a variety of host organisms.To be useful in a wide range of applications, it is essential that dsnS proteins are tolerant to a wide range of physiological  conditions. In comparison with our previous dsnP chassis, dsnS proteins bind consistently tightly to their RNA target over a broad range of pH, salt and temperature conditions. This makes this chassis a good basis for Physcomitrium patens . In general, PPR motifs distinguish between purines and pyrimidines much more effectively than they can distinguish between A and G or C and U (tufB sequence and the rpoA target (discounting the C/U mismatch adjacent to the editing site which is not in the region bound by the PPR motifs), two are purine mismatches (A/G) and only one is a purine-pyrimidine mismatch (A/C). In addition, the five nucleotides (CAAAA) that would align with the P2-L2-S2-E1-E2 motifs are also identical to those at the equivalent position of the intended target. Based on our prior results with synthetic editing factors (tufB transcript.As a demonstration of utility, we constructed a synthetic editing factor using a dsnS PPR array to target a specific cytidine within the UTR of its own transcript. We had previously constructed a similar editing factor using triplet arrays of P, L and S motifs modeled on natural PPR editing factors from plants . The prem patens , 36. MORm patens . We hypo editing . The spe C and U . Of the  factors , this isin  vivo affords an exciting route to repair or alter the protein content of an organism without permanently changing its genetics (Targeted RNA editing genetics . In pringenetics , add divgenetics , providegenetics  or regulgenetics . Most regenetics  or dCas1genetics . Howeverysab034_SuppClick here for additional data file.


Degradation of RBM39 by indisulam leads to exceptional response in high-risk neuroblastoma. Aberrant alternative pre-mRNA splicing plays a critical role in MYC-driven cancers and therefore may represent a therapeutic vulnerability. Here, we show that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires the splicing factor RBM39 for survival. Indisulam, a “molecular glue” that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to significant responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy. High-risk neuroblastoma has a poor prognosis and leads to a disproportionate number of cancer-related deaths in children, despite the use of increasingly intensive, multimodal therapies  are among the most common alterations in human cancer , we analyzed the 3000 genes most differentially expressed between low- and high-risk neuroblastomas among 498 primary neuroblastoma samples  were identified in a genome-wide CRISPR-Cas9 library screen by the Sanger institute  (fig. S1C). We further overexpressed MYCN in SK-N-SH cells and performed real-time polymerase chain reaction (PCR) and Western blot, which showed that RBM39 was induced at mRNA and protein levels by MYCN . These data suggest that RBM39 is a direct target of MYC.To determine cellular pathways in high-risk neuroblastoma   or C-MYC overexpression (SK-N-AS)  in comparison to vector controls , which are involved in a variety of key cancer pathways such as cell cycle (CDK4 and CCND3), apoptosis (BAX and BIRC6), DNA repair , tyrosine kinase receptor–mediated signal transduction , epigenic regulation , and mitochondrial metabolism (SDHC and SDHAF2), suggesting that RBM39 is a critical factor in the oncogenesis of neuroblastoma. Real-time PCR for specific exons spliced by RBM39 knockdown validated the RNA sequencing (RNA-seq) results . Western blot showed that the expression of these targets was reduced by RBM39 depletion . The splicing changes we observed for cells expressing each individual shRNA in both cell lines showed a large number of common splicing events and significant correlation with each other, as indicated by the Spearman correlation and Venn diagram analyses (fig. S4), suggesting that the observed alterations were specific to RBM39 suppression. Gene set enrichment analysis (GSEA) for the differentially expressed genes showed that RBM39 depletion significantly disrupts pathways that regulate RNA and protein homeostasis . Collectively, these data indicate that neuroblastoma has an inordinate dependency on RBM39-mediated RNA splicing, which may represent a vulnerability in MYC-driven high-risk neuroblastoma  screens (SK-N-AS) . RBM39 din vitro  and tumoin vitro . An analepletion . By compblastoma .RBM39 shRNA and those treated with indisulam showed a significant correlation . Pathway analysis of the genes with altered splicing by indisulam and RBM39 knockdown revealed that loss of RBM39 affected pathways involved in G2-M phase cell cycle, gene transcription, DNA repair, chromatin-modifying enzymes, metabolic pathways, and cancer genes (fig. S9). Real-time PCR and Western blot validated that the splicing events induced by indisulam were consistent with those induced by RBM39 knockdown .Indisulam and other related aryl sulfonamides represent a class of compounds known as “molecular glues” because of their ability to recruit the splicing factor RBM39 to the E3 ubiquitin ligase CRL4-DCAF15 for proteasomal degradation  and widerelation , in linerelation . In addirelation . Flow cy3 were dosed intravenously with 25 mg/kg for two 1-week cycles (5 days on and 2 days off). Although all tumors in all four models underwent an initial complete response following treatment, the time to relapse, following discontinuation of therapy, in neuroblastoma was substantially longer in comparison to the Rh30 and HCT116 models , rhabdomyosarcoma (Rh30), and colorectal cancer (HCT116). Mice harboring tumors of approximately 200 mm6 models . TogetheTo test whether the antitumor activity of indisulam in neuroblastoma is an RBM39-dependent effect rather than due to off-target activity, we engineered each of three neuroblastoma cell lines , using CRISPR-Cas9 knock-in, to create indisulam-resistant RBM39 variants (RBM39 G268V) . A glyciDCAF15 expression, and the degree of RBM39 degradation is proportional to the level of DCAF15 expression . We further compared DCAF15 expression between neuroblastomas and other types of pediatric cancers, among 2222 cases analyzed by RNA-seq (St. Jude Cloud)  cells (DCAF15 did not affect expression of endogenous RBM39 (fig. S12). However, indisulam-mediated RBM39 degradation did correlate with DCAF15 levels. DCAF15 KO reduced degradation, whereas DCAF15 overexpression enhanced degradation (DCAF15 and lower in DCAF15-silenced cells (DCAF15 expression levels determine the efficacy of indisulam with high levels of DCAF15 in neuroblastoma conferring more sensitivity to indisulam treatment.Relatively high pression . To direO) cells . The genradation . The effradation . The effed cells . TogetheMYCN and F1178LALK (F1178LTH-MYCN/ALK) by using ultrasound imaging to quantify tumor response. Human F1174LALK  is the most frequent somatic mutation in neuroblastoma and is associated with MYCN amplification, conferring a worse prognosis than MYCN amplification alone . Indisulam led to a dose-dependent regression of tumor growth, although animals treated with the lowest dose, 5 mg/kg, had evidence of early tumor relapse . The com2000 mm3 . Tumors 2000 mm3 , inlet. l tumors . TogetheDCAF15 expression levels. Among all tested animals, we did not observe a significant change in mouse body weight during treatment (To monitor target engagement in patient-derived xenograft (PDX) mouse models, we used Western blot analysis to determine the levels of RBM39 in tumor tissues as well as normal organs including heart, lungs, liver, and kidney 120 hours following indisulam administration (24 hours after the fourth dosing). In tumors, we observed a reduction in RBM39 at all three doses administered in two different PDX models . In contreatment . Althougreatment . These dSpliceosome-mediated pre-mRNA editing is an essential biological process that produces mature RNAs that serve as templates for protein synthesis. Alterations in RNA splicing pathways can lead to dysregulated gene splicing and a tumor-specific dependence  (23). RBMYCN/ALKF1178L mice, and tumors derived from C-MYC–overexpressing mice. We validated the on-target mechanism of indisulam in vitro and in vivo in several ways. First, we showed that cells expressing the RBM39 mutant G268V, which is incapable of binding indisulam but maintains its functional role in the spliceosome, were about 100-fold more resistant to indisulam in vitro than the parental cells and unresponsive to indisulam treatment in vivo. Second, genetic knockdown and pharmacologic degradation of RBM39 induced similar splicing changes, mainly leading to genome-wide skipped exons. Nevertheless, we noticed that the indisulam treatment induced more robust changes in splicing than the shRNA knockdown of RBM39 , which could be attributed to several reasons. First, indisulam might have other “off-target” effects. Second, indisulam may more potently destabilize the splicing complexes than shRNA knockdown due to insufficient RBM39 depletion. Third, the shRNA knockdown effect lagged behind indisulam-mediated degradation because it takes longer time for knockdown to effect in cells.The dependence of neuroblastoma on RBM39 provides a rationale to target the altered splicing machinery in neuroblastoma using an RBM39 degrader as a therapeutic approach. Targeted protein degradation enables conditional and temporal control over the levels of proteins. This approach has emerged as a new therapeutic modality owing to its potential advantages over traditional occupancy-based inhibitors with respect to dosing, side effects, drug resistance, and modulating “undruggable” targets , and 1% penicillin/streptomycin . CHLA20 was cultured in 1× Iscove’s modified Dulbecco’s medium  supplemented with 20% fetal bovine serum , 1% l-glutamine , 1% penicillin/streptomycin , and 1× insulin-transferrin-selenium . NGP, SKNDZ, CHP212, SK-N-FI, HS68, and BJ cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with penicillin/streptomycin , 2 mM l-glutamine , and 10% fetal bovine serum (FBS) . All cells were maintained at 37°C in an atmosphere of 5% CO2. All human-derived cell lines were validated by short tandem repeat (STR) profiling using the PowerPlex 16 HS System (Promega) once a month. In addition, a PCR-based method was used to screen for mycoplasma once a month using the LookOut Mycoplasma PCR Detection Kit  and JumpStart Taq DNA Polymerase  to ensure that cells were free of mycoplasma contamination.PDX models were propagated in CB17 severe combined immunodeficient (SCID) mice. KELLY, SIMA, BE2C, SK-N-AS, and SK-N-SH were cultured in 1× RPMI 1640  supplemented with 10% fetal bovine serum , 1% n = 3) and indisulam-resistant SK-N-AS tumors (n = 3) were excised and placed in a sterile tube containing phosphate-buffered saline (PBS) on wet ice during transport from the animal research facility to the research laboratory. Tumor samples were manually minced using a sterile scalpel and underwent an enzymatic digestion with collagenase IV  for 1 hour in a 37°C rotor . After digestion, cells were filtered using a 70-μm sterile strainer and cultured in RPMI medium with 10% FBS and 1% penicillin and streptomycin.The vehicle-treated SK-N-AS tumor (Indisulam was purchased from MedKoo Biosciences (#201540).The following antibodies were used: ß-actin , glyceraldehyde-3-phosphate dehydrogenase , RBM39 , c-Myc , MYCN , FLAG , ATM (D2E2) , EZH2 (D2C9) , CDK4 (D9G3E) , RET (C31B4) , and EED .et al. (Single-guide RNA (sgRNA) targeting RBM39 (5′-gaactgaagatatgcttcgt-3′) was cloned into the pSpCas9(BB)-2A-Puro (PX459) vector . For RBM39 G268V knock-in, SK-N-AS, BE2C, or SIMA cells were transfected  with PX459-RBM39 sgRNA and the single-stranded oligo 5′-GCTGCAGCAATGGCAAACAATTTACAAAAGGGAAGTGCTGGACCTATGAGGCTTTATGTGGGCTCATTACACTTCAACATAACTGAAGATATGCTTCGCGTGATCTTTGAGCCTTTTGGAAGAGTAAGTCCAGGTTCTTCAATGAATCTTCAGTAGGTTGTTGATCTGAGTATAACTACATAATTTCTGTATGTCTTGCT-3′. The oligonucleotide is engineered to introduce a synonymous mutation at R267, allowing us to confirm that the genomic sequence was edited with this template. Afterward, cells were exposed to 2 μM indisulam for 2 weeks to select for cells with RBM39 G268V knock-in. Cells that survived indisulam treatment were recovered and confirmed to have the correct G268V genomic conversion via amplicon sequencing. Amplicon sequencing of PCR products using genomic DNA as template revealed BE2C (62%), SK-N-AS (75%), and SIMA (55%) reads with the repair template sequence, consistent with a heterozygous effect. HCT116 cells expressing RBM39 mutations were previously described by Han One independent guide RNA targeting DCAF15 (5′-CGTGTCCCTCAAGAACATTG-3′) was cloned into lentiCRISPRv2 puro (Addgene #98290). Lentiviral packaging of the resulting plasmid was performed by cotransfecting the plasmid with psPAX2  and pMD2.G  into lenti-X 293T cells. SK-N-AS, BE2C, and SIMA cells were infected with medium collected from transfected lenti-X 293T cells. Cells stably expressing lentiCRISPRv2 with DCAF15 sgRNA were selected with puromycin (2 μg/ml) for 7 to 10 days to enrich for transduced cells.et al. (Full-length complementary DNA (cDNA) encoding DCAF15 with a 3xFLAG epitope at the C-terminal end was cloned into pLVX IRES Blast. The PAM (protospacer adjacent motif) site was mutated to prevent Cas9 from editing ectopic expression of 3xFLAG-tagged DCAF15 in SK-N-AS, BE2C, and SIMA cells generated by CRISPR-mediated deactivation of DCAF15. Lentiviral packaging of the resulting plasmid was performed by cotransfecting with psPAX2  and pMD2.G  into lenti-X 293T cells. The medium collected from transfected lenti-X 293T cells was used to infect SK-N-AS, BE2C, and SIMA cells. Cells stably expressing 3xFLAG-tagged DCAF15 were selected for 7 to 10 days with blasticidin (10 μg/ml). HCT116 cells were described by Han Total RNA was isolated from tissue samples as per the manufacturer’s instructions of the RNeasy Mini Kit . cDNA was prepared from 1 μg of total RNA using a cDNA synthesis kit according to the manufacturer’s protocol . RBM39 gene was amplified by PCR using the cDNA as the template along with the following primers  using the Phusion High Fidelity PCR Kit . The resultant PCR product was gel-purified using a Qiagen gel extraction kit  according to the manufacturer’s protocol. The DNA was sent for Sanger sequencing at Hartwell Center in St. Jude using the following primers: primer 288F, ACAGAAGTCCTTACTCCGGACC; primer 388R, ACTTTTGCTTCGGGAACGTCG;primer 602F, GTCGATGTTAGCTCAGTGCCTC. Primer 602F is used to detect the RBM39 mutation in the RRM2 motif.The TRC (The RNAi Consortium) lentiviral-based shRNA knockdown plasmids for RBM39 were purchased from Horizon Discovery . The lentiviral shRBM39 and shControl (pLKO.1) particles were packaged by Vector Lab at St. Jude. Briefly, human embryonic kidney (HEK) 293T cells were transfected with shRNA constructs and helper plasmids . The 48- and 72-hour posttransfection replication-incompetent lentiviral particles were harvested and transduced into cells with polybrene (8 μg/ml). Forty-eight hours later, puromycin (1 μg/ml) was added for selection for additional 48 hours before injection into mice or immunoblotting.g at room temperature for 2 min, 10 to 20 μl of the cell lysates were separated on 4 to 15% Mini-PROTEAN TGX Stain-Free Protein Gels from Bio-Rad and transferred to methanol-soaked polyvinylidene difluoride membranes (Millipore). Lysates for RBM39 G268V mutant cell lines and DCAF15 genetically modified cells were generated as previously described (Cells were washed twice with ice-cold PBS and directly lysed on ice with 2× sample loading buffer . On ice, cell lysates were sonicated once with a 5-s bursts at 40% amplitude output  followed by 25-min heating at 95°C. After the cell lysates were centrifuged at 13,000After removing medium, cells were washed with Dulbecco’s PBS (DPBS) without calcium or magnesium  and treated with 4% formaldehyde in PBS [paraformaldehyde (PFA)] for 20 min. Once PFA was removed, cells were stained with 0.1% crystal violet stain for 1 hour.50) was determined with GraphPad Prism using baseline correction , the asymmetric (five parameter) equation, and least-squares fit.Twelve-point dose responses were performed on 96-well assay plates with cell plating (1500 to 4000 cells per well) on day 1, compound addition on day 2, and survival measurement on day 5. Compounds were diluted in DMSO before adding to the cells. Final DMSO concentration was 0.5%. Cell survival assay was performed using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) that measures cellular adenosine triphosphate (ATP) content. Luminescence was recorded with an EnVision multimode plate reader (PerkinElmer). Median inhibitory concentration  for 5 days. Each concentration was tested in eight replicates. After completion of the treatment period, 10 μl of PrestoBlue reagent was added to each well and plates were incubated at 37°C with 5% CO2 for 30 min. Then, fluorescence (560-nm excitation/590-nm emission) was measured using a BioTek Synergy H1 microplate reader. GraphPad Prism was used to plot the curves and calculate the IC50 values.Cell viability was determined by PrestoBlue Cell Viability Reagent  according to the manufacturer’s protocol. Briefly, cells were plated in white 96-well plates  at a density of 1000 to 3000 cells per well and incubated at 37°C with 5% COCells  were plated in six-well culture plates and, next day, treated with indisulam for 48 hours. Cells were trypsinized and centrifuged at 1000 rpm for 5 min at 4°C and washed once with ice-cold PBS. The samples were then resuspended in the propidium iodide solution  and incubated at room temperature for 20 min. The cells were subjected to flow cytometric analysis by using an LSR II  flow cytometer, and the data were analyzed using BD FACSDiva .P value of <0.05. Then, GSEA was carried out using gene-level log2 fold changes from differential expression results against gene sets in the Molecular Signatures Database (MSigDB 6.2) (gsea2 version 2.2.3) for the data from GSE62564.Total RNA from cells and tumor tissues was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Paired-end sequencing was performed using the High-Seq platform with 100–base pair (bp) read length. Total stranded RNA-seq data were processed by the internal AutoMapper pipeline. Briefly, the raw reads were first trimmed  and mapped to human genome assembly (GRCh38) (STAR v2.7), and then the gene-level values were quantified  on the basis of GENCODE annotation (v31). Low-count genes were removed from analysis using a CPM (counts-per-million) cutoff corresponding to a count of 10 reads and only confidently annotated (level 1 and 2 gene annotation), and protein-coding genes are used for differential expression analysis. Normalization factors were generated using the TMM  method, counts were then transformed using Voom, and transformed counts were analyzed using the lmFit and eBayes functions (R limma package version 3.42.2). The significantly up- and down-regulated genes were defined by at least twofold changes and adjusted 6. Next, we averaged the junction-read vectors for samples of the same genotype. We further excluded those junctions for which the total normalized junction-read counts were smaller than 10. Then, we calculated the Pearson correlation coefficients for each pair of genotypes. The two-sided t tests were used to compute the significant levels of the correlations by the function chart.Correlation in R.After mapping RNA-seq data, rMATS v4.1.0 was used for RNA alternative splicing analysis by using the mapped BAM files as input. Specifically, five different kinds of alternative splicing events were identified, i.e., skipped exon, alternative 5′ splicing site, alternative 3′ splicing site, mutually exclusive exon, and intron retention. To keep consistent, the same GTF  annotation reference file for mapping was used for rMATS. For stranded RNA-seq data, the argument “--libType fr-firststrand” was applied. To process reads with variable lengths, the argument “--variable-read-length” was also used for rMATS. To select statistically significantly differential splicing events, the following thresholds were used: false discovery rate (FDR) < 0.05 and absolute value of IncLevelDifference > 0.1. For visualization, the Integrated Genomic Viewer (IGV) Genome Browser was used to show the sashimi plots of splicing events. To investigate the genome-wide correlations of differential splicing between two genotypes , we extracted splice junctions for all samples of both genotypes of interest from the STAR  > 0.1. Next, on the basis of these different splicing events, we used Venn diagrams to show their common and unique splicing events in the five splicing categories mentioned above.n = 3) in the 7500 Real-Time PCR System by Applied Biosystems (Thermo Fisher Scientific) using Power SYBR Green PCR Master Mix . ΔΔCT methods were applied to analyze the results. The following primers were used to perform the quantitative real-time PCR: GAPDH  and RBM39 .RNA was extracted using the RNeasy Plus Mini Kit  following the manufacturer’s protocol. cDNA was prepared in 20-μl reaction from 500 ng of total RNA using the Superscript IV First Strand Synthesis System . Real-time PCRs were run in triplicates  for 48 hours. For genetic inhibition, BE2C and SKNAS cells were transduced with shCtrl, shRBM39 (#188), and shRBM39 (#190) on day 1 and selected with puromycin (2 μg/ml) on day 2 for 24 hours. Total RNA was extracted after 48 hours using the RNeasy Kit (Qiagen). cDNA was synthesized using the SuperScript IV Reverse Transcriptase Kit . RT-PCR was performed using the Phusion High Fidelity PCR Kit  using cDNA as the template DNA. The PCR products were run on a 2% agarose gel and scanned using the Odyssey Fc Imaging System Fc (LI-COR Biotechnology). The following PCR primers were used: EZH2 , NTRK1 , CDK4 , and KIF20B .g for 10 min at 4°C after cell pellets were resuspended in lysis buffer . Nuclei were incubated with Tn5 transposon enzyme in transposase reaction mix buffer (Illumina) for 30 min at 37°C. DNAs were purified from the transposition sample by using a Mini-Elute PCR purification kit  and measured by Qubit. PCR was performed to amplify with High-Fidelity 2× PCR Master Mix [72°C/5 min + 98°C/30 s + 12 × (98°C/10 s + 63°C/30 s + 72°C/60 s) + 72°C/5 min]. The libraries were purified using a Mini-Elute PCR purification kit (Qiagen). ATAC-seq libraries were pair-end–sequenced on HiSeq4000 (Illumina) in the Hartwell Center at St. Jude Children’s Research Hospital, Memphis, TN, USA.Library preparations for ATAC-seq (assay for transposase-accessible chromatin using sequencing) were based on a published protocol with minor modifications  using BWA . DCAF15 expression data from various tumor tissues were downloaded from R2 program (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi), and DCAF15 expression data from RNA-seq data of various pediatric cancer tissues were downloaded from St. Jude Cloud (https://pecan.stjude.cloud/). The expression of RBM39 and clinical survival data were retrieved using R2 program using the dataset . Briefly, a 5-mm3 piece of tissue was added to 500 μl of 2× lysis buffer, homogenized for 30 s , heated at 95°C, and centrifuged at 12,000 rpm for 5 min at 4°C. Supernatant was further diluted 5×, sonicated for 30 s, and heated at 95°C. The sample was centrifuged at 13,000 rpm for 5 min and used as whole-tissue protein lysate.Tumor-bearing mice (SJNB11 and SJNB19 PDXs) were treated with indisulam  for five consecutive days via tail injection. At the end of the experiment, mice were euthanized by COComplete blood count was analyzed with the Forcyte analyzer hematology machine, according to the manufacturer’s instructions. Briefly, blood was collected in Eppendorf tubes containing 10 μl of 10% EDTA via retroorbital bleed using 200-μl heparinized capillary tubes . Blood samples were processed within 2 hours to avoid hemolysis. The number of leukocytes (white blood cell), erythrocytes (red blood cell), lymphocytes, neutrophils, monocytes, eosinophils, and platelets was counted. Proprietary lysing agent was added to liberate hemoglobin and ultimately convert it to cyanmethemoglobin to calculate the value.P values across multiple time points were adjusted for multiple comparison using the Benjamin-Hochberg method. Kaplan-Meier survival analyses were used with log-rank (Mantel-Cox) method in Prism program.Wilcoxon rank sum test (two-sided) was used to compare the volume between two groups at every time point.


Mesalazine  has been widely used to treat inflammatory bowel disease (IBD). However, it remains unclear about the underlying biological mechanisms of IBD pathogenesis and mesalazine treatment, which could be partially clarified by exploring the profiling of circular RNAs (circRNAs) using RNA-seq. A total of 15 mice (C57BL/6) were randomly assigned to three equally sized groups: control, dextran sulfate sodium , and DSS+5-ASA . We randomly selected three mice of each group to collect colon tissues for RNA-seq and then performed bioinformatic analysis for two comparisons: DSS vs. control and DSS+5-ASA vs. DSS. Comparisons of a series of indicators  verified the establishment of DSS-induced IBD mouse model and the effectiveness of mesalazine in treating IBD. We identified 182 differentially expressed circRNAs, including 55 up-regulated and 47 down-regulated circRNAs when comparing the DSS+5-ASA with the DSS group. These 102 circRNA-associated genes were significantly involved in the N-Glycan biosynthesis and lysine degradation. The network analysis of circRNA-miRNA-mRNAs identified an important pathway, i.e., chr10:115386962-115390436+/mmu-miR-6914-5p/Atg7, which is related to autophagy. The findings provide new insights into the biological mechanisms of IBD pathogenesis and mesalazine treatment, particularly highlighting the circRNA-miRNA-mRNA pathway. Inflammation has been involved in many chronic diseases including aging symptoms. Inflammatory bowel disease (IBD) is an idiopathic inflammatory disorder of gastrointestinal tract, including Crohn’s disease (CD) and ulcerative colitis (UC). Over the past 20 years, the incidence and prevalence of IBD have soared in developing countries in Asia , South America, the Middle East and Africa. The total number of IBD cases in China from 2005 to 2014 is about 350,000, and it is estimated that Chinese patients will reach to 1.5 million by 2025 , 7. As aCircular RNAs (circRNAs) are the novel star molecule, which forms covalent closed loops with different sizes and sources, and represent a class of abundant, stable and widely occurring RNA molecules in animals. As endogenous noncoding RNAs, circRNAs are more stable than linear RNAs, because of their closed loop structures , 9. TheyIn this study, we first demonstrated that an IBD mouse model was successfully established using dextran sulfate sodium (DSS) by assessing a series of indicators , and then administrated mesalazine and observed significant changes  in these indicators. We then used next-generation RNA-seq to explore the expression profiling and potential roles of circRNAs in the colonic inflammation of DSS-induced and mesalazine-treated mice based on the established mouse model. We also used qRT-PCR to further validate the differentially expressed circRNAs. We found an underlying pathway of circRNA-miRNA-mRNA relevant to the therapeutic process of mesalazine for DSS-induced IBD, suggesting the potentials of the circRNAs serving as new biomarkers for IBD progression. The workflow of this study is shown in As generally done in literature, changes in body weight, colon length and disease activity index (DAI)  were anaThe RNA-seq analysis showed that a total of 4460 circRNAs were detected in mice colonic tissues from the control, DSS and DSS+5-ASA groups. Most of these circRNAs were transcribed from protein coding exons and others were from introns, intergenic or antisense . Among tAmong the 4460 circRNAs, we identified 182 differentially expressed circRNAs, which were displayed by volcano plot and heat map with fold change≥2.0 and P≤0.05 , 3B. TheWhen conducting GO analysis, the most enriched biological process (BP) terms of the DSS group were regulation of vascular endothelial growth factor signaling pathway  domain, the most enriched terms were CCR4-NOT complex  terms, metal ion binding . These identical pathways were related to RNA degradation, endocytosis, fatty acid biosynthesis and metabolism, adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, N-Glycan biosynthesis and lysine degradation , 4D. TheFive cicrRNAs were selected from the 182 differentially expressed circRNAs for validation based on logFC and p-value , the seqThe prediction network result showed a network of circRNA-miRNA consisting of 65 circRNAs and 183 miRNAs .Furthermore, to explore whether circRNAs acted as “miRNA sponges”  for execTo the best of our knowledge, this study is the first to explore the circRNAs expression profiling in colon tissues from mesalazine-treated IBD mice by performing next-generation RNA-seq. Based on the DSS-induced IBD mouse model that we successfully established, we applied RNA-seq to explore the circRNAs expression profiling in colon tissues that were randomly selected from the control, DSS and DSS+5-ASA groups. We found a number of differentially expressed circRNAs and related pathways , particularly an important circRNA-miRNA-mRNA pathway (chr10:115386962-115390436+/mmu-miR-6914-5p/Atg7) related to autophagy. Furthermore, we performed primary validation of the identified circRNAs. Our results provide a new understanding of the mechanism of IBD pathogenesis and mesalazine treatment.The establishment and application of animal models, especially mouse models, are a bridge between basic research and clinical practice for disease research. Animal model is important for exploring molecular mechanisms of diseases, such as transplantation/induction tumor formation model for cancers , temporaIn this study, we detected 182 differentially expressed circRNAs that may include potential regulators and help, at least in part, explain the therapeutic mechanism of mesalazine for DSS-induced IBD. Together with the literature, we assume that mesalazine is most likely to treat IBD through playing anti-inflammatory role, e.g., blocking induction of nuclear factor kappa B(NF-κB) mediated by pro-inflammatory cytokines to enhance epithelial barrier function , 22. OurIt is widely recognized that mesalazine activates peroxisome proliferator-activated receptor (PPAR) gamma to attenuate colitis, which is closely associated with inhibition of NF-κB-dependent signaling. Results obtained in this study also suggest possible roles of circRNAs in the NF-κB-dependent pathway. Prior studies have suggested certain circRNAs may be involved in the regulation of NF-κB signaling pathway. For example, ssc_circ_009380 promoted activation of NF-κB pathway by binding miR-22 during Transmissible gastroenteritis virus (TGEV)-induced inflammation ; circUBAMeanwhile, we found eight enriched pathways based on KEGG analysis, which were fatty acid biosynthesis and metabolism, AMPK signaling pathway, N-Glycan biosynthesis and lysine degradation, RNA degradation and endocytosis. Short chain fatty acids (SCFA), such as acetate and butyrate, which are metabolized by gut bacteria have been reported to mitigate inflammation in IBD animal models . Recent . Previous studies have demonstrated the loss of Atg7 was related to increased intestinal inflammation [In addition, we predicted the circRNAs-miRNA-mRNA network in specific IBD-related pathways, including JAK/STAT and autophagy pathway. The results indicated the pathway of chr10:115386962-115390436+/miR-6914-5p/Atg7 was likely related to autophagy. As the novel circRNA, chr10:115386962-115390436+ was upregulated in colon tissues of mice in the DSS group when comparing with the DSS+5-ASA group. Some miRNAs have been identified to be involved in IBD-associated autophagy, such as miR-30C , miR-130ammation . Furtherammation . BuildinCurrently, a few literatures have reported the relationship between circRNA and IBD. Most of these studies selected peripheral blood from IBD patients and healthy individuals or inflamed and normal colorectal mucosa tissues from IBD patients as samples to perform RNA-seq and investigated the effect of circRNAs in IBD by comparing the different expression profiling of circRNA in IBD and normal group , 42. To Limitations of this study included the small number of mice for each group, which should be increased in follow-up experiments to verify our findings. In addition, more experiments were needed to verify the hypothesis resulted from this study. For example, mesalazine might play a therapeutic role in IBD by regulating the NF-κB pathway. Therefore, it was necessary to further study the effects of mesalazine on NF-κB activation-related factors , to improve the understanding of the therapeutic process of mesalazine for IBD.In summary, our study was the first to present the circRNA expression profiling in colon tissues from mesalazine-treated IBD mice using RNA-seq. We suggested a number of potential IBD-related circRNAs and pathways, particularly highlighting an important circRNA-miRNA-mRNA pathway related to autophagy. These findings, although require further validations, provide new insights into the molecular mechanisms of IBD progression and clues for further research.C57BL/6 mice  were purchased from Shanghai Sippr-BK Laboratory Animal Co. Ltd . All mice were housed under conventional condition. Fifteen mice were randomly divided into three groups (five mice for each group): control, DSS, and DSS+5-ASA group after an acclimatization period. All experiments were approved by the Animal Experimentation Ethics Committee at Shanghai University of Traditional Chinese Medicine.IBD was induced by administrated 2.5% DSS  in drinking water for seven days . The micTo assess the inflammation severity, ELISA kits  were used to measure the expression levels of serum IL-6 and TNF-α.Three colon tissue samples were selected randomly from each of the three groups. Total RNA was extracted using Trizol  reagent. A NanoDrop ND-2000 instrument  was used to measure RNA quantification and quality. The OD260 / OD280 ratio was used as an indicator of RNA purity. The ratio is required to be close to 2.0 for pure RNA (a ratio between 1.8 and 2.1 is acceptable). RNA integrity and gDNA contamination were tested by denaturing agarose gel electrophoresis. The 28S and 18S rRNA bands is fairly sharp and intense, while the 5S rRNA bands is smaller and more diffuse. DNA contamination of the RNA preparation is evident as a high molecular weight smear or band migrating above the 28S rRNA band. Degradation of the RNA is reflected by smearing of rRNA bands.in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq Sequencer following the manufacturer’s instructions.High throughput whole transcriptome sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech . In brief, rRNAs were removed from total RNA using Ribo-Zero rRNA Removal Kits  according to the manufacturer's instructions. Using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit , we constructed the CircRNA library and then quantified it using the Bio Analyzer 2100 system . Quality control was performed for the library. A total of 10 pM libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified Total RNAs were reversely transcribed into cDNA by a Gene Amp PCR System 9700  and qRT-PCR amplification was conducted in ViiA 7 Real-time PCR System . CircRNA expression level was defined as the comparative cycle threshold (Ct) and calculated by the double-standard curve method. Primers  used forPaired-end reads were harvested from Illumina HiSeq 4000 sequencer. After 3’ adaptor-trimming and low-quality reads removing by cutadapt software , the higThe circRNAs were divided into exon circRNA, intron circRNA or intergenic circRNA, etc., according to the alignment positions of the two ends of the circRNA. The identified circRNAs were then linked to the circBase database based on the genomic location of the circRNA for annotation. The newly identified circular RNAs were labeled with novel.Cloudseq's home-made miRNA target prediction software based on miRanda and TargetScan , 51 and All results were reported as the mean ± standard deviation (SD). GraphPad Prism 6.0 was used to analyze the data. The Mann–Whitney test (one side) was used for comparisons of relative expression levels between the control and DSS groups, the DSS and DSS+5-ASA groups . A P-value < 0.05 was considered statistically significant.Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Tables 5, 6, and 7


Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro. Severe acute respiratory coronavirus 2 (SARS-CoV-2) is an important human pathogen and the causative agent of coronavirus disease 2019 (COVID-19). Vaccines are available to prevent the spread of SARS-CoV-2, and several antiviral strategies, such as treatment with remdesivir or reconvalescent plasma, have received FDA approval or emergency use approval. However, the development of additional strategies remains necessary, because of the continuous emergence of variants of concern (VOC) and current antiviral treatments can only be delivered intravenously. A key target for novel drug screening is the RNA polymerase of SARS-CoV-2 ,4.SARS-CoV-2 is a betacoronavirus and contains a positive-sense, non-segmented RNA genome of around 30 kilobases ,6. The 5Various nucleoside analogues and small molecule inhibitors were identified as potential inhibitors of the SARS-CoV-2 nsp12/7/8 complex . One of In this study, we show that enisamium inhibits coronavirus growth on Caco-2 and normal human bronchial epithelial (NHBE) cells. We also demonstrate that enisamium inhibits the minimal viral RNA polymerase complex in a mini-genome assay. Molecular dynamics (MD) simulation analysis suggests that VR17-04 reversibly binds to the exposed cytosine or adenine bases, preventing incorporation of GTP and UTP into the nascent strand. Overall, these results suggest that enisamium prevents SARS-CoV-2 RNA synthesis by inhibiting nascent strand elongation.®) or enisamium chloride (laboratory code FAV00B) 6 h prior to infection. The cells were infected with SARS-CoV-2 at multiplicity of infection 0.01 for 1 h, and compound reapplied following virus removal. At 48 h post infection, the cytopathic effect was recorded by examination of the infected cultures by light microscopy and supernatant collected to quantify virus RNA by RT-qPCR as described previously, using nsp12-specific primers 5′-GTGARATGGTCATGTGTGGCGG-3′ and 5′-CARATGTTAAASACACTATTAGCATA-3′ [Confluent layers of Caco-2 cells in 96-well plates were treated with serial dilutions of enisamium iodide  was added to each well for RNA isolation. Total RNA was isolated from cells using Trizol per the manufacturer’s instruction. Two-step RT-qPCR was performed using HCoV-NL63 N gene-specific primers/probe , GAPDH was analyzed at the same time with GAPDH-specific primers (5′-GTTCGACAGTCAGCCGCATC-3′ and 5′-AGTTAAAAGCAGCCCTGGTGA-3′) by RT-qPCR and served as a reference gene for normalization.MatTek’s EpiAirway System  consisted of differentiated NHBE cells that were cultured to form a multilayered, highly differentiated model that closely resembles the epithelial tissue of the respiratory tract. The cells from a single donor (No. 9831) were used for assay consistency. The apical surface of the cells was exposed to a humidified 95% air, 5% CO5 HEK 293T cells were transfected with 200 ng pcDNA6.B-nsp7-flag, pcDNA6.B-nsp8-flag, and pcDNA6.B-nsp12-flag, and 25 ng linear pPolI-SARS-CoV2-NLuc-N plasmid using Lipofectamine 2000 (Thermo Fisher). Cells were collected 24 h post-transfection and analyzed using a Nano-Glo dual-luciferase reporter assay  according to the manufacturer’s protocol and a Glomax navigator microplate Luminometer (Promega). For each condition, three biological replicates were conducted.The SARS-CoV-2 mini-genome assay was performed as described previously . BrieflyThe VR17-04 (2 mg) was dissolved in 0.6 mL D2O/water solution (5% D2O) or DMSO. NMR experiments were performed at 500 MHz at 277 K. For NOESY experiments (noesygpph) 128 transients were collected for each free-induction decay, using a mixing time of 300 ms and 20 sec of relaxation delay. The NOESY experiment (matrix 1024 × 320 points) was zero-filled to 2K × 2K before Fourier transformation. Measurements were taken on a Bruker 500 HD NMR spectrometer equipped with a 5 mm BB probe.0 (C(Ar)H-C(Ar)-C(CH2)-N) φ1 (C(Ar)-C(CH2)-N-H), φ2 (H-N-C-O), φ3 (O-C-C(Ar)-C(Ar)OH/H). The lowest energy conformation of VR17-04 (φ3eclipsed) and enisamium were selected for further docking and for partial charge estimation  and two Mg+2 ions, whose positions were included in 7BV2. The two Zn+2 ions that were co-crystallized in 7BV2 were also included in the models. The co-crystallized inhibitor remdesivir monophosphate was removed from the complex, while its position and the contacts with the template RNA through the unpaired uracil base in the +1 position were used to guide the molecular docking. The unpaired uracil base was subsequently mutated in cytosine (Cyt) or adenine (Ade) in Pymol 2.3.4 , generating two different complexes, identified as nsp12/7/8(Cyt) and nsp12/7/8(Ade), respectively. Next, Gasteiger charges were calculated for both ligands  and the receptor complexes, and used as parameters of the docking simulation [7, 270,000. At each run the docking solutions were clustered using a tolerance RMSD = 2.0 Å. Three different docking simulations were run: VR17-04 and enisamium were docked on to nsp12/7/8(Cyt), while, for comparison, VR17-04 was further docked on nsp12/7/8(Ade). The docking solutions were selected based on two criteria. The first criterium was the possibility to form a Watson–Crick base-pair interaction between VR17-04 (or enisamium) and the unpaired cytosine or adenine. The second criterium was based on the possibility to figure out interferences of the ligand with the catalytic mechanism, for example by interaction with key residues of the nsp12/7/8 complex. The selected poses were further ranked by a preliminary MD simulation (approximately 50 ns) in explicit solvent, to predict the stability of the interaction, and to estimate the Poisson–Boltzmann free energy of binding, this last property was used to obtain a final selection and ranking of the poses.The docking simulation was performed using Autodock 4.2 software . The geomulation . In the −1 for each atom of the solute, and progressively reduced at 0.5 and 0.2 Kcal mol−1. The cell density equilibration stage was monitored by plotting the cell volume (Å3) vs. time (ns), until the cell volume fluctuations level off to a horizontal axis that corresponds to the average final volume of the cell. This stage required between 20 and 50 ns approximately and was further checked by the formation of a thin water shell that surrounded the face of the inhibitor molecule that was exposed to the empty catalytic cavity of the nsp12/7/8 complex. In the second stage of the MD simulation, the harmonic restraint was removed, and the inhibitor-nsp12/7/8 complex was allowed to equilibrate in the geometry and the relative position of their elements: inhibitor, template RNA, nascent strand RNA, pyr, Mg+2, and Zn+2. To monitor the equilibration of the position and orientation that VR17-04 (or enisamium) occupies in the +1 position of the catalytic cavity, the distances between the carbonyl oxygen of VR17-04 and the hydrogen (NH2-) of cytosine (or adenine), and between the hydroxyl group of VR17-04 and the nearby lone pair of cytosine nitrogen (or adenine), were plotted vs. simulation time. These distances provide a direct indication of the stability of the Watson–Crick base pair interaction that hold the inhibitor near the target base. The orientation of VR17-04 in the catalytic site of the nsp12 was also monitored by the coplanarity angle χ as defined by the following atoms in bold: CO-NH2-N-OH, in which CO and OH belong to VR17-04, while the remaining NH2 and N belong to the opposite cytosine (or adenine). Alternatively, the enisamium-cytosine pair requires the following atoms CO-NH2-N-CH to define χ; in this case CO and CH belong to enisamium, while NH2 and N belong to the opposite cytosine. In fact, values of this angle around ‘0’ indicate that the ‘base pair’ contact between VR1704 the cytosine (or adenine) is coplanar, a geometric condition favoring the Watson–Crick ‘base-pair’ interaction between VR17-04 and the base. VMD 1.9.3 [Explicit solvent MD simulations were run using NAMD 2.12  softwareMD 1.9.3  was usedIn a system that evolves in accord to the complex formation reaction .(1)L+R The free energy change is calculated knowing only the initial and final state of the system:The free energy decomposed in the following terms:The free energy change is then conveniently split:MME corresponds to the potential energy of the system, as described by the force-field; solG is the polar solvation energy, estimated by the Poisson–Boltzmann equation [nopolG is the non-polar solvation energy, estimated by the solvent-accessible surface area, a method included in the MMPBSA. T is the absolute equilibrium temperature, while MMS is the molecular entropy of the system. The sum of the first three terms on the right-hand side of the Equation (3) is conveniently defined as the Poisson–Boltzmann free energy of binding ΔPBBindG (see Equation (4)). To estimate the absolute value of the free energy of binding ΔBindG the molecular entropy change MMΔS is required. Since in this study two similar molecules, VR17-04 and enisamium, were compared for their ability to bind cytosine , or alternatively two bound states VR17-04-cytosine or VR17-04-adenine were inquired, the respective entropy changes ΔMMS are considered similar. In this condition the Poisson–Boltzmann free energy of binding ΔPBBindG could be used to rank the selected molecular recognitions and/or the poses of the docking experiment.In Equation (3) the equation ; Gnopol 50 for enisamium chloride in Caco-2 cells was 1.2 mM (~300 µg/mL), which is comparable to the inhibitory effect of enisamium on influenza A virus replication in Caco-2 cells [Previous experiments showed that enisamium A can eff-2 cells .50 of ~60 µg/mL, implying that enisamium can inhibit coronavirus replication in NHBE cells in vitro.To test if enisamium can inhibit coronavirus replication in NHBE cells, NHBE cells were incubated with enisamium iodide for 6 h and subsequently infected with alpha-coronavirus HCoV NL63. Analysis of the N gene RNA levels in infected NHBE cells showed a strong effect on viral RNA synthesis A with anPrevious experiments showed that enisamium can inhibit the influenza A virus RNA polymerase in vitro . To testeclipsed or trans conformations, respectively. In the eclipsed conformation, the hydroxyl and carbonyl groups of VR17-04 can form two hydrogen bonds using both a proton donor (H-O) and proton acceptor (O=C), potentially with a single interaction partner, while in the trans conformation, single hydrogen bonds could be made on both sides of VR17-04, potentially with two interaction partners. Quantum chemical calculations suggest that the eclipsed conformation of VR17-04 has a lower energy in unbound state than the trans conformation . We predict that the eclipsed conformation is compatible with a sequence-specific inhibition of the nsp12/7/8 RNA polymerase. Future activity and structural studies can be performed to further uncover the molecular mechanism by which VR17-04 and enisamium inhibit the SARS-CoV-2 RNA polymerase, or other RNA virus RNA polymerases.Our docking and MD simulations suggest that VR17-04 can bind a template cytosine or adenine base in the active site of the SARS-CoV-2 RNA polymerase, forming a Watson–Crick base pair interaction. This hypothesis is supported by our NOE experiments performed in water, which indicate that VR17-04 can adopt an Overall, our results strongly suggest that enisamium metabolite VR17-04 inhibits RNA synthesis by the SARS-CoV-2 nsp12/7/8 complex. Moreover, unlike remdesivir, enisamium does not require intravenous administration, which would be advantageous for its use outside a hospital setting. Together with observations that enisamium can inhibit other RNA virus infections, and DNA virus infections , these r


In non-small cell lung cancer (NSCLC), identifying the presence of sensitizing and resistance epidermal growth factor receptor (EGFR) mutations dictates treatment plans. Extracellular vesicles (EVs) are emerging as abundant, stable potential liquid biopsy targets that offer the potential to quantify EGFR mutations in NSCLC patients at the RNA and protein level at multiple points through treatment. In this study, we present a systematic approach for serial mutation profiling of 34 EV samples from 10 metastatic NSCLC patients with known EGFR mutations through treatment. Using western blot and droplet digital PCR (ddPCR), sensitizing  mutations were detected in EV-Protein, and both sensitizing and resistance (T790M) mutations were quantified in EV-RNA. EGFR mutations were detected in EV-Protein from four patients at multiple time points through treatment. Using EV-RNA, tumor biopsy matched sensitizing mutations were detected in 90% of patients and resistance mutations in 100% of patients. Finally, mutation burden in EV-RNA at each time point was compared to disease status, described as either stable or progressing. For 6/7 patients who were longitudinally monitored through treatment, EV mutation burden mirrored clinical trajectory. When comparing mutation detection between EV-RNA and ctDNA using ddPCR, EVs had a better detection rate for exon 19 deletions and the L858R point mutation. In conclusion, this study demonstrates that integrating EV analysis into liquid biopsy mutation screening has the potential to advance beyond the current standard of care “rule in” test. The multi-analyte testing allows future integration of EGFR mutation monitoring with additional EV-markers for a comprehensive patient monitoring biomarker. EVs are isolated from plasma collected from NSCLC patients harboring known EGFR mutations using ultracentrifugation. RNA and protein are extracted from the EV samples and tested for EGFR mutations using ddPCR and western blot, respectively. The widespread adoption of targeted therapies using small molecule tyrosine kinase inhibitors (TKIs) has greatly benefited the 10–30% of advanced non-small cell lung cancer (NSCLC) patients who have sensitizing epidermal growth factor receptor (EGFR) mutations in an otherwise challenging to treat cancer . This suDue to the invasiveness and inherently localized sampling of tissue biopsies, they are not frequently used for repeated monitoring, may not result in enough material for testing, and may miss mutations carried in other tumor regions . To addrExtracellular vesicles (EVs), lipid bilayer bound nanovesicles approximately 30–150 nm in diameter, are a promising alternative blood-based biomarker offering increased abundance and stability compared to ctDNA . They arGiven the tiny amount of cargo carried by these nano-sized vesicles, the development of techniques that can analyze these important carriers is an active area of research. To date, research has largely been limited to microRNA or protein to glean information about a patient’s disease, and to develop diagnostic and prognostic signatures . A finalSignificantly, mutation profiling in extracellular vesicles is an emerging field, with the first studies focusing on the detection of cancer-specific mutations . MutatioSeveral recent works have demonstrated the feasibility of using EV-RNA to detect EGFR mutations from blood  and bronThe study presented here builds upon these previous works by following a cohort of metastatic NSCLC patients to quantify the EGFR mutation burden carried in both EV-RNA and EV-Protein. Specifically, ddPCR was used to analyze the EV-RNA without pre-amplification while mutated protein content of EVs was profiled using western blot. Patients carried at least one sensitizing EGFR mutation  based on tumor biopsy and were receiving TKI therapy at the time of enrollment. Moreover, a subset of patients in this study already carried the resistance T790M mutation at the beginning of longitudinal monitoring. Resistance mutations would potentially decrease the efficacy of TKIs, although novel TKIs are being developed to target these mutations as well. Despite the predicted TKI sensitivity, patients had differing therapeutic responses, which is a well-known challenge of TKI therapies. As such, there is a need to develop an enhanced approach to predict who will have favorable outcomes, or who should receive an alternative therapy.To this end, in this study the cohort was longitudinally monitored for EGFR mutation burden carried by EV-RNA and EV-protein. This study expanded upon previous works by monitoring patients serially, allowing for time points before, during, and after progression events. Finally, expanding beyond only EV-RNA profiling highlights the potential benefits of dual-monitoring both EV-RNA and EV-protein; distinct roles for EV-RNA and EV-protein were revealed.TM Reagent. The Norgen Single Cell RNA kit (Norgen Biotek Corp.) was used to purify and isolate the EV-RNA, due to the miniscule amount of RNA contained in these samples. RNA was reverse transcribed and directly loaded onto the RainDropTM (RainDance Technologies) ddPCR system for analysis.To first establish experimental protocols and demonstrate the presence of EGFR mutations in EVs, EVs secreted from lung cancer cell lines with known EGFR mutations were tested for EGFR mutations. EV-RNA and EV-protein were tested using ddPCR and western blot, respectively. ddPCR offers a highly sensitive platform to directly quantify the number of mutant transcripts from bulk RNA without risk of pre-amplification bias , allowinn = 3) L858R droplets and 0 ± 0 (n = 3) T790M droplets (n = 5) droplets in healthy donor EV-RNA, similar to the background signal from the negative control cell line EV-RNA H1975. This assay simultaneously screens for 19 different deletion variants using pooled primers, resulting in increased background signal. A threshold for positive detection was determined based on the highest background signal observed among all negative control samples. This threshold for detection was used for all subsequent analysis for NSCLC patient EV-RNA.Matching the cells of origin, EV-RNA derived from H3255 carried a heterozygous L858R mutation , EVs frodroplets . HoweverCell line derived EVs also carried mutant EGFR protein, as demonstrated using western blot. Specific identification of the two activating mutant proteins was achieved. L858R was detected exclusively in H3255 and H1975 derived EVs , and exoProtein intensity for each marker was normalized to the total protein loaded into each lane using Bio-Rad’s Stain Free Gel technology. Each band was normalized to the total protein per lane, eliminating the need for housekeeping genes which are not equally present in all EVs due to their loading mechanisms and cells of origin . Stain FThe above established dual EV-RNA and EV-protein mutational profiling was applied to analyze EVs from blood plasma in a cohort of ten metastatic NSCLC patients with at least one known sensitizing EGFR mutation based on primary tumor biopsy. Patients were enrolled after consent and blood was collected under IRB approval. The cohort’s median age was 64 years  and was well distributed between male and female. Full patient demographics can be found in Seven of the ten patients had samples collected at multiple time points, termed visits. The time between each visit varied depending on patient care, however, the time ranged from 26 to 231 days (mean = 89 ± 40 days) across all patients. The visit notation is used throughout this study to highlight general trends on the utility of patient monitoring using EVs to be compared across patients, however, patient specific details and timelines are shown in While a plethora of EV isolation methods have been widely developed, including ultracentrifugation , microflEV-protein was isolated from the plasma of four patients over 2–4 visits. EV-protein was tested for sensitizing mutations exon 19 del or L858R based on initial biopsy. EGFR mutations in the EV-protein were detected using western blot in samples from 4/4 (100%) patients, two patients with exon 19 del mutations and two with L858R mutations. Patients L3 and L5 have moderately identifiable bands for exon 19 del, while having various levels of CD9 and similar GAPDH bands, Conversely, the L858R band is present in samples from patients L9 and L10, but less distinct compared to exon 19 del in L3 and L5, Epidermal growth factor receptor mutations found in EV-protein demonstrate the potential to use EVs as multiple cargo biomarkers. This finding marks the first demonstration of EGFR mutations detected in patient-derived EV-protein. Full western blots can be found in n = 33 total samples) collected from each of these patients at different time points (up to six visits) through their course of treatment. The mutation burden was evaluated in each sample, defined as the number of mutant EGFR droplets detected using ddPCR normalized to 5 mL of starting blood volume. Exon 19 del was detected in 7/8 patients, n = 8). Taken together, the positivity rate across all samples for exon 19 del was 78% (n = 18/23).In compliment, EV-RNA was isolated from 10 patients with multiple samples , Finally, mutation burden between EV-RNA, EV-Protein, and ctDNA were analyzed to determine if there was a correlation between any of the three factors. Using a simple linear regression, it was found that the concentration of mutation burdens between the analytes  were not correlated based off their n = 17 samples) or progressing (n = 22 samples) based on available clinical data corresponding to each blood draw visit following the guidelines established in Response Evaluation Criteria in Solid Tumors  n = 3 patients), n = 2 patients), Each patient’s progression was classified based on overall disease trajectory into one of three categories based on trend in EV-RNA burden over time: consistently progressing , To summarize the overall effectiveness of using EV-RNA as a correlate for disease outcome, the change in EV-RNA mutation burden was calculated between each sequential pair of time points. It was then determined whether the patient remained stable or had disease progression between the two time points in question. Promisingly, change in EV-RNA exon 19 del burden mirrored clinical trajectories at 12/14 time points across all five patients who had 2+ time points, with the definition of mirroring being shown in Using ddPCR and western blots, EVs isolated from lung cancer patient plasma were analyzed for EGFR mutations. In this pilot study, the utility of EV-RNA and EV-protein is demonstrated to not only screen for the presence of mutations, but to dynamically monitor patient disease status. Mutant EV-RNA was detected in 9/10 patients, and for 6/7 patients who were longitudinally monitored, mutant EV-RNA burden mirrored clinical trajectory. Within patients who had exon 19 del, ΔEV-RNA mutation burden strongly indicated disease trajectory, demonstrating that a single time point may be insufficient to assess patient status. The power of liquid biopsies enabled multiple time points from each patient to be collected.While evidence from this pilot study suggests the rate of increase in EV-RNA mutation burden may be linked with progression severity, larger studies are needed to investigate this. For the patients with exon 19 deletion, both patients who had consistent progressive disease succumbed to their disease and are now deceased. These patients had a similarly consistent increase in EV-RNA mutation burden. Conversely, the three patients with decreasing EV-RNA exon 19 del burden have remained clinically stable for an average of 192 ± 9 days after the final blood draw. These patients saw consistently decreasing EV-RNA mutation burden that mirrored their stable disease status. This was further highlighted by the finding that there is a significant difference in the exon 19 del ΔEV-RNA burden between time points between stable and progressing patients. These results indicate that changes in EV-RNA mutant burden may be an early indicator of clinical stability and perhaps even indicate disease progression before clinical monitoring methods.Of the two patients with L858R/T790M mutations, one patient’s EV-RNA burden mirrored disease trajectory, while the other did not. L9 has had sustained progressive disease 87 days after the final blood draw. Conversely, L10, after switching from a TKI to chemotherapy between visits 4 and 5, has remained clinically stable for 133 days after the final blood draw, despite an increase in L858R EV-RNA. The change from TKI therapy to chemotherapy during this study could impact the utility of targetable mutations carried in EV-RNA for patient monitoring. Without the use of TKIs specifically targeting mutant EGFR, an increase mutant burden may not indicate treatment resistance in the tumor. Therefore, while EV-RNA may mirror disease trajectory for patients receiving targeted therapy, this may not extend to patients receiving other treatment types, such as chemotherapy and further studies are needed to investigate this.Additional studies with larger cohorts are needed to validate these findings, however, this study presents initial evidence that increase in EV-RNA indicates progression for patients receiving targeted therapy. Future studies are needed to determine if EV-RNA can be used to detect progression prior to current techniques. Of interest, L6, only had two time points collected but saw an upward trend in their burden despite being clinically stable thus far, While EV-protein was detected in samples from four patients, there was not an observed correlation between EV-protein burden and EV-RNA burden and there were too few patients to make assessments of correlation to disease. This could be due to the differences in EV packing of RNA cargo to protein cargo, which are largely poorly understood, especially in the ever-changing physiological states found during cancer immunotherapies. Beyond EV packaging differences, interestingly, previous studies in cell lines have shown that when exposed to TKIs, EGFR mutations result in differential protein stability compared to wildtype ; TreatmeLiquid biopsies hold the potential to address spatial heterogeneity and longitudinal monitoring limitations. However, the only FDA-approved liquid biopsy test, the Cobas v2, for mutation detection, relies on ctDNA. While capable at detecting the presence of new mutations, this test is still currently considered a “rule in” test, with the recommendation of a tissue biopsy to confirm a mutant negative result . CompareThis work lays the groundwork for future studies to establish the utility of mutations found in EV cargo for patient care. In this novel proof of concept study, EVs were screened for previously identified EGFR mutations carried by each patient. Changes in EV-RNA correlated with disease trajectory; however, the clinical implications of EV-protein remain unclear. The utility of EV mutation monitoring warrants further investigations across additional mutations and cancer types. Further, the dual analysis of EV-derived cargo has the potential to go beyond monitoring and be used in lieu of a tumor biopsy for non-invasive screening for both sensitizing and resistance mutations in EGFR across a patient’s treatment course. This minimally invasive approach could be integrated into the standard of care enable more rapid identification of treatment resistance and allow for timely treatment changes, overall improving patient care.g for 15 min and frozen at −20°C.H1975, H3255, and H1650 cells were grown in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1% Antibiotic-antimycotic (Gibco). Cells were grown to 80% confluence before subculturing using 0.05% Trypsin-EDTA (Gibco). To prepare extracellular vesicles, cells were seeded at 3,000,000 cells/100 mm dish (Sarstedt) in complete media. 24 h after seeding, cells were washed three times with phosphate-buffered saline (PBS) pH 7.4 (Gibco) and incubated for 72 h in serum-free RPMI-1640 media (Gibco). Cell culture media (CCM) was centrifuged at 2,000 × All blood was collected following IRB (HUM00119934) approval, and all patients gave their informed, written consent to participate in the study. All patients had metastatic lung adenocarcinoma. The cohort of patients in this study had known EGFR mutations.Plasma was prepared for EV isolation using one of the following three methods and was stored in the −80°C freezer until use.Plasma prep 1 (Ficoll): Whole blood was collected in EDTA tubes. Samples were prepared using Ficoll-PaqueTM PLUS  following the manufacturer’s protocol. The plasma and leukocyte layers were collected for CTC isolation and effluents were centrifuged following the plasma prep 3 protocol.Plasma prep 2 (Dextran): Whole blood was collected in EDTA tubes. 1 mL of 6% dextran solution (w/v) was mixed into 5 mL whole blood. The sample sat 1–1.5 h at room temperature to allow the red blood cells sedimentation. The supernatant was collected for CTC isolation and the effluent was centrifuged following the plasma prep 3 protocol.Plasma prep 3: Whole blood was collected in EDTA tubes was centrifuged at 2,000 × g for 15 min at room temperature. The plasma supernatant was collected and frozen at −20°C for up to 30 days.g for 20 min to remove cellular debris. The supernatant was then ultracentrifuged at 100,000 × g for 90 min to pellet the EVs using 36 mL Polyethylene terephthalate (PET) tubes (Thermo Fisher Scientific). Excess tube volume was filled with sterile PBS pH 7.4 (Gibco). The extracellular vesicle-pellet was washed with PBS and centrifuged at 100,000 × g for 90 min. Extracellular vesicles were suspended in 100 μL PBS pH 7.4 or RIPA with protease inhibitor cocktail (Thermo Fisher Scientific) and frozen at −20°C. EVs stored in PBS were then used for nanoparticle tracking analysis along with RNA extraction and characterization, while EVs in RIPA were used for western blot analysis.Plasma prep or CCM was centrifuged at 12,000 × Following isolation, extracellular vesicles were quantified for size and concentration using NTA using Malvern’s NanoSight. Quantification was performed using five-30 s runs at a flow rate of 20 using the brightness setting of 15. The camera detection was set to a level of 4 for all runs. Runs were then averaged with the average and standard deviation between the runs being reported.TM Reagent (TRIzol) (Invitrogen) at a 1:10 ratio of extracellular vesicle suspension to TRIzol and incubated at room temperature for 5 min. A 1:5 ratio of chloroform (Sigma-Aldrich) to TRIzol was added and briefly vortexed to mix, then incubated for 2–3 min at room temperature. The sample was centrifuged at 12,000 × g for 15 min. The aqueous phase was collected and mixed in a 1:1 ratio with 70% ethanol (Sigma-Aldrich). Total RNA was purified using the Norgen Single Cell RNA isolation kit (Norgen Biotek Corp.). cDNA was prepared using SuperScript IV VILO Master Mix with ezDNase Enzyme (Invitrogen) following the manufacturer’s protocol. All purified RNA and cDNA products were handled in a PCR workstation to prevent contamination.Ultracentrifuged extracellular vesicles were lysed using TRIzolTM gene expression PCR reactions were prepared using TaqManTM Fast Advanced Master Mix (Applied Biosystems) in 96-well MicroAmp Fast Optical Plates (Applied Biosystems) and processed on a QuantStudio 3 (Applied Biosystems) using fast cycling conditions. Each mRNA: sample pair was analyzed in technical triplicates.Twenty microliter TaqManCell-free DNA (cfDNA) was isolated from the plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) following the manufacturer’s processing protocol. cfDNA was eluted into 15 μL for ddPCR mutation detection.TM ddPCR (RainDance Technologies). In brief 25 μL reactions were prepared using TaqManTM SNP Assay (Life Technologies), 2x TaqMan Genotyping Master Mix (Applied Biosystems), and droplet stabilizer (RainDance Technologies). Maximum cDNA was loaded into each dPCR reaction. The PCR reaction was loaded onto the Source Chip (RainDance Technologies) to for droplet generation and collected into an 8-tube PCR strip (Axygen). The PCR tubes were transferred to the thermocycler for 45 rounds of PCR amplification (Bio-Rad). The PCR tubes, containing the samples, were then transferred onto the Sense Machine (RainDance Technologies) where the fluorescence intensity of each droplet was measured.Epidermal growth factor receptor mutations were identified by using RainDropFor the point mutations, L858R and T790M, mutations were considered present based on the detection of one or more positive droplets within the pre-established gates based on positive EV-RNA controls. For exon 19 del, deletions were considered present based on the detection of one or more positive droplets above the threshold. A threshold for detection was determined based on the number of false positive droplets detected using EV-RNA negative controls. The maximum number of false positive droplets detected in any negative control (16 droplets) was used as the threshold for detection. All presented data is represented as the threshold subtracted from the total number of mutant positive droplets counted and any further normalization specified in the respective figure.TM Protein Assay Kit (Thermo Fisher Scientific cat #23235). Western blot loading was normalized by using 5 mL blood volume for extracellular vesicle isolation, loading the maximum protein (37.5 μL) in each lane, and using Bio-Rad’s Stain Free gels to allow normalization. Briefly, the protein was separated at 250 V for 30 min. Semi-dry transfer was then performed using Trans-Blot Turbo Transfer System (Bio-Rad) to a high fluorescence PVFD membrane . The membrane was imaged using Bio-Rad’s ChemiDoc to quantify total protein per lane. The membrane was blocked and incubated overnight with primary antibody in 5 mL of 5% bovine serum albumin (Sigma-Aldrich) in Tris Buffered Saline (TBS) (Bio-Rad) with 1% Tween 20 (Sigma-Aldrich) (TBST). The membrane was then washed thoroughly before incubating with HRP-secondary antibody in 3% non-fat milk in TBST for 90 min followed again by additional washes. Measurement was performed using SuperSignalTM West Pico PLUS Chemiluminescent Substrate  and SuperSignal West Femto  and imaged on the ChemiDoc.Extracellular vesicles were isolated from ultracentrifugation into 150 μL RIPA buffer , protein concentration was measured by Micro BCANormalization was performed following Bio-Rad’s Stain Free Gel analysis protocols. Briefly, following protein separation and transfer, the blot is imaged using Bio-Rad’s Stain Free Blot imaging setting to capture the total protein per lane. Using Bio-Rad’s Image Lab 6.0.1 software, the total protein in each lane is compared and a normalization coefficient determined. After blotting for specific proteins, each band is compared to the total protein of the lane, adjusted using the normalization coefficient, and quantified as a Normalized Protein Intensity.P values were calculated using unpaired, two tailed, t-tests.All analysis was performed in GraphPad Prism V9. The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the University of Michigan Institutional Review Board #HUM00119934. The patients/participants provided their written informed consent to participate in this study.EPu and SO designed the study, interpreted the data, and co-wrote the manuscript. EPu, AR, NC, and CS developed the protocols and performed the western blots and NTA. SO and EPr performed the ddPCR experiments. T-WL performed the SEM. MZ reserved the CTC isolation effluent for use in this study. NR and SN oversaw the study, managed ethical patient enrollment and sample collection, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.