N-isopropylacrylamide monomers, resulting in RNA bottlebrushes, hydrogels,and stimuli-responsive materials. This approach, readily applicableto both post-synthetic and nature-derived RNA, can be used to engineerthe properties of a variety of RNA-based macromolecular hybrids andassemblies providing access to a wide variety of RNA-polymer hybrids.Combining synthetic polymers with RNA paves the way forcreatingRNA-based materials with non-canonical functions. We have developedan acylation reagent that allows for direct incorporation of the atomtransfer radical polymerization (ATRP) initiator into both short syntheticoligoribonucleotides and natural biomass RNA extracted from torulayeast. The acylation was performed in a quantitative yield. The resultinginitiator-functionalized RNAs were used for grafting polymer chainsfrom the RNA by photoinduced ATRP, resulting in RNA-polymer hybridswith narrow molecular weight distributions. The RNA initiator wasused for the polymerization of oligo(ethylene oxide) methyl ethermethacrylate, poly(ethylene glycol) dimethacrylate, and  For example, the noncovalent approaches may be appliedto a broad range of RNA substrates regardless of the length, source,or structure of the RNA substrate for coupling. However, the weakand non-covalent interaction between nucleic acids and polymers oftenraises concerns about the stability of the complex as well as difficultiesin control over the number- and binding site of polymers.21 In contrast, due to the specificity of the coupling chemistries,covalent modification methods allow for chemically stable and preciseincorporation of pre-synthesized polymers, acrylate groups, or polymerization-initiatingmoieties into the predetermined sites in nucleic acids.23 However, coupling handles  must be incorporated into the nucleic acid through thesolid-phase synthesis or the use of an enzyme, which limits the scopeof the substrate and, thus, hinders practical and versatile application.25 Therefore, for the successful modification of RNA with polymers,we desired an approach that would be capable of both (i) direct and(ii) covalent modification of RNA, to circumvent the problems relatedto current synthetic methods. We also sought a universal approachthat would expand the scope of RNA substrates from short syntheticoligonucleotides to longer RNA transcripts or biomass RNA (bmRNA)extracted from natural sources.27The fabrication of the RNA-synthetic polymer hybrid materials hasbeen facilitated through one of the following approaches: (1) noncovalentattachment of pre-synthesized polymers with RNA by electrostatic interactions29 In addition, compared to other reagents that are known to reactwith nucleic acids , acyl imidazole reagents have significantadvantages, such as high reaction yield and site-selective incorporation,while generating a less toxic and biocompatible by-product  that allows RNA mapping even in vitro30 and in vivo.31To address thischallenge and to facilitate covalent RNA modificationin a universal and direct manner, we explored the acyl imidazole chemistry:the activation of a carboxylic acid with an imidazole leaving group,which could covalently react with 2′-hydroxyl (2′-OH)groups in RNA to form 2′-O-adducts see 1. Indeed34 resulted in the controlled growth of polymer from RNA with a narrowmolecular weight distribution.Inspired by SHAPE reagents for 2′-OH functionalization,particularly acyl imidazole chemistry, here we developed a polymerizationinitiator-functionalized acyl imidazole reagent, which enables covalentmodification of RNA in a post-synthetic  manner. Followed by the initiator integration, thesubsequent atom transfer radical polymerization (ATRP) process37 In ATRP, polymerizationis initiated from an alkyl halide initiator  through the reversible activation and deactivationcycles, typically mediated by a copper catalyst.39 It is important to note that we chose to incorporate the ATRP initiatorand graft from the RNA rather than pre-synthesized polymers to avoidpotential steric hindrance and purification issues associated withthe “grafting-onto” approach. Initiating polymerizationdirectly from the RNA allows for easier purification of the finalproduct (separation of high-molecular-weight RNA-polymer hybrids fromunreacted low-molecular-weight monomers), while expanding the rangeof architectural complexity by dramatically reducing steric hindrance.24ATRP is one of the most widelyused controlled radical polymerizationtechniques for the synthesis of polymer-biohybrids.40 we used the oxygen-tolerant photoinduced ATRP (photo-ATRP)techniquemediated by eosin Y (EYH2) photocatalyst and a copper complex(X–CuII/L) under green light irradiation.42 In this green-light-driven ATRP, the excited eosin Y photocatalysttransfers an electron to the X–CuII/L complex, thereby(re)generating the ATRP activator (CuI/L) required forpolymer growth. Compared to other ATRP methods, this photoredox/Cu-catalyzedATRP method is particularly advantageous for polymerization from nucleicacid initiators. This is due to the excellent oxygen tolerance ofthe dual catalysis, which allows for polymerization in low volumes(150–250 μL) without the need for deoxygenation processessuch as N2 purging or freeze-pump-thaw cycles. These processesare typically challenging for low-volume polymerization with limitedamounts of initiators and may risk mechanical degradation of the nucleicacid.43Among the various ATRP methods reported,44 acyl imidazole-based reagents are particularlyuseful for RNA modification because they often exhibit longer half-lives(over 30 min) with quantitative reaction yields. Thus, our syntheticstrategy relied on the coupling reaction between 1,1′-carbonyldiimidazole(CDI) and β-alanine ATRP initiator (Br-Ala) toobtain an ATRP initiator-acyl imidazole reagent (Br-Ala) due to the highersolubility of amides in aqueous buffers compared to ester-based ATRPinitiators.46 The simple mixing of CDI withthe Br-Ala in anhydrous DMSO successfully yielded the Br-Ala-functionalized acyl imidazole (Br-Ala-AI) reagent. The structure of the reagent was confirmed by 1H NMR spectroscopy . The hydrolysiskinetics of Br-Ala-AI under coupling conditions (20% v/v DMSO in water) was also investigatedto estimate the reactivity of the reagent and determine its half-life(S2). The half-life of Br-Ala-AI was38 min, indicating that the reactivity of Br-Ala-AI wascomparable to previously reported acyl imidazole reagents.44Compared to the other SHAPE reagentsfor 2′-OH modification , reagent 1A. Notab reagent 1, Br-Alaalf-life1C and S2Br-Ala-AI, a 21-mer RNA(RNA21) and a 21-mer DNA (DNA21) of similar sequence were treated with Br-Ala-AI in 20% v/v DMSO in nuclease-free water  the treatmentof Br-Ala-AI revealed that the peak for the initial RNAsequence at 6648.2 m/z disappearedafter the reaction, indicating efficient RNA functionalization. Themass spectrum in Figure S3A. Additionally, the normal distribution of the masses ofthe reaction products suggested that an average of six ATRP initiatorswere attached to the RNA21. In stark contrast, no dramatic changein the mass spectrum was observed when DNA21 was reacted with the Br-Ala-AI  methyl ether methacrylate,average Mn = 500] using EYH2 as the photocatalyst and CuBr2/TPMA [TPMA = tris(2-pyridylmethyl)amine]as the catalyst under the green light irradiation (520 nm) in phosphate-bufferedsaline (PBS) without deoxygenation.41 Afterthe polymerization, the resulting RNA21-pOEOMA500 was dialyzedthree times using 100K MWCO filters, passed through a Sep-Pak C18reverse-phase cartridge to remove the photocatalyst and unreactedOEOMA500, and analyzed by size-exclusion chromatographyequipped with multi-angle light scattering detector (SEC-MALS). Despitethe low RNA21 initiator concentration of 0.075 mM, a relatively highmonomer conversion of 25% and a clear shift of the SEC-MALS traceto the high molecular weight area were observed (ca. 0.45 mM).38 Thedisappearance of the initiator peak (elution volume of ca. 21.4 min) indicates a nearly quantitative initiation from the RNA21initiator, which is also confirmed by the good agreement between theabsolute molecular weight obtained from SEC-MALS  and the theoretical molecular weight . In contrast to the reaction with RNA21initiator, no monomer conversion was observed, when the Br-Ala-AI-treated DNA21 was used as an initiator , as shown in Figure S4. However, the monomer conversion with BiBA-AI-treated RNA21 was not different from that of control experiments . This is likely to bedue to the rapid hydrolysis of the BiBA-AI reagent inaqueous media  caused bythe short distance between the strong electron withdrawing group  and acyl imidazole.47Of note, we synthesized and tested anotherreagent using α-bromoisobutyricacid  could provide a powerful approach totailoring their properties.fcDNA21) or partially complementary DNA (pcDNA21) prior to Br-Ala-AI treatment. Onceannealed, the fcDNA21/RNA21 heteroduplex is completely hybridizedwith 2′-OH groups inaccessible to reactions, whereas the pcDNA21sequence annealed to SRNA21 results in a single-nucleotide mismatchin the middle of the heteroduplex (Table S1). We reasoned that 2′-OH groups at the locally perturbedstructure  would be more accessible to the Br-Ala-AI.52 The annealed heteroduplex was treatedwith the Br-Ala-AI according to the previously establishedprocedure. The reaction was conducted in MOPS buffer (pH = 7.5) for4 h. Selective DNA degradation was then performed using DNase I.51 The remaining RNA product was isolated by isopropanolprecipitation and filtration using a 3K MWCO filter and characterizedby mass spectrometry. Interestingly, a significantly lower numberof modifications in RNA21 was observed for the fcDNA21/RNA21 duplexand most of the RNA21 remained unmodified . Thiscan be observed through reverse transcription and subsequent polyacrylamidegel electrophoresis (PAGE).52We attempted to testthe control over the incorporation of theATRP initiator groups in RNA21 by introducing fully complementaryDNA , which is likely a result of unreacted residualinitiators (a peak at an elution volume of ca. 21.4min). The residual initiator peak originates either from unmodifiedRNAs . Consistent with the results of the DNA-guided acylation,the clear shift of the mass peak of the hairpin RNA  was inhibited when the hairpin RNA was protectedby a fully complementary DNA sequence . These results indicate that controlover the incorporation of ATRP initiators with selectivity withinRNA sequences could be achieved by using an appropriate helper DNA.This strategy would be useful to control the density of initiatorsin RNA sequences toward engineering new architectures in RNA-polymerhybrids.We also tested a hairpinRNA (RNA32), which contains a double-strandedstem and a single-stranded loop in an individual RNA strand. The stemin the hairpin was able to sequester and protect some of the 2′-OHsin the RNA in a double-stranded region  of 2′-OHfunctionalization by improving the water solubility of the acylatingreagents, tuning the reactivity of the acylating reagents, or usingnucleophilic catalysts .56 Nonetheless, the hydrolysis ofthe reagents under typical reaction conditions (10–30% v/v DMSO in aqueous buffer) could preventreaching the stoichiometric level of reaction that we desired.With both moderate acylation on single-stranded RNA and controlledacylation reactions on RNA/DNA duplexes to incorporate the initiatorswithin RNA sequences, we next explored the possibility of enhancingthe initiator incorporation to reach a nearly quantitative yield  that could be useful for RNA bottlebrushsynthesis and engineering.Br-Ala-AI  and accomplish quantitative functionalization,we carried out the reaction under water-free conditions , as shown in Br-Ala-AI in anhydrous DMSO was added to thecontainer. Two different reaction times were tested and after 4 and24 h of incubation with gentle shaking, the RNA products were isolatedby isopropanol precipitation, and subsequential filtration was performedusing a MWCO filter. We observed a significant difference betweenthe mass spectra taken after 4 h , whichcorresponds to the absorbance of nucleobases (ca. 7 nm)of the nucleobase absorption peak and an increase in a peak at 310nm (A310) were observed, indicating an acylation reactionon the nitrogenous bases.57 After 4 h ofreaction, the shape of the UV–vis spectrum remained largelysimilar to that of the untreated RNA21 . We performed PAGE of RNA21 and DNA21 strands. We observedthe retarded migration of RNA21  and DNA21  after the incorporationof the ATRP initiators into the oligonucleotides.56To evaluate our hypothesis, the RNA21 after the4 or 24 h treatmentwith leobases 4B. Interv/v DMSO in water, 1H NMR  as a result of the increased numberof initiators per RNA (and DNA) in anhydrous DMSO reactions. The SECpeak of the pOEOMA500-grafted DNA21, acylated in 100% DMSO , was observed in the high molecularweight region (elution volume of ca. 18.3 mL) asa result of polymer growth from the DNA. However, the polymerizationproduct obtained from RNA21, synthesized in 100% DMSO, was too viscousto be injected to and analyzed by SEC. This is likely due to the acceleratedtermination reaction between inter- or intramolecular polymer chainsinduced by densely-packed initiating residues, which occurs as theconversion increases. The optimization of the polymerization conditionsusing sacrificial initiators could solve the problem. The resultsof the polymerization from oligonucleotide initiators are summarizedin Table S2.We performed ATRP with DNA21 or RNA21 initiators synthesizedbythe water-free approach. An increased monomer conversion of 65% forthe RNA21 initiator  extractedfrom torula yeast . Using 1H NMR spectroscopy, the ratio of ribonucleotides to ATRP initiatorresidues incorporated into the bmRNA was estimated to be 4.25 acylationsper 10 ribonucleotides .Over the past decade, the potential of nucleic acids from biomassas a novel building block for the fabrication of sustainable biocompatiblematerials has been demonstrated.la yeast 5A. We fiications 5B, rightr-Ala-AI 5B, left,r-Ala-AI 5C. A chaI  were isolated by using 100KMWCO filter and a Sep-Pak cartridge as mentioned above and then analyzedby SEC-MALS. The shift of the monomodal SEC-MALS traces in bmRNA were controlled by simply changing the concentration of the bmRNA macroinitiator. SEC-MALS analysis of the bmRNA macroinitiator showed a relatively low dispersity (D̵) of 1.15 with a number average molecular weight  of 12.1 kDa . This cyanine-based dye exhibits highfluorescence enhancement upon binding to nucleic acids. We used SYBRGold with untreated bmRNA, bmRNAmacroinitiator, and bmRNA-pOEOMA hybrids from bmRNA macroinitiators and bmRNA-pOEOMA hybrids. In contrast, no fluorescence was observedfor pOEOMA initiated from a conventional ATRP initiator . Moreover, we also observedthe lower fluorescence intensity of the bmRNA macroinitiatoror polymer-functionalized biomass RNA . An aliquot of the polymerization productwas taken without further purification and combined with the ATRPcatalytic mixture for the second polymerization.53 After 30 min of the second polymerization reaction under green light,49% of monomer conversion was observed with a clear shifting of theSEC-MALS trace to the high molecular weight values indicating preservedchain end activity after the first polymerization (Table S3).One of the important features of controlled radical polymerizationperiment 5F. InitibmRNA and subsequent polymerization to the fabricationof macroscopic structures. As shown in 500 using the bmRNA macroinitiatorin the presence of a crosslinker .The polymerization reaction was performed in a 96-well plate usinga photoreactor (520 nm). As a result of polymerization, a macroscopichydrogel was obtained, as shown in bmRNA without Br-Ala-AI treatment was used instead of bmRNA macroinitiator, no polymerization occurred . We reasonedthat this would be a promising new approach for the fabrication ofnucleic acid-polymer amphiphiles.66 In addition to water-solublepolymers, such as polyethylene glycol (PEG), that provide degradationresistance, other polymers such as poly(N-isopropylacrylamide)(pNIPAM) can provide temperature-responsiveness to RNA-based biomaterials.Using N-isopropylacrylamide (NIPAM) as the modelmonomer, we performed the polymerization reactions in 80% v/v DMSO in PBS. The resulting bmRNA-pNIPAM hybrid was dialyzed in ice-cold water and transferredto a 0.5-dram vial. As shown in Figure S12A), the bmRNA-pNIPAM hybrids aggregatedand became a gel. In contrast, when bmRNA without Br-Ala-AI treatment was used instead of the bmRNA macroinitiator, no polymerization of NIPAM was observed and thesolution remained as a flowable liquid even after heating . These results demonstratethat novel materials can be fabricated from bmRNA,functionalized using our ATRP initiator reagent with a wide rangeof monomers under anhydrous polymerization conditions.Taking advantage of the increased solubility ofATRP initiator-functionalizedRNA in DMSO, we pursued polymerization from the v/v DMSO inwater), which can be further engineered with the aid of a suitablehelper DNA or by performing coupling reactions in the hydrolysis-limitedenvironment . We have shown that we can extendthe scope of substrates from short synthetic RNA oligonucleotidesto RNA sequences extracted from biomass. Treatment of the bmRNA with the Br-Ala-AI and subsequent polymerizationdemonstrated the controllability over the molecular weights of thepOEOMA500 grafts in addition to the living polymerizationbehavior. Synthesis of bmRNA-pOEOMA hydrogel andtemperature-responsive bmRNA-pNIPAM hybrids was alsodemonstrated in water and DMSO, respectively. Our novel and versatileapproach to RNA-polymer hybrids circumvents the challenges of othercoupling methods, including azide–alkyne cycloadditions, electrostaticinteractions, or hydrogen bonding. We anticipate that the direct anduniversal incorporation of acyl imidazole-based ATRP initiators andsubsequent polymerization will expand the scope of RNA substratesfor the combination with polymers and increase their utility as innovativebiomaterials. In addition, the high coupling yield and, thereby, improvedsolubility of RNA initiators in the organic-phase will vastly broadenthe choice of polymerizable monomers that would be interesting fornucleic acid therapeutics67 or pathogendetection.68We have developed a universal reagent forthe covalent functionalizationof RNA with one or more ATRP initiators. Up to 10 modifications ofthe 2′-OHs in a 21-mer RNA were achieved under general acylatingconditions (i.e., 20%


To explore the differential expression of placental specific gene 1 (PLAC1) and neurite guidance factor 1 (netrin-1) in colorectal cancer (CRC) liver metastasis and its predictive value.Paraffin specimens of primary CRC were selected, including 60 simple colorectal cancer specimens and 47 liver metastasis specimens. At the same time, 40 cases of normal colorectal mucosa were taken as the control group. The expression of PLAC1 and Netrin-1 in tissue was detected by immunohistochemistry (IHC). The correlation between PLAC1 and Netrin-1 expression and clinicopathological characteristics of patients with CRC liver metastases was analyzed. Logistic analysis was adopted to analyze the influencing factors of liver metastasis in CRC. A prediction model was established and ROC curve was used to detect the discrimination of the prediction model. The clinical value of PLAC1 and netrin-1 in predicting liver metastasis of CRC was analyzed using ROC curve. The relationship between the expression of PLAC1 and netrin-1 and the prognosis of CRC patients with liver metastasis was analyzed using Kaplan Meier survival curve.P < 0.05). Positive expression of PLAC1 in metastatic group was higher than that in non-metastatic group without significant difference (P > 0.05). The metastasis group had much higher positive expression of netrin-1 than the non-metastasis group (P < 0.05). The content of PLAC1 in the tissues of CRC with liver metastasis had a close relationship with differentiation degree and lymph node metastasis (P < 0.05). The expression of Netrin-1 in the tissues of CRC with liver metastasis was associated with Dukes stage, differentiation degree and lymph node metastasis (P < 0.05). Logistic regression analysis showed that Dukes stage, differentiation, lymph node metastasis, CEA, Alb and D-dimer were the independent risk factors for liver metastasis of CRC (P < 0.05). The model was constructed according to the regression coefficients and constant terms, and the discrimination of the prediction model was evaluated using ROC curve, with the AUC of 0.903 (95% CI 0.831 ~ 0.975), the sensitivity of 93.80%, the specificity of 80.00%, and the Jordan index of 0.738. The AUC of PLAC1 and netrin-1 alone and combined detection to predict liver metastasis of CRC were 0.805, 0.793 and 0.921, respectively. The survival time of patients with positive PLAC1 and netrin-1 expression were sharply shorter than that of the patients with negative expression (P < 0.05).The positive staining of PLAC1 and netrin-1 was mainly located in the cytoplasm by IHC detection. Positive expression of PLAC1 and netrin-1 in CRC tissues was markedly higher than that in normal colorectal mucosal epithelium (The expression of PLAC1 and netrin-1 was strongly increased in CRC with liver metastasis, which had a certain clinical value in predicting liver metastasis of CRC. Dukes stage, differentiation degree, lymph node metastasis, CEA, Alb and D-dimer were independent risk factors for liver metastasis of CRC, and the model based on these indicators had good discrimination for effectively evaluating the risk of liver metastasis in CRC. Colorectal cancer (CRC) is a malignant tumor with the highest incidence rate and mortality in the world, the incidence rate of which is gradually increasing with the change of people’s living habits and the increasing social pressure . The patPlacenta specific gene 1 (PLAC1) is a member of the Cancer/Testis Antigen (CTA) family, which is restrictively expressed in placental trophoblast cells. Previous studies found that , 5, abnoIn this study, the expression difference of PLAC1 and Netrin-1 in liver metastasis of CRC was detected and a prediction model was built, aiming to provide an important basis for clinicians to evaluate patients’ condition.A total of 107 patients who underwent surgical resection of primary CRC in our hospital during January 2016 to January 2017 were picked as the study subjects. According to whether there existed liver metastasis, there existed 60 cases of simple CRC tissue and 47 cases of CRC liver metastasis ones, all of which were obtained by surgery and pathology. The selection process of general materials was shown in Fig. Preparation process of tissue section : The sliHIC staining : The secThe clinical data of selected cases were collected, including age, gender, maximum tumor diameter, Dukes stage, degree of differentiation, lymph node metastasis, histological type, serum carcinoembryonic antigen (CEA), serum carbohydrate antigen 199 (CA-199), D-dimer and albumin . Logistic multivariate analysis was used to analyze the risk factors of liver metastasis in CRC patients.2 test. The measurement data were in accordance with the normal distribution through the normal distribution test, and were expressed in the form of  was used for calculation according to different observation indexes and data. The enumeration data was expressed in [cases (%)], and compared using χ P < 0.05). Positive expression of PLAC1 in metastatic group was higher than that in non-metastatic group without significant difference (P > 0.05). The metastasis group had much higher positive expression of netrin-1 than that in the non-metastasis group . Netrin-1 was significantly higher in CRC with liver metastasis who had Dukes stage C, low differentiation and lymph node metastasis than these with Dukes stage A + B, moderately high differentiation and no lymph node metastasis  had significantly higher positive expression of PLAC1 than these with moderately high differentiation and no lymph node metastasis (P < 0.05), but there existed no significant differences in gender, age, maximum tumor diameter, histological type and CA199 between two groups . The median survival period of patients with positive and negative PLAC1 expression was 25 months and 39 months, respectively. The median survival period of patients with positive and negative Netrin-1 expression was 23 months and 35 months, respectively. The survival time of patients with positive PLAC1 and netrin-1 expression were sharply shorter than the patients with negative expression  is one of the most common malignant tumors with complex pathogenesis in clinic. At present, it is believed that the occurrence and development of CRC may be the result of multiple factors. CRC has a high incidence rate and mortality rate and poor prognosis, which seriously affects the quality of life and even life health of patients . With thPLAC1 is a placental specific gene. Under normal circumstances, PLAC1 is mainly expressed in placenta and testis, and the expression level of PLAC1 is strongly increased when tumors occur . Some scIn the present study, Logistic regression analysis showed that Dukes stage, differentiation degree, lymph node metastasis, CEA, Alb, D-dimer, and positive expression of PLAC1 and Netrin-1 were the independent risk factors for liver metastasis of CRC, which indicated that positive expression of PLAC1 and Netrin-1 and clinicopathological features jointly promoted the occurrence of liver metastasis of colorectal cancer. The reason may be that Dukes staging is an important indicator of tumor biological behavior. With the postponement of Dukes staging, the lower the degree of tumor differentiation is, the higher the degree of malignancy is, the greater the risk of distant metastasis is, and the worse the prognosis of patients is. Previous studies have shown that , whetherIn general, Dukes stage, differentiation degree, lymph node metastasis, CEA, Alb and D-dimer were independent risk factors for liver metastasis of CRC. The expression of PLAC1 and Netrin-1 in tissues of CRC with liver metastasis was largely increased, which have important value in predicting liver metastasis of CRC. However, due to the limited research time and the sample size included in the study, the mechanism of PLAC1 and Netrin-1 affecting liver metastasis of CRC is still unclear, which will be further explored in our follow-up research.This study confirms that PLAC1 and Netrin-1 play an important role in liver metastasis of CRC, which makes it possible to detect some metastatic lesions hidden in the liver at an early stage and carry out terminal targeted treatment for such high-risk patients. This study proves that PLAC1 and Netrin-1 are related to the degree of differentiation and lymph node metastasis of CRC liver metastasis, and can better judge the possibility of liver metastasis, which has certain clinical research value. Through the combined detection method, the diagnostic efficiency of PLAC1 and Netrin-1 can be further improved, and the parallel test of PLAC1 and Netrin-1 can make the sensitivity reach 96.90 and the specificity increase to 85% when diagnosing early liver metastasis, which will be conducive to clinical judgment and has certain clinical application value. Applying the results of this study to the clinic can help the early detection of liver metastases and serve as a good indicator for targeted treatment of CRC with high metastatic tendency.


Recent studies have identified RNAs in cells capped with metabolites such as NAD+, FAD and dephosphoCoA. Whether RIG-I recognizes these metabolite-capped RNAs has not been investigated. Here, we describe a strategy to make metabolite-capped RNAs free from 5′ PPP dsRNA contamination, using in vitro transcription initiated with metabolites. Mechanistic studies show that metabolite-capped RNAs have a high affinity for RIG-I, stimulating the ATPase activity at comparable levels to 5′ PPP dsRNA. Cellular signaling assays show that the metabolite-capped RNAs potently stimulate the innate antiviral immune response. This demonstrates that RIG-I can tolerate diphosphate-linked, capped RNAs with bulky groups at the 5′ RNA end. This novel class of RNAs that stimulate RIG-I signaling may have cellular roles in activating the interferon response and may be exploited with proper functionalities for RIG-I-related RNA therapeutics.The innate immune receptor RIG-I recognizes 5′-triphosphate double-stranded RNAs (5′ PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5′ PPP-end with m  RIG-I (Retinoic Acid Inducible Gene-I) is a pattern recognition innate immune receptor that belongs to the DEA(X)D-box family of helicases. Its primary function is to surveil the cytoplasm for non-self RNAs arising from infecting viruses and other pathogens . RIG-I r7G), which can affect RIG-I’s ability to bind cellular RNAs. Previous studies have shown that RIG-I binds m7G Cap0 RNA comparably to 5′ PPP RNA; however, the combination of the m7G cap and a methylation event at the 2′ position of the first ribose, together called Cap1, reduces RIG-I affinity by approximately 200-fold . Bot. Bot7G),p Figure .+ and Coenzyme A were initially discovered in bacteria , and 1 μM T7 RNA polymerase, purified as previously described . Samples were run at a 200°C source temperature rather than 350–375°C to prevent RNA 5' caps detachment.The fluorescently labeled ss39 complementary ssRNA and the 5′ OH 5′ OVG ssRNA were chemically synthesized (Dharmacon).Escherichia coli strain Rosetta (DE3) (Novagen). The protein was purified using a series of chromatography columns as published previously . The ATPase reaction was measured after 20, 40, and 60 min of reaction times in ATPase buffer  at 25°C. Reactions were stopped at each time point using 4 N formic acid and analyzed by PEI-Cellulose-F TLC (Merck) developed in 0.4 M potassium phosphate buffer (pH 3.4). TLC plates were exposed to a phosphorimager plate, imaged on a Typhoon phosphor-imager, and quantified using the ImageQuant software. The molar [Pi] generated during the reaction time intervals was plotted against time and fit to a linear equation with 3 points, not including 0 min. The slopes (ATPase rate) were plotted as a function of RNA concentration and fitted to the following equation to obtain the RNA KD,app values: slope = katpase × [PR]/[Pt]; where [Pt] is total protein concentration; [PR] is the amount of RIG-I/RNA complex formed and [R] is the RNA concentration being titrated. Each point's associated error was calculated from the linear fit. The quadratic equation (Equation 1) was used to determine [PR] and estimate the KD,app and katpase. Reported errors for KD,app and katpase are fitting errors.ATP hydrolysis was measured at constant RIG-I (15 nM) and increasing RNA concentration (1–400 nM) in the presence of 2 mM ATP  and incubated with a constant 20 nM fluorescein-labeled dsRNA for 15 min at 25°C; 500 μM ATP was added prior to taking measurements.Fluorescence polarization measurements were performed on a Tecan Spark microplate reader in a 384-well black plate at 25°C. A monochromator set the excitation wavelength at 485 nM and the emission wavelength at 535 nm, with a 20 nm bandwidth. Purified RIG-I protein was serially titrated in 1× ATPase buffer  from the initial fluorescence polarization (F0) is proportional to the amount of protein–RNA complex (PR) and modified by a coefficient of complex formation (fc). Reported errors for KD are fitting errors.To obtain 50 value rather than KD.To measure the affinity of ssRNA, we used a competition binding assay where the unlabeled ssRNA was serially titrated in 1× ATPase buffer and incubated with a constant, pre-mixed 50 nM RIG-I and 20 nM fluorescent dsRNA for 15 min at 25°C. 500 μM ATP was added prior to taking measurements under the same conditions previously stated. Polarization values were plotted as a function of ssRNA concentration. Unlabeled dsRNA was used as a control to demonstrate competition for RIG-I binding against the fluorescent dsRNA. This data was fit to equations (2) and (3) to estimate the ICEMSAs were performed by incubating RIG-I (75 nM) and ds39 RNAs (25 nM) in ATPase buffer for 60 min at 4°C. 2 mM ATP was added to each RIG-I containing reaction 15 min before loading the sample into the gel. Loading buffer  was added to the samples loaded on a 4–16% gradient Native PAGE gel (Invitrogen) at 4°C. Gels were scanned at 532 nm using a Typhoon FLA 9500 laser-based scanner (GE).2 and 37°C, in DMEM with 10% FBS in 6-well plates to 60% confluence and cotransfected with firefly luciferase reporter plasmid (pLuc125/2.5 μg), Renilla luciferase reporter plasmid (pRL-TK / 500 ng), and a plasmid carrying myc-tagged WT RIG-I gene under the constitutively active CMV promoter (pcDNA-3.1/2 μg). The firefly luciferase gene is under the control of the interferon β promoter, and the Renilla luciferase plasmid is under the control of the constitutively active TK promoter. The plasmid transfections were carried out with X-tremeGENE HP DNA Transfection Reagent (Roche). Cells were replated in 96-well plates the next day at 2 × 104 cells/well density and transfected with indicated single-stranded and double-stranded RNAs  using Lyovec transfection reagent (InvivoGen). After 20 h, the activities of firefly and Renilla luciferases were measured sequentially with the Dual-Luciferase reporter assay (Promega). Each trial's number of replicates are shown in figure legends. Error bars represent the standard error of the mean (SEM).HEK293T cells were grown in 5% CO2 at 37°C in DMEM cell culture medium (Gibco) with 10% FBS in 6-well plates to 80% confluence and transfected with empty vector and myc-tagged WT RIG-I gene under the constitutively active CMV promoter (pcDNA-3.1/2 μg). Cells were replated in 12 well plates the next day at 1 × 105 cells/well density and transfected with indicated RNAs (50 nM in 110 μl volume) using 50 μl Lyovec transfection reagent (InvivoGen).HEK293T RIG-I KO cells were grown in 5% CO5 cells/well density and transfected with indicated RNAs (100 nM in 110 μl volume) using 50 μl Lyovec transfection reagent (InvivoGen).A549 and A549 RIG-I KO cells were maintained in RPMI cell culture Medium (Gibco) with 10% FBS. Cells were plated in 12-well plates at 1 × 10Cells were washed with PBS, and total RNA was extracted from each well immediately using RNeasy micro kit  20 h post RNA transfection for HEK293T RIG-I KO and 25 h post RNA transfection for A549 and A549 RIG-I KO respectively. Cells were lysed by adding 350 μl of cold RLT lysis buffer containing 1% (v/v) 2-mercaptoethanol (MilliporeSigma) and passing the lysate through 1 ml Syringe 5–6 times. Total RNA was extracted from the lysate supernatant following the manufacturer's specification. The purification included in-column DNase treatment using the RNase-free DNase Set. The yield and purity of the RNA were measured using a NanoDrop 2000c spectrophotometer . The total RNA extracted ranged between 708 ng/μl to 1540 ng/μl concentration .Complementary DNA (cDNA) was prepared using 1.5 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit  in a total volume of 20 μl reaction mixture following the manufacturer's specification.http://rdml.org/miqe.html , and then boiled for 10 min. Proteins were separated by SDS PAGE and transferred onto 0.2 μm PVDF membrane. The membrane was immunoblotted with anti-RIG-I  and anti-actin  antibodies at 4°C overnight, followed by HRP-conjugated secondary antibodies  for 1 h. Blots were developed by using ECL western blotting substrate  on a Biorad ChemiDoc system.in vitro transcription reactions are not contaminated with 5′ PPP RNA-ends. Previously published techniques of T7 RNA polymerase (T7 RNAP)-based transcription relied on purifying ADP-based metabolite-capped RNAs from 5′ PPP RNA , resulting from loss of the flavin moiety and one phosphate from the diphosphate linkage. These species could have been generated during the mass spectrometry analysis steps. RIG-I does not recognize the 5′ monophosphate RNA end, and if present in the sample, it should not generate a false positive , the metabolite-capped RNAs . The 5′ OH 5′ OVG binds with the weakest affinity , 8-fold worse than the weakest binding metabolite-capped dsRNA, FAD. Additionally, each metabolite-capped dsRNA showed similar rates of ATP hydrolysis and comparable Vmax values as 5′ PPP . In the ATPase assay, we titrate a small amount of RIG-I with increasing RNA, conditions that support mostly RIG-I monomers on RNA. On the other hand, we titrate a small amount of RNA in the polarization assay with increasing RIG-I concentrations, conditions that support RIG-I oligomerization , OAS1 (2′-5′-oligoadenylate synthetase 1) and ISG15 (Interferon stimulated gene 15) after ectopic expression of RIG-I in HEK293T RIG-I KO cells Figure C–E. In aThe reporter signaling assay, shown above, relied on overexpressed, ectopic RIG-I. To determine if endogenous levels of RIG-I would recognize the metabolite-capped RNAs, we used A549 cells. In agreement with our reporter assay in HEK293T, the 5′ PPP ds39 and all the metabolite-capped RNAs generated a robust antiviral signaling state in A549 cells, showing expression of IFNB1, OAS1, ISG15, and MX1 (MX dynamin-like GTPase 1) genes Figure . In addiTo confirm that the immune stimulation observed in A549 cells was RIG-I-dependent, we transfected A549 RIG-I KO cells with our RNA panel, monitoring the antiviral immune response using qPCR targeting IFNB1 Figure . Agreein7G RNA cap to a mature mRNA with Cap1 modification to evade RIG-I recognition and prevent an immune response   or triggering it   , can be s Figure . Likely,s Figure , 9. The the RNA) .O-ribose like in Cap1 RNA. However, whether existing methylation pathways work with metabolite-capped RNAs is unknown and needs further investigation. Other means to evade RIG-I could include preventing dsRNA formation, sequestering the 5′ RNA end through protein binding, burying the RNA group in a larger structure, such as the ribosome, or sequestering the RNA in organelles outside the cytoplasm, such as the mitochondria or nucleus. When leaking from damaged mitochondria or suffering mitochondrial DNA damage, human mitochondrial RNAs enter the cytoplasm and stimulate RIG-I and MDA5 (7G but have been observed to incorporate alternative caps (The role of the metabolite-capped RNAs in mammals is not well understood, but if such RNAs accumulate in the cytoplasm of healthy cells, RIG-I will recognize them as immunogenic. Sensitive RIG-I recognition of metabolite-capped RNAs that are endogenously made needs to be demonstrated. Still, if they are immunogenic, additional mechanisms would be required to prevent deleterious autoimmune responses without viral infection. Such mechanisms could include internal RNA modifications, including methylation to inhibit RIG-I signaling , 34 or mand MDA5 , 35, 36.ive caps . The app7G capped mRNA for protein translation and RIG-I evasion (An understudied context of alternative capping is the viral capacity for using metabolites to cap their RNAs. Only one description of virally derived, metabolite-capped RNAs is found in Dengue virus-infected mosquito cells . Dengue  evasion . Additio evasion . Recogni7G cap presents additional constraints on how metabolite-capped RNAs can exist in the cytoplasm of a healthy cell and new avenues to explore their applications for RNA-based technologies.Altogether, the discovery that RIG-I recognizes a broader range of capped RNAs than previously known mgkad518_Supplemental_FileClick here for additional data file.


Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3′CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3′CITEs that differentially affect the structure and translation of different umbraviruses.The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3′ structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3′ cap-independent translation enhancers (3′CITEs), as umbraviruses can have different 3′CITEs in the central region of their lengthy 3′UTRs, and most also have a particular 3′CITE (the T-shaped structure or 3′TSS) near their 3′ ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3′CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in  A final concentration of 15 mM of N-methylisatoic anhydride (NMIA) dissolved in dimethyl sulfoxide (DMSO) was added to the (+) folded RNA, while an equal volume of DMSO was added to the folded (–) negative modification control. The (+) and (–) RNAs were incubated for 30 min at 37 °C, ethanol precipitated and then resuspended in 11 μL ddH20 for reverse transcription.As previously described , RNA traPrimers used for SHAPE probing were: OPMV_R1: 5′-GGATGGGAGTGACCACCAC (positions 4235–4217), OPMV_R2: 5′-CAGAACCCTCAGTTTGACTAC (positions 4008–3988), PEMV2_R1: 5′-AGGAAACAGCTATGACC (positions 4319 to 4302), PEMV2_R2: 5′-CGCGTTTGTGATCTTTTTGG (positions 3959 to 3940). For primer extension reactions, 2.5 µM of PET-labeled and 6-carboxyfluorescein (FAM)-labeled oligonucleotides were used with 6 pmoles of unmodified RNA (for sequencing reactions) or NMIA- and DMSO-treated RNA (sample reactions). SuperScript III reverse transcriptase (Invitrogen) was used for reverse transcription reactions as previously described ,39. To ptumefaciens strain GV3101 and cultured in the presence of antibiotics. The cells were harvested and resuspended in resuspension buffer . For infiltrations of OPMV/PEMV2 WT and mutant constructs, an OD600 of 0.6 was mixed with the pothos latent virus p14-silencing suppressor construct  dATP-labeled DNA oligonucleotides complementary to OPMV positions 3591 to 3630, 3634 to 3672, and 3676 to 3715. For PEMV2, a mixture of three [α−32] dATP-labeled DNA oligonucleotides complementary to positions 2731 to 2771, 2969 to 3004, and 3229 to 3270 were used. After incubation and washing, nylon membranes were exposed to a phosphorimager screen and scanned using an Amersham Typhoon Biomolecular Imager. The raw images were processed using ImageJ software to produce gel images and quantify band intensities.OPMV and PEMV2 binary vector constructs were transformed into A. onstruct  at an ODArabidopsis thaliana were transfected with luciferase reporter transcripts using a polyethylene glycol-mediated transformation protocol, as previously described [6 protoplasts were transfected with 20 μg of OPMV/PEMV2 luciferase reporter transcripts and incubated under constant light for 18 h at 22 °C. The cells were lysed at 18 h post-transfection, and the luciferase activity was assayed with a dual-luciferase reporter assay system (Promega) using a Modulus microplate multimode reader (Turner BioSystems). To check for the stability of the luciferase RNA constructs in protoplasts, the RNA gel blots were conducted using the total RNA extracted from protoplasts at 18 h post-transfection after exhaustive washing was performed.Protoplasts prepared from callus cultures of escribed . Brieflyhttps://rna2drawer.app) accessed on 1 October 2022 [All of the RNA structures were generated using the on-line RNAcanvas web app  from the base of a putative scaffold that supports known BTE 3′CITEs was a region with three discontinuous conserved motifs capable of folding into a small hairpin with an internal loop . All of In addition, a second structurally conserved element was found to be 0 or 1 nt upstream of CAS in all BTE-containing umbraviruses and two non-BTE umbraviruses (PEMV2 and RCUV). This element consists of two similar-sized stem-loops (SLX and SLY) separated by 6 to 11 nt. SLX and SLY apical loops contain three to six residues capable of canonical base-pairing, thus forming a KL between the two stem-loops . For thrTo investigate if the predicted CAS and KL element structures were present in OPMV transcripts, full-length gRNA and sgRNA2 transcripts were synthesized in vitro, denatured by heat and renatured by cooling, and then subjected to Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) RNA structure probing . SHAPE rAgrobacterium tumefaciens-mediated infiltration of expression constructs into N. benthamiana. The conversion of the conserved CAS 6 nt apical loop into a UNCG-type tetraloop (mC1) decreased virus accumulation by 7-fold  increased 5′38 + U luciferase activity by 1.8-fold but did not significantly affect translation of 5′69 + U  with the upstream KL element and downstream BTE, full-length OPMV gRNA transcripts containing a subset of the CAS mutations were subjected to SHAPE structure probing D. The daAltering the CAS apical loop (mC1) did not significantly change residue reactivity in the KL element , suggesting that the KL element is not interacting with the conserved CAS apical loop. In contrast, residues on the 5′ side of the BTE scaffold lower stem that were normally reactive by SHAPE see A had sigCAS interior bulge mutation (mC3) caused a substantial increase in the reactivity of the residues at the mutation site, suggesting that this bulge sequence is paired in the WT gRNA transcript D. AdditiDeletion of the entire CAS hairpin (mC4) caused similar reductions in reactivity in the BTE lower stem and upper conserved motif as mC1 D. Four aWhen sgRNA2 transcripts with the CAS deletion (mC4) were subjected to SHAPE structure probing, both similar and unique changes in residue reactivity were evident. In the BTE, very similar reactivity reductions were found at the base of the BTE and in the upper conserved motif. Two additional bulge loop regions in the BTE were uniquely altered: the upper bulge (and closing base-pair) had three of seven residues with reduced reactivity while the large bulge near the BTE base had four residues with enhanced reactivity. In the KL element, one residue in the loops of the SLX and SLY hairpins had reduced reactivity, along with two residues between the two hairpins. Since gRNA and sgRNA2 share identical sequences in this location, these results suggest that this location does not constitute a structurally separate domain in gRNA and sgRNA2 transcripts but is interacting with sequences in the gRNA that are missing in the sgRNA2 or that assume a different conformation.GG G/A UCCUGGUAAACAGG), including a covariant change in OPMV when compared with other umbraviruses (CCC:GGGUC to 5′GAUCC:GGAUC). The BTE mutation G3808A, which should disrupt the putative interaction while maintaining a consensus 17-nt BTE signature sequence, reduced OPMV accumulation in N. benthamiana to near undetectable levels  despite containing the more conserved versions of the two motifs.As shown in aviruses A. To dete levels B. In conSince mutagenesis assays did not conclusively rule in (or out) an interaction between the two conserved motifs, transcripts containing the single and double mutations were subjected to SHAPE structure probing. Our reasoning was that disruption of an interaction between the two motifs should cause structural changes in the partner sequence that might be reduced by compensatory mutations. C3726U caused enhanced reactivity at the mutation site upstream of CAS, suggesting that this sequence is normally paired C. HoweveN. benthamiana, single and compensatory mutations were generated, and the levels were determined by RNA gel blots RNA plant viruses follow unconventional translational pathways often mediated by 3′CITEs present near the 3′ end of the viral genomes ,5. UmbraN. benthamiana  or SLX and SLY (sgRNA). Since these hairpins are phylogenetically conserved, and since compensatory mutations at the base of the CAS stem restored gRNA levels in vivo for both OPMV B and PEMTombusviridae  did not reveal the existence of hairpins with similar conserved features, suggesting that CAS may be uniquely associated with umbraviruses. Other hairpins upstream of 3′-proximal CITEs have been found in betanecroviruses TNV-D and beet black scorch virus [A search for hairpins with CAS-conserved features upstream of 3′CITEs in virus members of other genera in the ch virus , but thech virus .Tombusviridae [Umbraviruses whose interior 3′CITEs have not yet been identified  are predicted to have similar structured elements atop extended stems that likely constitute novel CITE classes . All umbsviridae ,53 and wsviridae , which g


Sarcoma is a heterogeneous group of mesenchymal neoplasms with a high rate of lung metastasis. The cellular mechanisms responsible for sarcoma metastasis remain poorly understood. Furthermore, there are limited efficacious therapeutic strategies for treating metastatic sarcoma. Improved diagnostic and therapeutic modalities are of increasing importance for the treatment of sarcoma due to their high mortality in the advanced stages of the disease. Recent evidence demonstrates that the exosome, a type of extracellular vesicle released by virtually all cells in the body, is an important facilitator of intercellular communication between the cells and the surrounding environment. The exosome is gaining significant attention among the medical research community, but there is little knowledge about how the exosome affects sarcoma metastasis. In this review, we summarize the multifaceted roles of sarcoma-derived exosomes in promoting the process of metastasis via the formation of pre-metastatic niche (PMN), the regulation of immunity, angiogenesis, vascular permeability, and the migration of sarcoma cells. We also highlight the potential of exosomes as innovative diagnostic and prognostic biomarkers as well as therapeutic targets in sarcoma metastasis. Sarcoma is a group of large heterogeneous and primary malignant tumors, accounting for 19–21% of all cancer-related deaths in children and adolescents due to their highly aggressive nature and systemic metastasis ,4,5. HowExosomes belong to a category of extracellular vesicles which play critical roles in intercellular information transmission ,17,18,19Exosomes are widely recognized as essential modulators of cancer metastasis, and are a source for identifying potential biomarkers and therapeutic targets for many malignant tumors ,27,28. SIn this review, we discover exosome-related mechanisms in sarcoma metastasis and discuss the potential of exosomes as diagnostic and prognostic biomarkers, as well as potential therapeutic targets for metastatic sarcomas in the future.Exosomes are extracellular vesicles excreted by mammalian cells through endosomes that are of vital importance in cell communication by transporting intercellular cargo . ExosomeThe tumor microenvironment (TME) is critical in facilitating sarcoma growth and metastasis, and an increasing body of literature illustrates that the pre-metastatic niche (PMN) is intimately linked with the tumor cells metastasizing . PMN is The tumor microenvironment is a complex internal environment on which tumor cells arise and depend, mainly composed of the tumor itself and some other non-tumor cells , as well as blood vessels, extracellular matrix, and biomolecules . The TMEThe PMN is a preformed microenvironment made by exosomes secreted by the primary tumor site before widespread metastasis ,53. ExosExosomes can induce metastatic sarcoma organotropism via the release and uptake of exosomal cargos . Highly The survival of cancer cells that metastasize from primary tumors to secondary sites depends upon the stroma microenvironment. Tumor-derived exosomes assist in this process by educating and remodeling stromal cells in the metastatic site to support tumor cell viability and metastatic dissemination ,65,66. FMSCs interact with cancer and other cells in the TME via paracrine factors and through exosomes to support tumor growth, progression, and metastasis . PreviouCAFs are unique reprogrammed stromal cells with roles in pre-metastatic niche formation and metastasis, which is a major component of cancer stromal cells that account for about 40∼50% of the total cell population in cancer . Tumor-cTAMs are of the anti-inflammatory (M2) subtype and permeate malignant tissues . Within Exosomes are important intercellular communicators, which do not only account for cells, but also for the ECM, which is an important constituent of non-transformed tissues as well as tumors . ExosomeThe immune system is a significant barrier to metastasis. For sarcomas to thrive in their new environment, it is of utmost importance for the PMN to protect the metastatic sarcoma cells from being apoptosed upon entering the metastatic site . ExosomeAngiogenesis is the formation of new blood vessels in capillaries or venules behind capillaries ,95. TumoExosomes derived from sarcoma cells or sarcoma-educated stromal cells play significant roles in metastasis by promoting the migration ability of sarcoma cells . JNK/p38Exosomes play an integral part in sarcoma metastasis, participating in sarcoma cell proliferation, invasion, and migration. With the in-depth research in this emerging field, the potential clinical value of exosomes in sarcoma metastasis has been gradually excavated. The surface markers on sarcoma-derived exosomes and internal components mirror many of the molecular features of sarcoma which it originates from. Meanwhile, the unique expression pattern and relatively stable content make exosomes promising candidates as novel biomarkers for sarcoma metastasis ,29,105. OS is the most common primary tumor of long bones affecting children and adolescents . Due to LCP1, an actin-binding protein, was found in serum exosomes from OS patients and the exosomal LCP1 level was correlated with pulmonary metastasis . ExosomaAnother study found that a new fusion protein, Rab22a-NeoF1, can be sorted into exosomes of OS cells to facilitate the lung PMN formation and consequently promote lung metastasis . To be mMicro-RNAs (miRNAs) are single-chain, endogenous, and small noncoding RNAs with a length of approximately 22–24 nucleotides . They reMiR-675 is overexpressed in cancer tissues from metastatic OS patients as well as metastatic OS cell lines . OS-deriA growing number of studies suggest that miRNAs can be delivered to OS cells by BMSC-derived exosomes during OS metastasis. Exosomes produced by BMSCs could stimulate sarcoma cells to express chemokines, in turn promoting the proliferation and migration of OS cells. For instance, BMSC-derived exosomes containing miR-769-5p could be taken up by OS cells and induce the EMT and lung metastasis of OS xenografts in nude mice by increasing E-cadherin expression, suppressing dual-specific phosphatase 16 (DUSP16), and activating the JNK/p38 MAPK signaling pathway . In addiIn addition to BMSCs, CAF-derived exosomal miR-1228, internalized by OS, could downregulate endogenous SCAI mRNA and protein levels in OS, thus promoting the proliferation and metastasis of OS ,125. TAMBesides the distinct miRNAs previously mentioned, cancer tissues or peripheral blood originating from metastatic OS patients have presented high levels of long noncoding RNAs (lncRNAs). LncRNAs constitute a novel class of non-coding transcript RNAs with unique characteristics . These RExosomal lncRNA linc00852 was found in cell culture fluid from OS cells, and exosomal lncRNA linc00852 could promote OS metastasis by increasing the expression of receptor tyrosine kinase AXL . MechaniBy acting as a miR-183-5p sponge and activating ERG expression, exosomal lncRNA PVT1 secreted from BMSCs stimulates the proliferation and metastasis of OS cells . The lncChondrosarcoma (CS) is the second most common primary bone tumor among all malignant bone tumors. Chondrosarcoma is a malignant tumor of cartilage origin that includes several subtypes depending on their histological characteristics and pathological sites. Unlike many tumors, chondrosarcoma is usually resistant to chemotherapy and radiotherapy. Exosomes have shown their ability to regulate human chondrosarcoma metastasis.In chondrosarcoma tissues and cell lines, lncRNA RAMP2-AS1 expression was upregulated . ElevateRhabdomyosarcomas (RMSs) are a heterogeneous group of malignant tumors and are the most frequent soft tissue sarcoma in children . There aMiR-486-5p, an effector of PAX3-FOXO1, promotes migration, invasion, and colony formation of rhabdomyosarcoma cells by regulating its paracrine effects in exosomes. . Human RLiposarcoma (LPS) is the most common soft tissue sarcoma, accounting for roughly 25% of all sarcomas in adults . DespiteThe critical functions of exosomes and their cargos in metastasis demonstrate their potential as therapeutic targets, although this field of research is still in its early stages. It has already been shown that exosomes can be utilized in medicine as therapeutic tools, and their lipid bilayer makes them perfect drug transport systems . SarcomaGiven the fact that sarcoma cells send out exosomes to encourage their metastasis and proliferation, the first idea is to inhibit the release of metastatic sarcoma exosomes. Recent studies have identified that the neutral sphingomyelinase inhibitor GW4869 functionalized by inhibiting the ceramide-mediated inward budding of multivesicular bodies (MVBs) to decrease exosome release ,140. So,Similar results can be obtained according to the same experimental process in LPS . ThroughAn alternative therapeutic approach aimed at inhibiting exosome function is by blocking their downstream signaling pathways. IL-6/STAT3 is an essential pathway involved in the progression of metastatic sarcoma, and IL-6 production via sarcoma exosomes made mesenchymal stem cells responsible for stimulating sarcoma metastasis . So, bloAs discussed earlier, the lung metastases of OS cells were promoted by the exosomal Rab22a-NeoF1 fusion protein, and this event could be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide . The oveAltogether, the mechanisms of blocking the downstream pathways of exosomes are regarded as significant therapeutic methods in resisting metastatic sarcoma. Moreover, the results might help to find new therapeutic targets and novel opportunities for preventing sarcoma metastasis.Exosomes have the potential to be excellent carriers in delivering anti-cancer biomolecules for metastatic sarcoma because of their unique properties, such as high biocompatibility, low toxicity, specificity, and stability ,144,145.Similarly, exosomal miR-195 was formed by normal human chondrocytes and delivered to OS cells . MiR-195The studies mentioned above show that exosomes are potential candidates in the treatment of sarcoma metastasis.The advantages of using exosomes as non-invasive methods for prognostic stratification and precise therapy are evaluated clinically. Since many promising results have been achieved in in vitro and animal models, the use of exosomes is considered to be one of the most hopeful approaches for metastatic sarcoma diagnosis and therapy. Notably, some clinical trials have already made important achievements. As shown by the Musculoskeletal Tumor Center of Peking University People’s Hospital, compared with healthy subjects or individuals with a 5-year progression of free OS, OS patients with lung metastases usually exhibited elevated exosomal programmed death ligand 1 (PD-L1) and exosomal N-cadherin levels in the serum . FurtherThese observations indicate that peripheral blood N-cadherin, PD-L1, SENP1, miR-25-3p, and miR-92a-3p levels may be used as clinical biomarkers for diagnosing sarcoma micrometastases and evaluating prognosis.The current rapid expansion of molecular biological techniques on exosomes evidently demonstrates that exosomes play essential roles in sarcoma metastasis. This review described the possible mechanisms of exosomes in sarcoma metastasis, including organotropism, intercellular transmission, ECM alterations, immunosuppression, angiogenesis, and cell migration. Secondly, several sarcoma-derived exosomal biomarkers, including miRNAs, proteins, and lncRNAs, were reported with a desirable diagnostic value and the potential of predicting prognosis for sarcoma metastasis. Finally, it was elucidated that exosomes can be attractive targets for the development of metastatic sarcoma therapeutic methods. Although positive results have been achieved toward investigating the relationship of exosomes and sarcoma metastasis, the clinical application of exosomes in sarcoma metastasis does not go deep enough. Future studies on exosomes and sarcoma metastasis may focus on the following aspects: (1) probing more detailed mechanisms of exosomes in sarcoma metastasis; (2) discovering additional exosomal biomarkers in sarcoma metastasis; (3) strengthening research on other metastatic sarcomas, such as liposarcoma and Ewing’s sarcoma; (4) establishing effective methods for the isolation, quantification, and analysis of exosomes in the body fluids of patients; (5) exploring new exosome-based therapeutic approaches for metastatic sarcoma treatment; and (6) accelerating the translation of basic science discoveries to clinical trials. The use of exosomes as biomarkers and therapeutic targets of sarcoma metastasis is already being explored, and more positive breakthroughs will be promising.


Trypanosoma brucei, with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 32 of which are stage-specifically expressed. We also present insights into the complexity of the T. brucei transcriptome, including alternative transcriptional start and stop sites and potential transcript isoforms, to provide a bias-free understanding of the intricate RNA landscape in T. brucei.Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay, to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized to date. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known, because of the inherent cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here, we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome  Phytomonas, Leishmania, and Trypanosoma are single-cell eukarya that parasitize plants and animals [trans-splicing, RNA editing, ribosomal RNA fragmentation, and mitochondrial tRNA import [trans-splicing of a capped, 39 nt long spliced leader (SL)-RNA to the 5′-end and by 3′-end polyadenylation. Second, transcriptional control of gene expression is, with only a few exceptions, not existent in trypanosomatids. The organisms regulate gene expression post-transcriptionally. This implies that RNA-driven processes, such as splicing, alternative splicing, RNA turnover, and translation, must be able to sense and respond to extrinsic and intrinsic cues to adjust and modulate cellular protein levels. While a comprehensive verification of such a scenario awaits confirmation, the currently accepted view is that changes in the transcriptome are brought about by RNA-binding proteins (RBPs) utilizing a large panel of RNA-interacting proteins [T. brucei rely on a nearly complete inventory of canonical eukaryotic noncoding (nc)RNAs. The panel of small ncRNAs (RNAs < 200 nucleotides (nt) in length) includes the small nuclear (sn)RNA of the trans-spliceosome [T. brucei [grumpy) is functionally characterized. Grumpy acts as a precursor for the C/D box-type snoRNA snoGrumpy, which is involved in a vital differentiation step in the lifecycle of T. brucei [Trypanosomatid organisms including the genera  animals , and as  animals , a signiA import . This RNproteins . RBPs ariceosome , the smaiceosome ,8, as weiceosome , and teliceosome  as well iceosome ,13. Smaliceosome . About 1. brucei ,16; howeT. brucei. This is in part due to the limited number of data but also due to the use of mainly short-read RNA sequencing (RNA-seq). Although RNA-seq has played a dominant role in transcriptome profiling in many organisms, the technique is not without limitations. Firstly, the protocol relies on reverse transcription (RT) to convert RNA into cDNA and on PCR amplification for library construction. Both enzyme treatments have been shown to introduce RT- and amplicon-specific biases [Although the pool of uncharacterized lncRNAs likely contains multiple regulatory lncRNAs, as well as binding targets for regulatory RBPs, it is not clear whether the annotated sequences represent the full complement of lncRNAs in c biases , especiac biases . Other sc biases  and ambic biases ,21. Lastc biases .T. brucei transcriptome using the Oxford Nanopore Technologies (ONT) nanopore-based RNA sequencing platform [T. brucei transcriptome including poly(A)-tail length variations, alternative splice-acceptor sites (SAS) and polyadenylation sites (PAS), alternative transcriptional start and stop sites, and potential transcript isoforms.To avoid these drawbacks, here we perform direct RNA sequencing (DRS) of the platform . ONT-DRSplatform . DRS perT. brucei brucei strain Lister 427 [v/v) fetal calf serum (FCS), 0.2 mM 2-mercaptoethanol and 100 U/mL penicillin/streptomycin . Parasites were grown at 37 °C in 95% air and 5% CO2 at ≥95% relative humidity. Insect-stage (procyclic) parasites were propagated in SDM-79 medium [v/v) FCS at a temperature of 27 °C. Parasite cell densities were determined by automated cell counting.ster 427  was usedster 427  suppleme9 medium  in the pβ-cyanoethyl)--phosphoramidite monomers. Full-length synthesis products were purified by reverse-phase high-performance liquid chromatography and verified by mass spectrometry. The following DNA sequences were synthesized: 18S_forward: CGGAATGGCACCACAAGAC; 18S_reverse: TGGTAAAGTTCCCCGTGTTGA; β-Tub_forward: TTCCGCACCCTGAAA-CTGA; β-Tub_reverse: TGACGCCGGACACAACAG; nt_4120.1_forward: TGCAAG-TGAAGGCACTGTGT; nt_4120:1_reverse: GCAACAAGCACGGAAGATGG; nt_1759.1_forward: TTGCGTGTGTGTTGGAATGG; nt_1759.1_reverse: ACGACTCAA-AGGAATGCCA; nt_17.3_forward: ACCCGT-AATTTGCGGACTGT; nt_17.3_reverse: TCCCTTCCGTGTATCGTTGT; nt_256.1_forward: GTGTGTATGGGCGTTTCGTG; nt_256.1_reverse: ACATACGCGTAGCCACACAA .Oligodeoxynucleotide primer molecules were synthesized by automated solid phase synthesis using 5′-Dimethoxytrityl-derivatised  supplemented with 20 mM glucose. Cells were shock-frozen in liquid N2, and stored at −20 °C. Total RNA was isolated by guanidinium acid phenol extraction [25-derivatized magnetic beads (NEB). RNA yields were quantified by ultraviolet (UV) spectrophotometry and the integrity of the isolates was electrophoretically analyzed in 2% (w/v) agarose gels. Poly(A)-enrichment was assessed using qRT-PCR, comparing the ratio of the amount of β-tubulin-specific mRNA over 18S rRNA to that of total RNA using the ΔΔCT method [Bloodstream-stage trypanosomes were grown to 10traction . PolyadeT method .2O and 2 µL of diluted cDNA were used in 10 µL qPCR reactions containing the Luna® Universal qPCR Master Mix (NEB) and 0.3 µM of gene-specific forward and reverse primer. PCR reactions were carried out in a StepOnePlus real-time PCR instrument  using a modified Fast Run protocol . After each run, SYBR green displacement curves were generated  with continual fluorescence measurement at 535 nm. PCR products were analyzed by electrophoresis in 10% (w/v) non-denaturing polyacrylamide gels in 90 mM Tris/B(OH)3 pH8.3, 2 mM EDTA buffer (TBE).Total RNA (200 ng) or poly(A)-enriched RNA (40 ng) was reverse transcribed using 5 U/µL of Superscript IV reverse transcriptase (Thermo Fisher Scientific) in a final volume of 10 µL in the presence of 2.5 µM oligo dT-primer or 1 µM gene-specific reverse (rev) primer. Reaction mixtures were diluted with 10–50 vol. ddHq-score > 7 were further processed.Sequencing libraries were generated from 1 µg poly(A)-enriched RNA following the Oxford Nanopore Technologies (ONT) SQK-RNA002 protocol. Yields were estimated fluorometrically  and DRS sequencing was performed on a MinION device in ONT-MinION R9.4 flow cells for 20–24 h. Base calling was carried out after the sequencing runs using Guppy 4.2.2  as part T. brucei TREU927 (v52), the mitochondrial maxicircle genome of T. brucei (NCBI M94286.1), and the sequence of the RNA calibration strand  from the Saccharomyces Genome Database (SGD). Only reads from primary alignments were further processed. Please note that strain TREU927 was chosen as a reference genome  ,30. For see also ) becauseT. brucei mRNA. For this, sequencing reads were first mapped to the reference genome. Reads that were uniquely mapped were then appended to the 39 nt SL-sequence, followed by a 2nd mapping step. Reads mapping to at least 10 nt of the SL-sequence were considered 5′-processed. Poly(A)-extensions were analyzed using nanopolish [Full-length reads were identified by taking advantage of the presence of both a 5′-spliced leader (SL)-sequence and a poly(A)-sequence at the 3′-end of every nopolish ,33. OnlyFull-length reads from all libraries were combined N = 835,426) and mapped to the reference genome (TriTryp v52). The alignment file was converted into a bed file using BEDTools v2.26.0 ,35, and ,426 and p-values were adjusted according to Benjamin and Hochberg, and statistical calculations were performed using R (version 4.0.3) and RStudio (version 1.1.453).Expression profiles and the analysis of differential gene expression (DGE) were performed using the Bioconductor packages RsubRead  and edge5 and 6.6 × 105 reads (q)-score ≥ 7. Both the read quality and the read lengths are comparable for the bloodstream and insect-stage libraries with a median length of 770 ± 57 nt, a median q-score of 10.2 ± 0.3, and p-values of 0.8 and 0.43. Between 92 and 98% of the reads were mapped to the reference genome of T. brucei TREU927 and the mitochondrial (maxicircle) genome of the parasite. The mapping frequencies (p = 1.5 × 10−4), which is due to the high level of variant-surface-glycoprotein (VSG) expression. Three to four percent of all reads map to this transcript. Since the specific VSG sequence is lacking in the annotated TREU927 genome, a higher number of unmapped reads is obtained. Contaminations with ribosomal RNAs are <1.4% and the genome coverage varies between 64% and 73%. A total of 79–84% of the annotated exons  are detected, with significantly more exons covered in the bloodstream-stage libraries (p = 0.0014). As mentioned above, this is due to the presence of reads for VSG and expression-site-associated-gene (ESAG) transcripts. The overall error frequency for all libraries adds up to about 8%. Gene-expression profiles within libraries from either developmental stage correlate with a Spearman rank correlation coefficient (ρ) of 0.9. Procyclic and bloodstream-stage libraries correlate with ρ = 0.75  monitors the transcript inventory of cells “directly”. This enables a straightforward mapping of gene boundaries and transcript termini, which are characterized by sharp changes in the genome coverage profiles . As a coIn living cells, full-length transcripts coexist with RNA degradation products, which result from cotranslational mRNA decay, nonsense-mediated mRNA decay, or nucleolytic RNA maturation processes. Since DRS starts from the 3′-end of poly(A)-tailed RNAs, this results in a 3′-end bias. Furthermore, DRS also suffers from a 5′-end ambiguity, because the sequenced RNA strands are released from the nanopore about 10–12 nt before they reach the 5′-terminus . Artifictrans-splicing and polyadenylation from polycistronic primary transcripts. Thus, mature transcripts are represented as polyadenylated reads with a spliced-leader (SL) sequence at the 5′-end. This unique feature was used to unambiguously identify full-length transcripts. Reads carrying the SL sequence were selected as detailed in p = 0.005) in the libraries derived from bloodstream-stage parasites -tail, and the extent of this signal can be used to calculate the tail length ,41. We uT. brucei genome. About 40% of the polyadenylation sites were assigned to annotated exons, the majority mapping to 3′ UTRs. The median poly(A)-tail length is 96 nt for annotated mRNAs. It is moderately higher for pseudogenic transcripts and lncRNAs (117 nt), but considerably shorter for rRNAs (26 nt) and mitochondrial transcripts (29 nt)  a. For th (29 nt) .The selection of full-length reads with both a 5′-SL and a 3′-poly(A)-sequence on the same transcript, enabled us to precisely map all splice variants or transcript isoforms, thereby improving the current transcriptome annotation. For that, we used all full-length reads from all libraries  and identified 18,406 exons and/or transcript isoforms, many of which are partially overlapping. A total of 30% of the transcripts/isoforms consist of only one read. The maximum number of reads for a single transcript is 6076, which encodes the ribosomal protein L11 (Tb927.9.7620). The transcript length distribution varies from 113 nt to 14,636 nt with a median of 1501 nt. The longest transcript  covers the complete coding sequence of the trypanosome axoneme component, hydin  , 7436 (>5%), or 7496 (>1%) are at least partially covered in our DRS data. Taking into account an uncertainty of ±20 nt in the mapping of transcript boundaries, 2319 observed transcripts match precisely their annotated counterpart. About 10% of the sequenced transcripts represent partial sequences of annotated exons. More than 45% of the annotated exons are completely covered, and include the 5′- and 3′-extensions (median length = 313 nt), which is indicative of incompletely annotated UTRs. Large discrepancies between the annotated and observed length of transcripts can also be attributed to splicing events, which result in truncated RNAs, or to aberrant trans-splicing and polyadenylation events that generate long di- or tri-cistronic transcripts. Representative examples of di-cistronic transcripts are shown in T. brucei, with no apparent clustering  and a 3′-UTR-localized lncRNA  were used for subsequent analysis. Interestingly, both search algorithms also predicted annotated protein-coding sequences as noncoding   .T. brucei nuclear genome consists of three chromosome classes: 11 diploid pairs of megabase chromosomes (0.9–5.7 Mbp), intermediate-size chromosomes (300–900 kbp), and minichromosomes (50–100 kbp) [T. brucei reference genome and the lncRNAs described in [The 100 kbp) . The preribed in   and the same holds for members of the retrotransposon-hotspot-protein multigene family (RHS). This is consistent with the observation that many RHS genes or RHS pseudogenes are associated with VSG and ESAG loci in sub-telomeric regions [Of 11,769 genes, 8355 were included in the analysis based on the criterion of ≥15 reads/gene in all libraries and ≥5 reads in either the procyclic- or bloodstream-stage libraries. A total of 417 genes are up-regulated in bloodstream-stage parasites, compared to procyclic trypanosomes, and 155 genes are down-regulated. As expected, and as shown in  regions . A list T. brucei 927 and T. brucei 427 might change the picture to some degree. In particular, intergenic, noncoding sequences are likely more susceptible to inter-strain variations. Finally, we experimentally confirmed the differential expression of four of the new lncRNAs  by qRT-PCR, which is shown in Importantly, of the 705 lncRNAs included in the DGE analysis, 109 are differentially expressed a. Only fT. brucei is organized as a macromolecular assembly of thousands of catenated circular DNA molecules, known as the kinetoplast. Two classes of DNA circles can be distinguished: maxicircles and minicircles. Maxicircles are <25 kbp in size and are present in <50 copies per network. T. brucei minicircles have a size of 1 kbp with thousands of copies per kinetoplast. Maxicircles code for key components of the mitochondrial ribosome and the mitochondrial electron-transport and chemiosmosis system; however, some of the protein-coding genes are stored in a cryptic fashion. To generate functional mRNAs, these sequence-deficient transcripts are post-transcriptionally edited, which is detailed in The mitochondrial genome in T. brucei maxicircle . The median length of the poly(A)-tails of all mitochondrial transcripts is about 30 nt, with minor variations between different transcripts. The fraction of polyadenylation and the length of the poly(A)-tails are comparable in both life-cycle stages  gRNAs are needed to fully edit a single pre-mRNA. The reaction proceeds from the 3′-end of the pre-edited mRNA to the 5′-end and as a consequence generates a multitude of partially edited mRNAs, next to fully edited transcripts and never-edited mitochondrial mRNAs.As outlined above, gene expression of most maxicircle genes relies on a posttranscriptional RNA editing reaction. In the process, sequence-deficient primary transcripts are remodeled into functional mRNAs by inserting and deleting exclusively uridine (U)-nt at specific sites in the pre-mRNAs. For some mRNAs, >50% of the mature sequence are the result of the processing reaction. The sequence information for editing is provided by a trypanosome-specific class of small (40–60 nt long), ncRNAs, termed guide RNAs (gRNAs) ,13. In TAs shown in T. brucei mitochondrial transcriptome, including all fully edited sequences, was used to monitor the efficiency of the processing reaction  no completely edited transcripts were detected, in accordance with the findings of Opperdoes and Michels, 2008 [Mapping all reads to the reaction . As expels, 2008 .T. brucei transcriptome by using direct RNA sequencing on nanopore arrays. This was carried out to circumvent the reverse-transcription and PCR-amplification steps of short-read RNA sequencing, to obtain strand-specific long-read RNA-sequence information. We analyzed the two major stages of the parasite lifecycle using native poly(A)-tailed RNA and generated up to 0.7 × 106 reads per library, with a median read length of 1500 nt and a maximal length > 14,500 nt. A special emphasis was put on the inventory of lncRNAs as potential regulators of gene expression. Of the 1491 lncRNA genes annotated in the reference genome, only 50% were confirmed by DRS. However, we identified 207 previously unknown lncRNAs, 32 of which are stage-specifically expressed with the majority (>96%) up-regulated in bloodstream-stage parasites. Strikingly, 40% of the newly identified lncRNA sequences localize proximal to a bloodstream-stage specific VSG, ESAG, or RHS gene, suggesting a co-regulation scenario perhaps by a similar mechanism. Furthermore, the DRS data provide insights into the complexity of the T. brucei transcriptome. This includes 1756 loci, which code for 5203 different transcript isoforms either representing splice variants of the same transcript or alternative or aberrant splicing products resulting in split or truncated exons or di- or poly-cistronic RNAs. In addition, it includes a poly(A)-tail length plasticity between different RNA classes with a median length of 96 nt for mRNAs, 117 nt for lncRNAs, and only 29 nt for mitochondrial mRNAs. Together, the data demonstrate that DRS can provide a more comprehensive and bias-free understanding of the T. brucei transcriptome. Provided that the current basecall accuracy and genome-coverage limitations can be overcome [T. brucei, which should accelerate our understanding of the RNA biology of the parasite.In this work, we generated a set of genuine RNA sequencing data of the