Trans-polydatin (tPD), the 3-β-D-glucoside of the well-known nutraceutical trans-resveratrol, is a natural polyphenol with documented anti-cancer, anti-inflammatory, cardioprotective, and immunoregulatory effects. Considering the anticancer activity of tPD, in this work, we aimed to explore the binding properties of this natural compound with the G-quadruplex (G4) structure formed by the Pu22 [d(TGAGGGTGGGTAGGGTGGGTAA)] DNA sequence by exploiting CD spectroscopy and molecular docking simulations. Pu22 is a mutated and shorter analog of the G4-forming sequence known as Pu27 located in the promoter of the c-myc oncogene, whose overexpression triggers the metabolic changes responsible for cancer cells transformation. The binding of tPD with the parallel Pu22 G4 was confirmed by CD spectroscopy, which showed significant changes in the CD spectrum of the DNA and a slight thermal stabilization of the G4 structure. To gain a deeper insight into the structural features of the tPD-Pu22 complex, we performed an in silico molecular docking study, which indicated that the interaction of tPD with Pu22 G4 may involve partial end-stacking to the terminal G-quartet and H-bonding interactions between the sugar moiety of the ligand and deoxynucleotides not included in the G-tetrads. Finally, we compared the experimental CD profiles of Pu22 G4 with the corresponding theoretical output obtained using DichroCalc, a web-based server normally used for the prediction of proteins’ CD spectra starting from their “.pdb” file. The results indicated a good agreement between the predicted and the experimental CD spectra in terms of the spectral bands’ profile even if with a slight bathochromic shift in the positive band, suggesting the utility of this predictive tool for G4 DNA CD investigations. Among the noncanonical secondary structures of DNA, G-quadruplex G4) is an appealing therapeutic target being found in specific regions of the genome such as telomeres and the regulatory regions of many oncogenes including c-kit, c-myc, and bcl-2  is an ap,12,13,14Trans-polydatin tPD, a, the 3-Circular dichroism (CD) spectra of Pu22, either unliganded or in complex with the tPD, were recorded at variable temperatures in a buffer containing a lower concentration of potassium ions than previously reported . CD spec2, and tPD-(Pu22)2 molecular systems.The effective binding of polydatin to Pu22 had been unequivocally shown by some of us by using various techniques including fluorescence ; howeverWith the aim to shed light on the possible mechanisms underlying the previously reported anticancer activity of tPD , we evalm = 64 °C) with respect to the unliganded Pu22 G4 reference (Tm = 62 °C), leading to a ΔTm of +2 °C  and 90 °C (unfolded), for Pu22 alone or in complex with tPD, was 1.99 and 2.23, respectively, again confirming a higher structuration degree of the quadruplex when bound by the ligand. Specifically, the highest difference in the ΔCD for the two systems was evidenced between 40 and 50 °C  presented dimeric forms corresponding to slower migrating bands, more evident in the former case and somewhat faint, but still detectable, in the case of Pu22 , purchased by Eurofins  in lyophilized and desalted form, was dissolved in nuclease-free bidistilled water and quantified by UV measurements of the absorbance at 260 nm at 95 °C using as extinction molar coefficient of 228,700 M2HPO4, and 1.8 mM KH2PO4, pH 7.4; Sigma Aldrich, Milan, Italy), using 2.5 µM DNA (Pu22), diluted from the stock solution in water, and 125 µM tPD (50 equiv. respect to the DNA), diluted from the stock solution in DMSO.Circular dichroism (CD) spectra were registered with procedures similar to previous literature reports  on a Jas265nm vs. T in the 40–90 °C temperature range) are representative of three independent experiments. Melting temperature (Tm) values were determined as the temperatures relative to the minima of the 1st derivative plots of the denaturation curves. All experiments were repeated at least three times and all spectra were recorded in triplicate.All G4-containing solutions were annealed by heating them at 95 °C for 5 min and then letting them slowly cool down to room temperature (over 16 h). The presented melting curves . More details on the HDOCK docking server and on the procedures for docking experiments can be found at http://hdock.phys.hust.edu.cn/ (accessed on 9 November 2021). We analyzed the top-ranked pose (Top-1) and the top-three ranked poses for the complexes predicted by HDOCK according to the energy scores provided by the program as explained in the Results section. Ligand/DNA complexes were visualized by Discovery Studio 2021 software  [We used the 3D structure of the Pu22 DNA with the PDB (Protein Data Bank) ID: 6AU4 . The 3D CA, USA)  that wasThe prediction of the CD spectrum of the monomeric Pu22 G4 structure was performed using the DichroCalc  web servHere, we described a combined approach including in silico (molecular docking) and experimental  analyses, through which we verified that tPD can interact with Pu22, a G4-forming sequence related to the promoter region of the c-myc oncogene, stabilizing this DNA structure. The tPD anticancer activity previously observed in vitro correlates with its stabilizing effects on this cancer-related target. The interaction of tPD with the parallel quadruplex has been proven by CD, showing changes in the CD spectrum of this DNA secondary structure under our experimental conditions, especially in the characteristic positive band centered at 263 nm. Moreover, slight thermal stabilization effects on the G4 by tPD have been revealed by CD melting studies. The binding with the DNA structure has been described in more detail in silico by molecular docking, which suggests that the interaction of tPD with Pu22 G4 may take place through partial end-stacking to the terminal quartet involving deoxynucleotides placed in the external regions of the G4 and the sugar moiety of the ligand. Finally, the exploitation of the DichroCalc web-based server, normally used for the prediction of CD spectra of proteins, for the computation of CD spectra of Pu22 revealed the feasibility of the method for the predictions of CD spectra of G4 DNA.


GBCdb will help to elucidate the potential biological roles of different RNAs and allow for the exploration of RNA interactions in GBC. These resources will provide an opportunity for unraveling the potential molecular features of Gallbladder carcinoma.Gallbladder carcinoma (GBC), an aggressive malignant tumor of the biliary system, is characterized by high cellular heterogeneity and poor prognosis. Fewer data have been reported in GBC than other common cancer types. Multi-omics data will contribute to the understanding of the molecular mechanisms of cancer, cancer diagnosis and prognosis. Herein, to provide better understanding of the molecular events in GBC pathogenesis, we developed GBCdb (The online version contains supplementary material available at 10.1186/s12859-023-05133-2. Gallbladder carcinoma (GBC), the most common cancer of the biliary tract , 2, has Gene mutations and aberrant signaling pathways play key roles in GBC tumorigenesis. Mutations in TP53, ERBB2/ERBB3 and KRAS genes are frequently detected in GBC and are associated with clinical outcomes and treatment efficacy . HER2 gehttp://tmliang.cn/gbc) to exhibit the GBC-associated RNA expression landscape and potential RNA interactions , along with experimentally verified GBC-associated genes obtained from the literatures  version 6.8 [For the obtained RNA dataset, differentially expressed RNA profiles were estimated with limma package . To redusion 6.8  and clussion 6.8  to undersion 6.8 .http://www.seer.cancer.gov) SEER*Stat database released in May 2022: version 8.4.1. The log-rank test was used to calculate the differences among the difference grades. p < 0.05 indicated statistical significance.To evaluate whether cancer grades had potential prognostic values in GBC patients, survival analysis was performed using the Surveillance, Epidemiology, and End Results (SEER) dataset using survival R package. All cases were obtained from the SEER Program (From the potential interactions among different RNAs, an RNA interaction network was constructed using Cytoscape 3.8.2 . The colUsing the collected data and primary analysis, we developed GBCdb to query and browse GBC-associated RNA expression profiles, RNA interactions and experimentally supported multi-omics data, TF-regulatory network, and etc.Additional file 1. GBC related datasets involved in this study.


Long noncoding RNAs (lncRNAs) are usually 5′ capped and 3′ polyadenylated, similar to most typical mRNAs. However, recent studies revealed a type of snoRNA-related lncRNA with unique structures, leading to questions on how they are processed and how they work. Here, we identify a novel snoRNA-related lncRNA named LNC-SNO49AB containing two C/D box snoRNA sequences, SNORD49A and SNORD49B; and show that LNC-SNO49AB represents an unreported type of lncRNA with a 5′-end m7G and a 3′-end snoRNA structure. LNC-SNO49AB was found highly expressed in leukemia patient samples, and silencing LNC-SNO49AB dramatically suppressed leukemia progression in vitro and in vivo. Subcellular location indicated that the LNC-SNO49AB is mainly located in nucleolus and interacted with the nucleolar protein fibrillarin. However, we found that LNC-SNO49AB does not play a role in 2′-O-methylation regulation, a classical function of snoRNA; instead, its snoRNA structure affected the lncRNA stability. We further demonstrated that LNC-SNO49AB could directly bind to the adenosine deaminase acting on RNA 1(ADAR1) and promoted its homodimerization followed by a high RNA A-to-I editing activity. Transcriptome profiling shows that LNC-SNO49AB and ADAR1 knockdown respectively share very similar patterns of RNA modification change in downstream signaling pathways, especially in cell cycle pathways. These findings suggest a previously unknown class of snoRNA-related lncRNAs, which function via a manner in nucleolus independently on snoRNA-guide rRNA modification. This is the first report that a lncRNA regulates genome-wide RNA A-to-I editing by enhancing ADAR1 dimerization to facilitate hematopoietic malignancy, suggesting that LNC-SNO49AB may be a novel target in therapy directed to leukemia. Remarkably, recent studies have reported that the unusual biogenic processes of small nucleolar RNAs (snoRNAs) generate a group of lncRNAs, named snoRNA-related lncRNAs6, adding a novel layer to lncRNA biogenesis. SnoRNAs are evolutionarily conserved ncRNAs which are 60–300 nt in length and function mainly as guide RNAs during site-specific rRNA modification9. They are mostly processed from introns of protein-coding or noncoding genes called “host genes”11. Two types of snoRNA-related lncRNAs, sno-lncRNAs with a snoRNA at both ends and SPAs (5′ snoRNA capped and 3′ polyadenylated lncRNAs), have been identified13. The high abundance of these lncRNAs indicates that they are unlikely to be precursors of snoRNAs, suggesting previously unrecognized regulatory potential of snoRNA sequences.Long noncoding RNAs (lncRNAs) constitute a class of noncoding transcripts longer than 200 nucleotides with no apparent protein-coding potential. They are emerging as potential key regulators in numerous biological processes across every branch of life, exhibiting a surprising range of shapes and sizes12, with only a small fraction characterized. For example, sno-lncRNA1-55 and SPAs12, encoded in the deletion chromosome region of Prader-Willi syndrome (PWS), a neurodevelopmental genetic disorder14, sequester multiple RNA-binding proteins away from their normal functional sites to affect mRNA metabolism and contribute to PWS pathogenesis. A sno-lncRNA named SLERT was reported to control rRNA synthesis by directly binding to the DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence to alter the conformation of DDX21 in cancer15. SnoRD86-cSPA is speculated to operate as a decoy for snoRNP core proteins, sequestering them away from the nuclear16. These studies suggested the potential functional diversity of these ncRNAs. Given their important roles in cellular processes, additional snoRNA-related lncRNAs will be of great interest to be characterized and investigated for their roles in the disease context.To date, a set of putative snoRNA-related lncRNAs have been identified in human cells18. With standard chemotherapies, the event-free survival of acute lymphocytic leukemia (ALL) is ~60%–80%, and only 35%–40% of younger (aged < 60 years)19 and 5%–15% of older (aged > 60 years) patients with acute myeloid leukemia (AML) survive more than 5 years20. Thus, there is an ever-present need to better understand the genetic and molecular mechanisms of leukemia biogenesis and progression. Recent advances in understanding the noncoding RNA (ncRNA) transcriptome have highlighted the importance of various ncRNA species in leukemia23. Interestingly, functional validation studies have indicated that snoRNAs are required for leukemogenesis26, which has challenged the view that snoRNAs merely function as housekeeping genes for the posttranscriptional modification of rRNAs. Among these snoRNAs, two snoRNAs, SNORD49A and SNORD49B, attracted our attention. Although they are classical C/D box snoRNAs, both function independent of rRNA 2′-O-methylation24, suggesting alternative mechanisms by which they play oncogenic roles in leukemia. Interestingly, they were predicted to generate a snoRNA-related lncRNA6. Thus, we questioned whether SNORD49A/B can also function as snoRNA-related lncRNAs and contribute to hematopoietic malignancy.Leukemia is the most prevalent and aggressive blood cancer and can be subdivided into different subtypes according to cell maturity (acute or chronic) and cell type (lymphocytic or myeloid)In this study, we identified a novel and highly expressed snoRNA-related lncRNA, named as LNC-SNO49AB, in leukemia patient samples. Sequence and structure analyses showed that LNC-SNO49AB contains two C/D box snoRNA sequences, SNORD49A and SNORD49B, and a 5′-end m7G and a 3′-end SNORD49A, which is a new type of snoRNA-related lncRNA. We further showed that LNC-SNO49AB directly binds to adenosine deaminase acting on RNA 1 (ADAR1) and promotes its dimerization and function to elevate the global rate of RNA A-to-I editing, thereby regulating hematopoietic malignancy. Our study reveals a pivotal role for a snoRNA-related lncRNA with a previously unknown structure, LNC-SNO49AB, in ADAR-mediated RNA editing and leukemia progression.SNHG29 and the 5′ end of second exon to the 3′ end of SNORD49A of SNHG29 and is 804 nt . As shown in Fig.  nt Fig. . We also nt Fig. . These r nt Fig. . We then nt Fig.  and is t nt Fig. . Unexpec nt Fig. , not at ody Fig. , indicature Fig. .Fig. 1ChGAPDH mRNA and lncRNAs, which have poly(A) tails  assays showed that LNC-SNO49AB could be enriched by FBL, which is similar to SNORD49A and SNORD49B, while lncRNA SNHG29 transcribed by its host gene showed a lack of enrichment of FBL binding  and two boxes away from their termini (boxes D’ and C’)8 Fig. . Typical8 Fig. , which aing Fig. . Consisting Fig. . In addiing Fig. , suggesting Fig. , indicat13. However, different from the previous findings12, SNORD49A and B are at the 3′-end and in the middle of LNC-SO49AB respectively, raising the question of how these two snoRNAs participate in LNC-SNO49AB formation. We sequentially deleted the C, D, C’ and D’ boxes of SNORD49B and SNORD49A to see which one is important for the lnc-snoRNA. We found that LNC-SNO49AB formation was completely eliminated only when deleting the D box of SNORD49A, the rest boxes did not show significant effects , with 78.95% sensitivity and 84.21% specificity at the optimal likelihood ratio   or global rRNA when silencing LNC-SNO49AB , namely, RI, RII and RIII, in the middle; and a catalytic deaminase domain in the C-terminus42. We next conducted an RNA pull-down assay using a series of Flag-tagged ADAR1 with deletions based on structural features to map its functional motif, which was associated with LNC-SNO49AB  in a short stretch of dsRNA, resulting in a premature stop to translation. Importantly, this dsRNA structure was recognized as an editing substrate by ADAR1, and RNA A-to-I editing converted the stop codon (UAG) into UIG, allowing the translation of mature hRluc. Thus, the expression of hRluc was editing dependent. The reporter vector was transfected with either pcDNA3.1 or pcDNA3.1-LNC-SNO49AB into 293T cells, and the frequency of A-to-I RNA editing was measured by luciferase activity. We found that LNC-SNO49AB overexpression produced more hRluc than the control  in the RNA A-to-I editing rate when LNC-SNO49AB was silenced  is required for LNC-SNO49AB biogenesis, raising the possibility that snoRNAs embedded in introns individually might mediate snoRNA-related lncRNA generation. Interestingly, in addition to snoRNAs, microRNAs, other types of small RNAs located within introns, were reported to form lncRNAs called lnc-pri-miRNAs66. Lnc-pri-miRNAs are flanked by miRNAs at the 3′ end and thus lack poly(A)-tails66. We believe that the total number and diversity of lncRNAs with small RNA-ends will continue to climb, because of more-sensitive RNA sequencing and new computational pipelines, especially the improvement in non-polyadenylated RNA-seq.As a class of newly identified stable lncRNAs with unique structures, snoRNA-related lncRNAs have attracted much attention. While most lncRNAs are processed to be 5′ m7G capped and 3′ polyadenylated, the ends of snoRNA-related lncRNAs contain snoRNA sequences69. Recent studies indicated that oligomerization is facilitated by timely coupling translation and subunit interaction 69. Here, our results demonstrated that LNC-SNO49AB promotes ADAR1 dimerization at the posttranslational level, as shown by (1) LNC-SNO49AB interacting with ADAR1 in nucleoli; (2) LNC-SNO49AB forming a functional structure with an ADAR1 dimer; and (3) silencing of LNC-SNO49AB diminishing the interaction between the ADAR1s, inhibiting dimerization. Thus, LNC-SNO49AB acts as an RNA scaffold to facilitate the association between ADAR1s forming dimers posttranslationally. Similar to LNC-SNO49AB, a few lncRNAs have also been reported to be involved in protein complex formation70. Although co-translational assembly is an efficient method, lncRNA-mediated homodimerization or multimerization may be an important mechanism for protein activity control at the posttranslational level in response to cellular stresses and cancer progression since lncRNA expression levels vary72. This work will inspire future investigations to dissect the effects of lncRNAs on protein assembly at different developmental stages and in various diseases.Assembly into macromolecular complexes is crucial for most proteins to become functional, which requires that subunits find each other precisely and efficiently in the crowded cellular environment75. Despite increasing evidence showing that the epitranscriptome contributes to oncogenesis through the determination of RNA function and gene expression diversity74, our understanding of how it can be dynamically controlled is limited. Two antisense lncRNAs, FOXM1-AS76 and PCA377, have been reported to specifically regulate the m6A and A-to-I editing of their sense protein-coding genes by recruiting the m6A demethylases ALKBH5 and ADAR1, respectively, showing that lncRNAs can give rise to the specificity of RNA modification. However, the epitranscriptome  has often been shown to be globally dysregulated in cancer80. Therefore, it is important to determine the global effects of the epitranscriptome and its associated machinery. In this study, we identified that targeting LNC-SNO49AB decreases the global A-to-I RNA-editing rate by impairing ADAR1 dimerization, revealing a previously unrecognized strategy by which lncRNAs regulate the global epitranscriptome in cancer. Notably, very recent study demonstrated that endogenous ADAR-mediated RNA editing can persistently edit the target RNAs with oligonucleotides AIMers in non-human primate liver and has enormous potentials for specific cancer therapies in the nucleic acid-editing level81. In this study, we revealed that LNC-SNO49AB can specifically regulate the activity of ADAR1, which may provide a novel insight into the precision regulation of ADAR1-based RNA editing therapeutic strategy. It may be promising method of combining AIMers and LNC-SNO49AB overexpression to improve the efficiency in this ADAR1-based approach.Cancer is driven by alterations in genomic information. The recently coined term ‘epitranscriptome’ describes RNA modifications, including RNA editing and methylation, that induce relevant changes to the transcriptomeIn summary, we identified a novel lncRNA, LNC-SNO49AB, with a 5′ m7G and 3′ snoRNA structure and revealed the mechanism of LNC-SNO49AB dependence on its ADAR1 interaction that promotes ADAR1 dimerization and elevates RNA A-to-I editing rates to enhance leukaemogenesis. These findings broaden our understanding of lncRNA diversity and provide a potential therapeutic target for leukemia treatment.2 atmosphere. Transcription was blocked by adding 2 μg/mL actinomycin D or dimethylsulphoxide (Sigma) as a control to the cell culture medium.Human leukemia cell lines RS4;11, SUPB15, CCRF-CEM, MOLM13, THP1, MV4-11, NB4, HL60 and human embryonic kidney cell line HEK293T were obtained from American Type Culture Collection (ATCC) and grown according to standard protocols. The primary cells were culture in IMDM (HyClone) supplemented with 10% FBS. All cells were culture at 37 °C in a 5% COThe clinical leukemia samples were obtained at the time of diagnosis and with informed consent from the First Affiliated Hospital of Sun Yat-sen University. Sample collection was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. The study was conducted in accordance with the Declaration of Helsinki. The detail clinicopathological characteristics of the patients were summarized in Supplementary Table ® RT reagent Kit with Gdna Eraser . Quantitative RT-PCR was performed using the SYBR Premix ExTaq real-time PCR Kit  according to the manufacturer’s instructions. Data were normalized to GAPDH expression as a control. The relative expression level for LNC-SNO49AB and other lncRNA or mRNA was determined using the 2−ΔΔCt method. The primers are listed in Supplementary Table Total RNA was extracted from bone marrow and cell samples using an Invitrogen™ TRIZOL according to the manufacturer’s instructions. All RNA samples were stored at –80 °C before reverse transcription and quantitative RT-PCR. RNA was reverse transcribed into cDNA with the PrimeScript® RLM-RACE Kit (Invitrogen™) according to the manufacturer’s instructions. Notably, in 3′ RACE assay, RNA was reverse transcribed by TransScript® miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech) to add polyA tails. PCR products were obtained and then cloned into Peasy-T3  for further sequencing.Total RNA from RS4;11 cells was extracted using TRIzol according to the manufacturer’s guidelines. The 5′ and 3′ ends of Cdna were acquired using a FirstChoiceCell proliferation was measured using Cell Counting Kit-8 . Cells were seeded at a density of 20,000 cells per well in 100 μL of complete medium in 96-well plates. Absorbance was measured by a VICTOR™ X5 Multilabel Plate Reader  at wavelengths of 480 and 630 nm at 0, 24, 48, 72, and 96 h. For the cell cycle and apoptosis assay, cells were collected and washed once by PBS. Cell pellets were resuspended in 0.5 μL of PI/Rnase staining buffer or Annexin V/PI staining buffer , respectively, and incubated for 30 min at room temperature. Cells were immediately measured and analyzed using flow cytometer . For CFU assay, leukemia cells were plated into methylcellulose at a density of 500–1000 cells per 1.5 μL methylcellulose per 35 mm dish. Colonies (> 50 cells) were scanned after 10 days in culture.15. Briefly, 2 × 107 NB4 cells were collected and suspended in 200 μL lysis buffer  and incubated on ice for 10 min. The lysate was centrifuged at 1000× g for 5 min. The supernatant (cytoplasmic extract) was transfered to a clean tube immediately. The insoluble pellet (nuclear extract) was suspended in 200 μL 340 mM sucrose solution containing 5 mM MgCl2. To obtain nucleoplasmic and nucleolar fraction, nuclei were broken by sonication until intact nuclei cannot be detected by microscope. 200 μL 880 mM sucrose solution containing 5 mM MgCl2 was added gently to the sonicated nuclei and then centrifuged 20 min at 2000× g. The supernatant was the nucleoplasmic fraction and the pellet was nucleolar fraction. All centrifugation steps were performed at 4 °C and cell samples and extracts were always kept on ice during experiment. Fractioned RNAs from the same number of cells were used for reverse transcription and qRT-PCR.The nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s instructions. Total RNA from whole-cell lysates or the nuclear and cytoplasmic fractions were isolated using TRIzol . Nucleoli isolation was performed as described with modification+ cell populations) were tail vein injected into the mice (5 × 106 cells for LNC-SNO49AB knockdown groups and 3 × 106 cells for overexpressed ones in 150 Μl PBS per mice). Here, PCDH refers to the overexpression vector, PCDH-MSCV-MCS-EF1-Puro-copGFP. For the control, 150 μL of PBS without cells was injected. Thirty days after inoculation, xenografted mice were sacrificed for analysis. Human cell engraftment (GFP+ cell populations) in bone marrow was evaluated by flow cytometry83. The remaining mice were used to perform the survival assay.NOD-SCID mice were maintained under specific pathogen-free conditions in the Laboratory Animal Center of Sun Yat-sen University. All experiments on animals were performed according to the institutional ethical guidelines for animal experiments. RS4;11 cells stably expressing sh-NC, sh-LNC-SNO49AB, PCDH or PCDH-LNC-SNO49AB  capillary transfer. Membranes were dried and ultraviolet-crosslinked. Pre-hybridization was done at 42 °C for 1 h and hybridization was performed at 42 °C overnight.Biotin-labeled RNA probes were prepared by adding biotin-labeled UTP (Roche) in in vitro transcription using TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific). The PCR primers for RNA probe are listed in Supplementary Table 6 cells of each line was reverse transcribed into cDNAs for qPCR analysis and the copy number was quantitated from the standard curve.A serial dilution of the plasmin Blunt-LNC-SNO49AB was used in qPCR to generate a standard curve. To measure the copy number of LNC-SNO49AB in leukemia cells, total RNA extracted from 1 × 1038. Briefly, recombinant proteins were expressed in E. coli strain BL21 [Transetta (DE3) chemically competent cell ]. In brief, 5 μL Luria-Bertani (LB) culture supplemented with 100 μg/μL ampicillin was inoculated with a single colony at 37 °C. After overnight growth, the culture was diluted 100-fold into 300 mL LB supplemented with 100 μg/mL ampicillin. Protein expression was induced in the presence of 0.4 mM IPTG at 16 °C overnight. Then the cell pellets were collected by centrifugation at 5000 rpm, 4 °C for 10 min and purified recombinant proteins using a His or Flag tag Protein Purification Kit (BeaverBeads™) according to the manufacturer’s instruction.Protein recombination and purification were performed as described previously6 293T cells were transfected into 5 μg indicated plasmid and collected after 48 h. All proteins for RIP were lysed with cell lysis buffer supplemented with Thermo Scientific™ Halt™ Protease Inhibitor Cocktail . To prepare antibody-coated beads, 50 μL Protein A/G magnetic beads were incubated with 3 μg antibody or control IgG in 500 μL wash buffer at 4 °C for 1 h. Then the beads were washed three times and mixed with the cell lysates in new tubes. The tubes were rotated at 4 °C overnight. Finally, RNA extraction from the beads was further collected by using Trizol according to the manufacturer’s instructions. Reverse transcription and qPCR were performed as previously described.In the RIP experiment, anti-FLAG, anti-HA, or anti-IgG antibodies were used along with an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17–701)  according to the manufacturer’s instructions. In HA-tagged Flag-ADAR1 fragments RIP assays, 1 × 10The LNC-SNO49AB sequence was cloned into the BLUNT plasmid with the tRSA tag at its 5′ end. RNA products were transcribed in vitro using the TranscriptAid T7 High Yield Transcription Kit  and were then purified using the GeneJET RNA Purification Kit . The RNA pull-down assay was performed using 50 pmol RNA for each sample with the manufacturer’s instructions of Pierce Magnetic RNA-Protein Pull-down Kit . Briefly, folded tRSA and tRSA-labeled LNC-SNO49AB were mixed with RS4;11 cell extract  in 400 µL RIP buffer and incubated at RT (room temperature) for one hour. Next, 50 µL of washed streptavidin magnetic beads was added to each reaction and further incubated at RT for another hour. Beads were washed briefly with wash buffer six times and then boiled in SDS loading buffer. Finally, the enriched proteins were resolved via SDS-PAGE and silver stained followed by mass spectrometry (MS) identification  and western blot.The 50 pmol tRSA-RNA products were first mixed with 1–3 μg recombinant proteins, and then the mixture was rotated at 4 °C for 1 h in RNA binding buffer according to Pierce Magnetic RNA-Protein Pull-down Kit. Beads were washed three times with RNA wash buffer and then boiled in SDS loading buffer. Finally, the enriched proteins were resolved via SDS-PAGE and analyzed western blotting.Total protein was extracted from cells using RIPA lysis buffer  with 1× complete ULTRA . Proteins were resolved by 10% or 12% BisTrispolyacrylamide gels and then transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% BSA for 1 h, and followed by the appropriate antibody overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Membranes were visualized with an enhanced chemoluminescence detection system.6 cells/sample) were obtained from RS4;11 cells. The next-generation sequencing in this study was performed by poly-A RNA-seq on an illumina noveSeq instrument. The sequencing depth of the RNA-Seq is up to 6 G in 150-base paired-end mode. For the data analysis, pair-end reads were adapter and quality trimmed using Trim Galore (v0.6.4), and high-quality reads were aligned to reference transcriptome (Gencode v34) and quantified using Salmon (v1.3.0) with fragment GC bias and positional bias corrections84. DESeq2 was used for normalizing gene expression level and identifying differentially expressed genes85. Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) were used to quantify the gene expression. False discovery rate (FDR) was used to compare the si-LNC-SNO49AB, si-ADAR1 and si-NC. A-to-I editing analysis was performed as described previously86. Briefly, we collected all mismatches between the aligned reads and the reference genome, discarding mismatches in read positions with quality Phred score < 30 and those located at sites reported as genomic SNPs in dbSNP (SNP build 135)87.The si-NC, si-LNC-SNO49AN, and si-ADAR1 samples , or 0.004 mM final dNTP (low dNTP sample), 1 U/μL AMV Reverse Transcriptase (NEB) and random primers. Template RNA was degraded by the addition of 1 μL RNase H (BioLegend) and 1 μL of RNase A (NEB). Following purification using VAHTSTM DNA Beads (Vazyme), cDNAs were converted into a dedicated library by VAHTS ssDNA library prep kit for Illumina (Vazyme). This approach allowed us to keep the strand-specificity of the library, so that each read started 1 nt downstream of the RT stopping point. Libraries were subjected to sequencing on the Illumina™ NextSeq 500 Sequencer. Then, we aligned the sequence data to GRCh38.p12 genome by bowtie2 v2.4.5 (default parameter), and normalized each same treatment sample by mapped reads count. Then, we made 5 bp windows by bedtools v2.30.0 and summed the total counts in the window. Finally, we calculate 2-OMe ratio following the previous method88.2-OMe-seq was carried out as previously described with some modificationsTo validate whether the identified editing sites are bona fide we performed regular PCR to amplify a selection of sites. We used SYBR Premix ExTaq real-time PCR Kit  for the PCR reactions. All Sanger sequencing was carried out by RuiBiotech .∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.Mann-Whitney test was used to analyze the LNC-SNO49AB level between patients with or without leukemia. Kruskal-Wallis test was performed to compare multiple patient groups, and the Dunn’s multiple comparisons test was used to analyze multiple comparisons. Fisher’s exact test was used to determine the significance of differentially expressed lncRNA and mRNA levels between two groups. Data are expressed as the means ± SEM or ±SD of three independent experiments. Kaplan–Meier method with a log-rank test was used to analyze the mice survival. Two-tailed tests were used for univariate comparisons. Throughout the text: ns, not significant; Supplemental Fig S1Supplemental Fig S2Supplemental Fig S3Supplemental Fig S4Supplemental Fig S5Supplemental Fig S6Supplemental Fig S7Supplemental Tab S1Supplemental Tab S2Supplemental Tab S3Supplemental Tab S4Supplemental Tab S5Supplemental Tab S6


The survival rate of oral squamous cell carcinoma (OSCC) is only 50% due to a high incidence of metastasis. Long noncoding RNAs (lncRNAs) play a crucial role in OSCC genesis and progression, although their potential role in the metastasis of OSCC remains unclear.  The transcriptome of 5 metastatic and 5 nonmetastatic OSCC samples were assessed by RNA sequencing. The biological functions and regulatory mechanisms of LEMD1-AS1 in OSCC were explored by in vitro and in vivo assays.  We identified 487 differentially expressed mRNAs (DEmRNAs) and 1507 differentially expressed lncRNAs (DElncRNAs) in OSCC with cervical lymph node (LN) metastasis relative to the nonmetastatic samples. In addition, both LEMD1-AS1 and its cognate LEMD1 were up-regulated in metastatic OSCC compared to nonmetastatic OSCC. Gain-of-function, loss-of-function, and rescue experiments indicated that LEMD1-AS1 upregulated LEMD1 to increase OSCC migration and invasion in vitro and in vivo. Mechanistically, LEMD1-AS1 stabilized LEMD1 and increased its mRNA and protein levels, and consequently activated the PI3K-AKT signaling pathway to facilitate OSCC metastasis.  We established the lncRNA-mRNA landscape of metastatic OSCC, which indicated that LEMD1-AS1 enhanced OSCC metastasis by stabilizing its antisense transcript LEMD1. Thus, LEMD1-AS1 is a potential biomarker for predicting metastasis, as well as a therapeutic target of OSCC. Oral squamous cell carcinoma (OSCC) is one of the most commonly diagnosed malignancies worldwide –3 and iscis- or trans-mechanisms [cis-acting antisense lncRNAs bind to genes in their vicinity, while the trans-lncRNAs modulate more distant genes on the same or even on different chromosomes. Furthermore, cis-antisense lncRNAs modulate gene expression at the pretranscriptional, transcriptional, and posttranscriptional levels through DNA–lncRNA, lncRNA–RNA, or protein–lncRNA interactions. lncRNA-RNA interactions in particular are common during cancer initiation and progression and involve hybridization of the sense and antisense sequences into RNA duplexes that regulate the posttranscriptional outcome. Zhao et al. reported that MACC1-AS1 promoted gastric cancer cell metabolic plasticity by stabilizing MACC1 mRNA [Long noncoding RNAs (lncRNAs) are noncoding transcripts more than 200 nucleotides in length , 7 and achanisms . The cisCC1 mRNA . In addiCC1 mRNA . Yuan etTo this end, we performed next-generation sequencing analysis of human OSCC tissues in order to map the differentially expressed RNAs and establish a lncRNA-mRNA interaction network. Accordingly, we identified lncRNA LEMD1-AS1 and its target gene LEMD1, which acts as an oncogene in multiple cancers, and analyzed their biological role in OSCC via functional assays. Our findings provide new insights into OSCC metastasis and identify a novel biomarker for prognostic prediction and targeted therapy.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE145272). A total of 487 differentially expressed mRNAs (DEmRNAs) (319 upregulated and 168 downregulated) and 1507 differentially expressed lncRNAs (DElncRNAs) (971 upregulated and 536 downregulated) were identified in RNA-seq data using |log2 fold change (FC)| > 1.0 and P value < 0.05 as the thresholds  from the top 50 DERNAs identified in 10 samples by qPCR. LEMD1-AS1, LEMD1, TBILA, and LINC01133 were significantly upregulated in metastatic versus nonmetastatic OSCC while PURPL was downregulated in the former .Since both LEMD1-AS1 and LEMD1 were upregulated in the metastatic OSCC samples relative to the nonmetastatic samples, we hypothesized that LEMD1-AS1 promotes OSCC metastasis by upregulating its predicted target LEMD1. To confirm our hypothesis, we detected the expression levels of both in additional OSCC samples by qRT-PCR. Consistent with the bioinformatics results, the metastatic tumors expressed higher levels of both LEMD1-AS1 and LEMD1, which showed a significant positive correlation . In addiin vitro  or normal control (OSC3-NC) OSCC cells were injected into the mice FOM, and while 37.5% of the OSC3-OE mice had cervical LN metastasis, the OSC3-NC group did not show any metastasis | > 1.0 and P value< 0.05 were set as the cutoff criteria.Total RNA was isolated from 5 patients with cervical LN metastasis and 5 patients without cervical LN metastasis using the TRIzol reagent  according to the manufacturer's instructions. The quality of RNA was evaluated by Qubit, Nanodrop, and Agilent 2100 Bioanalyzer. RNA sequencing libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit according to manufacturer's specifications. The rRNAs were then removed, and the remaining transcripts were purified and fragmented. First-strand cDNA synthesis was performed using random primers, followed by second-strand cDNA synthesis and end repair. The 3′ ends of the cDNAs were adenlylated and ligated to Illumina Truseq adaptors for PCR. The cDNA libraries were sequenced by Illumina Hiseq 2500. All bioinformatical analysis were performed by R and LncTar software. 2. BALB/c-nude mice (5-week-old) were obtained from the Experiment Animal Center of Central South University. All animal experiments were approved by the Institutional Animal Care Committee of Central South University (No.2019sydw0116).OSCC cell lines including UM1, UM2, OSC3, OSC19, and CAL27 were cultured in high-glucose DMEM  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 mg/ml streptomycin  at 37% in a humidified incubator containing 5% COΔΔCT– method normalized to that of GAPDH or 18sRNA. Primer sequences are listed in Total RNA was isolated using TRIzol reagent  as described above, and reverse transcribed to cDNA by HiScript III RT SuperMix . Real-time PCR was performed on QuaintStudio 7 Flex System  using the SYBR All-in-One qPCR mix , and relative gene expression was calculated using 2μl hybridization buffer at 37°C. The slides were washed with a gradient of hybridization wash buffer  at 42°C for 5 min, respectively, and air-dried. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI), the slides were imaged under a fluorescence microscope.RNA fluorescence in situ hybridization (RNA-FISH) was performed using a FISH kit  according to the manufacturer's instruction. Briefly, the suitably treated cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS, and subsequently blocked through prehybridization at 37°C for 30 minutes. The cells were then incubated overnight with 50 nM FISH probe (Ribobio Co.) in 100 The Ribo™ Smart Silencer targeting LEMD1-AS1 was obtained from RiboBio  and included three siRNA and three antisense oligonucleotides targeting different sequences. The siRNAs for human LEMD1 were designed and synthesized by RiboBio (China). Cells were transfected with the respective siRNAs using Lipofectamine3000 Reagent . In addition, OSCC cells were transduced with LEMD1-AS1 expressing lentivirus  with specific MOI (multiplicity of infection). The siRNA sequences are listed in Cell growth was monitored using the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium reagent  assay according to manufacturer's instruction. The transient and stable transfectants were seeded in 96-well plates at the density of 3000 cells/well with 5 replicates per sample. CCK-8 reagent was added to each well after 1, 2, and 3 days of culture, and the absorbance of each well was measured at 450 nm.μl pipette tip, and the dislodged cells were washed. The wounded regions were photographed at 0, 12, and 24 hours after scratching, and the scratch area was calculated by ImageJ software.Suitably treated cells were seeded in six-well plates and grown till confluency. The monolayer was scratched across the plate using a sterile 10 4 cells/well in 200 μl serum-free high-glucose DMEM. The lower chambers were filled with 400 μl DMEM supplemented with 20% FBS. After a 24 h culture, the migrated cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 30 minutes, and counted.Cells harvested 36~48 hours after transient or stable transfection were seeded into the upper chambers of a transwell insert , NY, USA) at the density of 8 × 10Protein was extracted by lysing the cells in RIPA buffer  supplemented with protease inhibitors and phosphate inhibitors, separated by SDS-PAGE and transferred onto a PVDF membrane. After blocking in 5% skimmed milk for 1 hour, the gels were incubated overnight with the primary antibodies , followed by the HRP-conjugated IgG secondary antibody. The bands were visualized using ECL Substrates . Tubulin was used as the loading control.RNA duplex formation between LEMD1-AS1 and LEMD1 was assessed with the RNase A+T cocktail  that can only digest single-stranded and not duplex RNAs. Briefly, the samples were incubated with the enzyme cocktail at 37°C for 30 minutes, and the remaining double-stranded RNA was extracted using RNeasy Kit  and analyzed by qRT-PCR.μM of the RNA synthesis blocker α-amanitin. The cells were harvested 0, 6, 12, 18, and 24 hours posttreatment and analyzed by qRT-PCR. The 18s RNA was used as the internal control since it is stable after α-amanitin treatment.OSC3 cells stably expressing LEMD1-AS1 or the empty vector were seeded into 6-well plates and treated with 50 5 OSC3 cells (n = 8 per group) in 100 μl DMEM into the floor of mouth (FOM) via submental to the space between the FOM muscles (around 5 mm). The mice were sacrificed on day 30 after implantation or when their weight was reduced to 16 grams (g) or less. The tongue and cervical LNs were collected and fixed in 10% paraformaldehyde immediately for HE staining. The volume of the orthotopic tumor was calculated as length × width2/2. All tissue staining results were examined by two expert pathologists.An orthotopic oral tumor model was established in mice by injecting control or LEMD1-AS1-overexpressing 2 × 10t-test and One-way ANOVA, respectively. Pearson's correlation coefficient was used to analyze the correlation between LEMD1-AS1 and LEMD1 expression levels. P value < 0.05 was considered statistically significant.All data were analyzed using the SPSS 22.0 statistical software package  and visualized with Graphpad Prism 7. The results were expressed as the mean ± SD of three experiments. Two or multiple groups were compared using two-tailed Student's


Renal cell carcinoma is one of the common cancers whose incidence and mortality are continuously growing worldwide. Initially, this type of tumour is usually asymptomatic. Due to the lack of reliable diagnostic markers, one-third of ccRCC patients already have distant metastases at the time of diagnosis. This underlines the importance of establishing biomarkers that would enable the prediction of the disease’s course and the risk of metastasis. LncRNA, which modulates genes at the epigenetic, transcriptional, and post-transcriptional levels, appears promising. The actions of lncRNA involve sponging and sequestering target miRNAs, thus affecting numerous biological processes. Studies have confirmed the involvement of RNAs in various diseases, including RCC. In this review, we focused on MALAT1 , RCAT1 (tumour promoter in RCC), DUXAP9 , TCL6 , LINC00342 (acting as an oncogene), AGAP2 Antisense1 (plausible predictor of RCC progression), DLEU2 , NNT-AS1 (sponge of miR-22 contributing to tumour progression), LINC00460 (favouring ccRCC development and progression) and Lnc-LSG1 (a factor that may stimulate ccRCC metastasis). Renal cell carcinoma (RCC) is the seventh most common cancer globally and incidence and mortality are constantly growing worldwide, especially in Western countries ,2. ThereGenetic studies have found that only 2% of human genomic DNA can be subjected to transcription into mRNA, while the remaining 98% can be transcribed into noncoding RNA ,20. WithRecently, lncRNAs’ function as vital regulators of numerous cellular processes has been discovered. They are believed to be involved in various physiological or pathological processes ,25. LncRTumour cells adjust their metabolic pathways to survive as well as to grow and progress in rough tumour microenvironments (TMEs). Therefore, they switch glucose metabolism from phosphorylation to aerobic glycolysis (Warburg effect) . The metThe occurrence of cancers is associated with mutations in DNA repair genes which results in genomic instability (GI) . GenomicAnother study has suggested that the lncRNA signature is closely correlated with distant metastasis in ccRCC and prognosis . This siHierarchical clustering analysis demonstrated that patients with progressive disease and nonprogressive disease have differing transcriptome levels, which allows identifying new prospective biomarkers . HoweverBelow, we mention some of the lncRNA with a potential role in the metastasis and prognosis of ccRCC.Recently, extracellular vesicles (EVs) have been attracting attention due to their potential role in tumour development and progression . They arRCAT1  has recently been recognised as an important tumour promoter in RCC . The expOwing to advancements in molecular biology and genetics techniques, targeting lncRNA seems to be a promising therapy. The use of antisense oligonucleotides and RNA interference (RNAi) therapy may in the future enable the control of tumour growth, development, and metastasis. The experiments involving the delivery of nanoparticle-mediated RNAi targeting other oncogenic lncRNA (DANCT) have brought encouraging results as they indicated effective cellular uptake, prolonged target silencing, as well as the lack of evident toxic side effects .LncRNA DUXAP9 (ENSG00000225210) is another molecule possibly related to tumorigenesis. According to studies, DUXAP9 is m6A modified and binds to IGF2BP2, which raises its stability . The intAnother study found that lncRNA DUXAP9 could directly interact with E3 ubiquitin ligase Cbl-b (Casitas B-lineage lymphoma proto-oncogene-b) which resulted in the diminished degradation of the epidermal growth factor receptor (EGFR) . EnhanceFor the first time, lncRNA TCL6 has been reported in T-cell leukaemia . It was According to suggestions, TCL6 may regulate the EGFR/AKT pathway in placental tissue . The resMany studies have demonstrated that INC00342 acts as an oncogene. They indicated the association between its level and cancer progression in different cancers. LINC00342 was found to sponge miR-19a-3p, which was associated with the modulation of NPEPL1 expression and contributed to the growth and metastasis of colorectal cancer cells . Li et aThis RNA, known also as PUNISHER ENSG00000255737, is located at 12q14.1 . Nakken The results of studies revealed that AGAP2-AS1 was coexpressed with HDGF and ANGPTL4, both of which were related to tumour angiogenesis ,130,135.DLEU2 is another long noncoding RNA whose aberrant expression was observed in various cancers . StudiesThe expression of NNT-AS1 has been demonstrated in various cancers. It can serve as a sponge of miR-22, thus contributing to tumour progression . AnotherIn ccRCC cells and tissues, the upregulation of LINC00460 has been reported . The rolcis) to trigger the transcription of nearby genes via the stimulation of chromatin looping [The expression of Lnc-LSG1 is prevalent in the cytoplasm, and to a lower extent, in the nucleus . This RN looping . In orde looping . ESRP2 w looping  demonstr looping  found th looping . Moreove looping .All the aforementioned studies indicated the plausible role of lncRNA in the progression of ccRCC and metastasis. Some of them appear to be useful as biomarkers, factors enabling the prediction of disease course (progressive vs. nonprogressive), or differentiation between ccRCC and healthy tissue. However, before the expression of selected lncRNA could be used in clinical practice, their reliability has to be confirmed in large trials. Currently, there are just a few available research articles presenting the role of lncRNA in ccRCC, and therefore, it is hard to draw solid conclusions.


The pristine material exhibitshigh melting (Tm = 230 °C) and glasstransition (Tg ≈157 °C) temperatures and offers robust mechanicalproperties between these temperatures. We show that this regime canbe substantially extended through plasticization. To achieve this,the MSP network was blended with diisodecyl phthalate. The weightfraction of this plasticizer was systematically varied, and the thermaland mechanical properties of the resulting materials were investigated.We show that the Tg can be lowered bymore than 60 °C and the toughness above the Tg is considerably increased.The assembly of ligand-functionalized (macro)monomerswith suitablemetal ions affords metallosupramolecular polymers (MSPs). On accountof the reversible and dynamic nature of the metal–ligand complexes,these materials can be temporarily (dis-)assembled upon exposure toa suitable stimulus, and this effect can be exploited to heal damagedsamples, to facilitate processing and recycling, or to enable reversibleadhesion. We here report on the plasticization of a semicrystalline,stimuli-responsive MSP network that was assembled by combining a low-molecular-weightbuilding block carrying three 2,6-bis(1′-methylbenzimidazolyl)pyridine (Mebip) ligands and zinc bis(trifluoromethylsulfonyl)imide(Zn(NTf While this frameworkaffords linear chain-extended polymers, it is also possible to createsupramolecular polymer networks from multifunctional monomers thatfeature more than two binding motifs.9 In contrast to classicpolymers or polymer networks, in which the individual monomers arecovalently linked, the supramolecular counterparts can be readilydisassembled into the constituting building blocks by applying a suitableexternal stimulus such as light or heat that sufficiently weakensthe noncovalent interactions.12 The reversibility of the assemblyrenders supramolecular polymers an attractive material class and impartsthem with ease of processing and recyclability, as well as intriguingstimuli-responsive behaviors. Particularly drastic responses to externalstimuli are observed for materials that are assembled from buildingblocks having a low molecular weight. The disassembly can be, forexample, accompanied by a substantial viscosity decrease, which translatesinto a distinct advantage when considering aspects of processing orrecycling.14 Moreover, the dynamic responseof supramolecular polymers to external stimuli can render them healable,21 enables reversible adhesion,25 and imparts shape-memory effects29 and many other types of functionalresponses.12Supramolecular, polymer-like chains ornetworks are formed when(macro)monomeric building blocks are assembled by means of directional,noncovalent interactions.35 There are many examples of supramolecular building blocks that containtelechelic linkers that are amorphous with a glass transition temperature(Tg) significantly below room temperature.37 The supramolecular self-assembly of such building blocks into polymersis then primarily governed by the association between the bindingmotifs, but such materials typically display a low stiffness and strength,similar to (thermoplastic) elastomers. When building blocks are employedthat form a glassy phase with a high Tg or semicrystalline domains with a high melting temperature (Tm), the mechanical properties are often governedby the characteristics of the polymer, which can in turn hamper theassociation of binding motifs and the chain extension.40The bulk mechanical properties and stimuli-responsivebehaviorof supramolecular polymers are significantly influenced by the natureof the molecular or macromolecular core that links the binding motifsand by phase separation effects, i.e., the formation of crystallineor glassy domains of the binding motifs, which act as additional physicalcross-links.41 or isophthalic acid–pyridine interactions.14 The assembly of such multifunctional buildingblocks furnished amorphous polymeric materials that display a veryhigh stiffness, viscoelastic flow in the melt, and complete disassemblyin solution, but they are generally very brittle.41 We recently reported a trifunctional low-molecular-weight buildingblock (TAB) comprised of a trialkylbenzene core equippedwith the well-established 2,6-bis(1′-methylbenzimidazolyl)pyridine(Mebip) ligand 2) furnishes a stiff,semicrystalline metallosupramolecular polymer (MSP) network (TAB:Zn) with high melting (Tm >200 °C) and glass transition temperatures (Tg > 140 °C). The mechanical properties of thisstiffand rather brittle MSP depend on the processing conditions, whichgreatly impact the crystallinity. The properties can also be variedover a large range via the co-assembly with a building block featuringthe same ligand but a low-Tg telecheliccore. On account of microphase separation into hard and soft domains,the resulting materials display, depending on the composition, higherstrength, toughness, or failure strain than either of the individualMSPs.Supramolecular polymers with a high stiffness at room temperaturecan also be accessed from low-molecular-weight building blocks thatfeature a high density of binding motifs, e.g., hydrogen-bonding 2-ureido-4[1H]-pyrimidinones) ligand 1.42 The TAB:Zn can also be modified by conventionalplasticization. This strategy is commonly employed for conventionalpolymers,50 but it has been rarely applied to supramolecular polymers.51 We surmised that the MSP formed from TAB and Zn(NTf2)2 is an ideal testbed to explorethe plasticization of a supramolecular polymer, since low-molecular-weightadditives can be expected to modify interchain interactions in theamorphous regions of this MSP and effectively lower the Tg of this phase without affecting the crystalline domains.53 Leibler and co-workers previously reported a hydrogen-bonded supramolecularpolymer that was plasticized with ca. 11 wt % of dodecane.51 The plasticizer lowered the Tg from 28 to 8 °C and increased the extensibilityof the material from 350% at 90 °C to above 500% at room temperature.We show here that the addition of diisodecyl phthalate (DIDP) can lower the Tg of TAB:Zn by more than 60 °C. Intriguingly, a high stiffness, strength,and extensibility are observed above Tg for the plasticized materials, which corroborates that the additionof additives is a viable pathway for tuning the bulk properties ofsupramolecular polymers. The heat-induced disassembly of the MSP networkmoreover enables its usage as a reversible debonding-on-demand adhesivewith a high stiffness and strength.Here, we report that the thermal and mechanicalproperties of theMSP network TAB, Figure S1). We adapted a previously reported procedure42 and carried out the Williamson ether synthesisto access 1,3,5-tris(undec-10-en-1-yloxy)benzene from 1,3,5-trihydroxybenzeneand 11-bromo-1-undecene in the presence of 18-crown-6. 1,3,5-Tris(undec-10-en-1-yloxy)benzenewas subsequently converted into the corresponding alcohol derivativeby hydroboration and oxidation. The reaction with 2,6-bis(1′-methylbenzimidazolyl)pyridin-4-ol under Mitsunobu conditions finally afforded TAB as an analytically pure, semicrystalline powder in an overall yieldof 36% (see the Supporting Information for details). After screeningdifferent plasticizers , which isbeneficial for the processing of the MSP at elevated temperaturesand the retention of the plasticizer in the material.The trifunctional low-molecular-weightbuilding block equippedwith Mebip ligands TAB, 1 was preTAB solutions (c =ca. 7 μmol L–1) in a chloroform/acetonitrilemixture (9:1 v/v) in the absence(DIDP relative to TAB (2)2) were added to the TAB solutionsand the changes in the UV–vis absorption spectra were monitoredat wavelengths correlated with the diagnostic bands of the metal–ligandcomplex.46 The spectra of solutions with and without DIDP show that the absorption band at 314 nm associated withthe Mebip ligand is shifted to 340 nm upon addition of Zn(NTf2)2, indicating the formation of the metal–ligandcomplexes. The corresponding plots of the absorption at 340 nm againstthe Zn2+/ligand ratio confirm the formation of 1:2 coordinationcomplexes at a stoichiometric metal-to-ligand ratio, even in the presenceof DIDP (DIDP to solutions containing a stoichiometricmixture (3:2) of Zn(NTf2)2 and TAB show only a dilution effect , and no complex formation was observed upon addition of aliquotsof a solution of Zn(NTf2)2 to a solution of DIDP . Thus, the dataconfirm unequivocally that the plasticizer does not interfere withthe metal–ligand complex formation.To probethe formation of the metal–ligand complexes inthe presence of the plasticizer, spectrophotometric titrations werecarried out with  absence2a,b and e to TAB 2c,d. Thuof DIDP 2b,d. MorTAB:Zn witha 1:2 metal-to-ligand (M/L) stoichiometry without and with the plasticizerwere prepared by solvent casting from solution. Solutions of TAB (c = 0.012 mmol mL–1) in chloroform were mixed with acetonitrile solutions containinga stoichiometric amount of Zn(NTf2)2 (c = 0.045 mmol mL–1), and the mixtureswere cast into poly(tetra fluoroethylene) molds. For plasticized samples(TAB:Zn_1–20%), chloroform solutions (2 mL) containingeither 1, 2.5, 5, 10, or 20 wt % of DIDP with respectto the MSP were added to stoichiometric mixtures of TAB and the zinc salt. Such mixtures were stirred for 30 min prior tosolvent casting. After solvent evaporation, samples were dried at50 °C for 24 h in vacuo to furnish rigid, transparent films withappreciable mechanical properties. To explore if samples can be thermallyprocessed at elevated temperatures without loss of the plasticizer,the thermal properties of TAB:Zn and TAB:Zn_1–20% were investigated by thermogravimetric analysis (TGA). The TGA traceof TAB:Zn indicates a 5% (w/w) mass loss above 350 °C, while an onset of mass lossis observed at ca. 200 °C for samples of TAB:Zn_1–20% . A comparison with the TGAtrace of neat DIDP suggests that the lower onset temperatureat which mass loss was observed for the TAB:Zn_1–20% samples corresponds to the evaporation of DIDP. Toelucidate the extent of mass lost at 220 °C, i.e., the temperatureat which the cast films were further processed (vide infra), samplesof neat DIDP, TAB:Zn, TAB:Zn_1%, and TAB:Zn_10% were subjected to isothermal measurementsat this temperature . The recordedtraces indicate that a slow weight reduction occurs for plasticizedsamples at this temperature, but the mass loss is limited to below0.5 wt % over a course of 2 min, suggesting that processing is possiblewithout a significant reduction of the plasticizer content.To explore the effect of the plasticizer on thethermal and mechanicalproperties of the MSP network, samples of TAB:Zn and TAB:Zn_1–5% were processed at this temperaturewith a pressure of 6 t for 45 s, while a pressure of 4 t and a compressionfor 30 s were sufficient to obtain uniform TAB:Zn_10–20% films. Unless noted otherwise, samples were slowly cooled betweenthe steel plates of the hot press over a course of 45 min. While thecompression-molded films of TAB:Zn and TAB:Zn_1–5% were transparent, films with 10 wt % or more of DIDP appeared turbid and their surface was greasy , indicating that processing at this temperatureleads to plasticizer sweating. Indeed, a comparison of the TGA tracesof solvent-cast and compression-molded samples corroborates the lossof plasticizer for TAB:Zn_10–20% samples , whereas the traces of TAB:Zn_1–5% recorded before and after compression molding are comparable. Thus,the maximum concentration of DIDP that can be incorporatedinto TAB:Zn appears to be limited to <10 wt %, andattempts to incorporate larger amounts lead to macrophase separation.While TAB:Zn_10–20% films were nonetheless characterized,the absolute weight fraction of DIDP in these samplesis lower than the nominal concentration quoted.To process the (plasticized) MSPs into films with a uniform thickness(ca. 170–200 μm), solvent-cast films were compression-moldedin a hot press at 220 °C . The first and second DSC heating tracesof compression-molded TAB:Zn samples display a reversiblemelting transition (Tm) at ca. 231 °C,and a corresponding crystallization transition (Tc) is observed in the cooling trace at ca. 180 °C(S5 and S1); however, no glasstransition is discernible. The DSC traces of TAB:Zn_1–5% mirror those of the neat TAB:Zn, whereas the tracesof samples containing 10 wt % or more DIDP display slightlyreduced Tm (ca. 220 °C) and Tc (ca. 176 °C) values (S5 and S1). A comparison of the meltingenthalpies shows that this value does not change significantly; i.e.,the extent of crystallinity in all samples remains the same throughoutrepeated heating and cooling experiments . Moreover, a comparison of the first DSC heatingtraces of compression-molded and solvent-cast samples shows a minorincrease in the Tm for the former, whichis most pronounced for TAB:Zn_5% samples . This observationis tentatively interpreted as an annealing effect, as previously observedfor neat TAB:Zn,42 and corroboratedby the fact that the first cooling and second heating traces of melt-processedsamples mirror those of solvent-cast samples . The DSC measurements, hence, indicatethat the semicrystalline nature of the MSP network is retained inthe presence of the plasticizer, even though the melting and crystallizationtemperatures are slightly lower if the DIDP content isabove 5 wt %. Moreover, taking the results from DSC and TGA measurementsinto account, the plasticizer appears to be largely retained in theMSP when samples are briefly heated above the onset of DIDP evaporation of ca. 200 °C, butprolonged heating at these temperatures leads to a gradual loss ofthe plasticizer.Compression-moldedand solvent-cast samples of CS5 and 1 and S1;s S5 and 1 and S1.DIDP on the morphology of the MSPs, melt-processed samplesof TAB:Zn and TAB:Zn_1–20% were subjectedto small- andwide-angle X-ray scattering (SAXS/WAXS) measurements .In agreement with the DSC measurements, Bragg reflections at largerscattering vectors (q ≈ 0.6–2.3 Å–1) corroborate the presence of some degree of crystallineorder. A comparison of the scattering profiles of TAB:Zn_1–20% samples indicates that neither the signals observed in the SAXSregime nor the peaks in the WAXS regime experience considerable changesupon plasticization (Table S2). Additional SAXS measurementsat smaller angles were carried out in order to explore if pure DIDP domains phase separate in compositions with high plasticizerconcentrations, but the scattering profiles show no indication ofa structure formation on larger length scales . These findings corroborate that the plasticizerprimarily if not exclusively resides in the amorphous domains, withoutimpacting the lamellar structuration or crystalline domains of theMSP.In order to further elucidate the influence of urements 3b. The sTAB:Zn and TAB:Zn_1–20% samples were characterized by subjectingcompression-molded films to dynamic mechanic analysis (DMA) measurements.The DMA trace of TAB:Zn displays a wide regime of highrigidity, with a storage modulus E’ of 2.53± 0.21 GPa at −80 °C and an E’of ca. 1.73 ± 0.16 GPa at room temperature ; a correspondingmaximum in the tan δ curve at around 157 °C marks the Tg of the semicrystalline MSP network . The DMA traces of TAB:Zn samples that were cooled slowly after processing show no clear glasstransition, and E’ remains high (above 200MPa) until the samples melt, reflecting a higher degree of crystallinity.The DMA measurements of plasticized samples, which were all cooledslowly after processing, show that the storage modulus at −80°C remains unchanged, irrespective of the DIDP content.However, the temperature at which the storage modulus of the materialstarts to drop is reduced, and the room-temperature modulus graduallydecreases , the latter isreduced to ca. 106 ± 3 °C for TAB:Zn_1% and90 ± 2 °C for TAB:Zn_5% . Experimental results for TAB:Zn_1–20% were, hence, compared to the expected plasticization by using the Tg of TAB:Zn (ca. 157 °C) anda previously reported value for DIDP (Tg ≈ –64 °C).54 Intriguingly, the corresponding plot of thecalculated Tg values against the weightfraction of the added plasticizer shows that the experimental valuesare significantly lower than expected for a DIDP contentof up to 5 wt %  plasticizedwith DIDP54 and the values calculated with the Fox equation. Even at the lowest plasticizerconcentrations, for which one can assume complete miscibility, weobserve a pronounced deviation from the behavior predicted based onthe Fox equation. We speculate that this is due to the semicrystallinenature of the MSP network, which leads to an increase of the effectiveconcentration of the plasticizer in the amorphous phase. Other pertinentfactors may include larger differences in the heat capacities of theplasticizer and polymer, a high plasticizer efficiency, the impactof the plasticizer on the dynamics of metal–ligand complexesin the solid state, and the fundamentally different characteristicsof supramolecular polymers.Importantly, the tan δ traces of plasticized samples showa distinct reduction of the % S9 and 1. Increa.157 °C S8, the lwith DIDP or di of ca. 1.29 ± 0.07 GPa, a tensile strength of 9 ± 1 MPa,and a strain at break of 0.8 ± 0.1% (E of TAB:Zn_5% = 0.95 ± 0.13 GPa), while no statisticallysignificant changes in the strain at break and toughness were observed at 95 and 105 °C (S12 and Table S3). Upon uniaxial tensile deformation at 95 °C, samples of TAB:Zn_5% display Young’s modulus of 0.44 ± 0.04GPa, a tensile strength of 11 ± 1 MPa, and a strain at breakof 5 ± 1%, while the neat TAB:Zn shows Young’smodulus of 0.78 ± 0.13 GPa, a tensile strength of 11 ± 1MPa, and a strain at break of 2.0 ± 0.3% at this temperature.The toughness is thus considerably increased, from 131 ± 35 kJm–3 for TAB:Zn to 366 ± 130 kJm–3 for TAB:Zn_5%. As expected, increasingthe temperature further to 105 °C for tensile testing does notlead to a significant change in the mechanical properties of the plasticizedmaterial.The mechanical properties of  S11 and 1. The st8 ± 0.1% 1. In agrobserved1. To furTAB:Zn and of samplesplasticized with 5 wt % of DIDP (TAB:Zn_5%) were explored in debonding-on-demand and re-bonding scenarios.Thus, thin films of the materials (thickness of ca. 100 μm)were placed on a stainless-steel substrate and heated to a temperatureof 230 °C . The supramolecularnature of TAB:Zn allowed for re-bonding by locally heating and re-joining the substrates. The re-bonded lapjoints display a shear strength of 3.2 ± 0.4 MPa (S14); i.e., the original bond strength was fully re-established.The measurements with TAB:Zn_5% reveal a shear strengthof 2.6 ± 0.4 MPa for freshly preparedsamples and of 2.8 ± 1.1 MPa for re-bonded lap joints (S14); i.e., no statistically significant differencewas observed in the room-temperature adhesive properties.To demonstrate the stimuli-responsive nature ofthe materials,the adhesive properties of neat 0.3 MPa 4cand th2)2 was successfully plasticized by the additionof DIDP. The plasticizer is miscible with the supramolecularpolymer in a concentration of up to ca. 5 wt %, and its incorporationreduces the Tg by more than 60 °Cfrom 157 ± 1 to 90 ± 2 °C.At ambient temperature, i.e., below the Tg, the plasticized MSP networks show a slightly lower stiffness butthe same strength and toughness as the unplasticized material. Whenplasticized samples were deformed at temperatures above the Tg, however, a significantly increased strainat break and toughness were observed. These findings show clearlythat plasticization of a metallosupramolecular polymer is a promisingstrategy to straightforwardly tune the mechanical properties of thesematerials. For the investigated platform, it would be desirable toincrease the miscibility of the two components, so that the Tg could be reduced further to below ambienttemperature, which would potentially allow one to harness the tougheningeffect under more practically relevant conditions.In summary, a stiff and brittle semicrystallineMSP based on thetrifunctional low-molecular-weight building block TAB and Zn. While mixtures of TAB:Zn and diethyl phthalate and DIDP affordedtransparent films, MSPs containing dioctyl terephthalate were turbid.Due to the higher boiling point of DIDP compared to diethylphthalate, the former was chosen for in-depth experiments. Other phthalatederivatives were not investigated due to their reported toxicity.To determine what plasticizerscould be incorporated in the here presented MSP materials, differentsmall molecules were investigated. Based on previously reported supramolecularpolymers,TAB solution (c = 6.71 μM in CHCl3/CH3CN 9:1 v/v) with aliquots of 25 μL of asolution of Zn(NTf2)2 (c =132 μM in CHCl3/CH3CN 9:1 v/v). The metal salt solutions were corrected fordilution effects by addition of an appropriate amount of the TAB solution. UV–vis spectra were also recorded duringthe titration of a solution of TAB containing 20 wt %of DIDP with respect to TAB with Zn(NTf2)2. Data were acquired upon addition of 25 μLaliquots of a solution of Zn(NTf2)2 (146 μM)and TAB (7.72 μM) in CHCl3/CH3CN (9:1 v/v) to a solution of TAB (7.72 μM) and DIDP in the same solventmixture. Additionally, UV–vis spectra were recorded duringthe addition of a solution of DIDP (2418 μM) inCHCl3 to a solution of TAB (7.20 μM)and Zn(NTf2)2 with an M/L ratio of 1:2 in CHCl3/CH3CN (9:1 v/v), and spectra were recorded during the addition of a solution ofZn(NTf2)2 (143 μM) in CH3CN to a solution of DIDP (190 μM)in CHCl3.To demonstratethe complex formation, UV–vis absorption spectra were acquiredduring titrations of a 2)2  in CH3CN was added to a solution of TAB in CHCl3 and stirred until a clear solution was obtained.The solution containing the mixture of TAB and Zn(NTf2)2 was cast into a poly(tetrafluoroethylene) (PTFE)Petri dish, the solvent was evaporated overnight in a ventilated hood,and the sample was dried in vacuo at 50 °C over a course of 24h to yield solvent-cast films of the MSP. For plasticized samples,a solution of the corresponding amount of plasticizer in CHCl3 was added to the solution containing the mixture of stoichiometricquantities of TAB and Zn(NTf2)2 and the mixture was stirred for 30 min. The solution containingall components was then cast into a PTFE Petri dish, the solvent wasallowed to evaporate under ambient conditions in a ventilated hood,and plasticized samples were further dried in vacuo for 24 h at 50°C.A solution ofZn or at 220 °C for 30 sat a pressure of 4 t (10–20 wt % of plasticizer) to obtainthin films with a thickness between 170 and 200 μm. The sampleswere typically cooled down to room temperature over a course of ca.45 min by leaving them in between the steel plates of the hot pressthat were removed from the press for cooling. Quenched samples wereprocessed at 230 °C for 45 s and a pressure of 6 t and immediatelyremoved from the hot steel plates of the hot press and placed betweencold ones for rapid cooling. Samples for adhesion tests were preparedin the same way, using a spacer with a thickness of 150 μm.2 was covered with a film of a thicknessof around 100 μm of the MSP sample without or with a plasticizer.Upon melting of the film, the second lap joint was placed on top ofthe first one with an overlap area of the same size as the MSP film.The lap joints were left on the hotplate for ca. 20 s and then fixedwith clamps and cooled down to room temperature. Samples preparedin this way were tested within 1 h afterpreparation. The same protocol was used to rebind the samples. Sheartesting was carried out at a strain rate of 1 mm min–1 on a Zwick/Roell Z010 tensile tester that was equipped with a 10kN load cell. All provided values are reported as the average of 3–4independent experiments, and all errors given are standard deviations.Adhesive properties were investigatedin shear testing of single lap joints at room temperature. Two stainless-steelsubstrates were used for the preparation of single lap joints. Onewas heated on a hotplate to a temperature of 230 °C, and an areaof ca. 10 × 10 mm


Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting SBT have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of SBT for the first time. Sequence analysis of the obtained viral genome revealed high similarity with several viruses belonging to the genus Marafivirus. The genome of the new virus is 6989 nucleotides (nt) in length according to 5′, 3′ RACE (without polyA-tail), with 5′ and 3′ 133 and 109 nt long untranslated regions, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1954 amino acids with the characteristic marafivirus non-structural protein domains—methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by 6 nt, encoding the coat protein (CP) with typical signatures of minor and major forms. Both CP forms were cloned and expressed in a bacterial expression system. Only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly.The agricultural importance of sea buckthorn (SBT;  Hippophae rhamnoides L., genus Hippophae L., family Elaeagnaceae) is an exceptionally valuable plant that is currently domesticated and cultivated in orchards, particularly in Europe, Canada, and the USA .,38.MarafDV model  85]. Tw. TwMarafnfection . Tymoviridae [136PFQW138), motif II (169YRYA174), and motif III (222GGPV225) (Tymoviridae [ORF2 was in the same reading frame (+2) as ORF1 but was separated by six nt. According to CDD search results, ORF2 encoded a product showing features of a tymovirus-like CP domain , thus possibly encoding for a CP of SBuMV. The first start codon of ORF2 (minor CP) began at 6002 nt, and the second (major CP) at 6275 nt; however, both ended at 6880 nt with a stop codon UAA. The minor CP was 292 aa long and had a predicted molecular mass of 31 kDa (p31), with proline-(29.21%) and serine-rich (15.73%) N-terminal (89 aa), which is similar to OLV-3 CP . This seoviridae . Major CGGPV225) . PFQ conoviridae . Accordioviridae .Marafivirus species also was with OLV-3 (Marafivirus [MT (61.5%), PRO (44.14%), RdRp (67.2%), and CP (66.03%) aa sequences of SBuMV shared the highest similarity to the corresponding domains of OLV-3 . Only Hth OLV-3 . These aafivirus .61LSGG64, which was also identified in other marafiviruses (929LTGA932) and between the HEL and RdRp domains (1298LSGA1301) were also present.The SBuMV RP N-terminal contains a 70 aa protein domain with a molecular mass of 7.5 kDa, which is separated from PR by a putative papain-like cysteine PRO cleavage site iviruses . AccordiCAUUUGCGUG 3′; QGHLR). Differences in this genomic feature have also been reported for other marafiviruses. For example, the PeVD and OLV-3 “marafiboxes” differed from the consensus “marafibox” by six and three nt, respectively  that is of utmost importance in the omics era .Maculavirus; GFkV), and SBuMV, a degenerate p43-like ORF interrupted by several stop codons was identified. Therefore, it is reasonable to hypothesize that the truncated MPs of these viruses are remnants of an ancestral MP that had degenerated during evolution as these viruses became restricted to phloem cells [Luteoviridae, which are confined to the phloem (vascular tissue) potentially due to their specialized phloem-specific MP. These proteins are translated from a single viral mRNA, sgRNA1, via initiation at more than a single AUG codon to express overlapping genes and by ribosomal read-through of a stop codon [Moreover, the functional annotation of the SBuMV genome was possible directly due to the fact that many other complete or partial marafivirus genomes have been previously sequenced and are publicly available in the biological sequence repositories. With the growing number of novel viruses being identified using HTS, it can be foreseen that HTS will soon become a conventional method for viral phytopathology and will be applied in diverse studies, complementing classical virological approaches that are slowly becoming obsolete without being complemented by relevant sequence data. A comparison of the marafiviruses uncovered thus far shows that despite having some variation within their genome organization, most of their features are similar. The diversity in the genome organization among the marafiviruses could be due to evolution as a result of adaptation to new host plants; however, sequencing, annotation, and validation errors might also play a role in exaggerating the reality ,92. Addiem cells . The N-top codon . To maxiop codon . The absop codon ,97. Initop codon . In seveop codon ,100,101.CP stop codon of Luteoviridae family was used for minor CP variant translation. The readthrough domain in the N-terminal region, which is conserved, is required for aphid transmission; however, the C-terminus, which is variable, appears to enhance aphid transmission efficiency and function in the phloem retention of the virus, thus influencing systemic infection, virus accumulation, and symptom development [CP genes were transiently expressed in Nicotiana benthamiana [CP mRNA and/or host RNA [Readthrough of the elopment . Probablelopment . In addielopment  suggesteelopment , and thielopment . On the elopment ,103. MRFthamiana ,104, shohost RNA ,103. N. benthamiana [Tymovirus) PRO as the closest structural homolog, followed by ovarian-tumor-domain-containing protein 3 (OTUD3) from Homo sapiens and OTUD1 from Saccharomyces cerevisiae. Additionally, deubiquitinating activity has been shown for BlVS, CSDaV, GsyV1, MRFV, OBDV, and OLV3 [Potyviridae family, use PRO cleavage that resembles a consensus sequence (Y/F/G)xG [The major CPs of marafiviruses are translated from sgRNA by a leaky scanning mechanism . The indthamiana ,103. Thethamiana . Translathamiana . Sequencthamiana . Compariand OLV3 . On the and OLV3 ,108. Theand OLV3 ,110. Theand OLV3 . PRO cleand OLV3 . Other pFor many ssRNA(+) viruses, viral gRNAs are capped, allowing for efficient translation in eukaryotic cells. Cellular mRNA capping enzymes of the host are located in the nucleus; thus, many viruses that replicate in the cytoplasm encode their own capping enzymes . MultiplAll positive-sense RNA viruses with a genome size of over 6 kb encode a putative RNA HEL, which is thought to be involved in the unwinding of a duplex during the viral RNA replication and, perhaps, translation as well . Seven ccis-acting RNA elements (REs) that have been shown to impact many essential viral processes, including protein translation, genome replication, and transcription of sgRNAs. For many viruses, REs are usually located in the 5′ and 3′ UTRs and/or internally within inter-cistronic regions. However, one drawback of this approach is that the size and/or location of the non-coding regions can have some limitations. To avoid such limitations, many plus-strand RNA viruses have evolved to position their REs within their coding regions [These RNA genomes perform the traditional role of being the blueprint for all viral proteins; however, they also contain  regions .One potential drawback of having a RE in a coding region is that the same RNA sequence region physically couples two or more distinct activities; therefore, one or both of the corresponding sequence functions may be compromised compared with a hypothetical free-standing localization. Additionally, in some cases, the relative location of REs within the genome of a given virus may not be optimal for their activity; therefore, compensatory measures may be required. One strategy used by many RNA viruses to deal with sub-optimally positioned REs is to eventually reorganize their relative location within the genome via intramolecular long-range RNA–RNA interactions to mediate translational initiation and viral RNA genome replication and form functional secondary RNA structures .Hordeivirus, family: Virgaviridae) has a poly-A region followed by a 3′ terminal sequence capable of folding into a tRNA-like structure that can be aminoacylated [The SBuMV genome contains several putative structural elements in both the 5′ and 3′ UTRs as well as in the coding regions. RNA structure predictions suggested that the 3′ UTR could form a tRNA-like structure similar to that of other viruses. For example, the barley stripe mosaic virus  translation by a previously undetected -1 PRF site in an internal region of the polyprotein was discovered, despite EMCV serving as a model virus for more than 50 years [trans-activated by viral protein 2A. Consequently, the frameshifting efficiency increases from 0 to 70%  over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins [Luteoviridae family) have three sgRNAs [The internal putative structural element—an additional “marafibox” motif at the beginning of a HEL domain in SBuMV—could be an extra signal sequence. A consensus sequence (GnGAnCGnnUCCUCUC) B was ide50 years . PRF is proteins . Howeverproteins . The secproteins . Severale sgRNAs .Pseudoknots, secondary structures resulting from an interaction of stems and loops, represent a structurally diverse group of functional regulatory elements. They play several diverse biological roles, such as forming a catalytic core of various ribozymes, self-splicing introns, and telomerase. Moreover, they play a critical role in altering gene expression by inducing ribosomal frameshifting in many viruses .To identify the symptoms of a disease potentially caused by SBuMV, SBT orchids have to be independently tested, and the results should be carefully analyzed considering the possible presence of other potential causative agents so that all of Koch’s postulates are fulfilled. Analysis of the SBuMV genome and protein sequences has raised more questions than it has answered. For example, is the 5′ end capped? Does proteolysis occur at the identified cleavage sites? Which is the vector for SBuMV? All these and many more questions would be answered in the near future, thus paving the way for exciting discoveries and novel knowledge on phytoviruses.In conclusion, here, we, for the first time, present a virus putatively infecting SBT. We characterized its full genome, and cloned and expressed minor and major forms of CP either individually or co-expressed. We also showed that major CP-derived virus-like particles self-assembled directly in the bacterial cells, and the majority of the expressed CPs were soluble. Our study suggests that the minor CP is not essential for the assembly of seemingly structurally intact viral particles, meaning that it can have other functions.