IGH/IGK clonality testing and analyzed the repertoire of immunoglobulin heavy chain (IGH) or immunoglobulin kappa light chain (IGK) gene usage in Korean patients with multiple myeloma (MM) for the first time.We assessed the applicability of next-generation sequencing (NGS)-based IGH and IGK genes was performed using IGH FR1 and IGK primer sets.Fifty-nine bone marrow samples from 57 Korean patients with MM were analyzed, and NGS-based clonality testing that targeted the IGH and IGK rearrangements were observed in 74.2% and 67.7% of samples from Korean patients with kappa-restricted MM, respectively (90.3% had one or both), and in 60.7% and 95.5% of samples from those with lambda-restricted MM, respectively (85.7% had one or both). In total, 88.1% of samples from Koreans with MM had clonal IGH and/or IGK rearrangement. Clonal rearrangement was not significantly associated with the bone marrow plasma cells as a proportion of all BM lymphoid cells. IGHV3-9 (11.63%) and IGHV4-31 (9.30%) were the most frequently reported IGHV genes and were more common in Koreans with MM than in Western counterparts. IGHD3-10 and IGHD3-3 (13.95% each) were the most frequent IGHD genes; IGHD3-3 was more common in Koreans with MM. No IGK rearrangement was particularly prevalent, but single IGKV-J rearrangements were less common in Koreans with kappa-restricted MM than in Western counterparts. IGKV4-1 was less frequent in Koreans regardless of light chain type. Otherwise, the usages of the IGH V, D, and J genes and of the IGK gene were like those observed in previous Western studies.Clonal IGH/IGK clonality testing ought to be applicable to most Koreans with MM. The overrepresentation of IGHV3-9, IGHV4-31, and IGHD3-3 along with the underrepresentation of IGKV4-1 and the differences in IGK gene rearrangement types suggest the existence of ethnicity-specific variations in this disease.NGS-based  IG) gene rearrangement begins at the earliest stages of B-cell development and involves recombination events between the numerous V, (D), and J genes, which result in the formation of unique V(D)J sequences that ultimately encode the antigen-binding region of the IG chain ) was 99.12% (7.25–100%). The detection rates of clonal IGH/IGK rearrangements and IGHV SHM according to light chain restriction pattern are summarized in The total read counts (median [range]) for IGH and/or IGK rearrangements, which demonstrated an applicability rate of 88.1% when using the IGH FR1 assay and/or IGK assay in Korean patients with MM. Forty of fifty-nine had clonal IGH rearrangements (67.8%) and forty-two of fifty-three had clonal IGK rearrangements (79.2%); adding the IGK assay increased the positivity rate of clonal IG gene rearrangement rate from 67.8% to 88.1%. Clonal IGH rearrangements and clonal IGK rearrangements were observed in 74.2% and 67.7% of samples from patients with kappa-restricted MM, respectively, and in 60.7% and 95.5% of samples from patients with lambda-restricted MM, respectively. Overall, 90.3% and 85.7% of samples from patients with kappa-restricted MM and lambda-restricted MM, respectively, had clonal IGH and/or IGK rearrangements. Including the IGK assay increased the positivity rate of clonal IG gene rearrangement from 74.2 to 90.3% and from 60.7% to 85.7% in kappa-restricted MM and lambda-restricted MM, respectively.Overall, 52 of 59 samples had clonal IGH rearrangements, IGHV SHM was observed in 85.0%, including in 91.3% of samples from patients with kappa-restricted MM and 76.5% of samples from those with lambda-restricted MM.Among all 40 samples exhibiting clonal IGH rearrangement, respectively (P = 0.227). When IGH and IGK rearrangements were analyzed together, the medians (ranges) of BM plasma cells as a proportion of all BM lymphoid cells were 76.4% (28.5–100.0%) and 64.5% (32.6–92.5%) in subjects with clonal IGH and/or IGK rearrangement and in those with no rearrangement, respectively (P = 0.138). No or very weak correlation between the quantity of IGH/IGK rearrangements and the proportion of BM plasma cells as a proportion of all BM lymphoid cells was observed: y = 0.084x + 70.969  in patients with IGH rearrangements or y = 0.231x + 59.423  in patients with IGH and/or IGK rearrangements when only the larger values of the proportion of IGH or IGK rearrangements were considered.Clonal rearrangement was not associated with the BM plasma cells as a proportion of all BM lymphoid cells as estimated on the BM aspiration slides. The medians (ranges) of BM plasma cells as a proportion of all BM lymphoid cells were 76.9% (28.5–100.0%) and 67.7% (32.6–94.5%) in samples with and without clonal IGH rearrangement repertoire, we analyzed the VDJ gene usage of 43 rearrangements from 39 samples with clonal IGH rearrangement .To profile the IGHV3 was the most frequently reported IGHV group gene (67.44%) followed by IGHV4 (20.93%), IGHV1 (4.65%), IGHV2 (4.65%), and IGHV7 (2.33%). IGHV5 and IGHV6 were not observed in our sample set. Twenty IGHV genes were identified  were the most frequently reported, followed by IGHV3-30 and IGHV4-31 . Comparing IGHV usage between our study and 3 previously published Western studies : 76.4 [28.5–100.0%] and 64.5 [32.6–92.5%]). Most previous studies did not investigate the association between clonal IG gene rearrangement and the proportion of BM plasma cells. Rustad et al. suggested that low tumor cell content was the main reason for the failure to identify a clonal V(D)J sequence; while samples with more than 5% plasma cells had a 97% clonality detection rate, those with less than 5% plasma cells had a detection rate of ~70% [IG gene rearrangement tests found that clonal rearrangements could be traced to dilutions as low as 2.5% [The presence and extent of clonal rearrangement and the proportion of BM plasma cells as a proportion of all BM lymphoid cells were not significantly correlated in our study. There was a huge overlap in BM plasma cells as a proportion of all BM lymphoid cells between patients with clonal 4.5 32.6–2.5%). MoIGH and IGK gene usage in Korean patients with MM has not been reported to date; although data from few Western studies exist [IGHV3-9, IGHV4-31, and IGHD3-3 were common in Korean patients with MM but not in Western patients; no IGK rearrangement was found to be more frequent in Korean patients than in Western counterparts, particularly among those with kappa-restricted MM. Moreover, the single IGKV-J rearrangement type was more frequent in Western patients with kappa-restricted MM than in Korean counterparts; IGKV4-1 was more frequent in Western than in Korean patients regardless of light chain type. These data suggested the existence of ethnicity-based differences in disease risk and manifestation [IGH and IGK repertoire distribution in our study showed a similar pattern to those of Western studies [IGH gene usage in MM was similar to that of normal plasma cells [IGH repertoire followed a similar pattern to that of the normal repertoire [IGHV4-34 gene was rarely observed in MM, which was consistent with older IG profiling studies in patients with this disease [IGHV4-34 gene is common in B-lymphoid malignancies, particularly chronic lymphocytic leukemia [IGHV3, IGHD2, and IGHD3, as well as of the IGHJ4 gene group, compared to the normal B-cell repertoire [IGKV-J rearrangement was the most common , followed by KDE . IGKJ4 was the most predominant , and the distribution of the IGK gene usage in our study was similar to those found in previous studies except that the most frequent IGK gene observed in the only available Western study that analyzed the IGK repertoire, IGKV4-1, was not observed among our patients [The repertoire of es exist , 8, 24, estation , 8, 24.  studies , 8, 24. ma cells . Hadzidi disease –31; in cIGH/IGK rearrangement test for the detection of clonality in Asians with MM for the first time, (ii) we analyzed the profiles of IGH and IGK gene usage in Asians with MM and compared them with those from previous Western studies for the first time, and (iii) we investigated the association between clonal IGH/IGK rearrangement and the proportion of BM lymphoid cells that were plasma cells. Nevertheless, there were a few limitations, some aforementioned, as follows: (i) the number of samples was relatively small, (ii) the majority of samples were from high-risk patients with a relatively high percentage of BM plasma cells, and (iii) IGH FR2 and IGH FR3 tests were not performed.The strengths of our study include that (i) we investigated the applicability of the NGS-based IGH and/or IGK rearrangements were detected in 88.1% of Koreans with MM at baseline, indicating that such tests are applicable to most Korean patients with this disease to determine clonality and to monitor those under treatment. The profile of IGH and IGK gene usage in Koreans with MM was similar to those found in Western MM studies. However, IGHV3-9, IGHV4-31, and IGHD3-3 were overrepresented among Korean patients, whereas IGKV4-1 was underrepresented. No IGK rearrangement was found to be more common in Koreans with kappa-restricted MM, although the IGKV-J rearrangement type was more frequent in Western counterparts. This implied the existence of certain ethnicity-based differences in the characteristics of patients with MM.In summary, we showed that clonal S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


They describe an antiparallel heterotrimeric coiled-coil complex between the carboxy tail of KHC and Tm1-I/C (aTm1), the atypical tropomyosin that is important for oskar mRNA transport in Drosophila oocytes. This interaction enhances direct binding between KHC and RNA. Their findings demonstrate the structural plasticity of the KHC tail as a platform for protein–protein interactions and reveal how a cargo adaptor protein can modify a motor–RNA interface to promote transport.The prototypic and ubiquitous microtubule motor, kinesin-1, uses a variety of adaptor proteins to facilitate the selective transport of diverse cargo within the cell. These cargo adaptors bind to the motor complex through interactions with the kinesin light or heavy chains (KLCs or KHCs). In this issue of  Kinesin-1 family microtubule motor proteins play a key role in intracellular transport in most cell types. They can engage diverse cargos including membrane-bound organelles (MBOs), proteins, and RNA, as well as many viruses and slide microtubules to control the organization of the microtubule network itself . To meetaTm1), that is important for oskar mRNA localization to the posterior pole of the Drosophila oocyte. They solve X-ray crystal structures of an antiparallel homodimeric aTm1 coiled coil in addition to a heterotrimeric complex consisting of two parallel KHCs and one antiparallel aTm1 chain. The structure of the trimeric complex is validated by mutagenesis and biochemical and in vivo RNA transport assays. The KHC–aTm1 complex is shown to bind RNA with higher affinity than KHC only, suggesting a new mechanism by which kinesin–cargo (RNA) transport can be modulated through stabilization by a KHC cargo adaptor. This is likely due to the positively charged binding surface formed in the trimeric KHC–aTm1 complex and possible stabilization of an extended helical region of the KHC tail. To our knowledge, this is the first structural characterization of a direct cargo adaptor–KHC interface, giving important new insight into the mechanism of RNA recognition.aTm1 immediately follows the sequences of KHC that are responsible for binding the KLCs , and salt bridges formed by adjacent residues (g and e positions). As the investigators noted, the finding that the aTm1 constructs crystallize as antiparallel coiled coils is in itself interesting; the received wisdom is that tropomyosins form exclusively parallel coiled-coil dimers , leucine (Leu [L]) in the core (heptad l dimers . Therefo complex C. Noneth repeats B. It is Together, these data form a picture of the KHC stalk as a dynamic and flexible platform for protein–protein interactions. This is in contrast to the rigid spacer-like properties with functions limited to oligomerization sometimes associated with coiled-coil domains. This could play an important role in the larger conformational changes associated with transition from a compact autoinhibited state to an extended active form of the motor, capable of motility on the cytoskeleton.


Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA.Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs.We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in The online version contains supplementary material available at 10.1186/s12915-021-01053-w. S-adenosyl-L-methionine- (AdoMet-) dependent methyltransferases, which offer a broad range of tagging chemistries that can be deposited in vitro and in vivo  (PerkinElmer) and T4 PNK (Thermo Fisher Scientific) following the manufacturer’s recommendations. For RNA modification with azide group, 0.2-10 μM of N21 RNA (random sequence 21-mer) was mixed with 0.1–0.2 mM Ado-6-azide and 2 μM of DmHen1∆C in the ssRNA modification reaction buffer containing 10 mM Co2+ in the form of CoCl2 salt, 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 5% glycerol, 0.2 mM DTT, 0.1 mg/ml BSA and 0.04 U/μl RiboLock RNase inhibitor (Thermo Fisher Scientific) and incubated for 30–60 min at 37 °C. Reactions were quenched with Proteinase K as described in Mickutė et al. , and annealed to a complimentary strand by incubating at 85 °C for 3 min with subsequent cool down by − 0.6 °C/min to 4 °C resulting in RNA/RNA or RNA/DNA duplexes with 2 nt 3′ overhangs. For RNA modification with azide group 0.2-10 μM of miRNA/miRNA* or miRNA/DNA substrate(-s) was mixed with 0.3–400 μM Ado-6-azide and 0.25-2 μM of AtHEN1 in the dsRNA modification reaction buffer ) and incubated for 1–2 h at 37 °C. Reactions were quenched with Proteinase K and RNA was precipitated as described above. Click reactions, reverse transcription and analysis on dPAG were performed as described earlier.AtHEN1 was expressed and purified as described in Osipenko et al. . All alk2.5 μM of miRXplore Universal Reference (Miltenyi Biotec) RNA was heated to 82.5 °C in the presence of 0.1 mM EDTA for 2 min and immediately placed on ice for 5 min. Then, RNA sample was split in two and libraries were prepared in duplicate for each 3′ alkyne-adapter. 2 μM of DmHen1∆C was used to modify 0.2 μM of miRXplore RNA in the ssRNA modification reaction buffer as described earlier. Proteinase K treatment and RNA precipitation was followed by click reaction during which 0.4 μM of RNA-azide was incubated with 10 μM of 3′ alkyne-adapter/RT primer and precipitated afterwards. 0.35 pmol of RNA-DNA conjugate was used for RNA library preparation according to the NEXTflex Small RNA-seq Kit v3 (PerkinElmer) starting from Step B with the following modifications: at Step D, ¼ dilution of NEXTflex 5′ 4 N adapter was used; at Step G, 18 cycles of PCR were performed; and Step H1 was proceeded with Step H2. The size distribution of the library was evaluated by capillary electrophoresis on the Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit (Agilent Technologies) and quantified via a qPCR reaction using KAPA Library Quantification Kit (Roche) on a Rotor-Gene Q instrument. All RNA libraries were pooled and sequenced at Lexogen facilities using NextSeq 500/550 High Output Kit to obtain 1 × 75 nt single-end reads.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads that were too short (less than 10 bases), had no adapters or contained Ns in the sequence were removed from further analysis using cutadapt [The quality of raw sequencing reads was evaluated using FASTQC program  was grown in 200 ml of BD™ Difco™ Lactobacilli MRS Broth at 37 °C until it reached six different growth points in early, mid and late exponential and stationary growth phases where equal amount of bacteria were collected from three biological replicates, mixed with 1/8 volume of ice-cold STOP solution (95% ethanol and 5% acid phenol), centrifuged and stored at − 80 °C. Frozen cells from all six growth stages were mixed and ground to a fine powder in liquid nitrogen. About 100 mg of ground powder was used for the total RNA extraction with RNAzol RT (RN 190)  according to the manufacturer’s recommendations. The quality of extracted RNA was evaluated using Agilent RNA 6000 Nano Kit (Agilent Technologies). For sRNA library preparation total RNA was treated with DNase I, RNase free (Thermo Scientific) following the manufacturer’s recommendations and recovered from the reaction using RNA Clean & Concentrator-25 Kit (Zymo Research). rRNAs were depleted using the modified RiboMinus Transcriptome Isolation Kit, bacteria (Invitrogen) where 2 μl of RiboMinus Probes and 2 μl of 100 μM mix of three probes specific to 5S rRNA were used  in 4 h in a 25 °C incubator rotating at 300 rpm. The eluate was collected using Corning Costar Spin-X Centrifuge Tube Filters (Merck) and RNA precipitated with 1/10 volume of 3 M NaOAc, pH 5.2:20 mg/ml glycogen (19:1) and 2.5 volumes of 96% ethanol. After treatment with RNA 5′ Pyrophosphohydrolase (RppH) (New England Biolabs) according to the manufacturer’s recommendations and further recovery with RNA Clean & Concentrator-5 Kit (Zymo Research) for mDOT-seq 1.6 μM of RNA was heated at 82.5 °C for 2 min in the presence of 0.1 mM of EDTA and immediately transferred on ice for 5 min. As with miRXplore Universal Reference library preparation, 0.2–0.3 μM of RNA were modified with azide group and 0.2 μM of azide-RNA was used for click reaction. Two picomoles of RNA-DNA conjugate was further processed according to the NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) starting from the Step B with couple modifications listed: at Step D, ¼ dilution of NEXTflex 5′ 4 N adapter was used; at Step E, incubation at 42 °C was extended to 60 min; at Step G, 15 PCR cycles were performed; and Steps F and H1 were carried out as described in alternative protocol for Preparing Libraries without Size Selection. For N libraries, 2 pmol of RppH treated RNA was processed according to the NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) with modifications in Steps E, F and H1 identical to mDOT-seq protocol except for Step G were only 12 PCR cycles were performed. The quality and concentration of libraries was evaluated as indicated above. All libraries were pooled and sequenced at Lexogen facilities using NextSeq 500/550 Mid Output Kit to obtain 2 × 75 paired-end reads.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low-quality reads or those having Ns were filtered out using cutadapt v2.9 [https://sourceforge.net/projects/bbmap/). Deduplicated dataset was quality (PHRED threshold 20) and length (minimum length 30) filtered using Trim Galore! v0.6.5 . Adaptor sequences were removed using UMI tools v1.0.1 [L. casei reference genome (NC_010999.1). Mapped data were subjected to APERO v1.0.3 [https://www.ebi.ac.uk/Tools/rna/infernal_cmscan/) [The quality of raw sequencing reads was evaluated using FASTQC program v0.11.9  . sRNAs’ cmscan/) . Visual cmscan/) . Custom cmscan/) , except cmscan/) .Pipeline for sequencing data analysis and identification of putative sRNAs is provided in the following scheme.V20-SDB semi-dry blotter (Fisherbrand). Blots were UV-cross-linked by irradiation at 254 nm, 25 J in a crosslinker (UVITEC Cambridge) and prehybridised for 2 h with a Hybridization buffer  BSA, pH 7.2). Membranes were incubated overnight at 42 °C with γ32P-ATP end-labelled oligodeoxyribonucleotides specific to certain sRNAs  by electroblotting at 5 V for 2 h using Additional file 1: Fig. S1. The chemical structures of alkyne-modified nucleotides and nucleosides. Fig. S2. Alkyne-adapter is efficiently attached to RNA-azide of nanomolar concentration. Fig. S3. cDNA synthesis through a conjugation linker using different reverse transcriptases. Fig. S4. Different miRNAs are efficiently modified using Ado-6-azide cofactor and AtHEN1. Fig. S5. Comparison of libraries prepared using different 3′ alkyne-adapters. Fig. S6. Effects of various factors on miRNA representation in mDOT-seq libraries. Fig. S7. Deviation of observed nucleotides frequencies from expected values in 3′-terminal section (16 positions) of identified miRNAs. Fig. S8. Evaluation of the miRNA quantification accuracy using different 3′ alkyne-adapters. Fig. S9. Sequence of 3′ alkyne-adapters applied for Lactobacillus casei sRNAs sequencing. Fig. S10. Spearman correlation among biological replicates of the L. casei libraries. Fig. S11. Classification of sRNAs into groups based on their genomic context. Fig. S12. The amount of predicted sRNAs with shorter 5′ or 3′ ends is similar among different libraries. Fig. S13. Putative 3′ UTR-derived sRNAs tend to be incorrectly identified in the N library. Fig. S14. Read coverage plots of experimentally verified sRNAs identified in all three libraries. Table S1. The number of miRNA species captured in sequenced libraries.Additional file 2. The data underlying published graphs.Additional file 3: Table S2. List of predicted sRNAs and their main features. Table S3. List of predicted sRNAs originating from more than one loci and their main features. Table S4. Promoters of predicted sRNAs. Table S5. Terminators of predicted sRNAs. Table S6. Detailed genomic context of predicted sRNAs. Table S7. Detailed genomic context of predicted sRNAs originating from more than one loci. Table S8. Structural characteristics of predicted sRNAs. Table S9. Conservation of predicted sRNAs in Lactobacillaceae family.Additional file 4: Table S10. The list of RNA and DNA oligonucleotides used for labelling of double-stranded substrates with AtHEN1. Table S11. The list of L. casei BL23 5S rRNA specific probes used for rRNA depletion. Table S12. DNA oligonucleotides used for Northern blot analysis.


RISC fractions isolated from tospovirus-infected N. benthamiana plants specifically cleaved naked, purified tospoviral genomic RNAs in vitro, but not genomic RNAs complexed with viral N protein. In planta RISC complexes, activated by a tobacco rattle virus (TRV) carrying tospovirus NSs or Gn gene fragments, mainly targeted the corresponding viral mRNAs and hardly genomic  RNA assembled into RNPs. In contrast, for the (+)ssRNA cucumber mosaic virus (CMV), RISC complexes, activated by TRV carrying CMV 2a or 2b gene fragments, targeted CMV genomic RNA. Altogether, the results indicated that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA is well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the findings made in this study are likely applicable to all viruses belonging to this group.Antiviral RNA silencing/interference (RNAi) of negative-strand (-) RNA plant viruses (NSVs) has been studied less than for single-stranded, positive-sense (+)RNA plant viruses. From the latter, genomic and subgenomic mRNA molecules are targeted by RNAi. However, genomic RNA strands from plant NSVs are generally wrapped tightly within viral nucleocapsid (N) protein to form ribonucleoproteins (RNPs), the core unit for viral replication, transcription and movement. In this study, the targeting of the NSV tospoviral genomic RNA and mRNA molecules by antiviral RNA-induced silencing complexes (RISC) was investigated,  Plants have evolved RNA silencing as the first line of defense against virus infection. Antiviral RNA silencing as well as viral counter-defense strategies have been extensively studied for ss(+)RNA plant viruses, but less for plant negative-strand (-)/ambisense RNA plant viruses (NSVs). Upon virus infection, RNA-induced silencing complexes (RISCs) are activated to bind and cleave invasive viral RNAs. Unlike ss(+)RNA plant viruses, genomic RNA molecules of plant NSVs are not naked and tightly associate with viral nucleocapsid protein into ribonucleoproteins (RNPs), the core infectious unit for NSVs. However, their viral mRNAs are not assembled into RNPs. Whether the assembly of genomic RNA into RNPs provides another counter-defense strategy to protect against antiviral RISC activity has not been determined for any NSV. In this study, using tospovirus as a representative plant NSV model system, it is demonstrated that antiviral RISC mainly targets viral mRNAs but not genomic RNA embedded in RNPs. Our findings suggest that the RNA targets of RISC differ between plant NSVs and ss(+)RNA plant viruses and that RNPs provide another counter-defense way for NSVs to  evade/escape from cytoplasmic RISC-mediated degradation. Tobacco rattle virus (TRV) and Tomato bushy stunt virus (TBSV) [in vitro. Exogenous addition of RNA containing siRNAs from plants infected with an unrelated virus would re-program the specificity of RISC to target the heterologous virus genome [Plants have evolved different layers of defense against pathogen invasions –3. One os (TBSV) –14, and s genome . Interess genome .Orthotospovirus, family Tospoviridae within the order Bunyavidales [Tomato spotted wilt virus (TSWV) is the type species of Orthotospovirus and categorized into the American clade [i.e. they encode two non-overlapping open reading frames (ORFs) on opposite strands. The M RNA encodes a precursor for the glycoproteins in the viral complementary (vc) strand, which is further processed into the mature Gn and Gc glycoproteins. These are required for virus particle maturation and presented as spikes on the surface of the particle envelope [Negative-stranded RNA viruses (NSVs) include not only dangerous medical important pathogens but also agronomical important plant pathogens. Some well-known NSVs include Ebola virus (EBOV), rabies virus, influenza A virus (FLUAV) and Rift Valley fever virus (RVFV) –17, and avidales , and conavidales ,22. Tomaan clade ,20. Tospan clade . The M aenvelope –26. In tenvelope –30. The envelope . The NSsenvelope –35. In benvelope . The N penvelope –39. Viraenvelope –42. The envelope ,44 and senvelope .in vitro. Furthermore, RISC targeting of viral mRNA and genomic RNA was also tested in planta. Results show that RISCs mainly target tospoviral mRNAs but not genomic RNA embedded within RNPs.Antiviral RNA silencing has been extensively studied for (+)ssRNA plant viruses ,6. HowevUnlike with ss(+)RNA plant viruses, viral genomic RNA of plant NSVs are not naked and always associated with N protein to form ribonucleoprotein (RNP) complexes ,48. We tNicotiana benthamiana using Hydroxyapatite column chromatography followed with Superdex S-200 column chromatography as described previously [N. benthamiana RISC complexes contain nonspecific nuclease activity to S RNA, RISC was also isolated from mock-inoculated N. benthamiana plants and incubated with DIG-labeled full-length S RNA transcripts of TSWV. As shown in in vitro with (heterologous) DIG-labelled RNA transcripts of GFP. The results showed that fractions containing RISC-nuclease activity against TSWV S RNA did not cleave GFP RNA  was incubated with DIG-labelled full-length M RNA of TSWV, the band of full-length M RNA was completely digested , carrying gene fragments of N and NSs, respectively, to a priori assemble antiviral RISC in planta, and subsequently challenged with TSWV  typical TSWV symptoms , a distinct representative of the European-Asian type tospoviruses. In analogy to TSWV, RISC was isolated from TZSV-infected leaves using Hydroxyapatite and Superdex S-200 HR gel column chromatography. Pilot experiments revealed that gel filtration fractions 2–7 contained RISC associated ribonuclease activity , a representative ss(+)RNA plant virus. CMV contains a tripartite RNA genome of which RNA2 encodes two proteins, 2a and 2b  gene sequences from the NSV tospoviruses TSWV or TZSV or the (+)ssRNA CMV. The results showed that whereas for CMV all genomic and subgenomic (m)RNA molecules were targeted by RISC, RISC primarily targets viral mRNA from the NSV tospoviruses in vitro and vivo. Genomic (v and vc) RNA complexed with N in vitro, or within RNPs from a natural infection were hardly or not targeted. Altogether, the findings indicate that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA molecules are well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the observations made in this study are likely applicable to all viruses belonging to this group. Whether, in addition, the formation of RNPs also presents a strategy of the virus to evade from antiviral RNAi remains to be debated.In plants RNA silencing is part of the host innate immunity system against viruses in which, during viral infection, RISC becomes programmed to specifically identify and degrade the invasive viral RNAs ,6. RISC d plants –14, and N. benthamiana did not target/degrade GFP RNA, while corresponding fractions isolated from mock-treated N. benthamiana did not cleave tospoviral RNA. Previous studies have reported on two different RISC complexes in Arabidopsis thaliana. One complex containing an AGO1 core and sized >500 kDa, and another complex containing AGO4 and sized 60–200 kDa [N. benthamiana plants two different peak fractions (fractions 4–9 and fractions 12–16) were also observed to exhibit antiviral RISC-activity. However, and in contrast, only one (fractions 2–7) was isolated from TZSV-infected N. benthamiana. RISC fractions isolated from TRV-infected N. benthamiana plants also distributed in two different peaks [N. benthamiana plant contained RISC-activity. Earlier studies on tombusviruses showed that the RISC-activity could only be detected in fractions collected from TBSV P19 mutant-infected plants but not from wild type virus infected plants [The sequence specific degradation by antiviral RISC was nicely demonstrated in this study as well, where purified antiviral RISC fractions from tospovirus infected –200 kDa . During nt peaks , while nN. benthamiana and tomato [Previous studies have shown that tospovirus-specific siRNAs (vsiRNAs) from all three RNA segments are generated during an infection of d tomato ,55. Consd tomato ,55. AlthFindings from this study are also in agreement with earlier studies on pathogen-derived resistance engineered against TSWV . TRV-indPhenuiviridae within the order Bunyavidales. Transgenic rice plants that express  sequences of the C3 gene, coding for the nucleocapsid protein, or the C4 gene, encoding the viral movement protein, protect against RSV infection, similarly as observed with tospoviruses. On the other hand, transgenic plants expressing  sequences of p3, encoding a suppressor of RNA silencing, or p4, encoding a non-structural protein of unknown function, did not provide resistance to RSV infection [Studies from other plant NSVs provide support for the idea that antiviral RISC targeting of tospovirus mRNAs, but not RNPs, widely applies to other/all plant NSVs, Rice stripe virus is a tenuivirus from the nfection . ProteinIn summary, using the segmented NSV tospovirus as model system, we have shown that RISC mainly targets viral mRNA and not genomic RNA. N, TSWV NSs, TSWV Gn and TSWV NSm was amplified, respectively, from the cDNA that was reverse transcribed from the purified total RNA of N. benthamiana leaves infected with TSWV lettuce isolate, then inserted into vector pTRV2 (YL156) to generate pTRV2-TSWV N, pTRV2-TSWV NSs, pTRV2-TSWV Gn and pTRV2-TSWV NSm.To generate TRV constructs carrying TSWV gene fragments, a 300 bp gene fragment from the N-terminal region of TSWV N, TZSV NSs, TZSV Gn, TZSV NSm and TZSV RdRp genes was amplified, respectively, from the cDNA that was reverse transcribed from total RNA of N. benthamiana leaves infected with TZSV, then inserted into vector pTRV2 (YL156) to generate pTRV2-TZSV N, pTRV2-TZSV NSs, pTRV2-TZSV Gn, pTRV2-TZSV NSm and pTRV2-TZSV RdRp.To generate TRV constructs carrying TZSV gene fragments, a 300 bp fragment from the N-terminal region of TZSV 2a and a 190 bp fragment from C-terminal region of 2b was amplified from cDNA of CMV Fny, then inserted into pTRV2 (YL156).For generating the constructs pTRV2-CMV 2a and pTRV2-CMV 2b, a 300 bp gene fragment from the N-terminal region of N protein gene was amplified from the cDNA of TZSV, inserted into downstream of the 6x His tag in bacterial expression vector pET28a digested with Nde I and EcoR I.Generation of constructs pET28a-TSWV N and the expression and subsequent purification of N protein has been described previously . To geneAll primers of used in this paper are listed in N. benthamiana plants were used in all agroinfiltration assays. The TSWV isolate from asparagus lettuce (TSWV-LE), TZSV isolate from tobacco and CMV Fny isolate were used in this study [N. benthamiana. For long-term storage, infected new N. benthamiana leaves were kept in a -80°C. For plant inoculation, about 1 g fresh infected leaves of N. benthamiana were ground in 10 mL PB buffer  and this crude extract was mechanically inoculated onto plant leaves. The virus-inoculated N. benthamiana plants were grown in a growth chamber at 25°C and 16 h light/8 h dark photoperiod.Six-week old is study . The TSWN. benthamiana leaf tissue was homogenized in liquid nitrogen and the powder was resuspended in 40 mL loading buffer . The homogenate was filtered through two layers of gauze and subsequently centrifuge for 15 min twice at 14,000 ×g at 4°C. Supernatant (40 mL) was loaded onto a 25×3.6 cm2 column packed with 50 mL Bio-Gel HT Hydroxyapatite . Chromatography separations were performed on ÄKTA avant 25 . After washing the column with 300 mL loading buffer, the bound proteins were eluted using a linear gradient of sodium phosphate buffer (pH 6.8) from 10–200 mM at a flow rate of 1.0 mL/min. Fractions were analyzed for the presence of active RISC by RNA cleavage assay and stored at -80°C. Active RISC fractions (0.5~1 mL) isolated from Hydroxyapatite column were further fractionated on Superdex 200 Increase 10/300 GL column  pre-equilibrated with elution buffer  at a flow rate of 0.5 mL/min. Every fraction (600 μL) was analyzed for RNA cleavage activity and active RISC fractions were stored at -80°C until further use.The isolation of RISC fractions from virus infected plants was performed as described previously with slight modification –14. Briein vitro Transcription kit . DNA templates was prepared by directly amplifying the full length TSWV S, TSWV M, TZSV S and TZSV M from full length cDNA clones of TSWV and TZSV [RNA transcripts were generated using the T7 Riboprobe and TZSV . The T7 R94A/R95A and pET28a-TZSV N were individually transformed into Escherichia coli strain Rosetta (DE3). To express the recombinant proteins, a 10 mL of overnight culture was transferred to a 1-liter culture and incubated at 37°C in a shaker incubator. When the optical density of culture at OD600 reached to 0.6, 0.1 mM isopropyl b-D-thiogalactopyranoside was added and incubated overnight at 20°C to induce protein expression. The purification of 6xHis-tag recombinant proteins was done using nickelnitrilotriacetic acid resin  as described [Protein expression and purification was performed as described . Construescribed .2. Control reactions were performed with elution buffer or with chromatography fractions from mock-inoculated plants. DIG-labeled RNA transcripts were resolved by electrophoresis on a 1% agarose gel and transferred to a Hybond-N+ membranes  using the Bio-Rad semi-dry transfer unit . DIG-labelled signals on the blots were detected by AP-conjugated anti-digoxigenin antibodies and visualized by incubation in the presence of BCIP/NBT  staining.RNA cleavage reactions were performed by incubating DIG-11-UTP-labeled RNA transcripts or DIG-labeled RNA complexed with N protein fractions from tospovirus-infected plants as described . Five μgTobacco rattle virus (TRV) in N. benthamiana was performed as described previously [Agrobacterium tumefaciens by electroporation [Agrobacterium carrying pTRV2-TSWV N, pTRV2-TSWV NSs, pTRV2-TSWV Gn, pTRV2-TSWV NSm, pTRV2-TZSV N, pTRV2-TZSV NSs, pTRV2-TZSV NSm, pTRV2-TZSV Gn, pTRV2-TZSV RdRp, pTRV2-CMV 2a, pTRV2-CMV 2b, or pTRV2-GUS control was equally mixed with Agrobacterium pTRV1 and adjusted the final concentration to OD600 = 0.25. After 2~4 hours incubation in the dark at room temperature, the mixtures were infiltrated into leaves of Nicotiana benthamiana plants to induce antiviral RISC formation to TSWV, TZSV and CMV. About 10 days after agroinfiltration, the infiltrated leaves of TRV-preinfected plants were mechanically inoculated with crude extract of TSWV, TZSV, and CMV, respectively, from freshly infected N. benthamiana. The phenotype of virus targeting by pre-assembled antiviral RISC derived from TRV constructs in N. benthamiana was monitored at 9–12 days post inoculation. The agroinfiltrated or virus-inoculated N. benthamiana plants were grown in a growth chamber at 22°C and 16 h light/8 h dark photoperiod.Agroinfiltration of eviously ,62. Indiporation . AgrobacN. benthamiana were determined as described [N. benthamiana at 9–12 days post virus inoculation. About 1 g leaf tissue was homogenized in 2 mL extraction buffer  containing protease inhibitor cocktail . Samples were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane . To detect TSWV, TZSV and CMV accumulation, the blots were incubated with specific antibodies to TSWV N, TZSV N and CMV CP, followed with a secondary antibody containing HRP-conjugated goat anti-rabbit or goat anti-mouse. The blots were developed using the ECL Substrate Kit . ECL signals were visualized using the ChemiDoc Touch Imaging System (Bio-Rad). Blots were also stained with Ponceau S and used as reference for sample loading and quantification. Rabbit polyclonal antibodies specific for TSWV N and TZSV N were produced in this laboratory. Mouse Monoclonal antibody against CMV CP was kindly provided by Prof. Jianxiang Wu at Zhejiang University. HRP-conjugated goat anti-rabbit or goat anti-mouse IgG were purchased from Thermo Fisher Scientific .Protein levels of TSWV, TZSV and CMV in escribed ,65. TotaN. benthamiana were determined using quantitative RT-PCR (qRT-PCR) [N, TZSV N and CMV CP . A series of ten-fold dilutions from 1 to 10−5 ng μL−1 of the RNA transcripts of TSWV N, TZSV N or CMV CP was prepared. The DNA amount of TSWV N, TZSV N or CMV CP in each dilution sample was represented as copies per μL, which was calculated using the following equation:The RNA levels of TSWV, TZSV and CMV in qRT-PCR) . Three pd CMV CP . Quantit2), was used to evaluate the standard curves. After the cycle threshold (Ct) of TSWV, TZSV and CMV in infected N. benthamiana was determined, the copy numbers of TSWV N, TZSV N and CMV CP in infected plants were determined from the standard curves based on the crossing points of the samples. The expression of Elongation factor 1-alpha gene  was used as an internal control. Each experiment was performed with three biological replicates.The logarithm of the gene copy number in each dilution sample (x axis) was plotted against the corresponding crossing point value (y axis) to generate the standard curve for TSWV N, TZSV N and CMV CP. The linear regression equation, along with the coefficient of determination  [N, NSs, Gn and NSm was inserted into a TRV vector. To prepare the template for DIG-labeled probes that do not overlap with the gene fragment of TRV constructs, a 450 bp, 500 bp, 500 bp and 500 bp gene fragment from the C-terminal region of TSWV N, TSWV NSs, TSWV GP and TSWV NSm, respectively, was amplified from the full length cDNA clones of TSWV [N and TZSV NSs, respectively, was amplified from TZSV cDNA; a 600 bp gene fragment from the C-terminal region of 2a without overlapping with 2b, and a 170 bp fragment from the C-terminal region of 2b from cDNA clones of CMV-Fny. Due to the small size of CMV 2b, the 2b probe overlapped with the 2b fragment (190 bp) inserted in pTRV2-2b. The T7 promoter was added into each forward primer to generate PCR products containing a T7 promoter. The purified PCR products were used as DNA template for probes. Strand-specific DIG-labeled probes to detect for TSWV N, TSWV NSs, TSWV NSm, TSWV GP, TZSV N, TZSV NSs, CMV 2a and CMV 2b, respectively, were synthesized using a DIG High Prime RNA labeling kit . The blots were developed using a DIG-High Prime Detection Starter Kit II  according to the manufacturer’s instructions.The genomic vRNAs, vcRNA and viral mRNA of TSWV, TZSV and CMV were detected by northern blot as described . To analare, UK) . A 300 bS1 FigIn vitro cleavage assay of naked genomic S RNA of TSWV by isolated chromatography fractions from TSWV infected N. benthamiana. Fractions from Superdex S-200 chromatography were incubated with digoxigenin (DIG) labelled in vitro full-length genomic S RNA of TSWV (100 ng). (B) Chromatography fractions isolated from mock-inoculated N. benthamiana plants were incubated with DIG-labeled full length genomic S RNA of TSWV (100 ng), and tested for their ability to cleave viral RNA. (C) Chromatography fractions isolated from TSWV-infected plants were incubated with DIG-labelled RNA transcript of GFP and tested on cleavage specificity.(A) (TIF)Click here for additional data file.S2 FigIn vitro cleavage assay of TSWV genomic S RNA complexed with N and NR94A/R95A mutant proteins by fraction 6 Click here for additional data file.S3 FigN. benthamiana plants using a 1 mL needle-less syringe. Ten days after agro-infiltration, these plants were rub inoculated with crude extracts from TSWV-infected leaves. The phenotype of TSWV challenged plants was monitored at 9 dpi.Agrobacterium cultures containing pTRV2-NSs, pTRV2-N, pTRV2-NSm or pTRV2-Gn were equally mixed with Agrobacterium cultures containing pTRV1 and infiltrated into 4-leaf stage (TIF)Click here for additional data file.S4 FigN. benthamiana plants using strand specific DIG-labelled N probe. (C) Detection of genomic vRNA and NSm mRNA of TSWV M segment targeted by antiviral RISC from TRV-NSm and TRV-GUS pre-infected plants by northern blot analysis using strand specific DIG-labelled NSm probe. (D) Genome M vcRNA and GP mRNA were detected in TRV-Gn and TRV-GUS pre-infected plants using strand specific DIG-labelled Gn probe. N. benthamiana plants were agroinoculated with the TRV constructs and after about 15 days, to allow for pre-assembly of antiviral RISC, the local leaves were challenged with TSWV. Samples were collected at 5 dpi from the TSWV inoculated leaves of TRV pre-infected plants. Genomic vcRNA (or vRNA) and viral mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show equal RNA loading of samples.(A) Detecting genomic vRNA and NSs mRNA of TSWV S segment targeted by RISC from TRV-NSs and TRV-GUS plants using strand specific DIG-labelled NSs probe. (B) Northern blot analysis of viral genomic vcRNA and N mRNA of TSWV S segment targeted by pre-assembled RISC from TRV-GUS and TRV-N in (TIF)Click here for additional data file.S5 FigN. benthamiana. Every fraction (600 μL each) was incubated in vitro with digoxigenin (DIG) labeled full length S RNA of TZSV (100 ng) to examine which fraction contained the RISC associated nuclease activity to viral S RNA.Sixteen Superdex S-200 fractions were isolated from TZSV infected (TIF)Click here for additional data file.S6 FigCMV RNAs encodes five open reading frames 2a is encoded by genomic RNA2 of CMV. 2b is encoded by subgenomic RNA2 of CMV. The 1a, 2a and 3a ORFs expressed from the genomic strands are denoted in blue, whereas 2b and CP, expressed from a subgenomic mRNA, are denoted in red.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Artemisia argyi is a traditional Chinese medicine with anti-inflammatory and anti-tumor activities. This study investigated the potential therapeutic benefits of Artemisia argyi essential oil (AAEO) in suppressing metastasis of HCC by targeting DEPDC1. Assessment of AAEO cytotoxicity was performed by MTT assay. Anti-metastatic effects of AAEO were investigated in vitro using wound healing and transwell assays. The HepG2 cells were transduced with lentiviral vector containing luciferase (Luc). A metastasis model of nude mice was established by tail vein injection of HepG2-Luc cells. The nude mice were treated with AAEO  or sorafenib (40 mg/kg). Metastasis of HCC cells was monitored via in vivo bioluminescence imaging. After treatment for 21 days, tissues were collected for histological examination and immunohistochemistry analysis. Gene and protein levels were determined by real-time quantitative PCR and western blotting. The results revealed that AAEO significantly inhibits the migration and invasion in vitro in a concentration-dependent manner. In vivo assays further confirmed that AAEO markedly inhibits HCC metastasis into lung, brain, and femur tissues and exhibits low toxicity. Our results suggested that AAEO significantly downregulates the mRNA and protein expression of DEPDC1. Also, AAEO attenuated Wnt/β-catenin signaling through reduction of Wnt1 and β-catenin production. Moreover, AAEO prevented epithelial-mesenchymal transition (EMT) by downregulation of vimentin and upregulation of E-cadherin. Furthermore, we found that DEPDC1 promoted HCC migration and invasion via Wnt/β-catenin signaling pathway and EMT. These results demonstrate that AAEO effectively inhibits HCC metastasis via attenuating Wnt/β-catenin signaling and inhibiting EMT by suppressing DEPDC1 expression. Thus, AAEO likely acts as a novel inhibitor of the DEPDC1 dependent Wnt/β-catenin signaling pathway.The tumor metastasis is the major hurdle for the treatment of advanced hepatocellular carcinoma (HCC), due in part to the lack of effective systemic treatments. DEPDC1, a novel oncoantigen upregulated in HCC, is thought to be a molecular-target for novel therapeutic drugs.  Hepatocellular carcinoma (HCC) is one of the most lethal cancers and the second most common cause of cancer-related deaths globally, due in part to the lack of systemic treatment options . SeveralDEPDC1, DEP  domain-containing 1 protein, is a novel oncoantigen upregulated in multiple types of cancers, including HCC . DEPDC1 Artemisia argyi Lévl. et Vant. is a famous plant species in traditional Chinese medicine used to control dysmenorrhea, abdominal pain, and inflammation , including abundant monoterpenes and their derivatives , sesquiterpenoids and a comparatively minor amount of aldehydes, ketones, phenolic and aromatic compounds . Therefore, AAEO likely acts as a novel inhibitor of the DEPDC1 dependent Wnt/β-catenin signaling pathway, and may provide a potential strategy for the treatment of HCC.Artemisia argyi leaves were collected from Zhumadian city, Henan province, China and identified by Prof. Dong Chengming from Henan University of Chinese Medicine. AAEO was obtained by an optimized distillation extraction method according to the Chinese Pharmacopoeia  was deposited at the herbarium of the Henan Institute of Medical and Pharmaceutical Sciences, Zhengzhou University.acopoeia . BrieflyThe fingerprint of AAEO  was cond2 incubator. The HepG2 cells were transduced with a lentiviral vector containing luciferase (Luc). Subsequently, HepG2-Luc was used for establishing an HCC metastatic model in nude mice. HepG2 and SMMC-7721 cells were transduced with lentiviral vectors, resulting in DEPDC1 overexpression (DEPDC1-OE) or DEPDC1 knockdown (DEPDC1-KO) HCC cells, respectively. Western blotting and qRT-PCR assays were subsequently performed to confirm the expression of DEPDC1 in these cells.HepG2 and SMMC-7721 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and streptomycin at 37°C in a humidified 5% CO3/well. After incubation for 24 h, the cells were treated with AAEO at different concentrations  for 72 h. Cell viability was measured by the 3--2, 5-diphenyltetrazolium bromide (MTT) test. The CC50 or IC50 was defined as the concentration required to inhibit cell proliferation by 50%.HepG2, SMMC-7721, or LO2  cells were seeded in 96-well plate at a density of 3 × 10DEPDC1-OE or DEPDC1-KO HCC cells were performed as mentioned above without AAEO treatment.HepG2 or SMMC-7721 cells were seeded in a six-well dish and incubated for 24 h. The monolayer was then scratched with pipette tips and washed with phosphate buffered saline (PBS). The cells were cultured in fresh medium containing AAEO at different concentrations  for 24 and 48 h. The rate of wound closure was examined and photographed by an inverted microscope . Wound healing assay of 4 cells/well. Following 24 h incubation, the cells were fixed in methanol and stained with crystal violet. Subsequently, the number of cells that had migrated or invaded was counted using an inverted microscope . Migration and invasion assays of DEPDC1-OE or DEPDC1-KO HCC cells were performed as mentioned above without AAEO treatment.Migration and invasion assays were performed using a transwell chamber (8 μm pores). Briefly, the chambers set into the 24-well cluster plates were coated with 20 μL matrigel and incubated at 37°C for 30 min for the cell invasion assay, while non-coated chambers were used for the cell migration assay. HepG2 and SMMC-7721 cells were resuspended in medium with different concentrations of AAEO  and seeded at a density of 2 × 10ad libitum. All animals were treated in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (China), and experimental procedures were approved by the Animal Ethics Committee of Zhengzhou University.Female nude mice , aged between 4 and 6 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The animals had free access to drinking water and feed 6) were intravenously administered via tail vein infusion. Two weeks post inoculation, 25 tumor-bearing nude mice were randomly divided into five groups (n = 5 per group). The tumor-bearing mice were intraperitoneally injected with AAEO  or given oral dose of sorafenib (40 mg/kg/day) for 21 days. Body weight was measured every 2 days, and bioluminescence imaging was performed using IVIS Lumina III . At the end of the study, all mice were sacrificed, and the tissues were collected for histopathological as well as immunohistochemical examination.To establish the metastasis model, HepG2-Luc cells  for histopathology.TMRT reagent kit (Takara). The primer sequences were as follows: DEPDC1: forward, 5′-CCATCATTGCAATAGCAGG-3′ and reverse, 5′-GAGCATACATATGTTCAAACTTC-3′; GAPDH: forward, 5′-CAGGAGG CATTGCTGATGAT-3′, reverse, 5′-GAAGGCTGGGGCTCATTT-3′. Quantitative real-time PCR was performed using SYBR Premix Ex Taq II (Takara) in an Applied Biosystems 12k machine . Polymerase chain reaction conditions were as follows: 95°C for 30 s for stage 1, 40 cycles at 95°C for 5 s, and 64°C for 34 s for stage 2. The relative expression was calculated using the 2Ct–ΔΔ formula.Total RNA of cells and lung tissues was extracted using RNAiso Plus  and reverse-transcribed using the PrimeScriptThe cells or tissue homogenate were harvested, lysed with lysis buffer for 30 min on ice, and centrifuged at 14000 rpm for 15 min at 4°C to obtain the protein. The western blot was performed according to the manufacturer’s specification. The primary antibodies used were anti-DEPDC1 , anti-Wnt1 , anti-β-catenin , anti-vimentin , and anti-E-cadherin .Immunohistochemistry was performed as described previously. In brief, tissues were fixed with 4% paraformaldehyde, paraffin embedded, and cut into 4 μm slices. Immunohistochemical staining was conducted according to the manufacturer’s protocol. The protein expression was evaluated using Image-Pro Plus 6.0 based on the integral optical density.1, and differences were considered statistically significant at p < 0.05. The differences between two groups were analyzed using a two-sided unpaired Student’s t-test. The differences among multiple groups were analyzed using one-way ANOVA, followed by Duncan’s multiple-range tests. All data are expressed as mean ± standard deviation (SD).Statistical analysis was performed using SPSS 20.0 in vitro and inhibits tumor growth in a HCC mouse xenograft model through induction of tumor cell apoptosis and blocking the cell cycle in the G2/M phase (unpublished data). In this study, AAEO exhibited low toxicity on a normal liver cell line (LO2) with a CC50 value of 749.6 ± 24.4 μg/mL. Moreover, it displayed significant inhibitory activity against HCC in a concentration-dependent manner, with IC50 values of 321.7 ± 3.8 and 322.0 ± 14.3 μg/mL for HepG2 and SMMC-7721 cells, respectively (10) AAEO for subsequent studies. Wound healing assay suggested that AAEO suppressed the migration of SMMC-7721 and HepG2 cells in a concentration-dependent manner. Moreover, transwell migration and invasion assays showed that both SMMC-7721 and HepG2 cells treated with AAEO displayed a marked decrease in migration and invasion in comparison to untreated cells , whereas it upregulated the expression of epithelial marker (E-cadherin) in SMMC-7721 and HepG2 cells . Consistft model . All theDEPDC1-KO HCC cells, while the mRNA and protein levels were increased in DEPDC1-OE HCC cells . In this study, we further investigated the anti-metastatic activity of AAEO on HCC.The tumor metastasis is the major hurdle for the treatment of advanced HCC, due in part to the lack of effective systemic treatments. Currently, the multi-kinase inhibitor sorafenib is the only systemic chemotherapeutic drug approved for the treatment of HCC. However, clinical data have indicated that sorafenib prolonged the median survival by approximately 3 months in patients with advanced stage HCC and acquired resistance to sorafenib over the course of therapy is likely . Additio cancers . Our pre cancers . Also, win vivo investigations showed that it exhibits potent anti-HCC metastasis activity in a dose-dependent manner. The inhibitory effect in the AAEO (115 mg/kg) group was similar to that in the sorafenib group (40 mg/kg), and the AAEO (230 mg/kg) group showed a higher anti-metastasis effect than the sorafenib group. Clinical data have indicated that the benefit of sorafenib is relatively low for patients with the worst prognosis, such as those with macroscopic vascular invasion or extrahepatic spread (Our results demonstrated that AAEO can significantly inhibit the migration and invasion of SMMC-7721 and HepG2 cells in a concentration-dependent manner. Further c spread . MoreoveCaenorhabditis elegans to mammals (DEPDC1 was first identified as a novel gene in bladder cancer, wherein it plays an essential role in the growth of bladder cancer cells (DEPDC1 is aberrantly upregulated in various tumors and is involved in the occurrence and development of HCC (DEPDC1. Moreover, in vivo metastasis assays confirmed that AAEO markedly inhibited the expression of DEPDC1 and reduced lung metastasis of HCC. The results from our study supported that DEPDC1 is likely to represent a potential therapeutic target of HCC (DEPDC1 is a highly conserved protein across many species, from  mammals . It is i mammals . DEPDC1 er cells . In recet of HCC , breast t of HCC , lung cat of HCC , and glit of HCC . A DEPDCt of HCC . Howevert of HCC .DEPDC1-KO reduced migration and invasion ability of HCC cells by downregulation of Wnt/β-catenin signaling and inhibition of EMT. In contrast, DEPDC1-OE promoted migration and invasion via upregulation of Wnt/β-catenin signaling and induction of EMT in HCC cells. Moreover, AAEO inhibited HCC metastasis via suppression of DEPDC1 dependent Wnt/β-catenin signaling pathway. These results supported DEPDC1 dependent Wnt/β-catenin signaling pathway is likely to play an essential role in HCC metastasis and represent a promising therapeutic target.The canonical Wnt pathway is one of the most frequently deregulated pathways in HCC. Evidence suggests that the canonical Wnt signaling pathway plays essential roles in cell cycle, trafficking, signaling, and migration in HCC . During Previous reports have demonstrated that DEPDC1 promotes cell proliferation, invasion, and angiogenesis in numerous cancers regulated by multiple signaling pathways. For example, DEPDC1 has been shown to activate NF-κB and E2F signaling pathways to accelerate cell cycle progression, and to promote the K-RAS and Wnt/β-catenin signaling pathways to drive tumor cell proliferation and metastasis . Also, DThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by the Animal Ethics Committee of Henan Institute of Medical and Pharmaceutical Sciences. Written informed consent was obtained from the owners for the participation of their animals in this study.YP and ZhZ conceived and designed the study. YL, YT, and NW performed the experiments. WZ, YW, YZ, ZlZ, JL, and FM analyzed the data. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Three combinations of modified ribonucleotides—2′O methyl-ATP (Am), N6 methyl-ATP (m6A), and pseudo-UTP (ψ)—conferred SINEUP activity to naked RNA. The best combination tested in this study was fully modified with m6A and ψ. Aside from functionality, this combination conferred improved stability upon transfection and higher thermal stability. Common structural determinants of activity were identified by circular dichroisms, defining a core functional structure that is achieved with different combinations of modifications.SINEUPs are a novel class of natural and synthetic non-coding antisense RNA molecules able to increase the translation of a target mRNA. They present a modular organization comprising an unstructured antisense target-specific domain, which sets the specificity of each individual SINEUP, and a structured effector domain, which is responsible for the translation enhancement. In order to design a fully functional   in vitro transcribed RNA. Common structural determinants of activity were identified by circular dichroisms.SINEUP RNAs are a new platform for therapeutics to increase endogenous protein expression within a physiological range. We identified a combination of modified chemical modifications to confer SINEUP activity to an  SINEUPs are a new class of antisense long non-coding RNAs (lncRNAs) able to enhance the translation of a target mRNA.4,,in vitro and in vivo and on ectopically expressed or endogenous transcripts, including in patient-derived cell models.,,,,The structure-activity relationship of both domains has been extensively investigated.,,Increasing understanding of the structural determinants of activity has allowed the progressive miniaturization of SINEUPs, with the aim of designing a fully functional, shorter version.in vitro synthesized exogenous RNA faces common roadblocks linked to its susceptibility to enzymatic degradation, its potential immunogenicity, and the efficiency of uptake by the recipient cell.,Drug delivery of Another important issue is the preservation of the functional secondary structure of the molecule.,Cells add chemical modifications to newly synthesized RNA molecules during and after transcription, complementing the mere sequence of RNA with a multifaceted layer of functionalities that define the intensively studied field of epitranscriptomics.Given the above considerations, when designing modular RNA drugs such as SINEUPs, it is important to define a set of chemical modifications for the general stabilization and de-immunization of the molecule but also to achieve a functional tertiary structure, which is crucial to preserve a correct network of interactions with other RNA molecules and proteins.in vitro transcribed (IVT) SINEUP on endogenous target mRNAs.In order to address these issues, and to design fully functional exogenously transcribed SINEUPs, we set out to study an optimized set of modifications that could maximize the activity of an exogenous in vitro through cell transfection and western blotting (WB) analysis of protein levels. This screening allowed the identification of a subset of exogenous modifications important for the functionality of the IVT RNA molecule. Then, structural analysis by circular dichroism (CD) unveiled structural determinants for activity. The experimental design adopted in this study is illustrated in IVT RNA containing different combinations of modifications were synthesized and screened in vitro transcribed SINEUPs, we used as a model molecule a validated SINEUP, named miniSINEUP-DJ-1,,In order to study the functionality of modified and unmodified in vitro in the presence or absence of cap analog reagents, in order to obtain co-transcriptionally capped or uncapped products. Capped and uncapped transcripts were used as templates for enzymatic polyadenylation or used without further modifications. It was therefore possible to study the effect of capping and polyadenylation independently and in combination. As a control, truncated delta-BD transcripts (ΔBD), lacking the binding domain and thus not functional, were also produced.MiniSINEUP-DJ-1 was transcribed All transcripts were purified and transfected into 293T/17 cells in equimolar amounts. We first optimized RNA transfection conditions by testing different transfectants and protocols. We eventually chose to use a cationic polymer transfectant (polyethyleneimine [PEI]), as it showed the highest transfection efficiency . As showin vitro transcripts did not show any translation enhancements, and the addition of capping or poly(A), alone or in combination, was not sufficient to restore SINEUP activity.In summary, unmodified Given the lack of activity of unmodified IVT SINEUPs, we sought to investigate the optimal mix of modifications to preserve and maximize SINEUP activity in IVT SINEUP RNA. We screened several combinations and proportions of natural modifications, introducing modified nucleotides co-transcriptionally, by  substitution of the respective unmodified nucleotide with a modified analog in the reaction mixture. In particular, modifications were tested first alone and then in combination. The most promising modifications active alone were then tested in a larger number of combinations with others. A total of 22 combinations of modifications was analyzed. With this strategy, we limited progressively the magnitude of the screening, in order to focus on the most favorable combinations. This initial screening returned three “active” combinations of modifications: 2′O methyl-ATP, N6 methyl-ATP, and pseudo-UTP, which were further screened.As shown in In order to rule out any possible non-SINEUP-related effect on DJ-1 protein level, we performed an additional experiment in which we transfected cells with scrambled sequences of the same length of miniSINEUP DJ-1 and containing the same active combinations of modifications, or unmodified. As shown in In order to better characterize the content of modifications, we performed mass spectrometry (MS) analysis of these transcripts. The results show that when using a ratio of Am to m6A of 99:1 in the IVT reaction mixture, the relative abundance of the two modifications in the final molecule was only 20:80. Importantly, IVT RNAs containing lower ratios of Am to m6A were not functional, similar to transcripts containing m6A alone, indicating that a threshold level of Am is needed for activity.Noticeably, although m6A 100% + ψ 100% IVT RNA was active, the corresponding transcripts modified with only one of these two nucleotide analogs (either m6A 100% or ψ 100%) were completely inactive A and 3B.The lack of activity of unmodified IVT SINEUPs could be due to reduced stability of the unmodified RNA.On the other hand, we noticed that the pattern from qRT-PCR on both RNA extracts from transfected cells A and fro,Circular dichroism spectroscopy was used as a tool to compare the conformations adopted in solution by the RNA molecules under investigation and to evaluate the possible role played by the structure on SINEUP activity  was very different from both the one containing m6A alone and that containing ψ alone . Indeed, the spectrum of the miniSINEUP containing m6A was very similar to that of the unmodified RNA. On the contrary, the spectrum of the RNA fully modified with ψ reflected the distortion of the helix attributable to this particular nucleotide,Tm) was determined by following the variation of the CD intensity at 270 nm as a function of temperature.Tm by about 15°C.Tm comparable with the unmodified one, while the RNA with m6A displayed a Tm increment of 5°C. Apparent melting temperature curves for all transcripts are reported in Studies of the thermal stability of the unmodified and the modified miniSINEUPs were also performed by means of CD, as structural stability is a crucial parameter when designing RNA for potential therapeutic applications C. The ap,,,,,RNA post-transcriptional modifications are widespread across the transcriptome, in both coding and non-coding regions.in vivo. To overcome these drawbacks, capping can be introduced co-transcriptionally  or post-transcriptionally (by a step of enzymatic capping with vaccinia virus capping enzyme).IVT RNA lacks the natural modifications present in RNA, as well as 3′ and 5′ end processing such as capping and polyadenylation, and it may thus result in both instability and inefficiency Several mixes of artificial and natural modifications have been used to address stability and immunogenicity roadblocks of exogenous IVT RNA, which still represent major hurdles for the design and delivery of RNA therapeutics.,In particular, 2′O methyl-ATP is often found in the extended cap structure in mRNA, where the 2′O sites of the first and second nucleotides adjacent to the cap are methylated.,,,,7G cap enhances transcript stability and promotes translation.Both N6 methyl-ATP and ψ are involved in post-transcriptional gene regulation.,Ψ is the most abundant modification in non-coding RNA (ncRNA).in vitro and in vivo.Our results show that the three modifications above are all capable of preserving SINEUP activity of IVT RNA, when present in the appropriate combination and ratio. Somehow unexpectedly, when used in combination , the effInterestingly, although neither ψ nor m6A modifications alone could restore the functionality of IVT miniSINEUPs, they did so when present in combination on the same molecule. The gain of activity was reflected in a marked structural change in the molecule, as evidenced by CD spectra of the mono- and double-modified transcripts. A previous workIn an attempt to correlate the structural properties brought to the miniSINEUP by a given nucleotide modification and the observed SINEUP activity, we speculated that the B-like form may be the most biologically active miniSINEUP. This consideration would explain why the SINEUP versions containing Am alone, or the combination of m6A and ψ, displaying almost identical B-like conformations from CD spectra, are biologically active despite the different modification patterns. Nevertheless, this would not fully justify the activity of the miniSINEUP modified with both Am and m6A.,These data suggest that other factors contribute to SINEUP activity of the modified IVT RNAs tested in this study. For instance, RNA modifications may modulate the interaction with RNA binding partners important for activityAnother important aspect to take into consideration is the fact that RNA changes its conformation when it is hybridized to another RNA or DNA molecule. Given that our miniSINEUP is a modular molecule, including an antisense RNA BD and a more structured ED, the structure of the isolated domains is definitely worthy of further investigation. For instance, it has been shown that DNA-RNA hetero-hybrids have a conformation that is intermediate between A-form RNA and B-form DNA, while when such hetero-hybrids include fully 2′O-modified RNA, the resulting conformation corresponds fully to A-form RNA.In summary, the results obtained using CD spectroscopy support the theory that a common secondary structure may be linked to comparable SINEUP activity. In addition, certain modifications (such as m6A) may also contribute to SINEUP activity by contributing to regulate the interactions with protein or RNA binding partners.The modularity of SINEUP molecules also aids in generalizing our results obtained on the model miniSINEUP DJ-1. Indeed, as already mentioned, the ED is universal for all the SINEUPs, whereas the BD is different and target dependent. As the BD is the unstructured module of the molecule, because of the hybridization with the target RNA, it is reasonable to infer that the functional secondary structure of the ED is a general feature of all SINEUPs, independent of their binding domain.In this study, we have defined different combinations of natural modifications that can be added to an IVT SINEUP RNA in order to reproduce the functionality of an endogenously transcribed SINEUP. Among the tested modifications, the combination of m6A and ψ was the best combination. Indeed, aside from retaining SINEUP functionality, it enhanced the half-life after transfection and conferred enhanced structural stabilization. Furthermore, by structural analysis through circular dichroism, we have been able to correlate structural features with SINEUP activity, identifying a common functional structure obtained by different sets of modifications. These results further support the notion of a modular arrangement of this class of ncRNAs, in which an unstructured domain (BD), operating by sequence-specific hybridization with a target, is paired to a structured domain (ED), with specific and functional tertiary folding and modifications, operating via the interactions with protein and RNA binding partner to achieve translation enhancement. Both the correct set of chemical modifications and a functional structural arrangement are therefore important in the design of fully functional, tertiary-folded RNA therapeutics, such as SINEUPs.in vitro transcription were all based on a pCMV6 backbone. MiniSINEUP-DJ-1Plasmids for in vitro using the MEGAscript T7 kit (Thermo Fisher Scientific). Modified nucleotides triphosphates  were purchased from TriLink Biotechnologies. In vitro transcription reactions were assembled according to recommendation from the kit manufacturer and incubated overnight (16 h) at 37°C. Co-transcriptional capping was performed by substituting part of the GTP in the reaction with cap analog m7G(5′)ppp(5′)G (Thermo Fisher Scientific). After transcription, capped transcripts were treated with 1 μL thermosensitive shrimp alkaline phosphatase  for 15 min at 37°C. All capped and uncapped transcripts were then treated with DNase I for 15 min at 37°C and immediately purified using the RNeasy Mini Kit (Qiagen). Post-transcriptional polyadenylation was performed on purified transcripts by adding 1 μL E. coli poly(A) polymerase  and 1 mM ATP and incubating at 37°C for 30 min. Polyadenylation reactions were stopped by purification with the RNeasy Mini Kit. All transcripts were checked for purity and integrity by UV-vis spectrophotometry and denaturing poly-acrylamide gel electrophoresis (PAGE).Unmodified and modified RNA molecules were transcribed 293T/17 cells, purchased from the American Type Culture Collection (ATCC), were cultured in DMEM high glucose (4.5 g/L D-glucose) with L-glutamine (Gibco), completed with 10% fetal bovine serum and 1% penicillin/streptomycin mix and 1% HEPES buffer (Gibco). 293T/17 cells were passaged 1:5 to 1:10. Cell lines were used within passage 10.RNA was transfected using polyethyleneimine   according to the following protocol. Cells were plated at a density of 250,000 per well in a six-well plate 24 h before transfection, in DMEM complete medium. The day after, immediately before transfection, medium was replaced with 1 mL Opti-MEM (Thermo Fisher Scientific) per well. A transfection mix was prepared containing 400 ng RNA in 160 μL DMEM without serum and antibiotics, at room temperature. PEI  was added to the reaction, and the tube was briefly vortexed for 1 s and incubated at room temperature for 10 min. The tube was vortexed again for 1 s and added to the cells. Cells were harvested at 48 h for western blot and qRT-PCR analyses.DNA control transfections were carried out in parallel using 1 μg plasmid DNA (pCS2-miniSINEUP-DJ-1 or pCS2 empty) and 3 μL Lipofectamine 2000 (Thermo Fisher Scientific) in 200 μL Opti-MEM. In this case, transfection medium was changed 6 h after transfection and replaced with 2 mL DMEM complete medium per well. Cells were harvested at 48 h.Cell pellets were lysed in lysis buffer (PBS + 1% Triton X100) with cOmplete protease inhibitor (Roche) on ice, briefly sonicated on ice, and centrifuged at maximum speed for 20 min at 4°C. Supernatants containing total lysates were collected on ice and quantified for total protein contents using the BCA assay kit (Thermo Fisher Scientific). Total lysate (10 μg) was loaded on NuPAGE 10% Bis-Tris, 1.5 mm, Protein Gel, 10-well (Thermo Fisher Scientific) and run at 120 V for approximately 90 min for SDS-PAGE. Gels were transferred to a 0.2 μm nitrocellulose membrane (Amersham) at 250 mA for 90 min. DJ-1 was detected with mouse anti-DJ-1 primary antibody (Enzo Life Sciences), and actin was detected with rabbit anti-β-actin primary antibody (Sigma Aldrich), both diluted 1:8,000 in 5% BSA in Tris-buffered saline-Tween 20 (TBST) and incubated overnight at 4°C. Horseradish peroxidase conjugated secondary anti-mouse and anti-rabbit antibodies were diluted 1:10,000 and incubated at room temperature for 1 h. Signals were detected using Pierce ECL plus detection reagent (Thermo Fisher Scientific) and read on ChemiDoc (Bio-Rad). Band intensities were calculated using ImageJ (NIH) and Image Lab (Bio-Rad) software. Data from a minimum of three independent experiments were analyed statistically using t tests.Total RNA was extracted using the RNeasy Mini Kit, and samples were then quantified using UV-vis spectrophotometry. DNase digestion was then performed adding 1 μL Turbo DNase I (Sigma Aldrich) and 1 μL 10× DNase I buffer to 800 ng RNA in a 10 μL reaction and incubating for 15 min at room temperature. DNase was inactivated by adding 1 μL DNase I stop solution and incubating for 10 min at 70°C. DNase-treated RNA  was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions, in a 20 μL reaction. qRT-PCRs were then set up, including 2 μL cDNA, 5 μL iTaq Universal SYBR Green Supermix (Bio-Rad), 0.4 μL forward primer, and 0.4 μL reverse primer, in a total volume of 10 μL. qRT-PCR was run on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using the following thermal cycling conditions: 98°C for 30 s, then 40 cycles at 95°C for 10 s and 60°C for 30 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference to normalize the results. Data from a minimum of three independent experiments were analyzed statistically using two-way ANOVA or t tests as appropriate.Isolated RNA was digested to component nucleosides with an enzyme cocktail of Benzonase, phosphodiesterase, and alkaline phosphatase (Merck), all used according to the manufacturer’s instructions. Nucleosides were separated by reverse-phase liquid chromatography; eluent A was 0.1% v/v formic acid in water, and eluent B was 0.1% v/v formic acid in acetonitrile, and a non-linear gradient of 2%–15% B resolved nucleosides across an Acquity HSS T3 C18 column (Waters). The eluent was sprayed into a 4500 triple quadrupole mass spectrometer (Sciex) and characterized using tandem mass spectrometry with a multiple reaction monitoring approach. Injection amounts were normalized by internal calibration with isotopically labelled uridine and quantification extrapolated from external calibration of a range of nucleoside standards using MultiQuant software (Sciex).2 flow of 4.0 L/min. RNA was defrosted immediately before the experiments and diluted in 10 mM phosphate buffer at pH 7.2 with 5 mM MgCl2 to reach a concentration between 100 and 150 ng/μL. CD spectra were acquired in duplicate or triplicate at 25°C and are reported as the average of at least 10 scans. All spectra were blanked for the buffer signal, corrected for the concentration and number of nucleotides, and expressed as molar ellipticity. The thermal denaturation analysis was performed monitoring the changes of the CD signal at 270 nm between 25°C and 100°C, using a 1°C/min gradient. The apparent melting temperature was derived by fitting the data according to the literature.Far- and middle-UV CD spectra of RNA molecules were recorded on a JASCO-1500 spectropolarimeter using a 1 mm path length quartz cuvette and a constant N


Non-coding RNAs are regarded as promising targets for the discovery of innovative drugs due to their abundance in the genome and their involvement in many biological processes. Phytochemicals (PCs) are the primary source of ligand-based drugs due to their broad spectrum of biological activities. Since many PCs are heterocyclic and have chemical groups potentially involved in the interaction with nucleic acids, detailed interaction analysis between PCs and RNA is crucial to explore the effect of PCs on RNA functions. In this study, an integrated approach for investigating interactions between PCs and RNAs were demonstrated to verify the RNA-mediated PCs functions by using berberine (BRB) as a model PC. RNA screening of a transcriptome library followed by sequence refinement found minimal RNA motif consisting of a cytosine bulge with U-A and G-U neighbouring base pairs for interaction with BRB. NMR-based structure determination and physicochemical analyses using chemical analogues of BRB demonstrated the importance of electrostatic and stacking interactions for sequence selective interaction and RNA stabilization. The selective interaction with a relatively small RNA motif based on a chemical structure of a planer heterocyclic highlights the biological activities of various PCs mediated by the interactions with particular functional RNAs. In addition, the systematic and quantitative investigations demonstrated in this study could be useful for the development of therapeutic chemicals targeting functional RNAs, based on the PCs, in the future. Currently, therapeutic drugs exist mainly in the form of target proteins . Howeveret al. identified the preferred RNA structure for a particular ligand from 3 000 000 combinations of RNA motif–ligand interactions , which are often derived from the secondary metabolites of plants, show a broad spectrum of biological activities, such as antioxidant, antibacterial and antitumor properties ,19. PCs 6 M−1 at 25°C. Subsequently, studies of BRB with different variants of RNA secondary structures showed that neighbouring base pairs of the C-bulge, which contains one wobble base pair, are essential for the interaction. An NMR study clarified that BRB induced a well-organized duplex by stacking between the base pairs at the C-bulge site. Further investigations using BRB derivatives demonstrated the energetic correlation between the thermodynamic stability of the RNA and the ligand binding affinity, highlighting the importance of electrostatic and stacking interactions for the stabilization of RNA. The systematic and quantitative approach used in the present study to determine the RNA structure motifs for a specific PC interaction explores a mode of potential biological activity of PCs other than that targets proteins and provides beneficial information for future RNA-targeting drug discovery based on PCs.Regarding the screening of RNAs that interact with small chemicals, we recently developed a technique for constructing RNA-capturing microsphere particles (R-CAMPs) ,35. R-CAAll DNA oligonucleotides used in this study were purchased from Fasmac Co., Ltd.  or Japan Bio Services Co., Ltd. . RNA oligonucleotides were purchased from Fasmac Co., Ltd. or GeneDesign, Inc.  as HPLC purification grade. BRB was purchased from Sigma-Aldrich Co. . Coptisine (COP), palmatine (PAM) and epiberberine (EBRB) were purchased from Nagara Science Co., Ltd. . For other chemical reagents, a grade appropriate for biochemical experiments was used.2, 5 mM DTT, 2 mM spermidine, 1 mM rNTPs, 0.01% Tween-20 and 1 U/μL T7 RNA polymerase . The transcription reaction and co-transcriptional capturing of nascent RNA transcripts on MPs were performed by incubating the reaction mixture at 25°C for 20 min. The transcription reaction was terminated by the addition of 800 nM competitive T7 promoter duplex (5′-GAAATTAATACGACTCACTATA-3′ and 5′-TATAGTGAGTCGTATTAATTTC-3′). After termination, the buffer solution in which R-CAMPs had been dispersed was replaced with 50 mM MES-LiOH (pH 7) containing 0.01% Tween-20 by three rounds of washing and centrifugation. The R-CAMPs were then kept on ice. Control MPs, which do not display RNA, were prepared by performing the same procedure without the addition of T7 RNA polymerase.Template cDNA library consisting of sequences derived from transcriptome RNA was prepared from total RNA of a human cervical cancer cell line (HeLa). A detailed procedure for preparing the cDNA library is described in supporting information. Preparation of R-CAMPs from the cDNA library was performed according to the previously established protocol with some modifications ,35. Deta2, 1 μM BRB and 100 ng/μl competitive RNA from bacteriophage MS2 , 0.1% DMSO and 0.01% Tween-20, and kept at room temperature for at least 60 min. The fluorescence signals of BRB on the R-CAMPs were analysed by fluorescence-activated cell sorting (FACS) using FACS Aria II . Single particles were distinguished by a fluorescence signal of AF647 chemically modified on the capture primer. The AF647 signal was detected using a 633-nm excitation laser and a 660/20-nm bandpass filter. The fluorescence signals of BRB on each particle were analysed using a 488-nm excitation laser and a 530/30-nm bandpass filter. R-CAMPs showing strong fluorescence of BRB were selected and sorted one-by-one into the wells of a 96-well plate  containing 10 μl of H2O.R-CAMPs or control MPs were resuspended in a buffer containing 50 mM MES-LiOH (pH 7), 100 mM KCl, 0.5 mM MgCl2O. Aliquots of the diluted samples (1 μl) were mixed with Thunderbird qPCR Mix  containing A-primer and B-trP1 primer (4 pmol). DNA amplification profiles were subsequently evaluated using the Mx3000P qPCR system to estimate the number of PCR cycles that showed signal saturation. Based on the number of PCR cycles showing saturation, template DNAs for the transcription of sorted RNAs were prepared by 20 or 24 cycles of PCR amplification using KOD-Plus-Ver.2. The second PCR products (1 μl) were diluted 10-fold in transcription buffer containing 40 mM Tris-HCl (pH 8), 8 mM MgCl2, 5 mM DTT, 2 mM spermidine, 1 mM rNTPs, 0.01% Tween-20 and 1 U/μl T7 RNA polymerase. The transcription of the sorted RNAs was performed at 37°C for 60 min. RNA samples in the transcription buffer were then diluted 10-fold in a buffer containing 50 mM MES-LiOH (pH 7), 100 mM KCl, 0.01% Tween-20 and 0.5 U TURBO DNase (Thermo Fisher Scientific), and then incubated at 37°C for 30 min. RNA samples (6.25 μl) were mixed with buffer (3.75 μl) containing 50 mM MES-LiOH (pH 7), 100 mM KCl, 2.67 μM BRB, 0.267% DMSO and 0.01% Tween-20. Although the mixed samples were crude, containing dNTPs, rNTPs, DNA and RNA polymerases, the mixed solution, containing 50 mM MES-LiOH (pH 7), 100 mM KCl, 0.5 mM MgCl2, 1 μM BRB, 0.1% DMSO and 0.01% Tween-20, was the same with the buffer at R-CAMPs sorting except for competitive RNA from bacteriophage MS2. The mixtures were incubated at 25°C for 60 min. The fluorescence signals of BRB in the mixtures were measured using a fluorescence microplate reader  at 365 nm excitation and 550 nm emission.DNA on sorted R-CAMPs was directly amplified in 96-well plates adding 10 μl of reaction solution for KOD-Plus-Ver.2  containing A-primer and B-trP1 primer (6 pmol). After 20 cycles of PCR amplification, the samples were diluted 100-fold with HDNA templates prepared by PCR for the transcription of sorted RNAs were purified using the QIAquick PCR Purification Kit . The purified DNA was mixed with A-primer, and the sequences were analysed using a contract service . Sequence alignment was performed on the Basic Local Alignment Search Tool (BLAST) website.2, 5 mM DTT, 2 mM spermidine, 1 mM rNTPs, 0.01% Tween-20 and 1 U/μl T7 RNA polymerase at 37°C for 60 min. Fluorescence analysis of BRB mixed with the RNA transcripts was performed using the same process used to evaluate the sorted RNAs.DNA templates for transcribing designed RNAs and their truncates were prepared by primer extension or PCR amplification using the oligonucleotides listed in 2, 0.1% DMSO and 0.01% Tween-20 in the presence of the indicated concentrations of KCl, and subsequently incubated at 70°C for 5 min and cooled to 25°C at 1°C min−1. The fluorescence intensity of BRB in the mixture was measured in a 384-well plate for a small volume sample  using InfiniteM200 PRO at 365 nm excitation and 550 nm emission after incubation at 25°C for 60 min. The observed binding constant (Kobs) between BRB and RNA at 25°C was calculated from changes in the fluorescence signals depending on the RNA concentration according to equation Variants of RNA duplexes were prepared by annealing the sense and antisense strands listed in 2HPO4 buffer (pH 7) with the addition of 100 mM NaCl, 1 mM EDTA and 10% 2H2O. The final RNA concentration of individual strands in 300 μl was 300 μM. 1D 1H and 2D NOESY and TOCSY spectra were acquired at 25°C utilizing excitation sculpting solvent suppression. The processing and assignment of spectra were performed using NMRPipe and CcpNmr software.NMR data of RNA and its complex with BRB were collected on an Agilent VNMRS 800 MHz NMR spectrometer equipped with a cryogenic probe. RNA oligonucleotides were dissolved in 10 mM Na−1Å−2 for hydrogen bonds, 20 kcal·mol−1Å−2 for NOE distance restraints, 100 kcal·mol−1rad−2 for backbone and 100 kcal·mol−1rad−2 for the χ torsion angle. All cross-peaks in imino-aromatic, imino-imino, amino-imino and amino-aromatic regions were classified as strong (2.5 Å), medium (3.5 Å) or weak (4.5 Å), with restraint ranges of ±1–1.5 Å. The dihedral angle χ was restrained to an anti range between 90° and 270° for all nucleotides. The restraints for the backbone dihedral angles included α (-120° to 120°), β (150° to 210°), γ (30° to 90°), ϵ (170°-300°) and ζ (-120° to 120°). Bulged nucleobases were excluded from backbone torsion angle restraints. Depending on the sugar pucker (derived from the TOCSY spectra), the δ torsion angle was restrained to the C3′-endo range between 55° and 115° or to the C2′-endo range between 130° and 190°. Ten structures were selected based on the lowest energy and subjected to energy minimization with a maximum of 1000 steps. Molecules were visualized using Chimera software.The molecular dynamics (MD) were calculated in AMBER 20 software using the ff99OL3 force field. The force-field parameters for BRB were derived from the RESP ESP Charge Derive Server. The initial linear structures of oligonucleotides, created with the LEAP module of AMBER, were subjected to MD with a gradual turning on of the restraints until a duplex was obtained. Subsequently, one BRB molecule was added 20 Å off the central axis of the duplex and used as a starting structure. A total of 100 structures were obtained in 1-ns restrained, simulated annealing simulations using the Born implicit solvent model with random starting velocities. In the first 200 ps of simulated annealing, the temperature was kept at 1000 K, followed by slow cooling in the next 400 ps down to 300 K and to 0 K in the last 400 ps. The force constants were 20 kcal·mol2HPO4 (pH 7), 100 mM NaCl and 1 mM EDTA to a concentration of 10 μM and placed in an ITC cell. RNA sample (100 μM) dissolved in a buffer containing 10 mM Na2HPO4 (pH 7), 100 mM NaCl, 1 mM EDTA and 0.1% DMSO was prepared by incubating at 70°C for 5 min, cooled to 25°C at 1°C min−1 and collected in an ITC syringe. The injection of RNA (2 μl) was performed 19 times at time intervals of 150 s. The thermogram obtained was normalized by subtracting that obtained by RNA titration into the buffer without ligand and fitted using MicroCal software following a single binding site model.The thermodynamic parameters for the interaction between RNA and ligands were determined using MicroCal iTC 200 . A 10 mM stock solution of ligands in DMSO was diluted 1000-fold into a buffer containing 10 mM Na2HPO4 (pH 7), 100 mM NaCl, 1 mM EDTA and 0.1% DMSO in the absence or presence of 10 μM ligand with a heating rate of 0.5°C min–1 using a Shimadzu-1800 UV/Vis spectrophotometer equipped with a temperature controller. The thermodynamic parameters were calculated from the melting curves following the two-state melting transition model of oligonucleotides (The melting of RNA oligonucleotides (2 μM) was measured by absorption at 260 nm in a buffer containing 10 mM Naleotides .In our previous strategy for the optimization of RNA aptamers using R-CAMPs, we used an artificially designed library containing randomized mutations at the region, which contribute to the interaction between RNA and small molecules, based on the tertiary structure of the aptamers ,35. HoweFigure Since each of the sorted R-CAMPs displayed both DNA and RNA clones on the surface, PCR amplification was performed to prepare the cloned template DNA. Unfortunately, not all well positions, where the R-CAMPs were sorted, showed an amplification profile when the amplification process was monitored by quantitative real-time PCR . A totalAll 42 RNAs, which were displayed on the sorted R-CAMPs, were individually transcribed from the amplified template DNA and subsequently mixed with BRB to verify the fluorescence signal of BRB in solution . All theThe RNA sequences of the eight samples were analysed and verified to determine which RNA transcripts encoded the sequences . As a reFour RNA samples sorted into the well positions A1, A10, C7 and C10, which showed a particularly strong fluorescence of BRB , were seKobs) of 2.79 and 0.98 × 106 M−1 for RNA-A and RNA-B, respectively  and uracil in RNA-H, at the 3′ neighbouring position , with exchanged base pairs at the 3′-end, and RNA-L (1.88 × 106 M−1), with exchanged base pairs at the 5′-end, were comparable with those obtained by RNA-A , palmatine (PAM) and epiberberine (EBRB)  was not so large for any of the ligands, the ligands were found to significantly enhance the thermodynamic stability of RNA-A at 25°C (ΔG°25 (melting)) by decreasing the enthalpy change of the melting (G°25 (melting) in the presence of the ligand compared to that in its absence (ΔΔG°25 (melting)). The ΔΔG°25 (melting) values for BRB, COP, PAM, and EBRB, were −2.0, −1.8, −1.3 and −1.0 kcal mol−1, respectively  Table ) were alstrength , was usestrength , the thestrength  37–39)..1H NMR s melting . Energet2HPO4 (pH 7), 100 mM NaCl, 1 mM EDTA and 0.1% DMSO, which was the same as the buffer used for the UV melting assay  obtained from the ITC thermograms was 0.80–0.91. The interaction of all BRB analogues was driven by a favourable enthalpic contribution (negative ΔH(ITC) value), likely due to stacking and electrostatic interactions. The order of the equilibrium association constant at 25°C obtained from the ITC profile (KA(ITC)) was BRB > COP > PAM > EBRB. These results indicate the importance of the dioxirane ring of BRB to enhance its interaction, despite not having a direct interaction with RNA-A  value) of the interaction. By comparing BRB and COP, the dimethoxy group of BRB on the other side of the dioxolane group was found to make a small contribution to enhance the affinity. Although the dioxolane group showed no direct interaction, it may contribute to the interaction via the rearrangement of the solvation state, such as hydrated water networks (G°25 (ITC), which was obtained by ITC using a theoretical equation of ΔG°25 (ITC) = ΔH(ITC) – TΔS(ITC), and ΔΔG°25 (melting)  in a buffer containing 10 mM NaA Figure . The connetworks . A lineaIn summary, we have demonstrated an integrated approach of physicochemical and structural investigations that enable insight into the interaction between particular RNA motifs and phytochemicals. The RNAs that interacted with medicinally important PCs, such as BRB, was screened using R-CAMPs, and quantitatively explored as an RNA motif of a single C-bulge with U-A and G-U neighbouring base pairs. NMR analysis elucidated the importance of the electrostatic and stacking interactions for stabilizing the RNA that was further demonstrated through the physicochemical analyses. Based on the results, not only BRB but also natural phytochemicals, which are planar heterocycles with electropositive atoms, such as derivatives of protoberberine and quinolinium ,58, haveAtomic coordinates and list of chemical shifts for the RNA-A/berberine structures have been deposited with the Protein Data bank and the Biological Magnetic Resonance Bank with accession numbers PDB ID 7A3Y, BMRB ID 34553.gkab189_Supplemental_FileClick here for additional data file.