Background: Ovarian cancer is the most lethal gynecologic malignancy. There is a lack of comprehensive investigation of disease initiation and progression, including gene expression changes during early metastatic colonization. Methods: RNA-sequencing (RNA-seq) was done with matched primary tumors and fallopian tubes (n = 8 pairs) as well as matched metastatic and primary tumors (n = 11 pairs) from ovarian cancer patients. Since these are end point analyses, it was combined with RNA-seq using high-grade serous ovarian cancer cells seeded on an organotypic three-dimensional (3D) culture model of the omentum, mimicking early metastasis. This comprehensive approach revealed key changes in gene expression occurring in ovarian cancer initiation and metastasis, including early metastatic colonization. Results: 2987 genes were significantly deregulated in primary tumors compared to fallopian tubes, 845 genes were differentially expressed in metastasis compared to primary tumors and 304 genes were common to both. An assessment of patient metastasis and 3D omental culture model of early metastatic colonization revealed 144 common genes that were altered during early colonization and remain deregulated even in the fully developed metastasis. Deregulation of the matrisome was a key process in early and late metastasis. Conclusion: These findings will help in understanding the key pathways involved in ovarian cancer progression and eventually targeting those pathways for therapeutic interventions. Ovarian cancer (OC) is the deadliest gynecologic malignancy and a highly heterogenous disease ,2,3. HigFor a more comprehensive understanding of the disease, one has to start from its tissue of origin. For many years, HGSOC was thought to originate from the ovarian surface epithelium. However, strong evidence for fallopian tube (FT) fimbria as the tissue of origin was uncovered when careful study of BRCA mutation carriers and later, HGSOC patients, revealed serous tubal intraepithelial carcinoma (STIC) in their FT fimbria as precursor lesions ,8,9. MorDue to these facts, a similar comprehensive approach comparing the transcriptome of fallopian tube with primary tumors and metastasis would be beneficial in revealing the oncogenic pathways altered, independent of genetic alterations. Since it has been demonstrated that accumulation of genetic changes is limited during metastasis , an RNA-RNA isolated from eight pairs of matched normal FT and primary tumors from OC patients were sequenced to identify the transcriptome changes during carcinogenesis. Similarly, RNA isolated from eleven pairs of matched primary and metastatic tumors were sequenced. Patient characteristics are included in A total of 2987 genes were differentially expressed in the primary tumor samples versus FT with a significance level of false discovery rate (FDR) < 5% and absolute fold change ≥ 2 , whereasGene set enrichment analysis (GSEA) was done for the hallmark gene sets to identify the deregulated pathways during OC progression. 15 gene sets were positively correlated with primary tumors versus FT at FDR < 25% while four gene sets were negatively correlated at FDR < 25%. The most significantly correlated gene sets in primary tumors versus FT were E2f transcription factor (E2F) targets (enrichment score (ES) = 0.804), G2M Checkpoint (ES = 0.776) and MYC targets V1 (ES = 0.692) C. Taken Other significant Hallmark gene sets positively correlating with primary tumors versus FT were mTOR complex 1 (MTORC1) signaling, MYC targets V2, DNA repair and oxidative phosphorylation . AdditioThe primary tumor versus FT and metastasis versus primary tumor data for the top deregulated genes B were fuAs the disease progresses from the formation of the primary tumor to the dissemination and development of the metastasis, it is expected to be associated with alterations in gene expression. Our analysis of sequencing data from patient specimens showed that the formation of primary tumors resulted in deregulation of 2987 mRNAs . The proEPYC, COL10A1 and PLPP4, which were upregulated in both metastasis versus primary and primary versus FT were all confirmed by qRT-PCR. All three genes led to poor patient prognosis when overexpressed (The common upregulated genes in metastasis and primary tumor include epiphycan (EPYC), collagen type X alpha chain 1 (COL10A1), fibronectin type III domain containing 1 (FNDC1) and podocan-like protein I (PODNL1), which are mainly extracellular matrix related genes. These findings further signify the importance of the tumor microenvironment in the progression of OC. The common downregulated genes in metastasis and primary tumor include anterior gradient protein 3 homolog (AGR3) and Sentan cilia apical structure protein (SNTN). Of the 2683 genes that were unique to primary versus FT and 541 genes unique to metastasis versus primary, the top 10 upregulated and downregulated ones are listed in xpressed A.Among the genes downregulated in both the datasets, AGR3 and STN were confirmed to be downregulated in metastasis by qRT-PCR, but collagen type XXVIII alpha 1 chain (COL28A1) did not validate this B. SimilaThe comparison of metastatic tumors with primary tumors of OC patients provides an end-point analysis of the differential gene expression occurring during metastasis. However, metastasis is a multi-step process and the knowledge of the regulation of the individual steps remains limited. The important, rate-limiting step of metastasis is the step of metastatic colonization . At thisSome of these changes may be transient, while others may be sustained and essential for the metastatic tumor development. Therefore, this dataset was compared with the differentially expressed genes in the end-point analysis of metastasis versus primary tumors to identify those genes essential for both early colonization, as well as for the advanced metastasis D. The 14In the current study, we did a comprehensive RNA-seq analysis of normal fallopian tubes, primary tumors and metastasis from OC patients, in combination with an organotypic 3D omental culture model for early metastatic colonization. While much information is available on the gene expression profiling of OC primary tumors compared to normal tissue, the information available about metastasis is still limited . TherefoIt has been well documented that the genes coding for ECM components undergo deregulation in tumor progression ,33,34,35Collagen acts as a scaffold in tumor ECM and the synthesis and degradation of collagen largely impacts the metastatic attributes of cancer cells. Collagen is found in abundance in the ECM ,41. The Focal adhesions mediate the contact between cell and extracellular matrix, helping anchor the cell to the substratum and promote motility, playing a major role in metastasis ,51,52,53Primary tumors differentially expressed 2987 genes as compared to the normal fallopian tubes. However, the process of metastasis resulted in changes in the expression of only 845 genes. Of these, 304 genes overlapped with those deregulated in primary tumors versus fallopian tubes. An interesting observation in our study was that these 304 common deregulated genes in primary tumors versus normal fallopian tube and metastasis versus primary tumors were not necessarily altered in the same direction . This imA key factor to consider is the different microenvironments of the primary tumor and the site of metastasis. Since gene expression changes can be regulated by the signals emanating from the microenvironment, this could be an important reason for the differences in metastasis. Such changes may not be reflected by genetic mutations in the cancer cells. The metastasizing cancer cells have to adapt to the new microenvironment and this is stimulated by the productive cross-talk with the microenvironment ,59. Our Dulbecco’s Modified Eagle Medium (DMEM), minimal essential medium vitamins, minimal essential medium nonessential amino acids, Trypsin and Penicillin–Streptomycin were obtained from Media Tech .Matched pairs of fresh frozen primary tumors and normal fallopian tubes from eight OC patients were obtained from the Indiana University Simon Cancer Center Tissue Bank. Similarly, matched pairs of fresh frozen primary tumors and metastasis from 11 OC patients were also obtained from the Indiana University Simon Cancer Center Tissue Bank. RNA was isolated from these tissues using miRNeasy kit  following the manufacturer’s protocol and submitted to the Center for Genomics and Bioinformatics core facility, Indiana University, Bloomington for library preparation and sequencing. The study was approved by the Institutional Regulatory Board of Indiana University (protocol numbers 1106005767 and 1606070934). Patient written consent was obtained and tissues were collected by Indiana University Simon Cancer Center Tissue Bank. Only deidentified patient specimens were provided by the tissue bank.Human HGSOC cell lines OVCAR8 were acquired from Ernst Lengyel at the University of Chicago and OVCAR4 was from Joanna Burdette, University of Illinois at Chicago. Kuramochi was procured from Japanese Collection of Research Bioresources. The cell lines used were genetically validated and tested to be mycoplasma free using respective services from Idexx BioResearch . The genetic validation was done using the CellCheck 16 (16 Marker STR Profile and Inter-species Contamination Test) and mycoplasma testing was done using Stat-Myco.5 cells) were mixed with 91 μg Collagen I  and seeded in a 10 cm dish and allowed to attach for 4 h. Thereafter, 3.6 × 106 HPMCs were overlaid on them to form a confluent monolayer. This 3D omentum culture mimicking the surface layers of the omentum was allowed to grow for 24 h so that the cells could secrete ECMs and other factors to form a complex microenvironment. GFP labelled OC cells were then seeded on the 3D omentum culture to mimic the initial steps of colonization and allowed to grow for 48 h. The cancer cells were then isolated by fluorescence activated cell sorting and compared to control cancer cells directly seeded on 10 cm tissue culture dishes and sham sorted. Total RNA was isolated using miRNeasy kit  and submitted for RNA-seq. Only those genes that were significantly differentially expressed (< 5% FDR and absolute fold change ≥ 2) in all three HGSOC cells were considered for further analysis. We chose to use Kuramochi, OVCAR4 and OVCAR8 as they have been reported as ‘likely/possibly’ HGSOC cell lines [Total RNA was isolated from a panel of HGSOC cell lines  grown in 3D omental cultures as described previously . Three ill lines . Moreovell lines ,63. TherLibrary preparation and next generation RNA sequencing was carried out at the Center for Genomics and Bioinformatics core facility, Indiana University, Bloomington. The library preparation was done using TruSeq Stranded mRNA HT Sample Prep kit  according to the manufacturer’s protocol and 8-nucleotide barcodes were added for multiplexing. The barcoded libraries were cleaned by bead cut with AMPure XP beads , verified using Qubit3 fluorometer  and 2200 TapeStation bioanalyzer , and then pooled. The pool was sequenced on NextSeq 500 (Illumina) with NextSeq75 High Output v2 kit .https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) and Metascape (metascape.org). Metascape is a web-based tool that has been under development since 2014 [10 . Interaction modules were also clustered on the basis of functional similarity by Metascape.Reads were adapter trimmed and quality filtered using Trimmomatic ver. 0.33 , with thnce 2014 . It enabGene set enrichment analysis was conducted using GSEA Desktop ver. 3.0, build 0160 . HallmarHeat maps were generated using the heatmap.2 function from R programming tools (gplots).All RNA-seq data have been made available to the public .Taken together, our RNA-seq data has generated a wealth of information in the form of crucial pathways and targets which can be further explored to unravel the molecular mechanisms regulating the OC metastasis. Overall, the results of this study emphasize the need to further explore the tumor microenvironment and extracellular matrix components in the progression of OC metastasis. OC patients typically present with the whole spectrum of metastasis ranging from early lesions to advanced metastasis. Targeting key components of the pathways identified in


Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.  Arenaviridae , three fragments such as the order Bunyavirals , or multiple fragments such as the Orthomyxoviridae . Examples of families of non-segmented negative-sense RNA viruses with a single-stranded RNA genome  include the Filoviridae , the Paramyxoviridae ), and the Pneumoviridae . Infection with negative-sense RNA viruses is associated with a broad spectrum of human diseases. Influenza and respiratory syncytial virus (RSV) are recognized as widespread global pathogens, while others, such as Ebola virus (EBOV), Lassa virus (LASV), or Crimean–Congo hemorrhagic fever virus (CCHFV) are known for sporadic outbreaks, their high epidemic potential, and their high mortality rates . NegativPrevention and treatment of infection with negative-sense RNA viruses remain challenging. The 2014 Ebola virus disease (EVD) outbreak in West Africa caused approximately 28,000 cases and 11,310 deaths and is a sober reminder of an unmet medical need . HoweverFavipiravir (T-705) is a nucleoside precursor that has been approved for treatment of pandemic influenza in Japan ,18,19. I50) in the submicromolar range -GTP  would reach its maximum after 30 min. The amount of RSV and h-mtRNAP was optimized to have the similar apparent activity as EBOV RdRp by testing various amounts of enzymes. This involved employing low-micromolar concentrations of NTP substrates in RNA synthesis reactions containing h-mtRNAP. RNA synthesis assay consisted of mixing  Tris-HCl , RNA primer (200 μM), RNA template (1 μM), [α-32P]-GTP (0.1 μM), various concentrations and combinations of NTP and NTP analogues, and EBOV RdRp (1–2 μL) on ice. Note that DNA templates were used in reactions containing h-mtRNAP. The reaction mixtures (10 μL) were incubated for 10 min at 30 °C followed by the addition of 5 μL of MgCl2 (5 mM). The reactions were stopped after 30 min by the addition of 15 μL of formamide/EDTA (50 mM) mixture and incubated at 95 °C for 10 min. The 3 μL reaction samples were subjected to denaturing 8 M urea 15% polyacrylamide gel electrophoresis to resolve products of RNA synthesis followed by signal quantification  through phosphorimaging . Incorporated nucleotide product fraction was plotted versus nucleotide substrate concentrations and fitted to the Michaelis–Menten equation using GraphPad Prism 7.0 .Data acquisition and quantification were also done as previously reported by us . To acco32P]GTP that serves as the first substrate in the reaction. The next site of incorporation, referred to as i, allows binding of ATP or the inhibitor remdesivir-TP. Full-length product formation requires the simultaneous presence of ATP and CTP. RNA synthesis was analyzed with three different concentrations of the natural substrates ATP and CTP   B. IncreaVmax and Km (Vmax/Km is a measure for the efficiency of AMP or remdesivir-MP incorporation and the ratio of Vmax/Km (AMP) over Vmax/Km (remdesivir-MP) defines the selectivity. Selectivity values of ~4 for EBOV RdRp and ~3 for RSV RdRp demonstrate that remdesivir-TP is almost as efficiently used as ATP. In contrast, human mitochondrial RNA polymerase (h-mtRNAP) discriminates against the inhibitor (We measured the efficiency of incorporation of the monophosphate (MP) AMP and remdesivir-MP at position i with a modified template that does not support base-pairing of adenosine analogues at position i+1. This template allowed us to study single nucleotide incorporations with both EBOV and RSV enzyme complexes A. Increax and Km . The ratnhibitor B,C. The nhibitor .m value. The maximum rate of product formation (Vmax) is almost identical at saturating concentrations of substrate. Hence, the obstacle can be overcome and chain termination is unlikely a possible mechanism of action for both enzymes. At most, the RSV enzyme seems to pause following incorporation of remdesivir-MP.The incorporated nucleotide analogue inhibitor can potentially affect incorporation of the next nucleotide. However, the presence of a 3′-hydroxyl group in remdesivir-TP would theoretically allow the nucleophilic attack on the α-phosphate of an incoming nucleotide. To quantify a possible kinetic effect of remdesivir on subsequent incorporation events, we terminated the primer with AMP and remdesivir-MP and measured rates of incorporation of the natural UTP substrate at position i+1 A. For EBWe next studied a potential distant effect of the incorporated remdesivir-MP. To this end, we devised longer templates that allowed synthesis of a 14-mer full-length product . To reduWe also studied whether delayed chain termination at position i+5 can be overcome with higher concentrations of the natural nucleotide substrate at position i+6 . Dose–reWe finally compared the inhibition pattern of remdesivir-TP with ara-ATP and 2′C-methyl-ATP . Ara-CTPRemdesivir or GS-5734 is a nucleotide analogue prodrug that is currently being evaluated in clinical trials for the treatment of EVD ,45. Prec50 values in cell-based assays. Remdesivir inhibits EBOV and RSV replication in cell-based assays with EC50 values of ~100 nM [50) of remdesivir are generally >100 µM for various cell types [50 values >100 µM [The broad spectrum of antiviral activities associated with remdesivir raises the question of whether delayed chain termination is a unifying mechanism of inhibition. Previous data have shown that the inhibitor is also incorporated by RSV RdRp . The inhll types ,47; howell types . Low inh >100 µM .In conclusion, this study provides evidence to show that delayed chain termination is a plausible mechanism of action of remdesivir against EBOV. Delayed chain termination has been described earlier in the context of inhibition of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) and the hepatitis B virus (HBV) ,50,51. A


Conversely, RNA secondary structure can inhibit the binding of a PPR protein to its specific binding site. The parameters that influence whether PPR-RNA or RNA-RNA interactions prevail are unknown. Understanding these parameters will be important for understanding the functions of natural PPR proteins and for the design of engineered PPR proteins for synthetic biology purposes. We addressed this question by analyzing the effects of RNA structures of varying stability and position on the binding of the model protein PPR10 to its atpH RNA ligand. Our results show that even very weak RNA structures  involving only one nucleotide at either end of the minimal binding site impede PPR10 binding. Analysis of binding kinetics using Surface Plasmon Resonance showed that RNA structures reduce PPR10’s on-rate and increase its off-rate. Complexes between the PPR proteins PPR10 and HCF152 and their respective RNA ligands have long half-lives (one hour or more), correlating with their functions as barriers to exonucleolytic RNA decay in vivo. The effects of salt concentration on PPR10-RNA binding kinetics showed that electrostatic interactions play an important role in establishing PPR10-RNA interactions but play a relatively small role in maintaining specific interactions once established.Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA sequences via modular 1-repeat:1-nucleotide interactions. Binding specificity is dictated, in part, by hydrogen bonds between the amino acids at two positions in each PPR motif and the Watson-Crick face of the aligned nucleobase. There is evidence that PPR-RNA interactions can compete with RNA-RNA interactions  PPR proteins consist of tandem degenerate repeats of approximately 35-amino acids, each of which forms a helical hairpin. Consecutive repeats stack to form an elongated superhelix that binds single-stranded RNA. The sequence specificities of PPR proteins are, to some extent, predictable and customizable. Each repeat binds a single nucleotide, with nucleotide specificity dictated by the identities of two amino acids: the sixth amino acid in a given PPR motif and the first amino acid in the next (denoted as the 6 and 1’ amino acids according to the nomenclature in ref -ATP. The binding reactions contained 5 pM RNA, 40 mM Tris-HCl pH 7.5, 140 mM NaCl, 10% glycerol, 4 mM DTT, 10 U RNAsin, 0.1 mg/mL BSA, 0.5 mg/mL heparin, and protein at the indicated concentrations. Unless otherwise noted, binding reactions were incubated for 30 minutes at 25°C. Results were imaged with a Storm phosphorimager and quantified with Image Studio Lite. Curves were fit to the data using a nonlinear regression curve fit using Prism software. The sequences of the atpH-related RNAs are shown in petB RNA used in HCF152 binding assays had the following sequence: 5’ GGUAGUUCGACCGUGGA-3’.Gel mobility shift assays were performed as previously described , with miBiotinylated RNAs with a standard 6-carbon linker were purchased from IDT, with the following sequences:atpH: 5’-GAUUGUAUCCUUAACCAUUUCUUUU-3’ biotin;biotin5’-AUUGUAUCCUUAACCAUUUCUUUUUUUUUGAAAGA-3’3’-6bp: 5’-ACAAUGUUUUUUAUUGUAUCCUUAACCAUUUCUUU-3’-biotin;5’-6bp petB: 5’-UGGUAGUUCGACGUGGAUUUU-3’-biotin.atpH RNA, lane 3 was labeled with 5’-6bp RNA, and lane 4 was labeled with 3’-6bp RNA. Lanes 1–4 were analyzed in series with the same injections of PPR10 and the resulting data were statistically analyzed using a ratio paired t-test. For experiments that examined the effect of salt concentration on PPR10 binding kinetics, lane 1 was left blank for background subtraction and lane 2 was labeled with the atpH RNA; these data were statistically analyzed using a students t-test. For analyses of HCF152 with petB, lane 1 was left blank for background subtraction, lane 2 was labeled with atpH RNA and lane 4 was labeled with petB RNA; these data were statistically analyzed using a students t-test. The range of protein concentrations in each experiment were injected in a random order with buffer injections every third injection to be used as a second background subtraction. Bound proteins were washed off the chip between protein injections using 0.02% SDS in HBS buffer. Data were analyzed using the Biacore Evaluation software.SPR streptavidin chips (GE Sensor Chip SA) were labeled with 5 response units (RUs) of biotinylated RNA by injecting RNA (1 pM) in HBS buffer  at a rate of 10 μL/s for 10 seconds. This yielded a maximum of approximately 25 Response Units (RUs) upon protein injection, the value suggested by Katsamba et al for analysis of high affinity interactions . We usedS1 FigatpH-related RNAs diagrammed in (A) Predicted and measured stabilities of the (PDF)Click here for additional data file.S2 FigpetB RNA ligand (left) and HCF152’s interaction with PPR10’s atpH RNA ligand (right). (B) SPR analysis of PPR10 interaction with the 3’-6bp RNA ligand  Specificity controls for PPR10 and HCF152 SPR assays. Sensorgrams are shown for the analysis of PPR10 interaction with HCF152’s gand see . The exp(PDF)Click here for additional data file.


Using the m8Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m This allowed us to see the effect of the incorporation of a m8Gm on RNA and allowed us to understand the Z-RNA structure. The Z-RNA stabilizer can also be used to investigate the interaction of the Zα domain and Z-RNA.Several studies have been reported on incorporating modified nucleic acid residues for the stabilization of Z-DNA ,19,20,21leobases . Based o8Gm-incorporating oligonucleotides by phosphoramidite chemistry, using 2′-O-methyl guanosine  between C1′H and 8CH3, we confirmed the syn conformation of m8Gm (We synthesized the muanosine . The ind of m8Gm .4 concentrations.Circular dichroism (CD) spectroscopy has been used to study the Z-form conformation ,22. A-RN2 in A-form does not transfer to the Z-form, even in the presence of 3 M NaClO4 (8Gm-incorporated r(CGC[m8Gm]CG)2 showed a Z-form CD spectrum with increasing concentrations of NaClO4. (4 concentration for r(CGC[m8Gm]CG)2 was 880 mM, lower than that of the native RNA (4090 mM) (8Gm]C[m8Gm]CG)2 containing two m8Gms greatly stabilized the Z-form, showing a Z-form CD spectrum even in the presence of 50 mM NaClO4 M NaClO4 a. m8Gm-i NaClO4. b. The mi4090 mM) . r/r(CGCACG), in which the underlined sequences can form a duplex containing only one m8Gm base. We compared its stability with native RNA r(CGCGUGCG)/r(CGCACG). Although the 6 base pairs duplex containing an AU base pair does not favor the formation of Z-RNA, it facilitated the A- to Z-RNA transition as compared to native 6 base pairs RNA   , indicat8Gm effect on the thermodynamic properties of Z-RNA, the melting temperature (Tm) and thermodynamic parameters were examined using CD melting experiments. (Tm) values of m8Gm-incorporated hexamer RNA increased by 6–15 °C (ΔTm), , , and octby 15 °C . Thermodtive RNA  suggestius study . The advcleotide ,27.8Gm]CG)2. A complete list of 1H chemical shifts was shown in 8Gm]CG)2 is Z-RNA. We observed the strong cross-peaks in 2D-NOESY spectrum from intranucleotide H1′/H8 NOEs of G2 and G6 and a special NOE between C1′H and 8CH3 of m8Gm4. This indicated the syn conformation of all three guanosine residues  allows for a cross-peak assignment of Z-RNA nonexchangeable resonances (1 to G6 for the Z-form can complete the pathway: C1(H6/H5′′)-G2(H8/H1′)-C3(H6/H5′′)-m8Gm4(8CH3/H1′)-C5(H6/H5′′)-G6(H8/H1′) (The NOE connectivity path between H6/H5′′ of C and H8/H1′ of G (8CHsonances a–c. Sequ(H8/H1′) a–c, indi2 resonance was assigned based on NOESY spectra for sequential connections between H6/H8 and H′2 of C and G (5(NH2) amino proton has cross-peaks (C5(NH2)/C1–H5) to the C1–H5 proton, and the C1(NH2) amino proton has cross-peaks to the C5–H5 proton (C1(NH2)/C5–H5) (The H′ C and G . Moreove C and G d. The 2D)/C5–H5) c. Such c31P-NMR spectroscopy to confirm these observations. We found that these sequences as shown in 4 from 5 mM to 7 M (We further used M to 7 M . These r8Gm]CG)2 was constructed based on the reported Z-form structure and NOE-constrained refinement [8Gm-contanied Z-RNA, the C8-methyl groups were hydrophobic and located in the outside of the helix, which is consistent with the induction of a methyl group that strongly contributes to the increased stabilization of Z-RNA.A model of r.We carried out CD experiments by a JASCO J-820 CD spectrophotometer . The melting curves were obtained by monitoring 285 nm. 0.3 mL samples at 0.15 mM base concentration with 0.1–7 M NaClO2O/10% D2O, 5 mM sodium phosphate buffer (pH 7.0), 50 mM or 3 M NaClO4. 2D NOE spectra were recorded with mixing times of 500 ms.We performed NMR experiments on a Bruker AV-400 M spectrometer . A special Micro Tube that is designed for use with reduced sample volumes was used . Dissolve RNA samples (1.0 mM) in 150 μL of 90% HThe model of RNA structure was manually generated based on the reported structure using the BIOVIA Discovery Studio 4.5 . Then molecular dynamics simulations were performed by the standard dynamics cascade in BIOVIA Discovery Studio 4.5 with some modifications. The structure was heating from 50 K to 300 K over 4 ps and equilibration at 300 K with 100 ps simulation time. The save results interval in the production step was 2 ps during 100 ps simulation time at 300 K. 10 best conformations generated by simulation were further energy minimized. The conformation with lowest energy was selected as shown in 8Gm and Cy3-labeled RNA (1 μM each) with Zα-GFP fusion protein (10 μM) in a buffer, 10 μg/mL BSA and 10% (v/v) glycerol, 100 mM NaCl, 10 mM dithiothreitol, 10 mM Tris-HCl (pH 7.0). Nondenatured polyacrylamide gel (8%) is used and performed at 80 V and 4 °C in 1× TBE buffer with 20 mM NaCl.We performed the experiments using RNA containing m


No comparison data have been reported on viral and epidemiological profiles of hospitalized children with severe acute respiratory infection (SARI) in Beijing or Shanghai, China.We collected 700 nasopharyngeal aspirates (NPA) from hospitalized children with SARI in Beijing (northern China) and Shanghai (southern China). Multiple respiratory viruses (including 15 common viruses) were screened by validated polymerase chain reaction (PCR) or real-time reverse transcription-PCR assays and confirmed by sequencing. Demographic data and the distribution of viral infections were also examined.Of 700 samples, 547 (78.1%) tested positive for viral infections. The picornaviruses (PIC), which included rhinovirus (RV) and enterovirus (EV), were the most common (34.0%), followed by respiratory syncytial virus (RSV) (28.3%), human bocavirus (HBoV) (19.1%), adenovirus (ADV) (13.7%), human coronaviruses (HCoV) (10.7%), influenza A and B (8.9%), parainfluenza virus (PIV 1–3) (7.9%), and human metapneumovirus (HMPV) (5.0%). PIC (RV/EV) and RSV were the most prevalent etiological agents of SARI in both cities. The total and age-matched prevalence of RSV, HCoV, and hMPV among SARI children under 5 years old were significantly higher in Beijing than in Shanghai. Different age and seasonal distribution patterns of the viral infections were found between Beijing and Shanghai.Viral infection was tested and shown to be the most prevalent etiological agent among children with SARI in either the Beijing or the Shanghai area, while showing different patterns of viral and epidemiological profiles. Our findings provide a better understanding of the roles of geographic location and climate in respiratory viral infections in hospitalized children with SARI. Acute respiratory infections (ARI) are associated with significant morbidity and mortality worldwide, particularly in children under the age of 5 years , 2. SeveBeijing, the capital of the People’s Republic of China, has a population of more than 21 million. Beijing is located in the North of China on the Pacific Ocean, which stands at the northern tip of the North China Plain. Beijing has a semi-humid continental climate in the warm temperate zone. The spring and autumn are relatively short when compared with the duration of summer and winter. The annual temperature is around 11.8 degree Celsius, January can be considered as the coldest month in Beijing for average temperature at − 4.6 degree Celsius, while July will be the hottest month in Beijing. Shanghai is the largest city in China, with a population of more than 25 million. The city is located in the southeast region of the country and has a subtropical monsoon climate. Shanghai lies on China’s east coast roughly equidistant from Beijing. Shanghai’s climate is classified as humid subtropical and experiences four distinct seasons. Summer temperatures at noontime often hit 35–36 °C (95–97 °F) with very high humidity. Winters are chilly and damp, with northwesterly winds from Siberia can cause nighttime temperatures to drop below freezing. In between, spring can feature lengthy periods of cloudy, often rainy, weather, while Autumn is generally mild to dry and sunny. The city averages 4.8 °C (40.6 °F) in January and 28.6 °C (83.5 °F) in July, for an annual mean of 17.1 °C (62.8 °F).We previously reported the viral etiology of 370 children hospitalized with SARI in Beijing between May 2008 and March 2010 based on an xTAG® RVP FAST assay . The preThe study protocol was approved by the hospitals’ Ethics Committees and the Chinese Center for Disease Control and Prevention. Participants received a document entitled “Written Informed Consent” describing the study’s purpose and their right to keep information confidential. Written consent was obtained from all of the participants or their guardians.SARI surveillance was conducted in Beijing Children’s Hospital in Beijing and the Children’s Hospital of Fudan University in Shanghai, China between May 2008 and March 2014. All of the patients included in the study were younger than 14 years of age and were diagnosed with SARI according to the World Health Organization (WHO) case definition of a history of symptoms for ≤72 h , 15. EliWe collected 700 nasopharyngeal aspirates (NPAs) between May 2008 and March 2014. There were 259 NPAs from inpatients admitted to Beijing Children’s Hospital, the largest pediatric hospital in northern China, and 441 from children admitted to the Children’s Hospital of Fudan University, the largest pediatric hospital in southeast China. It should be noted that several of the SARI patients in our study resided in areas around Beijing and Shanghai. Informed consent was obtained from the parents of all of the participants before samples were collected. NPAs were collected on the day of admission, placed in a viral transport medium, and stored at − 70 °C prior to analysis. Basic demographic and clinical data were obtained from a questionnaire completed on admission.Nucleic acid was extracted from the samples using QIAamp MiniElute Virus Spin kits  following the manufacturer’s protocol. cDNA was synthesized from 10 μL RNA eluted using the Promega Reverse Transcription System with random hexamer primers and avian myeloblastosis virus reverse transcriptase  as described previously , 16.All of the specimens were screened for Flu A/B, PIV1–3, RSV, PIC (RV/EV), and ADV using three validated multiple-nested polymerase chain reaction (PCR) assays , 17. TheP-values < 0.05 were considered to indicate statistical significance.Statistical differences were determined using the Chi-square test using SAS software (version 9.2). The study included 700 NPAs from pediatric cases diagnosed with SARI: 259 from the Beijing Pediatric Research Institute of the Affiliated Beijing Children’s Hospital, Capital Medical University , and 441 specimens from the Children’s Hospital of Fudan University in Shanghai . The male-to-female ratio did not differ between hospital samples. We noticed that the difference in the gender ratio (M/F) which is in the range of 1.5–1.8:1.for two groups,the reason need to be clarified on why there are more infected males than females for respiratory infections. The patients were divided into five groups according to age: 0 to 6 months (M), 7 M to 1 year (Y), 1 to 2 Y, 3 to 5 Y, and more than 5 Y (> 5 Y). The mean and median ages in months of the Beijing group were younger than those in the Shanghai group, with more cases in Beijing in the 0–1 Y group and fewer cases in Beijing in the >5Y group. The pediatric SARI cases from Beijing and Shanghai shown different clinical manifestations. The main symptoms found in our study group were cough and fever(≥38 °C), followed by wheezing and diarrhea, runny nose and cyanosis was also recorded. The pediatric SARI cases from Beijing presented a higher frequency of bronchopneumonia and lower Pneumonia when compared to those from Shanghai. A summary of the demographic and clinical characteristics of the participants is presented in Table P = 0.0012). The most frequently detected respiratory virus was PIC (RV/EV), with a prevalence rate of 34.4% (241/700), followed by RSV and HBoV . HCoV was detected in 75 patients (10.7%), ADV was detected in 96 (13.7%), PIV1–3 was detected in 55 (7.9%), Flu A/B was detected in 62 (8.9%), and HMPV was detected in 35 patients (5.0%). The comparison of the respiratory virus detection rates in Beijing and Shanghai revealed that the prevalence of RSV, HCoV, and HMPV were higher in Beijing than in Shanghai.The viral infection profiles are shown in Table P <  0.05). All of the age-matched distribution patterns for RSV in Beijing were higher than those in Shanghai, and most age-matched distribution patterns in Beijing were higher than those in Shanghai for both HCoV (except for the > 5 Y group) and HMPV (except for the 1–2 Y group). However, the prevalence of PIV 1–3 in Shanghai for the 7 M–1 Y group was significantly higher than that in Beijing. In addition, the peak of individual viral infection differed between Beijing and Shanghai: Flu A/B peaked in the > 5 Y group in Beijing. The ADV infections peaked in the 1–2 Y group in Beijing and in the 7 M–1Y group in Shanghai. The HMPV infections peaked in the 0–6 M group in Beijing and in the > 5 Y group in Shanghai. Moreover, RV/EV, HBoV, and PIV1–3 peaked in the 7 M–1 Y group in Shanghai.The distributions of viral infections in Beijing and Shanghai are shown in Fig. Comparisons of the seasonal patterns of respiratory viral pathogens in Beijing and Shanghai are shown in Fig. ARI is common in children and can cause mild-to-severe disturbances, including upper and lower respiratory infections, such as pneumonia, bronchiolitis, asthma, and acute respiratory distress syndrome , 2. Our P <  0.05). In addition, a comparison of our data with those from other studies collected during the same period in the same locations revealed that our infection rates differed from those of the other studies [Previous studies have reported that RSV was the primary cause of SARI in hospitalized children –30. In c studies , 19, 25.The age distribution patterns of the respiratory viral infections significantly differed in Beijing and Shanghai. In children less than 1 year old, the positive detection rates of several viruses  in Beijing were significantly higher than in Shanghai, whereas the infection rates of other viruses  in Shanghai were significantly higher in Beijing. Given the high co-infection rate in our study, we did not investigate associations between clinical characteristics and individual viral infections. A previous study found that RSV infections were more strongly associated with comorbidities and bronchiolitis than were non-RSV infections . HoweverPrevious investigation also reported that the profiles of respiratory viruses in different area and seasons may be influenced by weather conditions  and indoor crowding during the cold season , 31. In Notably, we observed that non-influenza respiratory viruses were common in hospitalized children with SARI. The detection rate of at least one virus was 78.1%. PIC (RV/EV), RSV, HBoV, ADV, and HCoV were the most common pathogens detected, whereas Flu A/B, PIV 1–3, and HMPV were relatively rare in our sample. The total and age-matched prevalence of RSV, HCoV, and HMPV among SARI children under 5 years old were significantly higher in Beijing than in Shanghai. Moreover, the seasonal distributions of the pathogens differed between the regions. We noticed that the mean and median ages in months of the Beijing group were younger than those in the Shanghai group, so age adjustments of the results should be used to clarify future findings based on future larger surveillance studies.To our knowledge, our study is the first to compare the profiles of multiple (about 15) viruses and their related epidemiological profiles in pediatric patients with SARI in China. Viral infection was tested and shown to be the most prevalent etiological agent among children with SARI in either the Beijing or the Shanghai area, while showing different patterns of viral and epidemiological profiles. The observed pattern of seasonal variation of respiratory viruses are complex between hospitalized children with SARI in Beijing and Shanghai, since the weather and temperature variations between the two cities. Our findings provide a better understanding of the roles of geographic location and climate in respiratory viral infections in hospitalized children with SARI. In addition, our findings provide baseline data for investigations of the burden of respiratory viral infections in Beijing and Shanghai. However, additional studies with larger patient populations are needed to clarify the roles of viral and bacterial pathogens in SARI cases and to evaluate the overall burden of respiratory pathogens in asymptomatic children .Viral infection was tested and shown to be the most prevalent etiological agent among children with SARI in either the Beijing or the Shanghai area, while showing different patterns of viral and epidemiological profiles. Our findings provide a better understanding of the roles of geographic location and climate in respiratory viral infections in hospitalized children with SARI.


The isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on‐column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real‐time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans. The AF itself can be separated in outer and inner AF, with the latter being a transition zone between the outer AF and the central NP. While there is a natural evolution of the IVD macroscopic structure and phenotype during life, IVD degeneration is considered to be an accelerated aging process of the IVD and has often been associated to low back pain.Considering the high amounts of proteoglycans present in cartilage, several protocols have been specifically developed for the successful isolation of RNA from cartilage.Our goal was to develop a method for isolating high‐quality RNA from all disc tissues that allows using small amounts of tissues (approximately 100 mg per sample). Indeed, the amount of tissue available can be limited when caudal discs from large animals or IVDs from small animals  are used. In addition, the number of samples to process from a single experiment can be relatively high when NP, inner AF and outer AF are collected from multiple discs. In this study, we compare 3 previously established methods  for the isolation of RNA from bovine disc tissue to a new protocol (predigestion and pulverization) that produces reliable amounts of high‐quality RNA. This new method is based on predigestion of the tissue with pronase prior to extraction with guanidinium thiocyanate‐phenol‐chloroform. We demonstrate that this method is valid for the isolation of RNA from all tissues of the IVD, namely NP, inner and outer AF.22.1n = 6). For RNA isolation, NP and inner and outer AF tissues from each disc were separated and chopped into small pieces (1−3 mm3) using a scalpel. The minced tissues were stored in 5 mL PBS/sample to avoid tissue drying until all samples were prepared. The tubes containing the minced tissues were briefly centrifuged  and PBS was aspirated.IVDs comprising cartilaginous endplates were harvested from bovine tails (10‐12 months old) obtained from a local abattoir within 2 hours of death and washed in phosphate buffered saline (PBS) containing 10% penicillin‐streptomycin  for 10 minutes, followed by a second wash in PBS with 1% penicillin‐streptomycin. Three bovine tails were dissected, and the 2 biggest discs were collected from each tail. IVD health state was confirmed by visual inspection by an experienced scientist. Thickness of AF , jelly behavior of NP (quick shape recovery following deformation induced with a spatula) and absence of vasculature ingrowth inside the IVD were assessed. Discs were cultured overnight in Dulbecco's modified Eagle's medium (DMEM 4.5 g/L glucose) with 2% fetal bovine serum , 1% insulin transferrin selenium and 0.2% Primocin . From each disc, NP, inner and outer AF tissues were collected and a piece (100‐150 mg) of each tissue type was randomly assigned to 1 of the 4 RNA isolation groups. Tissue from each disc and tail was treated separately (2.2n = 6 per tissue type) were digested using a two‐step enzymatic digestion (pronase followed by collagenase), as previously described.g for 2 minutes and washed twice with PBS, followed by a second digestion  at 37°C on a waving shaker set a 40 rpm using collagenase II  at a concentration of 150 U/mL in DMEM. The volume of collagenase II solution was 5 mL per 100 mg of original tissue wet weight. Digestion was terminated by adding FBS (0.5 mL FBS/5 mL solution) when the tissue appeared macroscopically nearly digested. The cell suspension was filtered through a 70 μm cell strainer, cells were washed twice (with 7 minutes centrifugation step at 300g in between) with DMEM and lysed in 1 mL of TRI Reagent and 5 μL of polyacryl carrier .Minced tissue samples (2.3n = 6 per tissue type) were digested at 37°C for 1 hour in DMEM with pronase using the same amounts and concentrations as specified above. The digestion was then stopped with FBS and the tissue was washed twice with PBS. Any remaining PBS was aspirated, and tissue was snap‐frozen and pulverized in liquid nitrogen with a custom‐made pestle device  were frozen and pulverized in liquid nitrogen with a custom‐made pestle device. The resulting powder was mixed with 1 mL TRI Reagent and 5 μL of polyacryl carrier and homogenized using a Tissue Lyzer as described above. Following centrifugation at 2000g for 2 minutes to remove remaining tissue debris, the supernatant was transferred to a 1.5 mL microcentrifuge tube and RNA isolation continued as described above.Minced tissue samples (2.5n = 6 per tissue type) were frozen and pulverized in liquid nitrogen with a custom‐made pestle device. The resulting powder was mixed with 1 mL TRI Reagent and 5 μL of polyacryl carrier and homogenized using a Tissue Lyzer. Following centrifugation for at 2000g 2 minutes to remove remaining tissue, the supernatant was transferred to a new 1.5 mL tube. RNA isolation continued as described above until phase separation: the resulting aqueous layer was mixed with pure ethanol (0.7 mL ethanol per 1 mL of acqueous phase), transferred to an RNeasy column (Qiagen) and cleaned according to manufacturer's instructions. RNA was eluted with 30 μL of RNase‐free water.Minced tissue samples . Since nucleic acids absorb at 260 nm, it is possible to calculate the concentration of RNA in a solution based on Beer‐Lambert's law. With a 1‐mm path length, an absorbance of 1.0 at 260 nm corresponds to 40 μg/mL of RNA. Contamination of RNA by other molecules  is assessed by measuring the absorbances at 230 and 280 nm. The ratios of the absorbances at 260 and 280 nm (260/280 ratio) and the ratios of the absorbances at 260 and 230 nm (260/230 ratio) are used as indicators of RNA purity.2.7n = 2 per group). The RNA concentration and purity (260/280 ratio) of the samples were determined spectrophotometrically and samples were sent to Microsynth AG  for RIN analysis. Samples were quantitatively assessed using an Agilent 2100 Bioanalyzer System and Agilent 2100 expert software .To complete the results obtained spectrophotometrically, a RNA integrity number (RIN) evaluation was conducted on a separate set of samples , 2.5 μM random hexamers, 0.4 U/μL RNase inhibitor, and 1.35 U/μL Multiscribe reverse transcriptase; Applied Biosystems, Forster City, CA, USA). For all samples, 500 ng of total RNA was transcribed in 20 μL reaction mixture and the resulting cDNA was diluted 1:4 Tris‐ethylenediaminetetraacetic acid (Tris‐EDTA) buffered RNase‐free water. For all samples, real‐time PCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems) with QuantStudio 6 Flex instrument (Applied Biosystems). The following genes were analyzed: collagen type Iα1 (COL1), collagen type IIα1 (COL2), aggrecan (ACAN), matrix metalloproteinase 3 (MMP3) and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4). The 18S ribosomal RNA (18S) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were used as endogenous controls. Primers and TaqMan probes were supplied by Microsynth or Applied Biosystems, using the same sequences as in previous studies.Samples with too low RNA quantity or quality were excluded from gene expression analysis. Criteria for inclusion were defined as follows: RNA concentration >7 ng/μL and 260/280 absorption ratio >1.6 . For all samples meeting these requirements, reverse transcription was performed using TaqMan reverse transcription reagents  for RNA yield, concentration, 260/280 and 260/230 ratios. A one‐way ANOVA with subject matching and Tukey post‐doc (P < .05 was considered significant) was performed on the Bioanalyzer data and corresponding Nanodrop data (data from different tissues using the same RNA isolation method were combined in one group). For gene expression analyses, since not all genes of interest were detected in all samples, a two‐way ANOVA without subject matching was performed with Tukey post‐hoc (P < .05 was considered significant).Since the tissues  collected from 1 disc were split into 4 groups (the 4 RNA isolation methods) and 6 discs were used in the study, a two‐way analysis of variance (ANOVA) with subject matching and Tukey post‐hoc was performed  value for each gene differed depending on the RNA isolation method used, especially in the case of NP tissue  are likely to be identified by all the extraction methods compared in this study.Threshold cycle (Ct) values of the anabolic genes collagen type I, collagen type II, and aggrecan, and catabolic genes 4The IVD is composed of 2 tissues, the central NP rich in proteoglycans and the AF rich in collagen. Ideally, the chosen RNA isolation method should allow the isolation of high‐quality, intact RNA from all tissue types. Several methods have been reported for the isolation of RNA from collagen‐rich tissues such as cartilage,In terms of homogenization methods of IVD tissues in preparation for RNA isolation, Peeters et al have shown that RNA of higher purity is obtained when goat NP tissue is first pulverized in liquid nitrogen and then suspended in lysis buffer rather than homogenized in lysis buffer directly.As for the lysis buffer, Peeters et al have shown that RNeasy Fibrous Tissue kit  yields RNA of higher purity (as attested by 260/280 ratio) compared to RNA isolation with TRIzol.Concerning the RNA extraction step, Lee et al have recommended to add a second phase separation and to use a high salt solution for precipitation.It is worth noting that only with the pronase predigestion step, the 260/230 ratio of RNA isolated from bovine NP was ~1.5, while even with extra phase separation and use of high salt solution the 260/230 ratio was <0.8 (RIN not reported).100 to 150 mg tissue per samplepredigestion in 0.2% pronase (5 mL/100 mg tissue) for 1 hour at 37°C on a shakerpulverization of IVD tissues in liquid nitrogenlysis in TRIzol homogenization 1 phase separationprecipitation with isopropanol and high salt solution2 washing steps with 70% ethanolIn summary, we recommend:Our results are similar to the data reported by Yu et al on RNA isolation from cells encapsulated in chitosan hydrogels, confirming that the use of an enzymatic pretreatment improves the purity of samples isolated with guanidinium thiocyanate.Besides RNA extraction directly from IVD tissues, complete tissue digestion leading to a single cell suspension has been widely used. In the present study, isolation of good quality RNA (RIN > 7) was possible with either complete matrix digestion or predigestion and pulverization. Between these 2 methods, more consistent RIN values were obtained with the predigestion and pulverization method, probably because the endpoint of complete digestion must be monitored and adapted , which can result in under‐ or over‐digestion, thereby affecting both RNA yield and quality. The complete digestion process is time‐consuming and critical in terms of stopping the digestion at the right time . Moreover, a long digestion process (>8 hours) might affect the expression of certain genes of interest.As an alternative method, tissue pulverization in liquid nitrogen followed by RNA extraction in guanidinium thiocyanate‐phenol‐chloroform and further purification of the aqueous phase with silica columns have been reported as a way to eliminate contaminants due to the coprecipitation of ECM components during RNA extraction.As for the gene expression, higher Ct values were observed for the endogenous control genes in the pulverization only group, which could be due to low yield and/or the possible presence of contaminants  which may affect both the reserve transcription and the RT‐PCR. For instance, it has been shown that proteoglycans can act as inhibitors of PCRs.In conclusion, this new combined method (predigestion and pulverization) improves the yield and quality of the RNA extracted from IVD tissues  compared to pulverization alone. As a guideline when a RIN analyzer is not available .Table S3 Average Ct values for RT‐PCR of endogenous control genes for the NP, inner AF, and outer AF tissues of bovine discs, according to the method used to isolate the RNA from each sample. Data are represented as mean ± SD; n = 3‐6.Table S4 Average dCt values for RT‐PCR of genes of interest for the NP, inner AF, and outer AF tissues of bovine discs, according to the method used to isolate the RNA from each sample. Data are represented as mean ± SD; n = 3‐6; n.d., gene not detected.Click here for additional data file.


Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.Oxazinomycin is a C-nucleoside antibiotic that is produced by  Streptomyces soil bacteria have been reported to produce C-nucleoside compounds such as pseudouridomycin, formycin, showdomycin and malayamycin . Compounds with O- and N-glycosidic bonds are widely present in metabolic pathways, but are enzymatically labile thereby facilitating the rapid recycling of metabolites and the base excision repair of the DNA . Many biayamycin .d-ribofuranosyl-l,3-oxazin-2,4-dione), also known as minimycin, is a C-ribonucleoside antibiotic first isolated from Streptomyces hygroscopicus (Streptomyces sp. 80432)  and a single subunit RNAP from human mitochondria.Oxazinomycin  . Tymiak imobilis , and theimobilis . OZM is imobilis ,16. OZM imobilis , OZM mayAll RNAPs responsible for transcribing cellular genomes are multisubunit enzymes that share homologous catalytic cores. The bacterial RNAP, a five-subunit complex, ααββ’ω, is the simplest model system used for studying the fundamental mechanistic properties of all multisubunit RNAPs. Eukaryotic cellular RNAPs consist of 12–17 subunits and contain homologs of all five subunits present in the less complex bacterial RNAPs . In contet al. who reported the stimulation of mitochondrial transcription by secondary structures in the nascent RNA . SeveraHPLC-purified DNA oligonucleotides were purchased from Eurofins Genomics GmbH . The PAGE purified ATTO680 labeled RNA primer was purchased from IBA Biotech . NTPs and Ψ triphosphate (ΨTP) were from Jena Bioscience . DNA oligonucleotides and RNA primers are presented in Streptomyces hygroscopicus subsp. hygroscopicus JCM 4712 was cultivated in the medium containing 4.0 % glucose, 2.0% soybean meal , 1.0% wheat embryo , 0.4% brewer’s yeast , 0.2 % NaCl, 0.2% KCl and 0.2% CaCO3 , the supernatant (1.7 l) was collected by centrifugation and pH was adjusted to 2.0 with HCl. Activated charcoal (20 gl−1) was added to the supernatant and the mixture was stirred overnight at 4°C. The charcoal was collected by filtration and the adsorbed compounds were washed out of the charcoal cake with acetone/H2O (1:1) (1 L). The combined extracts were concentrated to a small volume and the mixture was fractioned by silica gel chromatography, eluting first with acetone/toluene (2:8) and then with acetone/toluene (8:2). The fractions containing OZM were combined and evaporated to dryness under reduced pressure. The residue was dissolved in a small volume of water and OZM was finally isolated by RP-silica column chromatography  eluting with a gradient of 3–6 % MeCN/H2O. The fractions containing OZM were evaporated to dryness and further dried in a vacuum desiccator to yield 184 mg of OZM as a white solid. 1H NMR : δ 7.73 , 4.60 , 4.19 , 4.05 , 3.92 , 3.76 , 3.63 . 13C NMR : δ 163.0 (C-2), 154.6 (C-6), 149.7 (C-4), 114.2 (C-1), 83.1 (C-4′), 78.0 (C-1′), 73.5 (C-2′), 70.5 (C-3′), 61.3 (C-5′); HRMS (ESI) m/z: [M-H]- calculated for C9H10NO7- 244.0457; found 244.0465. The chemical structure validation data are presented in 2% CaCO3 . Precult−1, eluted with a gradient from 0.025 M triethylammonium acetate in H2O to 0.025 M triethylammonium acetate in acetonitrile/H2O, 1:1, over 25 min). The collected fractions were lyophilized to yield a triethylammonium salt of the OZM 5′ triphosphate. 1H NMR : δ 7.82 , 4.73 , 4.20 , 4.23 , 4.07 , 4.16 , 4.09 ; 13C NMR : δ 163.6 (C-2), 154.5 (C-6), 150.2 (C-4), 114.7 (C-1), 81.3 (C-4′), 77.6 (C-1′), 74.0 (C-2′), 69.9 (C-3′), 64.8 (C-5′); 31P NMR : δ – 10.74 , – 11.32 , – 23.36 ; HRMS (ESI) m/z: [M-H]- calcd for C9H13NO16P3- 483.9453; found 483.9451. The chemical structure validation data are presented in OZM  was dissolved in dry triethyl phosphate (TEP) (0.95 ml) under nitrogen atmosphere and the solution was cooled down to −10°C on an ice-salt bath. Freshly distilled phosphoryl chloride  was added dropwise and then dry 2,4,6-trimethylpyridine  in one portion. The reaction was allowed to proceed for 1.5 h at −10°C, then a dry solution of tributylammonium pyrophosphate  in MeCN (1.5 ml) and dry tributylamine  were added to the cooled reaction mixture, and the stirring was continued for 2 h at 0°C, and then for 18 h at room temperature. Triethylammonium acetate solution  and chloroform (2.5 ml) were added to the reaction mixture and stirring was continued for 30 min. The aqueous layer was separated and washed two times with chloroform (3 ml). NaI (80 mg) and acetone (35 ml) were added to the aqueous solution to precipitate the phosphorylated compounds and the mixture was vortexed for 15 min and cooled down to 0°C. The precipitate was collected by centrifuging the mixture and separating the supernatant. The OZM 5′-triphosphate was purified by RP-HPLC  RNAP was expressed in T7 Express qlysY/I cells from New England Biolabs  bearing the pVS10 plasmid (or pIA528 and pIA839 plasmids in case of β’N458S RNAP) and was purified by Ni-, heparin and Q-sepharose chromatography as described previously (Saccharomyces cerevisiae RNA polymerase II (Sce RNAPII) was purified from the S. cerevisiae strain SHy808  largely as described previously  lacking 213 N-terminal amino acids  supplemented with 1 mM β-ME, a tablet of EDTA-free protease inhibitors , 1 mg/ml lysozyme, incubated on ice for 30 min and disrupted by sonication. The lysate was cleared by centrifugation at 18 000 × g, 4°C for 30 min. The supernatant was supplemented with 10 mM imidazole and loaded onto a Ni-sepharose  column pre-equilibrated with Lysis Buffer. Protein was eluted using a step gradient  of imidazole in Lysis Buffer. The 250 mM imidazole fraction containing RNAP was further purified using Heparin and Resource-S columns in Buffer A  and Buffer B (Buffer A supplemented with 1.5 M NaCl). Hsa MT RNAP eluted at ≥50% and ≥30% Buffer B from Heparin and Resource-S columns, respectively. The fractions containing the purified protein were concentrated using Amicon Ultra-4 centrifugal filters , dialyzed overnight in Storage Buffer  and stored at −80°C.The human mitochondrial RNAP (y domain ) was expEscherichia coli GreA and GreB were expressed in E. coli T7 Express qlysY/I cells bearing the pIA578 (GreA) or the pIA577 (GreB) plasmid and purified by Ni-sepharose followed by gel filtration as described in Perederina et al.  were assembled by a procedure developed by Komissarova et al.  and with 2 μM of the non-template DNA for 20 min at 25°C. Sce RNAPII TECs were assembled as described above except that the incubation time after the addition of the non-template DNA was 10 min.a et al. . An RNA 4-EDTA and 0.2% Orange G). RNAs were separated on 16% or 25% denaturing polyacrylamide gels and visualized with an Odyssey Infrared Imager ; band intensities were quantified using the ImageJ software  or OZM triphosphate (OZM-chase) or ΨTP (Ψ-chase). The final concentrations of the TEC and NTPs were 0.5 and 100 μM each, respectively. The reactions were incubated for 5 min at 25°C and quenched with 40 μl of Gel Loading Buffer. RNAs were separated on 16% denaturing polyacrylamide gels, visualized and quantified as described above.2+ were 0.1, 2 and 9 mM, respectively. Aliquots (10 μl) were withdrawn at the indicated time points and quenched with 30 μl of Gel Loading Buffer. RNAs were separated on 16% denaturing polyacrylamide gels, visualized and quantified as described above.TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO  pre-equilibrated with TB0 buffer . RNA cleavage was initiated by mixing 10 μl of the pre-extended TEC with 90 μl of GreA in TB10 at 25°C. The final concentrations of the TEC, GreA and MgTime-resolved measurements of nucleotide addition were performed in an RQF 3 quench-flow instrument . The reaction was initiated by the rapid mixing of 14 μl of 0.2 μM TEC with 14 μl of 400 μM NTP. Both TEC and NTP solutions were prepared in TB10 buffer. The reaction was allowed to proceed for 0.004–60 s at 25°C and quenched by the addition of 86 μl of 0.45 M EDTA or 0.5 M HCl. HCl quenched reactions were immediately neutralized by adding 171 μl of neutralizing-loading buffer . RNAs were separated on 16% denaturing polyacrylamide gels, visualized and quantified as described above.Equilibrium levels of fluorescence were determined by recording the emission spectra of 6-methyl-isoxanthopterin (6-MI) (excitation at 340 nm) with an LS-55 spectrofluorometer  at 25°C. The fluorescence at the peak emission wavelength (420 nm) was used for the data analysis and representation. Preassembled TECs (1 μM) were diluted into 200 μl of TB10 buffer with or without NTPs and guanosine-5′-[-methyleno]triphosphate (GMPCPP). The final concentrations of the TEC, NTPs and GMPCPP were 0.1, 10 and 500 μM, respectively. Following a 2-min incubation at 25°C, 5 μl aliquots were withdrawn, quenched with 20 μl of the Gel Loading Buffer and subsequently analyzed in a denaturing PAGE to determine the RNA extension efficiency. The rest of the sample was transferred into a 16.160-F/Q/10 quartz cuvette  and the emission spectra were recorded.Measurements were performed in an Applied Photophysics  SX.18MV stopped-flow instrument at 25°C. The 6-MI fluorophore was excited at 340 nm and the emitted light was collected through a 400-nm longpass filter. The reaction was initiated by mixing 60 μl of 0.2 μM TEC with 60 μl of 2.5–500 μM of substrate NTP (both in TB10). At least three individual traces were averaged for each concentration of NTP.2+, thereby inactivating the free NTP substrate, but allows a fraction of the RNAP-bound NTP to incorporate into RNA after the addition of the quencher (Time-resolved uridine and Ψ incorporation data (HCl and EDTA quenched reactions) and the translocation time-traces were simultaneously fitted to a four-step model using the numerical integration capabilities of the KinTek Explorer software  KinTek ,89. The quencher . The latThe time courses of guanosine incorporation were fitted to a sum of exponential (modeled fast phase) and stretched exponential (modeled slow phase) functions. The time courses of the GreA facilitated cleavage of the nascent RNA were fitted to the stretched exponential function. The median reaction times were calculated as in Turtola & Belogurov . We usedStreptomyces hygroscopicus subsp. hygroscopicus JCM 4712 was cultivated in the medium described by Kusakabe et al. , RNA polymerase II from S. cerevisiae (Sce RNAPII) and the single subunit RNAP from human mitochondria (Hsa MT RNAP). Transcription elongation complexes (TEC) were assembled on synthetic nucleic acid scaffolds and contained a fully complementary transcription bubble flanked by 20-nucleotide long DNA duplexes upstream and downstream  in place of UTP. Incorporation of Ψ into RNA altered the electrophoretic mobility to a similar extent as did the incorporation of uridine Figure . QualitaP Figure .Eco RNAP and Hsa MT RNAP but not Sce RNAPII incorporated OZM in place of cytidine  incorporated into the template DNA downstream of the active site  and the normalized translocation curves Figure  should bEco RNAP.Finally, we took the opportunity to completely resolve the kinetics of Ψ incorporation Figure  and D. T−1 for guanine incorporation by the fast fraction (50–60%) of the TEC-U, TEC-OZM and TEC-Ψ. The slow fraction of the TECs that incorporated guanosine with the rate of ∼1 s−1 (40–50%) likely represented a population of paused TECs that is usually present in TEC preparations, though typically does not exceed 15–20% (We next investigated how the incorporation of OZM into RNA affects the rate of the incorporation of the next nucleotide. The assembled TECs were pre-extended with uridine (TEC-U) or OZM (TEC-OZM) or Ψ (TEC-Ψ) and challenged with 200 μM GTP in a quench flow instrument. The resulting guanosine incorporation time courses largely superimposed Figure  suggestid 15–20% ,88. The d 15–20%  did not d 15–20% .We measured the kinetics of GreA-facilitated RNA cleavage of the nascent RNA in the TECs assembled on the nucleic acid scaffolds and extended with the uridine and guanosine (TEC-U-G) or OZM and guanosine (TEC-OZM-G) Figure . These Tin vitro transcription reactions using two different NTP mixtures containing 100 μM ATP, GTP, CTP and UTP (U-chase) or OZM triphosphate (OZM-chase) for 5 min at 25°C. Incorporation of the OZM during transcription of a short template with a single thymidine positioned four base pairs downstream of the RNA primer  did not measurably impair RNA synthesis as expected from the single nucleotide addition measurement  or OZM triphosphate (OZM-chase) or ΨTP (Ψ-chase) for 5 min at 25°C. OZM did not affect transcription through the thymidine-free tract as expected but arrested a significant fraction of RNAP at the polythymidine sequences. Thus, approximately 2-fold more RNAP was retained at the polythymidine tracts following the 5-min incubation in the OZM-chase compared with the U-chase (Figure Considering that a single incorporated OZM had measurable effects on the lateral mobility of the nascent RNA within the TEC as both the 3′ end Figure , we rease Figure . The arre Figure  and B. Ge Figure  and B, be Figure . These re Figure  and multe Figure  OZMs: GrSce RNAPII was arrested at the four-thymidine tract and the single thymidine OZM-responsive arrest site similarly to the Eco RNAP (Figure Sce RNAPII. In contrast, no significant accumulation of the arrested RNAs within the four-thymidine tract was evident during transcription by Hsa MT RNAP (∼30% of RNAs correspond to the polythymidine tract in U-, OZM- and Ψ-chase lanes in Figure P Figure  except tE. coli and S. cerevisiae and a single subunit RNAP from human mitochondria. We show that the OZM triphosphate is a good substrate for Eco RNAP and is also efficiently incorporated into RNA by Sce RNAPII and Hsa MT RNAP. A detailed analysis of the translocation equilibrium suggested that the 3′ terminal and the penultimate OZM increased the lateral mobility of the nascent RNA within the RNAP and favored backward translocation by one or more registers. However, the above effects were apparently benign for the processive transcription of some sequences and the incorporated OZM was rapidly extended by the next nucleotide (Figures In this study, we characterized the inhibitory activity of the C-nucleoside antibiotic OZM on transcription by multisubunit RNAPs from  Figures  and 6A.  Figures ,97, wher Figures ,98, and  Figures .Our data also suggest that the incorporation of OZM significantly alters the properties of RNAs compared with the uridine containing RNAs. First, the incorporation of OZM into RNA resulted in a very small change in the electrophoretic mobility of the RNA compared to that upon uridine or pseudouridine incorporation. Second, long RNAs with incorporated OZM entered the denaturing PAGE gels poorly for some unknown reason. Third, from a purely theoretical standpoint, the free electron pair of the OZM ring oxygen and its partial resonance to the carbonyl group at C2 may lead to deviations in the secondary and tertiary structures compared to the native, uridine-containing RNAs. Most importantly, both the transcriptional effects of OZM and the unusual properties of OZM-containing RNA could be attributed to an oxygen atom in the position corresponding to the carbon five of the uracil ring rather than to the C-glycosidic bond. Thus, the ubiquitous C-nucleoside pseudouridine did not cause transcriptional arrest at the OZM-responsive sites and the pseudouridine-containing RNAs did not display the anomalous properties of the OZM-containing RNAs.We argue that OZM functions as a Trojan horse substrate. OZM enters the target organism in nucleoside form, becomes phosphorylated by the recipient’s kinases  and compin vitro and our data suggest that at a high OZM/U ratio in vivo, OZM should primarily act as a transcription inhibitor by arresting RNAP at the frequently encountered sequences such as three-four-thymidine tracts. However, at a low OZM/U ratio in the target cell, the probability of the incorporation of several successive OZMs is low and OZM may be permissive for transcription. Under these conditions, the post-transcriptional effect, such as altered RNA secondary structures or interference with translation may potentially contribute significantly to the inhibition of the cellular functions. For example, the targeted pseudouridinylation of the mRNAs has been shown to cause the read through of stop codons by the ribosome (in vivo.In this study, we assessed the effects of OZM on transcription ribosome . Accordigkz782_Supplemental_FileClick here for additional data file.


DHX36 is a DEAH-box helicase that resolves parallel G-quadruplex structures formed in DNA and RNA. The recent co-crystal structure of DHX36 bound G4-DNA revealed an intimate contact, but did not address the role of ATP hydrolysis in G4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 displays ATP-independent unfolding of G4-RNA followed by ATP-dependent refolding, generating a highly asymmetric pattern of activity. Interestingly, DHX36 refolds G4-RNA in several steps, reflecting the discrete steps in forming the G4 structure. We show that the ATP-dependent activity of DHX36 arises from the RNA tail rather than the G4. Mutations that perturb G4 contact result in quick dissociation of the protein from RNA upon ATP hydrolysis, while mutations that interfere with binding the RNA tail induce dysregulated activity. We propose that the ATP-dependent activity of DHX36 may be useful for dynamically resolving various G4-RNA structures in cells. DHX36 is a G-quadruplex (G4) resolving helicase that targets both DNA-G4 and RNA-G4. Here the authors use single molecule FRET measurements and show that DHX36 resolves RNA-G4 structures by a mechanism involving an ATP-dependent, highly repetitive and stepwise refolding of RNA-G4 that differs from its DNA-G4 structures resolving mechanism. Accordingly, DHX36 is found enriched in G4 loci in cells7. DHX36 activity in targeting and unfolding G4-RNA is implicated in many mRNA and long non-coding RNA processes11. DHX36 helps promote translation of mRNAs with G4 motifs by unfolding the G4 structure12. In agreement, DHX36 knockout impedes translation of mRNAs containing G4 motifs at both 5′UTR and 3′UTRs13. In addition, DHX36 regulates the G4-harboring p53 mRNA after oxidative damage and localizes G4-forming mRNAs to stress granules, suggesting diverse, yet G4-specific functions of DHX36. Furthermore, the human telomeric G4-RNA is resolved by DHX36, contributing to the telomerase activity and telomere regulation.15 Aided by such G4 specific-helicases, cellular G4-RNA structures are efficiently resolved16. Thus, G4-RNA structures are likely undergoing dynamic folding and unfolding cycles18.DHX36  is an RNA helicase that can resolve G-quadruplex (G4) DNA and G4-RNA with high specificity and affinity20 with a single-stranded (ss) tail16. The recent co-crystal structure of DHX36 bound to G4-DNA revealed that the resolved state entails a single guanine base pulled out of the folded G4 structure21, suggesting a partial rearrangement of G4 rather than a complete unfolding. Interestingly, we demonstrated that such activity by DHX36 is highly repetitive, monomer-driven and ATP-independent21. Structurally, the partial unfolding arises from rotation of the C-terminal domain and helicase core opening, resulting in pulling of one nucleotide out of the G4 registry. This is followed by subsequent refolding of G4. Cycles of such unfolding and refolding occur in succession, resulting in back-and-forth springing of the G4 structure between fully folded and partially disrupted states in an ATP-independent manner21.We and others have reported that DHX36 binds specifically to parallel conformation-G4-DNAsHere, we report a distinctly different mechanism by which DHX36 acts on G4-RNA substrates. Unlike on G4-DNA, DHX36 binding to G4-RNA induces a stably unfolded state without repetitive pulling on the substrate. The unfolding is followed by successive cycles of gradual, ATP-dependent and stepwise refolding of G4-RNA. Such ATP-independent unfolding and ATP-dependent refolding of G4-RNA is repeated multiple times before the protein dissociates from the G4-RNA substrate. Mutational analysis reveals that disruption of G4 contacts induces rapid dissociation of DHX36 from G4-RNA whereas the changes in ssRNA contacting domains perturb the ATP-dependent stepwise refolding of G4-RNA, leading to an erratic and dysregulated motion of DHX36 on G4-RNA. Together, these studies describe a detailed mechanism for the G4-RNA resolving activity of DHX36.20, likely due to the tight contact between DHX36 and the substrate21. We have demonstrated that the high FRET state from this position of FRET pair dyes represents the G4 folded state. Protein binding to single-stranded tail results in high to mid FRET (0.65) transition whereas the unfolding of G4 by DHX36 or RecQ family helicases induce further FRET decrease to 0.423. Based on this observation, we designed G4 constructs with an end-labeled short tail that allows DHX36 to bind and act on G4, and yields high FRET sensitivity21. As before, we employed a single molecule (sm) FRET assay to probe the DHX36 activity on both constructs.In order to compare the activity of DHX36 on G4-DNA- vs. G4-RNA, we prepared two FRET constructs in which the G4 sequence consists of four runs of G triplets separated by single thymine and single uracil nucleotide in G4-DNA and G4-RNA, respectively Table . The ss 24 and a short tail of nine nucleotides, bringing the two dyes into close proximity. We have shown previously that the non-G4 forming sequence or G4 forming sequences with chemically modified guanines yield significantly lower FRET, further confirming the high FRET arising from G4 folding28. The representative smFRET trace displays a steady high FRET signal, consistent with the sharp FRET histogram peak. Upon addition of 5 nM DHX36, the high FRET peak immediately shifts to a broad mid FRET (0.5–0.6)  as depicted by a sharp high FRET peak in the FRET histogram  Fig. , middle .6) Fig. . We inte19. The steady low FRET level reveals that G4 is stably engaged with DHX36, and partially disrupted by the protein. The measurements were taken after excess protein was removed by flowing buffer into the imaging chamber, confirming the tight binding of DHX36 on G4-RNA. When ATP was added, the FRET peak gradually shifted from 0.4 back to the 0.8 in 12 min, indicating that DHX36 slowly dissociated from G4-RNA  by itself Fig. , gray. URNA Fig. . The indRNA Fig. . To test21. Based on our experimental scheme in which the measurement is taken after washing out excess protein by buffer flow  and Cy5 (red) changing in a stepwise and anti-correlated manner Fig. , top and20. To test whether the G4 or the ssRNA tail is responsible for the ATP-dependent activity of DHX36, we prepared two RNA-DNA chimeras comprised of G4-RNA with ssDNA tail  to a mid FRET (0.6) and subsequent partial unfolding of G4 induces further FRET decrease to 0.4 state for all DSM and OB subdomain mutants21. Consistently, the 0.4 FRET value shown here represents the state in which DHX36 is engaged stably with both ssRNA and G4. In the presence of ATP, however, they rapidly dissociate from G4-RNA without exhibiting the ATP-dependent asymmetric motion , R856A and YLY900AAA (in the OB subdomain), interact directly with the ss nucleic acid tail Fig. 21. All tase Fig. . In summcis-annealing20. We hypothesized that the annealing between G4 and complementary strand positioned in the same molecule can only occur if DHX36 successfully resolves the G4 and thereby exposes the G4 strand. If the resolving activity is insufficient, G4 structure will refold , yet unevenly distributed. The enrichment of G4 forming sequences in promoters of oncogenes and regulatory genes strongly suggests their potential role in regulating transcription of important genes33. The presence of G4 in the human genome was confirmed by antibody probing and sequencing35 and more recently by BioTASQ18. Telomeric G4 is another site of G4 overrepresentation36. Therefore, both the promoter G4 and telomeric G4 are targeted for anticancer therapeutic by small molecules37. The recent co-crystal structure of DHX36-G4-DNA clearly demonstrated the nature of intimate contact between the protein and a parallel G4 structure21. DHX36 binding requires two parts; parallel G4 and a ss tail of at least 8–9 nucleotides20. Our previous and current findings indicate that upon engaging with the G4-DNA, DHX36 repetitively pulls out one guanine and releases for G4 to resume folding, resulting in a tug of war-like motion. This partial unfolding and refolding cycle is ATP-independent and continues for many cycles without dissociation from the substrate (over 20 min). Nevertheless, the motion discontinues when a C4 complementary strand anneals with the G4 strand20, suggesting that the partial unfolding induced by DHX36 is sufficient to disrupt the G4 structure to allow for annealing with the complementary strand. Such activity can lead to restoring of the duplex DNA configuration, which can contribute to genome integrity.DHX36 is unique in its ability to resolve both G4-DNA39. Therefore, stable folding of G4-RNA can potentially modulate translational activity unless resolved by helicases such as DHX36. Indeed, DHX36 deletion led to heart defect and embryonic lethality in mice due to G4-RNA formed in 5′UTR of Nkx2-5 mRNA which requires DHX36 activity for translation13. Here, we present a mechanism by which DHX36 resolves G4-RNA in an ATP-dependent and repetitive manner. The mechanism is unique in several aspects. First, the binding of DHX36 which does not depend on ATP, leads to unfolding of G4-RNA and the binding is stable against buffer wash. This reflects an inherent binding mode which is built to twist open the G4 structure. Second, ATP hydrolysis leads to slow, gradual, and stepwise refolding of G4-RNA (FRET recovers to the level of folded G4-RNA). This may represent ATP binding in a closed core conformation (FRET 0.5), hydrolysis of ATP to ADP (FRET 0.7), ADP release (FRET 0.8) followed by phosphate release which springs back to unfolded G4 (FRET 0.4), yet our experiment cannot directly probe such motion. Determination of such detailed mechanism is warranted for future study. Third, the asymmetric ATP independent unfolding followed by ATP dependent refolding of G4-RNA is driven by a monomer DHX36 and repeated many times in succession. Fourth, such repeated cycles of activity leads to dissociation of DHX36 from G4-RNA substrate. Fifth, the ATP-dependent refolding of G4-RNA arises from the RNA tail rather than the RNA-G4. If such activity occurs in cellular RNA, for example at a parallel G4 formed in 5′UTR, DHX36 monomer will maintain contact with the RNA-G4 while dynamically modulating the structure until it encounters a cognate molecule such as ribosomes that need to gain access the 5′ side of the mRNA. This unique mechanism can potentially serve to; (i) resolve G4 structure, (ii) prevent the reformation of G4, (iii) protect the G4 from degradation, (iv) shield G4 from other proteins, (v) keep the G4 in dynamic state, and (vi) release from G4 structure eventually.Unlike the G4-DNA which forms in duplex DNA, G4-RNA forms in single stranded context such as mRNA and long noncoding RNA. In particular, the G4-RNA forming sequences are highly enriched in 5′UTR, 3′UTR and the 5′ side of the first intron of numerous genes21, yet the same mutant remained bound to RNA-G4 even after the buffer flow, indicating a different DSM-G4 interaction between DNA and RNA. Despite its fast dissociation upon ATP hydrolysis, the DSM mutations led to efficient annealing between the G4 and the complementary C-rich strand. For K76G N77G K78G mutant, the protein appears to detach from the substrate immediately after the flow of ATP , heating to 85 °C for 2 min, and gradually cooled at the rate of 2 °C/min until it reached 50 °C, followed by 5 °C/min decrease until it reached 4 °C on a thermocycler.All RNA and DNA substrates are listed in Table  oC for overnight to reach OD of 1.2. The cell lysate was treated with 2× Sigma protease inhibitor mixture, 0.01 mM PMSA and 15 mg/ml leupeptin and applied to TALON cobalt bead by means of hexahistidine tag (Clontech). After washing three times with SSC (4×) with BME (0.5 mg/ml) containing buffer, the protein was eluted from the column by elution buffer containing 0.7 M histidine, pH 6.0, 8.6 mM BME and 1× Sigma protease inhibitor mixture. For second phase of elution, CMYC G4 DNA bound streptavidin paramagnetic beads were prepared as instructed . The eluant was applied to CGSPB and washed two time with cold SSC (4×) with 0.1% lactalbummin and 0.5 mg/ml BME. The elution was performed by adding ATP containing buffer. The purified enzyme stock was stored at −80  °C21. Single-molecule reactions were performed with 10 nM DHX36 in RNAse free buffer containing 50 mM Tris-HCl, pH 7.6, 50 mM KCl, 50 mM NaCl, 0.5 mM MgCl2, 10 percent glycerol and 1 mM ATP or other ATP analogs including ATPγS, AMP-PNP, ADP-AlF4. For ATP titration measurements ATP concentrations ranging from 10 μM to 4 mM ATP was used.Wild type and mutant DHX36 were purified by transforming RHAU encoding plasmid  into BL21 (DE3) E. coli strain and inducing the protein expression by IPTG at 1440. Partial duplex RNA or DNA molecules were annealed and immobilized on the PEG-passivated surface via biotin–NeutrAvidin interaction at the concentration range of 10–50 pM. Excess molecules were washed away with RNase free T50 buffer. An oxygen scavenging system  was added to the imaging buffer for all smFRET measurements to minimize photobleaching of dyes. All experiments and measurements were carried out at room temperature (23 °C ± 1 °C). We used custom-built prism based total internal reflection fluorescence microscope for all single-molecule FRET assays. A solid-state 532 nm laser (Spectra-Physics) was used for smFRET measurement. Emission signal was separated by a dichroic mirror (cut off = 630 nm) and detected by an EMCCD camera (Andor). Data were recorded with a time resolution of 50–300 ms and analyzed with scripts written in interactive data language (IDL) to give fluorescence intensity time trajectories of individual molecules.Single-molecule FRET experiments were carried out on quartz slides (Finkenbeiner). To minimize nonspecific interaction of DNA, RNA or protein the quartz slides , For Prism type TIR. G. Finkenbeiner Inc.) and coverslips were passivated by treating with polyethylene glycol (PEG). Methanol, acetone, and potassium hydroxide were used to clean the slides and coverslips. Further, slides were burned, treated with amino silane, and coated with a mixture of 98% mPEG  and 2.0% biotin PEG Matlab scripts have been used to analyze FRET efficiency, E, calculated as the intensity of the acceptor channel divided by the sum of the donor and acceptor intensities. To generate sm FRET histograms, we have collected data from over 6000 molecules and were fitted to Gaussian distributions using Origin software 8.0. For histogram analysis, we take short (3–5 s) movies from 20 different sm imaging surface with each movie yielding approximately 300–400 FRET values. We combine all 6000–8000 FRET values to build one histogram. The initial four frames taken from 300–700 ms duration are taken from each of ~6000 molecules are taken to build each smFRET histograms. Dwell times were analyzed by obtaining time intervals corresponding to each FRET state. TDP was created by Origin 8.0 software. The mean values with standard error of means were plotted.Ten microliters of GQ-containing ssRNA with 3′Cy3 Table  was heatFurther information on research design is available in the Supplementary InformationReporting SummarySource Data