Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1–1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1–1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1–122) are necessary and sufficient for specific vault RNA1–1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1–1 further reveals a central flexible loop within vault RNA1–1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein–protein interactions critical to the activation of specific autophagy. Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The human autophagy receptor p62  guides selective macroautophagy of intracellular cargo to maintain homeostasis in situations of proteotoxic stress or starvation . FollowiIn this process, p62 recognizes ubiquitinated cargo via its carboxy-terminal ubiquitin-associated (UBA) domain  and N-arSince the affinity of p62 monomers for specific cargo and the ATG8-like proteins is rather modest, protein oligomerization is essential for achieving high avidity protein interactions while maintaining selectivity . OligomeSupplemental Fig. 3; We recently discovered that the small noncoding vault RNA1–1 directly binds to p62 and regulates p62 oligomerization and hence function in autophagy . Vault RTo identify the region of p62 that is required for RNA binding in a systematic manner, we generated a series of FLAG-HA tagged p62 truncation constructs A, and as1–122; aa 1–122) significantly increases normalized RNA binding above the level of wild-type p62, suggesting a negative modulatory function of the ZZ domain on RNA binding. However, the PB1 domain alone (aa 1–102) or the ZZ domain with the linker (aa 102–172) display little if any RNA-binding capacity  may play a regulatory role  protein C,D. Addicapacity C,D. Thes1–122 displays specific and maximal vault RNA1–1 binding compared to full-length p62 . In contrast, p62 association with the other vault RNA paralogs is not significantly changed. These findings reflect the prime role of vault RNA1–1 in p62 riboregulation as previously observed  conditions, we performed native immunoprecipitation followed by RT-qPCR of copurified RNA (RIP). In this assay, p62observed  and hint1–122—the PB1 oligomerization domain with its adjacent carboxy-terminal linker—accounts for specific vault RNA1–1 binding to p62. This result is unexpected in light of the previously identified ZZ domain mutant R139A/K141A, which showed reduced RNA binding and implicated the ZZ domain as a critical region for the RNA interaction . Yet, their RNA-binding capacity is not significantly changed . In addition to the in cellulo experiments, we used maltose-binding protein (MBP)-tagging to generate recombinant p62 with low oligomerization potential .We next generated FLAG-HA tagged mutant constructs of p62 to (i) assess, whether RNA binding depends on oligomerization, and (ii) to narrow down the RNA-binding interface in the context of full-length p62. To prohibit PB1-dependent oligomerization, we mutated the negatively charged OPCA motif A introduotential  and test1H, 15N-HSQC NMR spectroscopy with the minimal construct p621–122 which we forced into a monomeric form by the before-characterized triple mutation (D69A/D71A/D73A). We recorded spectra with and without equimolar amounts of in vitro transcribed full-length vault RNA1–1 to assess binding , confirming the direct involvement of these residues in RNA binding. In contrast, the RNA-binding capacity of the linker mutant MBP-p62 R106A/R107A . These results favor the interpretation that mutations targeting the linker and ZZ domain foster multimeric forms of p62 in cellulo that exclude RNA from binding to the autophagic receptor.In contrast, R22 and R96 appear to extend the RNA-binding interface without making a strong contribution to multimerization in the absence of RNA binding. Mutations in the linker region or ZZ domain of p62 both resulted in reduced RNA binding and increased presence of multimers D,E. To dSupplemental Fig. 4; The tertiary structure of vault RNA1–1 is presently unknown. Previous secondary structure analyses using RNase H probing were useful but limited in resolution . We applSupplemental Fig. 3). Overall, our data implicate nucleotides within the central loop of vault RNA1–1 as specific p62 contact points.Next, we conducted RNase A footprinting to identify vault RNA1–1 residues that are protected by p62 binding . When coSupplemental Fig. 5). We compared reintegrated wild-type vault RNA1–1 with two mutants that alter either the central loop triplet U36/U37/U38 alone (M1) or in combination with residues A46/C47 . As expected, the central domain mutants M1 and M2 tend to show reduced p62 binding in cellulo compared to the wild-type vault RNA1–1 . This conclusion is further supported in vitro by EMSAs, where the mutations in the central loop of vault RNA1–1 compete less well for p62 binding than the wt counterpart . This assay also shows that the central loop region of vault RNA1–1 alone is not sufficient for p62 binding in vitro  and reveals the importance of the 3D structure of vault RNA1–1 for p62 binding.To validate the functional importance of the central flexible loop region of vault RNA1–1 for riboregulation of p62, we generated a HuH-7 Flp-IN vault RNA1–1 KO cell line with a single FRT integration site. This KO cell line thus allowed the stable and isogenic reintegration of vault RNA1–1 variants expressed from the endogenous promoter  are both necessary and sufficient for maximal and specific vault RNA1–1 binding. Mutational analyses pinpoint to K7 and R21 as critical amino acids that are necessary for RNA binding  was shown to exert negative auto-regulation through direct interaction with the ZZ domain  that showed strongly diminished RNA binding and increased p62 oligomerization D,E. InteZ domain . In lighZ domain , we hypoZ domain . The excZ domain .Supplemental Fig. 6B; Supplemental Fig. 6B).As proposed before, our new data further corroborate that vault RNA1–1 primarily inhibits p62 oligomerization. Our new data also suggest that vault RNA1–1 may not be able to disrupt p62 oligomers that are formed when cargo binds to the ZZ domain. Integrating all available experimental evidence, we now suggest a refined mechanistic model for riboregulation of p62 by vault RNA1–1: under physiological, nutrient-replete conditions, vault RNA1–1 inhibits p62 oligomerization by binding the critical hinge points K7 and R21. When starvation reduces cellular vault RNA1–1 levels, p62 oligomerization via K7 and R21 is facilitated, stimulating autophagic flux  of vault RNA1–1, we identified nucleotides required for p62 binding , 4. We iThe p62/vault RNA1–1 binding interface may well be targeted by regulatory modifications that potentially influence riboregulation. For example, K7 has previously been found to be ubiquitinated by TRIM21 . Like vaOur data also demonstrate that autophagic clearance can be modulated by experimental changes of intracellular vault RNA1–1 levels . ModulatIn addition to its function as an autophagy receptor, p62 provides a platform for key cellular signaling pathways . While wWith hundreds of newly identified RNA-binding proteins , riboregThis work provides a structure-function analysis to identify critical residues of p62 and vault RNA1–1, respectively, for the specific interaction between the two partners and their function in the riboregulation of p62 in mammalian autophagy. As such, it answers what distinguishes vault RNA1–1 from the other three human vtRNAs, and how vault RNA1–1 achieves control of p62 oligomerization via K7 and R21. In the future, it will be important to examine this interaction at atomic resolution using state of the art structural technologies.Since our experiments have been conducted with cultured human cells, it will also be interesting to examine in vivo models  expressing the mutant RNAs and forms of p62, respectively, which we have defined in this work. The investigation of animal models and clinical samples in the context of viral infections, cancer or neurodegenerative disorders will advance our understanding of the functional importance of p62 riboregulation in organismal function and disease.Supplemental Table 1.Antibodies and reagents used in this study are listed in l-glutamine , and 100 U/mL penicillin/streptavidin  at 37°C and 5% CO2. The cells were routinely passaged 2–3 times per week. For this purpose, the cells were dissociated through the addition of Trypsin-EDTA . HuH-7 Flp-In cell line stocks and their derivatives were cultured in medium containing zeocin  or Hygromycin B Gold  depending on expressed marker genes. The parental hepatocellular carcinoma HuH-7 Flp-In cell line  was first established in HuH-7 cells (derived from a hepatocellular carcinoma of a human aged 57 yr) and their derivatives were cultured in DMEM containing 1 g/L glucose supplemented with 10% heat-inactivated FCS , 2 mM Restriction-free cloning was performed according to Supplemental Table 2; http://crispor.tefor.net; Version May 2017), the highest-scoring candidates ordered from Sigma-Aldrich, annealed, and ligated into BbsI linearized pSpCas9(BB)-2A-GFP/RFP/Cer constructs . Combinations of the generated plasmids were nucleofected into HuH-7 Flp-In cells using the SF Cell Line 4D-Nucleofector X Kit according to the manufacturer's guidelines, with 1Mio cells and 1 µg of total plasmid DNA in a 100 µL setup running program FF137. As a negative control, a mixture of all parental plasmids was used. A fluorescence-activated single-cell sort of double/triple-positive cells was performed 48 h after nucleofection. Upon expansion, the clones were tested for vault RNA1–1 deletion by PCR from genomic DNA using locus spanning primers . Cell lines lost the transiently transfected plasmids after outgrowth as checked via FACS.CRISPR/Cas9 genome editing was based on Transfections were performed using Lipofectamine 3000 according to the manufacturer's guidelines. For cell-based assays, XIE62-1004-A , the protein concentration determined by Bradford assay and the input material adjusted accordingly (1–2 mg/IP). Per IP, 25 µL anti-HA magnetic bead slurry was washed twice with PBS prior to addition to the samples. The IPs were performed for 1–2 h at 4°C with constant rotation. Following, the samples were washed six times with 1 mL of Trit-Lysis buffer and the reaction tubes exchanged after every second wash. For pH elution, 50 µL of 0.1 M glycine pH 2 were added per condition and incubated for 5 min at room temperature. The eluates were transferred into a new reaction tube and neutralized through addition of 7.5 µL of 1 M Tris-HCl pH 8.5.For IPs, one confluent ∅ 15 cm dish per IP served as starting material. The cells were washed twice with ice-cold PBS. The buffer was aspirated completely and the cells lysed on ice through the addition of 750 µL Trit-Lysis buffer  supplemented with cOmplete Protease Inhibitor Tablets  and 5 µg/mL RNasin . The cells were collected through scraping and homogenized by pipetting. Lysates were cleared by centrifugation for 10 min at 13,0002O.For protein analysis, 1% of input material and 15%–20% of elution were analyzed by western blotting. For RNA analysis, 5% of input material and 70% of elution were processed by the addition of 400 µL RNA lysis buffer (Zymo) and RNA extraction using the Zymo Quick-RNA MicroPrep RNA Extraction Kit (Zymo), following the manufacturer's guidelines, including the DNase I on-column DNA digest. Elution was performed with 15 µL RNase-free H2. Subsequently, the cells were lysed on ice with 0.75–1 mL PNK lysis buffer , and transferred into 1.5 mL reaction tubes. The lysates were homogenized by sonication on ice  and cleared by centrifugation at 16,000g for 10 min, 4°C. The protein concentration was measured by Bradford assay and the input material adjusted accordingly (1–2 mg/condition). Following, the lysates were treated with 5 ng/µL RNase A and 2 U/mL Turbo DNase for 15 min at 37°C and 1100 rpm. Per condition, 25 µL anti-HA magnetic bead slurry was washed twice with PBS prior to addition to the samples. The IPs incubated for 1.5–2 h at 4°C under constant rotation. Subsequently, the IPs were washed three times with PNK lysis buffer and three times with PNK wash buffer . Radioactive labeling of retained RNA was performed on beads in PNK wash buffer containing 0.1 µCi/µL [γ-32P] ATP, 1 U/µL T4 PNK, and 1 mM DTT for 15 min at 37°C and 850 rpm. After another four washes with PNK wash buffer, the proteins were eluted by addition of 50 µL 0.1 M glycin pH 2 for 5 min and eluates neutralized with 7.5 µL 1 M Tris-HCl pH 8.5. The samples were complemented with 4× sample buffer containing 200 mM DTT, heated to 70°C for 3 min, resolved by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were rinsed, dried, and a radioactive signal was detected with a phosphorimaging screen for 3–72 h. Following, the membrane was used for western blot analysis. Image analysis was performed with Fiji Image J 2.0.0 (https://imagej.net/Fiji). The relative PNK signal was calculated and normalized to the relative western blot signal to account for variation in IP efficiency.For the PNKs, one 80%–90% confluent ∅10 cm or ∅15 cm dish per condition served as starting material. The cells were washed twice with ice-cold PBS, the PBS was aspirated completely, and the cells were UV-crosslinked at 150 mJ/cmFor standard protein analysis, cells were washed twice with ice-cold PBS, the buffer aspirated completely and cells lysed through the addition of RIPA lysis buffer . Following, the cells were collected by scraping and protein concentration measured by Bradford assay. The lysates were mixed with 4× NuPage LDS Sample buffer supplemented with 200 mM DTT before denaturation at 70°C for 3 min. Typically, 5–15 µg total protein were resolved by SDS-PAGE on 4%–15% TGX Precast gels using 1× Laemmli running buffer . The proteins were transferred onto PVDF or nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad) and transfer efficiency assessed by Ponceau Red staining. The membranes were blocked in PBS-T 5% milk  for 1 h at room temperature. Primary antibodies were diluted in PBS-T 5% milk and added to the membrane overnight at 4°C under constant shaking. After three PBS-T  washes, each for 5 min, the membrane was incubated with HRP-conjugated secondary antibody in PBS-T 5% milk for 1 h at room temperature. Following three PBS-T washes, the western blots were developed using ECL on a Bio-Rad ChemiDoc MP Imaging System with auto-capture function.+ membrane with 0.8 mA/cm2 for 2.5 h in 0.5× TBE. Following, RNA and membrane were crosslinked by UV exposure at 150 mJ/cm2.Typically, 15–20 µg of total RNA were mixed with 2× RNA gel loading dye , denatured for 5 min at 95°C, loaded onto a denaturing 8% polyacrylamide gel , and separated for 1–2 h at 350 V in 0.5× TBE. A semidry blotting apparatus was used to transfer the RNA onto a Hybond N32P-labeled DNA antisense oligonucleotide probe for overnight incubation at 50°C . The DNA probe was labeled with PNK and purified over a Chroma Spin chromatography column  according to the manufacturer's guidelines. Finally, the membrane was washed three times with a high stringency buffer  and three times with a low stringency buffer , before visualizing the radioactive signal by 4–16 h exposure to a phosphorimaging screen.The membrane was prehybridized  for 1 h at 50°C before the addition of a radioactively Protein expression was performed according to 15NH4Cl as the sole nitrogen source at 22°C O/N upon induction with 0.5 mM IPTG. The cells were lysed in a buffer containing 50 mM Tris pH 8, 750 mM NaCl, 10% glycerol, 20 mM imidazole, and cOmplete, EDTA-free Protease Inhibitor Cocktail using a microfluidizer . Subsequently, the protein was purified from the cleared lysate by Nickel affinity chromatography  in the same buffer. The affinity tag was removed by 3C protease cleavage , dialysis, and an additional passage over the HisTrap column. As a final purification step, oligomers were removed by gel filtration on a Superdex S75 10/300 in a buffer containing 20 mM MES pH 6.5, 100 mM NaCl, 0.2 mM TCEP, and 0.05% NaN3.The oligomerization deficient p62 truncation p62 was cloned into pCoofy4 (Ref: PMID: 23410102) using restriction-free cloning , transfo3 were titrated with full-length vault RNA1–1 in the same buffer. At each titration step, an apodization-weighted sampled HSQC was collected  according to the manufacturer's guidelines. RNA was gel purified and phenol-chloroform extracted, dissolved in water and its concentration measured by QuBit assay (Thermo).For biochemical assays: pUC57-T7-vaultRNA1–1 plasmids were linearized and used for in vitro transcription of RNA using the MEGAshortscript Kit  with 2, 2 mM Spermidine-HCl, 40 mM DTT, 0.1 mg/mL BSA, 7.5 mM each NTP, 800 units/mL RNasin, 10 units/mL IPP, 25 µg/mL template DNA, and 10,000 units/mL T7 RNA polymerase and incubated 8 h at 37°C. The RNA was gel purified, extracted by electrophoresis, precipitated, and its concentration measured by NanoDrop.For NMR: pUC57-T7-vaultRNA1–1 plasmids were linearized and used for in vitro transcription of RNA in a large-scale reaction containing 100 mM HEPES-KOH pH 7.5, 10 mM MgCl2. The EMSA reactions typically contained 10 nM–2 mM protein, 10 nM radioactively labeled RNA , 150 nM nonlabeled bacterial tRNA, 1 mg/mL of BSA, 10 mg/mL RNasin, 5 mM DTT, 0.5 mM PMSF, 2.5 mM MgCl2, 100 mM KCl; 20 mM HEPES pH7.9; 0.2 mM EDTA, and 20% glycerol. In the case of competitive EMSAs, the binding reaction was competed with nonlabeled RNA at a typical range of 0.1 to 2 µM that was added to the protein together with the labeled probe. Reactions were incubated for 10 min at room temperature, loaded onto a native 5% acrylamide gel, and run overnight at 70 V in 0.5× TBE. Subsequently, the gel was dried for 1 h at 80°C and signal visualized by exposure to a phosphorimaging screen.Before the reaction, RNA was denatured for 2 min at 95°C and subsequently refolded in the presence of 2.5 mM MgCl32P-γUTP (Hartmann) and purified over a Chroma Spin chromatography column. Prior to RNase footprinting, the RNA was denatured for 10 min at 65°C and allowed to refold by ramping down 1°C/30 sec to room temperature. Per condition, 8 µL containing 20 µM 3′-end 32P-labeled RNA (∼1600 cps) and 6 µM protein were assembled in binding buffer , and incubated for 10 min at room temperature. RNase A (5 ng/µL) was prediluted  in H2O and 2 µL dilution added per reaction. The digest was incubated for 15 min at room temperature. Following, the RNA was extracted by Phenol-Chloroform extraction using TRI Reagent and dissolved in 2× RNA gel loading dye. For the alkaline ladder, 2 µL containing 20 µM 3′-end 32P-labeled RNA (∼400 cps) were mixed with 8 µL 50 mM NaHCO3 pH 9.2, incubated at 95°C for 5 min and supplemented with RNA gel loading dye. The samples and ladder were loaded onto a denaturing 8% polyacrylamide gel  and separated at 42 W (50°C–55°C) in 0.5× TBE. The gel was fixed for 10 min in a bath of 6% acetic acid and 10% EtOH, dried on Whatman filter paper in a vacuum gel dryer at 80°C for 40 min, and the radioactive signal visualized by exposure to a phosphorimaging screen.In vitro transcribed RNA was dephosphorylated with Fast Alkaline Phosphatase (FastAP) according to the manufacturer's guidelines and purified over a Zymo Quick-RNA Miniprep column. Subsequently, the RNA was radioactively labeled in a T4 PNK reaction with 2O, denatured at 95°C for 2 min and directly transferred on ice. The RNA was allowed to fold for 20 min at 37°C upon addition of 12 µL 2× folding buffer . Subsequently, 12 µL protein (15 µM) in 1× folding buffer was added to the reaction and incubated for 10 min at 37°C before splitting the reaction into 4 × 8 µL each. For the modification reactions, 92 µL Mod. buffer A for DMS  or Mod. buffer B for CMCT  were added. For DMS treatments 1 µL of 16×, 4×, or 2× DMS diluted in EtOH was added to the reactions, immediately mixed and incubated for 15 min at 37°C. For CMCT treatments, 5, 10, or 15 µL of CMCT solution (42 mg/mL in H2O) were added to the reactions, immediately mixed and incubated for 15 min at 37°C. The reactions were stopped on ice, RNA extracted with TRI reagent according to the manufacturer's guidelines and EtOH precipitated. Modified residues were detected by primer extension reaction using a radiolabeled DNA oligonucleotide (T4 PNK reaction and purification over a Chroma Spin 10 chromatography column) complementary to the 3′-end of the RNA. For primer extension, 1 µL of 10 µM radiolabeled primer was annealed to 2 µL of extracted RNA and used in a 5 µL reaction with AMV reverse transcriptase according to the manufacturer's guidelines. For the sequencing lanes, in vitro transcribed RNA was used in primer extension reactions that contained respective ddNTPs. The reactions were stopped with 20 µL Stop buffer  and the cDNA EtOH-precipitated and separated on a denaturing 8% polyacrylamide gel  at 42W in 0.5× TBE. Subsequently, the gel was fixed for 10 min in a bath of 6% acetic acid and 10% EtOH, dried on Whatman filter paper in a vacuum gel dryer at 80°C for 40 min, and the radioactive signal visualized through exposure to a phosphorimaging screen.Per compound, four probing reactions were performed with one untreated control and three treated samples of varying concentrations. Therefore, 20 pmol in vitro transcribed RNA were diluted in 12 µL HSupplemental Table 2. The analysis was performed on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR system in the default standard two-step cycling run. Relative expressions were calculated with the ΔΔCT method. The mean Ct value was used when normalizing to multiple housekeeping genes.Reverse transcription was performed with 500 ng of total RNA or 7 µL of purified RNA from IPs in a 10 µL reaction using the Maxima First Strand Synthesis kit  according to the manufacturer's guidelines. Control reactions that did not include the reverse transcriptase (-RT) were included for each sample. The obtained cDNA was diluted 1:20–1:50 in nuclease-free water and assembled in 10 µL qPCR reactions with 5 µL cDNA, 4.6 µL Fast SB SYBR Green Master Mix, 0.2 µL each primer (10 µM). Primer sequences can be found in www.graphpad.com). Details for each experiment can be found in the corresponding figure legend. Image analysis was performed with Fiji Image J 2.0.0 (https://imagej.net/Fiji). Structure representations were generated using PyMOL Molecular Graphics System 2.0.7 (https://pymol.org). NMR analysis was performed with NMRPipe (https://www.ibbr.umd.edu/nmrpipe/) and NMRFAM SPARKY (https://nmrfam.wisc.edu/nmrfam-sparky-distribution/).Statistical analysis was performed with GraphPad Prism 8.3.1 (279; Supplemental material is available for this article.


Application of electron cryomicroscopy (cryo-EM) has greatly expanded our understanding of the atomic structures of mature pathological amyloid fibrils, but little is known at the molecular level of the initiation of fibril formation. RNA has been shown to be one cofactor in the formation of fibrils of tau protein and is also known to bind to other proteins, including TDP-43, FUS, and HNRNPA2, which form pathological inclusions. Our cryo-EM structure of recombinant tau protein with RNA reveals a 36-residue, C-terminal fibril core bound to RNA which runs parallel to the fibril axis. We speculate that this structure could represent an early step in the formation of tau fibrils. In neurodegenerative diseases including Alzheimer’s and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here, we present the 3.4-Å resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryomicroscopy (cryo-EM). The structure reveals the familiar in-register cross-β amyloid scaffold but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNase is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients’ brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature. The two pathological hallmarks of Alzheimer’s disease (AD) are extracellular Aβ plaques and intracellular tau neurofibrillary tangles that accompany neuron loss in the brain . The depCofactors are necessary to form and stabilize tau fibrils in vitro and in vivo. In vitro, fibrillization of recombinant tau requires cofactors such as heparin, RNA, or arachidonic acid –7. Recen814RNA is implicated as a cofactor in the formation of fibrils of RNA binding proteins (RBPs) associated with amyotrophic lateral sclerosis, such as FUS, TDP-43, and hnRNPA1. These RBPs play important roles in gene expression by forming ribonucleoprotein complexes and participating in RNA processing steps including alternative splicing, stress granule formation, and RNA degradation , 21. RBP22RNA also induces tau to condense into a separate phase similar to RBPs  despite To learn how RNA interacts with tau at the atomic level and triggers fibril formation, we determined the cryo-EM structure of the fibril of recombinant full-length tau induced by RNA. With this structure and associated biochemical experiments we addressed several questions: 1) Which tau residues compose the RNA binding site? 2) Why is the C-terminal region of tau poorly ordered in tau fibrils extracted from autopsied brains of patients with tauopathies? 3) How does RNA facilitate reversibility in fibril assembly and modulate fibril stability? 4) What role does RNA play in nucleating or deterring  formatioTo test whether RNA can act as a cofactor to assist the fibril formation of full-length tau , we incubated tau40 in the presence of total RNA extracted from mouse liver. We chose RNA from mouse liver because liver has less tau protein than other organs . In the Next, we examined whether the tau-RNA fibrils have the capability to seed tau aggregation in HEK293 tau biosensor cells that express YFP-tagged tau-K18 . As showTo test the role of RNA length on RNA-induced tau fibril formation, we performed in vitro fibrillation experiments using total RNA or RNA treated with RNase (termed predigested RNA). Predigested RNA was incubated with tau40 monomers, but no amyloid fibrils formed even after 60 h of shaking . In contSI Appendix, Fig. S1A). Fewer and shorter fibrils are formed in the presence of digested RNA . We did not observe any fibril in the presence of 5% seeds (2.5 µM of sonicated tau40 fibrils) and absence of RNA , consistent with the critical role of polymerized RNA in supporting fibril formation. Similarly, no fibrils were observed when both RNA and seeds were predigested by RNase . We speculate that digested RNA would not have been sufficient to produce short tau fibrils if it were not for the small amount of RNA that accompanied the fibril seeds, consistent with the observation that seeding of monomer in the presence of digested RNA and digested seeds lacks even short fibrillar structures. These results suggest that polymerized RNA appears to be an essential constituent of self-assembling and seeding-active tau fibrils.Fichou et al.  reportedSI Appendix, Fig. S2), consistent with previous studies that reported the binding of polyA RNA to tau-K18 and tau-K19 fibrils . The protofilaments are composed of flattened tau molecules stacked in parallel, in-register β-sheet alignment . The fibril core spans 36 residues near the C terminus of tau, from Glu391 to Ala426, and encompasses five β-strands   classification: one fibril polymorph is twisted, and the other lacks a twist. The twisted species was the more abundant, accounting for 75% of the fibrils. The species lacking a twist accounted for the remaining 25% of fibrils and was not suitable for structure determination. The twisted tau-RNA fibril exhibits a pitch of 829 Å. It is composed of two protofilaments related by a pseudo 2 Val420) A and E. Val420) 8, 16, 36.Stripes of residual cryo-EM density run along the length of the two tau protofilaments, resembling RNA strands on the fibril surface C and D. 37The interaction of RNA and side chains in our tau-RNA fibril is reminiscent of the interaction between side chains and unidentified cofactors in recent brain-derived fibril structures. The proximity of residual density near two positively charged side chains (Arg406 and His407) in our tau-RNA fibril matches the pattern seen in cryo-EM maps of ex vivo α-synuclein fibrils . For exaWe note that density for RNA is relatively weak. One explanation for this weakness is the potential mismatch in distance between repeating units of tau and RNA. Tau molecules stack in a protofilament with a repeat distance of 4.8 Å, whereas the preferred base stacking distance of RNA nucleotides is 3.3 Å. As a result, the nucleotide bases stack with less aromatic overlap than normally observed in canonical A-form RNA structures. The lack of overlap probably limits the stability of the RNA–tau interaction.To determine whether RNA is integrated in the fibril architecture or plays only a catalytic role in amyloid fibril formation, we exposed mature fibrils to enzymatic digestion. Tau-RNA fibrils were digested by mixing the fibrils with RNase at 1:0.6 and 1:3 molar ratios (fibrils:RNase) for 2 h at 37 °C. As shown in −1 chain−1, −0.39 kcal mol−1 residue−1) is similar in value to other reversible amyloids such as hnRNPA2 LCD  and FUS    , 43  B and C. sidue−1) , 44. In sidue−1) , and thesidue−1) C.The relative ease of dissolving tau-RNA fibrils may be partly attributed to the lack of a sizable hydrophobic core. Note the paucity of strongly stabilizing residues , and thrSI Appendix, Fig. S5).To test the capability of tau fibrils in the presence of RNA to disassemble upon raising the pH value, tau fibrils were incubated with 30 mM CAPS at pH 9.5 for 3 h at 37 °C. The abundance of tau fibrils in electron micrographs decreased approximately fivefold at pH 9.5, supporting the validity of the hypothesis that pH elevation deprotonates Asp418 and Asp421 and facilitates tau fibril disassembly  and its mated sheet face . Steric zippers B1 and B2 are composed of a surface displaying residues Pro397, Val399, and Thr403 mated with a complementary surface displaying residues Thr414, Gly415, and Ile417. Steric zipper C differs from the preceding zippers because it mates together identical β-sheets from separate protofilaments (homotypic). β-sheet residues Leu408, Asn410, and Ser412 mate with identical residues of the opposing protofilament. In agreement with our cryo-EM model, the potential for forming such a homotypic steric zipper was predicted by our ZipperDB database. Rosetta calculates energetic stabilities for homotypic hexapeptide segments. The segment 408-LSNVSS-413 exceeds the −23 kcal/mol threshold for identifying amyloid fibril-forming segments .The cryo-EM reconstruction of the tau-RNA fibril reveals tight-fitting interfaces between its β-sheets that entirely exclude water molecules. Such stable structural motifs consisting of mated β-sheets are termed steric zippers. Our RNA-induced tau fibril is stabilized by five steric zippers, referred to as A2, and C A. StericSI Appendix, Fig. S6). The hydrophobic interactions between the side chains of Val411 and Val420 stabilize steric zippers B1 and B2 and help assemble the RNA binding site. The tau-RNA structured core, residues His407 to Ser422, adopt a beta-arch motif, formed by multiple copies of antiparallel protein strands (β4 and β5), which stack in layers perpendicular to the fibril axis .Notice that the polyanion RNA interacts with tau basic residues of Arg406 and His407, thereby stabilizing the β-arch motif (residues 408 to 421) at the C terminus of the ordered tau segment . Tau-K18+/K19+ include an additional eight-residues C terminal to tau-K18 in order to encompass all residues found in the core of AD fibril structures (residues 306 to 378). All three of these constructs lack the C-terminal domain of tau that is ordered in our full-length tau-RNA structure. EM micrographs show that addition of RNA facilitates fibril formation of 50 or 100 µM tau-K18 . Interestingly, the yield of fibrils generated by tau-K18 with RNA is lower than the yield from tau40 (with RNA). The tau-K18 fibril yield increases when seeded by tau40-RNA fibrils, indicating these C terminus-containing seeds can coax the C terminus–deficient tau-K18 monomers into fibrils. In addition to tau-K18, we found that the constructs tau-K18+ and tau-K19+ have a greater amount of fibril formation in the presence of RNA compared to tau-K18. EM data also indicates that seeding with tau40-RNA can lead to faster and more abundant fibril formation . These data suggest that RNA may be able to bind to regions in the microtubule binding region of tau and promote fibril assembly. The fact that residues from K18, K18+, and K19+ are not found in our full-length tau-RNA structure indicates the strongest RNA–tau binding site is that visualized in our structure: Arg406 and His407.To test whether RNA may induce tau fibril formation by binding to other regions of the tau molecule than visualized in our tau-RNA structure, we tested fibrillation of three truncated versions of tau: tau-K18 (residues 244 to 372 with repeat 2), tau-K18+ (244 to 380 with repeat 2), and tau-K19+ (244 to 380 without repeat 2)  was expressed in pET28b vector in BL21-Gold E. coli. All proteins were purified as previously described (Human wild-type tau-K18 (residues 244 to 372), human tau-K18+ (residues 244 to 380 of 4R), and human tau-K19+ (residues 244 to 380 of 3R tau) were expressed in a pNG2 vector in BL21-Gold escribed –53.E. coli cells grown in LB to an optical density of 0.7 at 600 nm. Cells were induced with 0.5 mM IPTG for 3 h at 37 °C and lysed by sonication in 20 mM MES buffer (pH 6.8) with 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, and HALT protease inhibitor before addition of NaCl 500 mM final concentration. Lysate was boiled for 15 min and then clarified by centrifugation at 32,000 × g for 15 min and dialyzed to 20 mM MES buffer (pH 6.8) with 50 mM NaCl and 5 mM DTT. Dialyzed lysate was purified on a 5 mL HighTrap SP ion exchange column and eluted over a gradient of NaCl from 50 to 600 mM. Proteins were polished on a HiLoad 16/600 Superdex 75 pg in 10 mM Tris (pH 7.6) with 100 mM NaCl and 1 mM DTT and concentrated to ∼20 to 60 mg/mL by ultrafiltration using a 3-kDa cutoff.For human tau-K18, tau-K18+, and tau-K19+ purification, BL21-Gold E. coli cells grown in TB to an OD600 = 0.8. Cells were induced with 1 mM IPTG for 3 h at 37 °C and lysed by sonication in 50 mM Tris (pH 8.0) with 500 mM NaCl, 20 mM imidazole, 1 mM beta-mercaptoethanol, and HALT protease inhibitor. Cells were lysed by sonication, clarified by centrifugation at 32,000 × g for 15 min, and passed over a 5-mL HisTrap affinity column. The column was washed with lysis buffer and eluted over a gradient of imidazole from 20 to 300 mM. Fractions containing purified tau40 were dialyzed into 50-mM MES buffer (pH 6.0) with 50 mM NaCl and 1 mM beta-mercaptoethanol and purified by cation exchange. Peak fractions were polished on a HiLoad 16/600 Superdex 200 pg in 1× PBS (pH 7.4), 1 mM DTT, and concentrated to ∼20 to 60 mg/mL by ultrafiltration using a 10-kDa cutoff.For human tau40 purification, Tau40 was expressed in pET28b with a C-terminal His-tag in BL21-Gold 2O.Wild-type mouse liver RNA was isolated with RNA TRIzol Reagent  according to manufacturer’s protocol. Briefly, 100 mg of mouse liver was homogenized in 1 mL TRIzol reagent, extracted by 0.2 mL chloroform, precipitated by 0.5 mL isopropanol, and dissolved in 100 μL RNase-free HRecombinant tau40 was diluted at 50 µM in 20 mM ammonium acetate, pH 7.0, and incubated with 400 µg/mL of RNA. The amyloid fibril formation was examined using negative stain transmission EM after 2 d of shaking at 37 °C.Recombinant tau40, tau-K18, tau-K18+, and tau-K19+ were diluted individually to 50 μM in 20 mM ammonium acetate, pH 7.0, 10 μM ThT, and mixed with 400 µg/mL of RNA. Protein was liquated to three replicate wells of a 384-well plate (Thermo Scientific Nunc), and plates were incubated at 37 °C for 60 h with shaking. The fibrillation was confirmed by negative-stain transmission EM.SI Appendix, Fig. S1. The digestion of RNA was performed by incubating RNA or tau-RNA seeds with RNase at 4:1 ratio at 37 °C for 6 h.Digestions of RNA were carried out by incubating RNA or tau-RNA seeds with RNase A  in 20 mM ammonium acetate, pH 7.0, as shown in g for 1 h and followed by washing twice with RNA free water. Fibrils was treated with RNase A at 1:0.6 and 1:3 molar ratio (fibrils:RNase) in 20 mM ammonium acetate, pH 7.0, for 2 h at 37 °C.Tau fibrils were centrifuged at 159,000 × Negative-stain transmission EM samples were prepared by applying 4 μL of solution to 400 mesh carbon-coated formvar support films mounted on cooper grids . The grids were glow-discharged for 30 s before applying the samples. The samples were incubated on the grid for 1 min and then blotted off with a filter paper. The grids were stained with 4 μL of 2% uranyl acetate for 2 min and washed with an additional 4 μL of 2% uranyl acetate and allowed to dry for 10 min. The grids were imaged using a T12 (FEI) election microscope.2 in a humidified incubator. Tau RNA-fibril seeds were sonicated in a cup horn water bath for 5 min and then mixed with 1 volume of Lipofectamine 3000  prepared by diluting 1 μL of Lipofectamine in 19 μL of OptiMEM. After 20 min, 10 μL of fibrils were added to 90 μL of tau biosensor cells. The number of seeded aggregates was determined by imaging the entire well of a 96-well plate in triplicate using a Celigo image cytometer (Nexcelom) in the YFP channel. Aggregates were counted using ImageJ  cryo-EM center. The microscope was operated with 300 kV acceleration voltage and slit width of 20 eV. Movies were acquired using superresolution mode with a nominal physical pixel size of 1.06 Å per pixel−1 (0.53 Å per pixel in superresolution movie frames) with a dose per frame of ∼1.3 e−/Å2. Forty frames were recorded for each movie . Automated data collection was driven by EPU automation software package. Motion correction and dose weighting was performed using Unblur and contrast transfer function estimation was performed using CTFFIND 4.1.8 . The cryo-EM dataset was collected on 300 kV Titan Krios (FEI) microscope with a Gatan K3 camera located at the Stanford–SLAC Cryo-EM Center (SND 4.1.8 . All fibND 4.1.8 . We usedND 4.1.8 , 59. ParND 4.1.8 . The 3D The refined map was sharpened using phenix.auto_sharpen at the resolution cutoff as indicated by half-map FSC . We usedThe stabilization energy was calculated for each residue by the sum of the products of the area buried for each atom and the corresponding atomic solvation parameters. The overall energy was calculated by the sum of energies of all residues, and different colors were assigned to each residue in the solvation energy map.Supplementary File


Flaviviridae, which is mostly transmitted via tick bites. However, transmission by consumption of virus-contaminated goat raw milk and goat raw milk products has also been described. Only a few methods have been reported for the detection of TBEV in food so far. Here, we compare different virus extraction methods for goat raw milk and goat raw milk cream cheese and subsequent detection of TBEV-RNA by RT-qPCR. Langat virus (LGTV), a naturally attenuated TBEV strain, was used for artificial contamination experiments. Mengovirus and the human coronavirus 229E were compared to assess their suitability to serve as internal process controls. Out of three tested extraction protocols for raw milk, sample centrifugation followed by direct RNA extraction from the aqueous interphase yielded the best results, with a recovery rate (RR) of 31.8 ± 4.9% for LGTV and a detection limit of 6.7 × 103 LGTV genome copies/ml. Out of two methods for cream cheese, treatment of the samples with TRI Reagent® and chloroform prior to RNA extraction showed the best RR of 4.7 ± 1.6% for LGTV and a detection limit of 9.4 × 104 LGTV genome copies/g. RRs of Mengovirus and LGTV were similar for both methods; therefore, Mengovirus is suggested as internal process control virus. The developed methods may be useful for screening or surveillance studies, as well as in outbreak investigations.Infection with the tick-borne encephalitis virus (TBEV) can cause meningitis, meningoencephalitis and myelitis in humans. TBEV is an enveloped RNA virus of the family  Flavivirus within the family Flaviviridae  belongs to the genus ae ICTV, . It is aae ICTV, . The cliae ICTV, . Until nae ICTV, . The subae ICTV, . In contae ICTV, .Ixodes ricinus for the European subtype and Ixodes persulcatus for the Asian subtypes /ml , a naturally attenuated TBEV strain . Goat raw milk cream cheese was purchased at an organic-food market in Berlin, Germany. Until use, sample aliquots were stored at − 20 °C.2. The LGTV-containing supernatant was collected and aliquots of this LGTV stock were stored at − 80 °C until further use. Mengovirus strain vMC0  was propagated in HeLa-cells (ATCC® CCL-2™) according to the ISO 15216-1 annex E  with 1 × NEAA, 2 mM L-glutamine, and 0.1 µg/ml gentamicin (hereafter referred to as complete DMEM) supplemented with 10% FBS. For HCoV-229E propagation, an 80% confluent cell monolayer was incubated with the virus for 1 h at 34.5 °C. Subsequently, the inoculum was removed, complete DMEM supplemented with 5% FBS added and incubated at 37 °C and 5% CO2 for 30 h. Thereafter, the culture flask was frozen once at -80 °C and thawed at 37 °C. Cell debris was removed by centrifugation at 1000 × g for 20 min at 4 °C. The supernatant (“HCoV-229E stock”) was stored at − 80 °C until use.All cell culture reagents and media were purchased from Pan Biotech  unless otherwise indicated. LGTV  was propagated in the human lung carcinoma cell line A549  which was maintained in Eagle’s Minimal Essential Medium  supplemented with 10% fetal bovine serum (FBS), 1% non-Essential Amino Acids (NEAA), 1% L-glutamine and 1% gentamicin. Briefly, a 90% confluent A549 cell monolayer was inoculated with LGTV and incubated for three days at 37 °C and 5% COx E ISO, . HCoV-222. After 3 h, the inoculum was removed and the cell monolayer was covered with 3 ml overlay medium (previously held in a water bath at 40 °C). The overlay medium contained 1% agarose  in 1 × EMEM without phenol red (stock solution 2 ×) and supplemented with 10% FBS, 1% NEAA, 1% l-glutamine and 1% gentamicin. The plates were incubated at 37 °C and 5% CO2 for 72 h and cells were thereafter fixed with 10% paraformaldehyde  overnight at 4 °C. Subsequently, the solution and solid overlay was removed and 1% crystal violet  solution was added. After washing, plaques were manually enumerated and a virus titer of 7 × 105 PFU/ml was determined for the LGTV-stock. For titration of HCoV-229E stock infectivity, Huh-7 cells (6.25 × 105/well) were seeded in 6-well plates in complete DMEM with 10% FBS for 24 h. Subsequently, medium was removed, 500 µl of tenfold virus sample dilutions in DMEM were added to the wells and incubated for 1 h at 34.5 °C and 5% CO2 while manually rocking the plates every 10–15 min. After removing the inoculum, the cell monolayer was covered with 3 ml of the same overlay medium as described for the LGTV plaque assay. The cells were fixed at 4 °C for 1 day after adding 1 ml 10% paraformaldehyde solution  to the agarose plug. Thereafter, paraformaldehyde was aspirated and discarded, the agarose plug removed and cells stained with 1% crystal violet. Cells were washed with water, dried, and stored in the dark until plaques were manually counted. A virus titer of 2.3 × 106 PFU/ml was determined for the HCoV-229E stock.For titration of LGTV stock infectivity, A549 cells were seeded into 6-well plates  at a density of 300,000 cells/well and were incubated for one day. Thereafter, the cells were inoculated with duplicates of a tenfold serial virus dilution (1 ml/well) in MEM at 37 °C and 5% CO2 for 1 h, the virus dilutions were removed and replaced with medium with additives  was visible in the wells with less diluted virus. The Spearman and Kärber algorithm  along with 10 µl of Mengovirus (106 TCID50/ml) or 35 µl HCoV-229E (2.3 × 106 PFU/ml) as process control were added to 140 µl goat raw milk or to 0.14 g or 0.1 g  raw goat cream cheese directly before the experiment. As a recovery rate (RR) control (inoculum), 140 µl PBS were mixed with 4 µl of LGTV and either 10 µl of Mengovirus or 35 µl HCoV-229E, respectively.For generation of artificially contaminated samples, 4 µl of LGTV . Subsequently, 10 ml of a 10% (w/v) PEG/NaCl-solution was added, followed by incubation on a rotator at 4 °C for 1 h. Thereafter, the solution was centrifuged at 10,000×g for 30 min at 4 °C, and the supernatant was discarded. The precipitate was resuspended in 500 µl PBS and subsequently 500 µl of a butanol/chloroform (1:1) solution was added, followed by mixing. After centrifugation at 14,000 rpm for 15 min at 4 °C, 140 µl of the aqueous phase was aspirated and viral RNA was extracted with the QIAamp Viral RNA Mini Kit, as described above. For method 3, 140 µl of the contaminated milk sample were centrifuged at 4 °C and 6000×g for 10 min. The aqueous interphase was aspirated and 140 µl (120 µl of aqueous interphase filled up with PBS) was used for viral RNA extraction using the QIAamp Viral RNA Mini Kit.Three different methods were tested for virus and RNA extraction from goat raw milk, which are schematically shown in Fig. g and 4 °C for 10 min, and the RNA was extracted from 140 µl (120 µl of aqueous interphase filled up with PBS) using the QIAamp Viral RNA Mini Kit. Method 5 is based on an extraction protocol for viral RNA from food with high content of fat and protein (sausages)  was transferred into a new tube and filled up with PBS to 280 µl. Subsequently, RNA was extracted with the QIAamp Viral RNA Mini Kit using the double amount of lysis buffer and reagents before loading to the column as recommended by the manufacturer for 280 µl samples.For virus and RNA extraction from goat raw milk cream cheese, two methods were compared which are schematically shown in Fig. The RNA samples were analyzed for the presence of TBEV-RNA by RT-qPCR as previously described  × 100%, where ΔCq = Cqmatrix − Cqinoculum , and m is the slope of the inoculum control virus RNA standard curve.The recovery rate (RR) for each virus was calculated according to ISO 15216-2 ISO, . Brieflyα = 0.05). An unpaired Student’s t-test (α = 0.05) was used to determine differences between RRs of the two virus extraction methods from goat raw milk cream cheese. Statistical analyses were performed using SigmaPlot Version 14 .Differences between the RRs of the three extraction methods from goat raw milk were determined by one-way analysis of variance (ANOVA) with Bonferroni correction  and used for in vitro transcription with the MEGAscript in vitro transcription system . The transcribed RNA was purified using the Monarch RNA purification kit (New England Biolabs) and quantified by using Qubit™ RNA High Broad Range Assay Kit  and the Qubit 4 fluorometer . Molecule copy numbers were calculated based on the determined RNA mass and the theoretical molecular weight of the LGTV RNA standard molecule. The LGTV RNA standard stock solution contained 6.38 × 1012 copies/µl. RNA was extracted from the LGTV stock (from cell culture as described above) using the QIAamp Viral RNA Mini Kit and subjected to RT-qPCR for TBEV-RNA detection as described above, along with a tenfold dilution series of the LGTV RNA standard stock solution. The LGTV stock had a concentration of 2.35 × 109 genome copies/ml.An LGTV RNA standard was generated and was used for determination of the viral genome copy number per ml of the LGTV stock. Briefly, a gBlocks™ gene fragment of the 5`UTR sequence (nucleotide 52–176 in NCBI reference sequence of LGTV: NC_003690.1) of LGTV (125 bp) was obtained from Integrated DNA Technologies  and cloned into pCR4-TOPO using a TOPO TA cloning kit  according to the manufacturer’s instructions. DNA of the resulting plasmid was purified using the QIAGEN Plasmid Mini Kit , linearized with the restriction enzyme 7–6.7 × 102 LGTV genome copies (2 × 104–2 × 10–1 PFU) per ml goat raw milk and 9.4 × 107–9.4 × 102 LGTV genome copies (2.8 × 104–2.8 × 10–1 PFU) per g goat raw milk cream cheese were used. The milk samples inoculated with the LGTV dilutions were then administered to extraction method 1 and 3, whereas that of cream cheese were tested with method 5. Negative controls consisting of goat raw milk and goat raw milk cream cheese without LGTV inoculation were included in each analysis. Each inoculation level was performed in triplicate for the respective food matrix. RNA was analyzed by RT-qPCR for TBEV-RNA detection as described above. The detection limit was defined as the lowest genome copy number, for which all three subsamples tested positive.Samples of 140 µl goat raw milk and 0.1 g goat raw milk cream cheese were inoculated with 4 µl of tenfold serially diluted LGTV solutions in water. Contamination levels of 6.7 × 10In order to compare extraction protocols for goat raw milk, two very different protocols were selected, tested and compared Fig. A and B. As shown in Fig. For TBEV detection in goat raw milk cream cheese, two different extraction methods were selected, tested and compared Fig. A and B. 4 LGTV genome copies (corresponding to 20 PFU) per ml of goat raw milk and 6.7 × 103 LGTV genome copies (corresponding to 2 PFU) per ml of goat raw milk for method 3. For method 5, the detection limit was at 9.4 × 104 LGTV genome copies (corresponding to 28 PFU) per g of goat raw milk cream cheese.Based on the results for RRs of LGTV-RNA presented above, methods 1 and 3 for goat raw milk and method 5 for goat raw milk cream cheese were selected for further characterization regarding determination of detection limits. Samples of both matrices were contaminated in triplicates with tenfold dilution series of LGTV and analyzed for the presence of LGTV-RNA using the selected methods, respectively. As shown in Table Transmission of TBEV via consumption of goat raw milk and goat raw milk products has been described repeatedly  with a complex elution/concentration method involving PEG precipitation and chloroform/butanol cleanup (method 2). PEG-based precipitation has already been successfully used for TBEV concentration from cell culture supernatant  was compared with a more complex method using TRI Reagent® extraction and chloroform cleanup (method 5). It was previously shown that the latter method is most suitable for analysis of sausages  and goat raw milk cream cheese (method 5). Mengovirus can be recommended as an internal process control for monitoring the whole analytical process. Future studies should focus on the evaluation of the methods with naturally contaminated field samples. The developed methods can be useful for screening or surveillance studies, as well as in outbreak investigations.


Further, considerations regarding the quality control of circRNA vaccines are highlighted herein, and the main challenges and problem-solving strategies in circRNA vaccine development and quality control are outlined to provide a reference for circRNA vaccine-related research.Owing to the success of linear mRNA coronavirus disease 2019 (COVID-19) vaccines, biopharmaceutical companies and research teams worldwide have attempted to develop more stable circular RNA (circRNA) vaccines and have achieved some preliminary results. This review aims to summarize key findings and important progress made in circRNA research, the  Coronavirus disease 2019 (COVID-19) vaccines are currently the first vaccines to be rapidly and successfully developed and applied through multiple technological routes following the publication of a pathogenic sequence . These vin vivo compared with linear RNAs  Exonic circRNAs contain only exons, account for more than 80% of all identified circRNAs, and can be generated by lariat-driven circularization or direct back-splicing circularization. Base-pairing between the two flanking introns, which contain reverse complementary sequences, brings together a downstream 5′ ss and an upstream 3′ ss and facilitates back-splicing reactions to produce circular RNAs. , 67–69.According to the published studies, factors, such as sequence characteristics, protein regulators, and transcription stage, can affect the efficiency of back-splicing circularization , 41, 67.According to several studies, circRNAs exist in various types of tissues in different species and possess multiple biological functions, such as the regulation of gene transcription and protein encoding , 71–74. circSEP3 (derived from exon 6 of SEPALLATA3) causes transcriptional termination through R-loop formation with cSMARCA5 (derived from exon 15 of SMARCA5) (circRHOT1 recruits Tat-interactive protein 60 kDa (TIP60) to the nuclear receptor subfamily 2 group F member 6 (NR2F6) promoter, thereby promoting cell proliferation, migration, and invasion in hepatocellular carcinoma through the induction of proto-oncogene expression  . circRHOpression .circHIPK3 (derived from exon 2 of homeodomain interacting protein kinase 3 (HIPK3)) can bind to miR-124 and inhibit its activity, thereby preventing miR-124 from inhibiting human cell proliferation . For instance, feration ; and cir124-2HG . Studiesradation .circBART2.2, after infecting host cells, which can be recognized by the RIG-I receptor of the cells  produces the circRNA, he cells . Thereafhe cells –85.via internal ribosomal entry site (IRES)-mediated or m6A-induced ribosome engagement site (MIRES)-mediated translation initiation instead of 5’ cap-dependent translation initiation  and localized to the cytoplasm, contains m6A modifications and performs E7 oncoprotein translation  is used for the direct removal of the β and γ phosphates  Chemical synthesis: cyanogen bromide (BrCN) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) induces covalent bond formation between the 5’-terminal phosphate and 3’-terminal hydroxyl groups of linear RNAs to produce circRNAs. The occurrence of side reactions, such as the formation of 2’-5’ phosphodiester bonds during the circularization reaction, is a limitation of this approach. Further, chemical bonds in RNA oligomers are usually less effective than those in DNA analogs  as delivery systems. In mice separately immunized with doses of 10 μg/animal and 50 μg/animal, the serum neutralizing antibody titers at 5 weeks post-boost were approximately 10 in mice .Seephetdee et al. adopted the group I intron splicing method for RNA circularization to formulate a COVID-19 circRNA vaccine using the spike (S) protein of the original COVID-19 strain as the antigen-binding construct and including multiple mutations . Mice administered two immunizations of the vaccine at a dose of 5 μg/animal exhibited certain neutralizing activity against the Alpha, Beta, Delta, and Omicron variants and pseudoviruses. IFN-γ responses were also elicited in T cells .Wang et al. formulated a COVID-19 circRNA vaccine using the group II intron splicing method for RNA circularization. When used for mice immunization, the vaccine elicited strong RBD-specific memory B cell responses and balanced Th1/Th2 cellular immune responses. The serum IgG titer of mice immunized with the circRNA vaccine was 10-fold that of mice immunized with a linear mRNA vaccine. Further, the circRNA vaccine exhibited good neutralizing activity against pseudo-SARS-CoV-2 and effectively blocked the binding of three types of receptor-binding domain (RBD) mutants  to the hACE2 receptor on 293T cells. The COVID-19 circRNA vaccine could perform continuous expression in cells for 6 days, while expression by the self-formulated mRNA vaccine only lasted for 2 days .The use of the T4 dt intron splicing method for RNA circularization causes the generation of “splicing scar” sequences, with different sequence lengths leading to different topological structures. A study revealed that differences in the constituent elements and compositions of circRNAs affected the translation efficiency, with vector topological structure, untranslated region (UTR), and IRES playing the most critical roles , 59. Thrvia IRES-mediated translation pathway. Similar to the linear mRNA vaccines, the circRNA vaccines are also translated into proteins in bodies to induce the robust humoral and effective cellular immunity, among which liposome and others could play the role in vaccine adjuvants. Some studies indicated that circRNA vaccines could continue to translate into proteins longer than the linear mRNA vaccines and even up to about 7 days  RNA circularization; and (3) Vaccine encapsulation using delivery systems, such as LNPs. Therefore, the acquisition of high-quality plasmids is of primary importance  Synthesis of linear RNA molecules through portance . The indproducts . In the products , 24. HowH of 7.5 . FinallyH of 7.5 Figure 4H of 7.5 .Presently, two COVID-19 linear mRNA vaccines have already been marketed; however, the quality control processes for these vaccines are relatively mature. The World Health Organization (WHO) issued a guidance document on regulatory considerations regarding the evaluation of the quality, safety, and efficacy of messenger RNA vaccines for the prevention of infectious diseases –97. Furtvia routine pathways, which eliminates the need for 5’ cap and 3’ poly(A) tail structures in the RNA molecules used to formulate circRNA vaccines  related to the unique characteristics of circRNA vaccines must be emphasized. Circularization rate is the most important CQA of circRNA vaccines and has been measured by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) in previous studies , 24. TheCircRNA vaccines may serve as a powerful tool for tackling future emerging major infectious diseases or frequent viral diseases and may be developed into therapeutic vaccines for tumors. Biopharmaceutical companies and research teams worldwide have therefore diverted considerable attention and research efforts to the development of circRNA vaccines. Currently, research on circRNAs remains in the preclinical stage due to various issues in research and development, production, quality control, and safety. Recently, researchers proposed that a blueprint for quality by digital design (QbDD) to support rapid RNA vaccine process development, manufacturing and supply . AccordiFuture directions for the various aspects of circRNA vaccines are as follows:Research and development: linear mRNA vaccines exhibit a “self-adjuvant” effect , 16, 106Production: Attempts can be made to optimize the reaction conditions to achieve enhanced circularization efficiency. Under laboratory conditions, a re-execution of the circularization step with circularized RNA contributes to higher circularization efficiencies. Therefore, the development of a recircularization process and the incorporation of such process into vaccine production can be attempted . The simQuality control: As mentioned earlier, the molecular weights of circRNAs are similar to those of the corresponding linear RNA precursor molecules. Consequently, the two types of RNA molecules exhibit close peak appearance times during CE or HPLC to determine the RNA circularization rate. Accordingly, it is difficult to effectively distinguish between the two , 24. FurSafety: Organisms contain an abundance of circRNAs that play important roles in regulating gene transcription, expression, and cellular signal transduction pathways, with certain circRNAs capable of protein expression , 121. WhYB and ZL conceived the framework and main text of this review article. YB and DL wrote the draft. QM and ZL reviewed the manuscript. QH and JL searched the literatures. All authors contributed to the article and approved the submitted version.


The usage of pesticides is deemed essential to ensure crop production for global food security. Conventional chemical pesticides have significant effects on ecosystems. Nanopesticides are increasingly considered an emerging alternative due to their higher efficiency and lower environmental impacts. However, large knowledge gaps exist in the public perceptions and willingness-to-pay (WTP) for nanopesticides. Thus, we conducted a regional survey of pesticide users and food consumers on perceptions and WTP for nanopesticides across China. We found that 97.4% pesticide users were willing to pay for nanopesticides, with a main price from 25% to 40% higher than for conventional pesticides. Experience with applying pesticides, income, familiarity with and attitude toward nanopesticides, and trust in industries were significant determinants of WTP. Although the public were not familiar with nanopesticides, they had positive attitudes toward their future development and supported labeling nanoscale ingredients on products. Pesticide users presented high trust levels in governments and industries, while 34% of food consumers neutrally or distrusted industries in selling and production. This study highlights the socioeconomic and technological aspects of nanopesticides, which could provide guidance for industries to develop market strategies and for governments to design relevant regulation policies effectively, contributing to crop yield improvement and sustainable agriculture. The rapid growth of the global population, which is predicted to reach nearly 9.8 billion people by 2050, requires food production to increase by 50% compared to the levels in 2012 . ApplicaWith the rapid development of nanotechnologies, nanopesticides have been increasingly anticipated for the agrochemical sector, including nanoemulsions, nanocapsules , and inorganic engineered nanoparticles  . ComposeCurrently, research on nanopesticides has been mainly focused on their performance , mechaniFactors that could affect the public perceptions and WTP of nanotechnologies or emerging agri-food technologies depend on complicated mechanisms and multiple aspects. For example, socio-demographic factors  were shown to be related to understanding the risks and benefits of nanotechnologies ,23. FamiTo investigate public perceptions of and WTP for nanopesticides, we conducted a regional survey of pesticide users and food consumers across China, with almost 400 valid responses. China, as a large agricultural country with 23.6% of its workforce employed in the agricultural sector in 2020 , is the The survey was conducted between 24 July 2020 and 5 August 2020 via face-to-face interviews with a questionnaire. We stratified the survey sites into the western, middle, and eastern parts of China. The locations included the countryside located in two municipalities directly under the central government , eight cities of two autonomous regions  and 56 cities in 11 provinces ; D is the minimum expected difference, which is specified here subjectively to reflect the difference between the upper and lower limit of an expected CI ; and p is a pre-study estimate of the proportion to be measured. We set the CI at 95 percent that yielded critZ = 1.960, assumed D = 10% (0.1), and estimated p = 0.9 . Based on these assumptions, Equation (1) yielded a sample size of N = 138. Therefore, considering possible invalid responses, we expanded the survey scale and the final 232 valid responses of pesticide users met the requirements of the sample size.More specifically, the appropriate sample size was estimated before investigation by calculating the equation of simple random sampling with substitution ,37:(1)N=Ten independent variables were selected to evaluate public acceptance of emerging technologies. Factors related to socio-demographic information  and public perceptions of nanopesticides  were investigated. In addition, we included experience of applying pesticides and the associated quadratic term to examine the possible incremental or diminishing effects.The dependent variables included (a) the decision to spend money on nanopesticides when the price was lower than that of conventional pesticides. If respondents indicated unwillingness, they would end the questionnaire. Otherwise, respondents were asked specific follow-up questions about (b) the price ranges of WTP for nanopesticides .As illustrated in The WTP questions were designed using the contingent valuation method (CVM), a mature tool used to estimate public WTP for environmental goods and services in the marketplace ,38, and The survey data were analyzed using the Stata programming software. A descriptive statistical analysis of 232 pesticide users was conducted. In order to explore the influencing factors of WTP for nanopesticides, the Heckman model was firstly used to test whether there would be sample selection bias  if we exiy depends on a selection variable iz, such incidental truncation may result in a missing data problem of iy and biased coefficient estimation using standard regression techniques . In order to resolve this potential bias, the Heckman model was introduced and assumed a two-stage relationship (Equations (A1)–(A3) in Sample selection bias may arise when values of dependent variables are missing or unobserved caused by another process  ,47. For s exists . If rho s exists . OtherwiTo further examine the significance levels of different independent variables for 226 samples, the interval regression model was used as a preferred method when the outcome was measured as interval data, left-censored data, or right-censored data ,49. Othei had a WTP for nanopesticides By using the interval regression model, we assumed that each respondent ero mean .i was intreg in Stata, and the specific Equations (A4)–(A8) were illustrated in We did not observe uestions . TherefoThe ordinary least squares (OLS) and ordered logistic models were used to identify the robustness and credibility of the interval regression model. Specifically, we converted the interval data, left-censored data, and right-censored data of WTP into point data of the interval median, upper limit value, and lower limit value, respectively, to estimate the OLS regression model. Meanwhile, nine price ranges of WTP  were conIn addition, the relative influence importance of different variables was compared using standard beta coefficients. The plots of the public’s perspectives on nanopesticides were created using the online OmicShare Tools .Among all 395 samples, 163 food consumers answered the survey, except for questions regarding the experience of applying pesticides and the WTP for nanopesticides. A total of 232 pesticide users responded to all the questions. The descriptive statistics of the pesticide users were summarized in As shown in Not surprisingly, based on the above positive attitudes, most pesticide users (97.41%) were willing to spend money on nanopesticides. Only six pesticide users (2.59%) would not like to spend any money on nanopesticides, even if the price was lower than that of conventional pesticides . The higAs shown in As shown in On the other hand, pesticide users with higher income would be willing to pay more for nanopesticides compared to individuals with lower income. For every 100,000 RMB  increase in annual income, the WTP price range would increase by 1.39% , which wFamiliarity with and attitude toward nanopesticides were found to positively and significantly influence the price range of WTP for nanopesticides . A senseIn addition, the price range of WTP for nanopesticides could be notably improved with the rise of the trust level in industries . GeneralGender, age, and education level were not significant determinants of WTP. Although labeling preference was also not a significant influencing factor of pesticide users’ WTP for nanopesticides , the parThe standardized beta coefficients in the regression model were further calculated to examine which variables contributed most to the interval regression model , with a Based on the results of the interval regression model, we estimated the actual WTP for distinct pesticide users’ profiles . For exaAs discussed above, 163 food consumers  also participated in the survey. We analyzed all responders’  perspectives on nanopesticides to assess the overall perceptions .In general, nearly half of the survey participants were not familiar with nanopesticides  for nanopesticides and public perceptions from both pesticide users and food consumers perspectives. The findings provide key information for industries and governments to improve marketing strategies and regulations for the large-scale future application of nanopesticides, thus ensuring crop production for global food security and maintaining agricultural sustainability.As this study demonstrated, nanopesticides were highly accepted by pesticide users, and 97.4% were willing to spend money on them. The main price range (%) pesticide users were willing to pay for nanopesticides was 25–40% higher than that for conventional pesticides. The experience of applying pesticides had a greater influence on the WTP for nanopesticides than the other variables. Familiarity, trust in industries, attitude, and income were also positive and significant determinants of WTP for nanopesticides. The general public’s familiarity level with nanopesticides was low. Nevertheless, both pesticide users and food consumers supported the future development of nanopesticides quite strongly. Most of the participants agreed that nanopesticides must include labels indicating that the product contains nanocomponents. Pesticide users generally trusted governments and industries, while a few food consumers had neutral or distrust levels in industries for selling and production.Based on our findings, we suggest that governments should take label requirements into account when developing regulations. The related knowledge of nanopesticides should also be provided to the public via media, lectures, and training programs. In addition, governments should take responsibilities for optimizing relevant regulatory frameworks, such as the standard code of nanopesticides for entering markets, the legality of industries, and the use and recycling of nanopesticides.The current study is also subject to certain limitations, as survey results were based on Chinese samples. It would be important to conduct local studies in different countries with larger sample sizes, since public responses may vary with cultures and traditions . Moreove


Unfinished Nursing Care (UNC) concept, that express the condition when nurses are forced to delay or omit required nursing care, has been largely investigated as tasks left undone, missed care, and implicit rationing of nursing care. However, no summary of the available evidence regarding UNC antecedents has been published. The aim of this study is to identify and summarise antecedents of UNC as documented in primary studies to date.A systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was conducted. MEDLINE, CINAHL, SCOPUS, and PROSPERO databases were searched for quantitative studies reporting the relationships between antecedents and UNC published after 2004 up to 21 January 2020. The reference lists of secondary studies have been scrutinised to identify additional studies. Two reviewers independently identified studies and evaluated them for their eligibility and disagreements were resolved by the research team. The quality appraisal was based on the Joanna Briggs Institute Critical Appraisal tools, according to the study designs. A data extraction grid was piloted and then used to extract data. The antecedents that emerged were thematically categorised with an inductive approach.Fifty-eight studies were included; among them, 54 were cross-sectional, three were cohort studies, and one was a quasi-experimental study. They were conducted mainly in the United States and in hospital settings. The UNC antecedents have been investigated to date at the (a) unit , (b) nurse , and (c) patient levels .At the unit level, it is highly recommended to provide an adequate staff level, strategies to deal with unpredictable workloads, and to promote good practice environments to reduce or minimise UNC. By contrast, at the nurse and patient levels, there were no clear trends regarding modifiable factors that could decrease the occurrence of UNC. The map of antecedents that emerged can be used to design interventional studies aimed at changing research from merely descriptive to that which evaluates the effectiveness of interventions.The online version contains supplementary material available at 10.1186/s12912-022-00890-6. Unfinished Nursing Care (UNC) is an overarching term encompassing several concepts  that expWithin the overarching UNC term , there aA few years later, Kalisch  introducAccording to the state of the research in this field, UNC antecedents were considered in a linear relationship , 14, witThe following research questions were addressed: (a) What antecedents have been investigated to date as associated with the UNC? (b) What is the direction of the relationships between such antecedents and the UNC that has been documented to date?Therefore, the aims of the study were to (a) map factors, predictors, correlates, or linked factors – hereafter, ‘antecedents’, and (b) summarise the direction of their relationships with UNC. A systematic review of the literature was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines .The Sample, Phenomenon of Interest, Design, Evaluation, Research type (SPIDER)  methodolIn a preliminary phase, the International Prospective Register of Systematic Reviews (PROSPERO) database was checked to determine whether there were ongoing systematic reviews about the antecedents of UNC. Then, MEDLINE, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and SCOPUS were searched.There were included those primary studies: (a) investigating antecedents of the UNC in adult care settings (patients ≥ 18 years of age); (b) providing measures of relationships between the investigated antecedent(s) and the occurrence of the UNC; (c) employing quantitative designs and reporting the abstract, (d) published in English, German, Greek or Italian, the languages accessible to the research team (see authors); (e) from 2004 up to 21 January 2020, when the Task Left Undone concept, included in the UNC overarching term, was first established  and the UNC phenomenon); and the main findings regarding the relationships between the antecedent(s) and the occurrence of UNC.The entire research team then approved the extraction grid via an online meeting. Three researchers piloted the grid independently with three studies, and all authors agreed that no changes were needed at the end of the pilot. Then, the same three researchers independently extracted the data and agreed upon.The data extracted were thematically analysed  using anThe 58 studies were evaluated for their methodological quality with the Joanna Briggs Institute Critical Appraisal approach. This was selected according to its capacity to guide the development of high-quality systematic reviews addressing policy and practice interventions . First, The 58 studies included . Subsequently, the process of missed nursing care concept development firstly reported among US studies, was researched also across the world, especially to Europe . An impoTo date, both antecedents and the occurrence of UNC have been studied mainly from the point of view of nursing staff as a self-assessment, perceptions that can be influenced by several biases. Moreover, some studies have examined perceptions of both nurses and nurse’s aides , 48, 77;The study designs were largely cross-sectional in nature, with mainly convenience samples and a great variance in the participation rates, that all might have introduced biases in the evaluation of both antecedents and the UNC occurrence. In addition, antecedents and UNC occurrence have been largely measured at the same time point, thus assuming that the former has influenced the latter whereas control variables and/or confounding factors  were not investigated. These issues have been reported also by Griffiths et al.  regardinLongitudinal, pre- and post-study designs, or comparative studies are encouraged to increase the strength of evidence, by quantifying also the benefits of reducing/minimizing unfinished care and the costs, feasibility and long-term sustainability of implemented interventions. However, study designs should be considered in light of the complexity of the nursing care and the issue under study: UNC occurs in the real world of nursing across the world as a multifactorial phenomenon. Assessing precise antecedents might be difficult—moreover, designing interventional studies manipulating for example, the work environment, or the number of staff might be not feasible given the complexity of the turbulence of environments, and the challenges of the long-term implementation. Therefore, an in-depth discussion regarding the research issues in this field is required, analogously to that already developed in the context of nursing staffing and outcomes .Conceptual articles have highlighted that UNC is influenced by patient care demand, resource allocation, and relationship/communication issues  as well At the unit level, the staff adequacy as measured with different methods  and the occurrence of UNC have mainly reported conflicting findings. Some authors also included variables that are not usually measured, such as the nurse’s personality and the country of origin , 73 and Patient characteristics as antecedents of UNC have been poorly investigated. According to the available studies, clinical instability , 80 can This systematic review has several limitations. Despite the rigorous approach, some studies might be missed for different reasons and among other, the fact that no quantitative measures (Cohen’s kappa coefficient) were used to evaluate the agreement across researchers regarding the study inclusion. Moreover, according to the limitations applied in the languages  a potential publication bias might have been introduced. Furthermore, the timeframe was limited by including only studies published after 2004, the year when the first concept pertaining to the UNC was established . HoweverIn performing the data extraction, some antecedents might have been neglected in favour of providing a comprehensive map of those investigated to date. Commonalities across antecedents by using an inductive approach  were seaTo the best of our knowledge, this is the first systematic review summarising the antecedents of the unfinished nursing care. Several studies have been conducted throughout the world, indicating a clear interest in this research field. However, the available evidence has mainly been collected with cross-sectional designs, performed at the hospital level, and describing nursing staff perceptions as collected with different tools. Hence, more robust studies are needed in this field challenging the multifactorial nature of the UNC where assessing precise antecedents might be difficult.Several antecedents of UNC were investigated to date at the unit, nurse, and at the patient levels. At the unit level, (a) structural factors such as an adequate staff levels and strategies to deal with unpredictable variations in the workloads, and (b) process factors, as investing in good work environments for nurses, are highly recommended to minimize/reduce the occurrence of the UNC. At the nurse and patient levels, no clear trends emerged regarding modifiable factors.The antecedents emerged can be used to design interventional studies in the field that are also aimed at changing the patterns of research from merely descriptive to evaluate the effectiveness of interventions targeting some modifiable factors. This endeavour could minimise and/or reduce the UNC and, ultimately, ameliorate patient, nurse, and system outcomes. Future studies should also consider community settings and involve more robust measures by using different sources of data to identify additional meaningful factors that could contribute to explain the UNC. However, an in-depth discussion regarding the research issues in this field is recommended in order to design studies capable to add value and, therefore, to inform policy-makers shaping nursing services.Additional file 1.


To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out in vitro RNA synthesis at low temperatures (4–25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3’-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for in vivo use.RNA research and applications are underpinned by  The column was washed with 10 volumes of elution buffer, first containing 20 mM imidazole and then containing 50 mM imidazole, and the majority of the His-tagged VSW-3 RNAP was eluted by elution buffer containing 100 mM imidazole. Collected eluates were concentrated to 2 mL with Ultra-15 Centrifugal Filter units (Millipore) and loaded onto a 200-mL preparative Superdex S200 column for gel filtration chromatography. Finally, VSW-3 RNAP was dialysed against buffers containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, and 50% glycerol and then stored at −20°C. The protein concentration of VSW-3 RNAP was determined by using a Bradford protein quantitative kit (Bio-Rad), and protein samples collected via Ni2+ affinity purification and gel filtration chromatography were analysed by 10% sodium dodecyl-sulphate polyacrylamide gel electrophoresis and stained with Coomassie blue (Bio-Rad).The predicted coding sequence for VSW-3 RNAP and its Y578F mutant (Table S1) was inserted into a pCold vector harbouring an N-terminal His-tag using Gibson Assembly cloning technology . After t2, 5 mM DTT, and 2 mM spermidine]. Reactions were incubated at 25°C for 12 h; then, 1 μL of 6× DNA loading buffer and 5 μL of reaction products were loaded onto a 1.5% agarose gel and electrophoresis was conducted at 100 V for 30 min. The gels were stained with ethidium bromide and visualized using a UVsolo Touch system .DNase and DNA polymerase contamination in purified VSW-3 RNAP was examined in reactions containing 50 ng/μL copepod green fluorescent protein (copGFP) DNA template and 2 μM VSW-3 RNAP, in the presence or absence of a 4 mM dNTP mix in IVT buffer  at 30°C for 10 min. Then, 10 μL of the mixtures were mixed with 5 μL native loading dye and analysed by 12% native Tris-borate-EDTA (TBE)-PAGE. Electrophoresis was conducted at 120 V for 60 min, and the DNA products were stained with ethidium bromide and visualized using a UVsolo Touch system. The grey values of gel bands were quantified using ImageJ software. Promoter binding percentages were calculated as the molar ratio between the bound DNA and the total DNA at each enzyme concentration.To determine the 5’ boundary of the VSW-3 RNAP promoter, we constructed dsDNA templates containing the putative VSW-3 RNAP promoter with gradual truncations at the 5’ end and the downstream 40 bp (Table S1). These dsDNA templates containing DNA with various lengths of putative VSW-3 promoters  were incubated with 0, 1, 2, or 3 μM VSW-3 RNAP in IVT buffer , we screened various enzyme concentrations  at 20°C overnight for optimal RNA yield. Finally, we screened incubation temperatures  in combination with various incubation times in the presence of 0.15 μM VSW-3 RNAP for maximal RNA yield.Using the optimal IVT conditions , and incubated with the dsRNA-specific J2 mAb (English & Scientific Consulting) for 30 min at 25°C. The membranes were washed two times with TBS-T buffer, reacted with hydrogen peroxidase-conjugated donkey anti-mouse Ig (Jackson Immunology), washed two times, and then detected with an ECL detection reagent (Thermo). The ECL signals were captured on an X-ray film (Kodak) through photosensitive development.dsRNA  used as a quantitative standard and 200 ng of the In addition, copGFP RNA synthesized by VSW-3 RNAP was used as a template (to replace the DNA template) in T7 and VSW-3 IVTs, and the products were compared to the untreated copGFP RNA and copGFP dsRNA  (Table S2) in 5x RNA annealing buffer ) using 1.5% agarose gel electrophoresis and dot-blot analysis, as described in a previous section.The newly discovered VSW-3 RNAP is an ssRNAP from Napahai Lake in the southwestern plateau of China with an average annual temperature of 5°C. It has a homology of 31% to T7 RNAP, and the distance tree analysis revealed that VSW-3 RNAP has a different evolutionary distance and direction compared with T7, SP6, Syn5, or KP34 RNAP . As the For bacteriophage T7 and SP6, transcription initiation mainly depends on a 17 bp promoter sequence that appears several times in their genomes . HoweverUsing a linearized pUC19 plasmid with the predicted VSW-3 promoter inserted as a transcription template, the IVT activity of VSW-3 RNAP was demonstrated . We anal2+ and DTT, and higher concentrations of NTPs required a corresponding higher Mg2+ concentration in the transcription buffer . Although the buffer containing 9 mM Mg2+ and 1 mM DTT supported an optimal yield of cas9 RNA in the presence of 4 mM of NTPs , it was unstable and the yield of cas9 RNA dropped significantly when the buffer was stored at −20°C for a few days . Finally, we established an efficient and stable (more than 6 months) IVT buffer for VSW-3 RNAP .We found that the IVT yield of VSW-3 RNAP was significantly affected by the concentration of Mgfew days . Higher in vitro, with the highest yield at 25°C (VSW-3 RNAP produced RNA over a wide range of temperatures (4–37°C)  at 25°C . At lowe at 25°C , similar at 25°C . Maximum at 25°C . A workicas9 RNA  and T7 RNAP  were comparable, while for the synthesis of long RNAs such as cas9 RNA , tdTomat2 and 5 mM DTT) was also optimal for T7 RNAP to achieve high yields in the presence of 4 mM NTPs, compared to the routine buffer  . Under this condition, the T7 RNAP reached its maximum yield in 1 h, and further extending the incubation did not increase the yield but decreased the amount of run-off products . We also compared the pUC19-RNA yield of VSW-3 and T7 RNAP IVTs at 20°C  and consistently found that the maximum yield of VSW-3 RNAP IVT was comparable to that of T7 RNAP IVT (The optimized transcription buffer for VSW-3 RNAP (16 mM MgClRNAP IVT .During the production of pUC19-RNA (Table S3), we observed a byproduct shorter than the run-off RNA for both T7 and VSW-3 RNAP . 3’-RACE of the terminus of the shorter RNA and structural analysis of the upstream sequence revealed that this byproduct was due to the transcription termination by both enzymes at a T7 class I  terminatcas9 RNA , we found that there was a group of terminated RNA products synthesized by T7 and Syn5 RNAP. According to the markers in the agarose gel, the terminated RNA products were estimated to be 1500–1600 nt in length (cas9 RNA (There are two classes (class I and class II) of transcription terminators known to partially stop the transcriptional elongation of T7 RNAP. The class I terminator forms a stem-loop structure, and the class II terminator typically contains a 7 nt sequence (5’-ATCTGTT-3’) ,20,30. Dn length  gel. By cas9 RNA  bottom. cas9 RNA , and A ccas9 RNA .T7 RNAP retains RdRp activity  and cataWe examined the RdRp activity of T7 and VSW-3 RNAP that used the HPLC-purified sgRNA product of VSW-3 RNAP as a template . ActivitWe also examined the 3’-termini of long IVT products. Among the five colonies picked for both VSW-3 RNAP and T7 RNAP sequencing following 3’-RACE, only one sequencing result matched the actual eGFP RNA sequence (978 nt) for both VSW-3 RNAP and T7 RNAP . VSW-3 RNAP produced eGFP RNA transcripts shortened by one or more nucleotides , while T7 RNAP consistently produced transcripts with an extension of one or more 3’ nucleotides .The self-templated 3’-extension of the T7 IVT products resulted in the formation of the loop-back dsRNA  and S4, in vitro and in vivo ..in vivo Click here for additional data file.


Picornaviridae family. RNA-dependent RNA polymerase (RdRp) of RNA viruses is highly conserved. Compounds that bind to the RdRp active site can block viral replication. Herein, we combined double virtual screenings and cell-based antiviral approaches to screen and identify potential inhibitors targeting FMDV RdRp (3Dpol). From 5596 compounds, the blind- followed by focus-docking filtered 21 candidates fitting in the 3Dpol active sites. Using the BHK-21 cell-based assay, we found that four compounds—NSC217697 (quinoline), NSC670283 (spiro compound), NSC292567 (nigericin), and NSC65850—demonstrated dose-dependent antiviral actions in vitro with the EC50 ranging from 0.78 to 3.49 µM. These compounds could significantly block FMDV 3Dpol activity in the cell-based 3Dpol inhibition assay with small IC50 values ranging from 0.8 nM to 0.22 µM without an effect on FMDV’s main protease, 3Cpro. The 3Dpol inhibition activities of the compounds were consistent with the decreased viral load and negative-stranded RNA production in a dose-dependent manner. Conclusively, we have identified potential FMDV 3Dpol inhibitors that bound within the enzyme active sites and blocked viral replication. These compounds might be beneficial for FMDV or other picornavirus treatment.Foot-and-mouth disease virus (FMDV), an economically important pathogen of cloven-hoofed livestock, is a positive-sense, single-stranded RNA virus classified in the  Picornaviridae family, which consists of both animal and human pathogens, such as enterovirus, rhinovirus, and poliovirus. The FMDV genome is single-stranded RNA with positive polarity. Viral RNA synthesis during replication and transcription is achieved by RNA-dependent RNA polymerase (RdRp) encoded by 3D, so-called 3Dpol naphthalen-5-one at position 2. This spiro compound has been reported to predictably interact with hepatitis C virus E2 envelope glycoprotein (HCV E2). It could bind HCV E2 protein as detected by surface plasmon resonance -4-yl)diazenyl)-2-hydroxybenzoic acid) is a compound in the NCI diversity set III. Thus far, its antimicrobial effect has not been reported in the available databases. Therefore, we further investigated the physicochemical properties of this compound. The predicted model based on the SwissAMDE analysis  , NSC670283 (spiro compound), NSC292567 (ionophore antibiotic), and NSC65850 inhibited FMDV replication by binding to 3D