Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.  •RNA derived from double-strand breaks (DSBs) can promote TIRR/53BP1 complex dissociation•RNAPII-transcribed hairpin RNA binds to the same residues on TIRR as 53BP1•TIRR/53BP1 separate in proximity to the DSB•Dicer and Drosha are required for TIRR/53BP1 dissociation Cells constantly experience different types of DNA damage, including toxic double-strand breaks (DSBs). RNA is an important player in DSB repair. Ketley et al. show that RNA transcribed at DSBs promotes separation of the repair protein 53BP1 from its negative regulator TIRR, contributing to our understanding of DSB repair. DNA double-strand breaks, where both strands of the DNA double helix are broken, are considered to be one of the most harmful types of DNA damage . Double-Over the last decade, the importance of various RNA classes, including long non-coding RNAs (lncRNA) and microRNAs (miRNA), in the repair of DSB lesions has been demonstrated . In partin vitro and in vivo approaches, we show that TIRR/53BP1 dissociation is promoted by RNAPII transcription and RNA molecules. We further show that TIRR can bind single-stranded RNA and hairpin RNA structures and that hairpin RNA can directly separate the TIRR/53BP1 complex in vitro and in vivo. Furthermore, we show that TIRR is also recruited to damaged DNA sites; however, recruitment of a mutated TIRR, which cannot bind 53BP1 (K10E), is impaired, suggesting that TIRR and 53BP1 are in proximity to chromatin while still bound as a complex, and their dissociation occurs at the DSB itself. We also develop a technique to study the interactions of proteins with RNA de novo transcribed at sequence-specific DSBs; fluorescence in situ hybridization proximity ligation assay (FISH-PLA). Using the FISH-PLA technique, we find that TIRR and 53BP1 are in close proximity to DSB-derived RNA, implicating nascent RNA in TIRR/53BP1 dissociation. These results not only uncover a previously uncharacterized mechanism of TIRR/53BP1 dissociation in response to DSBs but also highlight the diverse and crucial roles of RNA in the DNA-damage response. In addition, cancers with HR deficiencies are sensitive to treatment with poly (ADP-ribose) polymerase inhibitors (PARPis)  . Resista(PARPis) . Therefoin vivo PLA  RNaseH1, which digests the RNA:DNA hybrids, or the binding and catalytic mutant (WKKD) RNaseH1 , which translocates to the nucleus after addition of 4-hydroxytamoxifen (4-OHT), after which AsiSI cuts in a sequence-dependent manner to induce site-specific DSBs  of ∼100 μM. The binding affinity of 53BP1 to TIRR has been shown to be around 1 μM  are important in the modeled interaction of TIRR with the RNA. Together, these data suggest that RNA can directly compete for TIRR interaction with 53BP1, and there is considerable overlap between residues involved in RNA interaction and 53BP1 interaction on TIRR.Understanding how TIRR influences 53BP1 function is crucial for understanding DNA repair and the mechanisms of cancer progression. Interestingly, we also found that TIRR and 53BP1 can be dissociated by different drugs, which can lead to the production of DNA DSBs as part of their mechanism of action, including cisplatin A and S8BDe novo transcription has been observed at DSBs occurring at different regions of the genome, including genic and intergenic regions .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Monika Gullerova (Reagents generated in this study can be made available on request.AsiSI-ER U2OS cells (a gift from Gaëlle Legube) were cultured in DMEM with 1% L-glutamine, and 10% FBS, with and without antibiotics in 5% CO2 at 37°C.HeLa Wild Type, TRex Flp-IN shTIRR HeLa, TRex Flp-IN shGFP HeLa, TRex Flp-IN TIRR-GFP HeLa, TRex Flp-IN GFP HeLa , 53BP1-GFP U2OS (a gift from Matthias Altmeyer), and Ionising Radiation induced damage was delivered at the indicated doses using the Gravatom (GRAVITRON RX 30/55).Short interfering RNA (siRNA) for gene expression knockdown was performed using Lipofectamine RNAimax according to the manufacturer’s instructions. Cells were seeded in antibiotic free media, and forward transfected 24 h later with 20nM siRNA and incubated for 48 h unless otherwise stated. siRNA used; ON-TARGETplus Non-targeting Control (Dharmacon), ON-TARGETplus Human DGCR8 siRNA (Dharmacon), ON-TARGETplus Human DICER1 siRNA (Dharmacon), ON-TARGETplus Human DROSHA siRNA (Dharmacon).For transfection of the Cy3 labeled RNA hairpin, the RNA was annealed at a concentration of 10μM in 1X annealing buffer  by heating at 98°C for 3 min, followed by slowly cooling to room temperature. RNA was transfected using Lipofectamine RNAimax with either 20nM (low) or 100nM (high) RNA concentrations.Plasmids were transfected using Lipofectamine 3000 according to the manufacturer’s instructions. Cells were seeded and 24 h later forward transfected with 1-5μg of plasmid DNA. Cells were harvested 24–48 h later. Plasmids ppyCAG_RNaseH1_WT and ppyCAG_RNaseH1_WKKD were a gift from Xiang-Dong Fu (Addgene plasmid #111905 and #111906).RNA and DNA oligonucleotides were purchased from either IDT or Sigma. RNA sense (5′ → 3′): GGA CAA GAU GCC UUA UGU GG; RNA Antisense: CCU GUU CUA CGG AAU ACA CC; RNA Hairpin: GGA CAA GAU GCC UUA UGU GG ACU UCA CCA CAU AAG GCA UCU UGU CC; RNA Beacon: CGC UCC CAA AAA AAA AAA CCG AGC G; DNA sense: GGA CAA GAT GCC TTA TGT GG; DNA Antisense: CCT GTT CTA CGG AAT ACA CC; DNA Hairpin: GGA CAA GAT GCC TTA TGT GG ACT TCA CCA CAT AAG GCA TCT TGT CC; Cy3 Labeled RNA Hairpin: Cy3 GCC GGG CGA AGA AUG AAA ACG GC.Protein samples in 1X Laemmli Buffer (Alfa Aesar) were sonicated for 10–30 s, followed by incubation for 5 min at 98°C. Samples were then loaded in 4-15% Mini-Protean TGX gels (Biorad) and electrophoresed in Tris-Glycine running buffer (Biorad) at 100V for 1 h. Gels were transferred onto nitrocellulose membranes for 1 h 30 min at a constant voltage of 15V. Successful transfer of protein to the membrane was visualised using Ponceau S (Merck). Membranes were blocked in 5% milk or BSA in PBS with 0.1% Triton X-100 (PBST) at room temperature for 1 h. Membranes were then incubated with primary antibodies overnight at 4°C. Membranes were washed 3 times in PBST and then incubated with a secondary antibody for 1 h at room temperature. Membranes were washed 3 times in PBST, then visualised using ECL and Amersham Hyperfilm ECL film. Some membranes were processed using the Super Signal Western Blot Enhancer kit (Thermo Fisher) according to the manufacturer’s instructions.Cells were plated on glass cover slips. Cover slips were washed 3 times in cold PBS and fixed in cold 4% paraformaldehyde (Alfa Aesar) in PBS for 10 min. Cover slips were then washed 3 times in cold PBS and permeabilised with cold 0.2% Triton X-100 in PBS (PBST) for 10 min. Slides were then washed 3 times in cold PBS and blocked in 10% FBS in PBS for 2 h at 4°C. Primary antibodies were diluted to the appropriate concentration in 10% FBS in PBS and incubated overnight at 4°C in a humidified chamber. Cover slips were then washed 3 times in PBST. Coverslips were incubated for 2 h at RT with secondary antibodies diluted in 0.15% FBS in PBS. Coverslips were then washed 3 times in PBST in the dark. Coverslips were then washed a final time in PBS before mounting on slides with Fluoroshield Mounting Medium with DAPI (Abcam). Slides were imaged using an Olympus Fluoview FV1200 confocal microscope with a 60X objective lens.2, RNAse T1 (Thermo) 1:200 (v/v), ShortCut RNAseIII (NEB) 1:200 (v/v) in PBS) for 2 h at RT  according to the manufacturer’s instructions. Briefly, cells were seeded on coverslips, fixed and permeabilized according to the immunofluorescence protocol. Cover slips were blocked in PLA blocking buffer in a humidity chamber for 1 h at 37°C. Primary antibodies were diluted in PLA antibody diluent and incubated with coverslips in a humidified chamber at 4°C overnight. Cover slips were then washed 3 times in PLA buffer A at RT, then incubated with PLA probes for 1 h at 37°C. Cover slips were washed 3 times in PLA buffer A, then incubated with PLA ligase in ligation buffer for 30 min at 37°C. Coverslips were washed 3 times in PLA buffer A and incubated with PLA polymerase in polymerase buffer for 100 min at 37°C in the dark. Cover slips were washed 3 times in PLA buffer B in the dark, and twice in 0.01X PLA buffer B. Coverslips were mounted on slides using PLA DAPI solution and imaged using an Olympus Fluoview FV1200 confocal microscope with a 60X objective. Images were visualised using Fiji  and anal2, 0.5% NP40, Pierce Universal Nuclease for Cell Lysis (Thermo Fisher), Protease inhibitors and Phosphatase inhibitors (Roche)) for 30 min at 4°C on a wheel. Lysates were centrifuged at 17,000g for 10 min at 4°C. 1.5x volume of Co-IP dilution buffer ) was added to the supernatant. GFP-Trap magnetic beads (Chromotek) were washed 3x in cold dilution buffer and added to the lysate on a wheel at 4°C for 1.5 h. The beads were then washed 3x in cold Co-IP dilution buffer and samples were eluted using 2x Laemmli buffer (Alfa Aesar) by heating at 95°C for 10 min.Cells were harvested from in 10cm dishes by washing 3x in cold PBS, followed by scrapping in PBS, and centrifuged at 500g for 5 min. The cell pellet was lysed in 5 volumes Co-IP lysis buffer  or RNA lysis reagent from the Monarch Total RNA Miniprep Kit (NEB) as per the manufacturer’s instructions. For TRIzol extraction, TRIzol was added to cells, vortexed, and chloroform was added to the TRIzol sample according to the manufacturer’s instructions (200μL of chloroform per 1mL of TRIzol Reagent) and vortexed, before centrifugation at 12,000g for 15 min at 4°C. The aqueous phase was collected and used in the Monarch Total RNA Miniprep Kit (NEB) as per the manufacturer’s instructions. RNA was eluted in water and quantified using the Implen Nanophotometer N60.2. To select for cells with similar expression levels, cells were systematically chosen using a 488-nm laser setting. DNA damage was induced by a 405nm laser and recruitment of the fluorescently labeled proteins was monitored by live cell imaging. All images were quantified using FIJI software. Fluorescent intensities were normalised against intensities measured in undamaged parts of nuclei.Cells were seeded in 35 mm glass bottom dishes. Before laser stripping, cells were pre-sensitised using Hoechst 33342 (10μg/mL) at 37°C in phenol red free media. Cells were imaged using an Olympus SoRa spinning disc confocal microscope using the 405nm FRAP module in a chamber at 37°C and 5% COin vitro RNA separation assays for TIRR and 53BP1 with increasing concentrations of nuclear irradiated RNA, 1μg of recombinant Flag-TIRR and 1μg recombinant HA-53BP1Tudor were incubated together with 20μL of Anti-FLAG M2 magnetic beads (Sigma) per sample in TGEN150 buffer ) for 1 h at 4°C on a wheel. The supernatant was removed and the beads washed 1X in TGEN150 buffer. Nuclear RNA from HeLa cells 1h post 10Gy IR was extracted using TRIzol followed by Monarch Total RNA Miniprep Kit. Increasing amounts of RNA  was added to the beads in constant volume of TGEN150 buffer with Ribolock (Life Technologies) and incubated at RT on a wheel for 1 h. Beads were washed 2X in TGEN150 buffer, before being eluted in 2X Laemmli SDS sample buffer (Alfa Aesar) by boiling at 95°C for 10 min. Eluant was collected and ran on a Western blot to visualise binding.To perform siSI U2OS Cells were plated on glass coverslips and 24 h later incubated with α-amanitin (2μg/mL) where indicated. 24 h after addition of α-amanitin, Tamoxifen (4-OHT) was added at a concentration of 500nM where indicated for 4 h. Coverslips were then washed 3 times in cold PBS and fixed in cold 4% paraformaldehyde (Alfa Aesar) in PBS for 15 min. Coverslips were then washed 3 times in cold PBS and permeabilised with cold 0.5% Triton X-100 in PBS (PBST) for 10 min. Slides were then washed 3 times in cold PBS and blocked in blocking buffer , 20μg/μL salmon sperm DNA (Life Technologies), 0.5% Triton X-100, 2% BSA, 1U/μL RNAsin Plus (Promega)) at 37°C for 1 h. Probe incubation buffer  was heated to 98°C for 3 min and cooled on ice for 10 min before addition of 1% Triton X-100 and 1U/μL RNAsin Plus . Coverslips were incubated with probe incubation buffer for 3 min at 80°C, and then at 37°C overnight. Coverslips were washed 3x in SSC-T buffer  and 2x in PBS, before blocking for 1 h at 37°C with PLA blocking buffer. Primary antibodies were diluted to the appropriate concentration in PLA antibody diluent and incubated overnight at 4°C in a humidified chamber. Cover slips were then washed 3 times in PLA buffer A at RT, then incubated with minus rabbit PLA probe (Sigma) for 1 h at 37°C. The PLA protocol was then followed according to the manufacturer’s instructions using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma).ADS1: RBMXL1 (promoter) hg19, chr1:89,458,643DS2: VSTM2B (CDS) hg19, chr19:30,019,487Sequences of FISH-PLA Probes can be found in Rosetta™(DE3) E. coli cells from a glycerol stock were grown in TB supplemented with 8g/L glycerol, 100μg/mL kanamycin and 34μg/mL chloramphenicol at 37°C overnight. The overnight culture was used to inoculate three 2L bottles, each containing 1L TB supplemented with 8g/L glycerol, 50μg/mL kanamycin and approximately 200μL 204 Antifoam A6426 (Sigma). The culture was at 37°C until the OD600 reached ∼1.0. The culture was down-tempered to 18°C over a period of 1 h before target expression was induced by addition of 0.5mM Isopropyl β-d-1-thiogalactopyranoside (IPTG). Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation . The resulting cell pellet was stored at −80°C.Cells were resuspended in IMAC wash1 buffer , supplemented with 6000 U Benzonase (Merck) and three tablets of Complete EDTA-free protease inhibitor (Roche Applied Science) and lysed by sonication. The cell suspension HisTrap 5mL column was equilibrated with IMAC wash1 buffer, and the size exclusion column ) was equilibrated with GF buffer . Purification of the protein was performed on an AKTA prime system . The filtered lysate was loaded onto the HisTrap column and washed with IMAC wash1 buffer. Bound protein was eluted from the IMAC column with a gradient increase in the imidazole concentration. The eluted protein was injected into a size exclusion column and fractions containing the target protein were identified by SDS-PAGE. Fresh TCEP was added to a final concentration of 2 mM. The protein was concentrated using a Vivaspin 20 centrifugal filter device  and the identity of the protein was confirmed by mass spectrometry.32P, 10μM of RNA or DNA was incubated with T4 polynucleotide kinase  in T4 PNK buffer (NEB) and incubated for 1 h at 37°C. After reaction completion, 30μL of H2O was added to samples to reach a final volume of 50μL, and reactions were ran through a G25 spin column (VWR) to remove free nucleotides. For single stranded substrates, 50μL of water was added to the sample to reach a final concentration of RNA/DNA of 100nM. For hairpin or double stranded substrates, 40μL of water and 10μL of 10X annealing buffer  was added for a final concentration of 100nM. RNA and DNA substrates were annealed at a concentration of 100nM in 1X annealing buffer by heating at 98°C for 3 min, followed by slowly cooling to room temperature.For 5′ end labeling of DNA and RNA substrates with 2, 0.01% Tween 20). Reactions were carried out in a final volume of 10μL. Reactions were incubated for 15 min at 37°C, before adding native agarose gel loading solution (Thermo Fisher) to 1X final. Reactions were run on 0.5X TBE 6% native polyacrylamide gels in 0.5X TBE running buffer (Life Technologies) for 3 h at 100V. Gels were then dried on Whatman paper for 2 h at 80°C, and imaged using autoradiography film overnight at −80°C.EMSA assays were performed to assess the binding of protein to RNA and DNA substrates. 10nM of radiolabeled RNA or DNA substrate was incubated with increasing concentrations of recombinant protein ) as indicated in 1X EMSA buffer -L-lysine hydrate reagent (Sigma) dissolved in 50 mM borate buffer (pH 8.5) was chemically bound to the SPR CM4 sensorchip  using an amine coupling kit . 6–211 TIRR-His (0.1 μM) was injected via NTA-Capture coupling on the SPR surface during 200s at 5 μL/min to reach ∼600 RU of protein immobilised ). After equilibration of the Biacore system in running buffer , DNA and RNA substrates were injected in multi-cycle kinetics over TIRR-His protein in a dose-response up to 200μM with 100s association and 100s dissociation at 30 μL/min. Binding curves were analyzed using Insight Evaluation Software and fitted when possible with a 1:1 steady-state affinity model.Corresponding RNA or DNA substrates were annealed 95°C for 5 min in annealing buffer  and slowly cooled to RT. All experiments were conducted on a Biacore 8K + Device and were performed at 25°C. 1536 well plates (Corning) were thawed at RT and centrifuged at 1800rpm at RT and placed on a rotating Cytomat hotel of a robotic platform. Solutions of proteins  and of antibodies  were prepared in PPI buffer . An EL406 (Biotek) was used to dispense solutions of proteins into 1536 well plates and incubated at RT for 15 min. Antibodies were then added and left to incubate for 2 h at RT. PPI buffer alone was used to measure background signal. Readings were then taken on a Tecan Spark:Measurement 1Excitation filter: 340(20)nm/Emission filter: 620(10)nm/MirrorDichroic: 510Lag time: 100μs/Integration time: 300μs/Number of reads: 3/Gain: 170/Z-Position: 20500 [μm] Measurement 2Excitation filter: 340(20)nm/Emission filter: 665(8)nm/MirrorDichroic: 510Lag time: 100μs/Integration time: 300μs/Number of reads: 3/Gain: 160/Z-Position: 20500 [μm]Raw data files are processed with a local tool (Screener Utilities) before implementation in Genedata Screener for QC validation, data pre-processing and data analysis. Raw data are uploaded into Genedata Screener and plates or wells that had technical issues are masked. Raw fluorescence of the donor (620 nm) and acceptor (665 nm) is recorded and the HTRF ratio is calculated. The HTRF ratio is normalised on each plate independently using the median of control wells: 100% inhibition (no target proteins in DMSO) and 0% inhibition (proteins with HTRF antibodies in DMSO).6D0L (3COU). In PyMol(6D0L). The information on the positions of the missing residues of the TIRR protein in the 6D0L structure were taken from the PDB structure 6CO1, and placed by overlapping the two structures using PyMOL. Both systems, RNA-TIRR monomer and RNA-TIRR dimer, were placed at the center of a truncated octahedral box of TIP3P  were se In PyMol, residue In PyMol with theof TIP3P  water moof TIP3P  added toof TIP3P , thermalof TIP3P  in the Nof TIP3P  and periof TIP3P . We usedof TIP3P  and the of TIP3P , followeof TIP3P  and BIGNof TIP3P  suite ofof TIP3P . The simof TIP3P . The eleof TIP3P ). The RNof TIP3P  and RNACof TIP3P . For the∗, p ≤ 0.01 = ∗∗, p ≤ 0.001 = ∗∗∗, p ≤ 0.0001 = ∗∗∗∗.Statistical analysis was performed using GraphPad Prism 9. The Kolmogorov-Smirnov normality test was performed to test for a normal distribution. If data met the requirements of a normal distribution, an unpaired t test was performed. If data did not show a normal distribution, for a comparison of two groups, a Mann-Whitney test was performed. For comparison of more than two groups, a Kruskal-Wallis with Dunn’s multiple comparisons test was performed. p > 0.05 = ns, p ≤ 0.05 =


We considered similarity with Arabidopsis thaliana plastid transporters, transcriptional co-regulation with genes encoding core plastid metabolic pathways and with genes encoded in the mitochondrial genomes, inferred evolutionary histories using single-gene phylogeny, and environmental expression trends using Tara Oceans meta-transcriptomics and meta-genomes data. Our data reveal diatoms conserve some of the ion, nucleotide and sugar plastid transporters associated with plants, such as non-specific triose phosphate transporters implicated in the transport of phosphorylated sugars, NTP/NDP and cation exchange transporters. However, our data also highlight the presence of diatom-specific transporter functions, such as carbon and amino acid transporters implicated in intricate plastid-mitochondria crosstalk events. These confirm previous observations that substrate non-specific triose phosphate transporters (TPT) may exist as principal transporters of phosphorylated sugars into and out of the diatom plastid, alongside suggesting probable agents of NTP exchange. Carbon and amino acid transport may be related to intricate metabolic plastid-mitochondria crosstalk. We additionally provide evidence from environmental meta-transcriptomic/meta- genomic data that plastid transporters may underpin diatom sensitivity to ocean warming, and identify a diatom plastid transporter (J43171) whose expression may be positively correlated with temperature.Diatoms are an important group of algae, contributing nearly 40% of total marine photosynthetic activity. However, the specific molecular agents and transporters underpinning the metabolic efficiency of the diatom plastid remain to be revealed. We performed  As a re2 concentration and carbon metabolism systems  not known in plant plastids  ; nitraters (SLC) ; and nucrs (SLC) .2 availability, and temperature)   directlyerature) , and interature) .Phaeodactylum tricornutum, with complete models realised for > 99% of its probable encoded genes via transcriptomic and proteogenomic reannotations  (https://wolfpsort.hgc.jp/) using a plant reference dataset  , and whe dataset . LocalizA. thaliana plastid transporters were searched by SUBA (https://suba.live/), and manually checked in the literature if not cataloged. For P. tricornutum, plastid transporters that have been experimentally verified by GFP were identified from the literature (P. tricornutum homologues (identified by genome-to-genome RbH) to plastid transporters experimentally identified from the purified plastid proteome of the diatom Thalassiosira pseudonana  are noted in terature , alongsieudonana . All 67 P. tricornutum genome   of P. tricornutum genes in T. pseudonana microarray data  Pportal/) , and , cryptomonads and haptophytes   database, and transcript abundance estimates were calculated from the meta-transcriptomes (MetaT) database. Complete details of homologue sequences, taxonomic annotations, and environmental distributions of each diatom transporter meta-gene, as inferred by Ocean Gene Atlas website, are provided in the linked osf.io repository https://osf.io/89vm3/ in the folder “Transporters”The relationships between ersion 1  on the O-atlas/) . The FAS-05. The Tara Oceans sequences were searched against the reference dataset by BLASTp with the –max_target_seqs 1 threshold applied, and only sequences with diatom plastid-targeted best hits were retained. A second BLASTp was performed against the complete protein sequence annotation models from the P. tricornutum genome using the same criteria, and only sequences that obtained best hits with the specific queried gene ID were retained. The intersection of each set of retained genes were selected and combined with the reference dataset, and aligned using MAFFT  and different depths . Absolute correlation coefficients were calculated by Pearson correlation analysis between the sum MetaT total relative abundance, the sum MetaG total relative abundance , and measured environmental factors for each station, following data from PANGAEA . A two-tFinally, environmental parameters which were shown to be strongly positively or negatively correlated in MetaT compared to MetaG data (MetaT-MetaG) in at least three of the ten combinations of different size fractions and depth conditions , were inferred to be concluded to significantly related to the expression of the transporter. To allow a more intuitive view of the results, five pigment concentration parameters related to photosynthetic potential ; and netA. thaliana and P. tricornutum were compared using reciprocal BLASTp-BLASTp searches. A total of 77 A. thaliana plastid transporters were defined from the literature (P. tricornutum plastid transporters were found based on (i) the presence of a transport-associated KEGG, GO or PFAM domain  in the P. tricornutum proteome, but only 20 of these proteins have at least one homologue that was predicted to contain plastid targeting sequences in P. tricornutum using ASAFind or HECTAR as above  essential for the import of NTPs from the cytoplasm into plastid  which exhibit a wide range of substrate specificities, including non-bile acid organic compounds such as pyruvate, steroids, methionine-derived glucosinolates (GSL) and exogenous substances  retrieved BLASTp results with the A. thaliana TPT, glucose 6-phosphate transporter (GPT), xylulose 5-phosphate transporter (XPT), and phosphoenolpyruvate transporters  and phosphoenolpyruvate (PEP)  , whereaste (PEP) . An ABC P. tricornutum transporters that have not previously been annotated. These include J39274 which was annotated within the version 3 P. tricornutum genome as an Na+/H+ transporter by CDD search and PFAM; but in our search shows more similarity to KEA family transporters. We note that J39274 has recently and independently been functionally characterised as the diatom KEA3  apparently displayed a different localization in each species , mitochondria (M), plasma membrane (PM) and endoplasmic reticulum (ER) , six P. tricornutum plastid transporters  were found to be homologous to plastid transporters    and NTT1eudonana , The cloediction .P. tricornutum homologues of A. thaliana transporters did not show exclusive plastid localization, but were also inferred to localize to the PM/ER . The P. tricornutum homologues of a thylakoid ADP/ATP carrier protein (AT5G01500), implicated in both ATP/ADP and 3’-phosphoadenosine 5’-phosphosulfate (PAPS) transport across the A. thaliana plastid envelope   , and memA. thaliana homologues of 36 P. tricornutum plastid transporters included the plastid (C), but also the plasma membrane (PM), mitochondria (M), Golgi body (G) and vacuole (V) ; a transporter of unknown function (EG02514); a multidrug and toxic compound extrusion family/MATE efflux family protein (J35587); a glycolipid transporter (J36726); and a sodium/bile acid transporter (J4286)  linked to all five pathway categories , and a triose phosphate transporter (J24610) . The majtegories 3. None A. thaliana, we wished to understand the deeper evolutionary conservation and gene transfer events underpinning the origins of P. tricornutum plastid transporters. First, to assess the immediate evolutionary conservation of each transporter, we identified homologous sequences of all 70 P. tricornutum plastid transporters by RbH amongst a previously assembled composite library of more than two hundred species  were found to possess the greatest number (63) of homologues of P. tricornutum plastid transporters  were inferred to be conserved across, but unique to diatoms  and meta-genomes (MetaG) abundance against sampled environmental variables at each depth and size fraction. Krona Plots concerning the nearest homologue from cultured species of all phylogenetically identified diatom transporter meta-gene are provided in P. tricornutum, reflecting its rarity in environmental samples  were found to be associated with multiple P. tricornutum plastid metabolism pathways , showed a strong positive correlation (0< P< 0.05 observed between a parameter and transporter expression abundance across ≥ three combinations of depth and size fraction) with two transporters, and strong negative correlations (-0.05< P< 0 observed between a parameter and transporter expression abundance across ≥ three combinations of depth and size fraction) with five transporters  are shown in Phaeodactylum.One transporter, J43171, encoding an ion channel protein with an EF-hand calcium-binding motif, uniquely showed a weak positive relationship with temperature in the ans data B. This P. tricornutum, alongside environmental sequence data from Tara Oceans, to close the knowledge gap concerning the plastid transporters associated with diatoms, and other related algal groups with four-membranes surrounded plastids derived from the secondary endosymbiosis of red algae . Complementing more detailed studies from other groups on specific proteins . These additional transporters may be best revealed by targeted proteomics either of isolated diatom chloroplasts  also had strong correlations to primary production in Tara Oceans data  and pH decreases (ocean acidification), will most likely individually or synergistically influence diatom photosynthesis, respiration  and primse to pH Figure 5We see contrasting relationships between the expression parameters of different diatom plastid transporters with CDOM. The physiological links between transporter expression and this environmental parameter, which is influenced by both primary production, photodegradation and decomposition , and in In conclusion, our holistic study is significant to broader studies of diatom plastid metabolic, evolutionary and environmental functions, allowing the pinpointing of targets linked to key physiological and environmental processes for functional and experimental characterization.https://osf.io/89vm3/ in the folder “Transporters”.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: osf.io repository SL and RGD conceived the project and designed research; SL, MS, and RGD performed research; SL wrote the paper; CB, RGD, and GF contributed to funding acquisition; all authors contributed to the article and approved the submitted version.This work was supported by grants from the Chinese Scholarship Council  to SL, a CNRS Momentum Fellowship (awarded 2019-2021), and an ANR JCJC (ANR-21-CE02-0014-01) to RGD, an ANR Collaborative grant (ANR-19-CE20-0020) of France to CB, and a European Research Council Advanced Grant  to GF.Tara Oceans series.SL acknowledges a Chinese Scholarship Council PhD student . RD acknowledges a CNRS Momentum Fellowship (awarded 2019-2021) and an ANR t-ERC (ChloroMosaic). MS and GF acknowledge the support by the European Research Council (ERC) Chloro-mito (Grant No. 833184). CB acknowledges an ANR collaborative grant . The authors thank Morgane Roquais and Juan Pierella Karlusich (IBENS) for assistance in the retrieval of Tara Oceans homologues for Phatr3_J43171. This is article # ABCDEF in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-pyridinium chloride ) with sodium dicyanamide (Na(CN)2N) resulted in anion exchange between Cl− and (CN)2N− to yield a new Zincke salt, salt((CN)2N−). Reactions of salt((CN)2N−) with piperazine, specifically (R)-(−)- or (S)-(+)-2-methylpiperazine under eco-friendly conditions, such as in aqueous solution, in the absence of a catalyst, and at room temperature, resulted in pyridinium ring opening to yield ionic high-molecular-weight polymers with 5-2,4-dienylideneammonium dicyanamide units or chiral 5-(2-methylpiperazinium)-2,4-dienylideneammonium dicyanamide units, namely, polymer2N−), polymer2N−), and polymer2N−). UV-Vis measurements revealed that the π-conjugation system expanded along the polymer chain due to the orbital interaction between the electrons on the two nitrogen atoms of the piperazinium ring. Circular dichroism (CD) measurements revealed a helical conformation of the main chain in polymer2N−) and polymer2N−). The reaction of polymer2N−) with p-phenylenediamine (PDA) caused recyclization of the 2,4-dienylideneammonium unit and resulted in depolymerization to yield N-(4-aminophenyl)pyridinium dicyanamide. Cyclic voltammetry analysis suggested that the polymers obtained in this study undergo electrochemical oxidation and reduction.Reaction of  2N−) with (R)-(−)- or (S)-(+)-2-methylpiperazine under eco-friendly conditions resulted in a yield of ionic helical polymers.Reactions of salt((CN) The existence of aliphatic cycles in the polymer main chain usually prevents expansion of the π-conjugation system along the polymer chain. We reported the synthesis of ionic polymers—polymer with piperazines in water yielded a mixture of unidentified products. We expect that the introduction of a hydrophilic anion into a Zincke salt may enable reaction with an amine in water, and incorporation of electron-accepting groups may contribute to the formation of an intramolecular hydrogen bond with the amine group in the intermediate adduct, which maintains the boat form of the piperazinium ring. Based on this hypothesis, we employed a Zincke salt with a dicyanamide anion, salt((CN)2N−), in this study. The reaction of salt((CN)2N−) with piperazines in water without a catalyst at room temperature yielded ionic polymers with high molecular weights. To the best of our knowledge, this is the first report of a non-catalytic synthesis of ionic polymers with expanded π-conjugated system along the polymer chain via the reactions under environmentally-friendly conditions. The eco-friendly synthesis of such polymers will lead to new methods for the development of functional materials.The development of non-catalytic syntheses of polymers with expanded π-conjugated system along the polymer chain in water at room temperature is attractive because it offers many advantages with regard to cost, safety, and environmental concerns. The reaction of salt was prepared according to the literature.3 Other reagents were purchased and used without further purification. Solvents were dried, distilled, and stored under N2. Reactions were carried out using standard Schlenk techniques under nitrogen.Salt and GPC measurements were conducted with a SHOKO science DAWN HELEOS II and a TOSO HLC-8220 with polystyrene gel columns (Shodex LF-804) using DMF containing 0.06 M LiBr as an eluent, respectively. Cyclic voltammetry was performed on a DMSO solution containing 0.10 M , 163.1235. Found, 171.0923, 163.1234.After the acetone solution (3 mL) of modelN-pyridinium chloride ) with sodium dicyanamide resulted in anion exchange between Cl− and (CN)2N− to yield N-pyridinium dicyanamide 2N−)) (2N−) with piperazine, R-(−)-2-methylpiperazine, S-(+)-2-methylpiperazine and 2-methylpiperazine in water at room temperature led to ring-opening of the pyridinium ring to afford polymer2N−), polymer2N−), polymer2N−) and polymer2N−), in 71%, 34%, 36% and 21% yields, respectively (−) with piperazine in water at room temperature resulted in unidentified products.Reaction of CN)2N−)) . The reaectively . However2N−) was synthesized by the reaction of 1-phenylpiperazine with salt((CN)2N−) in 74% yield 4% yield . The resN,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and N-methyl-2-pyrrolidone (NMP). Polymer2N−), polymer2N−) and polymer2N−) were partially soluble in methanol but polymer2N−) was insoluble in the solvent. Model2N−) was soluble in acetone, MeOH, DMF and DMSO. The weight-averaged molecular weights (Mw's) of NMP solutions of polymer2N−), polymer2N−) and polymer2N−), as determined by light scattering method, were 53.2 × 104, 3.95 × 104 and 13.7 × 104, respectively. The Mw value of polymer2N−), as determined by GPC, was 3.27 × 104 (Mw/Mn = 1.46).The polymers obtained in this study were completely soluble in polar organic solvents such as ηsp/c) of the DMSO solutions of the polymers are summarized in ηsp/c values of the polymers in DMSO increased as their concentrations, c, were reduced 2N−), polymer2N−) and polymer2N−) changed from 0.78, 0.62 and 0.56 g−1 dL (c = 0.10 g dL−1) to 0.95, 0.74 and 0.71 g−1 dL (c = 0.050 g dL−1) through values of 0.85, 0.66 and 0.64 g−1 dL (c = 0.071 g dL−1). These results suggest that the polymers behaved as polymeric electrolytes in dilute solutions.4 The ηsp/c values of polymer2N−) and polymer2N−) were roughly the same, although the Mw value of polymer2N−) was considerably higher than that of polymer2N−). The results imply that polymer2N−) has a stiffer structure than polymer2N−) in solution. It has been reported that helical polymers show greater reduced viscosities due to their stiff structures when compared to random coil polymers.5The reduced viscosities 2N−), polymer2N−) and model2N−) in DMSO-d6. The peak assignments are shown in the figure. The 1H NMR spectra of the DMSO-d6 solutions of polymer2N−) and polymer2N−) were similar, and each spectrum showed three signals, at approximately δ 7.9, 7.6, and 6.1 in a 2 : 1 : 2 integral ratio, arising from the protons in the penta-2,4-dienylideneammonium group. The 1H NMR spectra of the DMSO-d6 solution of polymer2N−) also showed three signals corresponding to the penta-2,4-dienylideneammonium group at almost the same positions corresponding those of polymer2N−) in a 2 : 1 : 2 integral ratio, arising from the protons in the penta-2,4-dienylideneammonium group. These observations suggest that the π-electrons are delocalized along the penta-2,4-dienylideneammonium group. The integral ratio of the peaks corresponding to the methyl group and piperazinium protons of the polymers supports the structures shown in c′′, Hb′′ and Ha′′ of model2N−) in a 2 : 2 : 1 integral ratio suggests that the π-electrons are delocalized across the aminopenta-2,4-dienylidene group. The 13C NMR data also support this view, showing three signals arising from the penta-2,4-dienylideneammonium group  with PDA caused recyclization of the 2,4-dienylideneammonium unit and resulted in the formation of N-(4-aminophenyl)pyridinium dicyanamide (APD) and N-phenylpiperazine  with PDA also caused recyclization of the 2,4-dienylideneammonium unit of the polymer and resulted in depolymerization to yield APD perazine . The forield APD .N-phenyl-2,4-pentadienylideniminium chloride is converted spontaneously and quantitatively to N-phenylpyridinium chloride and aniline in solution.3 However, the ring-closure of the 2,4-pentadienylideniminium group by reaction with an amine has not been previously reported. The assumption that the above reactivity is due to the presence of dicyanamide anions in the polymer and model compound is confirmed by the result that polymer and model did not react with PDA. Pyridinium salts are an important class of compounds that are used as initiators of cationic polymerization,8 cationic surfactants,9 non-linear optical materials,10 and phase transfer catalysts.11 The reactive pyridinium salts could be used as starting species for functional materials such as dyes, nonlinear optical polymers, and polymer catalysts. However, reports on such reactive pyridinium salts are limited because of difficulty in synthesis of them.12 In fact, APD could not be obtained by the reaction of salt(Cl−) with PDA. In contrast, the ring-closure reaction yielded APD. 2N−). Nucleophilic addition of one of the amine groups of PDA to the carbon atom (Ca) bonded to the piperazinium ring occurs first. The fact that the electron density of Ca is lowest among the 2,4-dienylideneammonium carbons is confirmed by the appearance of the 1H NMR signal at the lowest magnetic field position among the 2,4-dienylideneammonium protons.It has been reported that 5-anilino-N-(4-aminophenyl)-2,4-pentadienylideniminium unit to yield N-(4-aminophenyl)pyridinium dicyanamide and N-phenylpiperazine. The reaction of N-penta-2,4-dienylidene-1-N-arylinium chloride with piperizine to provide N-piperizinium chloride has also been previously reported.6,7The newly formed C–N bond exchanges between the aminophenyl and piperazinium rings, followed by the ring-closure of the 5-piperizine-13 However, the piperazinium and 2-methylpiperazinium rings of the polymers obtained in this study primarily assume the boat form, which contributes the expansion of π-conjugation system, as revealed by the following UV-Vis measurements. The intramolecular hydrogen bond between the nitro group and the NH group in the intermediate adduct seems to play an important role in the predominant formation of the piperazinium ring with the boat form in the polymers, as illustrated in Scheme S1.−), polymer and polymer converted from the boat to chair form when solutions of each were allowed to stand in air.1,2 The possibility that the UV-Vis spectral changes were attributed to decomposition of the polymers could be ruled out from the result that the viscosities of the polymer solutions did not change with time. Whereas such interconversion of the piperazinium and 2-methylpiperazinium rings of the polymers with dicyanamide anions was not observed. In other words, the polymers can remain their extended π-conjugation system in solution. We speculated that this result may be the intramolecular interaction between the nitrogen atom of the piperazinium ring in the boat form and a CN group of the dicyanamide anion, as illustrated in It is known that piperazine derivatives usually assume the energetically stable chair form rather than the boat form.H-Me;(CN)2N−), polymer2N−), polymer2N−) and model2N−). The UV-Vis data are summarized in λmax) of the DMSO solutions of the polymers appeared at longer wavelengths than did that of model2N−), suggesting extension of π-conjugation across the aminopenta-2,4-dienylidene groups and through the 2-methylpiperazinium and piperazinium rings. The λmax's of polymer2N−) in DMF and methanol were 473 and 475 nm, respectively. These wavelengths were essentially the same as that in DMSO. The change in the UV-Vis spectra over time of freshly prepared MeOH solutions of polymer, polymer and polymer at 30 °C in air corresponds to the two-step conversion of 2-methylpiperazinium and piperazinium rings from the boat to the chair form via a half-chair intermediate.1,2 When the 2-methylpiperazinium rings of the polymers adopt the chair conformation, the distance between its two nitrogen atoms is too large to allow through-space interaction; therefore, there is no expanded π-conjugation in these polymers. We proposed a possible reaction mechanism for the predominant formation of the thermodynamically unstable boat form of piperazinium ring in the polymers at the initial stage.1 However, the UV-Vis spectra of solutions of polymer2N−), polymer2N−) and polymer2N−) in DMSO were almost unchanged when the solutions were allowed to stand in air. The UV-Vis spectra of DMF and methanol solutions of polymer2N−) were also unchanged when the solutions were allowed to stand in air. This result suggests that the 2-methylpiperazinium and piperazinium rings did not change their conformations in solutions and maintained the π-conjugation lengths of the polymers.2N−) at 472 nm in the presence of an equimolar amount of PDA decreased with time when the solution was left open to air r Fig. S6. The decR-Me;(CN)2N−) and polymer2N−). The CD spectrum of polymer2N−) displays a relatively strong positive Cotton effect, with zero-crossings centered at the π–π* transition of the polymer chain 2N−) is consistent with a similar trend for the polymer.2 Similarly, polymer2N−) showed a relatively strong negative Cotton effect. These Cotton curves suggest that the main chains of polymer2N−) and polymer2N−) have oppositely oriented, highly ordered structures, such as right- and left-handed helical conformations. The ellipticities for the Cotton curves of polymer and polymer gradually decreased with time when the methanol solutions were allowed to stand in air. These decreases are presumably due to time-dependent dynamic conformational changes in the polymers; the helical conformation of the polymers disappeared as the 2-methylpiperazinium ring converted from the boat to the chair form via a half-chair intermediate form. Whereas such decrease in ellipticities for the Cotton curves was not observed in the CD spectra of polymer2N−) and polymer2N−). The results are consistent with the fact that the UV-Vis spectra of the DMSO solutions of the polymers were almost unchanged with time.4N]BF4 are summarized in R-Me;(CN)2N−) and model2N−). Polymer2N−) and model2N−) underwent electrochemical oxidation of the piperazinium ring and reduction of the CN groups of the dicyanamide anion. The oxidation peak potential of polymer2N−) was lower (Epa = 0.71 V vs. Ag+/Ag) than that of model2N−) (Epa = 1.32 V vs. Ag+/Ag) because of the longer π-conjugation length of the polymer. The oxidation peak potential of polymer2N−) was the essentially same as that of polymer2N−). The dicyanamide anions of the polymers, except for polymer2N−), and the model compound participated in two-step electrochemical reductions at approximately −0.6 V and −1.3 V (vs. Ag+/Ag). The reason for the disappearance of the reduction peaks in polymer2N−) is unclear.Cyclic voltammetry (CV) measurements suggest that the polymers and model compound obtained in this study underwent electrochemical oxidation of the 2-methylpiperazinium and piperazinium rings and reduction of the dicyanamide anions. The oxidation and reduction potentials of the polymers and model compound in DMSO containing 0.10 M [EtN-pyridinium dicyanamide with chiral 2-methylpiperazines in water at room temperature. UV-Vis measurements revealed that the 2-methylpiperazinium rings of the polymers with dicyanamide anions did not undergo conformational changes in solution, in contrast to those of the polymers with chloride anions. This increased stability contributed to the preservation of the π-conjugated length and helical conformations of the polymers in solution. The polymers obtained in this study reacted with PDA, resulting in depolymerization. The results obtained in this study indicate that Zincke salts with dicyanamide anions can be used to synthesize stable ionic helical polymers under environment-friendly reaction conditions. Other polymers were synthesized in a similar manner.In conclusion, ionic helical polymers were obtained from the reaction of There are no conflicts to declare.RA-008-C8RA05686B-s001


Advisory boards play a key role in guiding and informing research programs, including occupational health surveillance. It is important to evaluate the effectiveness of these advisory boards. This report details the organization of the Risk Information System for Commercial (RISC) Fishing Technical Advisory Board (TAB), the approach taken to evaluate the TAB, and the results of the evaluation. The RISC TAB was formed to provide advice and recommendations to the study team and informed the development and use of the safety surveillance system.The evaluation approach was informed by limited previous literature on advisory board assessments. This evaluation was conducted in Year 5 of the 6‐year project. A review of the meeting notes, materials and correspondences, and study progress was conducted internally to document input from the board and associated actions. To obtain member perspectives, we surveyed the TAB and discussed it in a subsequent TAB meeting.The RISC Fishing TAB members constitute a wide variety of commercial fishing safety stakeholders. The internal analysis identified the main project aspects and 14 of the proposed changes from the TAB that have either been implemented or are in progress in the project. Ten of the 15 TAB members responded indicating a positive experience on board organization and conduct.Evaluation of advisory boards is an essential part of a research program. A process is outlined in this report to inform future efforts to document measurable ways to inform projects based on advisory board feedback and reflections. This report details the operation of the RISC TAB, the approach taken to evaluate the TAB, and the results of the evaluation. Our report can help to improve the utilization of future advisory boards that will benefit OSH research projects.2The purpose of the RISC TAB is to identify relevant commercial fishing industry issues and priorities and to help translate surveillance findings into tailored, nimble responses for hazard assessment needs of important fishery safety stakeholders. The RISC TAB provides advice and recommendations to the study team and informed the development and use of the safety surveillance system. Project‐specific topics shared with the TAB included the: (1) database architecture; (2) data elements and coding; (3) data entry and query interfaces; (4) reporting elements; (5) report contents and data visualization; (6) priorities for hazard assessments; and (7) dissemination materials and methods .RISC TAB members were recruited based on their interest, ability to represent key stakeholders, and availability to commit time to the project. As this project is currently restricted to Oregon and Washington, all members represented commercial fishing interests in those states. The RISC TAB members constitute a wide variety of commercial fishing safety stakeholders , public health surveillance practitioners, and OSH researchers and practitioners Table .To date, nine semiannual meetings have been held over the 5 years of the project with meetings occurring both virtually and in person. During the pandemic, all meetings were virtual, but because the TAB was already well established, it continued to function effectively. Meetings were conducted in a professional but comfortable manner and facilitated by the project Principal Investigators. At the onset, TAB expectations were set with appropriate ground rules  Center's outreach core.3At the time of the evaluation, the RISC TAB had 13 of the original 16 members serving continuously since 2017 with a retention rate of 83%. All current board members have been attending meetings for at least the last 3 years.Analysis of meeting notes, materials, and correspondences identified six main project aspects that were presented at advisory board meetings: (1) Data sources; (2) Database variables; (3) Data coding; (4) Advisory Board Members; (5) RISC Fishing System Tool; and (6) Project Outreach and Deliverables. From this the research team was able to track what was presented at each meeting, the feedback collected, and if/how changes were implemented based on the feedback given by TAB members. Fourteen of the proposed changes have either been implemented or are in progress including the addition of EMS data, broadening the scope of searches in state trauma registries to identify more cases for inclusion, adding additional variables, and strategies for stakeholder engagement with the RISC Fishing System tool as well as outreach.Ten of the 15 TAB members responded to the survey with a response rate of 67%. Respondents included a fishing community member, safety professionals from Alaska Marine Safety Education Association and American Seafood Company, Fisheries Extension Faculty from Oregon State University/Oregon (OSU/OR) Sea Grant, a fisheries economist from the National Oceanic and Atmospheric Administration (NOAA), epidemiologists from NIOSH, safety researchers from PNASH, and a Coastal Shellfish Manager from Washington Department of Fish and Wildlife. Respondents have been in their current position anywhere from 2 to 24 years with an average of 10.8 years. Seven respondents indicated that they would be willing to serve on the advisory board again for subsequent OSU/PNASH Commercial Fishing Safety and Surveillance projects, one said “maybe” and two left the question unanswered. The TAB agreed with or was neutral with every statement in the Likert scale evaluation (see Table n = 1) and fishing community members (n = 3). More participation from the USCG was mentioned by one advisory board member.The open‐ended questions generated thoughtful responses. While two board members said the board was not missing stakeholders and it was well rounded, there were suggestions of additional groups/expertise that included an emergency physician who works in a commercial fishing community (n = 10) response was networking, sharing ideas, respective work in health and safety with other board members, and “making a connection with others working in the area.” One additional concept in response was related to the updates on the progress of the project and “having the opportunity to voice support and provide advice.”When asked about what the board enjoyed about being on the advisory, a common theme found in every  indicated they felt their feedback was incorporated into the project. “I feel that the chair does a good job of soliciting input and including it in the design and implementation of projects.” Most board members  thought serving on the board did not directly help in their own work but that the collaborations and connections of the board members did help them.Almost all of the board members  until …… and moved all meetings to be virtual in the Summer of 2020. Four board members cited that more in‐person meetings would have improved their experience, but acknowledged it was not possible with COVID.Four board members responded to our question if there was an unexpected outcome of their involvement in the advisory board. One stated that nothing came to mind, and two mentioned the Fishermen First Aid and Safety Training, a commercial fishing‐specific training that was developed with the help of injury statistics taken from the RISC Fishing project.ergonomics of captain's seats and operations,development of commercial fishing industry tools to share solutions and strategies across the PNW,research into how the insurance could influence fishermen's health and safety,what key drivers of risk‐taking such as weather, bar closings, season‐opening, and season timing could have to alleviate risk,sleep deprivation,hearing loss,boatyard hazards,other fishermen's health issues,safety management systems for small fishing vessels.Six board members provided ideas for unaddressed or new fishing safety ideas for future projects. This included:While extensive notes were taken at all TAB meetings, they did not necessarily capture feedback from board members in a way that could inform the future evaluation. Tracking changes to the project based on TAB member feedback would be more straightforward if an evaluation form and feedback processes were built into the TAB meetings. Future projects with advisory boards should take this into consideration and provide opportunities for board members to contribute directly to the written record through surveying and a more formal evaluation process.4By conducting our technical advisory board evaluation internally with the notes, materials, and correspondence review, we were able to readily see when feedback helped to shape our project. Externally, by conducting the survey of the TAB members, we learned directly from them what was effective for their engagement as well as opportunities for improvements and future research directions.The consistency of the TAB members has allowed for rich discussions and feedback and provides the research team with an invaluable sounding board who is well versed in the particulars of the RISC Fishing project.The internal review shows that the TAB members are an invaluable resource and have greatly contributed to the direction of the project as well as provided insight into their fields and how the RISC Fishing system could be useful to a variety of stakeholders. The survey of TAB members also indicated that they felt that their participation was useful and valuable in forming connections with other stakeholders and learning about what is happening in terms of commercial fishing safety in other fields.Though advisory boards are common, not much focus has previously been put on formally evaluating their effectiveness. Using the process outlined in this report as a template for future advisory board evaluations could lead to unintended positive outcomes and guide the process to be more focused on measurable ways to inform projects based on TAB feedback and reflections.Amelia Vaughan: Conceptualization; data curation; formal analysis; methodology; project administration; visualization; writing—original draft. Viktor Bovbjerg: Conceptualization; validation; writing—review and editing. Solaiman Doza: Writing—review and editing.  Laurel Kincl: Conceptualization; formal analysis; funding acquisition; methodology; project administration; supervision; validation; writing—review and editing. All authors have read and approved the final version of the manuscript. Amelia Vaughan had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.The authors declare no conflict of interest.This manuscript is an honest, accurate, and transparent account of the study being reported; no important aspects of the study have been omitted, and any discrepancies from the study as planned  have been explained.


The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site  of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise. RepurpoAntiviral drugs can be categorized into two kinds: those that act directly against the pathogen, by targeting some essential component of its viral machinery , versus those that act indirectly, by targeting some component of the host’s cellular machinery required for infection . Approaches to drug discovery, whether the identification of novel therapeutics or the repurposing of existing ones for different or additional contexts, can similarly be divided into two types. The first seeks to determine a druggable target, whether an antigen or a molecule in the infected organism with which an antigen interacts; the second attempts, given a known or putative target, to find a suitable drug against it. The criteria for such suitability obviously include efficacy and safety. Here, focusing on direct acting antivirals, we discuss each kind of approach in the light of findings by us and others, in a “bad news first” fashion, concluding with positive reassessments and recommendations. pro): these enzymes are indispensable for the SARS-CoV-2 replicative cycle and most viral inhibitors target either polymerases or proteases, making for straightforward repurposing and a greater likelihood of discovering a “best in class” inhibitor. The RNA synthesis active site (AS1) within RdRp is particularly attractive, because the chemistry of the nucleotide addition reaction involved is well-understood and the active sites are conserved across RNA viruses, yet different from cellular enzymes -GTP , and different concentrations of competitors were added to start the reaction. We used commercial remdesivir triphosphate , 2′-Azido-2′-deoxyguanosine-5′-triphosphate , 2′-Azido-2′-deoxyuridine-5′-triphosphate , and GTP . Diphosphates and triphosphates of AT-527 and sofosbuvir were synthesized at Gilead Scientific. For small molecule inhibitors, 10 μL reaction containing 0.5 μM nsp12, 5 μM nsp9 in NMPylation buffer was incubated with inhibitor at indicated concentration at 37 °C for 5 min prior to the addition of 25 μM GTP+[α32P]-GTP. Sources and structural analysis of rifapentine and rifabutin were described in [For competition assays with nucleotide analogs, 10 μl reactions containing 0.5 μM nsp12 and 5 μM nsp9 in NMPylation buffer  for 3 min at 37 °C. Following the addition of the RNA scaffold  and NTPs , reactions were incubated for 15 min and quenched with an equal volume of 2× stop buffer .An RNA oligonucleotide GA1 (5′ -AAAAGAAAAGACGCGUAGUUUUCUACGCG- 3′) with Cyanine 5.5 at the 5′-end was obtained from Millipore Sigma. Prior to the reaction, the RNA was annealed in 25 mM HEPES, pH 7.5, 50 mM KCl by heating to 75 °C and then gradually cooling to 4 °C. Reactions were carried out at 37 °C. 500 nM nsp12 variant, 1 μM Nsp7, 1.5 μM Nsp8, and inhibitors were incubated in the transcription buffer  in transcription buffer was incubated with inhibitors at indicated concentrations at 30 °C for 5 min. Then 100 nM GA1 RNA scaffold was added. After 10 min incubation, reactions were mixed with 10X loading buffer  and ran on a 3% agarose gel in 0.5× TBE on ice. The gel was visualized by Typhoon FLA9000 .5 cells/well and allowed to attach overnight. Cells were infected with SARS-CoV-2 WA1 strain at MOI 0.01 for one hour at 37 °C before the inoculum was removed, wells were washed twice with PBS, and increasing concentrations of compounds or DMSO as vehicle control were added to the wells. Infection was allowed to proceed for 24 h before harvesting supernatants. Viral titers were determined by tissue culture infectious dose 50 (TCID50) using the Reed and Muench method [Vero E6 cells were plated in 48-well plates at 1 × 10h method . Protein samples were heated for 4 min at 95 °C and separated by electrophoresis in 4–12% SurePAGE gels . RNA samples were heated for 2.5 min at 95 °C and separated by electrophoresis in denaturing 9% acrylamide (19:1) gels . The gels were visualized and quantified using Typhoon FLA9000  and Image Quant. All assays were carried out in triplicates. The means and standard deviation (SD) were calculated by Excel .


Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and ‘G–U wobbles’ on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response.Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the  In plants, the RNA silencing process is a crucial component of the immune response against pathogens such as viruses. Silencing is induced by double stranded (ds) elements of viral RNA genomes and/or mRNAs, dsRNA viral replication intermediates or dsRNAs that are generated from viral RNA templates by host RNA-dependent RNA polymerases (RDRs) ,2. The dTombusviridae). p19 binds siRNA duplexes with high affinity, and siRNA binding was found to depend on the size but not on the sequence of the RNA molecule -methionine  was added to the reaction. The samples were separated by 15% (w/v) SDS-PAGE and the labeled proteins visualized by phosphorimaging . For initial screening of miRNA interactions, ca. 30 pmol of radiolabeled AtmiR162 or AtmiR168a was added to the translation reaction and incubated for 60 min at 25 °C. The samples were mixed with 0.25 v/v of loading dye and analyzed by PAGE on a 6% TBE gel under non-denaturing conditions. Bands corresponding to bound and free radiolabeled RNA were detected by phosphorimaging  and quantified by ImageQuant software . All measurements were done at least in triplicate. Data were expressed as the mean and standard deviation, and the results were statistically analyzed using Student’s t-test.The in vitro translation of p19 variants was performed in 50% (nditions . Briefly


Rift Valley fever virus (RVFV), a bunyavirus, has a single-stranded, negative-sense tri-segmented RNA genome, consisting of L, M and S RNAs. An infectious virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes composed of encapsidated viral RNA segments. The antigenomic S RNA, which serves as the template of the mRNA encoding a nonstructural protein, NSs, an interferon antagonist, is also efficiently packaged into RVFV particles. An interaction between Gn and viral ribonucleoprotein complexes, including the direct binding of Gn to viral RNAs, drives viral RNA packaging into RVFV particles. To understand the mechanism of efficient antigenomic S RNA packaging in RVFV, we identified the regions in viral RNAs that directly interact with Gn by performing UV-crosslinking and immunoprecipitation of RVFV-infected cell lysates with anti-Gn antibody followed by high-throughput sequencing analysis . Our data suggested the presence of multiple Gn-binding sites in RVFV RNAs, including a prominent Gn-binding site within the 3’ noncoding region of the antigenomic S RNA. We found that the efficient packaging of antigenomic S RNA was abrogated in a RVFV mutant lacking a part of this prominent Gn-binding site within the 3’ noncoding region. Also, the mutant RVFV, but not the parental RVFV, triggered the early induction of interferon-β mRNA expression after infection. These data suggest that the direct binding of Gn to the RNA element within the 3’ noncoding region of the antigenomic S RNA promoted the efficient packaging of antigenomic S RNA into virions. Furthermore, the efficient packaging of antigenomic S RNA into RVFV particles, driven by the RNA element, facilitated the synthesis of viral mRNA encoding NSs immediately after infection, resulting in the suppression of interferon-β mRNA expression. Rift Valley fever virus (RVFV) is transmitted by mosquitoes and is endemic in sub-Saharan Africa and several countries in the Middle East Asia. Human infection occurs from the bite of infected mosquitos or from direct transmission of the virus from infected animal tissues or blood. Human disease manifestations include transient incapacitating febrile illness , encephaPhlebovirus, family Phenuiviridae, order Bunyavirales, and is an enveloped, negative-sense RNA virus that has a tri-segmented RNA genome, consisting of L, M and S RNAs  at their termini and the genomic and antigenomic S RNA carry an intergenic region between the N and NSs genes Figure 1Bunyavirales order and some members in the Arenaviridae family do not encode a matrix protein. It is hypothesized that the cytoplasmic tail of Gn functions as a matrix protein surrogate through its interaction with the viral RNPs and that this interaction drives the packaging of viral RNAs into those bunyaviruses lacking a matrix protein  and 1% penicillin-streptomycin. Huh7 cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% FBS and 1% kanamycin . BSRT7/5Renilla luciferase (rLuc) open reading frame (ORF) in place of the NSs ORF in the S segment. The rescued viruses were amplified once in Vero E6 cells, titrated by plaque assay, and used for virus infections. For virus infection, cells were inoculated with virus for 1 h at 37˚C and then washed three times with phosphate buffered saline (PBS) before fresh medium was added.All viruses used in the study were derived from MP-12 stain of RVFV and generated by an established reverse genetics system . RVFV-rLA recombinant PCR-based method was employed to generate the pProT7-S Δ19 construct, which expresses S Δ19 RNA carrying a 19nt deletion between nucleotide positions 1656 and 1675 in the 3’ NCR of antigenomic S RNA, by using the T7-driven plasmid pProT7-S encoding the antigenomic S RNA as a template. Sequencing of the S Δ19 plasmid construct confirmed the absence of unwanted mutations. The reverse genetics system to rescue RVFV S Δ19 was described as previously . BrieflyPlaque morphology of RVFV and RVFV Δ19 were determined in VeroE6 cells by plaque assay analysis used previously . BrieflyHuh7 cells were infected with RVFV or RVFV Δ19 at a multiplicity of infection (MOI) of 1.0 or 0.01 at 37°C for 1 h. After virus absorption, cells were washed three times with PBS and growth medium was added. For the growth curve at an MOI of 1.0, culture supernatants were harvested at 0 h, 8 h, 12 h, 16 h, and 20 h p.i. For the growth curve at an MOI of 0.01, culture supernatants were harvested every day up to 4 days p.i. Virus titers were measured by plaque assay. The points in the growth curves are shown as mean +/- standard deviation from three independent experiments.p < 0.05 (GraphPad Prism 9).Student’s t-test was used for growth curve analysis of RVFV and RVFV Δ19. Significant difference was defined as Culture supernatants harvested from virus-infected cells were purified as described previously . Briefly2, followed by lysis of the UV-crosslinked cells using a high stringency SDS lysis buffer  and heating of the lysate to 65°C for 5 min. Radioimmunoprecipitation assay (RIPA) correction buffer  was then added to the lysates. Samples were passed through a QIAshredder spin column (Qiagen) twice to reduce viscosity. Lysates were then subjected to RNAse digestion using 30 µg RNAse A and 325 units of RNAse T1 for 3 h at 37°C. Total RNA was extracted with Trizol following the manufacturer’s instructions.Huh7 cells were inoculated with RVFV at an MOI 3. After 8 h p.i., cells were mock-irradiated or irradiated with 254-nm-wavelength UV light at 250 mJ/cm2, followed by lysis of the UV-crosslinked cells using a high-stringency SDS lysis buffer  and heating of the lysate to 65°C for 5 min. The high stringency lysis buffer condition disrupts the interaction of Gn with N and L proteins, thereby eliminating the possibility of indirect co-immunoprecipitation of viral RNAs with Gn protein through its interaction with N protein and/or L protein  to obtain fragmented RNAs smaller than 200 nt. RNA was transferred to the Next Generation Sequencing Core facility at The University of Texas Medical Branch for adapter ligation and cDNA library construction. Library was sequenced using the NextSeq 550 Illumina system and bioinformatics analysis was performed by the Next Generation Sequencing Core facility. Reads were mapped to RVFV MP-12 genome  and visualized using Integrated Genome Viewer. Enriched regions (peaks) in the immunoprecipitated samples were normalized to enriched reads mapped to RVFV-infected total RNA.Reverse transcription (RT) of viral RNAs was performed using a strand-specific assay as described previously . BrieflyTotal RNAs were extracted from virus-infected cells using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The same amounts of total RNAs were subjected to Northern blot analysis using digoxigenin (DIG)-labeled RNA probes that specifically bind to the genomic L, M, S segments, and NSs mRNA . A DIG-lIntracellular lysates were subjected to SDS-PAGE analysis using a 4 to 20% gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). For the detection of Gn, N and NSs proteins, mouse monoclonal anti-Gn antibody (R1-4D4), at a 1:3,000 dilution, rabbit polyclonal GST-N peptide antibody (R1-P1-GST-N) , at a 1:Previously, we reported the presence of a direct interaction between the envelope glycoprotein, Gn, and RVFV RNAs . FurtherWe performed CLIP-seq analysis using two independent samples. Briefly, the UV-irradiated and RNAse-treated extracts from RVFV-infected cells were subjected to co-immunoprecipitation analysis using an unrelated anti-H2K antibody  control. The preNo prominent sequence coverage that aligns with the minimal NCRs of most of the viral RNAs were detected, except for the 3’ NCR of the antigenomic S RNA, which had a prominent number of sequencing reads  that has a 19 nt deletion in the 3’-NCR of the antigenomic S RNA using a reverse genetics system   would exhibit an altered profile of viral RNA packaging, be less virulent and potentially, could be developed as a safer, live-attenuated RVFV vaccine.RVFV Δ19, lacking a part of this prominent Gn-binding site in antigenomic S RNA, was competent for S RNA replication and transcription, as S RNA replication and NSs mRNA transcription occurred in RVFV Δ19-infected cells 6. RobusWe showed the early accumulation of IFN-β mRNA in RVFV Δ19-infected cells, but not in RVFV-infected cells Figure 6https://www.ncbi.nlm.nih.gov/bioproject/PRJNA925541/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BT and SM conceptualized the study. BT, KT and KN performed experiments. BT, KT, KN and SM analyzed the data. BT and SM wrote the manuscript with contributions from KT and KN. All authors contributed to the article and approved the submitted version.


We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase  Flaviviridae, genus Flavivirus. The virus is neurotropic and causes tick-borne encephalitis, which affects primarily adult population within European and North-Eastern Asian countries  is an enveloped single-stranded positive-sense RNA virus from the family ountries . While Tountries , 3. TherTBEV encodes three structural and seven nonstructural (NS) proteins. Among the NS proteins, NS3 is a multifunctional protein that comprises two functional domains: a protease and a helicase. The chymotrypsin-like serine protease is located with the N-terminal region (172 amino acids) and is responsible for viral polyprotein processing. This domain is connected by a short flexible linker to the C-terminal helicase domain , which exhibits several activities: NTPase, RNA helicase, and RNA 5′-triphosphatase. The RNA helicase activity uses energy from NTP hydrolysis for unwinding of dsRNA replication intermediates, whereas RNA 5′-triphosphatase removes the terminal γ-phosphate from the 5′-triphosphate end of the positive-sense ssRNA before mRNA capping by the methyl transferase domain of NS5 protein  , 7 and cSeveral structural studies of flavivirus helicases from Dengue virus (DENV), Zika virus (ZIKV), yellow fever virus, Kunjin virus, and Japanese encephalitis virus revealed high structural conservation among them , 13, 14.i.e., in the absence of RNA) suggest that the ATPase rate-limiting step is phosphate release, which is allosterically stimulated by RNA. Indeed, ssRNA binds to NS3H with nanomolar affinity and stimulates its ATPase activity, whereas ssDNA inhibits the ATPase activity without directly competing with RNA. We explored ssRNA binding by modeling and MD simulations and identified conserved RNA-binding residues. We examined the allosteric roles of these residues by site-directed mutagenesis and functional assays. These results and simulations suggest that RNA binding is relayed to the ATPase active site via a conserved residue within domain 1, whereas RNA affinity is modulated by conformational changes associated with opening and closing of the ATP-binding cleft.Here, we biochemically characterized NS3H from TBEV and obtained structures of key intermediates along the ATPase cycle . While the overall structure of the TBEV NS3H is closely related to that of DENV and ZIKV, structural variation is observed in different nucleotide states. Trapping of ATP hydrolysis products within the crystal and the negligible basal activity (A). NS3H hydrolyzed all four NTP substrates with comparable turnover . This demonstrated that NS3H exhibits little specificity for different NTP substrates, as shown previously for DENV helicase . The Michaelis–Menten constant Km = 125 ± 15 μM and turnover kcat = 8.8 ± 0.2 s−1 of NS3H are similar to those of DENV helicase in the presence of short 5′-UTR RNA fragment , it can partially inhibit it in a dose-dependent manner at micromolar concentrations (D). However, these micromolar concentrations are much higher than the affinity for ssRNA (apparent KD = 49.7 ± 9.3 nM), which was determined by fluorescence anisotropy–based binding assay (E). Using the same approach, we observed that ssDNA partially competes with RNA binding at ∼20-fold molar excess of apparent KD for ssRNA substrate. Longer ssDNAs inhibited RNA binding more effectively (F), suggesting that the longer DNA might bind to several weak nonspecific binding sites. Altogether, these results indicate that the binding of the helicase to RNA substrate stimulates ATP hydrolysis, and DNA may act as a noncompetitive inhibitor via nonspecific binding. This specificity of NS3H toward RNA substrate is different from HCV helicase  with the overall RMSD of 0.8 Å against our apoNS3H calculated using DALI server   .Table 1C202SGKT206 sequence forming the P-loop region of NTPase site with residue K205 known to recognize the β- or γ-phosphate of ATP in homologous helicases  includes the arginine fingers (R463 and R466), which are important in the energy coupling during the NTP hydrolysis .The NTPase site is situated at the interface between domains 1 and 2. It encompasses Walker A and Walker B motifs identified in domain 1 and motif VI in domain 2. Walker A motif consists of Gelicases . Walker  in Ref. ). Motif drolysis , 25 and drolysis . The empdrolysis , whereasdrolysis   a post-hydrolysis intermediate, ADP–Pi–Mn2+-bound state; (iii) a product dissociation intermediate with Pi released and ADP–Mn2+ are still bound to the protein; and (iv) nucleotide-free (apo) product release state, which is ready to bind ATP.Three ternary complexes of NS3H with AMPPNP–Mniviruses , 16. Her2+- and ADP–Mn2+-bound NS3H structures were obtained via cocrystallization of apoNS3H with corresponding nucleotides, whereas the ADP–Pi–Mn2+ complex was obtained as a result of ATP hydrolysis during crystallization, that is, Pi remained trapped within the structure. The nucleotide complexes crystallize in P41212 space group with one molecule in the asymmetric unit similar to the apoNS3H (A) when the nucleotide complexes were compared with apoNS3H. Upon nucleotide and divalent ion (Mn2+) binding, the P-loop swings toward the bound nucleotide (B) with an inward orientation of K205 side chain to coordinate the phosphate group  to accommodate γ-phosphate. This motion is absent in the ADP–Mn2+ structure.The AMPPNP–Mn apoNS3H . Hence,  apoNS3H A when thcleotide B with ante group . In AMPP apoNS3H B to acco2+ complex (B), the triphosphate moiety of AMPPNP interacts with P-loop residues K205 and T206 via hydrogen bonds and with residues D290 and E291 of Walker B motif and Q459 of motif VI via water-mediated coordination. One of the arginine fingers, R463, and residue G420 (motif V), also interacts with γ-phosphate of AMPPNP. The 3′-OH group of the ATP ribose C3′ endo ring pucker is hydrogen-bonded with the main chain amide nitrogen in between residues T206 and H207. One potentially catalytic water molecule coordinates with the side chains of E291 , Q459 (motif VI), and γ-phosphorus atom in location suggested by AMPPNP-bound DENV helicase (2JLR) , whereae (2JLR) , it adope (2JLR) , which ie (2JLR) .2+ complex (C) was captured following the cocrystallization of NS3H and ATP, implying that ATP was hydrolyzed during crystallization or within the crystal and that the subsequent Pi release is extremely slow given the lengthy crystallization protocol. This suggests that phosphate release is the rate-limiting step of the ATPase cycle (in the absence of RNA) and is prevented by the closed conformation of the ATP-binding site. The low basal ATPase activity, as measured by phosphate release assay in the absence of RNA, further indicates that this step is facilitated by RNA binding and might be coupled to RNA translocation. This is similar to the proposed HCV mechanism . Thus, the strong interactions of the γ-phosphate are driving nucleoside triphosphate deeper into the binding pocket prior to hydrolysis. Hydrolysis then leads to irreversible phosphate separation, producing a long-lived post-hydrolysis state before Pi and ADP release from the NTPase site.The NS3H–ADP–Pi–Mn complex C was capechanism . Howeverechanism . We obse complex D. Thus, 2+ complex (D), the arginine fingers do not interact directly with any of the phosphate moiety of ADP. Residue R463 coordinates a water molecule, whereas the side chain of residue R466 moves away from the hydrolysis site. In addition, the 3′-OH group of the ribose C3′ endo ring pucker interacts with the main chain carbonyl oxygen of R466. Therefore, this structure captured a state with diminished diphosphate interactions that may facilitate ADP release.In the NS3H–ADP–Mn complex D, the ar2JLU (Eint) were computed for frames along the trajectory using generalized Born surface area method. Frames with diminished Eint may represent states in which weak interactions between the protein and RNA facilitate relative movement and enable translocation. In addition, structural comparison between states exhibiting high and low Eint, respectively, may reveal how protein conformational changes modulate RNA affinity.Our data and previously published study  show tha2JLU ) with bo2JLU . RNA intA, left panel) and sample multiple conformations. Likewise, RNA is bound with widely different Eint suggesting a substantially dynamic association . Overall structural differences are illustrated by comparing selected frames with low (Eint = −150 kJ/mol) and high affinity (Eint = −350 kJ/mol) conformations, respectively, with the starting apoNS3H structure (A). The overall disposition of protein secondary structure elements and motifs is similar between high RNA affinity and apoNS3H structures, whereas the low-affinity conformation differs substantially. With the exception of the P-loop, which assumes conformation seen in nucleotide-containing structures in the presence of RNA, larger conformational changes are mostly confined to domain 2. However, the α2 helix in the low-affinity structure moves closer to the β-sheet core and opens the nucleotide-binding pocket. In domain 2, the α6 helix shifts away from the RNA-binding groove. In the low-affinity structure, this shift is further accentuated by a relative rotation of the whole domain 2. This rotation and translation of α7 helix lead to a widely open nucleotide-binding cleft in the low-affinity structure, whereas in the high-affinity structure, the α7 helix only rotates around its axis. Slight repositioning of domain 3 further opens the RNA-binding cleft in the low-affinity structure.Both protein and RNA quickly depart from the initial model as demonstrated by RMSD , the ssRNA backbone is anchored by interactions between polar or charged residues along the edge of the groove . The polar group interactions are augmented by van der Waals contact from hydrophobic groups lining the groove . In addition, there are several base-specific contacts. The G2 base, which is conserved within the 5′-UTR of most flaviviruses , similar to that observed in the corresponding DENV crystal structure . The 5′ end of the RNA backbone is anchored in approximately the same place, but the interacting residues are different. In the high-affinity structure, the G2 5′ phosphate is held in place by K605 (domain 3) and S393 . The former interaction is replaced by K394 , and the S393 contact is weakened in the low-affinity structure. The conserved G2 base is shifted in position and only held by D296, and the backbone and base positions downstream are displaced and held by fewer contacts in the low-affinity structure , we have designed several NS3H mutants having residues R231, T270, R274, D296, and K394 substituted with alanine (A). All mutants were produced as soluble proteins, and their ATPase activity and RNA-binding affinity were examined (kcat reduction), whereas R274A exhibited approximately fourfold reduction. The R231A mutant was almost inactive (∼20 fold reduction), preventing us from obtaining meaningful Km values.Using MD simulation, we have identified NS3H RNA-binding residues . Based oFig. S2examined . MutatioAs expected, mutations disrupting protein–RNA backbone contacts  reduced RNA-binding affinity . Howeverkcat) and Km leaving the catalytic efficiency (kcat/Km) unchanged  is associated with formation of modest salt bridge between K394 side chain and RNA backbone phosphate in the low-affinity state  for DENV  and requires much higher ssDNA concentrations (micromolar range) than RNA binding , thus suggesting that DNA does not directly compete with RNA for the binding cleft. This is further supported by only partial binding competition even at high DNA concentrations .Biochemical data show that ssDNA acts as an inhibitor of RNA-stimulated ATPase activity. The inhibition is incomplete D and req41 suggests that binding to multiple low-affinity surface sites might be involved. The electrostatic surface charge distribution . As controls, two MD simulations were done: one without the bound DNA and another starting from the last frame of the NS3H–ATP–DNA complex but with the bound DNA removed. Analysis of ATP interaction energy distributions obtained from the stationary portions of these stimulation trajectories  revealed that DNA binding along the ATPase active site reversibly decreases the ATP interaction energy by about twofold (C). A similar effect is observed when ssDNA41 is placed across the cleft (C). In this case, the DNA, which in the initial model, is represented by a long straight B-type helix, folds and bends, and interacts with multiple positively charged patches  and adding a short (hexanucleotide) DNA with B-form backbone running along the ATP-binding cleft. The initial model was subjected to MD and rapidly converged to a stable ensemble of configurations A. As con twofold C. A simhe cleft C. In thiB and S8B). Such displacement would be enough to disrupt the precise alignment of the catalytic residues, water, and the γ-phosphate and may lead to inhibition of the ATPase cycle. DNA binding may also interfere with nucleotide exchange by sterically blocking access to the ATPase site, and the longer DNA also partially occludes the RNA-binding groove, explaining the observed partial and weak competition between the two nucleic acids.Given that ATP binding is dominated by polar and electrostatic interactions of the triphosphate moiety and the adenine base is highly mobile (as seen in MD simulations), it is not surprising that the DNA-induced decrease in interaction energy is associated with displacement of the triphosphate moiety within the ATPase active site B and S8Be.g., nucleotide or sulphated sugar) likely via direct competition with RNA (A–C). Similarly, near complete inhibition of the ATPase activity can be achieved by high salt concentration, highlighting the prominent role of electrostatic interactions in RNA and ATP binding (D).We compared the effect of DNA to another polyanion heparin, which is often used to compete with nucleic acid binding. Unlike ssDNA, heparin can effectively and fully inhibit RNA-stimulated ATPase activity at comparable micromolar concentrations . In comparison, full ATPase inhibition is achieved at 10 μM heparin, which corresponds to 30 μM negative sulfate charge. This indicates that heparin effectively competes with RNA for the binding site, whereas ssDNA fails to do so and must exert its effect by a different mechanism. This is consistent with the much lower binding affinity of the short ssDNA  when compared with that of the equivalent RNA oligomer . Partial competition with RNA is only seen for longer ssDNA suggesting that ssDNA exploits multiple weaker binding sites, which might augment the overall affinity and either partly occlude the RNA-binding groove  or even force a portion of ssDNA to adopt otherwise unfavorable extended conformation within the RNA-binding site. The latter would be consistent with partial activation of the ATPase at high concentration of long DNA . The different mechanism of inhibition by heparin and ssDNA, respectively, is likely because of their different polymer behavior. Heparin, with its high charge density, is likely to prefer extended configurations in a fashion similar to the stiffer RNA  and thu2+, -ADP–Pi–Mn2+, -ADP–Mn2+, and the apo form of the protein. The overall structure of TBEV helicase is highly similar to that of other flaviviruses such as DENV and ZIKV, suggesting similar ATP hydrolysis and RNA translocation mechanism. However, contrary to the mechanism proposed for DENV helicase  from TBEV strain HYPR, hereafter referred as NS3H, was amplified from pUC57-HYPR-FRAGII  using Q5 polymerase and gene-specific primers . The PCREscherichia coli BL21-CodonPlus (DE3)-RIPL competent cells (Agilent Technologies). Briefly, transformed competent cells were grown at 37 °C in LB media supplemented with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol until A600nm reached 0.5. The cells were chilled for 30 min at 4 °C, and the protein production was induced with 1 mM IPTG followed by incubation for 20 h at 18 °C or 5 h at 30 °C. The cells were harvested by centrifugation at 4000g for 30 min at 4 °C and stored at −80 °C prior to further use.Recombinant NS3H proteins (wildtype and mutants) were produced in g for 60 min at 4 °C, and collected supernatant was loaded on HisTrap FF column (Cytiva) equilibrated in buffer A. Histidine-tagged NS3H was eluted using 70% of buffer B . Fractions containing recombinant NS3H were pooled together, and the elution buffer was exchanged for buffer C  using Amicon Ultra-15 spin columns with 30 kDa cutoff (Merck). To remove the residual nucleic acids bound to NS3H, the sample was loaded into HiTrap Heparin HP column (Cytiva) pre-equilibrated in buffer C, and NS3H protein was eluted using 1 M NaCl. Finally, eluted NS3H was concentrated using Amicon Ultra-15 with 30 kDa cutoff spin columns and loaded into a Superdex 200 Increase 10/300 GL (Cytiva) pre-equilibrated in buffer C for final polishing. The purity of the protein was analyzed using an SDS-PAGE gel and MALDI-TOF mass spectrometry.Cells were resuspended in buffer A  and lysed using LM20 Microfluidizer Processor (Microfluidics). The resulting cell lysate was clarified by ultracentrifugation at 40,0002+ complex were obtained by cocrystallization of NS3H (2.2 mg/ml) with 5 mM MnCl2 and 5 mM ADP (Sigma–Aldrich) in 0.1 M Bis–Tris propane, pH 6.5, 0.2 M sodium acetate trihydrate, 20% w/v PEG 3350, and 10% v/v ethylene glycol. Crystals for NS3H–AMPPNP–Mn2+ complex were obtained by cocrystallization of NS3H (2.2 mg/ml) with 5 mM MnCl2 and 5 mM AMPPNP (Sigma–Aldrich) in 0.1 M Bis–Tris propane, pH 6.5, 0.02 M sodium–potassium phosphate, pH 7.5, 20% w/v PEG 3350, and 10% v/v ethylene glycol. With the same condition, NS3H was cocrystallized with 5 mM ATP (Thermo Fisher Scientific) and 5 mM MnCl2. For data collection, every single crystal was fished out and flash cooled in liquid nitrogen without any additional cryoprotection. X-ray diffraction intensities were collected on BL14.1 at BESSY II electron-storage ring operated by the Helmholtz–Zentrum Berlin  at 18 °C. The protein in buffer C (2.2 mg/ml) was crystallized using Ligand-Friendly Screening HT-96 (Molecular Dimensions) for the initial screening, and protein crystals grew in 0.1 M Bis–Tris propane, pH 6.5, 0.2 M sodium acetate trihydrate, 20% w/v PEG 3350, and 10% v/v ethylene glycol. Crystals for NS3H–ADP–Mnlin (XDS  with theMOLREP (CCP4i2 suite (6ADW) as a starting model. The ADP–Mn2+ and AMPPNP–Mn2+ ternary complexes were solved using the apoNS3H structure as the starting model. Where applicable, translation/libration/screw (TLS) groups were initially identified using TLS motion determination   (The apoNS3H structure was solved by a molecular replacement with the program MOLREP  from thei2 suite  using ZImination  followedmination  and inteLicense) . The quaLicense) . RefinemLicense)  and UCSFLicense) .41  as follows:Vmax is the maximum velocity at given enzyme concentration, Km is the Michaelis–Menten constant, Et is the molar enzyme concentration , kcat is the turnover number, and S is the concentration of ATP substrate.ATPase activity of NS3H was monitored using EnzChek Phosphate Assay Kit , enabling the quantification of Pi release, which is translated as the rate of ATP hydrolysis by NS3H. The assay was done in a final volume of 200 μl in a 96-well plate according to the manufacturer's instructions. Briefly, 6.25 nM NS3H was mixed with individual assay components in a reaction buffer supplied by the kit and with either RNA (poly(A), 1.4 mM; Merck) or ssDNA41 1 mM;  and incu12 . The reaction mixtures (100 μl) in triplicates were incubated for 45 min at 30 °C prior to measurements in Thermo Fisher Scientific black 96-well immunoplates using Synergy H1 microplate reader. Anisotropy (r) values were calculated as follows:KD) between NS3H and RNA was determined by nonlinear curve fitting of fluorescence anisotropy data to  were added to neutralize the overall net charge. All simulations were performed for a minimum of 0.5 μs or longer until stable configuration was achieved (verified by stable RMSD for over 100 ns). ApoNS3H structure  was used, and residues missing from the density maps  were modeled using PyMOL (12 complex (PDB code: 2JLU)   with AMBng PyMOL . Short (e: 2JLU)  and prot6 or long ssDNA41  were also performed to explore the effects of DNA on ATP binding. Relative binding energies were computed for subset of the simulation frames using the generalized Born surface area method with inclusion of the Still’s model (Models of heterologous nonspecific surface binding were constructed using ssDNA ssDNA41  using in’s model  for comp’s model . Superpo’s model .7OJ4 (apoNS3H), 7BLV (NS3H–ADP–Mn2+), 7BM0 (NS3H–AMPPNP–Mn2+), and 7NXU (NS3H–ADP–Pi–Mn2+). All remaining data are present within this article.The atomic coordinates and experimental structure factors were deposited in the PDB under accession codes: This article contains The authors declare that they have no conflicts of interest with the contents of this article.