The novel coronavirusSARS-CoV-2 is the causative agent of theCOVID-19 outbreak that is affecting the entire planet. As the pandemicis still spreading worldwide, with multiple mutations of the virus,it is of interest and of help to employ computational methods foridentifying potential inhibitors of the enzymes responsible for viralreplication. Attractive antiviral nucleotide analogue RNA-dependentRNA polymerase (RdRp) chain terminator inhibitors are investigatedwith this purpose. This study, based on molecular dynamics (MD) simulations,addresses the important aspects of the incorporation of an endogenouslysynthesized nucleoside triphosphate, ddhCTP, in comparison with thenatural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTPspecies is the product of the viperin antiviral protein as part ofthe innate immune response. The absence of the ribose 3′-OHin ddhCTP could have important implications in its inhibitory mechanismof RdRp. We built an in silico model of the RNA strand embedded inRdRp using experimental methods, starting from the cryo-electron microscopystructure and exploiting the information obtained by spectrometryon the RNA sequence. We determined that the model was stable duringthe MD simulation time. The obtained results provide deeper insightsinto the incorporation of nucleoside triphosphates, whose molecularmechanism by the RdRp active site still remains elusive. In fact, despite the vaccination,the continuous specific mutations occurring hide it from the immunesystem so that the use of antiviral therapy is mandatory to preventfuture outbreaks of rapidly spreading coronaviruses.The world is currentlyin a state of pandemic emergency due tothe spread of a disease called COVID-19 caused by a new viral etiologicagent, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2).2 However,very recently, interesting studies have appeared in the literatureon RNA-dependent RNA polymerase (RdRp),4 which is theobject of the present investigation.In thefight against coronavirus, a great deal of computationalstudies on the molecular targets of SARS CoV-2 have been reported,and in particular, major efforts have been devoted to provide atomisticdetails of the spike (S) protein and proteases, which have receivedmuch attention from the computational chemistry community. An interestedreader can find a fulfilling overview of excellent computational investigationsin the recent review of Gao et al.5 and to remove mismatched nucleotidesduring genome replication and transcription. In fact, the incorporationof nucleoside analogue molecules into viral RNA can lead to increasedmutation rates and to the failure of the virus through “lethalmutagenesis”.6 The RdRp complex,one of the largest proteins in the viral genome (932 residues), hasmultiple non-structural protein (nsp) units and, in particular, isorganized in the nsp12 core catalytic unit, in the nsp7–nsp8heterodimer, and in the nsp8 subunit, with a stoichiometry ratio of1:1:2.7 These three non-structural proteinsalong with the nsp13 helicase are the “core” of thereplication transcription complex. The category of antiviral drugstargeting the RdRp includes remdesivir,9 a nucleotideanalogue adenosine mimic that showed efficacy against the Ebola infectionand has been successfully repurposed for the treatment of SARS-CoV-2infection such that it is the only drug approved against SARS-CoV-2by the US Food and Drug Administration, on August 10, 2020.10 Several studies have proposed remdesivir toinduce delayed chain termination15 or to cause the pausing and backtracking of polymerase,paving the way to the use of other nucleotide analogues.16In detail, SARS-CoV-2 isa positive RNA virus, which for its proliferationrequires RdRp, a key enzyme that regulates the replication and transcriptionof the viral genome. For this reason, RdRp has become the validatedtarget for the development of drugs against the COVID-19 disease,which can be hindered by the capacity of the human coronavirus toproofread18 and its easy synthesis of ddhCTP has recently been described.19 Its chain terminator effect for RNA-dependentpolymerases of multiple members of the Flavivirus genus has been furtherreported,20 and it has been recently demonstratedthat the SARS-CoV-2 polymerase can incorporate this cytosine analoguewell.16 Drawing inspiration from the evidenceon ddhCTP21 and the computational work on remdesivir,22 a comparative in silico molecular modeling study (ddhCTP vs CTP)has been carried out on RdRp of SARS-CoV-2, to gain more insightsinto the incorporation of nucleoside triphosphates , an analogue of CTP, is a novel antiviral nucleotide-likecompound produced by enzyme viperin as part of the innate immune response,ates see 1, which tionsee 2.16However, the absence of 3′-OH precludes further nucleotideincorporation from the RdRp enzyme, which is consequently stalledwhen the RNA chain moves through the polymerase and ddhCTP is processed.Another advantage of ddhCTP, with respect to other nucleoside analogueinhibitors, is that it does not require any particular metabolic transformations,differing from the case of other prodrugs (remdesivir and its derivatives).16The choice of the in silico investigationof the endogenous productddhCTP as an antiviral drug represents a good strategy to developother compounds in more reduced times than those needed to obtainnew therapeutic molecules from the scratch. Furthermore, ddhCTP representsan example of how the location of the modification in the ribose differentlyfrom that in other antiviral nucleotide analogues can affect the catalyticpathway used for incorporation.In the present study, classical molecular dynamics (MD) simulationsare used to investigate the early steps of the RdRp working mechanism and with a partial bound template primer RNA and RDV-TP. The crystallographicstructure concerns the subunits nsp7 and nsp8 and the catalytic nsp12bound to a template primer RNA duplex, in complex with favipiravirribonucleoside triphosphate (favipiravir–RTP).26 Due to the different pyrimidine-like nature of ddhCTP withrespect to that present in the above-mentioned crystallographic structure,26 to build the proper biologically significant RNA fragment, a carefulmodeling process was required, coupling the crystallographic structuraldata and nucleotide sequence information obtained by mass spectrometry.27The investigationstarted from a cryo-EM22 which is crucial for the nucleotide triphosphatebinding, differently to other computational investigations on theremdesivir–SARS-CoV-2 RdRp interaction.30Our study additionally includes the RNAtemplate primer similarly to the work by Arba et al. on remdesivir,4 In addition, it is not always trivial to findout experimentally the number of Mg ions present in the active siteas well as the identity and the number of their coordination ligands.31Among the available crystallographic structures,our choice fellon the one that presented a complete structural composition  and the two magnesium ionsessential for the RdRp activity, as very recently adopted.32The nsp12 structure contains a right-handRdRp domain  and a specific N-terminal extension domain (residuesfrom Asp60 to Ala250), which adopts a nidovirus RdRp-associated nucleotidyltransferase(NiRAN) architecture see 1.32The polymerase and NiRAN domains are connectedby an interfacedomain (residues Leu251 to Ser397). A further N-terminal β hairpin inserts into the cleavage blocked by theNiRAN domain and the palm subdomain in the RdRp domain.33 and consists of threesubdomains of a “cupped right hand”: a finger subdomain, a palm subdomain(residues Thr582 to Pro627 and Ala688 to Gln815), and a thumb subdomain(residues His816 to Thr929).Thepolymerase domain adopts the conserved structure of the viralpolymerase family34 Most of these motifs are shared with other polymerases, indicatingthe importance of these structural elements in their enzymatic function.35This right-hand architecture inturn evidences seven conservedstructural motifs: motifs A–E located in the palm subdomainand motifs F and G in the finger subdomain.36The active site, contained in the palmdomain, is formed by residuesSer759, Asp760, and Asp761 of motif C.2+), which play the major role in catalysis,33 while two other metal ions (Zn2+)play a structural role to stabilize the enzyme. One of the zinc ionsis coordinated to amino acid residues His295, Cys301, Cys306, andCys310 in the N-terminal domain, while the second is bonded to Cys487,His642, Cys645, and Cys646 residues in the finger domain.37 As mentioned above, the selected RdRp crystalstructure incorporates the favipiravir inhibitor, which is a purinenucleic acid analogue derived from pyrazine carboxamide (6-fluoro3-hydroxy-2-pyrazinecarboxamide).38The carboxylate groups of these aspartates anchor a pair ofdivalentmetal ions (Mg27 The spectrometry study showed that the requiredbase sequences for ddhCTP and CTP are UAAAAU (5′ ≥ 3′)and AUUUUAGU (3′ ≥ 5′), for the primer and template,respectively.The design of the RNA double strand lengthand the type of basesfor the cytosine analogue was then required. The selection of theRNA sequence has been based on matrix-assisted laser desorption ionizationtime-of-flight experiments on CTP-like RdRp inhibitors.39 has been usedto obtain the final model  relaxation of the complexes. 20Na+ counter ions were added to neutralize the system forboth RdRp:RNA:ddhCTP and RdRp:RNA:CTP cases.The parametersof CTP were obtained by adopting AMBER 94 and AMBER 99 force field++ server,42 with a salt concentration of0.15 M.37 The Amber force fields FF14SB43 and RNA.OL344 wereused for the protein and RNA, respectively. The ZAFF force field,45 specifically parameterized for zinc-containingsystems in its common coordination in proteins, was selected for twoZn2+ ions, each of them coordinated to three Cys unitsand one His unit, as specified above. These ions only play a structuralfunction. The Mg ions were treated with the Li et al. force field.46 It is well-known that classical force fieldparameters for Mg2+ ions are challenged in MD simulationswith RNA filaments. Our choice follows the guidelines on site-specificstructural features for Mg2+ ions in RdRp:RNA, as suggestedby Casalino et al.47 and in agreement withother computational polymerase studies.49 The systemwas solvated in a orthorhombic box (12 Å from the protein) ofTIP3P water molecules.50The RdRp RNA protonationstate was prepared at physiological pH by using the H2), using 5000 steps of the steepest descent algorithm, followed by5000 steps of the conjugate gradient algorithm. In the second minimizationstep, we released the restraint on hydrogen atoms, with the thirdand fourth minimizations being conducted with and without restraintsto the protein backbone atoms, respectively. We carried out a progressiveheating phase from 0 to 310 K for 200 ps using the Langevin thermostatin the NVT ensemble, with a time step of 0.002 ps.The production phase of 300 ns for RdRp:RNA and that of 300 ns forboth RdRp:RNA:ddhCTP and RdRp:RNA:CTP complexes were performed underan integration step of 2 fs using the coupling SHAKE algorithm withthe NPT ensemble at 1 bar pressure using the Berendsenbarostat.51 The particle mesh Ewald summationmethod52 was employed for the electrostaticpotential, and the long-range electrostatic interactions were calculatedwith the 12 Å cutoff distance. Trajectories for RdRp:RNA andits complexes with ddhCTP and CTP were saved every 0.2 ps and analyzedthrough the ptraj module.53 Overall, 0.9 μs of simulations was conducted (300 ns for eachcomplex).The solvatedstructures were first minimized by applying harmonicrestraints on all atoms of the enzyme ,the propensity for a given residue or region to move, the root meansquare fluctuation (RMSF), and the evolution of hydrogen bonding networks.The secondary structures were assigned using the DSSP algorithm.39 to provide a sampled and energeticallyaccessible conformational ensemble. After removing overall rotationsand translations by RMS fitting the Cα atoms’ positionsof the trajectory, the average linkage clustering algorithm was appliedas implemented in cpptraj to identify 10 representativeconformation clusters of the protein. The four most populated structureswere used as a starting point for the docking procedure. The describedprocedure has been successfully adopted in a number of investigationson different enzymes.57The RMSD-based clustering of the wholetrajectories was performedaccording to the relaxed complex scheme protocol, as implemented inAmber16,cpptraj module of Ambertools 16 to extractthe large-scalecollective motions occurring in MD simulations of RdRp:RNA, RdRp:RNA:CTP,and RdRp:RNA:ddhCTP.39Principal component analysis (PCA) was performedusing the 39 selecting the per-residue decomposition scheme. The value of the igb flag equal to 5 associated to a salt concentration of0.1 M was used. For the calculations, 200 frames from each MD trajectoryin the last 100 ns were analyzed.Binding freeenergies between the binary complex RdRp:RNA and ddhCTPor CTP were calculated by solving the linearized Poisson–Boltzmannequation using the MM Poisson–Boltzmann surface area (MM-PBSA)method, as implemented in the Amber16 code,33.1Figure S1). Such an observed behaviorcan be further evinced by the visual inspection of the protein structuresobtained from the hierarchical clustering procedures .As the initial part of the investigation, 300 nsof the MD simulationon the RdRp:RNA complex was performed. The RMSD plot calculated forall the backbone atoms of the amino acid residues shows that the systemreached the equilibrium after a few nanoseconds of the simulation,as confirmed by the calculated average value of 1.49 ± 0.21 Å. High peaks were observed atVal405, Lys430, Thr644, Val737, and Thr850, which corresponded toloop regions of the protein, and at Leu895 and Glu903 in the proximityof carboxyl ends. Altogether, the preliminary analysis of the setof results on the structural stability of the RdRp:RNA complex allowedus to consider the in silico designed model to be reasonably stable.In addition, the RMSF of eachindividual residue of the non-structural proteins and RNA was calculatedto characterize the local structural fluctuations in each subunit. Furtherhigh interaction occupancies occurred between P:U20, T:G10, T:U12,P:A17, and P:A18 and Asp760, Ser682, Arg569, Arg585, and Arg836, respectively,of the different subunits with an occupancy range of 77–100,as shown in Table S2 related to nativecontacts.The structural homogeneity of the nucleic acid is ensuredby hydrogenbond interactions occurring with the positively charged residues ofthe domains, such as Arg569, Lys849, and Arg858  for the MgA–OW and MgB–OW pairs. In particular, the first andsecond hydration spheres for the two metal ions were identified, withhigher peaks observed in the case of MgB . The most frequent presence of watersin proximity of MgB, with respect to MgA, canbe ascribable to the P:U20 nucleotide located in proximity of thelatter, which occupies the room for solvent molecules. The presenceof H2O molecules has been further confirmed by the visualinspection of structures collected from the hierarchical clusteringanalysis, supplying an average number of four and five molecules inproximity of MgA and MgB, respectively (Table S3).In the selected crystal structure, the active siteMg3.2Table S4. The most energeticallyfavored conformers, −11.4 and −11.3 kcal/mol for ofRdRp:RNA:CTP and RdRp:RNA:ddhCTP, respectively, confirm that the introductionof ddhCTP can be considered feasible and, thus, can be allowed.The average binding affinities  obtainedfor the best docking poses of ddhCTP and CTP into the SARS-CoV-2 RdRpare reported in After the docking of the ligands, 300 ns of MD simulations were furtherperformed on both RdRp:RNA:CTP and RdRp:RNA:ddhCTP ternary complexes.The RMSD plot reveals that the system reached the equilibrium, withaverage values slightly higher than that of the respective RdRp:RNAsystem ,finger (Ser425), and thumb (Met902) subdomains.In the MD simulationsof RdRp:RNA:ddhCTP, the major fluctuationswere further observed in the interface subdomain, as evidenced bythe peaks obtained for Gly337 and Arg365 residues exposed to the solvent,in addition to fluctuations for the NiRAN (Asp62 and Asp107) and finger subdomains see 4B.In analogy to the observation obtainedfor the RdRp:RNA complex,PCA revealed that for both ternary complexes, nsp12 and nsp8 are theregions of the system involved in more detectable movements duringthe MD simulations and that the RNA molecule conformation is not affectedby any important structural rearrangement see 3.Figure S6.The orientation of the substrates is mainly ensured by the presenceof cation–phosphate interactions, occurring with differentcombinations between the phosphate arms of CTP and ddhCTP with MgA and MgB. The distances of each Mg ions with Pα, Pβ, and Pγ wereanalyzed to characterize the binding mode of the phosphate moietiesof ligands during the simulation, and the relative distributions aredisplayed in Figure S7. In the case ofCTP, the plot analysis revealed that oxygens of the Pα group bridge both cations, while those of Pγ coordinatemainly the MgB . A different behavior of the Pα group can be observed in ddhCTP as far as the MgA isconcerned. In fact, it is shifted at distances around 5 Å.In the binding site, CTP and ddhCTPligands are stably locatedduring the entire simulation, as can be observed by the analysis oftheir RMSD plots reported in B, the distributions of Pα andPγ results are very similar for both ligands, whilePβ in ddhCTP is longer than that in CTP. In additionto the phosphate groups of CTP and ddhCTP, MgA and MgB complete their hexa-coordination  with the side chain of two consecutive Asp residues inmotif C, Asp 760 in a μ fashion, as confirmed by a measureddistance of ca 2 Å for metal–OAsp760 distances.Finally, a lower number of water molecules coordinating MgA and MgB lie atdistances of 4.48 ± 0.12 and 4.96 ± 0.15 Å for CTPand ddhCTP, respectively . Theobserved value is consistent with that required by the reaction mechanismof two metal ions (<4 Å). It was suggested that during thereaction, a shrinking of this distance may occur, leading O3′to become closer to Pα and consequently facilitating the nucleophilicattack. The little longer distance in ddhCTP than that in CTP couldaffect the O3′–Pα alignment for catalysisduring the nucleophilic attack, in the reactive Michaelis–Mentencomplex.65During the simulations,MgCTP/ddhCTP–C1′G:T, N1CTP/ddhCTP–N1G:T, and N3CTP/ddhCTP–C1′G:T. Outcomes on the analysis of thesedistances are linked to the fidelity of DNA polymerases66 and can be extended to RNA polymerase due to the many similaritiesbetween the two classes of enzymes.69In order to analyze the interaction involving the cytosinenucleobase,which is the same for both ligands, attention was focused on threemain geometrical parameters involving the pairing with guanosine nucleobase,such as C1′CTP–C1′T:G, N1CTP–N1T:G, and N3CTP–N1T:G fit results described in a previous work.48 More interestingly, the inclusion of ddhCTP led to an increaseof ca 2 Å for all the considered distances , thus suggesting that in the presence of the endogenousinhibitor, the fidelity of the enzyme is further affected and thaterrors in the course of the duplication can take place already inthis step, thus concurring to the stalling of the enzyme.In the case ofthe CTP ligand, the analysis of the distributiondistances revealed that the nucleobases lie in an unpaired-like conformation,which can be linked to the low fidelity behavior of the RNA polymerasesaccordingly to the mechanism. Indeed, the calculated average valuesof 11.73 ± 0.47, 6.72 ± 0.59, and 5.98 ± 0.10 Åfor C1′Figure S11).Dueto the modification present at the 3′ position of theribose ring of ddhCTP, the analysis of the RDF and visual inspectionof clustered structures were conducted on the interactions involvingthe ribose group, focusing on the hydroxyl groups at C2′ andC3′ positions of CTP in comparison with that of ddhCTP. Inthe case of the CTP molecule, the O3′ atom established hydrogenbonds with the phosphate group of U20 and water molecules, while O2′was mainly involved in interactions with solvent molecules. In theabsence of O3′–H group in the ddhCTP-containing system,the interaction with U20 phosphate was observed to occur with theO2′ atoms (Tables S5−S7), suggesting asimilar interaction network in the binding site. However, the analysisdid not provide any relevant information about the interactions involvingthe ribose hydroxyl groups and the amino acid residues. The calculationof ΔGbind, carried out accordingto the MM-PBSA scheme revealed some differences for the ligands. Indetail, the binding free energy of ddhCTP was determined to be 15kcal/mol higher than that of CTP (Table S8).Fromthe contact analysis, effected for both CTP and ddhCTP, itcan be evinced that residues 618−621 and 551 (motif F) are implicated in interactions with the ligands with the active site environment.Energy decomposition analysis of every residue involvedin non-covalentinteraction with CTP and ddhCTP showed that, for the former, a strongerstabilization of the tertiary complex is observed due to the electrostaticinteraction of Arg553, Arg555, Lys621, and Asp761 side chains (seethe Gbind (lower affinity) canbe linked to the absence of the hydroxyl groupat the C3′ position, which causes a loss of three hydrogenbond interactions from ddhCTP species. In the course of the dynamics,both CTP and ddhCTP, finally, assume a reacting like conformation.This can be evinced, in particular, by analyzing the MgA–O3′P:U20 and the O3′P:U20–PαNTP distances, which are the reaction coordinates involved in the reactioncatalyzed by the enzyme.In addition, thehigher Δ16A very similar distribution of valueswas observed, see 4Nucleotideanalogues represent a powerful tool for fighting theviral infections and their consequences. In the present work, theinteraction of the RdRp protein, involved in the spreading of theSARS-CoV-2 disease, and the endogenously synthesized ddhCTP, the productof the viperin antiviral protein, has been investigated. Such a moleculediffers from CTP, its natural precursor, by the presence of a doublebond and the absence of a 3′-OH group in the sugar ring. However,it is reputed that the missing hydroxyl group can favor the stallingof the enzyme and, consequently, the replication of viral RNA. Thestudy has been conducted in the mean of all-atom MD simulations, onthe RdRp:RNA system and on each RdRp:RNA:NTP complex, with NTP equalto CTP or ddhCTP, and has focused on the recognition step of the RdRpcatalytic mechanism.A and MgB present a hexa-coordinationsphere, composed of Asp618, Asp760, Asp761, and water molecules.From the analysis of the binary complex,it is determined thatthe RNA is kept in the active site by interaction with a number ofresidues, such as Ser682 and Arg585, and that in the absence of othersubstrates, Mgα and Pγ of CTP and ddhCTP are directly involved in the coordination of MgA and MgB, while Pβ mainly interactedwith the positively charged Lys551 and Arg553, facilitating the bindingof the ligands in the active site. Importantly, Lys789 has been identifiedas a crucial residue bridging Asp618 and the Pγ groupof both ddhCTP and CTP.The comparative analysis of the conformational behavior of ddhCTPand CTP, overall, indicated high similarity between the two molecules.In detail, phosphate groups PThe hydroxyls groups of CTP at C2′and C3′ positionsestablished hydrogen bond interaction with solvent molecules and U20of RNA, respectively, while in the case of ddhCTP, the interactionwith nucleic acid is ensured by the 2′-OH group.CTP–C1′T:G, N1CTP–N1T:G, and N3CTP–N1T:G distances.The analyzed results are in agreement with the low fidelity of RdRp,which was slightly more affected by the presence of the ddhCTP molecule.Finally, both CTP and ddhCTP showed pro-reactive conformation, witha good alignment of MgA–O3′P:U20 and theO3′P:U20–PαNTP distances observed forboth the ligands.Theinteractions of the cytidine group of both nucleosides withguanine of the RNA chain were evaluated in terms of C1′The results described above shed light onthe relevant aspectsof the incorporation of CTP and ddhCTP species in the RdRp, layingthe foundation for the rational design of the SARS-CoV2 vaccine andantiviral targets and providing further insights that were not accessiblethrough experiments. Altogether, the available results from experimentsand the rationalization from computational investigation can thusbe very helpful for stimulating and driving future experimental investigations.AutoDock version 4.2 was adoptedto perform molecular docking calculations.All MD simulations were carried out using the AMBER16 package. TheAmberTools package was further adopted in the preparation of the parametersand input for MD simulations. Density functional theory (DFT) calculations,to parametrize ddhCTP, were carried out using Gaussian 09 ver. D01.Trajectory analysis was performed using the AMBER cpptraj module.The preparation of the structures and the images was carried out usingVMD software, version 1.9.3, and Chimera software. The full workflowis reported in the “


Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity. Accurate, end-to-end reverse transcription is required for faithful copying of RNA into cDNA molecules. Despite the critical importance of this reaction in biotechnology and medicine, the speed and behavior of reverse transcriptase (RT) enzymes on mixed sequence RNA templates have not been extensively characterized, particularly for new families of RT enzyme with altered properties, such as enhanced processivity. Conventional RT enzymes (such as the SuperScript series) are primarily derived from retroviral RTs, and these tend to pause or stop upon encountering certain types of RNA sequences, such as repeats , and wheIn addition to the challenges posed by the diversity of RNA templates, the actual speed along an RNA template at which RT enzymes move and copy RNA are not well known. However, in the few instances in which the RT speed has been characterized, it can be context-dependent. For example, the R2 RT has a maximal speed of 29 ± 9 nt/s on poly(rA) templates, but the speed reduces to 7 nt/s on templates with heterogeneous sequences . In thatMarathonRT (MRT) is a structurally characterized RT that is encoded within a eubacterial group II intron , where iIn this study, we report the length-dependent velocity and homogeneity of MRT and other RT enzymes in the presence of diverse heterogeneous templates, some of which have been engineered to contain stable structural elements that explicitly test the ‘power’ of the enzyme to pass through stable barriers. We show that MRT carries out synchronized, single-pass primer extension of highly structured templates at a constant speed of 25 nt/s, regardless of position along the RNA template, and it can pass through a diversity of stable RNA substructures without pausing or even slowing down. We find that a related group II intron RT can also traverse stable barriers within the templates, but it has a different intrinsic velocity. This robust behavior of group II intron RTs contrasts with that of commercially optimized retroviral RNA enzymes, which have low processivity, and are blocked upon encounter with stable RNA template structures.Eubacterium rectale  to make a reaction volume of 19 μl. After pre-incubation of the reaction mixture at 42°C for 1 min to allow the RT enzymes to bind to the primer-template, 1μl dNTP mix  was added to initiate the reaction . The reaction buffer for MRT contains 50 mM Tris–HCl pH 8.3, 200 mM KCl, 2 mM MgCl2, 5 mM DTT and 20% glycerol, as previously optimized for MRT , and the FAM-labeled gel was directly scanned by the Typhoon FLA 9500 scanner under fluorescence channel. Resulting data were analyzed and quantified using QuantityOne software (Bio-Rad).The basic RNA template used for this assay is the HOTAIR lincRNA 2148 nt), which was prepared by 8 nt, whicription  was labe for MRT . For SSIHOTAIR RNA and 5′-FAM labeled RT primer  were use2, 5 mM DTT and 20% glycerol) at room temperature for 10 min and then 42°C for 1 min. Subsequently the dNTPs, in combination with the respective trap molecule, were added to initiate the single-cycle reaction. The final concentration for the dNTPs was 0.5 mM, and the final concentration for each of the traps was described in the legend  and aligned to the reference sequence using ngmlr 0.2.7 with the parameter for Oxford Nanopore reads, -x ont , following manufacturer's protocol. Subsequently, double stranded DNA products were prepared for Nanopore sequencing using Oxford Nanopore Ligation Sequencing Kit and Native Barcoding according to manufacturer's protocol. A unique barcode was used for each sample. Final libraries were pooled and sequenced on a FLO-MIN106, R9.4.1 flow cell in a MinION device, using MinKNOW software v. 19.12.5 for device control and data collection. Output was base-called using Guppy  . The res, -x ont . ThereafBriefly, reverse transcription was performed with MRT, TGIRT and SSIV at 42°C, 60°C and 55°C for 5, 10 and 5 min, respectively as described. Four RT primers were used to cover the entire length of the GCSL HOTAIR template . cDNA prhttps://github.com/wyppeter/RTEventsCounter) (stop). The intensity corresponding to the GCSL stop site and that of longer extension products were calculated IGCSL,  to remove Illumina adaptor sequences and aligned to the GCSL template with HISAT2 (v2.1.0). The stop information was extracted with software RTEventsCounter.py (Counter) . To reduWhen RT efficiency is evaluated by users or developers for most applications, the enzyme is incubated with primer-template for a fixed period of time and the relative amount of primer extension is quantified, without considering the diverse physical processes that contribute to efficient primer extension on a given template. This becomes problematic when working on exceptionally long or complex templates, as it is important to know the actual speed and constraints on the polymerase so that one can gauge appropriate reaction times. In addition, most templates contain at least some short regions of stable secondary structure or repeat sequences and it is important to know how and whether these are traversed by the enzyme. Finally, an important metric of efficiency are the changes in speed or processivity that a polymerase displays during the initial, middle, and final stages of a template-copying reaction, as this will affect the number of times each base being copied as a function of position. To evaluate all of these attributes, we developed a method for monitoring the behavior of RT enzymes in real time as they travel along a test template.32P- or fluorophore-labeled primer that had been annealed to a long RNA template containing diverse sequences and secondary structures. After incubation of RT enzyme with primer-template, the primer extension reaction was initiated with a rapid pulse of dNTPs. The reaction was stopped at a series of short time intervals .Remarkably, time-courses of MRT primer extension revealed a number of unexpected behaviors. First, the population of advancing enzymes was observed to be highly synchronized Figure , migratir2 > 0.98)  Figure .The constant speed of MRT in this context is particularly significant given that HOTAIR is structurally complex, containing local regions of stable secondary structure and repetitive sequences . Most poThis behavior is in sharp contrast with the apparent behavior of retroviral RTs, which are obstructed by certain sequences and secondary structures during primer extension, displaying pauses, stops or bursts of dissociation at different types of template heterogeneities ,20. To dWhile electrophoresis is a rapid method to determine cDNA distributions and velocity, the calculated sizes are limited to resolution on the gels. Therefore, we wanted to use an orthogonal approach to estimate distribution of cDNA products at the reaction time points. Due to the large size of the RNA, we utilized Oxford Nanopore sequencing, a method that allowed us to use a single RT primer for sequencing the various length cDNAs at all time points. While we cannot determine single nucleotide resolution with precision, we were able to sequence individual cDNAs produced at each time point which allowed us to obtain distribution plots for the products Figure . Upon inTo determine elongation velocity from the global nanopore data, we calculated the distribution peak maximums at each time point and subsequently plotted these values against reaction time. Notably the calculated velocity by sequencing, 26.6 nt/s Figure , matchedDuring the velocity experiments described above, an excess of MRT enzyme was used to carry out the reactions, which is classically defined as a ‘multiple-cycle reaction condition’. This condition is typical for routine primer extension reactions using reverse transcriptases ,13. The To determine if the velocity we calculated is for a single MRT molecule copying in a single pass, we evaluated the velocity of MRT under single-cycle conditions, in which an excess of trap was added upon initiation of primer extension as described previously  . Before G = –33.32 kcal/mol), the mouse mammary tumor virus (MMTV) gag-pro shift site pseudoknot  as the template. The homogeneity of velocity along the length of this RNA suggested that the group II intron-encoded RTs can readily unwind RNA structures formed within the HOTAIR template. But none of the HOTAIR substructures represent unusually difficult, thermodynamically stable obstacles and so we wanted to provide a more challenging test. To systematically evaluate the ability of RT enzymes to open and copy a diversity of stable RNA substructures, we selected a set of stable RNA secondary and tertiary substructures as tests for the unwinding power of the enzymes. These RNA substructures were then imbedded into HOTAIR RNA to create challenge templates for reverse-transcription Figure . These Reudoknot , EscheriI intron  . While both enzymes are derived from group II encoded RTs, differences in the absolute value of their speed may reflect different inherent properties of each enzyme, as they are not identical. They share only 43% sequence identity and 62% sequence similarity. TGIRT is derived from a thermophilic organism, and may therefore have evolved enhanced binding affinity to RNA template-primer to improve thermostability . This maPfu and Deep Vent DNA polymerases, whose extension speeds were determined to be 25 and 24 nt/s on M13 ssDNA, respectively, at their optimal extension temperatures of 75°C. Remarkably, MRT is quite comparable in speed to these polymerases and our results suggest that a non-obstructed extension speed of ∼25 nt/sec is some type of ‘sweet spot’ in the efficiency of single-chain polymerases. The universality governing their rate-limiting mechanism merits further exploration.Unlike other RT enzymes, which can only be examined under multiple-cycle conditions , it is pThe observed kinetic behavior of MRT has important mechanistic implications. The constant, unperturbed speed of MRT along mixed sequence templates implies that the enzyme behaves much like a translocative helicase, which strips away obstacles in its path (whether its protein or base-paired RNA/DNA). The constant velocity of the enzyme, regardless of position along the template, also implies that the protein itself does not undergo rate-limiting conformational sampling as it travels or encounters features in the template, and that the active structure is highly stable, which is not surprising given the bioinformatic stability search that was used to discover the enzyme . Such prThe specific velocity of an RT enzyme has important practical implications for its use during primer extension reactions and its application as a biotechnological tool. Our findings demonstrate that, even for the most processive RTs available, one must be cognizant of the inherent enzyme velocity when planning primer extension experiments, particularly when performing reactions on templates that are kilobases in length. This is important because viral RNA genomes, long noncoding RNAs and full-length mRNAs are the focus of intense current research, and many of these transcripts are often very long, exceeding kilobases in length. Given the velocities we have measured, the reaction time for a 10kb template would be at least 7 min for end-to-end coverage by MRT at 42°C and 40 min for TGIRT at 60°C. Furthermore, while most reverse transcription protocols suggest an incubation of 1 hour at the optimal reaction temperature our data suggests one can significantly reduce this time for MRT, which streamlines long, multistep protocols. Additionally, MRT functions under mild reaction conditions, with an optimal reaction temperature of 42ºC for full activity whereas for TGIRT, the optimal reaction temperature is 60ºC. A recent study has shown that RNA is relatively stable at 45ºC (depending on magnesium ion concentration and pH), but the rate of degradation increases by nearly 10-fold at 60ºC . Using MNaturally-occurring RNAs contain a multitude of mixed nucleotide sequences, including repeat sequences and regions of stable RNA structure. While previous studies suggest that template secondary structures can induce premature RT stops, a systematic characterization of RT response to different types of RNA structural states and obstacles has never been done until now. To address this issue, we designed a series of stable ‘challenge structures’ (ranging from stable RNA stems to entire tertiary structures) and then created a set of ‘test templates’, in which we imbedded each ‘challenge structure’ at the same position in the center of a long RNA template, placing them well in the path of the elongating polymerase Figure . SpecifiWe observe that engineered template structures have minimal impact on the group II intron encoded RTs (MRT and TGIRT), but they block even the most highly optimized retroviral RT (SSIV). Surprisingly, the obstacle that caused the biggest disruption to the RTs in this study was not the imbedded tRNA or an intact group II intron, but a relatively small stem-loop that is rich in G-C pairs (GCSL). To obtain a better understanding of where the RT enzymes stop relative to the position of the stem loop, we utilized a high-throughput, single nucleotide resolution approach to determine exact stop sites induced by the GSCL for all three RT enzymes . The majBombyx mori R2 element have only demonstrated this type of processive, synchronized primer extension on homopolymeric poly(rA) templates and, even then, observed speeds were not uniform, ranging from 10–15 nt/s and 9.5–19.5 nt/s, respectively (One of the most striking features of the data obtained in this study is the remarkably synchronized pattern of primer extension by group II intron RTs, despite the heterogenous sequence content of the template. Previous studies using retroviral HIV RT and R2 RT from ectively ,29. In fOur engineered ‘obstacle course,’ combined with our ability to visualize the speed and synchrony of an RT population at all positions, resulted in a methodology unique amongst studies of polymerases, and as such, it provided fresh insights into response of polymerases before, during and after encounters with template secondary and tertiary structure. For example, when monitoring the extending cDNA population that makes it past the GCSL structure, we observe the same synchronized pattern of extension and constant speed as seen in the original template, which has never been observed for any enzyme. Single molecule studies suggest that retroviral enzymes slow down upon encountering structures , but theThis study has important practical ramifications for choosing an RT enzyme that is best-suited for certain types of transcriptome-wide analysis. Conventional retroviral RT enzymes severely limit the number and quality of reads that can be obtained on many types of natural RNA templates, therefore biasing the resultant transcriptomic datasets. Application of template-insensitive, processive group II intron RTs will enable researchers to capture the full spectrum of RNA transcript abundance, providing a wealth of information on novel gene targets and, potentially, leading to the discovery of new cellular RNA molecules. In addition to practical insights, this study underscores the microscopic diversity of RT behavior, demonstrating that each type of RT enzyme displays a unique pattern of behavior that arises from its specific role in biology and which lends itself to different types of technological applications. Despite the simplicity of their single-chain structures, we show that RTs like MRT are fast, processive, powerful enzymes that are largely insensitive to environmental conditions. The creative exploitation of these properties will lead to new insights into biological mechanism and new tools for exploring the expanding universe of functional RNA molecules.The sequencing data have been deposited to GEO under accession number GSE195979.gkac518_Supplemental_FileClick here for additional data file.


Healthy and protected coral reefs help island systems in the tropics thrive and survive. Reef passages link the open ocean to lagoon and coastal areas in these ecosystems and are home to an exceptionally diverse and abundant marine life, hosting emblematic species and fish spawning aggregations. Their multiple benefits for the islands and their peoples  remain yet understudied. Drawing from qualitative interviews with fishers, scuba divers, and surfers along the coast of Grande Terre in New Caledonia, this study highlights the multi-faceted importance of these keystone places. It shows that reef passages are locally deemed ‘communication zones’ between coastal and oceanic spaces and species, and have significant un(der)explored ecological and socio-cultural roles. Understanding and protecting these ecological and cultural keystone places will strengthen both the reef ecosystems and the people dependent on them.The online version contains supplementary material available at 10.1007/s13280-022-01762-8. The United Nations’ Sustainable Development Goal (SDG) 14 – Life below water – aims to support and encourage action to conserve and sustainably use the oceans. This SDG is entangled with the global target to expand conservation areas to 30% of the Earth’s surface by 2030. With this 30% target focusing on “areas particularly important for biodiversity” CBD , coral rCarangoides chrysophrys) use reef passages to move between feeding areas, with their predictable movements often being related to both tidal flows and lunar cycles  and are regularly generating new knowledge on these biologically rich and complex places. Indeed, this knowledge suggests that multispecies FSA sites should be treated as “ocean bright spots” (Pittman and Heyman In addition to fish and marine species (such as endangered sea turtles) at different life stages, various other living and non-living entities may transit through reef passages: nutrients, sediments, plastics, or chemicals from hinterlands  or conservation-minded . The population of New Caledonia is multi-ethnic. About 40% of inhabitants are Kanak, about 25% Europeans, and the rest have various origins, including Oceanian , Indonesian, and Vietnamese . Locations for interviews were chosen next to passages that were best known to the interviewees. From 93 reef passages .Thirty-two semi-structured qualitative interviews were conducted in 2021 on the North and West coast of New Caledonia’s Grande Terre—at Poum, Koumac, Ouegoa, Voh, Koné, Pouembout, Nouméa, Dumbéa, Païta and Bourail  was adjusted over the course of the study. This interview guide aimed to collect data on: (1) what reef passages  represent for different stakeholder groups, and what are their social-ecological roles according to the latter; (2) which reef passages are the most frequented within the survey area and what activities are carried out there; and (3) what biophysical conditions and species are observed by the interviewees at these passages.Interviewees were selected by opportunity- and snow-ball sampling, with interviews being carried out face-to-face (at the most convenient meeting points for the respondents) or, due to the Covid-19 pandemic, via Messenger call. The interviewees were fishers , scuba divers (n = 4), and surfers (n = 2), with several respondents practising several of these activities.Informed consent was obtained prior to all interviews, and we followed the ethical guidelines suggested by the International Society of Ethnobiology ISE . All intReef passages in New Caledonia are well known to many local residents, whatever community they come from. The respondents mainly talked about the passages closest to their homes, and therefore the ones they have access to most often, but it also happened that they mentioned a strong experience with another passage from times when they were younger. For many, reef passages are the remarkable breaks in the barrier reef that delimit the New Caledonian lagoon, and for some, they also include the openings in the fringing reefs, which were deemed by the interviewees to have the same ecological role but on a smaller scale. These natural passages in the barrier and fringing reefs are differentiated from channels on the reef flats created by humans to facilitate their crossing .“‘fausses-passes’, i.e., ‘false passages’. These, much shallower and therefore not navigable at all tides, can often be seen, for example, as coral ‘collapses’ at the level of the barrier reef, or where so-called spurs and grooves .People who frequently used the different types of passages also had detailed knowledge of their morphology , for instance:“If you look closely, there are passes that are very, very narrow, like the Koné pass, it's a pass that's really bad, it's twisted, the big boats will never be able to enter if they don't break or … it's really annoying whereas Voh's pass is quite straight.” .Conditions are often more dangerous in these narrow and winded passages compared to the coastal or seaward sides of the fringing and barrier reefs, and require excellent knowledge of (particularly strong) currents, adapted vessels and good weather:“La Gazelle (reef passage) is a beautiful piece of work! It's the only pass I've crossed, and I was really scared. Because it all depends on how the current arrives […] and it's very impressive when the swell is strong.” .Reef passages were attributed ecological roles by the respondents, as gathering and transit zones from the ocean to the lagoon (and vice versa) for people, animals, water, and the objects and particles the latter carries.Reef passages are often reported to be located in front of large rivers, in the prolongation of the river mouths, and most respondents also made a direct link with rivers as the origin of these formations, like an extension of the rivers at sea.“The passes are almost in front of the rivers; it is the fresh water that must prevent the coral from growing.” .As such, reef passages were described as key places for exchange between terrestrial and oceanic waters.All respondents said that the water carried by the tides through reef passages provides a permanent source of nutrients, hence making these passages attractive places of feeding and reproduction for marine animals, which was often seen as beneficial also for the health and species richness of the nearby adjacent reefs.“For me a pass is a break in the coral reef that allows some big fish or rather mammals, maybe whales, dolphins, etc., … to be able to enter the lagoon for what they have to do, and yeah I think it allows the exchange of nutrients between the real ocean and then the lagoon…” .Acanthocybium solandri and Scomberomorus commerson) in Népoui’s passage or speckled blue groupers (Epinephelus cyanopodus) in Dumbéa’s passage. The ‘false’ passage of Île Verte was also mentioned as the only known FSA site for napoleon wrasse (Cheilinus undulatus).Fishers also regularly pointed out the importance of passages for the spawning aggregations of several fish species, such as king mackerels .“After the big rains, it’s very dirty, especially around the FADs [Fish Aggregating Devices] which are generally in front of the passes, you find everything from the woods … It’s dirty.” .For all interviewees, reef passages were important places for a number of reasons, but first and foremost because they mark the links between Kanak clans, between the visible and invisible worlds, between the living and the dead. Reef passages are pathways to the afterworld for the spirits of the dead, both embodied and accompanied by different animals throughout their journey. In the words of one of our interviewees, these passages are therefore sacred and must not be disturbed but highly respected.For us, for example, in the Aijé-Arö area, the pass in Bourail is the passage of the dead for the entire area. Before, we used to say that when a dead man passed through, you could hear a noise when he took the channel, the passage to get to the bottom of the pass. […] They [the deceased] change, when you're in the river, we'll say you're an eel; […] the eel leaves, it changes, it becomes small, it goes to spawn and then it goes into the channel; by changing, it becomes another totem animal. That's why it's full of fish, why there are turtles; the turtle, the shark are totem animals that are linked to the sea clans. […] It's said that the shark also accompanies the dead. Basically, it's as if you go out and you're a remora  and then there's the shark that will accompany you and then you join another world. After all it's a cycle and you'll always come back anyway.” (Resident of Houaïlou).“Legends were mentioned by some interviewees . A ‘gesture’ towards reef passages, i.e., an offering  that one makes to the sea, sometimes accompanied by a few words, was said to placate the spirits therein, and to preserve those who go there.“The passes are sacred; it is the passage to the world of the dead, the spirits of the dead are there, we avoid disturbing them, we have a fear when we fish there, we don't go alone, always a small gesture to ask the old people for forgiveness and to look after us. .This gesture was also a way of remaining humble towards the sea, yet not anymore observed by all interviewees.“I don't make a gesture but my grandparents did at the time, it's not everywhere, it's just at the Amos pass, we have a false pass on the side, we call it taboo, so as it's taboo, you have to make a gesture when you leave and when you arrive, but I don't do it, I used to do it with my grandparents when I was a child.” .For important customary/community events (such as weddings in Poum), residents were allowed to access the passage to fish for specific species such as napoleon wrasse, to be consumed during the feast, even if this passage was considered sacred or ‘taboo’ and fishing was forbidden there for the rest of the year. Hence, in a taboo zone the activities are not entirely and always forbidden, but they have to be carried out with respect and sometimes only after getting authorisation by the clan elders.Finally, people talked of reef passages as ‘openings to the world’. They have indeed, for a long time, allowed trade and ensured the transport of people (and thus of news) as well as of products and raw materials from the outside to the ‘inside’ of the island, and vice versa. Many shipwrecks have occurred near these places, and some passages are therefore named after famous ships or navigators of the time.The interviews confirmed that reef passages were used mainly by: a) fishers , b) scuba divers (by boat) and, to a lesser extent, c) surfers.3The interviewees reckoned that reef passages are suitable for different types of fishing . However, deemed dangerous and sometimes very remote from the shore, reef passages were not necessarily seen as the best place for all these fishing techniques. Besides for fishing within these passages, fishers also used them for transit to get to the FADs in the open sea. Several fishers mentioned making a gesture (see above) at the passage to ensure a smooth navigation and a good catch.Spearfishing with underwater guns is a very popular form of selective fishing in New Caledonia. In general, this activity is practised by a minimum of three people, two in the water and one on the boat to ensure the safety of divers. Spearfishers like to fish at passages because there are always fish readily available there. However, fishing in a passage requires good physical ability and favourable weather conditions. Interviewed spearfishers had an excellent knowledge of their fishing grounds, knowing the distribution of consumable species and the (geo)morphology of the area.“…The right side of Uitoé [passage] when you look out to sea is big steps, things where the groupers can rest quietly, they have space, they have each other you see, so it's a grouper spot. After that, you have spots on the left of Uitoé with more coral, as there are coral descents with a little bit of caves, you can find big groupers hiding, like big mother groupers, all the species of groupers … the big blue groupers in the passes, the big parrotfish on the left; you will find some on the right but less … as it will be more of a drop off, and straight into the pass, if you are lucky, you will come across some jacks (Carangidae) that sometimes come out to see what you are doing, a kingfish if you are lucky…” .Trolling is a fishing practice that consists of towing a line with a bait, lure or live fish behind a moving boat at a slow or medium speed. In New Caledonia, fishers who practice this type of fishing usually have a boat with an engine of long-distance capacity, adapted to big game fishing.“At the central pass (Poindimié), I look for kingfish, rainbow tuna, and coral grouper. And I often catch bonito jacks, mackerel, and barracudas. I go to the pass to target these big fish that are not found in the lagoon. […] It's a channel between the coral reef, with sandy bottoms from 20 to 50 m depth approximately, with some shallow waters with pebble potatoes [little coral heads] down to 3–4 m, and on the exit of the channel, it drops quite quickly to a hundred metres deep. With a map of the seabed, you can see these shallows that often have higher fish concentrations, you pass close to these shallows for trolling, you jig4 on the edge of these shallows.” .Some anglers  go specifically to reef passages to target certain species . Some fishers in Bourail mentioned that they would not usually go fishing in the ‘false’ passage of Poé but go there sometimes to get shelter from wind and waves.“There are much easier areas [to catch fish]. But the good thing about this area is that it's protected from the swell and the wind, so it's quite useful when the conditions are not great for several days in a row." .Scuba diving is a widely practised activity—by both men and women—in New Caledonia, where there are about 20 diving centres visited by New Caledonians as well as tourists. Divers often look for places where they can find the highest biodiversity and therefore many different animals to observe, without having to dive very deep or travel very far. Reef passages are therefore often favourite dive spots, especially when they attract iconic species , and host FSAs, for instance of groupers:“At the big Kélé bend we have a school of between 20 and 30 female grey sharks for the most part. […] We will soon arrive at the end of the year, so the grouper breeding season is mainly in December, and we will have a gathering of hundreds of groupers, so it will be really beautiful, we will have a lot of jacks, barracudas …” .In New Caledonia, surfing is not as widely practised as fishing or scuba diving, which are very popular regardless of location, community affiliation, and age. According to the Caledonian surfing league, which had 400 members in 2016, there were half as many women as men. Seven surfing clubs“The swell when it comes in, it rushes into this channel  (Bourail passage) too, so the swell from the open sea is formed into this channel. When the wave arrives, it meets the reef on the right, it breaks and as it breaks, it rolls off the reef; as it rolls off, it gives a break zone which allows you to surf, otherwise it would just be a wave that crashes. The waves depend on the swell, the swell has to be from the west, and from the size of the swell obviously, up to 0.1 m to 1 m; […] the more it is west, the more it comes in and it comes in at the beach too.” .One spot at Gouaro was mentioned as the least dangerous, narrowest spot, which makes it better for beginners, and which is always surfable, depending on the tides. Another well-known spot is in proximity to the Ouano passage, 10 km away from the shore of la Foa where competitions are usually held.“The Ouano pass is super beautiful, it can go very far, you can have 300–400 m of zone from the take off6to the end of the wave, it is world famous even, there are foreigners who come to surf here, it is a super quality wave!” .Although the surf spots are quite explicit, they are sometimes frequented by spearfishers, and the proximity of people ‘hunting’ underwater can disturb some surfers, particularly regarding the risk of shark attacks."In Dumbéa, for example, it's well known that you have the false pass 1 and the false pass 2 […] To spot it, you see a lot of surfers who are often on it, but then there are also fishermen. So, there too, there are conflicts, especially when there were shark attacks last year, and I know that the surfers who were there on the false pass spots were fighting against the fishermen who were fishing in the false passes. Because they said that it would attract sharks and the others said well, we don't care, we've always fished there, it's not going to change now. So, I know that it was a bit tense for a while around the false passes.” .Similarly, scuba diving activities can occur in parallel with the fishers’ activities, which can also lead to tensions.“On the other hand, I've seen fishermen in the passes, for example, at the ‘shark faille’, where you're scuba diving next to them and it's very dangerous, it's forbidden to fish, you're in the reserve, and the guys are with their spearguns, waiting in the current and shooting. So I went to see them because they were diving, it's dangerous scuba diving, at the ‘shark faille’ there's not good visibility so you don't know with their spearguns if they recognize you're diving; so no, it's a potential risk of accident, of food stimulation for, well there are greys [grey sharks] that turn into the pass, and well yeah, afterwards you go to see them, and they say: here, I'm at home and you're not, and that's it … “ .These two quotes point towards tensions, potentially due to: a) perceptions by some surfers and scuba divers that fishers’ activities present a danger for other reef passage users, or at least attract dangers ; and b) fishers having a particularly strong attachment to the passages and a consideration of themselves as more legitimate users than surfers and scuba divers. These above quotes also hint towards a possible mismatch between the statements (mainly of surfers and scuba divers) that reef passages need better protection and fishers’ perceptions that there are already enough protected areas . For example, in the case of the ‘false pass’ of Île Verte near Poé, a protection of the passage would only be accepted in exchange for the removal of the (touristic) Poé protected area.There are already too many reserves at Bourail, if you listen to them [non-fishers] there's never enough. There is no need for the ‘false pass’ reserve. The interest of a reserve is the protection of several habitats, there is no proven ecological interest here, the spawning area, the last report I read it was not more a spawning area than other places. The interest of the current reserves, close to inhabited areas, is protection from direct over-exploitation but the downside is we must go further. We forget that people don't have the money for petrol, where will they go next? In my opinion, the ‘false pass’ reserve was originally designed to favour the diving clubs who were fed up with being next to the hooks. But anyway the ‘false pass’ is too deep for us hunter divers and it's not practical for the seine.” ."Carcharhinus amblyrhynchos), sharpnose , hammerhead (Sphyrna mokarran), bull (Carcharhinus leucas), and blacktip sharks (Carcharhinus melanopterus). The passages were indeed mentioned as those places where chances are highest to encounter big sharks and sometimes several dozen individuals.All interviewees highlighted the species richness of reef passages, including that of large predators. Sharks were mentioned exceptionally often, most frequently grey reef ."One of the testimonies collected from a Kanak fisher shows particularly well how important the shark is from both an ecological and a socio-cultural point of view:“The shark has always existed, it's part of our totems, if it's there it's because it has a job to do, and especially in the zone of the passes, you see we say that it's there in the path of the dead because it accompanies, but because there's also food there, so unconsciously we'll say well, it's the path of the dead, but when you look at the thing, it's full of nutrients inside […] It's a cycle that already exists, but yet the old guys of us [elders and ancestors], they didn't do science.” .Kanak fishers recognized the local ecological knowledge of sharks built by their ancestors and noticed the concordance of this knowledge with the scientific knowledge of which they became aware only later.The knowledge of New Caledonian reef passages held by the coastal stakeholders interviewed in this study was substantial and multifaceted, in terms of the different names that existed for the different types of passages, their morphological features, currents, and fauna. This mirrors results of in-depth studies in other parts of the South Pacific region, for example the Solomon Islands  these places. This abundance of sharks, together with the protected area discussions, caused some tensions—between scuba divers and fishers, as well as between surfers and fishers . A holistic lens is needed for timely solutions to these tensions, including considerations that integrate the recent ‘shark crisis’ following lethal shark attacks on fishers and surfers in New Caledonian waters.To protect New Caledonia’s reef passages as keystone places therefore seems to be not only a question of simply increasing the number of protected areas (and closed-off space), but rather to also change the character and quality of the existing protection, thereby increasing the level of respect and acceptance of protection criteria. Accounting for local priorities and forms of usage of marine areas such as reef passages , including immaterial values people assign to these places, can help to engage local people, acknowledge their connection to marine places, and ultimately contribute to marine conservation . Modern myths may include those conveyed by scuba divers (told as stories on the dive boats taking them to and from the passages and back on land with friends and family), often surrounding type, size, and behaviour of sharks and other remarkable keystone species, as well as currents, visibility, and encountered intricacies of the reef morphology. Tourist fishers may contribute their stories of the exhilarating battle with the fish, the profusion of various fish species, or the intricacies of navigating these dangerous areas; and surfers may contribute their own stories on these aspects, as well as on specific wave conditions in the reef passages. Accounting for the importance of these different local myths, knowledges, and philosophies, it seems important to contrast them with each other, in order to a) further illustrate the complexity of the afore-mentioned tensions, yet b) create a shared space (i.e. with the reef passage as boundary object) to jointly tackle the management and protection of reef passages and their local use(r)s.S3), this study is a first step highlighting the necessity of, and providing the foundation for, extended research on reef passages also in other countries across the tropical belt. Our study adds knowledge on both the ecological and socio-cultural significance of these keystone places, which should be more explicitly considered under existing and future management and conservation measures. Accounting for the multiple uses, knowledges, and values of reef passages indeed has the potential to increase the social-ecological efficacy and meaningfulness of these measures, including formal regulations.Therefore, whilst we have identified a gap between how scientific references to reef passages are scarce and scattered, and how reef passages appear of multifold ecological and cultural importance in New Caledonia ; areas through which everything from the outside enters the islands and affects societies in either a ‘good’ or ‘bad’ way .Inhabitants frequently use reef passages for fishing, scuba diving and surfing. Here, high-value pelagic fish can be caught near-shore, daily, without having to venture out into the open sea. Scuba divers can encounter many animals (including sharks) without having to dive very deep or travel very far, whilst surfers can find good spots, adapted to their various preferences and skills.The study has shown that, in New Caledonia, reef passages are considered ‘communication zones’ between coastal and oceanic spaces and species, and have significant yet un(der)explored ecological and socio-cultural roles. In our view, inter- and transdisciplinary research on reef passages is all the timelier as many people depend on them for food security and livelihoods; at the same time, they are increasingly threatened by climate change, pollution, and overexploitation. Further research on reef passages will thus contribute to global conservation agendas, aiming at interdisciplinary and inclusive procedures  surrounding SDG 14 (Life below water), and specifically the target to protect 30% of the Earth’s surface by 2030 (and resistances to it). For the ‘Large Ocean Island States’ of the South Pacific, these 30% will invariably cover to a large extent seascapes, potentially including reef passages.Supplementary file1 (DOCX 41 KB)Below is the link to the electronic supplementary material.


While conventional nuclease protection methods rely on long (~30 nt) oligonucleotide probes that preclude the global assessment of consensus motif modification stoichiometry, we investigated a series of ion-tagged oligonucleotide (ITO) probes and found that a benzylimidazolium-functionalized ITO (ABzIM-ITO) conferred significantly improved nuclease resistance for GGACU targets. After optimizing the conditions of the nuclease protection assay, we applied the ITO and MALDI-MS-based method for determining the stoichiometry of GG(m6A)CU and GGACU in RNA mixtures. Overall, the ITO-based nuclease protection and MALDI-MS method constitutes a rapid and promising approach for determining modification stoichiometries of consensus motifs.The reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control the translational output of the cell. Although characterization of modification occupancy at consensus motifs can be accomplished using RNA sequencing methods, these approaches are generally time-consuming and do not directly detect post-transcriptional modifications. Here, we present a nuclease protection assay coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to rapidly characterize modifications in consensus motifs, such as GGACU, which frequently harbor N6-methyladenosine (m Newly discovered roles for post-transcriptional modifications in regulating protein translation ,2 and RN6A) [6A can also be enzymatically removed from these consensus sites to mediate gene expression [6A and its writer and eraser enzymes has been linked to tumorigenicity of cancer cells [RNA modifications are often deposited by enzymatic writers at predictable sequence motifs ,18,19. F6A) ,21. In a6A) , m6A canpression ,24,25. Ipression ,11, and pression . Dysreguer cells , neurodeer cells , and adder cells . Character cells ,32,33,34m/z) [Direct detection and quantification of RNA modifications is often accomplished using mass spectrometry (MS) techniques ,37,38,39m/z) ,41,42,43m/z) . MALDI-Mm/z) ,45,46. Fm/z) . These nIn order to improve hybridization with target nucleic acids, chemical functionalization of oligonucleotide probes can provide enhanced melting temperatures and selectivity for complementary sequences . Recentl6A)CU targets, thus facilitating the analysis of modification stoichiometry at these sites. A series of alkylimidazolium and benzylimidazolium ITOs were investigated to enhance the stability of short target RNA sequences during digestion with single-strand-specific mung bean nuclease (MBN), revealing that a benzylimidazolium-functionalized ITO (ABzIM-ITO) maximized the liberation of the GGACU motif from full-length RNA. After optimizing the ITO-based nuclease protection assay, we investigated the effect of sequence context on the detection of GGACU motifs. Unlike conventional DNA probes, we found that the ABzIM-ITO showed no preference for terminal or internal placement of the GGACU motif sequences across RNAs, suggesting that the ITO hybridization probe is suitable for the unbiased evaluation of RNA modification occupancy at these sites. As a proof of concept, the ITO and MALDI-MS-based approach was applied for determining modification stoichiometry in heterogeneous pools of modified and unmodified RNAs. Overall, our results demonstrate that nuclease protection assays can be improved by using ITO hybridization probes and MALDI-MS to rapidly characterize RNA modification stoichiometries within short RNA sequences.In this study, we report a MALDI-MS-based nuclease protection assay that utilizes ITO hybridization probes to protect GGACU and GG or Dharmacon  and used without further purification . Mung beThe ITOs used in this study have been reported previously and were prepared by thiol-ene click reactions using published procedures ,50. BrieAfter the reaction, the mixture was diluted with 2 µL of nuclease-free water and 4 µL 7 M urea and subjected to denaturing polyacrylamide gel electrophoresis (PAGE). Unreacted thiolated oligonucleotide was separated from the ITO product using a 15% polyacrylamide gel prepared with 7 M urea. The gel was run at 130 V in tris-borate–EDTA buffer for ~45 min, and the bands were visualized by UV shadowing at 254 nm. The ITO band  was excised, transferred to a 1.5 mL tube, and crushed with a pipet tip. The ITO was eluted from the gel with 40 µL of nuclease-free water overnight, and the resulting ITO concentration determined by NanoDrop spectrophotometry.−1) was added, and the reaction was digested at 15 °C for 30 min.In a 0.2 mL PCR tube, 1 µL of 10X mung bean nuclease reaction buffer and 10–50 pmol of target RNA were combined with ITO probe (2:1 probe to target ratio) and water to a total volume of 9.5 µL. The oligonucleotides were annealed using the following temperature program: 65 °C for 3 min, hold at 4 °C. Next, 0.5 µL of mung bean nuclease . C18 pipet tips were pre-conditioned with 3 × 10 µL washes of 50/50 ACN/HA 0.5 µL aliquot of the sample was deposited on an MTP-384 polished steel target (Bruker), immediately followed by 1.0 µL of 0.5 M 3-HPA matrix prepared in 50/50 ACN/water. The sample was allowed to dry completely at room temperature.m/z range with a Bruker smartbeam-II laser set to “Ultra” (diameter of ~70 μm) and 83% power. The instrument was calibrated using a peptide calibration standard mixture (Bruker). Each spectrum resulted from 10,000 total laser shots generated across 10 manually selected positions, where each position was sampled with 1000 shots at a frequency of 1000 Hz. The instrument specific settings included pulsed extraction time of 120 ns, accelerating voltage of 25 kV, extraction voltage of 22.65 kV, lens voltage of 6.8 kV, and reflector voltage of 26.4 kV.MALDI-MS was performed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer operated in reflectron-positive mode over a 700–5000 http://rna.rega.kuleuven.be/masspec/mongo.htm, accessed 1 April 2022). For each sample, the stability ratio (SR) was calculated using Equation (1):fI1, fI2, and fnI are the peak intensities for the 1st, 2nd, and nth most intense degradation fragments with signal-to-noise (SN) ratios ≥10, and targetI is the peak intensity for the intact target sequence (GGACU or GG(m6A)CU) with SN ≥ 10 following digestion of the sample with single-strand-specific MBN.Mass spectra were processed using Bruker flexAnalysis 3.4, and peaks were manually annotated and verified with Mongo Oligo Mass Calculator v2.06 (6A consensus motif (6A consensus motif (GGACU) installed within a 15 nt synthetic RNA standard (Nuclease protection coupled with MALDI-MS is a high-throughput approach for characterizing RNA modifications that relies on an oligonucleotide probe complementary to the RNA sequence of interest and a single-strand-specific nuclease that cleaves the unhybridized regions of RNA molecules. Nuclease protection assays have thus far only been implemented with relatively long oligonucleotide probes ~20–50 nt) that cannot be used to globally interrogate the modification status of small consensus motifs across the transcriptome ,55. In o nt that us motif . This sestandard .Our first goal was to identify ITO probes that minimized the amount of degradation fragments produced from the target GGACU sequence while maximizing the amount of target sequence liberated from the RNA standard. ITO probes were annealed with the RNA standard (2:1 probe–target ratio) and incubated for 30 min with MBN at 30 °C according to the nuclease manufacturer’s instructions. After purifying the samples with C18 pipet tips, the samples were spotted with 3-HPA and subjected to MALDI-MS detection. The MALDI-MS platform permitted the rapid (~10–20 s per sample) and simultaneous detection of the consensus motif as well as the degradation fragments resulting from unsuccessful protection of the motif A–F. For 6A consensus motif studied here.We then investigated the effect of different digestion temperatures to identify conditions that both facilitated nuclease function and stabilized the ABzIM-ITO probe and the target RNA duplex. We tested the nuclease protection assay using 15 °C and 10 °C for the digestion step to compensate for the low melting temperature of the probe–target duplex (~10 °C) . SRs forNext, we compared SRs obtained when performing digestion with different amounts of MBN in the sample mixture. We rationalized that since degradation fragments had already been observed using 5 U of MBN, decreasing the amount of MBN to 2.5 U might improve the SR. In contrast to our expectations, we found that the lesser amount of MBN resulted in a lower signal intensity for the target peak, which in turn produced a substantially lower SR . This reWe then investigated the effect of installing the ion tag moiety on the 3′ or 5′ end of the ITO probe. Since the ITO group was appended to the 3′ end of the ABzIM-ITO probe in previous experiments, we anticipated that the enhanced target stability during nuclease digestion was due to π-π stacking interactions with the 5′ terminal guanosine of the GGACU motif. To test this hypothesis, a 15-mer RNA standard possessing a 5′ terminal GGACU motif was subjected to nuclease protection with either a 3′ or a 5′ functionalized ABzIM-ITO probe A. Signif8 and C10) produced significantly higher SRs compared to the un-tagged and/or no-probe control samples  whose functional groups cannot engage in stacking interactions. The SRs obtained from 3′ alkyl-functionalized ITOs were not significantly different from protection by an un-tagged probe or the no-probe control B, suppor samples B. The 5′6A occupancy at these sites.Another motivation for studying the effect of the sequence context of the consensus motif on the nuclease protection assay is that RNA modifications can be deposited both internally and near the 5′ termini of RNA biopolymers ,59,60. T6A)CU sequences and asked whether the 3′ ABzIM-ITO probe would confer nuclease resistance to the m6A-modified RNA. After accounting for the changes in the m/z of the target and degradation peaks originating from the modified RNA CU targets, respectively (6A)CU was calculated from three times the noise of a blank analysis and was 1.8 pmol.We then aimed to apply the ITO and MALDI-MS method for the determination of RNA modification stoichiometry at the consensus motifs. First, we designed 15-mer RNA standards with either internal GGACU or GG across the stoichiometries tested . While typical approaches for determining modification stoichiometries at these sites rely on RNA sequencing methods that include time-consuming library preparation steps and indirect detection by immunoprecipitation or chemical derivatization, the coupling of nuclease protection and MALDI-MS affords rapid analysis and direct detection of any RNA modification that alters the m/z of the resulting RNA sequence. This feature MS detection is important for the discovery of unknown RNA modifications or known modifications in unexpected sites. To improve nuclease protection assays for the analysis of short target sequences, we investigated a series of hybridization probes including alkylimidazolium- and benzylimidazolium-functionalized ITOs. We found that the ABzIM-ITO maximized the liberation of target GGACU motifs for detection by MALDI-MS, likely due to enhanced π-π stacking interactions with the target RNA sequence. Unlike conventional un-tagged hybridization probes, the ABzIM-ITO did not show any bias toward protection of internal or terminally positioned motif sequences, which is an important feature for the unbiased evaluation of modification stoichiometry across multiple sites in RNA molecules. When applied for the determination of modification stoichiometries in consensus motifs, the results from the ITO and MALDI-MS-based approach strongly agreed (R2 = 0.97) with the ratios of modified and unmodified RNAs added to the sample. In addition, since dozens of samples can be subjected to nuclease protection and purification within ~45 min, the detection methods for the resulting digest must have sufficient sample throughput to keep pace. While LC-MS of RNA oligonucleotides require ~30–60 min per RNA digest, the MALDI-MS analysis times reported herein are rapid and only require ~10–20 s per digest, thus providing a high-throughput alternative for characterizing modified and unmodified RNA fragments generated from nuclease protection.In this study, we report a nuclease protection assay that leverages ITO hybridization probes and MALDI-MS to characterize RNA modification stoichiometries in a short consensus motif that endogenously harbors m6 neurons in the mouse hippocampus, ~105–106 mRNAs per cell, and an estimated ~3–5 m6A modification sites per transcript [6A sites in specific mRNA transcripts. The application of longer ITO hybridization probes would be particularly interesting for transcripts containing multiple m6A consensus motifs. While such an approach would be limited by transcript abundance, it is conceivable that high-abundance mRNAs would be assessable.This study establishes a foundation for subsequent investigations into global RNA modification stoichiometry in complex samples that contain numerous targeted and untargeted RNA sequences. One of the advantages of the nuclease protection assay is that it dramatically reduces the complexity of RNA sequences within a sample via single-strand-specific digestion, potentially allowing for the rapid analysis of modification stoichiometries even in total RNA samples. In our experiments, we found that the LOQ for this approach was 10 pmol of RNA molecules that each contained one motif sequence. We expect that further improvements in the LOQ of the method will allow the characterization of RNA modification events in successively smaller samples, such as subregions of the mammalian central nervous system. For example, with ~10anscript , a two-f1A) [A limitation of our method is that it appears to have the best performance when a purine nucleobase is present at the 5′ terminus of the consensus motif. However, it is conceivable that consensus motifs bearing purine nucleobases at their 3′ ends could be sufficiently protected by a 5′-functionalized ITO probe, allowing adaptation of the ITO-based method for a variety of consensus motifs such as UGUAR (R denotes purine) for pseudouridine (Ψ)  or GUUCR1A) . Impleme


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic that broke out in 2020 and continues to be the cause of massive global upheaval. Coronaviruses are positive-strand RNA viruses with a genome of ~30 kb. The genome is replicated and transcribed by RNA-dependent RNA polymerase together with accessory factors. One of the latter is the protein helicase (NSP13), which is essential for viral replication. The recently solved helicase structure revealed a tertiary structure composed of five domains. Here, we investigated NSP13 from a structural point of view, comparing its RNA-free form with the RNA-engaged form by using atomistic molecular dynamics (MD) simulations at the microsecond timescale. Structural analyses revealed conformational changes that provide insights into the contribution of the different domains, identifying the residues responsible for domain–domain interactions in both observed forms. The RNA-free system appears to be more flexible than the RNA-engaged form. This result underlies the stabilizing role of the nucleic acid and the functional core role of these domains. Coronaviridae. The family belongs to the order Nidovirales, which includes viruses with single-stranded RNA genomes of positive polarity . I. I2 supeSP8 unit . InteresNidovirales order, which is the N-terminal zinc binding domain  that coordinates three Zn(II) ions; a helical “Stalk” domain (101–150); a β-barrel domain ; and two “RecA-like core helicase domain” subdomains , which can be referred to as the catalytic core of the helicase, since it holds the residues known to be responsible for nucleotide binding and hydrolysis [The high-resolution structure of NSP13 was resolved in 2020 at 1.94 Å resolution in the RNA- and ADP-free form C 19]. F. F19]. FAlthough NSP13 is a highly conserved protein—in fact, it differs from SARS-CoV by only one amino acid —its exac2 supercomplex. Therefore, two 10-μs-long sets of atomistic molecular dynamics simulations were implemented to examine the different conformational behaviors of NSP13 separated by the RTC complex in the presence or in the absence of an RNA fragment at the microsecond timescale. In addition to the classical structural observations, it was deemed interesting to explore the specific contact frequencies occurring between the single helicase domains. In other words, emphasis was placed on the residues involved in the mutual interactions of individual domains. The latter analysis was carried out in the aim of obtaining detailed information on the individual residues that change their interaction partners from the RNA-free to the RNA-engaged form of the SARS-CoV-2 helicase.The aim of this work was to investigate NSP13 from a structural and dynamical point of view by taking advantage of the recent structures of the protein in the RNA-free  and in tThe structural stability of the two helicase systems was tested during the simulations by calculating the deviations of each structure from the starting coordinates and the fluctuation of each residue with respect to the average structure after superimposition on the Cα atoms.N-terminal from the RMSD calculation. The coordination geometries of the three Zn(II) ions did not change during both simulations. The 2A domain remained extremely stable in the RNA-engaged form  of the trajectories was calculated for the Cα atoms to measure the flexibility of the protein chain C. The daA cluster analysis was performed for both the RNA-free and the RNA-engaged system in accordance with the GROMOS algorithm . For eacThe main difference in the conformation of the representative structure of the first two clusters in both systems C is the Motion correlations between various sub-parts of NSP13 can be characterized by a calculation of the covariance matrices of the amino acids’ displacements. The corresponding maps computed using the coordinates of the Cα atoms are shown in To summarize, the main differences observed in the covariance maps are as follows: (i) the ZBD moves as a single entity in the RNA-free form, while is divided in two sub-domains experiencing a reciprocal anticorrelated motion in the RNA-engaged form; (ii) the Stalk domain’s motions are anticorrelated with the 1A domain in the RNA-free form and are correlated in the RNA-engaged form; and (iii) the collective intradomain motions of Domain 1A are less correlated in the RNA-engaged form, meaning that the domain moves less as a single entity. The exact opposite can be observed for the Stalk domain, the motions of which appear to be more concerted in the presence of the RNA fragment.In order to characterize the main motions characterizing NSP13 in the two different forms studied in this work, the two trajectories were further analyzed using principal component analysis (PCA). In general, the dynamic behavior of NSP13 in both states could can be classified into at least 20 collective motions without any one of them being truly predominant over the others. The projection of the first two principal components, PC1 and PC2, on the structures of the RNA-free and RNA-engaged NSP13 was then analyzed. In the case of the RNA-free NSP13 C, the fiThe conformational space revealed by the first two covariance eigenvectors was also inspected, and the resulting maps were used to search for significative sub-states of the conformational populations in the two systems. The results presented in this manuscript are in excellent agreement with what has been obtained previously from a model of NSP13 from SARS-CoV-2 obtained by starting with the homologous protein from SARS-CoV and then inserting an ADP molecule and the nucleic acid fragment by modeling . Our resThe contacts among the five NSP13 domains were analyzed by calculating inter-residue distance matrixes. In particular, the strategy adopted was to calculate the normalized inter-residue contact frequencies, defining two residues as being in contact when the distance between any two heavy atoms was less than 3.5 Å for at least the 50% of the simulation time. The emerging picture is reported in The comparison of the interactions between the 1B and 1A domains  reflectsAn analysis of the contacts between Domains 1A and 2A during the simulations  showed tIn the RNA-engaged form of NSP13, the RNA fragment appeared to establish several stable interactions with residues belonging to the Stalk, 1B, 1A, and 2A domains . The lisFinally, the interactions of the ADP molecule present in the RNA-engaged form were also inspected. The ADP molecules formed extremely stable interactions  with the nearby residues, which were Pro283, Pro284, Gly285, Thr286, Gly287, Lys288, His290, and Lys320 in the 1A domain, together with Arg443, Lys465, Glu540, and Lys569 in the 2A domain. The position and the coordination geometry of the Mg(II) ion was stable throughout the entire 10 µs of the simulation. The ADP molecule was bound in a pocket between Domains 1A and 2A, probably helping to stabilize the interaction between these two domains, in agreement with what was observed in Section 2.3.2The model on which the simulations of the SARS-CoV-2 helicase in the RNA-free form were based was the crystallographic structure determined at a resolution of 1.94 Å with two subunits in the asymmetric unit (PDB ID 6ZSL). Generally, the electron density was of high quality, except for the lack of resolution of Residues 95–102, 186–193, 203–206, and 593–601 for Chain A and for Residues 1, 337–339, and 594–601 for Chain B. To correct these unresolved areas, homology modeling was performed using the Modeller 10.0 software  so ChainThe starting structure for the helicase in the RNA-engaged form was the cryo-EM-resolved structure with a resolution of 3.10 Å of the SARS-CoV-2 replication–transcription complex bound to the NSP13 helicase-NSP13(2)-RTC-engaged class (PDB ID 7RDY). After this, only the helicase with the bound RNA fragment and the ADP molecule were retained.The open-source web server H++  was used+ ions by using the genion tool included in GROMACS 2020.1. Analogously, additional Na+ and Cl− ions were placed in the water box to achieve physiological ionic strength (150 mM). The systems were energy-minimized with the “steepest descents” algorithms for 1500 steps and then equilibrated at 300 K and 1 atm by performing 1 ns of gradual annealing using GROMACS 2020.1. Next, to equilibrate the solvent around the protein, positional restraints were imposed on the protein, and each system was equilibrated for 50,000 steps with a time step of 2 fs at 300 K. The system’s pressure was coupled to a Berendsen [The resulting structures were then placed in a truncated octahedral water box, where a solvent buffer zone of 10 Å was set by using the tools included in the GROMACS 2020.1 suite ,37,38. Terendsen  barostaterendsen . The LINerendsen  was usederendsen  was usederendsen . In a prerendsen ,45. For The trajectories were analyzed by using GROMACS tools and in-house Python3 scripts, making use of the MDTraj 1.9.8  and mdciIn this work, extensive all-atom plain MD simulations were used to study the helicase (NSP13) of SARS-CoV-2 in its RNA-free form and RNA-engaged form. What emerged from the analyses was the main contribution of Domains 1A and 2A, the functional cores of the helicase, which make a major contribution to the stability and flexibility of the system of which they are part. Our simulations also highlighted the role of Domain 1B, which is responsible for forming the cavity in which the single RNA strand is lodged. The role of the ZBD domain as a domain supporting the functional activity of the helicase was also observed. The study showed a difference in the stability of ZBD domain between the RNA-free and RNA-engaged forms. As explained above, the ZBD is involved in the interaction with the other NSPs of the RTC. The differences highlighted during the simulations suggested that the presence of RNA can modify the dynamics of this domain, leading to the major stability of the interactions and promoting the activity of the complex itself. These results can be useful, not just to unveil the dynamics of NSP13 in the presence or absence of RNA, but also to define the main residues responsible for the stability of the system, representing putative regions to consider as targets for the development of new drugs. In particular, the NSP13 region defined by the residues found in contact with the RNA fragment  in the RAfter these observations, the analyses moved on to a more structural level, investigating the interactions existing among the various domains in both systems under examination. The residues responsible for the domain–domain interactions were identified, outlining a profoundly different picture between NSP13 in the RNA-free and RNA-engaged forms. Looking ahead, it would be inspiring to run a molecular dynamics simulation of the unfolding of the RNA’s double helix to observe what the arrangement of the protein domains interacting with the RNA would be.