This review is an accompaniment to a Special Issue on “Retroviral RNA Processing”. It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay. Viruses is devoted to Retroviral RNA Processing. We will focus on new developments in this field and will include representatives of several types of retroviruses. Topics in this Special Issue include regulation of HIV-1 alternative splicing during viral replication [This Special Issue of lication . In addilication . New livlication . Imaginglication . In addilication . Unfortulication ,8,9,10.One of many unusual features of orthoretroviruses is that they package two identical copies of their approximately 10 kb, single-stranded RNA genome; thus, the RNA genome in virions is diploid ,12,13,146A and m5C residues.Cleverly, the viral cDNA reverse transcript generates transcriptional regulatory sequences at each end of the genome in its Long Terminal Repeat (LTR) . The LTRRetroviral gene expression uses a “hybrid” model, involving both unspliced and spliced mRNAs. The unspliced full-length primary transcript is the mRNA that generates Gag and Gag-Pol polyproteins. This primary transcript is also the genomic RNA that is packaged into viral particles as dimers. The Gag and Gag-Pol polyproteins are subsequently cleaved by the viral-encoded protease after budding from the cell. The Gag polyprotein is processed to generate the capsid proteins: matrix, capsid, nucleocapsid, and additional, virus-specific small Gag proteins. The retroviral enzymes are generated from the Pol polyprotein and comprise protease, reverse transcriptase, and integrase.env mRNA, encoding the viral glycoproteins on the surface of the virion. Other singly spliced mRNAs include RSV src, HIV vpu and vif, and PFV pol. In addition, multiply spliced mRNAs encode accessory proteins, including HIV-1 Tat and Rev. However, a large fraction of the primary transcripts must remain unspliced for genomic RNA and Gag–Pol mRNAs, so splicing needs to be tightly regulated [The gene(s) at the 3′ end of the genome is expressed from subgenomic mRNAs, generated by splicing of the primary RNA transcript. All retroviruses generate a singly spliced egulated .gag and pol gene products need to be synthesized from the same mRNA. (5) Since the unspliced mRNA has a long 3′ UTR, it is a target for nonsense-mediated RNA decay (NMD). This must be inhibited to retain stable full-length mRNA for viral replication.This hybrid model generates several interesting RNA processing problems that retroviruses need to overcome: (1) Primary RNA transcripts must be sorted into packaged and translated RNAs, although they are thought to be essentially identical in sequence. (2) The translated mRNAs must be sorted into unspliced and spliced fractions, and the alternative splicing must be regulated to generate numerous products in the complex viruses. (3) The unspliced and incompletely spliced intron-containing RNAs must be exported to the cytoplasm, contrary to the normal rules of eukaryotic mRNA processing. This is necessary because introns perform a double duty as coding sequences. (4) Different amounts of This review will discuss numerous examples where different retroviruses use different methods to solve these problems. This Special Issue on Retroviral RNA Processing focuses on research investigating how retroviruses usurp cellular machinery to process their RNAs and express their genes.Foamy viruses are the most ancient retroviruses and comprise the subfamily Spumaretroviruses. Endogenous spumaviruses date back to the first jawed vertebrates (Coelecanths) over 450 million years ago ,18. Theygag, pol, and env genes. In addition, genes for two accessory proteins, Tas and Bet, are encoded at the 3′ end of the genome, making these complex retroviruses. Unusually, an internal promoter within the env gene is expressed early after infection to generate the accessory proteins from spliced mRNAs. Tas is a transcriptional activator that binds DNA, both in the LTR and at the internal promoter. It is expressed early after infection, and its expression leads to a switch from use of the internal promoter to the 5′ LTR promoter. This induces the expression of the viral structural genes later in infection. The Bet protein is an antagonist of APOBEC3 and prevents packaging of this deaminase [The spumaviruses’ mode of RNA processing has some interesting differences from most orthoretroviruses (reviewed in ). Like aeaminase . It is iThe full-length foamy virus RNA transcript functions as the mRNA for the N-terminal Gag protein. In contrast to the orthoretroviruses, Pol is expressed from a singly spliced mRNA and not as a Gag–Pol fusion polyprotein . Foamy vpol and env mRNAs occurs via the CRM1 pathway, but its use is novel because there is no homolog of the HIV-1 Rev regulatory protein. The cellular shuttle protein HuR binds the foamy viral RNA and interacts with CRM1 via the bridging proteins ANP32A and ANP32B [Nucleocytoplasmic export of the foamy unspliced RNA and spliced (but intron-containing) d ANP32B . HuR alsIn major contrast to orthoretroviruses, reverse transcription of the packaged RNA dimer begins in the cytoplasm before it exits the cell and is thought to be completed before the virus infects a new cell . In thisenv (reviewed in [env/U3 junction [The Betaretrovirus mouse mammary tumor virus (MMTV), like foamy viruses, has an internal promoter and splice donor (SD) in iewed in ). In MMTjunction .After integration into the host genome, proviruses are transcribed by host RNA polymerase II. Unspliced primary RNA transcripts can escape splicing and be exported from the nucleus to become packaged genomic RNA or translated mRNA. How and where is this selection between packaging and translation made? Alternative RNA structures in the 5′ UTR have been proposed to distinguish genomic from translated RNAs ,27,28,29gag AUG translation initiation codon. In contrast, the translated mRNA favors translation with an exposed cap and AUG, while the DIS is sequestered. The presence of a TMG cap on HIV RNA [Interestingly, a small difference in the sequence of the 5′ end of HIV-1 RNA has been observed between packaged genomic RNA and translated mRNA. The packaged RNA has a single transcribed G residue at its 5′ end, while the mRNAs have two or three transcribed G residues ,31,32. IDifferences in transcription initiation sites and the presence of TMG caps have not been observed for retroviruses other than HIV-1. However, the TMG cap appears to be associated with CRM1-mediated export, so may be found in other complex retroviruses. Other factors that might help distinguish between RNA destined for packaging or translation include RNA dimerization, RNA modification, and binding to Gag or cellular splicing factors or ribosomes.HIV-1 RNAs appear to be exported to the cytoplasm as monomers and to dimerize during virion assembly at the plasma membrane ,34. In cThe Parent lab has previously observed that trafficking of the Rous sarcoma virus (RSV) Gag protein through the nucleus is necessary for efficient genomic RNA packaging . Further7G cap, presumably because they are not exported by the CRM1 pathway. Other viral RNAs exported by CRM1 would be predicted to have TMG caps.All retroviral RNAs are transcribed by cellular RNA polymerase II and capped cotranscriptionally by the cellular capping enzymes. Interestingly, some HIV-1 RNAs have been observed to have a TMG cap, probably due to their interactions with Rev, TGS1 (methyl transferase), and CRM1 ,39. Unli6ACU [In addition to cap modifications, retroviral RNAs are also modified internally, much like cellular mRNAs. RSV genomic RNA was first shown in 1977 to have an average of 12 m6A residues with a consensus sequence of RGm6ACU ,41,42. O6A [5C [6A of HIV RNA enhanced Gag protein expression and viral replication [6A at two sites in the RRE was shown to enhance its interaction with Rev [5C to HIV-1 RNA enhanced ribosome binding and translation [6A residues were observed in virion RNA than in cellular HIV-1 RNA. Greatly increased (>10 fold) modification at m5C and 2′O-Me sites was also seen in genomic RNA in comparison to total cellular mRNA [6A, have been shown to inhibit innate immune responses to HIV-1 RNA [HIV-1 RNA is also methylated to generate numerous m6A ,44,45 an6A [5C  residueswith Rev . The addnslation . Intereslar mRNA . ImportaIV-1 RNA ,48.6A binding proteins (YTHDF proteins) also decreased HIV-1 reverse transcription [6A reader protein YTHDF3 is packaged into HIV particles and acts as a restriction factor, reducing infectivity at the level of reverse transcription. However, the packaged HIV-1 protease cleaves this reader protein, increasing infectivity of HIV [Overexpression of mcription . Recently of HIV .Most cellular genes have multiple large introns that are removed during splicing, and small exons that encode proteins. However, viruses have to be more economical with their genetic material and cannot afford to discard their introns. Thus, retroviral introns are also coding sequences. In addition, retroviruses must retain a large portion of their unspliced RNA for mRNA for Gag and Pol polyproteins, as well as genomic RNA. Thus, splicing is incomplete. Over-splicing prevents viral replication ,50. A geLike most cellular mRNAs, retroviral RNAs are spliced to generate subgenomic mRNAs, which are exported to the cytoplasm and translated by cellular ribosomes. The simplest retroviruses, such as avian leukosis virus (ALV), have a single spliced mRNA encoding the Env glycoproteins on the surface of the virion. The Env polyprotein is cleaved to generate SU (surface) and TM (trans membrane) proteins; these interact with cellular receptors to facilitate entry.gag gene, located about 300 nts downstream from the 5′ splice site. The NRS acts as a decoy 5′ splice site that forms a nonfunctional spliceosome, thus inhibiting splicing [myb [The avian retroviruses (ALV and RSV) have a negative regulator of splicing (NRS) sequence within the splicing . The NRSsplicing . These bing [myb ,55.The complex retroviruses, such as HIV-1, generate additional viral proteins by alternative splicing . HoweverHIV-1 latency is regulated at both transcriptional and post-transcriptional levels, as discussed in this Special Issue by Pasternak and Berkhout . There aRetroviral RNA is polyadenylated, but the process is not efficient, and readthrough of about 15% of the ALV transcripts into downstream cellular genes is observed . Mutatioenv and mediates export through the cellular CRM1 pathway  in iewed in ). Rev haiewed in . Completenv gene. MMTV, a betaretrovirus, encodes an export protein called Rem as part of the N-terminus of Env [Other complex retroviruses use similar export mechanisms. HTLV-1 encodes a Rex protein that binds a Rex responsive element in the s of Env . Althougs of Env .cis-acting viral sequences to interact with host export proteins. A constitutive transport element (CTE) was initially observed in the 3′ end of another betaretrovirus: the Mason-Pfizer monkey virus (MPMV) [The simple retroviruses, which lack accessory proteins, use s (MPMV) . The CTEs (MPMV) . Interess (MPMV) .pol gene, and the packaging sequence [Recently, Mougel et al.  proposedsequence ,66,67. Ssrc gene that is important for RNA export to the cytoplasm and likely analogous to the CTE [RSV has two copies of a direct repeat (DR) sequence flanking the  the CTE ,69. Thes the CTE  similar  the CTE . Gag and the CTE .RSV Gag proteins shuttle between the nucleus and the cytoplasm. Leptomycin B (an inhibitor of CRM1-mediated export) inhibits this shuttling, and Gag is retained in the nucleus. There is evidence that Gag may interact with RSV full-length RNA in the nucleus and export it for packaging in a CRM1-dependent manner . Thus, igag gene, while other retroviruses have protease as part of pol. Three upstream open reading frames have been identified in the avian retroviruses, which appear to promote virus production [All retroviruses express the 5′ terminal Gag polyprotein from an unspliced primary RNA transcript. The avian retroviruses include protease at the 3′ end of the oduction . The Hacoduction .Moloney MLV encodes Gag proteins with two different N-termini. Glycogag synthesis is initiated at a CUG that is 264 nt upstream of the AUG that initiates normal Gag protein synthesis. Glycogag promotes viral core stability and protects the core from APOBEC3 and other cytosolic sensors of viral nucleic acid during reverse transcription . It alsogag is pol in all retroviruses. pol is also translated as a polyprotein. In most retroviruses (with the exception of the foamy viruses) a Gag–Pol readthrough product is synthesized. In most orthoretroviruses, including RSV and HIV, a (−1) frameshift shifts the reading frame from gag to pol to generate a Gag–Pol polyprotein. A slippery sequence just upstream and a structured pseudoknot sequence downstream of the termination codon are involved in the frame shift [pro and a second into pol. The next gene downstream of me shift . Some regag termination codon to generate an in-frame Gag–Pol polyprotein. The MLV gag–pol readthrough may be the precursor of the frameshift at gag–pol in other orthoretroviruses; it also involves a slippery sequence upstream and pseudoknot downstream of the gag termination codon [pol from a singly spliced mRNA, so they do not make a Gag–Pol protein but instead a Pol polyprotein [In contrast, murine leukemia viruses do not frameshift but can read through the on codon . The foayprotein .All of these readthrough or frameshift events are relatively rare (5%) so that much less Pol is translated than Gag. This promotes appropriate stoichiometry since many more capsid structural proteins are needed in a virion than enzymes. In the case of two frameshifts in MMTV, each occurs about 25% or less of the time.gag gene of RSV were seen in 1988 to be associated with degradation of viral unspliced RNA [gag termination codon were resistant to degradation, while those >100 nt upstream were not [Premature termination codons (PTCs) in the iced RNA . Surpriswere not . This RNwere not .gag termination codon was necessary for stable RNA [gag led to stabilization of the RNA, supporting the hypothesis that this sequence could stabilize a termination codon that would otherwise lead to degradation [gag gene, generating a 4–7 kb 3′ UTR. Even if readthrough into pol occurs, the 3′ UTR is still about 3 kb. In contrast, most cellular 3′ UTRs are less than 1 kb in length. In fact, longer 3′ UTRs have been associated with NMD [Further, a sequence immediately downstream of the normal able RNA ,83. Thisable RNA . Insertiwith NMD .The RSE from RSV was shown to stabilize an unstable human globin construct with a SMG 3′ UTR in human cell lines . Furthergag gene, the RNA underwent NMD and was present at about 10% of normal levels. Interestingly, the genomic RNA in virions was also present at a reduced level, suggesting it underwent NMD before packaging. Since NMD is coupled to translation, this suggests that the packaged RNA was previously translated [While it is thought that retroviral RNAs are either packaged or translated, there is evidence that RSV RNA may be translated and then packaged. When premature termination codons were inserted into the RSV anslated .Another mechanism is used to evade NMD by HTLV-1 . Both viIt is not clear how other retroviruses manage to evade NMD, but they all form aberrant unspliced mRNAs with long 3′ UTRs. One possibility is that the TMG cap of HIV-1 RNA exported with Rev/CRM1 may also inhibit NMD, although this has not been investigated. NMD involves interactions with the CBP80/CBP20 nuclear cap-binding complex , and theStudies of retroviral RNA processing have taught us a lot about cellular RNA processing, especially about RNA nuclear export ,63, spec


The Musashi (MSI) family of RNA-binding proteins, comprising the two homologs Musashi-1 (MSI1) and Musashi-2 (MSI2), typically regulates translation and is involved in cell proliferation and tumorigenesis. MSI proteins contain two ribonucleoprotein-like RNA-binding domains, RBD1 and RBD2, that bind single-stranded RNA motifs with a central UAG trinucleotide with high affinity and specificity. The finding that MSI also promotes the replication of Zika virus, a neurotropic Flavivirus, has triggered further investigations of the biochemical principles behind MSI–RNA interactions. However, a detailed molecular understanding of the specificity of MSI RBD1/2 interaction with RNA is still missing. Here, we performed computational studies of MSI1–RNA association complexes, investigating different RNA pentamer motifs using molecular dynamics simulations with binding free energy calculations based on the solvated interaction energy method. Simulations with Alphafold2 suggest that predicted MSI protein structures are highly similar to experimentally determined structures. The binding free energies show that two out of four RNA pentamers exhibit a considerably higher binding affinity to MSI1 RBD1 and RBD2, respectively. The obtained structural information on MSI1 RBD1 and RBD2 will be useful for a detailed functional and mechanistic understanding of this type of RNA–protein interactions. Examples of evolutionary conserved RBDs are the RNA recognition motif (RRM), the heterogeneous ribonucleoprotein (hnRNP) K-homology (KH) domain, and the C3H1 zinc-finger (ZF) domain. These bind to a relatively restricted set of the primary RNA sequence space, often utilizing additional contextual traits such as RNA secondary structure or base compositional context for additional specificity2.RNA-binding proteins (RBPs) are key regulators of numerous cellular processes, mediating different aspects of co- and posttranscriptional gene expression. They contain well-defined RNA-binding domains (RBDs) that confer sequence- and/or structure-specificity for endogenous target RNAs3. They have been initially identified in the central nervous system, where they are involved in the regulation of Notch signaling by binding to the mRNA of its antagonist Numb4. While MSI proteins are typically expressed in stem cells5, they are absent in differentiated tissue. Being evolutionarily conserved among invertebrates7 and vertebrates8, there has been emerging evidence that MSI proteins mediate biological processes that regulate the initiation and progression of various cancer cells, including colorectal, breast, lung, and pancreatic cancers, as well as leukemias and glioblastoma9.The Musashi (MSI) protein family comprises a group of RBPs that act as translational regulators and are involved in the maintenance and self-renewal of neuronal progenitor and stem cells12 the sequence identity of the regions containing the two RRMs in mouse MSI1 and MSI2 is remarkably high at 86%13, suggesting a common RNA target motif. For MSI1, this has been determined as (G/A)UnAGU (n = 1–3) by an in vitro selection approach (SELEX)4. NMR titration experiments with a series of RNA oligomers revealed that MSI1 RBD1 and RBD2 bind to GUAG and UAG motifs with high affinity14. These data are in line with cross-linking and immunoprecipitation (iCLIP) studies, which revealed the trinucleotide sequence UAG in a single-stranded structural context, predominantly in the 3'UTRs of mRNAs, as a core Musashi binding element (MBE)16. Likewise, quantitative fluorescence anisotropy assays confirmed the binding specificity of the UAG trinucleotide, while nucleotides outside this core MBE have limited contribution to the overall binding free energy17.The MSI gene has been duplicated in vertebrates, resulting in the two paralogs Musashi-1 (MSI1) and Musashi-2 (MSI2), each containing two ribonucleoprotein (RNP)-type RNA recognition motifs (RRMs) in their N-terminal regions, followed by a poly(A)-binding protein region. While the structures of the mouse MSI1 and MSI2 RRMs have been solved18. ZIKV is a mosquito-borne Flavivirus (MBFV) that has been circulating for decades in Africa and Asia, often being misdiagnosed as dengue. During a ZIKV outbreak in the Americas in 2015–2017, an unexpectedly high number of congenital malformations coupled with intrauterine growth restrictions, placental damage, and microcephaly has been associated with ZIKV infections19. While MBFVs are typically horizontally transmitted between arthropod vectors and vertebrate hosts, the capacity for transplacental passage aligns ZIKV with a handful of other MBFVs, including West Nile virus (WNV) and Powassan virus (POWV), that have been shown to cause placental infection and fetal neuropathology20. The presence of UAG-containing MBEs in the 3'UTRs of Flavivirus genomes, together with in vivo data revealing that MSI not only interacts with ZIKV RNA but also enhances viral replication, has led to the understanding that MSI is involved in ZIKV-induced neurotropism18. It has been hypothesized that MSI might stabilize viral RNA, thereby maintaining a sufficient RNA level that is not translated but subjected to purposeful exoribonuclease degradation21. The latter results in the production of short flavivirus RNA (sfRNA), which modulates cellular mRNA decay22 and antiviral interferon response24. While these findings highlight the instrumental role of MSI in virus-associated cytopathicity, the biochemical foundations and mechanisms of the MSI-mediated congenital neuropathology remain elusive.A different aspect of MSI pathobiology has been recently elucidated, i.e., the role of MSI proteins as host factors in viral infections, specifically their capacity to promote Zika virus (ZIKV) replication25. We have recently addressed the question as to whether other Flaviviruses have a similar MSI-mediated neurotropic potential to ZIKV by analyzing the affinity of Musashi binding elements (MBEs) in 3'UTR regions to appear in a single-stranded structural context, which is a requirement for efficient MSI–RNA interaction21. To this end, we have shown that the structural accessibility of MBEs along viral RNA molecules can be used as a proxy for predicting MSI–RNA interactions, thereby assessing the neurotropic potential of viruses. By employing a thermodynamic model of RNA folding based on the ViennaRNA package26, we computed the average opening energy that is necessary to keep specific MBEs in an unpaired structural context, rendering them accessible for MSI RRM–RNA interaction. Our data highlighted that MBEs in the 3' untranslated region (3'UTR) of ZIKV are highly accessible for MSI binding, thereby corroborating earlier studies that addressed the neurotropic potential of flaviviruses and alphaviruses20.Computational prediction of the structural accessibility of RNA binding motifs is a promising approach for the characterization of RNA-protein binding sites. This idea has been applied to several eukaryotic RBPs, resulting in the observation that target site accessibility almost always increases the ability to predict sequence-specific RBP–RNA binding13 and 5X3Z14), as shown in Fig. 21.Here we follow up on this idea and model the 3D structure of MSI RBDs with Alphafold2. Subsequently, we investigate MSI–RNA association complexes, employing molecular dynamics (MD) approaches to gain more insight into the molecular traits of this type of RNP binding. Specifically, we focus on the published MSI1 RBD1–RNA complex and MSI1 RBD2–RNA complex  approach for highly accurate protein structure prediction. In combination with MMseqs228, a program for protein sequence search within large databases and generation of high quality protein sequence alignments, Alphafold2 is capable of simulating high accuracy structures for a wide range of proteins, for which structural data are unavailable. Here, we performed predictions for MSI1 RBD1 and MSI1 RBD2 using ColabFold29, which couples MMseqs2 and AlphaFold2 in publicly available notebooks that can be executed on the Google Cloud infrastructure. We were specifically interested in determining the protein structures in the apo form and comparing these to structures available through PDB. The sequences of MSI1 RBD1 and MSI1 RBD2 were retrieved from PDB IDs 2RS2 and 5X3Z. The first candidate structure (model 1) of both RBDs from ColabFold was selected as the initial conformation to assess GUAGU binding to both RBDs by MD simulations.AlphaFold230 was used to construct complexes with three alternative RNA pentamers  by modifying the central trinucleotides. The protonation states of RNA–protein complexes were computed using the PDB2PQR server at pH 7.4. The AMBER ff14SB and chiOL3 (OL3) force fields30 were employed for protein and RNA, respectively. According to standard procedures, the missing hydrogen atoms of each system were added by the LeaP module. The added hydrogen atoms were then minimized for 1000 steps by steepest descents (SD) and subsequently by 3000 steps of conjugated gradient (CG). Subsequently, solvation of each system was performed by TIP3P water molecules31 of approximately 6800 atoms for RBD1 and 7300 atoms for RBD2 in a periodic box at a distance of 12 Å apart from the protein surface, resulting in a box dimension of 63 × 70 × 62 Å3, and 70 × 66 × 63 Å3, respectively. The systems were neutralized using Na + counter ions. Periodic boundary condition with isothermal-isobaric ensemble (NPT) ensemble and a step-size of 2 fs for the simulation time were applied. The water molecules and ions were then minimized with 1000 steps of the steepest descent (SD) and continued with 3000 steps of the conjugate gradient (CG) method. The entire system was fully minimized in the last step by the same minimization process. All bonds with hydrogen atoms were constrained using the SHAKE algorithm32. MD simulations under periodic boundary conditions were performed five times for all systems using the AMBER16 software package30.The top five NMR structures of MSI1 RBD1/2:GUAGU complexes were retrieved from PDB IDs 2RS2 and 5X3Z. The LEaP module of AMBER1633. The interactions between protein and RNA were visualized and analyzed using Discovery Studio Visualizer34. Additionally, the solvated interaction energy (SIE)35 method was applied to estimate the binding affinities of MSI1 RBD1/2 RNA complexes, and to determine the binding contribution of each nucleotide. SIE is an end-point physics-based scoring function that approximates the binding free energy from the force–field non-bonded interaction terms, continuum solvation, and configurational entropy linear compensation35. For each individual simulation, the SIE binding free energy of the complex was calculated over 200 snapshots from the last 20 ns  using the equation:The MD simulation started by heating the system from 10 to 310 K. Next, the system was equilibrated at a constant temperature of 310 K. 100 ns MD simulation was performed under NPT conditions at 1 atm and 310 K. The last 20 ns MD trajectories were taken for structural and energetics analyses. Root-mean-square displacement (RMSD) and distance between the centers of mass of protein and RNA were calculated by the cpptraj module of AmberTools16Ec) and van der Waals interactions (∆EvdW), the electrostatic solvation contribution (∆GR), reaction field energy, and nonpolar desolvation energy. ∆Ec and van der Waals interactions of the bound state were calculated with AMBER ff14SB and OL3 molecular mechanics force fields. The electrostatic solvation contribution was carried out using the continuum dielectric model with a solute interior dielectric constant and a solvent dielectric constant. The reaction field energies were considered by the Poisson equation with the boundary element method program. The nonpolar desolvation was estimated by a linear proportionality with the change in the solute molecular surface area35. Note that the global proportionality coefficient associated with the loss of conformational entropy upon binding (α) is 0.104758, while the solute interior dielectric constant (Din) is 2.25. The molecular surface area coefficient (γ) is 0.012894 kcal/mol−1 Å−2, ΔMSA(ρ) is the difference in molecular surface area between the bound and free state of the protein and constant (C) is − 2.89 kcal/mol−1. These parameters were optimized by fitting to the absolute binding free energy37 The binding affinity values of the canonical RNA motif (GUAGU) and three modified RNA motifs  with MSI1 RBD1 and MSI1 RBD2 from the SIE method were taken from the 200 snapshots of the last 20 ns of the five models of each system . For the amino acids involved in each nucleotide binding of the four RNAs, ΔGbind,res calculations based on the MM/PBSA method were performed on the same series of 1000 snapshots.The binding affinity prediction was estimated by summation of Coulomb interactions  are used to determine the validity of the Alphafold2 results . For MSI1 RBD1, the core structure is covered by approximately 600 sequences at each position, while there are only approximately 100 sequences in the C terminal region . Likewise, the model confidence at each position increases up to 90% and drops to 70% and 40%, respectively, at the flexible loops and C-terminal. Interestingly, all predicted structures of the five models of MSI1 RBD1 exhibit a similar structure, except in the C terminal region. A similar situation is found for the MSI1 RBD2 models, except that the model confidence at the two terminals is lower to some extent . The core structure of the predicted models MSI1 RBD1/2 is comparable to the experimentally solved structures (PDB IDs 1UAW and 5X3Y), in particular, RBD2 with the RMSD value 1.777 and 0.837 Å, respectively , which has been extracted from the corresponding NMR structure. The root-mean-square displacement (RMSD) during the simulation was evaluated from the geometric coordinates of all atoms of the complex, as well as from the RBD site with respect to those of the initial structures. As shown in Figure S3A, the RMSD values of the predicted MSI1-RBD1:GUAGU increase up to ~ 5.0 Å during the first 20 ns, then decrease to ~ 3.1 Å with a fluctuation of approximately 0.5 Å until the end of the simulation. For MSI1-RBD2 , RMSD increase is found within the first 20 ns and maintained at around 6.0 Å with a fluctuation at 1.0 Å up to 100 ns. The RBD site exhibits a much lower RMSD of ~ 1.0–1.7 and ~ 2.0–2.3 Å in both systems, respectively. This implies high fluctuation at the protein terminals, especially at the C terminal end, as well as flexible loops, and the 3' end of the GUAGU pentamer .Gbind results of MSI1–RBD1  and MSI1–RBD2  are comparable, and the Coulomb interaction plays a significant role in RNA binding, approximately 2–3 times higher than the vdW interaction  in Figure S5. To this end, nucleotides of the pentamer triplet cores were adjusted using the NMR structure to obtain starting geometries for MD. The last snapshots from all simulations were superimposed and are depicted in Figure S6, while the root mean square fluctuation (RMSF) of protein residues is shown in Figure S7. MSI1-RBD1/2 in complex with the canonical RNA GUAGU show the highest stability among all complexes, i.e., the pentanucleotide is well accommodated within the RBD site.In addition to studying the MSI1-RBD1/2 in complex with GUAGU, which has been obtained from NMR structures, we set out to explore three alternative RNA pentamers, i.e., GUUGU, GGAGU, and GAUGU, by MD simulations. The overall tightness of the MSI1-RBD1 and MSI1-RBD2 in complex with these four RNA pentamers bound was assessed by radius of gyration  in the GUAGU model, Lys177 (16%) in the GUUGU model, Phe154 (61%) in the GGAGU model, Phe112 (87%), and Phe154 (70–71%) in the GAUGU model. One electrostatic interaction was also discovered for the phosphate group between the third and fourth unit of the RNA in the GUAGU, whereas two electrostatic interactions were found in the GUUGU, GGAGU, and GAUGU.21. To investigate the properties of the RNA–protein association complexes, we performed molecular dynamics simulations and computed the interaction energies by the SIE method.Musashi genes have attracted considerable interest as regulators of stem and progenitor cell characteristics. In the present study, we evaluated the three-dimensional structures of the MSI1 in complex with RNA. To this end, we studied the association of MSI1 RNA-binding domains 1 and 2 (RBD1 and RBD2) with different RNA motifs. We investigated the canonical RNA motif GUAGU, as well as the alternative motifs GUUGU (good binding affinity), GGAGU (weaker binding affinity), and GAUGU (unfavorable binding affinity). We compared the protein structures without RNA from the PDB with Alphafold2-predicted geometries and found that protein structures of the binding domains are highly similar, therefore no conformational changes on the protein occur upon binding of the RNA. In addition, our results corroborate earlier findings that MSI1 RBD1 and RBD2 structures are remarkably similar, despite variation in the underlying primary sequence17, the central trinucleotides of the RNA pentamers  are more rigid than the flanking nucleotides. Moreover, the flanking nucleotides lack interaction with MSI1 RBDs13, suggesting that MSI1–RBD1 and RBD2 require the central trinucleotides for recognition. Our MD simulations show that the central trinucleotides of the RNA motifs exhibit a significantly lower distance to the MSI1 RBDs than the enclosing nucleotides. Thus, the central trinucleotides play an important role in the interaction of MSI1–RBD1 and RBD2 with RNA.In agreement with earlier resultsWe identified key residues for MSI1–RBD1 binding, specifically Phe23, Trp29, Phe63, Phe65, Phe96, Arg98 and Arg99 interacting with nucleotides. Our MD simulations are consistent with the fact that Phe23, Phe63 and Phe65 are conserved among all models and interact with A3 and G4 of the pentanucleotides. For MSI1–RBD2, Lys110, Phe112 Gly115, Phe152, Phe154, Gln185, and Lys187 are in contact with the nucleotides. Stacking interactions between evolutionarily conserved phenylalanine  and non-conserved residue tryptophan , phenylalanine (Phe96:RBD1 and Gln185:RBD2) of MSI1 and the aromatic bases and ribose rings of the RNA contribute to target recognition within MSI1.The SIE calculations lead to the following conclusions: Assessment of the contributions to the overall binding free energy of individual nucleotides of the GUAGU and GGAGU motifs shows that the central core nucleotides have the largest interaction energies, with A3 and G4 nucleotides exhibiting the most pronounced contribution. The flanking nucleotides contribute significantly less. Our calculations show that for RBD1, the GUUGU motif possesses the largest binding free energy, followed by GUAGU. While this appears counterintuitive, it is in line with earlier data that assessed opening energy z scores at the level of RNA secondary structures. RBD2 on the other side has overall smaller interaction energy, with the GUAGU motif showing the highest binding affinity for all pentamers.Calculated decomposition energies clearly show the contributions of individual amino acids to the complexation of the RNA. For RBD1, we should highlight Phe23, Phe63 and Phe65 because of their substantial interaction with the core motif. In analogy, Phe112, Phe152 and Phe154 of RBD2 show a strong interaction with the core trinucleotides.In summary, we show here the feasibility of MD and SIE calculations to investigate the selectivity of RNA–protein interaction complexation. Further studies are warranted, such as the binding of a longer RNA chain that includes both binding motifs of the two RBDs of Musashi proteins. MSI1 plays a particularly important role in brain development, and increased expression of Musashi proteins in patients infected with Zika virus during pregnancy has been associated with microcephaly. A better understanding of the interaction of the MSI proteins with RNA, in particular, Zika virus RNA is required to address the issue of inhibiting Zika virus replication in infected patients without affecting brain development.Supplementary Information.


Models of consumer choice that assume rational decision processes are too simplistic, as they ignore intuitive processes and combinations of intuition and rationality. In dual process theory, System 1 processes are intuitive, fast, require low cognitive effort, and involve autonomous systems, while System 2 processes are deliberative, slower, reflect greater cognitive effort, and involve controlled attention. The dual process framework facilitates understanding of decision processes that may be diverse and complex. Based on response time as an indicator of System 2 use, we fill gaps in the tourism and choice experiment literatures by i) assessing the dimensionality of a decision style scale and its role in predicting System 2 use and ii) assessing whether researcher interventions, such as instructions, can promote System 2 use. The study is based on survey-based choice experiment responses of 483 domestic and international visitors across two Norwegian nature-based tourism destination contexts. Each visitor completed four choice experiment tasks for a total of 1,932 choice occasions. Results indicated diversity in extent of System 2 use. The decision style scale was multidimensional with both the intuitive and rational subscales predicting response time. We encourage inclusion of decision style scales–and specifically multidimensional scales–in future tourism choice and choice experiment applications. Statistically significant coefficients for instructions and unhurriedness suggest potential for researchers to increase System 2 processing in survey tasks. We encourage future use of this intervention, especially when survey tasks are intended to replicate “real world” decisions that rely heavily on System 2 use. There is recognition within both the applied discipline of tourism and the core discipline of economics that models of consumer choice are too simplistic when they assume deliberative and rational decision processes, ignoring intuitive processes and the combination of intuition and deliberation –5. Dual The extent to which System 2 is used for a given choice task may depend on multiple factors, including situational characteristics, such as the difficulty of the task, and personal characteristics, such as the respondent’s disposition to engage in reasoning or the respondent’s prior experience with similar choices , 8. For For example, if decision style (disposition) affects System 2 use, an understanding of individual differences in decision style may facilitate customization of marketing approaches across consumers and consumption contexts. In addition, survey respondents may be less likely to engage in an extensive System 2 process in survey tasks than in the “real world” consumption decisions those survey tasks are intended to replicate. In such cases, instructions or other catalysts for System 2 use during survey tasks may enhance the accuracy of survey results relative to real world decisions.A review of the literature indicates limited application of dual process theory in the tourism and choice experiment fields , 9. PachDual process theory is a framework for understanding choice processes. As with other theoretical frameworks, it provides a structure that can stimulate and guide research in order to build knowledge. Based on response time as an indicator of System 2 use, we fill gaps in the tourism and choice experiment literatures by i) assessing the dimensionality of a decision style scale and its role in predicting System 2 use and ii) assessing whether researcher interventions, such as instructions, can promote System 2 use. The study is based on survey choices of domestic and international visitors in two Norwegian nature-based tourism destination contexts, but the approach is relevant to other tourism and choice experiment contexts.The present study is based on the serial default-interventionist model within dual process theory . In thisMuch dual process theory research involves limited tasks with normatively correct responses and the potential for varying degrees of System 2 processing. For example, a classic task is the bat-and-ball cognitive reflection test : “A bat Choice experiments are widely used in tourism and other fields to understand preferences across choice options , to assess the importance of the attributes that characterize each option, and to estimate willingness-to-pay for attributes and packages of them –17. The Choice experiments differ from traditional dual process theory laboratory tasks. The consistency of responses across multiple choice experiment tasks can be evaluated , 19, butTask and time differences should be considered when applying a dual process theory model developed from controlled laboratory studies to choice experiment studies. Nonetheless, there is recognized diversity in tourist and choice experiment decision making processes , 9, withThe degree to which System 2 is utilized in a given decision making process is not measured directly; rather, it is assessed indirectly, if imperfectly, via indicators , 9, 21. An alternative indicator is response time, the time taken to provide a response to the task. Elapsed time may be high in the System 2 evaluation and rationalization processes that lead to reporting of A1, such that longer response time potentially reflects System 2 rationalization of A1 rather than reasoning and generation of A2. Nonetheless, response time is commonly used in the psychology and, more recently, choice experiment literatures , 23. ResVarious factors potentially affect the extent of System 2 use. Though the present study is based on dual process theory, there is a related recognition of variability in response strategies in the survey research literature, using a continuum from satisficing (consistent with System 1) to optimizing (consistent with System 2).The extent of System 2 has been linked to personal characteristics, including an individual’s inclination to engage in slow, deliberative processing , 26; thiOne might expect choice experiments to fall into the rational task category given the assumption that individuals systematically consider attributes and levels . HoweveDe Bekker‑Grob and colleagues , 13 incoWith respect to additional personal characteristics, motivation inherent in an individual’s disposition may be complemented by motivation due to an individual’s familiarity with, and interest in, the decision task . Trip chDemographic characteristics may affect both motivation and ability. For example, IQ, education, and similar characteristics play a role in System 1, but in particular may reflect the capacity to inhibit the A1 default response and generate a new response via System 2 . CognitiLikewise, there may be conflicting forces with respect to age. Individuals in higher age categories may experience decreased working memory and inhibition control, with an increased age deficit particularly impactful for complex tasks such as choice experiments . This maThe selectivity hypothesis posits that females tend to process information more comprehensively than do males . In the With respect to researcher-affected factors, there often is a goal of increasing the extent of System 2 processing (in the survey research literature: increasing optimizing over satisficing), so an important question is whether researchers can increase this extent. One potential mechanism for doing so is to inform respondents of the importance of the decision–or of engaging in deliberative processing , 39. As Extensive use of System 2, either to rationalize A1 or to generate A2, typically requires more time than quick reporting of A1. Thus, the researcher goal may be to alleviate the time pressure that might limit System 2 processing , 8. ThisWhen assessing the contribution of personal or researcher-affected factors, it is important to control for additional factors that might affect response times. Choice experiments typically involve responses to multiple choice tasks, with respondents potentially becoming more time efficient as they progress through the tasks, due to learning about the tasks and about their preferences across attributes . In addiThough not well evaluated within a dual process framework, the screen size of the device through which the survey is completed  potentially affects response time. On the one hand, smaller devices may increase the use of heuristics to simplify choice tasks that may be visually both intensive and extensive. On the other hand, the challenge of task completion on smaller devices may reduce feelings of fluency and thus catalyze increased use of slower System 2 processing . In addiLastly, some response patterns may reflect the degree of System 2 processing and affect response time. Examples of survey satisficing behavior include straightlining or non-differentiation across multiple survey or choice tasks, with outcomes including selecting the same value across all items in a Likert scale or the same option across all choice tasks , 9, 40 , researcher-affected factors (instructions and unhurriedness), and control factors.Evaluation of personal characteristics may help researchers and product marketers understand individual differences in tourist and choice experiment decision making. Evaluation of researcher-affected factors may help researchers increase the use of System 2 processing when conducting choice experiments. Evaluation of control factors may provide a richer understanding of System 2 processing, as well as reduce the potential effect of confounding factors.Specific research questions are as follows, with response time serving as the indicator of System 2 processing. Expectations are based on previous research, described in Section 1.2.1a. Is respondent decision style (disposition) in the tourism choice context multidimensional?Decision style is expected to be multidimensional, with separate factors (dimensions) for the intuitive and rational subscales.1b. Do both intuitive and rational dimensions of decision style predict System 2 processing?Intuitive decision style is expected to negatively correlate with response time, while rational decision style is expected to positively correlate with response time.2. Do instructions and respondent unhurriedness predict System 2 processing?Instructions and unhurriedness are expected to increase response time.3. Do past trip experience, education, age, and gender  predict System 2 processing?a priori expectations about the other factors.Female respondents are expected to have longer response times. Due to potential countervailing effects, there are no 4. Do task order, utility difference, screen size, and satisficing behavior  predict System 2 processing?a priori expectation about the effect of screen size. Satisficing behavior is expected to reduce response time.Later tasks are expected to involve less response time than earlier tasks. Tasks with larger utility difference are expected to involve less response time than those with smaller utility difference. Due to potential countervailing effects, there is no This study was based on surveys of nature-based tourism visitors to the Trysil and Hardanger areas of Norway . Trysil The surveys included choice experiments of destination package choice, with activities representing a primary attraction at each site. The Norwegian Centre for Research Data (NSD) evaluated the handling of personal data in this project  as being in accordance with data protection legislation.Study participants were recruited on-site with a short self-administered registration survey during the summer season of 2017. They were then invited to complete the full survey online in 2018. Response rates for these on-site samples were 52 percent for Trysil (mountain bikers) and 45 percent for Hardanger (hikers).In addition, the tourism board in Trysil (Destinasjon Trysil) asked persons on their mailing list  to complete the online survey if they mountain biked in Trysil in 2017. A total of 847 (8 percent) responded, but only 383 of these were confirmed as bikers. Of those, 224 (58 percent) completed the survey. A drawing-based incentive of 1000 euros was provided for survey completion, applied to the on-site and Destinasjon Trysil samples combined.For the choice experiments, survey participants were asked to assume they were planning their next trip and deciding whether to book a package for the trip. We then presented participants with introductory information and four sequential choice tasks comprised of two alternative packages, each containing specified levels of characteristics (attributes). We asked participants to assume they only had a choice between the two presented package options or a neither (opt out) option. The choice experiment results are reported elsewhere , but an A d-efficient experimental design with 24 choice sets was created in Ngene version 1.2  and usedThe primary analysis was of response time as a function of personal characteristics, researcher-affected characteristics, and controls. The variables are described here, with survey wording presented in Tables The respondent-level variables  reflect between-subjects data. The choice-level variables  reflect within-subjects data. The decision style analysis (results in nd) was the elapsed time between clicking the continue arrow at the bottom of the first choice task screen to clicking the continue arrow at the bottom of the second choice task screen.The choice experiment introduction and each of the four choice tasks were presented on separate screens. Response time is the elapsed time in seconds from i) when the respondent clicked to proceed to the screen to ii) when the respondent clicked to proceed to the next screen. Thus, the response time for the second task (Order 2Because decision style (disposition) may vary across decision objects , intuitiTrip experience was measured as the mean of i) the number of sites at which the respondent had participated in biking (for Trysil) or hiking (for Hardanger) in the previous year and ii) the number of years the respondent had engaged in biking or hiking. Education, gender, and age were analyzed as dummy variables, with males and the lowest level of education and age used as reference categories.The choice experiment task included an introduction, with half the respondents selected at random being presented an additional paragraph specifically requesting that respondents take their time because the destinations may make investment and management decisions based on their responses. In addition, we asked respondents to report their affect while completing the survey, using a semantic differential format. One of the five word pairs was hurried and unhurried (relaxed).Several variables were used as controls. The order within the set of four choice tasks was recorded for each task. Utility difference calculation followed Olsen et al.  and was The trip experience and screen size variables were transformed  to preserve two significant digits in the presentation of coefficients . The disA conservative listwise approach was utilized, with all four choice observations for a respondent deleted if there was a missing value for any variable in the model. For example, all four observations were removed if a respondent did not report education or spent more than 600 seconds on one of the four choice tasks. This led to 1,232 choice observations (308 respondents) for Trysil and 700 choice observations (175 respondents) for Hardanger.The response time model was analyzed with the Mplus version 8  two-leveExploratory factor analysis results for the modified Decision Styles Scale are shown in 2 for the choice level (utility difference and the three order variables) was 0.33. The R2 for the respondent level  was 0.19. Thus, a combination personal characteristics, researcher-affected factors, and task characteristics is relevant for understanding, and explaining variation in, response time.Model results are presented in Response time increased with increasing levels of rational decision style and decreasing levels of intuitive decision style. Females tended to spend more time responding. Time on task increased with motivational instructions and when respondents felt unhurried.With respect to task characteristics, respondents spent less time on each choice as they proceeded through the four choice tasks. They also spent less time on choice tasks involving larger utility differences. Respondents completing the choice tasks with a larger device did so more quickly than those with smaller devices. Lastly, respondents selecting all the same option completed the tasks relatively quickly.This study responds to the call for empirical evaluation of the dual process framework in the context of tourism and broader choice contexts , 5, as wWith respect to the four research questions (Section 1.3), results were consistent with all prior expectations for coefficient signs, which were based on previous research. For Research Question 1a, a modified version of the Decision Styles Scale  had goodFor Research Question 1b, both decision style subscales significantly predicted response time, with respondents high in intuitive decision style completing choice tasks relatively quickly and respondents high in rational decision style taking more time. This was consistent with the expectation that respondents high in rational decision style engage in more System 2 processing than do those low in rational decision style. The present study appears to be the first to use a decision style scale in a specific tourist choice context  and theWith respect to Research Question 2, unhurriedness and instructions to increase motivation and System 2 processing both predicted response time. While recognizing the limits of encouraging System 2 use in non-laboratory tasks, results were consistent with the broader literature indicating the potential to increase deliberative processing through instructions , 40. MorThe present study did not manipulate unhurriedness when completing the survey, but results suggest the potential benefit of researchers encouraging respondents to complete the survey when they have time to do so in a relaxed manner. Researchers may facilitate this goal by ensuring the duration of the overall survey, or of the choice task component specifically, is reasonable given the context .With respect to the role of personal characteristics beyond decision style (Research Question 3), female respondents utilized more time in completing the choice tasks, consistent with the selectivity hypothesis . CoefficThis study fills a gap in the literature and introduces a multidimensional decision style scale to the tourism and choice experiment fields. Present results indicate the importance of using multidimensional measures in future studies.In addition, the study illustrated how researchers may be able to promote System 2 use in survey contexts. We do not assume that System 2 is inherently better than System 1, but we expect that some “real world” decisions are more likely than others to involve System 2 processing. Insofar as research is intended to replicate “real world” decisions, such as to generate willingness-to-pay estimates for policy making, interventions such as instructions potentially increase System 2 use in the survey context to better match System 2 use in “real world” contexts–thereby contributing to more informed policy making.Most fundamentally, this study contributes to the tourism and choice experiment literatures by extending the promising, but so far relatively limited, use of dual process theory. Personal characteristics have been a significant focus of the tourist decision making and marketing literature , 52, andThe specific inclusion of a decision style scale when evaluating tourist decisions can enhance both theory development and the effectiveness of destination marketing by helping illuminate the “black box” of the diverse processes involved in decision making, thereby allowing more inclusive and customized tourism marketing approaches. For example, the mix of marketing approaches can be customized to potential customers who make decisions quickly and with presumed reliance on System 1 processes, those who engage in substantial information processing (System 2), and those with varying combinations of these approaches. As McCabe, Li, and Chen [3 p 12] note, marketing messages across different contexts can be aligned with decision processes; they observe that avenues for future research include the “timing of campaigns, type of information provided, pricing strategy, and use of discounts and promotions to elicit specific types of decision strategies.”This study focused on dual process theory in the specific context of a survey-based choice experiment of nature-based tourism destination choice in Norway. We encourage replication to better understand how System 2 use, and the factors predicting it, varies across choice contexts, including across diverse types of tourist choices and across non-tourist choices. Relative to the present trip choice context, some tourist decisions may rely more heavily on System 1 processing, with the intuitive decision style playing a positive role. Conversely, other decisions may rely more heavily on System 2, with the rational decision style playing a positive role. It will be important for future research to assess and model decision style in a multidimensional format unless a unidimensional format is indicated, such as by a single-factor solution in exploratory factor analysis.The inclusion of decision style as a predictor of System 2 use fills a gap in the literature. However, substantial variance in response time remains to be explained. Therefore, we encourage further exploration of potential predictors to explain individual differences in System 2 use.The present study focused on response time as the outcome, rather than as a predictor of response quality in choice experiments. In the latter context, response time has been used in various forms, including as a criterion for membership in latent classes , while pChoice experiments are often conducted via surveys, with interest in response time motivated in part by concern that estimates of preferences and willingness-to-pay may be affected by “professional” respondents in internet panels . There iS1 Data(XLSX)Click here for additional data file.


Avsunviroidae) and viroid-like satellite RNAs share self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity strands. Using a homology-independent method based on the search for conserved structural motifs of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it was not found again in the original citrus source when repeatedly searched in the following years, suggesting that it was actually not directly associated with the plant. Therefore, the possibility that this novel self-cleaving viroid-like RNA is actually associated with another organism , in turn, transiently associated with citrus plants, is proposed.Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs reported in plants only so far. Some viroids (family  Viroids and viroid-like satellite RNAs (Vd-LsatRNAs) are small, infectious, non-protein-coding and circular RNAs reported in plants only so far ,2,3,4. TPospiviroidae and Avsunviroidae. Members of the family Pospiviroidae adopt a rod-like or quasirod-like conformation of minimal free energy, which contains a central conserved region (CCR) involved in their nuclear replication mediated by an asymmetric rolling circle mechanism [Avsunviroidae lack the CCR but contain in both polarity strands the structural elements needed to form hammerhead ribozymes (HHRzs) [According to structural, biological and functional features, viroids are classified in the families echanism . This reechanism . Viroids (HHRzs) . These r (HHRzs) ,13. HHRz (HHRzs) . Both vi (HHRzs)  previous (HHRzs) . Sequenc (HHRzs) .Apiognomonia erythrostomam) infecting cherry leaves [Whether viroids or Vd-LsatRNAs also exist in organisms other than plants is not known. However, indirect evidence was provided for two small circular RNAs containing HHRz in each polarity strand (cscRNA1 and cscRNA2), which have been isolated from cherry leaves ,18. Theyy leaves ,22,23,24y leaves . These fPospiviroidae have been identified in plants by high-throughput sequencing and the following search for de novo assembled contigs with sequence similarity to viroids previously annotated in databases [Avsunviroidae share little-to-no sequence identity. Instead, relationships between viroids of this family are based on structural features, such as the quasirod-like or branched secondary structure, tertiary interactions  and the type of hammerhead ribozymes embedded in both polarity strands of their circular RNAs [Avsunviroidae, apple hammerhead viroid, and a potential new viroid of the same family, grapevine hammerhead viroid-like RNA, were identified using HTS-based methods that are sequence homology-independent and identify potential novel viroids and/or viroid-like RNAs by looking for de novo assembled contigs with terminal direct repeats, a hallmark of potential circular RNAs [In the last few years, several new viroids of the family atabases ,28,29,30lar RNAs . Therefolar RNAs ,32.Avsunviroidae and other viroid-like RNAs, we focused our attention on HHRz, a specific hallmark of these infectious agents to develop a homology-independent method to search for HHRzs in reads or de novo-generated contigs from HTS libraries. By searching in the RNA preparations from a citrus tree grown in the field, a novel Vd-LRNA containing one HHRz in either polarity strand was identified. The molecular and biological features of such a novel circular RNA, tentatively named citrus transiently-associated hammerhead viroid-like RNA 1 (CtaHVd-LR1) are presented and discussed here.In an attempt to identify novel viroids of the family Citrus reticulata plant grown in Southern Italy (isolate 14A) and showing the typical symptoms of this disease was analyzed. Very young green sprouts were collected in spring 2018 and stored at −80 °C. A sample of the collected material (5 g) was extracted with buffer-saturated phenol and the preparation was enriched in double-stranded (ds) RNAs partitioning the nucleic acids by chromatography on non-ionic cellulose CF-11  with STE ) containing 16% ethanol [In the frame of a study aimed to identify the potential causal agent of cristacortis, a virus-like disease of still unknown etiology, a  ethanol . Followi ethanol , using STrimmed and high quality filtered reads from the sequenced RNAseq library were assembled de novo using SPAdes , and to dsRNA enriched preparations were reverse transcribed using Superscript IV reverse transcriptase  and random primers following the manufacturer’s instructions. PCR amplification of the cDNA was performed using 2 μL of the reverse transcription reaction, specific primers  at a finNorthern blot hybridization assays were performed as reported previously . BrieflySpeI or NcoI and transcribed with T7  or SP6 RNA polymerase (New England Biolabs) to obtain HVd-VL1 RNA transcripts of plus and minus polarity, respectively. After degradation of the DNA template with RQ DNase , the in vitro transcription reaction was analyzed in a 5% PAGE under denaturing conditions (see above). The full-length monomeric RNAs and the 3′ self-cleavage products generated during the in vitro transcription were eluted from the acrylamide gels by excising the band and grinding with one volume of water-saturated phenol and one volume of elution buffer  [Monomeric transcripts of both polarity strands were obtained by in vitro transcription of the pGemT-easy plasmid containing the full-length cDNA sequence of CtaHVd-LR1 (variant 3). The recombinant plasmid was linearized by digestion with .5% SDS) . The RNAFor the analysis of RNA self-cleavage, the eluted RNAs were resuspended in 1 mM EDTA pH 6, boiled for 2 min, snap-cooled on ice and then incubated at 40 °C for 1 h in self-cleavage buffer . The self-cleavage reaction was analyzed in 5% PAGE under denaturing conditions as indicated above. The 5′ terminal sequence of the eluted 3′ self-cleavage RNA products was determined by 5′ RACE as described  using thAvsunviroidae and most viroid-like RNAs contain one HHRz in either RNA polarity strand. The active conformation of these catalytic motifs assume a conserved secondary structure, with typical loops and stems. By comparing all natural HHRzs known so far, we developed a specific pattern syntax , which was composed of a sequence fragment of 550 nt followed by a partial direct repeat of 101 nt  in the plus polarity strand that, however, does not show any significant similarity with proteins in GenBank and does not contain any confidently predicted domain, motif and feature according to a search in SMART . These fAvsunviroidae [Circular RNAs can be separated from the respective linear forms by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions that determine a delayed migration of circular RNAs with respect to the linear ones . To asceviroidae  and otheviroidae . Indeed,viroidae .The existence of CtaHVd-LR1 in the original RNA preparations used for generating the RNAseq library was initially confirmed by RT-PCR using specific primers designed in the contig . With alIn the proposed rod-like structure, the central region contains the HHRzs of the two polarity strands that were located one in front of the other. Interestingly, most polymorphic positions did not modify the proposed rod-like secondary structure because they were co-variations, conversions of canonical into wobble base-pairs or were located into loops . It is wThe CtaHVd-LR1 hammerhead ribozymes have a central catalytic core surrounded by three hairpins, two of which (hairpin I and II) are closed by small loops . The sel2, most of the transcript self-cleaved, generating the expected RNA fragments  polarity strand had the expected nucleotide at the 5′ terminus. In the (+) polarity strand, an extra nucleotide was observed at the 5′ terminus of the 3′ fragment generated by the (+) HHRz . HoweverSince HHRzs have also been reported in circular RNAs that have a DNA counterpart integrated in the host genome  ,6,7,8, wWhile RT-PCR assays performed using RNA preparations from the original sample of sprouts collected in 2018 from the isolate 14A were always positive, regardless of the primer pair  used, alIn line with the results reported above, the attempts of transmitting CtaHVd-LR1 to two grapefruit and two sour orange seedlings by grafting bark tissues from stems collected from the isolate 14A in early spring 2018 also failed, as confirmed by the negative results obtained when samples from the inoculated seedlings were tested for the next four years by RT-PCR. In contrast, in all the inoculated seedlings, CiVA was detected by RT-PCR already six months post-inoculation and during the following years, confirming that this virus was effectively graft-transmitted to the indicator plants and that the infection was stably maintained over time.Altogether, these data support the view that CtaHVd-LR1 was only transiently associated with the citrus isolate 14A in 2018 and, possibly, that this circular RNA is not able to infect this host. Instead, considering the results of this study, the alternative possibility that CtaHVd-LR1 was infecting another organism transiently associated to the isolate 14A appears more feasible.The identification of CtaHVd-LR1, by looking for conserved motifs of HHRzs, showed that homology-independent methods based on the identification of structural elements may be an effective method to find new viroid-like RNAs. With respect to the previously reported homology-independent methods, the one used in this study does not rely on the effective de novo assembly of the full genome plus terminal repeats of the viroid or viroid-like RNA for its identification. Indeed, when a HHRz is identified in a read that is not assembled in a contig with terminal repeats, adjacent primers of opposite polarities can be designed in the read of interest to check, by RT-PCR, whether the HHRz is part of a potential circular RNA that can be further characterized by sequencing full-length cDNAs and by northern blot hybridization assays.Chrysovirus, Partitivirus and Totivirus [Chrysovirus, Partitivirus and Totivirus. The fact that CtaHVd-LR1 was not found in the same isolate in the following years strongly supports the conclusion that it was only transiently, and possibly indirectly, associated with citrus plants. In this respect, the possibility that it could be an infectious agent of an organism other than citrus is quite likely. In conclusion, our study highlights the high sensitivity of HTS approaches associated with homology-independent bioinformatics tools to identify novel viroid-like RNAs. At the same time, we showed that the finding of a novel viroid-like RNA in a plant RNAseq library must not be considered as evidence of a direct association of the former with the latter. As also previously reported for plant viruses [By showing that CtaHVd-LR1 is a circular RNA endowed of an active HHRz in either polarity strand, we provided solid evidence that it is indeed a novel viroid-like RNA, confirming the reliability of the searching method. The detection of circular RNA forms of both polarity strands strongly support that this RNA replicates through the symmetric pathway of a rolling circle mechanism previously reported for several viroids and viroid-like RNAs . The biootivirus ,21,22,23 viruses ,52, the


The fast rate of viral mutations of SARS CoV-2 result in decrease in the efficacy of the vaccines that have been developed before the emergence of these mutations. Thus, it is believed that using additional measures to combat the virus is not only advisable but also beneficial. Two antiviral drugs were authorized for emergency use by the FDA, namely Pfizer’s two-drug regimen sold under the brand name Paxlovid, and Merck’s drug Lagevrio. Pfizer’s two-drug combination consists of nirmatrelvir, a protease inhibitor that blocks coronavirus ability to multiply and another antiviral, ritonavir, that lowers the rate of drug clearance to boost the longevity and activity of the protease inhibitor. Merck’s drug Lagevrio (molnupiravir) is a nucleoside analogue with a mechanism of action that aims to introduce errors into the genetic code of the virus. We believe the armament against the virus can be augmented by the addition of another class of enzyme inhibitors that are required for viral survival and its ability to replicate. Enzymes like nsp14 and nsp10/16 methyltransferases (MTases) represent another class of drug targets since they are required for viral RNA translation and evading the host immune system. In this communication, we have successfully verified that the MTase-Glo, which is universal and homogeneous MTase assay can be used to screen for inhibitors of the two pivotal enzymes nsp14 and nsp16 of SARS CoV-2. Furthermore, we have carried out extensive studies on those enzymes using different RNA substrates and tested their activity using various inhibitors and verified the utility of this assay for use in drug screening programs. We anticipate our work will be pursued further to screen for large libraries to discover new and selective inhibitors for the viral enzymes particularly that these enzymes are structurally different from their mammalian counterparts. The recent pandemic outbreak of COVID19 caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) was first reported in Wuhan, China in late 2019 resulting in infection of about 550 million and death of over 6.3 million world-wide and over 87 million infections with over one million deaths in the USA as of July 2022 (WHO). The infection by this virus is highly transmissible and can lead to severe pneumonia and death. The SARS-CoV-2 is an enveloped virus and belongs to the beta coronavirus family containing a large and complex positive-sense single stranded RNA genome (~30kb) which is similar to the other known six human coronaviruses . The virus binds to human ACE2 receptor with high affinity, compared to the other CoVs, via its spike protein (S) followed by membrane fusion leading to the release of its genomic RNA that replicates and produce higher number of subgenomic RNAs. The viral RNA is then capped to limit its degradation and enhance its translation in the host cell  UUG UUA GUC UA 3’; Oligo #2: 5’ {Gppp.}AG UUG UUA GUC UA 3’; Oligo#3: 5’ {N7-MeGppp.}AG UUG UUA GUC UA 3’. Additional Oligo RNAs were obtained from IDT Technologies, Inc : Oligo RNA 0011: SAH standard curve was prepared in the MTase reaction buffer to assess the linearity of the assay during enzyme titrations in order to calculate the amount of SAH produced at each concentration of enzyme used. The 1μM and 10μM SAH concentration stock were made first in MTase reaction buffer. To perform ½ dilution factor of SAH, 200μl of SAH solution (1μM or 10μM) was added to well A1 and 100μl of MTase buffer was added into well A2 to well A12 in a regular 96-well plate. A serial twofold dilution was performed by transferring 100μl from well A1 to well A2 and mix by pipetting up and down. Then, 100μl was transferred from well A2 to well A3 and the transfer/mix process was repeated from well A3 through well A11. SAH solution was then discarded and well A12 contained only buffer. Once standards are prepared, 4μl of each of the 12 points was transferred to the assay plate and SAH was detected using the MTase-Glo™ Assay following the manufacturer’s procedure. Briefly, 1μl of 0.5X MTase-Glo™ Reagent was added then the plate was mixed for 1–2 min and incubated at room temperature (23° C) for 30 min. Then, 5μl MTase-Glo™ Detection Solution was added and the plate was mixed again before incubating it for another 30 min to be used by luciferase/luciferin reaction. Luminescence was recorded on a plate reading luminometer.Generally, all MTase reactions were performed in MTase reaction buffer using 384 low volume solid white assay plates described previously .For SARS-CoV-2 MTase reactions were performed at 37°C in 4μl for 90min with a proper plate seal. However, for the kinetic studies to determine substrate Km values, the reactions were performed at room temperature (23°C) in 4μl, followed by addition of 1μl of TFA to a final concentration of 1% to stop the reactions. Then, 5μl MTase-Glo™ Detection Solution was added and the plate was mixed again before incubating it for another 30 min to be used by luciferase/luciferin reaction and Luminescence is recorded.50 of inhibitors for SARS-CoV-2 MTases, reactions were performed in 5μl mixture containing 1X MTase reaction buffer, with or without 0.4% DMSO, substrate RNA and either 1ng nsp14 or 60ng nsp10/nsp16 per reaction containing 5μM, 25μM, or 50μM of SAM, and serial dilution of small molecules to be tested at each SAM concentration. Reactions were incubated at 37°C for 90 minutes. After incubation time, MTase-Glo assay protocol was applied before luminescence was recorded and IC50 values were determined. To test P29 polypeptide for inhibiting MTase activity, reactions containing 1ng of nsp14 were carried out same as previously mentioned for small molecule inhibitors. However, with nsp 10 and nsp16 study, the nsp10 and nsp 16 were tested individually or in combination. Inhibition of nsp16 by P29 was carried out by pre-incubating P29 with or without nsp16 for 10min to 15min at room temperature (23°C) before mixing reactions with nsp10 at the following nsp16:nsp10 ratios: 0μM (1:0), 0.5μM (1: 1), 1.0μM (1: 2), 2μM (1: 4), and 3μM (1: 6) and reactions were started by adding substrates (oligo RNAs and SAM) with MTase-Glo Reagent. The reactions were incubated at 37°C for one and half hour. before SAH was detected using MTase-Glo assay protocol.For determination of ICAll low volume 384-well and regular 384-well assay plated were read using infinite 500 or M1000PRO instruments from Tecan Ltd. . For plotting, analysis of data and calculation of MTases biochemical parameters, both Microsoft Excel and Prism from GraphPad Software  were used. IC50 values were determined by using a nonlinear regression fit to a sigmoidal dose response (variable slope).To demonstrate that the assay we developed can determine the activity of SARS-CoV-2 MTases (nsp14 and nsp10/16) and is amenable to high throughput screening for potential MTase inhibitors that may progress forward to clinical testing, it was necessary to establish the sensitivity and linearity of the assay performance.As we have already demonstrated, the assay principle is based on the detection of S-adenosyl homocysteine (SAH), a universal product of all MTases using S-adenosylmethionine (SAM) as the universal substrate. The assay principle is depicted in Since the enzyme nsp14 is known as a capping RNA MTase by methylating the 5’Guanisine triphosphate, we checked its activity using multiple capped and uncapped RNA substrates, in order to find out whether the presence of methylated capped RNA will be as a good substrate as the unmethylated uncapped RNA. For this, we titrated nsp14 in the presence of two groups of substrates. The first group consists of only dinucleotides and the second group contained longer oligonucleotide that are either uncapped or methylated and capped).50 μM SAM and 40 μM Oligo RNAs, SARS-CoV-2 nsp14 MTase generated the highest activity with the uncapped RNA #1 substrate (G (5’) ppp(5’) A RNA) with as little as 5 ng of nsp14 producing a maximum activity. However, when we used the analog substrate RNA# 2 (m7 G(5’) ppp(5’) A) which is identical to RNA#1 but capped, it was a poor substrate with as high as 80 nanogram of nsp14 generating only one sixth of its activity compared to nonmethylated RNA#1 substrate. This suggests that nsp14 prefers uncapped substrates which is expected since its main function is to methylate the 5’G nucleotide. To test whether the nature of the second nucleotide following the first G nucleotide has any effect on the enzyme preference for the substrate, we titrated nsp14 as described above but using the RNA substrate G (5’) ppp(5’) G (RNA#3) instead of G(5’)ppp(5’)A and as a control, its capped version m7 G(5’) ppp(5’) G (RNA#4). It is apparent that the enzyme methylated the oligonucleotide when G in the penultimate nucleotide with similar high activity as when A was in the penultimate position. This means that the nsp14 enzyme loses its stringent selectivity of methylating only the noncapped oligo when the penultimate nucleotide was G albeit at lower rate than with the other noncapped oligonucleotide shown in As shown in To understand fully the substrate requirement for the enzyme, we tested its activity using longer oligonucleotides. The enzyme was again titrated in the presence of an uncapped substrate, a methylated substrate on the 5’G nucleotide, or methylated at the second position nucleotide. As shown in To assess the affinity of nsp14 towards its substrates, we measured the nsp14 methyl donor and acceptor substrate apparent kd values. We compared the activity of the enzyme towards short and long oligonucleotide substrates using different concentrations of both substrates in the presence of constant concentration of the enzyme and the methyl donor SAM (50μM) at 23°C for 30min. It appears that the enzyme prefers the short sequence since apparent kd for the substrate is 1.01 μM and 1.74 μM for the short vs the long oligonucleotides, respectively .We further tested the enzyme for its affinity towards the other substrate (SAM). As shown in Finally, because this MTase assay can be used with a wide range of substrates and substrate concentrations, we compared the enzyme selectivity towards RNA oligonucleotides as well as oligopeptides that are known to be substrates for other MTases. We used a full-length histone H3 and full-length histone H4 that are known substrates for protein MTases. As shown in Since SARS-CoV-2 MTase nsp10/16 MTase heterodimer is known to methylate the ribose sugar of the penultimate nucleotide of RNA substrate, we used methylated capped oligonucleotide (Oligo#3) and other substrates to assess the enzyme activity. Similarly, to nsp14, we observed that the enzyme shows high selectivity towards small oligo and other oligonucleotides that we tested for nsp14  but not Moreover, the results in It has been documented that replication and translation of RNA viruses in eucaryotic cells requires capping of RNA by capping viral MTase (cap-0) followed by methylation of capped RNA at the 2’-ribose of the penultimate nucleotide resulting in the formation of cap-1 structure that mimics cellular mRNA and thus preventing recognition of the viral RNA by host innate immunity , 28. As As discussed earlier, although the human homolog of nsp16 (CMTr1) does the same reaction as the, viral counterpart nsp16 does not require another nsp protein (nsp10) for enhanced activity. Using molecular dynamics simulations, it has been shown that nsp10 binding to nsp16 shifts its conformation to be active , 28, 31.Effect of nsp10-derived peptide on nsp10/16 MTase activity. Since nsp10 is required for maximal activity of nsp16, it was suggested that a peptide derived from nsp10 can be used as inhibitor of nsp16 MTase activity (30–32). We synthesized P29 polypeptide that was reported to inhibit MTase activity of nsp16 and tested its potential inhibitory effect. We found that 200 μM of P29 inhibited the MTase activity of nsp16 (0.5μM) by 20% and 300 μM inhibited MTase activity by 40%. The percent inhibition was similar when using nsp10: nsp16 ratio of 4:1 and 6:1  and its ammonium salt shows antiviral activity in vitro against coronaviruses such as SARS, MERS and SARS-CoV-2, and thus proved useful in scientific research into novel antiviral drugs to combat these diseases. Similarly, the compound sinefungin has been frequently used as MTase inhibitor due to its competitive inhibition with SAM substrate [50 of 2.41, 5.02, and 11.37 μM at 5, 25, and 50 μM SAM concentrations respectively, indicating that sinefungin inhibits nsp14 in a competitive manner towards SAM substrate. When, we tested the effect of sinefungin on MTase activity of nsp14 using the other oligonucleotide Oligo1 substrate to assess its potency . Using Oligonucleotide 3 and different concentrations of SAM, the activity was determined using 0, 50 μM, and 100 μM sinefungin. The results in In this communication, we have successfully demonstrated the utility of MTase Glo, which is universal and homogeneous MTase assay to determine the activity of the two SARS- CoV-2 MTases, nsp 14 and nsp10/16. Furthermore, we have carried out extensive studies on those enzymes using different RNA substrates and tested their activity using various inhibitors and verified the utility of this assay for use in drug screening programs. We anticipate our work will be pursued further to screen for a large library to discover new and selective inhibitors for the viral enzymes particularly that these enzymes are structurally different from their mammalian counterparts.


The nitroxide TPA  is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached. The light sensitive 2‐nitrobenzyloxymethyl group can be removed in high yield by short irradiation at 365 nm. Based on this approach, a doubly spin‐labeled 27mer neomycin sensing riboswitch was synthesized and studied by PELDOR. The overall thermal stability of the fold is not much reduced by TPA. In‐line probing nevertheless detected changes in local mobility. The nitroxide TPA  has been protected with a light sensitive 2‐NBOM group and connected to a uridine phosphoramidite building block. Spin‐labeled RNA can be obtained from this compound by standard protocols of oligonucleotide synthesis and used for EPR studies after light‐induced deprotection. Attaching this radical to cytidine residues as shown in structure 2 is known to destabilize RNA duplexes by 5–6 °C per modification and up to 10 °C when present in loop‐closing base pairs.2. In contrast, TPA [4 combines good spectroscopic properties with low impact on duplex stabilities. The synthesis of TPA labeled RNA,[Although compound cetylene)58, 593. As in the case of compound 1, the photolabile 2‐NBOM group protected the nitroxide and allowed us to assemble RNA strands by unmodified reaction cycles.Afterwards, chain extension was resumed on the synthesizer.3 is shown in Scheme 5 was converted into Weinreb amide 7 by bromination (6) and Favorskii rearrangement. The protected nitroxide 11 was then obtained by N‐oxidation (8) and reaction with 1‐(chloromethoxymethyl)‐2‐nitrobenzene. This alkyl chloride was prepared in situ from compound 10, accessible by Pummerer rearrangement from 2‐nitrobenzylic alcohol 9. The protection of the nitroxide is thought to involve recombination with alkyl radicals generated by reduction of the chloromethoxy‐methyl chain. Such radicals may attack CC triple bonds. We therefore preferred to introduce this group one step later, by DIBAL reduction and conversion of aldehyde 12 with the Ohira‐Bestmann reagent (13). Uridine 14 was transformed by a series of standard procedures into the tritylated and 2’ silylated 5‐iodouridine 19. Sonogashira cross coupling with alkyne 13 and phosphitylation completed the synthesis of compound 3.The synthesis of phosphoramidite 21 and 23 . The photolabile nitroxide protection was then removed by irradiation of an aqueous solution for 20 min .As a first test, we investigated the palindromic 18mer RNAs 22 a eliminated CH2O and was transformed into the TPA‐labeled RNA 23 by spontaneous air oxidation. The cleavage of light sensitive groups from hydroxylamines can break the weak N−O bond leading to amines. The corresponding product 24, however, was not detected in the mass spectrum of the crude mixture after irradiation of 21 . Palindromic sequences form duplexes as shown in Figure 23 the duplex structure prevails . When HPLC‐purified RNA 22 was irradiated, oxidized under air and annealed as shown above, such samples of nitroxide labeled RNA could be directly used for PELDOR experiments without additional purification steps  is reduced to 79.8 °C in case of 23. Thus two modifications in the duplex cause a ΔTm of only 1.7 °C (Table S2).Upon heating to 90 °C for 70 min, the resulting hemiacetal 25, modified in positions 14 and 26.The EPR investigation of the neomycin‐responsive riboswitch25 as described above for RNA 22 from 2’‐OTBDMS protected phosphoramidites and compound 3. After desilylation and purification by HPLC , the 2‐NBOM groups were removed by irradiation for 20 min . The sample was then heated under air to 90 °C for 70 min to eliminate CH2O and to generate the final TPA labeled riboswitch 25 , identical to the material previously reported by Krstić et al.We have now synthesized riboswitch m measurements with in‐line probing as a method which can sensitively record more local effects that may be overlooked if just Tm values are considered. Towards this end the riboswitch analogs 26 and 28 were synthesized, purified and, in case of 28, irradiated to liberate the TPA spin labels. Both RNAs carry a T10 tail and an alkyne linker required for the synthesis of the dye labeled analogs 27 and 29. The Tm of 26 (49.5 °C) nevertheless came close to the value of 50.5 °C reported for the unmodified 27mer RNA.28 slightly reduced the overall stability . Addition of neomycin increased for both RNAs the values by almost 15 °C. As in the case of the 18mer RNA 23, these data confirm the low impact of TPA on general duplex stabilities.An important issue in EPR studies is the possible impact of sterically demanding spin labels on the structure and dynamics of the target molecule. Duplex melting temperatures are the traditional parameters to assess the overall stability of RNA folds. We have combined T2+.N2 displacement at carbon, this reaction has the stereoelectronic precondition of an in‐line alignment of nucleophile, electrophile and leaving group not given in regular helical duplexes. Cleavage is therefore restricted to positions of increased conformational mobility such as bulges and loops. In riboswitch RNAs 25–29 the first five nucleotides are part of a stable stem structure. Accordingly, not much cleavage is seen for these nucleotides upon treatment of the dye‐labeled RNA 27 with Mg2+  and after the nucleotides following U(18). Along this line, we also found that brief annealing of PELDOR samples at 0 °C before freezing resulted in a sharper spin‐spin distance distribution within RNA 25 .The presence of TPA labels in RNA C Figure  trace C:4 and 2 in Figure 3 now offers access to TPA labeled RNAs in a faster and more reliable way. Light induced liberation of nitroxide radicals[Increased rigidity and reduced steric perturbation of nucleic acid structures are the advantages of TPA spin labels over TEMPO‐modified RNA , in‐line probing still detected undeniable local effects. Accordingly, it is highly recommended to combine EPR studies of RNAs with additional secondary structure mapping to rule out major spin label‐induced perturbations.The rigid Ç label and even TEMPO do not significantly change the base pair arrangements in simple RNA duplexes.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.