Sindbis virus (SINV) can infect neurons and cause encephalomyelitis in mice. Nonstructural proteins are translated from genomic RNA and structural proteins from subgenomic RNA. While visualization of viral proteins in living cells is well developed, imaging of viral RNAs has been challenging. RNA aptamers that bind and activate conditional fluorophores provide a tool for RNA visualization. We incorporated cassettes of two F30-scaffolded dimers of the Broccoli aptamer into a SINV cDNA clone using sites in nsP3 (genomic RNA), the 3′UTR (genomic and subgenomic RNAs) and after a second subgenomic promoter resulting in 4–28 Broccoli copies. After addition of the cell-permeable 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI-1T) conditional fluorophore and laser excitation, infected cells emitted green fluorescence that correlated with Broccoli copy numbers. All recombinant viruses replicated well in BHK and undifferentiated neural cells but viruses with 14 or more Broccoli copies were attenuated in differentiated neurons and mice. The signal survived fixation and allowed visualization of viral RNAs in differentiated neurons and mouse brain, as well as BHK cells. Subgenomic RNA was diffusely distributed in the cytoplasm with genomic RNA also in perinuclear vesicle-like structures near envelope glycoproteins or mitochondria. Broccoli aptamer-tagging provides a valuable tool for live cell imaging of viral RNA. Togaviridae family, is an enveloped plus-strand RNA virus that can cause seasonal outbreaks of rash and arthritis in humans and encephalomyelitis in experimentally infected mice3. On entry, nonstructural replicase proteins (nsPs) are translated from the capped and polyadenylated 11.7 kb genomic RNA as two polyproteins (P123 and P1234) to initiate infection. P123 and the nsP4 RNA-dependent RNA polymerase induce spherules at the plasma membrane and replicate the full-length minus-strand RNA in early replication complexes as a template for synthesis of plus-strand RNAs4. P123 is subsequently processed by the nsP2 protease to nsP1 and P23 and then to individual nsPs leading to cessation of minus-strand synthesis. With the switch to stable plus-strand replication complexes genomic and subgenomic (sg) RNAs are synthesized6. sgRNA is translated to produce the structural proteins E1, E2 and capsid required for assembly of virions containing genomic RNA12.Sindbis virus (SINV), an alphavirus in the 16. The RNA aptamer Spinach2 within a tRNA scaffold was incorporated into a cDNA clone of the TE strain of SINV to produce recombinant viruses that expressed viral RNA tagged with the aptamer17. Spinach2 tagging allowed visualization of SINV RNA in live cells as green fluorescence after addition of the non-toxic cell-permeable conditional fluorophore DFHBI-1T ((Z)-4--2-methyl-1--1H-imidazol-5(4H)-one) and excitation at 488 nm17. However, this system had several limitations. First, stop codons are present in all frames of tRNA-Spinach2 precluding insertion into viral protein coding regions and limiting the RNA tagging sites. Therefore, for SINV the Spinach2 aptamers were only inserted into the 3′UTR and/or after an engineered second subgenomic promoter and did not allow differentiation between viral genomic and subgenomic RNAs. Second, the signal was too dim for visualization in a conventional epifluorescence microscope and even with laser-scanning confocal microscopy, the signal remained insufficiently bright for visualization in cells with restricted replication and background autofluorescence, such as differentiated neurons.To aid in studies of the regulation of viral RNA synthesis, we previously applied RNA aptamer technology to develop tagged fluorescent SINV RNA. RNA aptamers can bind and activate a conditional fluorophore that emits fluorescence in response to excitation with light at specific wavelengths19. Unlike the Spinach2 tRNA scaffold, the new more stable F30 Broccoli scaffold is not subjected to intracellular cleavage and accommodates two sets of dimeric Broccoli (dBroccoli)21. The F30-2xdBroccoli  is compact (234 bases) and contains no in-frame stop codons. After binding to the Broccoli RNA aptamer and laser excitation, the fluorophore DFHBI-1T emits green fluorescence similar to green fluorescent protein (excitation/emission = 472/507)21 23 were used. F30-2xdBroccoli, a double dimeric Broccoli (4 copies in the F30 scaffold) from the pET28c plasmid (Addgene 66843) was inserted into at least 1 of 3 restriction sites: SphI engineered into the 3′UTR17, BstEII just downstream of the second subgenomic promoter23, and SpeI in nsP324  with Broccoli-tagged SINV RNA, BHK cells were infected with TEUTR4Br and TEds10Br followed by FISH probes detecting sequences in E1 and E2  with only rare discrepancies for TEds10Br  could differentiate the signal from autofluorescence in only a few of the brightest cells0Br Fig. . TEds10B0Br Fig. .Figure 926  and TEds6Br (genomic and subgenomic) were imaged 16–20 h after infection Fig. . Broccol26 Fig. . Genomic26 Fig. . Co-stai26 Fig. .Figure 117. The current study has shown that labeling SINV RNA with multiple copies of the F30-2xdBroccoli aptamer overcomes many of the obstacles previously encountered with the Spinach2 aptamer. F30-2xdBroccoli is smaller and much brighter than tRNA-Spinach2 and, unlike the tRNA scaffold, the F30 scaffold is not a target for intracellular cleavage and does not contain stop codons21.Live cell imaging of viral RNA provides many advantages over fixed cell imaging at single time points, but has been difficult to accomplish. Previous studies of SINV RNA labeled with the Spinach2 aptamer were limited by the availability of insertion sites and brightness of the signal17, insertion of 4 Broccolis into the 3′UTR did not compromise virus replication. At this site, as well as after a second subgenomic promoter, the RNA that is visualized is primarily subgenomic RNA because of its greater abundance. However, also like Spinach2, insertion of too many Broccoli copies (14 or more) impaired virus replication in differentiated cells and in vivo.Lack of stop codons in the cassette allowed insertion into the genomic RNA region that encodes the nonstructural polyprotein. Viruses with 4–8 Broccolis in the C-terminal coding region of nsP3 replicated well and allowed visualization of genomic RNA. Like Spinach25 molecules/undifferentiated cell27). Multiple copies of Broccoli inserted into the nsP3 coding region, permitted visualization of genomic RNA (~1.6 × 105 molecules/undifferentiated cell28) and viral RNA after infection of less permissive differentiated neural cells  type I, which is formed at the plasma membrane and contains the viral replication complex in spherulesin vitro and in vivo. The real-time measurement of the fluorescent signal intensity is faster, cheaper, easier and more informative than FISH or RT-qPCR. This technique is also suitable for high throughput anti-viral drug discovery that targets viral RNA replication.In summary, the production of virally encoded fluorescent RNA aptamers will facilitate new approaches for study of viral RNA dynamics 26. SINV cDNA clones TE22 and TEds23 were the background for all aptamer-tagged recombinant viruses. After sequencing to confirm the correct insertions and orientations, each constructed plasmid was linearized with XhoI (New England Biolabs) and transcribed into mRNA by Sp6 mMessage mMachine (Ambion). Recombinant viruses were recovered by transfection of mRNA into BHK cells and collection of supernatant fluids after 24 h as previously described17. Titers of virus stocks were determined by plaque assay in BHK cells.SINV Toto E2-mCherry was a gift from Richard Kuhn 2 in DMEM-10% FBS. AP7 odora cells , derived from rat olfactory neurons and immortalized with a temperature-sensitive SV40 T antigen35, were either passaged as cycling cells (cAP7) in DMEM-10% FBS at 33 °C 7% CO2, or were differentiated for 7 days into neurons at 39 °C 5% CO2 (dAP7) by adding 1 μg/ml insulin (Sigma), 100 μM ascorbic acid (Sigma), and 20 μM dopamine (Sigma) into the medium as previously described36. Cells were infected with the recombinant viruses in DMEM-1% FBS at multiplicities of infection (MOI) of 5 to 100. Replication was assessed in BHK, cAP7, and dAP7 cells after infection at an MOI of 5 by assaying supernatant fluids collected in triplicate by plaque assay in BHK cells.BHK-21 cells were cultured at 37 °C 5% CO2 were kept at the same level as the culture condition for each cell type with a heated chamber at 24% humidity. ImageJ Software was used for image analysis.Cells were grown in 35 mm dishes, glass bottom 24-well plates (MatTek), or 8-well chamber cover glasses (Lab-Tek). Infected cells were imaged 30 min after replacing the culture media with phenol-red-free imaging medium  containing 20 μM DFHBI-1T (Lucerna), 25 mM HEPES (Gibco) at 37 °C, 1% FBS, 1% PS and 2 mM glutamine. Autofluorescence was assessed relative to SINV TE (no aptamer)-infected cells and was minimized by adjusting the laser power and gain in each experiment. The laser scanning confocal microscope  with A 488 nm laser excitation was used to activate the Broccoli-DFHBI-1T complex. In dAP7 cells, the true signal was differentiated from autofluorescence with the lambda scan that captured an emission spectrum in each pixel. The Broccoli-DFHBI-1T signal had an emission peak at 507 nm and appeared green in lambda mode, while the autofluorescence signal had no peak, extended from green to far red in its spectrum, and appeared yellow. In time-lapse experiments, temperature and COBHK cells in a 96-well glass-bottom plate (Cellvis) infected with TEUTR4Br or TEds10Br were fixed with 3.7% formaldehyde in RNase-free PBS at room temperature for 10 min. After washing twice with RNase-free PBS, 5 min each, cells were permeabilized with 70% ethanol at 4 °C for 1 h. Ethanol was replaced with washing buffer  for 5 min. Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight. Each well was then washed twice at 37 °C, 30 min each, with washing buffer. After another 2 washes with RNase-free PBS, 5 min each, imaging medium containing DFHBI-1T was added. The SINV RNA FISH (red) and Broccoli (green) signals were analyzed using the Co-localization Analysis plugin in ImageJ.Three to six C57BL/6 mice, 4–6 weeks old, were infected intracerebrally with 1000 pfu/20 μL PBS of each recombinant virus. Three days after infection, brains were collected and the left anterior quadrant was homogenized with 1 ml PBS in Lysing Matrix A tubes in a FastPrep-24 machine  at 6 m/sec speed for 40 sec to make 10% brain homogenates for plaque assays on BHK cells. All mouse studies were performed according to protocols approved by the Johns Hopkins University Animal Care and Use Committee.


Lys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication.HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNA Lys3 with the complementary primer binding site (PBS)  protein, which is produced after maturation of the virion proteins (The HIV-1 5′ untranslated region (5′UTR) consists of a series of structural elements that coordinate various viral replication events. The hallmark event of reverse transcription initiates on the 5′UTR by annealing of tRNAproteins .Lys3 during reverse transcription initiation (Lys3 onto the PBS-segment. The process is mediated by the interactions between the anticodon binding domain of LysRS and the tRNA-like element (TLE) within the PBS-segment  co-assembles with the HIV-1 genomic RNA (gRNA) and bolsters virion infectivity ,17. RHA Lys3 annealing, the PBS-segment RNA serves as a scaffold to interact with viral and host factors, including aforementioned NC, tRNALys3, LysRS, RHA and possibly other yet to be characterized factors , and contained a GFP ORF driven by a CMV promoter . The template DNAs of TLE, PAS stem, hairpin-control and tRNALys3 were custom synthesized (IDT). The nucleotide specific 2H labeled samples, including A2RGUR-, A2RCRU-, A2RCRUR-, A2RGR-, AC- and AG-PBS-segment  were prepared by incorporating corresponding deuterated and protonated rNTPs in T7 transcriptions as previously described  in T7 transcription.All of the RNA constructs, including 5′UTR, 5′UTRescribed ,27. Fullescribed . 13C/15N2). The RNA samples were then incubated at 37°C for 30 min prior to EMSA and ITC experiments.The PBS-segment RNA used for biophysical studies was refolded by preparing the RNA in 10 mM Tris–HCl, pH 7.5, incubating at 95°C for 3 min, snap cooling on ice, and then mixing with salts to reach final salt concentrations of binding buffer A . The recombinant N-terminally His6-tagged dsRBD1 was expressed in Escherichia coli BL21(DE3)-pLysS cells (Invitrogen) treated with 0.3 mM IPTG for 4 h at 37°C. To express 15N labeled dsRBD1, the bacteria were grown in the LeMaster and Richards minimal medium with 15NH4Cl (0.5 g/l) as the sole nitrogen source. The harvested cells were resuspended in lysis buffer  with lysozyme, sonicated and centrifuged. The supernatant was applied to a Cobalt column , and the protein was eluted with lysis buffer containing additional 150 mM imidazole. To remove the His6-tag, the protein was digested overnight at 4°C with TEV protease (1 mg protease per 20 mg of His6-dsRBD1) and dialyzed in low-salt buffer . The protein was then subjected to ion exchange and size exclusion chromatography, and stored in storage buffer at –80°C. The plasmid for recombinant NC (HIV-1 NL4–3 strain) expression was a kind gift from Dr Michael F. Summers . The NC protein was expressed and purified as described elsewhere (The plasmid for recombinant RHA expression was a kind gift from Dr William Clay Brown (University of Michigan). The recombinant RHA contained N-terminal Hisescribed  and the lsewhere . The pRTlsewhere  for reco5 cells per well in six-well plates overnight and then co-transfected with 500 ng of pNL4–3-CMV-EGFP or mutants together with 100ng pMD-G (AIDS Reagent Program), which encodes the vesicular stomatitis virus glycoprotein, VSV-G, for pseudotyping. The mixtures were added dropwise to the cells and incubated for 6 hrs in 5% CO2 at 37 °C, rinsed, and cultured in 2 ml of 10% FBS DMEM. The supernatant was harvested 48 h post-transfection by centrifugation at 1000 rpm for 10 min at 4°C, and the vector virus preparations passed though 0.45 μm filters. The transfected cells were collected and fixed with 4% of paraformaldehyde, and transfection efficiency was monitored by detection of EGFP on an Accuri C6 Flow Cytometer . The Gag p24 was quantified by ELISA using pre-coated MicroFluere plates .293FT cells (Invitrogen) were propagated in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS). Wildtype and mutant viruses were produced by transfection as previously described . Briefly5 of TZM-bl cells in 12-well plates. The infected cells were collected and fixed with 4% paraformaldehyde 24 hrs post-transduction, and the percentage of cells expressing EGPF was measured using the BD Accuri C6 Flow Cytometer . The infectivity was calculated as the percentage of EGFP-expressing cells in mutant virus infected cells compared to that of the wild type (WT).Equivalent amounts of VSV-G-pseudotyped vectors viruses (250 ng of Gag p24) with or without mutations in the PBS-segment were used to transduce 2 × 105 cells in 0.5 ml of RPMI medium) were seeded in 12-well plates and incubated with equivalent cell-free medium containing vector viruses normalized to 200 ng of Gag p24 and spinoculated for 2 h, washed twice in RPMI medium and cultured for 4 h. The inactivated virus control sample was heated to 100°C for 5 min, cooled to room temperature, centrifuged at 12 000 rpm for 1 min, and treated with DNase I for 1 h. The infected cells were collected and washed twice with 1× phosphate buffered saline and DNA was extracted in the QIAamp DNA blood mini kit (Qiagen). Cellular DNA (600 ng) was evaluated by qPCR with specific primer sets that detect early and late products of HIV-1 reverse transcription (MT4 lymphocytes (5×10cription  31). Th. Th5 celLys3, and 600 pmol NC (∼6 nucleotides per NC molecule) in 20 μl of buffer B . The mixture was incubated at 37°C for 30 min, and the reaction was terminated by incubating with 0.5% SDS and 0.2 mg/ml Proteinase K at 37°C for 30 min. The RNA was purified by phenol-chloroform extraction and electrophoresed on 10% polyacrylamide gels.The annealing reaction was initiated by mixing 20 pmol PBS-segment, 20 pmol tRNALys3 annealed on PBS-segment and mutants in the presence of NC. To label tRNALys3 on the 5′-terminus, GMP-primed tRNALys3 was synthesized by mixing GMP with rNTPs in T7 transcription, and the RNA was fluorophore labeled with a Cy3-NHS ester (Lumiprobe) (Primer extension assays were performed with 5′-Cy3-labeled tRNAmiprobe) ,32. The Lys3 to prime reverse transcription, 2 ng of extracted RNA was mixed with dNTP (10 mM each), 5× First-Strand Buffer (Invitrogen), 0.1 M DTT and RNaseOUT™ (40 units/μl), and the mixture was incubated at 42°C for 2 min. Primer extension was initiated by adding 1 μl (200 units) of SuperScript™ II RT (Invitrogen), and the reaction was incubated at 42°C for 50 min. To quantify the (-)cDNA products, 2 μl of cDNA generated from the previous step was mixed with the primer/probe set targeting the RU5 region . To quantify the total gRNA, the primer/probe set . To detect if plasmid DNA were carried over through RNA extraction, the same amount of RNA (0.2 ng) and primer/probe set targeting the gag region were used in qPCR. Both qPCR and RT-qPCR were performed on CFX96 Real-Time PCR Detection System (Bio-Rad).To check the tRNA placement on the WT and mutant viral RNAs, 200 μl of WT and mutant pseudotyped viruses were harvest from transfected 293FT cells and treated with Dnase I and DpnI at 37°C for 90 min. The viral RNA was extracted with TRIzol (Invitrogen). To check if the viral RNA was pre-annealed with tRNA5 region  for qPCRrobe set  targetin11, pH 7.5 and 1 mM MgCl2 at 37°C for 30 min. Two-dimensional 1H–1H NOESY and 1H–1H TOCSY data for control RNA fragments, and 1H–1H NOESY data for fully protonated and site-specific deuterated PBS-segment RNA and in complex with dsRBD1 were collected in D2O  at 308K. 1H–13C HMQC data were collected for 13C/15N labeled control RNA fragments. One- and two-dimensional imino proton spectra were collected for the PBS-segment RNA and control RNAs (200–300 μM) prepared in buffer containing 10% D2O + 90% H2O. HNNCOSY data were collected for 13C/15N labeled TLE stem loop RNA. 1H–15N TROSY data were collected for 200 μM dsRBD1 titrated with 0, 54, 108, 136 and 160 μM of the PBS-segment RNA in binding buffer B  in 10% D2O and 90% H2O at 318 K. All the NMR data were collected on a Bruker Avance III 800 MHz spectrometer equipped with TCI cryoprobe . The NMR data were processed by NMRPipe (The PBS-segment and its control fragment samples were prepared (200–300 μM) and pre-incubated in 10 mM Tris-d NMRPipe  and anal NMRPipe .q  was 0.005–0.85 Å–1. The sample passed through the FPLC column and was loaded to a flow cell for SAXS measurements. The flow cell is a cylindrical quartz capillary 1.5 mm in diameter and 10 μm wall thickness. The exposure time was set to 1 s to reduce radiation damage and data were collected at every other second. The 2D scattering images were converted to 1D SAXS (I(q) versus q) curves through azimuthally averaging after solid angle correction and then normalizing with the intensity of the transmitted X-ray beam flux, using the beamline software. The SAXS data were then processed using ATSAS suite 2.8 and 3.0 (Rg) was determined by the Guinier plot in its linearity region and pairwise distribution was plotted using GNOM  at 0.75 ml/min flowing rate. SAXS data of the mutant RNA samples were collected at beamline 12-ID-B of Advanced Photon Source at Argonne National Laboratory, using an in-line AKTA micro FPLC setup with a Superdex 75 Increase 5/150 GL size exclusion column. The wavelength, λ, of X-ray radiation was set to 0.9322 Å. Scattered X-ray intensities were measured using a Pilatus 2M detector. The sample-to-detector distance was set such that the detecting range of momentum transfer  and 3.0 . The rading GNOM . The ab  DAMAVER .2 values were selected for further molecular dynamics (MD) refinement. For each of the 10 structures, 1 ns MD simulation was performed in 1 mol/L NaCl solution under 300 K and 1 bar with NMR restraints using Amber software package  structures were reduced to 1.35–1.5. The 10 structures of the lowest energies were deposited to the RCSB protein databank (PDB: 7lva).The PBS-segment structure was initially calculated using NMR-derived restraints by CYANA . Standar package , and thesoftware  was usedComputational mutation scanning was performed using the Vfold2D RNA structure folding model  to prediΔTAR-PolyA or 5′UTRΔPBS) and recombinant RHA at concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4 μM in binding buffer A. To compare RHA binding to PBS-segment and TAR-PolyA, 0.5 μM RNA was incubated with 0, 1, 2, 3 and 4 μM of RHA. The mixtures were incubated at 37°C for 10 min and resolved on 1.5% agarose gels stained with 0.5 μg/ml ethidium bromide. The gels were imaged using Gel Doc XR+ (Bio-Rad). EMSA experiments were repeated three times and a representative gel is shown.EMSA was carried out by preparing mixture solutions of 0.5 μM RNA  were summarized from three experiments . Heat of dilution titrations were performed by titrating the dsRBD1 protein into a matching buffer in the same experimental settings. Both protein and RNA samples were prepared in binding buffer B. The baseline was corrected by subtracting the heat of dilution, and the data were fitted using ‘one-site’ non-linear least square regression for hairpin-control and ‘two-site’ non-linear least square regression for PBS-segment. The statistics of thermodynamic parameters . Distance restraints were also set for residue Y43 that should not be within 5 Å distance of RNA because no chemical shift perturbation was observed in the TROSY titration. Any qualifying candidate must satisfy all the specified restraints, resulting in a total of 69 modeled complex structures for further evaluation with experimental data. The best model with lowest energy was further optimized with UCSF Chimera  and 1040 PBS-segment RNA structures that had been relaxed in MD simulations, a free docking by our in-house MDockPP program  was carr Chimera .º angular interval was used to sample the relative orientations between the dsRBD2-Core and PBS-segment RNA. The resulting 54,000 potential binding poses were then filtered with three constraints: (i) The α1 helix of the dsRBD2 interacts with the RNA, (ii) Region 3 residues  interact with the RNA and (iii) The dsRBD2-Core does not have any steric clash with the dsRBD1 domain when both bind to the RNA. For constraints (i) and (ii), a 7Å distance cutoff was used to define the proximity to the RNA. Namely, the constraint (i) was met if at least one atom in a residue was within the cutoff distance to the RNA for every residue in the α1 helix. The constraint (ii) regarding K235 or K236 was likewise determined. The constraint (iii) was based on the fact that both dsRBD1 and dsRBD2-Core bind to the RNA simultaneously in physiological condition. The assessment of this constraint was based on the buried solvent accessible surface area (SASA) between the dsRBD1 and the dsRBD2-Core. The SASA was calculated using NACCESS (dsRBDSASA1 + dsRBDSASACore2− − dsRBDSASAdsRBD2−Core1+)/2, where 2 was used to determine if two molecules have atomic clashes. The dsRBD1 and the dsRBD2-Core were deemed to have no atomic clash if the calculated buried SASA was below this threshold value.Using Modeller , the str NACCESS . The bur1H–1H NOESY spectrum as that of the PBS-segment RNA. Imino proton resonances of the PBS-segment were very broad  were prepared for NMR data collection and assignments of non-exchangeable protons. This strategy enabled us to assign residues in and near the tRNA annealing arm that are not covered by the control RNA fragments. In addition to imino proton resonances, we also relied on the sequential and long-range NOEs that involve adenosine H2 to obtain secondary structure information within the PBS-segment  125–223 was synthesized with two terminal G-C pairs for efficient transcription Figure . NMR assry broad . The basry broad . Additiory broad . Assignm-segment  ..1H–1H NO2H labeling strategies  was performed. A total of 1,908 PBS-segment containing sequences (C125–G223) were aligned using ClustalX2  single nucleotide mutations, to exhaustively predict structures for each mutant, and learned that different mutations can cause different structural changes. Critical nucleotides whose mutation cause disruption of the folding were identified. Three major types of structural changes were observed: (i) in the central three-way junction region, (ii) the TLE stem loop and (iii) the tRNA-annealing stem. Some of mutant structures maintain the WT three-way junction global fold while some form an alternative elongated stem-loop structure. We found that the disruption of the central three-way junction can result in folding changes in both the TLE stem loop and the tRNA annealing arm. The mutations that lead to the disruption of the central three-way junction are distributed mostly in the lower stem of TLE (U135–A140 and U172–G178). The result shows the TLE stem is stable as long as the lower stem is maintained. Mutations in the upper stem of TLE only cause local structural variations, but do not change the overall three-junction structure. Similarly, single point mutations in PAS and the tRNA annealing arm lead to only local changes without disrupting the three-way structure of the PBS-segment. In summary, the predicted low-tolerance nucleotides reside in the lower stem of TLE, while majority of the nucleotides outside of this region can be mutated , and their shapes are different compared with the WT PBS-segment  disrupt the TLE loop, A157G and ΔUC-loop (C151CUUUUA157 was substituted by CGAGAG), (ii) perturb tRNA annealing arm, G212C/U213C and A214U and (iii) disrupt the three-way junction structure, G137C, A140C and C173G. To avoid possible disruption of tRNA annealing and reverse transcription initiation, none of the mutation sites selected are predicted to be involved in inter-molecular interactions with tRNAx models ,64–66. Vx models  suggestsx models . Consist-segment .env gene of the reporter virus was replaced by an EGFP open reading frame and thus EGFP was produced when the cells become transduced  were transduced with the pseudotyped virions in cell-free medium containing 200 ng of Gag p24 by spinoculation. Six hrs post-infection, cellular DNA from the transduced MT4 cells was isolated and analyzed by qPCR using primer pairs to amplify the early and late products of reverse transcription (P < 0.001) cDNA synthesis, we then examined the reverse transcription activity of these mutants in lymphocytes. MT4 lymphocytes (5 × 10cription . The res) Figure . The synLys3 annealing or RT loading, we examined tRNALys3 annealing by EMSA, compared the in vitro primer extension efficiencies, and quantified the tRNA placement on viral RNA in virions. The WT and mutant RNAs were annealed with tRNALys3 in the presence of NC. None of the mutations disrupted formation of an RNA: RNA duplex, and the migration rates of the mutant complexes on a polyacrylamide gel were similar to the WT ssDNA. No difference in the primer extension efficiency between the WT and mutants was observed . We then investigated tRNALys3 placement on WT and mutant RNAs using RNA extracted from viral particles produced from transfected 293FT cells. The RNA was mixed with an RNase H activity reduced RT  and dNTPs. If the mutations in the PBS-segment do not affect tRNALys3 placement on gRNA, the co-purified tRNALys3 will serve as a primer to generate (–)cDNA when supplemented with RT  and with PBS-segment deleted (5′UTRΔPBS). These 5′UTR constructs have the 3′-half of the AUG hairpin truncated to favor RNA dimerization  and PBS-segment (33 kDa), RHA preference towards the PBS-segment was observed , the titration was carried out in a high salt buffer and at high temperature  to reduce non-specific electrostatic interactions between protein and RNA. As a result, the affinity between PBS-segment and dsRBD1 was weakened and thus fast exchange regime was observed   . To idenn Figure , as we pn Figure . Chemican Figure . The pron Figure .With the guidance of the experimental data, a data-driven molecular docking study was exploited to model the structure of the PBS-segment: dsRBD1 complex. The crystal structure of RHA dsRBD1 (PDB: 3vyy) was used to dock the PBS-segment structure using an in-house program MDockPP . Protein10 complex, in order to have all the three regions to interact with RNA, region 1 is slightly distant from the RNA so that region 2 could be close. In the case of the PBS-segment, due to the angle between the TLE lower stem and PAS, dsRBD1 is closer to RNA with regions 1–3 all in contact with RNA. This leads to formation of additional hydrogen bonds between dsRBD1 and PBS-segment as compared to a straight (GC)10 RNA shown in the crystal structure e Figure -B.N = 5.2 ± 0.2) at Kd = 18.5 ± 1.1 μM, indicative of non-specific electrostatic interactions  and a second set of weak binding sites (Kd2 = 45 ± 8.1 μM) -driven, but the enthalpy change associated with PBS-segment binding is significantly greater than hairpin-control binding  (Kd can be accurately determined but △H can be underestimated (△H for these binding sites are less negative than the dsRBD1:hairpin-control interaction (10 or hairpin-control)  and TLE lower stem (nt 134–141 and 171–178), and is enclosed by a CG pair and a UUCG tetraloop Figure . The isos Figure . The ITC) Figure . The affs Figure . Assumins Figure . Both thcontrol) . For a lstimated . The seceraction . In summ) Figure , B. Reco10 RNA reveals that it binds to RNA similarly as dsRBD1, with equivalent α1 helix (region 1) and loop connecting β1- β2 (region2) binding to two successive minor grooves and α2 helix (region 3) binding to the major groove in between . Cr between  51). RH. RH10 RNe Figure , and doce Figure . Region Solving the structure of the PBS-segment has been challenging as the tRNA annealing arm is highly metastable thus making crystallization difficult and traditional characterization by NMR impractical. Metastable residues in the tRNA annealing arm were proposed to be unstructured by chemical probing, enzymatic probing, SAXS and computational modeling . In our The identified three-way junction structure is reinforced by phylogenetic analysis of HIV sequences in patient samples, which document conserved pairings at the bottom of TLE Figure . In lineLys3 onto PBS requires a chaperone, which can be the NC domain of Gag polyprotein or the processed mature NC protein . These dsRBD1: dsRNA crystal structures indicate that dsRBD1 binds to straight A-form dsRNA helices without sequence specificity.DsRBDs exist in many proteins and influence various steps of RNA metabolism. Some of the proteins have more than one dsRBD, and the contribution of each dsRBD to target RNA selection is usually not equal. MLE helicase facilitates the incorporation of the roX lncRNA into the dosage compensation complex via its dsRBDs interacting with the roX lncRNA. Studies revealed that dsRBD2 has 10-fold greater affinity than dsRBD1, and dsRBD2 plays the major role in recognition of the R2H1 and SL7 stem loops of roX lncRNA ,79. BothEfforts have been made to investigate the sequence and shape recognition of dsRBDs in directing the proteins to their target RNAs. In general, dsRBDs use conserved residues in region 1, 2 and 3 to interact with two consecutive minor grooves in dsRNAs ,80–85. S10 complex and our dsRBD1: PBS-segment model. In addition, K16, T18, Y21, K29, N23 and E41 participate in H-bonds with PBS-segment, which explains the Kd and enthalpy differences between dsRBD1 binding to a straight helical RNA and PBS-segment. Some of these residues are unique in the dsRBD family (Here we show that dsRBD1 preferentially binds to the PBS-segment RNA by recognizing the TLE and PAS stems which are not co-axial stacked in the three-way junction structure. ITC data demonstrate that the dsRBD1: PBS-segment interaction is of higher affinity compared to binding to a straight A-form helical RNA structure (hairpin-control, Figure D family , and theIn summary, we show that the structure of the PBS-segment prior to tRNA annealing folds into a three-way junction structure that is important for viral infectivity. The biophysical studies of the interactions between RHA dsRBD1 and the PBS-segment provides a plausible explanation for the recruitment of a host factor beneficial for the late stage virus replication. Although we cannot exclude the possibility of other yet-to-be discovered molecular interactions involving PBS-segment, our solution structure of the PBS-segment provides the structural basis for further investigation of the biomolecular interactions that occur on the scaffold of the PBS-segment.Atomic coordinates for the PBS-segment RNA structure have been deposited into the RSCB Protein Data Bank under the accession number 7LVA. Chemical shifts have been deposited to the BMRB under accession number 30868. SAXS data for PBS-segment have been deposited to the SASBDB under accession number SASDJU7.gkab342_Supplemental_FileClick here for additional data file.


Immune checkpoint inhibitors (ICIs) have become one of the standard treatment options for advanced lung cancer. However, adverse events (AEs), particularly immune–related AEs (irAEs), caused by these drugs have aroused public attention. The current network meta-analysis (NMA) aimed to compare the risk of AEs across different ICI–based regimens in patients with advanced lung cancer.We systematically searched the PubMed, EMBASE, and Cochrane Library databases (from inception to 19 April 2021) for relevant randomized controlled trials (RCTs) that compared two or more treatments, with at least one ICI administered to patients with advanced lung cancer. The primary outcomes were treatment–related AEs and irAEs, including grade 1–5 and grade 3–5. The secondary outcomes were grade 1–5 and grade 3–5 irAEs in specific organs. Both pairwise and network meta-analyses were conducted for chemotherapy, ICI monotherapy, ICI monotherapy + chemotherapy, dual ICIs therapy, and dual ICIs + chemotherapy for all safety outcomes. Node–splitting analyses were performed to test inconsistencies in network. Sensitivity analyses were adopted by restricting phase III RCTs and studies that enrolled patients with non–small cell lung cancer.Overall, 38 RCTs involving 22,178 patients with advanced lung cancer were enrolled. Both pooled incidence and NMA indicated that treatments containing chemotherapy increased the risk of treatment–related AEs when compared with ICI-based regimens without chemotherapy. As for grade 1–5 irAEs, dual ICIs + chemotherapy was associated with the highest risk of irAEs (probability in ranking first: 50.5%), followed by dual-ICI therapy (probability in ranking second: 47.2%), ICI monotherapy (probability in ranking third: 80.0%), ICI monotherapy + chemotherapy (probability in ranking fourth: 98.0%), and finally chemotherapy (probability in ranking fifth: 100.0%). In grade 3–5 irAEs, subtle differences were observed; when ranked from least safe to safest, the trend was dual ICIs therapy (60.4%), dual ICIs + chemotherapy (42.5%), ICI monotherapy (76.3%), ICI monotherapy + chemotherapy (95.0%), and chemotherapy (100.0%). Furthermore, detailed comparisons between ICI–based options provided irAE profiles based on specific organ/system and severity.In consideration of overall immune–related safety profiles, ICI monotherapy + chemotherapy might be a better choice among ICI–based treatments for advanced lung cancer. The safety profiles of ICI–based treatments are various by specific irAEs and their severity.https://www.crd.york.ac.uk/prospero, identifier CRD42021268650 Lung cancer remains the leading cause of global cancer mortality, with approximately 1.8 million deaths annually  , 3. ReceWith a dramatic increase in the availability of ICI drugs and their superior efficacy, a substantial proportion of patients with lung cancer are administered these agents. Nonetheless, concerns regarding unique treatment–specific toxicities owing to their pharmacological mechanisms, namely immune–related AEs (irAEs), associated with ICI regimens are growing . IrAEs aPrevious meta-analyses have examined the risks of irAEs associated with ICI therapy; however, most of them mainly involved patients with all types of cancers –13. In ahttps://www.crd.york.ac.uk/prospero/#searchadvanced), and reported in accordance with the PRISMA  guidelines  independently screened all titles and abstracts, and further assessed potentially eligible full text based on the aforementioned criteria.The primary outcomes of this study were overall safety outcomes, viz. treatment–related adverse events and immune-related adverse events as defined in each study Table S3The methodological quality of the included trials was assessed using the Cochrane Collaboration Risk of Bias Tool . Low, mo2I-test, with a value > 50% representing considerable heterogeneity  with 95% confidence intervals (95% CIs) using a random–effect model. Statistical heterogeneity was assessed using the ogeneity . A sensiogeneity , 25. Furogeneity . When a ogeneity . For outogeneity . Transitogeneity . All datOur initial search yielded 1,725 records from databases and 914 records from the ClinicalTrials.gov platform; 2,535 records were excluded following screening of titles and abstracts. The remaining 104 full–text articles were reviewed, and 66 articles were excluded for reasons depicted in The included RCTs satisfied three tool items, viz. random sequence generation, incomplete outcome data, and selective reporting. Twenty–seven open–label trials did not meet the item of allocation concealment or blinding of participants and personnel, resulting in a high risk of bias in the study assessment. Overall, 27 studies exhibited a high risk of bias, and 11 studies were considered to have a low risk of bias. The details of the quality assessment are presented in vs. chemotherapy), while there was a reduced risk when compared with dual ICIs + chemotherapy . Other results were consistent with those observed in grade 1–5 AEs.Direct comparisons were conducted to assess safety profiles among five treatments options Table S72I: 38.2%–95.0%), except for two pairs . Overall, the general heterogeneity in individual irAEs was low to moderate.In terms of irAEs, the use of ICI monotherapy  or ICI monotherapy + chemotherapy  was associated with a significantly higher risk of developing these manifestations than chemotherapy. Dual ICIs therapy had a similar risk of grade 1–5 irAEs  but a superior risk of grade 3–5 irAEs  compared with ICI monotherapy. Detailed data in specific irAEs are listed in vs. chemotherapy (P = 0.018)  , ICI monotherapy + chemotherapy , dual ICIs therapy , as well as dual ICIs + chemotherapy  . ICI monotherapy + chemotherapy showed a higher risk of causing grade 1–5 AEs than chemotherapy , while no significant difference was seen when compared with that of the ICIs + chemotherapy group . Similar results were observed in grade 3–5 AEs. The ranking probabilities based on the five treatment groups are also depicted in The pooled incidence for five treatments showed the following rankings: ICI monotherapy + chemotherapy had the highest incidence of AEs , followed by dual ICIs + chemotherapy , chemotherapy , dual ICIs therapy , ICI monotherapy   and grade 3–5 events . Among ICI therapeutic schedules, ICI monotherapy + chemotherapy seemed safer than ICI monotherapy , dual ICIs therapy , and dual ICIs + chemotherapy , with one ICI being observed to be safer than two ICIs combination with regards to grade 3–5 irAEs . In aspect of safety ranking, dual ICIs + chemotherapy was associated with the worst ranking for grade 1–5 irAEs (probability: 50.5%), followed by dual ICIs (47.2%), ICI monotherapy (80.0%), ICI monotherapy + chemotherapy (98.0%), and finally chemotherapy (100.0%). The risk of experiencing grade 3–5 irAEs was ranked from high to low as follows: dual ICIs (60.4%), dual ICIs + chemotherapy (42.5%), ICI monotherapy (76.3%), ICI monotherapy + chemotherapy (95.0%), and chemotherapy (100.0%) , or ICI monotherapy + chemotherapy   and irAEs of any grade, and those receiving dual ICIs may have the highest risk of developing serious irAEs. There were differences observed in the toxicity spectra among the five treatment therapies. However, these results should be carefully interpreted because of the limited number of studies on groups receiving dual ICIs with and without chemotherapy.Recently, developments in lung cancer patients with advanced stages of the disease have shown immunotherapy as a promising treatment option. However, the expanded use of ICIs has resulted in noticeable growth in adverse events, particularly irAEs , 68. WitP = 0.004)  . However= 0.004) . Further= 0.004) –18. Nota= 0.004) . The stuIt is well known that the incidence of overall adverse events during ICI monotherapy is lower than that for conventional chemotherapy, while exposure to immunotherapy increases the risk of irAEs, as seen in the present study. Interestingly, further analyses of ICI–based regimens found that ICI monotherapy + chemotherapy decreased the risk of grade 1–5 and grade 3–5 irAEs compared with ICI monotherapy and dual ICIs therapy. This trend was also observed in pneumonitis and myocarditis (grade1–5 and grade 3–5), as well as grade 1–5 hyperthyroidism, hypothyroidism, thyroiditis, severe skin reaction, hepatitis, myositis based on ranking. Consistent with our study, Chen et al. reported that the use of an ICI with chemotherapy led to less pneumonitis than use of ICI monotherapy or dual ICIs combination . One posSome studies have pointed out that the incidence of irAEs in patients on a combination of two ICIs was higher than that observed with ICI monotherapy. In the present study, we did not observe any statistical differences between these two groups in terms of overall grade 1–5 irAEs, let alone for rare irAEs, such as thyroiditis, diabetes, myocarditis, nephritis, hepatitis, and myositis. Even between dual ICIs + chemotherapy and ICI monotherapy (both grade 1–5 and grade 3–5 irAEs), significant difference was not detected. However, this trend can be seen from the ranking either in grade 1–5 irAEs or grade 3–5 irAEs. Given the fact that a limited number of studies directly compared dual ICIs (with or without chemotherapy) with ICIs monotherapy, these results should be interpreted with caution. Therefore, more high–quality RCTs are needed to investigate the incidence of irAEs among different ICI–based regimens.Our results provide possible safety speculations for clinical decision–making to tailor the best immunotherapy strategy for each patient with lung cancer. For instance, administration of an individual ICI or dual ICIs plus chemotherapy was reported to have a significantly lower risk of pneumonitis (grade 1–5 and grade 3–5) than ICI monotherapy and dual ICIs therapy and could perhaps be preferred in selected cases of lung fibrosis or severe chronic obstructive pulmonary disease. In addition, ICI monotherapy was associated with the lowest risk for both diarrhea and colitis among ICI regimens and could perhaps be preferred in selected patients in whom gastrointestinal irAEs could be a concern. Of course, these results should be proven in prospective registries or cohorts to better understand the safety of novel ICI–based options in this subset of patients.The major strength of this study was to depict a full view of the safety profile of ICIs at different levels . Second, owing to the different toxicity spectrum based on cancer types, the study focused on a specific population of patients . Third, except for combination of ICIs and targeted agents, we included all available ICI–based regimens to aid clinicians to tailor the ICI strategy for individual patient with lung cancer.However, several limitations of this study need to be acknowledged. First, the included RCTs used different terms to describe irAEs. In clinical settings, AEs or irAEs are usually recognised and reported depending on the evaluation of the physician and are diagnosed based on their experience. Therefore, the identification of irAEs might not be completely accurate and might lead to bias in the assessment. In addition, few studies reported irAEs during the entire ICI monotherapy maintenance as separate outcome, making it difficult to investigate irAEs during this period. Second, the included studies showed heterogeneity in terms of subtype of cancer, pharmacological strategy, follow–up time, and other factors. As we focused on outlining the entire safety profile of ICI agents, subgroup analyses based on patients’ histology, specified ICIs and kind of chemotherapy were not performed. However, we performed sensitivity analyses and meta-regression in pairwise meta-analysis, as well as sensitivity analyses in NMA, to control for these possible confounders. Third, inconsistent results between direct and indirect comparisons were observed. Unlike direct comparison, network meta-analysis included both direct evidence and indirect effects from the other studies. Because of above-mentioned heterogeneity among RCTs, integrated results possibly underestimated or overestimated the actual results. Fourth, we did not obtain access to comorbidity data, which might be high–risk factors for certain irAEs. Lastly, we did not have the resources to review non-English publications. However, we enrolled studies identified following a comprehensive search of broad databases and are thus confident that this study covered the majority of trials in these special patients. Given aforementioned limitations, further studies are needed to confirm our findings.In summary, this network meta-analysis contributes to clarifying the frequency and characteristics of adverse events during ICI treatment in patients with advanced lung cancer. We found that ICI monotherapy + chemotherapy had the best immune–related safety profile, followed by ICI monotherapy, dual ICIs therapy, dual ICIs + chemotherapy for grade 1–5 irAEs, and ICI monotherapy, dual ICIs + chemotherapy, dual ICIs therapy for grade 3–5 irAEs. The safety ranking of ICI-based choices is modulated by specific irAEs and severity.The original contributions presented in the study are included in the article/Z-CG, X-YC, and H-WL are the guarantors of the entire manuscript. Z-CG and Y-DY contributed to the study conception and design, critical revision of the manuscript for important intellectual content, and final approval of the version to be published. J-JC, JF, and Y-JS contributed to the data acquisition, analysis, and interpretation. All authors contributed to the article and approved the submitted version.This study was supported by the Ren Ji Boost Project of National Natural Science Foundation of China (RJTJ-JX-001), the Research Funds of Shanghai Health and Family Planning Commission (20184Y0022), Clinical Pharmacy Innovation Research Institute of Shanghai Jiao Tong University School of Medicine (CXYJY2019ZD001), and Shanghai “Rising Stars of Medical Talent” Youth Development Program – Youth Medical Talents – Clinical Pharmacist Program [SHWJRS (2019) 072].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The coronavirus pandemic has escalated rates of anxiety in the general U.S. population. Understanding how factors associated with coronavirus anxiety at the start of the pandemic differed among populations hardest impacted by coronavirus anxiety is key to effectively remediating negatively associated health outcomes and to better understand how to address concerns of the public at the start of a global pandemic. This study was a secondary analysis of data from a cross-sectional online survey of 1165 Prolific users between 13 and 15 March 2020. Data were collected from a stratified sample of U.S. adults aged 20 or older and currently living in the United States. The sample was stratified for age, gender, and race. Coronavirus anxiety was assessed as the dependent variable, alongside three independent variables: coronavirus crisis perception, perceived economic risk of coronavirus, and general self-efficacy. Multiple linear regression assessed the associations between the independent variables and coronavirus anxiety. Interactions between independent variables and two sociodemographic variables  were also explored. The models were adjusted for age, gender, race, education, employment, and income. The average age of participants was 45.6 ± 15.7. The majority (76%) identified as White, approximately half identified as female and reported obtaining a bachelor’s degree or higher. Coronavirus crisis perception and perceived economic risk of coronavirus were positively associated with coronavirus anxiety . General self-efficacy was negatively associated with coronavirus anxiety . Gender and race both moderated the association between coronavirus crisis perception and anxiety. Race moderated the association between perceived economic risk and coronavirus crisis perception. These results provide a foundation to further explore cognitive factors in subgroups disproportionately affected by anxiety during the pandemic. In December 2019, the coronavirus identified as SARS-CoV-2 prompted a global pandemic as many fell ill to COVID-19 across the globe. By early 2020, increasing COVID-19 rates in the United States affected employment rates, social interactions, and social norms across the nation. Unsurprisingly, increased anxiety, stress, fear, and depression were more frequently observed during the pandemic compared to months prior . Two dayPast work has primarily explored demographic factors and pre-existing health diagnoses associated with increased anxiety during pandemics ,8,9. PreIn selected forms of anxiety, an individual may have elevated perceptions of risk while underutilizing coping mechanisms . Risk peAn important cognitive factor to understand in the context of coronavirus anxiety may be perception of the crisis. Perception of the health impact of coronavirus was associated with higher anxiety among people with high-risk type 2 diabetes . Risk peMany stressors may contribute to anxiety symptoms. For example, financial and economic hardship has been associated with anxiety and stress . In ApriSelf-efficacy is another cognitive factor that may be associated with coronavirus anxiety. The association between lower self-efficacy and increased general anxiety symptoms has been primarily observed among samples of frontline health workers in Wuhan, China, where the pandemic began ,29. It iThe present study explored cognitive factors related to coronavirus anxiety potentially modifiable through public health messaging or future interventions. The hypotheses were: (1) Higher coronavirus crisis perception will be associated with higher coronavirus anxiety; (2) Higher economic risk perception of coronavirus will be associated with higher coronavirus anxiety; and (3) Lower general self-efficacy will be associated with higher coronavirus anxiety. This study further assessed how these associations may have differed among populations at higher risk of coronavirus anxiety . Additional hypotheses were: (1) Female gender moderates the associations between coronavirus crisis perception, economic risk perception, general self-efficacy and coronavirus anxiety; and (2) White racial status moderates the associations between coronavirus crisis perception, economic risk perception, general self-efficacy and coronavirus anxiety. For the purposes of this study, White racial status refers to non-Hispanic White individuals.From 13 to 15 March 2020, online surveys were collected from the Prolific platform. Eligibility criteria included adults who were 20 years old or older, living in the United States, identified as U.S. citizens, and registered on Prolific. The sample was stratified by age, gender, and race. Data from the parent study are available via the online OSF platform . The priN = 1165). Analyses were derived from a dataset used to answer other research questions [The dataset featured more than 50 variables measured by a total of 378 unique items. The dataset was planned from the offset to be utilized for numerous hypotheses in international collaborations examining COVID-19 in the context of behavioral psychology , religiouestions , but resA 10-item coronavirus anxiety scale  . Responses were summed and averaged. Higher scores reflected higher coronavirus crisis perception. Example items are: “I think the world is facing an unprecedented health emergency because of coronavirus” and “I think coronavirus is not a serious threat to the world” (reverse coded). A 1-item measure for perceived economic risk of the coronavirus was used: “I think coronavirus will be a disaster for our economy”. A higher score indicated higher perception of risk that the coronavirus poses for the U.S. economy. An 8-item self-efficacy measure was adapted from the New General Self-Efficacy Scale . ResponsSix covariates  were used in the analyses. Age in years was self-reported by participants. Participants were asked to indicate their gender as (1) female, (2) male, or (3) other. Responses were then dummy-coded for analysis. Race and ethnic data were collected as (1) Asian, (2) African American, (3) Hispanic, (4) Native American or Native Hawaiian, (5) White, or (6) other. For this specific study, race categories were recoded as (0) other and (1) non-Hispanic White. Education level was collected categorically as (1) less than a high school diploma, (2) high school diploma or equivalent , (3) some college, no degree, (4) associate degree, (5) bachelor’s degree, (6) master’s degree, (7) professional degree, (8) doctoral degree, (9) vocational training or trade, and (10) other school, with a text space to specify. For this study, responses regarding educational status were recoded into (0) associate degree or less and (1) bachelor’s degree or higher. Employment status was collected as (1) employed full time (40 or more hours a week), (2) employed part time up to 39 h a week), (3) unemployed and currently looking for work, (4) unemployed and currently not looking for work, (5) student, (6) retired, (7) homemaker, (8) self-employed, and (9) unable to work. For this specific study, responses were recoded as (1) employed full time (40 or more hours a week and (0) other. Lastly, annual gross household income was collected as (1) I do not have personal income, (2) less than USD 20,000, (3) USD 20,000 to USD 34,999, (4) USD 35,000 to USD 49,999, (5) USD 50,000 to USD 74,999, (6) USD 75,000 to USD 99,999, (7) USD 100,000 up to USD 114,999, (8) USD 115,000 up to USD 129,999, (9) USD 130,000 or more, and (10) I do not wish to respond. Gross household income responses were treated as continuous. “I do not wish to respond” was treated as missing. All measures for this study are available via the OSF platform [ h a weekp-value of 0.05 or less was considered significant for bivariate tests.Analyses were conducted using Stata software (Version 15.1). Preliminary data screening checked data for outliers beyond three standard deviations, skewness, and kurtosis. Outliers observed in the distribution of continuous variables were not excluded because they were all within a feasible range. Robust standard errors were used to address deviations from normality. The income variable contained <2% of missing data. Case-wise deletion was used for missing observations since total missing is below the recommendation for imputation . Variancp-values of 0.05 or less were considered significant for all tests. Standardized coefficients are reported. Model fit was assessed using the mean-adjusted R-square value. The computed multiple regression model is as follows.To test the three initial hypotheses, one multiple linear regression model assessed the associations between anxiety and the three cognitive factors, adjusting for covariates of age, gender, race, education level, employment status, and income. To test directional hypotheses, one-tailed i represents 1165 survey responses.In this equation, p-values of 0.05 or less were considered significant for all interaction model tests.To explore differences across gender and race in the multilinear model, 6 additional models included interaction terms between cognitive factors  and coronavirus anxiety. Predicted probabilities of coronavirus anxiety by cognitive factor were calculated and graphed among significant interactions to explore the moderation effect of gender and race. A one-tailed The primary study was designed and conducted in accordance with the Declaration of Helsinki for research involving human subjects. The Ethics Review Panel (ERP) at the Université of Luxembourg approved the primary study protocol and procedures. Digital informed consent was obtained by participants prior to the start of the online survey from the primary study. This study was a secondary analysis of data collected in the primary study and deemed exempt by the Univeristy of California, Irvine Institutional Review Board.The average age of survey respondent was 45.6 ± 15.7 years, and the majority identified as non-Hispanic White (76%). Respondents in this sample were highly educated, with 53% attaining a bachelor’s degree or above. Approximately 32% of participants were employed full-time at the time of the study. Females and non-White participants reported higher coronavirus anxiety. R-square value indicated that the covariates explained approximately 32% of the variance in coronavirus anxiety.The single multiple regression model adjusting for six covariates supported all hypotheses . The firThree interaction models were run to evaluate the effect of gender on the association between each cognitive factor  and coronavirus anxiety. The first interaction model showed statistical significance between females and males, indicating the slope of the relationship between coronavirus crisis perception and coronavirus anxiety differed by gender . In the second and third models, gender was not a statistically significant moderator between the association of economic risk perception  or self-efficacy  on coronavirus anxiety.Next, three interaction models were run to evaluate the effect of race on the association between each cognitive factor  and coronavirus anxiety. The first interaction model showed statistical significance between White and non-White participants, indicating a difference in the relationship between coronavirus crisis perception and coronavirus anxiety by race . In the second model, race also moderated the association between economic risk perception  and coronavirus anxiety. Race was not a statistically significant moderator between self-efficacy and coronavirus anxiety .The results of significant interaction models are displayed in This study explores various cognitive factors associated with coronavirus anxiety among subgroups disproportionately affected by anxiety in the general U.S. population. Using data from the start of the pandemic, our results indicate coronavirus crisis perception and perceived economic risk of coronavirus were positively associated with coronavirus anxiety and general self-efficacy was negatively associated with coronavirus anxiety when controlling for age, gender, race, education, employment, and income. Female gender and White race moderated the association between coronavirus crisis perception and coronavirus anxiety. Similarly, White race also moderated the association between economic crisis perception and coronavirus anxiety.These findings support the RPA framework by showing perceptions and efficacy are tied to health outcomes such as anxiety. Using the RPA framework to simultaneously measure risk perception and self-efficacy, standardized coefficients suggest crisis risk perception carries a stronger association with coronavirus anxiety compared to economic risk and self-efficacy. To potentially alleviate coronavirus anxiety among the general population, coronavirus health messages should address how protective health behaviors reduce the harmful impacts of pandemics on a global level.The coronavirus crisis perception measure used as an independent variable in this study was a more homogenous measure focused on a subjective assessment of the virus on a global scale, in contrast to prior risk-perception scales evaluating the SARS pandemic . A crossStudies have documented the association between financial and economic hardship and mental health outcomes, including those related to the coronavirus pandemic ,48,49,50Growing empirical support suggests self-efficacy may buffer some of the negative effects of mental health . PreviouStrengths of this study include early reporting on the outcome of coronavirus-specific anxiety measures that captured a composite of cognitive and somatic dimensions. This study builds on prior coronavirus studies ,29,42 byGeneralizations from the study need to be tempered by the fact that the Prolific online survey respondent population presents a bias toward female, young, highly educated, and White participants. In addition, respondents chose to participate, indicating they may be interested in the topic and may be systematically different from other populations . SimilarThis study provides initial insight into coronavirus anxiety in the general U.S. population. After controlling for covariates, this study found associations between higher coronavirus crisis perception and higher coronavirus anxiety, higher perceived economic risk and higher coronavirus anxiety, and lower self-efficacy and higher coronavirus anxiety. Our study has practical implications to psychiatry. The World Psychiatric Association outlines the role of psychiatrists in advocating for government interventions that reduce stress and suicide in the general population . Our stu


RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity. RNase MRP is a ribonucleoprotein (RNP) consisting of a highly structured RNA molecule (MRP RNA) and multiple protein subunits. The nucleolar RNP is structurally very similar to the precursor tRNA (pre-tRNA) processing complex RNase P ). RNase RMRP gene, which is transcribed by RNA polymerase III resulting in a 267 nucleotide transcript. The RNP contains at least seven protein subunits . CHH is rarely observed in the general population but occurs with a relatively high incidence in the Amish  and Finnish  populations. Patients affected with CHH are characterized by skeletal dysplasia, leading to short-limbed dwarfism, and hypoplastic hair. CHH is a pleiotropic disease and other symptoms, such as immunodeficiency, Hirschsprung’s disease, childhood anaemia and predisposition to specific cancers, are also frequently observed.RMRP. These mutations localize between the TATA box and the transcription start site of RMRP and include duplications, triplications and insertions. Mutations in the promoter region of RMRP lead to reduced transcription and decreased levels of MRP RNA [Multiple mutations causing CHH have been reported, and these can be divided in two groups . The fir MRP RNA . Promote MRP RNA .in vivo MRP RNA levels is missing. For some mutations, it was demonstrated that they affect the cleavage of the cyclin B2 mRNA or the precursor 5.8S rRNA substrates in human cell culture, whereas others lead to a decreased binding of a subset of the RNP protein components [The effect of CHH-mutations on the MRP RNA expression in CHH-patients has been described for a small number of patients ,9,11. Inmponents ,12,13.Even though a number of CHH-mutations have been studied on RNA expression levels, substrate cleavage and protein binding, a comprehensive overview of the effect of CHH-mutations on MRP RNA expression and stability, RNP composition and cleavage activity is lacking. Currently, no tool is available to isolate active RNase MRP complexes. Due to the high similarity to RNase P, immunoprecipitation of RNase MRP leads to a mixture of these two RNases. Therefore, we developed a method to specifically isolate RNase MRP, using the RNA streptavidin aptamer tag, S1m . FurtherAll PCR reactions were performed using the Phusion HSII polymerase (Thermo Scientific) in a two-step protocol. Oligonucleotides used are listed in Supplemental Table 1.The S1m aptamer sequence, flanked by 5’-BamHI and 3’-BglII restriction sites, was made as described previously . PCR mutMRP RNA mutations were introduced into the S1m-tagged DNA constructs by PCR-based mutagenesis.Adherent HEK293T cells were cultured in DMEM supplemented with penicillin/streptomycin and 10% FBS. Transfections were performed 18 to 24 hours after seeding of the cells once the cells reached 20% confluency. For transfection, branched polyethylenimine (PEI):DNA complexes were used in a 1:3 w/w ratio. For the transfection of a single well of a 12 well-plate, 1 µg of DNA was used and 48 hours after transfection the cells were harvested by trypsinization. For the transfection of cells cultured in petri dishes the amounts were adapted in accordance with the surface areas.2, 100 mM KCl, 10% glycerol, 0.1% NP-40 and Complete protease inhibitor (Roche)). To remove insoluble material the lysate was centrifuged for 11,000 x g at 4°C for 10 minutes. Subsequently, the lysate was diluted twice in lysis buffer without NP-40, resulting in a final NP-40 concentration of 0.05%. The lysate was added to 40 μL of packed pre-equilibrated streptavidin-Sepharose beads  and allowed to bind for 16 hours at 4°C. Subsequently, non-bound material was collected and the beads were washed three times with lysis buffer containing 0.05% NP40.In order to purify the S1m-tagged RNPs for determining the RNP composition and activity, HEK293T cells in three 15 cm petri dishes were transfected and lysed after 48 hours by sonication in 2 mL of lysis buffer  MRP RNA signals were used to quantify the relative amounts of bound material. For the activity analyses of mutant S1m-RNAs equivalent amounts of mutant S1m-RNA were used.As a positive control a mouse monoclonal anti-Rpp20 antibody  was coupled to protein-A-agarose beads, followed by immunoprecipitation of RNase MRP/P from a HEK293T cell lysate, as described previously [32P-labeled viperin Vip 401–500 mRNA fragment and precursor tRNA were generated by in vitro transcription, as previously described [in vitro transcribed using T7 RNA polymerase in the presence of 32P-labeled UTP. The viperin substrate was transcribed from a PCR product generated from a plasmid containing the human viperin cDNA. For the tRNA substrate, a linearized plasmid with yeast tRNASer was used as a template for in vitro transcription. Beads containing S1m-RNPs or anti-Rpp20 immunoprecipitated material were incubated with the radiolabeled substrates in a buffer containing 20 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 50 μg/mL BSA, 16.7 μg/mL E. coli tRNA and 4.2 Units/mL RNasin for 1.5–2 hours at 37°C for both the pre-tRNA and the viperin mRNA fragment substrate. The cleavage reactions were stopped by the addition of Trizol, the RNA was isolated, and the reaction products were analysed by electrophoresis in an 8% polyacrylamide/8 M urea gel followed by visualization by phosphorimaging.The escribed . Briefly+. After separation on a denaturing polyacrylamide gel, RNA was transferred to Hybond N+ membranes by electroblotting in 18.4 mM NaH2PO4 and 6.5 mM Na2HPO4 buffer. After drying the blot, RNA was fixed to the membrane by UV-irradiation at 700 mJ/cm2. Blots were blocked at 65°C in pre-hybridization buffer  for one hour. After blocking, radiolabeled antisense probes were added, followed by an overnight incubation. Subsequently the blots were washed twice with 1x SSC, 0.2% SDS and, depending on the remaining radioactivity, once or twice with 0.1x SSC. The signals were detected using Phosphor screens and visualized with the Typhoon imager. Expression levels of the S1m-tagged MRP RNAs were normalized to that of the U3 small nucleolar RNA (snoRNA). Within each experiment the level of the wild-type S1m-MRP RNA was set at 100%. Expression levels of 5’-S1m-MRP RNAs (except 154 G > U) were measured in quadruplicate and that of the 3’-S1m-MRP RNAs in triplicate.Cells were seeded in 12 well plates and transfected with (mutant) S1m-tagged RNase MRP constructs. After 48 hours the cells were harvested and total RNA was isolated using Trizol reagent (Ambion). Ten μg of total RNA was fractionated by electrophoresis in a 6% polyacrylamide/8 M urea gel and blotted on Hybond Nin vitro transcription of linearized plasmids as described elsewhere [Radiolabeled antisense probes for RNase MRP RNA, RNase P RNA and the U3 RNA were generated by lsewhere .After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes by electroblotting. The blots were incubated with antibodies as described previously . The folSince all known protein components of human RNase MRP are shared with RNase P, the available antibodies do not allow the selective isolation of either RNase MRP or RNase P. In order to study the effect of CHH-mutations on the protein composition of the RNase MRP complex, we developed an isolation procedure specific for RNase MRP based on the RNase MRP RNA component.Previously, Li and Altman introduced a streptavidin-binding RNA aptamer (S1) into the RNase P RNA to isolate active RNase P complexes from HeLa cells . LikewisHEK293T cells were transiently transfected with the S1m-tagged RNase MRP and P RNAs constructs and the expressed RNAs were isolated using streptavidin beads. All three S1m-tagged RNase MRP RNAs as well as S1m-tagged RNase P RNA were successfully isolated from the cell lysates . Moreover, Western blot analysis of the isolated material showed that at least a subset of the RNase MRP/P proteins were associated with the MRP and P RNAs . In case of RNase P, all six of the ten known protein subunits that were analysed  were found to bind to the S1m-P RNA. In case of RNase MRP, hPop1 and Rpp25 associated with the S1m-MRP RNAs and also weak signals were observed for Rpp38 in the material isolated with 5’- and 3’-S1m-tagged MRP RNAs. Interestingly, Rpp38 only appeared to associate with the MRP RNAs carrying the S1m tag at the 5’ or 3’ terminus, but not with the internally tagged RNA. As a negative control, an antibody reactive with the U1 and U2 snRNP-associated proteins U1A and U2B” was used. These proteins were indeed not detectable in the material isolated with the S1m-tagged RNAs.To investigate whether the complexes formed with the S1m-tagged RNase MRP and RNase P RNAs were functionally active, we incubated the isolated complexes with established substrates for both enzymes, a viperin mRNA fragment for RNase MRP and pre-tRNA for RNase P. The viperin mRNA fragment appeared to be cleaved by the 5’- and 3’-S1m-tagged RNase MRP complexes, but not by the internally tagged complex. Likewise, the pre-tRNA substrate was, as expected, specifically cleaved by the S1m-RNase P complex . IncubatThe assembly of functional RNase MRP complexes on S1m-tagged MRP RNA in transfected HEK293T cells indicates that this system can be used to assess the effects of mutations on biochemical properties of RNase MRP in an RNase P-independent fashion. It is also important to note that due to the insertion of the S1m-tag the size of the RNA is increased, which means that northern blotting of RNAs extracted from transfected cells allows discriminating between the endogenous MRP RNA and the S1m-tagged exogenous RNA.Eleven different RNase MRP RNA mutations that are found in CHH patients were introduced in the 5’- and 3’-S1m-tagged MRP RNA. Since the internally S1m-tagged MRP RNA forms an inactive complex, we did not pursue analyses with this variant. The selected mutations were spread across the MRP RNA molecule and except for the 70A>G, 94A, 95 G> DEL and 218A>G mutations, all are located in double-stranded regions of the predicted secondary structure. We investigated the effects of these CHH-mutations on the expression level of the RNA. To this end, we expressed the S1m-tagged wildtype and mutant MRP RNAs in HEK293T cells and assessed their expression levels by northern blot hybridization . The resIn vitro studies have shown that the 40 G > A substitution leads to reduced binding of Rpp25 to this part of the MRP RNA [To show that isolated S1m-MRPs can be used to study the association of specific proteins with MRP RNA mutants and the enzymatic activity of such mutant RNase MRPs, a selection of 3’-S1m-MRP mutants was used to investigate the binding of Rpp25 and the cleavage of the viperin mRNA fragment substrate. Rpp25 is known to bind to the P3 region . Although not all known protein subunits were confirmed to be associated with the MRP RNAs, the 5’- and 3’-S1m-tagged versions were catalytically active, as demonstrated by viperin mRNA fragment cleavage in vitro. Compared to the cleavage of the pre-tRNA substrate by RNase P, the efficiency by which the viperin mRNA fragment is cleaved by RNase MRP is low. As a consequence of this low efficiency, studying the effect of RMRP mutations on the cleavage activity using this substrate is difficult, especially when quantitative data are desired. For five mutants with CHH-derived mutations at various locations in RMRP virtually no cleavage was observed, but it should be noted that the signals obtained with the wild type RMRP are rather weak, which means that a partial reduction might already lead to signals below the detection threshold. Nevertheless, these data are in line with the reduction of catalytic activity of RNase MRP by CHH-associated mutations in the RNA component.Previously, it was shown that viperin mRNA is cleaved by immunoaffinity purified material obtained with anti-Rpp25 and anti-hPop4 antibodies, which will contain a mixture of RNase MRP and RNase P complexes . More reThe low cleavage activity of the 5’- and 3’-S1m-MRPs may be explained by the inefficient association of some protein subunits. It is possible that the S1m aptamer interferes with the binding of some proteins. Although the function of the individual protein subunits is unknown, they might be important for the catalytic activity of the complex. From a more quantitative point of view, it should be noted that transiently transfected cells expressing the S1m-tagged RNAs do not provide meaningful information on the ratio between the tagged and corresponding endogenous RNAs. Based upon northern blot hybridization signal intensities obtained with data from several experiments, the levels of tagged RNAs were estimated to be similar to those of the endogenous RNAs. However, it should be taken into account that during transient transfection only a subset of cells will express the transgene and that the levels of transgene expression may differ between individual cells. Therefore, the generation of stably transfected cell lines is required for more quantitative analyses.in vivo is dependent on methylation of the RNA. To more accurately quantify the effects of RMRP mutations on the cleavage activity, a substrate that is more efficiently converted by S1m-tagged RNase MRP is highly desired.Also, the immunoprecipitated RNase MRP/P complexes displayed a relatively low cleavage activity for the viperin mRNA fragment when compared to cleavage of pre-tRNA. In addition to suboptimal particle assembly, this may also be explained by differences in the catalytic activity between RNase MRP and RNase P or by suboptimal folding of the viperin mRNA fragment. Recently, RNase MRP has been implicated in the cleavage of mRNAs containing methylated adenosine, m6A, the most abundant internal modification in RNA . Endoribin vitro cleavage activity. Nevertheless, the MRP RNA can be isolated using streptavidin beads and appeared to be bound by hPop1 and Rpp25, just like the 5’- and 3’-S1m-tagged MRP RNAs. Although in theory the S1m-tag at the internal position might interfere with the enzymatic activity, it is more likely that improper particle assembly is responsible for this phenomenon. Indeed, Rpp38 did not detectably associate with the reconstituted complex, in contrast to the particles that were formed when the S1m-tag was positioned at the 5’- or 3’-end of RMRP. Since the internal tag was inserted in a region that is known to be involved in Rpp38 association [The internally S1m-tagged RNase MRP did not show any ociation , the tagThe generation of S1m-tagged MRPs was aimed at studies on the effects of mutations on the biochemical properties of this complex. We have shown that this approach not only allows the assessment of expression levels, protein binding and cleavage activity but that also the effects of mutations can be studied. The majority of the CHH-related RMRP mutants were expressed at significantly lower levels compared to the wildtype RNA. It is not yet known whether the lower expression levels are due to decreased RNA stability or transcription efficiency, but it is likely that the mutations destabilize the RNA. The binding of Rpp25 to the mutants was as expected based on the previously determined interaction between Rpp25 and the P3 region and therefore the 3’-S1m-tagged MRP provides a tool to analyse the binding of proteins to RMRP mutants, at least for Rpp25, Rpp38 and hPop1.in vitro, as shown by the cleavage of viperin mRNA. Furthermore, the S1m-MRP complexes can be used to study the effects of CHH-associated RMRP mutations on its expression level, RNase MRP protein composition and the enzymatic activity.We conclude that the use of the S1m-aptamer leads to successful isolation of RNase MRP. The method we developed represents the first experimental approach that allows for the highly selective purification of human RNase MRP in the absence of RNase P. This selectivity of S1m-MRPs isolation greatly facilitates the validation of putative RNase MRP substrates Click here for additional data file.


In the article by Chen et al., 1 State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, China2 Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, China3 NYU‐ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai, China


RNA structural research lags behind that of proteins, preventing a robust understanding of RNA functions. NMR spectroscopy is an apt technique for probing the structures and dynamics of RNA molecules in solution at atomic resolution. Still, RNA analysis by NMR suffers from spectral overlap and line broadening, both of which worsen for larger RNAs. Incorporation of stable isotope labels into RNA has provided several solutions to these challenges. In this review, we summarize the benefits and limitations of various methods used to obtain isotope-labeled RNA building blocks and how they are used to prepare isotope-labeled RNA for NMR structure and dynamics studies. RNA is a dynamic macromolecule that has many biological functions, including gene regulation ,2,3,4,5,2H, 13C, 15N, and 19F) labeling of RNA have revolutionized the analysis of RNA by NMR and opened the door to functional understanding.NMR is a competitive RNA structure determination technique that accounts for 35% of the structures in the NDB, as compared to the 8% of protein structures in the PDB B. HoweveThe main approaches to obtaining isotope-labeled RNA are enzymatic or chemical syntheses. For the enzymatic approach, almost all methods are based on DNA template-directed T7 RNA polymerase (RNAP)-based in vitro transcription (IVT) using labeled ribonucleoside 5′-triphosphates (rNTPs) ,29,30,31In this review, we detail the benefits and limitations of these four methods . With la2H, 13C, 15N, or both of the latter) is enriched; nucleotide-specific labeling is when every nucleotide of a certain type  is enriched; atom-specific labeling is when every atom of a certain type  is enriched; and position-specific labeling is when an individual nucleotide  is labeled. In the latter case, the type of label that is incorporated site-specifically can be uniformly or atom-specifically-labeled. Thus, these labeling categories are not mutually exclusive. Throughout this review, we follow the IUPAC/IUB guidelines for RNA atom numbering ,38. When2H, 13C, and/or 15N isotope labels from a commercial source. As of July 2021, Cambridge Isotope Laboratories (CIL), Sigma-Aldrich, Cassia LLC, Silantes, and INNotope are the major suppliers of isotope-labeled rNTPs, whereas Silantes and INNotope are the only suppliers of isotope-labeled RNA amidites. Unfortunately, these products can be prohibitively expensive. Uniformly-labeled 2H, 13C, 15N, 2H/15N, and 13C/15N-labeled and atom-specifically 2H, 13C, 2H/13C-labeled rNTPs are available for $800–5600 per 100 μmol or 50 mg , harvesting cells and extracting RNA, hydrolyzing RNA to rNMPs, and converting those to rNTPs. Triphosphate conversion can be achieved by chemical -sodium acetate labeled purine C4 and C6 (>90%), pyrimidine C2 and C4 (>95%), and ribose C3′ (~75%) [E. coli strains deficient in enzymes involved in the tricarboxylic acid cycle (DL323) [13C-glycerol sources [13C2]-AMP was created with an 80% yield in K10-1516 cells grown with [2-13C]-glycerol. Importantly, Hoogstraten and co-workers measured longitudinal (R1) and rotating-frame (R1ρ) relaxation rates in -AMP and uniformly 13C-labeled AMP to demonstrate that 13C–13C dipolar couplings lead to over-estimated relaxation rates [Hoffman and Holland modified the biomass method to make atom-specifically-labeled rNTPs . In thei′ (~75%) . Among o (DL323)  or the o (DL323)  were gro sources . While mon rates .13C]-pyruvate , 15N-ammonium chloride (15NH4Cl), and 13C/15N-L-serine enabled atom-specific labeling. As shown in 15NH4Cl. Similarly, 13C/15N-L-serine labels C2, C4, C5, C8, and N7, and NaH13CO labels C6. Finally, 13C-D-glucose provides the label for the ribose carbons  and C6. Williamson and co-workers were the first to describe the de novo biosynthesis of isotope-labeled purine rNTPs . Their a2 via decarboxylation of 6-phosphogluconate to ribulose-5-phosphate during PRPP production links the isotope label of the C1 of glucose to C6 in the purine nucleobase. If ribose labeling is not required, and only purine C6 labeling is needed, then PRPP must be made directly from unlabeled ribose. Similarly, care must be taken to prevent isotopic dilution from atmospheric CO2, if both 13C-ribose and 13C6-purine labeling are required. Additionally, the C2 and C8 positions are labeled together or not at all. Finally, C6 and ribose labeling are limited by commercial sources of D-glucose.While this methodology is very powerful, it also comes with a number of drawbacks. The use of labeled glucose can limit the potential labeling patterns if both ribose and nucleobase labels are desired. For example, production of CO13C2]-ATP in a 57% yield and 26, 24, and 27 enzymes helped synthesize uniformly 13C, 15N, 13C/15N-labeled GTP in 66, 24, and 42% yields, respectively -UTP with a 25% yield; and batches of 15, 16, and 16 enzymes helped synthesize uniformly 13C, 13C/15N, 15N--labeled UTP with 40, 45, and 30% yields, respectively -UTP to its CTP counterpart with a 48% yield. The utility of their UTP and CTP labels was demonstrated for the same 30 nt RNA model system. This labeling scheme reduced spectral crowding even more than before, owing to ribose deuteration. To summarize, a total of 16 biosynthetic enzymes were used for the efficient one-pot synthesis of UTP (and CTP) over 3–4 days with yields of up to 45% . Specifiectively . AdditioChemo-enzymatic labeling is a hybrid approach that we developed, taking inspiration from the Tinoco  and Will13C3-1,3-15N2]-UTP and -CTP in 90 and 95% yields, respectively, by coupling -uracil to -ribose -adenine or [8-13C]-guanine with either [1-13C], or [2-13C], or -ribose to yield the desired rNTPs in 70–95% yields -ribose . These a reports ,44,49. E% yields . These a% yields . Additio% yields ,31,56, a% yields ,59. ThisIt is worth noting that there are other approaches to enzymatically coupling nucleobase and ribose sources. Indeed, the synthetic method of Serianni and co-workers begins with the chemical synthesis of inosine, followed by its phosphorylation by purine nucleoside phosphorylase (PNPase) (EC 2.4.2.1) to give α-D-ribofuranosyl 1-phosphate sodium salt (αR1P) . The αR113C2]-AMP which was then dephosphorylated to adenosine and used as the entry point for amidite synthesis - and [6-13C-5-2H-]-uridine and -cytidine, and - and [8-13C]-adenosine and -guanosine TOM and tBDMS amidites for use in 1H- -UTP and [5-2H]-CTP enzymatically . . 101]. HAnother unique ligation strategy employs RNAse H and hammerhead (HH), Varkud satellite (VS), and hepatitis delta virus (HDV) self-cleaving ribozymes. This approach dramatically reduces 3′- and 5′-end heterogeneities and has therefore been embraced as a popular method to segmentally label large RNAs for NMR studies. Two such examples have come from the Puglisi  and LukaBuilding on this work, Wijmenga and co-workers developed an efficient two-step ligation method to selectively label central positions of large RNAs . The utiThe final two RNA preparation methods rely on enzymatic incorporation of position-specific isotope labels within RNA. These approaches hold promise for enabling site-specific NMR measurements of large RNAs, and therefore combine the benefits of both enzymatic and chemical SPS preparation methods. Wang and co-workers developed a powerful but laborious method to obtain position-specific isotope-labeled RNA ,109,110.To facilitate the use of this technique for the synthesis of larger RNAs, Liu and co-workers recently characterized the sequence requirement for the initiation step which is crucial for an efficient synthesis of RNA at optimal yields . From thIn an attempt to make position-specifically-labeled RNA more accessible, Schwalbe and co-workers developed a chemo-enzymatic synthetic approach. Therein, a single modified nucleoside-3′,5′-bisphosphate is incorporated to the 3′-end of an RNA fragment followed by DNA-splinted ligation to complete the desired internally labeled RNA sequence  116]. T. T116]. We have presented a detailed overview of the various methods for obtaining isotope-labeled rNTPs  and how However, RNA structural biology is moving toward larger and larger RNAs, especially as cryo-EM gains in resolution and popularity ,118. Nev