Picornaviral IRES elements are essential for initiating the cap-independent viral translation. However, three-dimensional structures of these elements remain elusive. Here, we report a 2.84-Å resolution crystal structure of hepatitis A virus IRES domain V (dV) in complex with a synthetic antibody fragment—a crystallization chaperone. The RNA adopts a three-way junction structure, topologically organized by an adenine-rich stem-loop motif. Despite no obvious sequence homology, the dV architecture shows a striking similarity to a circularly permuted form of encephalomyocarditis virus J-K domain, suggesting a conserved strategy for organizing the domain architecture. Recurrence of the motif led us to use homology modeling tools to compute a 3-dimensional structure of the corresponding domain of foot-and-mouth disease virus, revealing an analogous domain organizing motif. The topological conservation observed among these IRESs and other viral domains implicates a structured three-way junction as an architectural scaffold to pre-organize helical domains for recruiting the translation initiation machinery. Picornaviruses use modular RNA domains in their internal ribosome entry sites (IRESs) for translation through non-canonical, cap-independent mechanisms. Here the authors report the crystal structure of domain V from the IRES of hepatitis A virus (HAV) ssRNA genome, suggesting that the functional homology among different types of picornaviral IRESs is structure-based. For most eukaryotic mRNAs, translation initiation involves the interaction of translation initiation factors with a 5′-cap structure in the mRNA followed by the recruitment of the 40S ribosomal subunit, which then scans the mRNA to find a suitable start codon. In contrast, many viral genomes and a subset of cellular mRNAs bypass this canonical mechanism of translation initiation and use a non-canonical, cap-independent mechanism that involves the cis-acting RNA elements. These RNA elements located at the 5′-UTRs are usually known as internal ribosome entry sites (IRESs)5. During translation initiation, RNA domains in an IRES either interact with the 40S ribosome directly or recruit the ribosome through the interaction with the translation initiation factors in a cap-independent manner8. Similarly, cap-independent translation of many plant viruses involves RNA elements located near or within the 3′-UTRs of their genomes, termed cap-independent translational elements (3′-CITEs)11. Despite significant differences in size and location within viral genomes compared to IRES elements, 3′-CITEs essentially play roles analogous to IRES elements in recruiting translation initiation factors or the ribosome subunits11. The CITEs “circularize” the viral genome presumably by base-pairing interactions with the 5′ end, thereby priming the genome for translation initiation11. Here, we report the structure of a key RNA domain from an IRES that likely interacts with initiation factors, domain V of the IRES of hepatitis A virus (HAV), which is a member of hepatovirus genus from picornaviridae family.RNA structures in the 5′ and 3′ untranslated regions (UTRs) of an mRNA play fundamental roles in initiating and regulating translation, and influence nuclear export, cellular localization and the stability of the transcript15. Such diversity occurs even within the picornaviridae family, and thus picornaviral IRESs have been classified further into five types based on their predicted RNA secondary structures and their translation factor requirements, with the HAV IRES studied here belonging to type III. Moreover, some picornaviral IRESs such as those classified into type IV have secondary structure elements more similar to the IRES of HCV from Flaviviridae family than to IRESs within their own family. Consequently, correlating the structural organization of RNA domains in various types of picornaviral IRESs to their biological role remains a challenge, limiting our understanding of the mechanism of IRES dependent translation compared to the canonical translation.Despite a conserved biological function, IRESs diverge significantly in their primary sequences, secondary structures, and requirements for trans-acting translation initiation factors. For example, in hepatitis C virus (HCV) or cricket paralysis virus (CrPV) the IRES recruits the ribosome directly without the pre-recruitment of translation initiation factors, whereas many picornaviral IRESs recruit the ribosome through their interaction with translation initiation factors20. Recent high-resolution structures of dicistrovirus and HCV-like IRESs in complex with the ribosome, enabled by advances in cryo-electron microscopy, have yielded significant insight into how IRES structure relates to its biological function8. The ability of these IRES elements to recruit the ribosome directly has facilitated the acquisition of particles appropriate for structural studies with cryo-electron microscopy. In contrast, little high-resolution structural data has emerged for picornaviral IRESs which contain relatively long, highly flexible RNA elements21 and engage the translation initiation factors and the ribosome via a multistep, dynamic assembly process14. For picornaviral type II IRESs, a recent NMR structure of the J–K domain from the encephalomyocarditis virus (EMCV) represents the only high-resolution structure reported to date22. Here, we investigated the crystal structure of the corresponding domain of a type III picornaviral IRES–dV of HAV25. Compared to picornaviral type I and type II IRESs, the HAV IRES drives translation relatively inefficiently and imposes distinct requirements on cellular translation initiation factors27, which has rendered study of HAV IRES more challenging.Many biochemical, biophysical, and structural probing approaches have been used to elucidate the secondary structure of IRES domains and to understand the interaction of those domains with their cognate initiation factors28. The ~735-nt 5′-UTR contains six modular domains comprising highly organized RNA secondary structures that include the IRES elements designated as domains II to VI  and type II (EMCV J–K domain) IRESs both interact with the initiation factor eIF4G. Moreover, a recent biochemical and biophysical study suggested that polio virus (PV) and HAV IRES domains may share a common mechanism of binding and utilizing translation initiation factors such as eIF4G and eIF4F for viral translation31. However, it is not clear how these domains might perform similar roles during the viral translation despite having highly dissimilar primary sequences and predicted secondary structures.The IRES studied here occurs within the 7.5 kb positive-sense ssRNA HAV genome, which consists of a single open reading frame flanked by highly conserved 5′ and 3′ UTRs VI Fig.  and 229.don Fig. . Based o22. In particular, these domains exhibit striking similarities in the organization of the three-way junction by an analogous motif with an A-rich loop closed by a lone pair stem32 (designated A-rich motif or AL hereafter) and in the location of the bulges within the analogous helices. Such structural homology allowed us to obtain a three-dimensional structural model for the corresponding domain (dIV) in foot-and-mouth disease virus (FMDV) IRES using computational approaches. Consistent with the previous biochemical observations33, the FMDV dIV structure revealed a topological homology with EMCV J–K domain or HAV dV, including the similar structural configuration within the three-way junction. The helical junction architecture organized by an AL motif within these three-way junctions approximately resembles those suggested by computational models for some 3′-CITES but organized by a pseudoknot34. Given the diversity of primary sequences and secondary structures among picornaviral IRESs and 3′-CITEs, such similarity has shed light on the presence of analogous architectural strategies for organization of RNA domains that recruit translation initiation factors. Moreover, the dV structure, which represents the first high-resolution crystal structure of an RNA domain from any picornaviral IRES, will facilitate efforts to understand the structural organization and functional roles of essential RNA domains within the picornaviral IRESs.We employed a chaperone-assisted RNA crystallography approach to obtain the high-resolution structure of dV from HAV IRES. We obtained an antibody fragment (Fab) that binds to the RNA with low nanomolar affinity, crystallized the RNA in complex with the Fab and solved its structure at 2.84-Å resolution using the Fab as a molecular replacement model for the initial phasing. Unexpectedly, we observed that this IRES domain folds into a Y-shaped structure that topologically resembles the previously reported NMR structure of the J–K domain from EMCV IRES29 referred to hereafter as HAV domain V or dV. This construct lacks domain VI (nt 681–705 of the WT sequence), which is a small stem-loop inserted near the bottom of the P1 helical stem  and upstream of the pyrimidine-tract. They contain ~125 nucleotides that form extensive secondary structures Fig.  for phag37. To identify Fabs that specifically bind to dV, we performed selections using three reduced codon libraries, termed YSG, YSGR, and YSGRKX. The YSG and YSGR libraries contained constant sequences in CDRs-L1 and -L2 derived from the parent Fab4D5 sequence, and both libraries contained binary degenerate codon diversity in CDR-L3, -H1, and -H2, encoding equal proportions of Y and S at specific positions. The two libraries differed in their CDR-H3 diversity. In the YSG library, seven residues from the parent Fab4D5 CDR-H3 were replaced with diversified loops of variable lengths (6–17 residues) in which each position was a mixture of 20% Y, 15% S, 15% G, and 50% Z, where Z represents an equimolar mixture of all other natural amino acids except for Y, S, G, and C. In the YSGR library, each CDR-H3 position encodes 38% Y, 25% S, 25% G, and 12% R. In the YSGRKX library all six CDRs were diversified38. CDR-L1 and -L2 contained equal proportions of Y and S, CDR-H1 and -H2 contained equal proportions of Y, F, and S, and CDR-L3 and H3 encoded 25% Y, 15% S, 10% G, 12.5% R, 7.5% K and 30% X, where X represents all other amino acids except C, I, and M. Additionally, CDR-L1, -L3, -H1 and -H3 were designed to have variable loop sizes: CDR-L1 (5–6 residues), -L3 (2–8 residues), -H1 (3–8 residues), and -H3 (4–17 residues).Previously, we have used phage libraries displaying Fabs derived from the humanized Fab4D5 framework bearing reduced codon diversity in their complementarity determining regions (CDRs) to select Fabs that specifically bind to RNA targets of interest. We subsequently employed these Fabs for RNA crystallization and structure determination35. Using the YSGR library we obtained several clones that showed a positive RNA binding response in a phage ELISA assay. We expressed these Fabs individually as soluble proteins in E. coli using a phagemid expression vector, purified them by affinity and ion-exchange chromatography to obtain RNase free Fabs, and tested their binding affinity for the dV using a filter binding assay. The lowest Kd we observed for the expressed clones was 290 ± 10 nM . As our previously used Fab crystallization chaperones had RNA binding affinity in the low nanomolar range (10–100 nM), we proceeded with affinity maturation of this Fab using error-prone PCR as described previously36. However, we observed no significant improvement in the affinity for the RNA target and did not pursue this Fab further. Using the YSGRKX library we observed that a single clone was 20,000 times enriched after four rounds of selection. Following expression as soluble protein and purification by affinity and ion-exchange chromatography, we found that this Fab bound to dV with good affinity, with a dissociation constant (Kd) of 44 ± 8 nM as determined by a filter binding assay in 10 mM tris, pH 7.5, 50 mM KCl, 10 mM MgCl2 buffer at 23 °C  compared to the dV construct  minus all CDR loops as a search model, highlighting another benefit of Fabs for structure determination. Except for the CDR loops, sequences of the scaffold-domain of the Fab HAVx and Fab BL3-6 (PDB code: 4KZE) are identical; however, due to the flexibility of the Fab elbow angle39, we searched for the constant domain first followed by the variable domain rather than the intact scaffold. RNA was built unambiguously by modeling individual nucleotides into the electron density map obtained after the initial molecular replacement. After iterative rounds of model building and refinement at 2.84-Å resolution, the final values of Rfree and Rwork were 25.2% and 18.6%, respectively. The structural model of the HAVx–dV complex from the final refinement along with the 2|Fo|− |Fc| electron density map is shown in Supplementary Fig. To test the ability of Fab HAVx to serve as a chaperone for the RNA crystallization, we set up crystallization trials for the RNA constructs HAV593-684 (or dV), HAV593-706 and HAV593-720 in complex with the Fab HAVx using the hanging drop vapor diffusion method. In 22 out of 480 conditions screened, we observed crystals only for the Fab HAVx–dV complex. We observed no crystals in analogous trials using only the RNA, underscoring the usefulness of the Fab in assisting the dV crystallization. Four conditions were further optimized for pH, precipitant and salt concentration to grow larger crystals using the hanging drop vapor diffusion method. Within 1 week, we observed robust growth of large crystals in 0.2 M ammonium sulfate, 0.1 M HEPES, pH 7.5, 25% PEG 3350 at room temperature that diffracted to 2.84-Å resolution. To solve the crystal structure of Fab–dV complex, we obtained the initial phases by molecular replacement using the previous crystal structure of Fab BL3-6  between the P1 and P2 helices, adopting a lone-pair trinucleotide loop (LPTL) structure. The A-rich trinucleotide loop is closed by a single, non-canonical U611•A615 pair  do engage in base-pairing interactions  was not expected—the nucleotides involved (U611–A615) were instead proposed to contribute base-pairing interactions on the 5′-side of the P1 helix  and an overall distance distribution that follow the crystal structure closely  analysisχ2 = 244.1) from the profile predicted from the crystal structure  suggest that the standalone RNA adopts a more extended conformation in solution compared to the crystal  and 496 Å2 (38%), respectively (PDBePISA)6. For comparison, this interfacial area exceeds that observed for Fab-protein complexes (on average 777 ± 135 Å2)42 and Fab BL3-6–hairpin RNA epitope complex (821 Å2)43 but approximates that observed for Fab2–P4P6 RNA complex (1316 Å2)35. For RNA binding, Fab HAVx predominantly uses arginine, serine and tyrosine residues from four of the six CDRs, L1, L3, H2, and H3 to mediate stacking, electrostatic and hydrogen bond (direct and water-mediated) interactions , suggesting that this interaction likely has little influence on the stability and conformation of the complex. Supplementary Note Within the binding interface involving Fab CDRs, Fab-RNA interactions bury a total of 1315 Åons Fig. . Additioons Fig. . While mons Fig. . HoweverL motif containing a LPTL structure analogous to those observed previously in many rRNA and tRNA structures32. The AL motif  to engage in base-pairing interactions with U662-G666 to extend the P1b helix  Fig. . This 5-tif Fig.  stacks dtif Fig. . In contL motif in the dV structure forms a single non-canonical U•A pair to close a UAA trinucleotide loop  to mediate A-minor type tertiary interactions  within the GTPase center of E. coli 23S rRNA (PDB code: 1QA6)45. In contrast to the Hoogsteen base-pairing in the dV AL motif, in the ribosomal domain the U and A residues closing the trinucleotide loop form a Watson-Crick base-pair 35, the three adenines adopt essentially the same configuration that the three adenines in the AL motif (A613–A615) adopt, with the A’s stacked and oriented analogously for minor groove interactions  within the AL motif to uridines (U770–U775) abrogates eIF4G binding22. Similarly, constructs with A771U or C696A–G729U mutations, which prevent A771•C696–G728 base triple formation, do not engage the HEAT-1 domain, implicating the AL in a functionally significant structural role that determines the spatial arrangement of the helices around the three-way junction and pre-organizes the J–K domain for recruiting the translation initiation factors22. Consistent with the secondary structure homology between the EMCV J–K and FMDV dIV, previous UV-crosslinking and mutation analysis have also shown that deletions or mutations within the corresponding A-rich motif from FMDV IRES abolishes eIF4G binding and reduces IRES activity51.Despite some differences in the identities of the interacting nucleotides, the global organization of the three-way junction and the ARES Fig. –1522. Th P1 Fig. . Circula dV Fig. –15. HoweL motifs54. For example, previous biochemical assays have shown that EMCV J–K domain and FMDV domain IV (dIV) have homologous secondary structures that bind eIF4G similarly33. Nevertheless, modest rearrangement of the FMDV dIV secondary structure, particularly around the three-way junction, reveals the capacity to form an AL motif, yielding a revised secondary structure that improves homology between J–K domains, particularly in the J and St stem regions immediately flanking the junction, but remains consistent with the biochemical probing data 56. Specifically, we seeded simulations with the three-way junction as well as a two-way junction within the J helix from the EMCV J–K domain, threaded with the FMDV sequence and re-optimized. We then solved the remaining junctions and apical loops using stepwise Monte Carlo and used ensembles of those best models to seed a final FARFAR simulation. As expected, the computed model revealed topological homology with EMCV J–K domain and HAV dV, including the similar structural configuration of the three-way junction 22 revealed unexpected structural homology in which an AL motif organizes the topology of the respective three-way junctions  binds to eIF4G (aa 682–1599) with Kd = 239 ± 10 nM and eIF4E binding to eIF4G generates an even higher affinity complex that binds with Kd = 94 ± 3 nM. For EMCV, Lomakin et al.59 reported that the isolated J–K domain binds to eIF4G HEAT-1 domain (aa 643–1076) with Kd = 5 nM and 170 nM with and without the eIF4A, respectively. In addition, recent structural studies demonstrated that the HEAT-1 domain binds between the St and K domains22. A-rich motif deletion, mutation to a U-rich motif or mutation to perturb the tertiary interactions with the J and K helices abrogate HEAT-1 domain binding to the J–K domain. Each of these mutant constructs retains the secondary structure of the St, J, and K subdomains, implicating a structural role for the AL motif in modulating the spatial arrangement of the helices around the three-way junction22. Consistent with a functionally significant role of the AL motif, these mutations also have deleterious effects on translation and infectivity of the EMCV60. The dV from PV IRES (nts 448–555) binds to eIF4G (aa 557–1599) and eIF4G–eIF4E complex with Kd values of 276 ± 21 nM and 49 ± 2 nM, respectively31. However, analogous data for the isolated HAV dV are not available.The presence of a common motif across picornavirus IRES subtypes implicates a biologically significant function. Consistent with an important contribution from these domains to translation initiation factor recruitment, nucleotide truncations within HAV dV and EMCV J–K domain decrease or abolish in vitro translation of reporter constructs driven by the corresponding IRES elements. For HAV, the truncations of nts 638–739, 670–739, 638–694, 523–628, and 628–734 have a clear inhibitory effect on translation62. Addition of isolated EMCV J–K domain or PV IRES dV inhibited in vitro translation of a reporter construct, presumably through sequestration of eIF4G by the isolated viral domain  binds eIF4E and eIF4E-eIF4G complex with Kd = 58 ± 16 nM and 48 ± 21 nM, respectively64. Analysis of the secondary and computed 3-dimensional structures PEMV2 and PMV (panicum mosaic virus) 3′-CITEs revealed a pre-organized three-way junction that bears some resemblance to EMCV J–K domain or HAV dV except that a pseudoknot rather than an AL motif dictates the overall junction architecture  and the locations within the genome (5′- or 3′-UTR), implicates a structured three-way junction with preorganized helical domains as an effective platform for recruiting translation initiation factors. Viral domain structural information provides valuable input for developing algorithms to predict RNA structures and search for new IRESs and IRES-like RNAs using bioinformatic tools53, testing functional hypotheses, and designing structure-based therapeutics67.RNA elements within the 3′-UTR of some plant viruses (3′-CITEs) have been implicated in the recruitment of initiation factors for cap-independent translation68, Fab HAVx recognizes an asymmetric bulge within a helix, thereby offering an alternative RNA motif for grafting into an RNA construct to be crystallized with the Fab chaperone. Given the diversity of sequence composition, conformation and length of CDRs, Fabs, in general, could serve as a unique and versatile scaffold with the potential to bind to a wide variety of RNAs . The first two nucleotides of the reverse primer contained 2′-OMe modifications to reduce transcriptional heterogeneity at the 3′ end69. RNA was prepared by in-vitro transcription for 3 h at 37 °C in buffer containing 40 mM Tris-HCl pH 7.9, 2 mM spermidine, 10 mM NaCl, 25 mM MgCl2, 10 mM DTT, 30 U/ml RNase Inhibitor, 2.5 U/ml TIPPase, 4 mM of each NTPs, DNA template 30 pmol/ml, 40 μg/ml homemade T7 RNA polymerase70. Transcription reactions were quenched by adding 10 U/ml RNase free DNase I  and incubating at 37 °C for 30 min. After the Phenol/Chloroform/Isopropanol, pH 4.3 extraction, the RNA was purified by denaturing polyacrylamide gel electrophoresis. The corresponding RNA band was visualized by UV shadowing and excised from the gel. RNA was eluted overnight at 4 °C in 10 mM Tris, pH 8.0, 2 mM EDTA, 300 mM NaCl buffer. The buffer for eluted RNA was exchanged 3 times for pure water using 10 kDa-cutoff size-exclusion column . RNA was collected, aliquoted and stored at −80 °C until further use.DNA templates for transcription reactions were prepared by PCR amplification of ssDNA oligomer with T7-promotor sequence purchased from integrated DNA technologies  was hybridized with the biotinylated DNA oligonucleotide, 5′-Biotin-ACC GGG GAT CCT CTA GAG TC and this RNA–DNA hybrid were immobilized on the streptavidin-coated magnetic beads via biotin-streptavidin interaction. For the first round, 500 nM of RNA was immobilized by using a predetermined amount of beads required for complete immobilization and then incubated with 1013 cfu (colony forming units) of phages for 15 min in 1 ml of selection buffer, PBS , 0.05% Tween 20, 2.5 mM EDTA, 12.5 mM MgCl2, pH 7.4 supplemented with 0.1 mg/ml BSA, 0.1 mg/ml streptavidin, and 1 unit/µL RNase inhibitor . The solution was then removed, and the beads were washed twice with the selection buffer. In subsequent rounds, purified phage pools were first incubated with streptavidin beads in the selection buffer for 30 min, and the supernatant was used for the subsequent selection on a King Fisher magnetic particle processor . The 1011 cfu of Phages were incubated for 15 min with  of the RNA in 100 µl of the selection buffer, supplemented with 0.1 mg/ml BSA, 1 unit/µl RNase inhibitor, and 1.5 µM X-module DNA–RNA hybrid as a competitor. Streptavidin magnetic beads were then added to the solution for 15 min to allow the capture of the biotinylated RNA construct together with the bound phages. The beads were then blocked with 50 µM biotin, washed five times with the buffer, and eluted in 50 µL of elution buffer . The biotinylated RNase A was removed from the resulting phage library by incubation with streptavidin beads. After each round of selection, recovered phages were amplified as described previously36. After 3rd and 4th round of selection, phages were sequenced.Phage display to select a Fab that binds the RNA of interest was performed by following similar strategy as described elsewhererd or 4th rounds were tested for binding to target RNA using phage ELISA. For the phage ELISA assay, the RNA construct was immobilized through the x-module via biotin-neutravidin interactions, and ELISA assay was performed according to published protocols71. Clones that showed a positive binding response in a phage ELISA assay were reformatted for soluble protein expression with the introduction of a stop codon on phagemids using Q5 site-directed mutagenesis kit . Fabs were expressed and purified first on a small scale (100–250 mL culture) as described elsewhere71. No affinity tag was employed in the purification, which is described briefly below. Binding affinity of each clone with the HAV593-684 RNA lacking the x-module was determined by filter binding assay. Fabs that bound the RNA with desirable affinity were then expressed on a larger scale (4-liter culture) according to published protocols55. Both small and large scale Fab production methods essentially followed similar steps and yielded pure and RNase free Fabs. Briefly, phagemids with stop codon were transformed into 55244 chemical competent cells (www.atcc.org) and directly inoculated a starter culture with 100 µg/ml ampicillin. This overnight culture was then used to inoculate 2xYT media and grown for 24 h at 30 °C. Culture was centrifuged at room temperature, cell pellet was resuspended in the same volume of CRAP-Pi media71 with 100 µg/ml ampicillin and grown for 24 h at 30 °C. Collected cell pellets were lysed in PBS buffer, and Fab proteins were purified using the AKTAxpress fast protein liquid chromatography (FPLC) purification system  as described previously36. The lysate in PBS buffer (pH 7.4) was loaded into a protein A column, the eluted Fab in 0.1 M acetic acid was dialyzed back into the buffer PBS (pH 7.4) and loaded into a protein G column. The eluted Fab from protein G column in 0.1 M glycine (pH 2.7) dialyzed into 50 mM NaOAc, 50 mM NaCl buffer (pH 5.5) and loaded into a heparin column. Finally, the eluted Fab in 50 mM NaOAc, 2 M NaCl (pH 5.5) was dialyzed back into 1x PBS (pH 7.4), concentrated, and analyzed by 12% SDS-PAGE using Coomassie Blue R-250 staining for visualization. Aliquots of Fab samples were tested for RNase activity using the RNaseAlert kit . The aliquots of Fab samples were flash frozen in liquid nitrogen and stored at −80 °C until further use.Enriched output clones from the 343. Briefly, ~ 20 pmol of RNA was 5’-32P radiolabeled and purified by denaturing polyacrylamide gel electrophoresis. A constant amount of radiolabeled RNA was incubated at 50 °C for 10 min in a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 10.1 mM MgCl2 and 0.1 mM EDTA. The sample was cooled to room temperature for 10 min and incubated for 30 min with Fab HAVx ranging from 2 nM to 2 µM in a final volume of 40 µL. The Bio-Dot apparatus from Bio-Rad was assembled by placing a BA85 nitrocellulose filter  at the top and Hybond filter at the bottom  and wells were pre-equilibrated with 100 µL of selection buffer. The Fab–RNA complex was applied and washed 2 times with 100 µl of the selection buffer at a time. Both filters were air dried, exposed to Phosphor-Imager screens, scanned with a Typhoon Trio imager  and the amount of RNA retained in each of the filters was quantified by using Image Quant software (Molecular Dynamics). The dissociation constants were calculated by fitting the data of fraction of RNA retained in the nitrocellulose membrane versus the concentration of the Fab to the equation:F represent the fraction of bound RNA at a given concentration of the Fab, Kd is the dissociation constant and F0 and Fmax are the minimum and maximum fractions of the bound RNA, respectively.The binding constants of selected RNA clones and related mutants were determined by nitrocellulose filter binding assay as reported previously2 and 0.1 mM EDTA buffer. For refolding, RNA was heated at 90 °C for 1 min in water, added the appropriate volume of 10× folding buffer and then incubated at 50 °C for 15 min in 1× folding buffer followed by incubation at room temperature for 5 min and in ice for 5 min. The refolded RNA was then incubated with 1.1 equivalents of the Fab at room temperature for 30 min and concentrated to 6 mg/ml using 10 kDa-cutoff, Amicon Ultra-15 column (www.emdmillipore.com). The formation of Fab–RNA complex was confirmed by native polyacrylamide gel electrophoresis (nPAGE). To decrease the number of nucleation events, Fab-RNA complexes were then passed through 0.2 μm cutoff, Millipore centrifugal filter units ((www.emdmillipore.com). A Mosquito liquid handling robot  was used to set up high-throughput hanging-drop vapor-diffusion crystallization screens at room temperature using commercially available screening kits from Hampton Research, Sigma and Jena Bioscience. Several trials were reproduced in larger 1 complex μL + 1 μL well hanging drops on siliconized glass slides. Crystals appeared and grew to full size within 2–3 days at 22 °C in a drop with well condition 0.2 M ammonium sulfate, 0.1 M HEPES, pH 7.5, 25% PEG 3350. For cryoprotection, drops containing suitable crystals were brought to 30% glycerol, keeping all other compositions same. Crystals were immediately flash-frozen in liquid nitrogen after being fished out from the drops and taken to Argonne National Laboratory for collecting the X-ray diffraction data.An aliquot of RNA sample was refolded in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 10.1 mM MgClhttps://rapd.nec.aps.anl.gov/rapd/). Initial phases were obtained by molecular replacement with previously reported structure of Fab BL3-6 (PDB code: 4KZE or 3IVK) as the search model using Phaser on Phenix72. Except for the CDRs, sequences of Fab BL3-6 and Fab HAVx are identical. Iterative model building and refinement were performed by using COOT73, and Phenix package72. RNA was built by modeling the individual nucleotides into the electron density map obtained from the molecular replacement. During the refinement, default NCS option in Phenix was selected. Most of the water molecules were automatically determined by Phenix during refinement. Some water molecules were added manually for the positive electron density in the map based on their possibility to form hydrogen bonds with protein or RNA residues. However, we cannot rule out the possibility that at this modest resolution these densities belong to ions like Mg2+, K+, Cl− etc. Solvent-accessible surface area and area of interaction were calculated using PDBePISA (http://www.ebi.ac.uk/pdbe/pisa/). All structure related figures were generated in PyMOL  and figure labels were edited in CorelDraw .The X-ray diffraction data sets were collected at the Advanced Photon Source NE-CAT section beamlines 244-ID-B and 24-ID-C. All the datasets were then integrated and scaled using its on-site RAPD automated programs  data set and merging with ALMERGE, extrapolating to infinite dilution. SAXS curves were calculated from the crystal structure atomic coordinates and fit to the experimental data using the FoXS. The bead model molecular envelopes were constructed with DAMMIF. The details regarding the SAXS data collection, scattering-derived parameters, and programs used for the data analysis with associated references are presented in Supplementary Figs. SAXS experiments were conducted on the SIBYLS beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory following similar protocols described elsewherehttps://www.github.com/everyday847/FMDV_homology), along with detailed explanation of each modeling sub-step. All modeling was conducted with Rosetta 3.1074. The model from the NMR structure of EMCV (PDB code: 2NBX)22 was obtained and the central three-way junction was excised, renumbered to match the FMDV numbering (A:692–696 A:728–733 A:766–777 becomes A:317–321 A:365–370 A:394–405), and threaded. The same process yielded a model of the J-arm’s central two-way junction (EMCV residues A:698-708 A:716–726 become A:323–333 A:353–363). We conducted two simulations to generate models of the full FMDV Domain IV. First, we directly conducted a FARFAR simulation55 using the inferred secondary structure previously discussed, seeded with the fixed three-way junction described above; we generated 10,000 models and clustered the lowest-energy 1000 with a cluster radius of 2.0 Å heavy-atom RMSD. Second, we identified several individual junctions and hairpin loops that merited high-resolution modeling: two-way junctions in the St and K helices, as well as the hairpin terminating the J helix. We set up stepwise Monte Carlo simulations56 for these individual motifs. We generated 10,000 models of each junction, clustered the lowest energy 1000 models with a cluster radius of 2.0 Å heavy-atom RMSD, and took the ten lowest energy cluster centers as representative models of those junctions. Proceeding with those models, we seeded a FARFAR simulation55 with each of those “libraries” of starting conformations, which generated 40,000 models, and we clustered the lowest energy 1000 models with a cluster radius of 2.0 Å heavy-atom RMSD. We took the most populated cluster center among the ten lowest energy clusters as the single best model of the interaction.All input files and scripts necessary to conduct the modeling are provided as GitHub repository (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


RNA interference (RNAi)-based antiviral defense generates small interfering RNAs that represent the entire genome sequences of both RNA and DNA viruses as well as viroids and viral satellites. Therefore, deep sequencing and bioinformatics analysis of small RNA population  allows not only for universal virus detection and genome reconstruction but also for complete virome reconstruction in mixed infections. Viral infections (like other stress factors) can also perturb the RNAi and gene silencing pathways regulating endogenous gene expression and repressing transposons and host genome-integrated endogenous viral elements which can potentially be released from the genome and contribute to disease. This review describes the application of small RNA-omics for virus detection, virome reconstruction and antiviral defense characterization in cultivated and non-cultivated plants. Reviewing available evidence from a large and ever growing number of studies of naturally or experimentally infected hosts revealed that all families of land plant viruses, their satellites and viroids spawn characteristic small RNAs which can be assembled into contigs of sufficient length for virus, satellite or viroid identification and for exhaustive reconstruction of complex viromes. Moreover, the small RNA size, polarity and hotspot profiles reflect virome interactions with the plant RNAi machinery and allow to distinguish between silent endogenous viral elements and their replicating episomal counterparts. Models for the biogenesis and functions of small interfering RNAs derived from all types of RNA and DNA viruses, satellites and viroids as well as endogenous viral elements are presented and discussed. Nicotiana benthamiana plants inoculated with potato virus X  using RNA blot hybridization, and their abundance was found to gradually increase in the time course of viral infection  and its RNAi-deficient mutant lines, and discusses conservation of these mechanisms in other plant species and families. Finally, the review presents models for biogenesis and possible functions of siRNAs derived from endogenous viral elements (EVEs) which represent host genome-integrated counterparts of extant or ancient episomal DNA viruses of the families Caulimoviridae and Geminiviridae and of some RNA viruses, and highlights characteristic differences in siRNA size, polarity and hotspot profiles between a silent EVE and its episomal copy that can potentially be released from the host genome to cause systemic and transmissible infection.This review article compiles and summarizes growing evidence demonstrating a universal power of small RNA-omics for diagnostics of all types of plant viruses and for exhaustive reconstruction of viromes of various cultivated and non-cultivated plants. Plant virus diagnostics and genome assembly by high-throughput sequencing of other target nucleic acids such as long single-stranded or double-stranded RNA and, in the case of DNA viruses, circular viral DNA enriched by a rolling circle amplification method have been previously reviewed . Furtherbona fide siRNAs generated by the plant RNAi machinery (described below), have been reported for circular single-stranded (ss)RNA viroids replicating in both chloroplasts (Avsunviroidae) and nuclei (Pospiviroidae) and for viral families with six types of genomes : (i) positive-sense ssRNA , (ii) negative-sense ssRNA , (iii) double-stranded RNA , (iv) positive-sense ssRNA replicating via DNA intermediate by reverse transcription , (v) single-stranded DNA  and (vi) double-stranded DNA replicating via RNA intermediate by reverse transcription [dsDNA-RT: Caulimoviridae]  associated with helper ssDNA viruses of Geminiviridae (Supplementary Table Alphasatellitidae) associated with helper ssDNA viruses of Geminiviridae and Nanoviridae , monocots (n = 10) and ANITA grade basal angiosperms (n = 2) are represented with at least one plant species. The basal angiosperms are represented with Amborella trichopoda (family Amborellaceae) that generated small RNAs from three endogenous caulimovirids of a tentative genus Florendovirus (Caulimoviridae) and water lily (Nymphaeaceae) that accumulated small RNAs from (i) cucumber mosaic virus , (ii) an uncharacterized virus from the genus Cytorhabdovirus (Rhabdoviridae) and (iii) an uncharacterized, likely endogenous, caulimovirid (Cycadaceae), moss Physcomitrella patens (Funariaceae) and green algae Chara coralline (Characeae), which all accumulated small RNAs from putative endogenous caulimovirids (Marsileaceae) that accumulated small RNAs from turnip yellows virus   .Caulimoviridae have been identified in the genomes of gymnosperms, ferns and club mosses (Florendovirus (Caulimoviridae) identified in the genomes of angiosperms  and Pseudoviridae (Ty1/Copia) which can give rise to siRNAs as has been reported for the angiosperms A. thaliana  small RNAs with 5′U derived from both strands of a (-)ssRNA bunya/phlebo-like virus  and from both strands of an LTR Copia retrotransposon (Pseudoviridae), which is indicative of a functional antiviral RNAi response  and rice tungro bacilliform virus , only RTBV-derived siRNAs could be readily identified by deep sequencing, while RTSV-specific reads were negligible and comparable to those in a control “virus-free” plant  could be identified by de novo assembly only in the long RNA dataset, because of low abundance of the viral reads in the small RNA dataset , Potyviridae (turnip mosaic virus) and Virgaviridae (oilseed rape mosaic virus) spawn highly abundant siRNAs constituting 30–70% of total  small RNA sequencing reads, whereas their suppressor protein-deficient mutant derivatives spawn siRNAs of much lower abundance that correlates with strongly reduced titers of viral genomic RNA  whose siRNAs constituted ca. 90% of total small RNA reads in symptomless N. benthamiana -derived siRNAs constitute ca. 50% of total small RNA reads in A. thaliana at late stages of infection  for another 5 viruses. Likewise, small RNA-omics has been successfully used for reconstruction of the complex viromes of vegetatively cultivated sweet potatoes  and RNA (Virgaviridae) viruses the host genome filtering allowed for de novo assembly of complete viral genomes, albeit this step was implemented after small RNA assembly into contigs by a short read assembler, followed by assembly of all the unmapped contigs by a long read assembler  in N. tabacum is reportedly 10 times higher than that of quantitative RT-PCR  with extremely low titer (below immune detection) and symptomless in sweet potato could be assembled by sequencing viral small RNAs that covered the complete reference genome at an average 470 reads per nucleotide  coinfecting a potato plant could be de novo assembled into strain-specific small RNA contigs only for a 1 kb 5′-portion of the virus genomes which share 75% nucleotide identity. The remaining portions of the genomes sharing >87% identity merged into one chimeric small RNA contig and could be separated only by a reference-based approach  with 82–87% identity in co-infected tomatoes (Potato virus X (Potexvirus) with 80% identity in a co-infected potato (Virgaviridae) enabled construction of a fully biologically active infectious clone of the virus  strains or genetic variants of the same virus when they share high sequence identity. Thus, two strains of approach . Likewistomatoes  and two d potato  could bed potato . Thus, she virus .Barley stripe mosaic virus  by sequencing small RNAs extracted from a 700 years-old seed of barley, with 99.4% of the contemporary virus reference genome being covered by small RNA contigs. Likewise, Cilevirus, unassigned family) and citrus leprosis virus nuclear type  from herbarium specimens of orange fruit peals collected in 1967 and 1948, respectively. The genome sequences of these isolates were found to be respectively 99% and 80% identical to those of contemporary isolates of the two viruses that had previously been identified by sequencing and de novo assembly of small RNAs from fresh citrus samples  family proteins. Both miRNAs and siRNAs are then loaded onto Argonaute (AGO) family proteins to guide the resulting RNA-induced silencing complexes (RISCs) to complementary target RNA and/or DNA molecules (reviewed in trans-acting siRNAs (tasiRNAs) and epigenetically activated siRNAs (easiRNAs) and their biogenesis requires miRNA-directed cleavage of target mRNA or other Pol II transcripts and then involves complementary RNA synthesis by RNA-dependent RNA polymerase (RDR) family protein(s) (RDR6 or RDR1), followed by DCL4- and/or DCL2-mediated processing of the resulting dsRNA into 21-nt and 22-nt siRNAs. Secondary siRNAs are loaded onto AGO1 or AGO2 clade proteins to post-transcriptionally repress their target RNAs through in cis or in trans cleavage and degradation  can interfere with HEN1-mediated methylation of miRNAs and viral and endogenous siRNAs, likely through binding and sequestering small RNA duplexes produced by DCLs  and ssDNA  viruses do not interfere with HEN1 activities in A. thaliana  small RNAs are methylated at 2′OH of 3′-terminal nucleotide by the methyltransferase HEN1, which protects small RNA from degradation. Notably, suppressor proteins of some (+)ssRNA viruses  small RNAs onto specific AGOs is determined largely by the small RNA 5′-terminal nucleotide and length, but also other factors  and turnip mosaic virus  lead to activation of DCL4- and RDR1-dependent production of endogenous 21-nt siRNAs from multiple protein-coding genes. These virus-activated siRNAs (vasiRNAs) are incorporated into AGO1 and AGO2 RISCs and silence the corresponding host genes post-transcriptionally  and, to a much lesser extent, in the eudicot N. benthamiana and the monocot Oryza sativa, have been reviewed in great detail strand bias of viral small RNAs has been reported for some (+)ssRNA viruses such as cucumber mosaic virus  in tomato and N. benthamiana but not N. tabacum, and citrus tristeza virus  in four out of nine different Citrus species hosts, and for some members of Tombusviridae and Virgaviridae in some hosts. On the other hand, (+)ssRNA viruses of Tymoviridae often exhibit (-)strand bias in viral siRNAs  but not for the helper virus itself that spawned 22-nt and 21-nt siRNAs from both strands in N. benthamiana -derived small RNAs in N. benthamiana was initially attributed to DCL-mediated processing of secondary structures of single-stranded viral genomic RNA -infected A. thalianadcl2 dcl3 dcl4 triple mutant plants have a strong (+) bias and a much broader size range, unlike those predominantly 21-nt and 22-nt viral siRNAs produced from both strands in control wild-type plants ssRNA, (-)ssRNA, dsRNA], 21-nt and 22-nt small RNAs are produced from both strands of the viral genome without any strong bias in most cases ssRNA, ssDNA and dsDNA-RT viruses. Nonetheless, analysis of the viral siRNA size, strand bias and coverage profiles for (-)ssRNA and dsRNA viruses revealed similarities to those of (+)ssRNA viruses  and the dsDNA-RT virus Cauliflower mosaic virus  using combined blot hybridization, small RNA-seq and biochemical approaches in A. thaliana and its RNAi-deficient mutant lines  and its suppressor-deficient mutants have been investigated with a main focus on involvement of components of RdDM and histone modification machinery in antiviral responses -infected cells -infected cells  in A. thaliana (Tungrovirus) in O. sativa  and genomes of other plant species have not been conclusively shown to be involved in biogenesis of endogenous or viral siRNAs, although a tomato Ty-1/Ty-3 RDR gene is implicated in defense against tomato yellow leaf curl virus  and amplification of viral siRNAs (Caulimoviridae (Badnavirus have the (-)strand discontinuity at a very short distance from the Pol II start site, which may not be compatible with efficient production of a dsRNA decoy , whereas AGO1-associated 21-nt and 22-nt viral siRNAs were readily detectable in this pathosystem  and cotton leaf curl Multan betasatellite (Tolecusatellitidae), similar to their helper viruses of Geminiviridae (Geminiviridae and Nanoviridae and their satellites (Alphasatellitidae and Tolecusatellitidae), dsRNA precursors of viral siRNAs may also arise from pervasive sense and antisense transcription mediated by Pol II on multimeric linear dsDNA byproducts of recombination-dependent replication  and Pseudoviridae  that spawn 21-nt, 22-nt, and 24-nt siRNAs as discussed above, EVEs of the families Caulimoviridae (dsDNA-RT), Geminiviridae (ssDNA) and Bromoviridae [(+)ssRNA] have been characterized by small RNA sequencing , some of its EVE copies integrated in the genome of Petunia hybrida are arranged in tandem repeats, which allows for Pol II transcription of complete pgRNA followed by reverse transcription, giving rise to episomal viral dsDNA  and asymmetric (CHH) sites, which is a clear signature of RdDM , virus-derived siRNAs of 21-nt and 22-nt classes are more abundant than those of 24-nt class  and other Dioscorea species appear to express the viral replication protein Rep  proteins spawns siRNAs covering both strands of the integrated sequence, with 21-nt and 24-nt species being the first and the second most abundant  is integrated in the genome of Glycine max (soybean) ssRNA virus Cucumber mosaic virus  , while E species . In both species , which i species . Notably species . Based ohpsiRNAs  and the hpsiRNAs , the CMVhpsiRNAs  . The distinguishing features of silent EVEs of Caulimoviridae and Geminiviridae as well as RNA viruses would be predominantly 24-nt siRNAs of lower abundance which might be more broadly distributed along the reference genomes of their episomal counterparts and which would represent only those sequences of a reference episomal virus genome that are integrated in the host genome. Furthermore, siRNAs derived from inverted repeats of EVEs (if any) are expected to be more abundant and likely enriched in 22-nt and 21-nt classes in addition to 24-nt class. Further research using small RNA deep sequencing is needed to investigate size, 5′-nt identity, polarity, and hotspot profiles of EVE-derived siRNAs as well as the mechanisms of epigenetic silencing and activation of the infective EVEs giving rise to episomal viruses.Virus infections (like other stress factors) and especially activities of viral suppressor proteins might potentially perturb the RdDM machinery and lead to activation and release of transposons and infective EVEs which would contribute to disease severity. For the purpose of virus diagnostics and virome reconstruction the distinguishing features of episomal dsDNA and ssDNA viruses would be (i) high abundance viral siRNAs of both 21–22 nt and 24-nt classes, reflecting activities of the components of both post-transcriptional and transcriptional silencing machineries, and (ii) coverage of the entire circular viral genome sequences with siRNAs with characteristic hotspots within ORFs rather than Pol II promoter regions  in tomato  in cucurbits  in sweet cherry  in N. benthamiana -derived 21-nt siRNAs and less abundant 22-nt siRNAs are generated by DCL4 and DCL2, respectively, in the cytoplasm of infected peach cells. Likewise, DCL4 and DCL2 have been proposed to mediate the biogenesis of predominantly 21-nt siRNAs and less abundant 22-nt siRNAs derived from both strands of apple hammerhead viroid (tentative Pelamoviroid) in apple  has revealed that 21-nt, 22-nt, and 24-nt viroid siRNAs are generated by DCL4, DCL2 and DCL3, respectively (dcl2 dcl3 dcl4 triple knockdown lines (A. thaliana, with both DCL4 and DCL1 contributing to production of 21-nt viral siRNAs (N. benthamianardr6 knockdown plants (A. thaliana AGOs agro-expressed in N. benthamiana followed by small RNA sequencing (de novo RNA-directed DNA methylation of the transgenes containing viroid sequences (In the case of nuclear viroids, their replication in the nucleus via an asymmetric rolling circle mechanism (likely mediated by Pol II; reviewed in ectively . DCL1 mawn lines . This isl siRNAs . Interesl siRNAs  (Figure n plants . This suquencing . This inquencing , althougquencing . Notablyequences . Whetherequences .Dianthus caryophyllus retroviroid-like element integrated in the genome of carnation could be identified by small RNA sequencing and assembly (Interestingly, assembly , suggestA. thaliana imply that in those species of land plants that lack some of the paralogs of the DCL, RDR, or AGO family genes (As described above, all families of land plant viruses and viroids spawn characteristic small RNAs whose deep sequencing and bioinformatics analysis allows for virus identification and virome reconstruction. The small RNA size, polarity and hotspot profiles are indicative of virus interactions with components of the plant small RNA-generating RNAi machinery and allow to distinguish between exogenous viruses and silent EVEs, some of which can potentially be released from the plant genome to establish systemic and transmissible infections. Based on the conservation of the small RNA-generating RNAi machinery in all land plants and eukaryotic algae, the small RNA-omics approach is universal for diagnostics of known viruses, identification of viruses or virus-like agents associated with diseases of unknown etiology, and exhaustive reconstruction of viromes of any plant species. Specialized but partially redundant functions of DCLs, RDRs, and AGOs in both endogenous and antiviral siRNA pathways revealed in the model plant ly genes , other rly genes . As mentNotably, viral siRNA size and hotspot profiles of those propagative RNA viruses that replicate both in the plant and the insect vector differ substantially . Unlike MP wrote the manuscript, prepared the figures, and the Supplementary Tables and Lists.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stevia rebaudiana, a perennial herb native to northeastern Paraguay, has gained immense attention globally over the recent decades due to the natural sweetness of its leaves. Like in most plants, this particular species contains high amount of secondary metabolites, thus rendering the isolation of high quality and quantity RNA extract for molecular applications rather challenging. An effective, high-yield and high-quality RNA isolation protocol for this economically important plant species was devised here based on the cetyltrimethylammonium bromide (CTAB) extraction method, with an additional genomic DNA (gDNA) removal step. DNA and other contaminants that may affect downstream applications were effectively removed. Our results exhibited that RNA samples isolated from the leaves and stems of Stevia rebaudiana using this improvised method are high in integrity and quality with RNA integrity number (RIN) of more than 8 and low in contaminants. Stevia rebaudiana (Bertoni) is an herbaceous perennial plant of the Asteraceae family that is mostly grown in tropical and subtropical climatic regions . Although considerable amount of research has been carried out on this plant, studies investigating genetic background such as the molecular mechanisms controlling the biosynthesis of the diterpenoids and other secondary metabolites that give Stevia rebaudiana its commercial value are still limited. This may partly due to the difficulties in isolating high quality nucleic acid samples from this plant. regions . The pladiseases . The expStevia rebaudiana contains high amount of secondary metabolites including alkaloids, flavonoids, glycosides, saponins and tannins (Ribonucleic acid (RNA) extraction from plant tissue is always challenging. Plants contain polysaccharides and secondary metabolites such as polyphenolic compounds that could interfere with the quality and quantity of the RNA extract by co-precipitating with or binding to RNA and resulting in poor yields , 70% and 100% ethanol (ice-cold), RQ1 RNAse-Free DNAse , and DEPC-treated and -autoclaved distilled water. All chemicals used were of molecular grades.All glass apparatus, mortar and pestle, and plastic wares were pre-treated with 0.1% DEPC (diethylpyrocarbonate) overnight and then autoclaved at 121°C for 45 min before being used for RNA isolation. All solutions were prepared with distilled water pre-treated with 0.1% DEPC. The extraction buffer used for this study contained: 2% CTAB (cetyltrimethylammonium bromide), 100 mM Tris-HCl (pH 8), 2M NaCl, and 25 mM EDTA (ethylenediaminetetraacetic acid). The other solutions and reagents used include: PVP , β-mercaptoethanol, chloroform: isoamyl alcohol (24:1), 8M lithium chloride . The 1.5 mL tube was shaken a few times for mixing and centrifuged at 16500 ×g for 10 min at 4°C. Then, the supernatant was transferred into a new 1.5 mL tube and 1/3 volume of 8M LiCl2 was added, mixed well and the sample was precipitated at −20°C overnight.Prior to pre-warming at 65°C in dry bath, 0.014 g of PVP and 14 μL of β-mercaptoethanol were added to 686 μL of extraction buffer. In the meantime, 0.1 g of frozen leaf (or stem) samples were ground into fine powder using pre-chilled mortar and pestle in the presence of liquid nitrogen. The fine powder was then transferred into a 1.5 mL microcentrifuge tube and 700 μL of pre-warmed CTAB extraction buffer containing PVP (2% w/v) and β-mercaptoethanol (2% v/v) were added immediately. The sample was vortexed twice, each time for 10 s, and immediately incubated at 65°C for 20 min with gentle mixing by inverting the 1.5 mL tube every 5 min. An equal volume of chloroform: isoamyl alcohol (24:1) was added into the 1.5 mL tube containing the sample, and vortexed until the mixture was homogenous. The mixture was then centrifuged at 16500 ×Following overnight precipitation, the sample was centrifuged at 16500 ×g for 20 min at 4°C and the RNA pellet was washed twice with 70% ice-cold ethanol and finally with ice-cold absolute ethanol. The resultant RNA pellet was air-dried for 10 min and then resuspended with 30 μL to 50 μL of DEPC-treated water depending on pellet size.g for 5 min at 4°C. Following centrifugation, the supernatant was transferred to a new 1.5 mL tube and 1/10 volume of 8M LiCl2 was added, mixed well and incubated at −20°C overnight.For the removal of gDNA, RNA sample was treated with RQ1 RNAse-Free DNAse (Promega) in a 1.5 mL microcentrifuge tube according to the manufacturer’s instructions. The 10 μL DNAse digestion reaction was set up with: 1 μL of RQ1 RNase-Free DNase 10X Reaction Buffer, 1μL of RQ1 RNase-Free DNase and 8 μL of RNA sample. Following an hour of incubation at 37°C, the solution was placed on ice and then topped up with to a volume of 100 μL DEPC-treated water. An equal amount of chloroform: isoamyl alcohol (24:1) was added, and the mixture was hand-shaken to mix prior to centrifugation at 16500 ×g for 15 min at 4°C and the pellet was washed twice with 70% ice-cold ethanol and finally with ice-cold absolute ethanol. The resultant pellet was air-dried and then dissolved with 10 μL to 15 μL of DEPC-treated water. The dissolved sample was then stored in −80°C freezer until further use.Following overnight precipitation, the sample was centrifuged at 16500 ×The quality of the extracted RNA sample was checked by agarose gel electrophoresis (1% agarose gel), measurement of absorbance ratios at 230 nm, 260 nm and 280 nm using NanoDrop spectrophotometer . RNA integrity was determined using the Agilent 2100 Bioanalyzer . Concentration of RNA was quantified using a Qubit fluorometer .The quality of extracted RNA samples was first examined via gel electrophoresis . Based oRNA samples were isolated from three replicates of each of the leaf and stem tissues. In order to assess sample purity and quality with high accuracy, the absorbance ratios of 260/280 and 260/230 of total RNA were analysed in combination with overall spectral quality using a NanoDrop spectrophotometer . All theRNA integrity was then evaluated using the Agilent 2100 Bioanalyzer. All the RNA samples extracted from both leaves and stems had RIN values of more than 8 . In our study, we modified their protocol as follows: (1) the addition of PVP to the CTAB buffer, (2) higher centrifugation speed used in this study (16500 ×g), (3) inclusion of the usage of 100% ethanol at the end of washing step, and (4) an additional gDNA removal step. These modifications yielded high quality RNA samples in our study (with RIN > 8 and both absorbance ratios > 2).The CTAB protocol modified in this study was based on a reported protocol by 2 precipitation was carried out since LiCl2 precipitates RNA readily at certain concentrations but not DNA and protein  and Spectrum Plant Total RNA Kit , had been successfully used to isolate RNA from the leaf tissues of Stevia rebaudiana with CTAB as basis.Thus, conventional methods such as the acid guanidinium thiocyanate-phenol-chloroform extraction method developed by Stevia rebaudiana by Stevia rebaudiana effectively.The integrity of RNA sample isolated in our study was validated via agarose gel electrophoresis and subsequently assessed using Agilent 2100 Bioanalyzer. The bioanalyzer takes into account the entire electrophoretic trace of RNA samples and RNA qualities were classified into RINs of 1 (most degraded profile) to 10 (most intact profile)  that are suitable for subsequent molecular applications such as RNA sequencing.The modified CTAB-based extraction protocol used in this study was effective and reliable in isolating RNA samples from leaves and stems of


This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions  In early female embryos genes on both X chromosomes are expressed at normal levels. As cell differentiation and development proceed, each cell randomly selects and coordinately silences one of the two X chromosomes . Chromosin cis, circumscribing a discrete nuclear subdomain that overlaps with Xi chromatin in the interphase cell nucleus [Studies in the 1990s proposed a long non-coding (lnc) RNA, conserved in human and mouse, and transcribed specifically from the Xi, as having a central role in regulating the X inactivation process –7. Intri nucleus . The beh nucleus ,7. Appro nucleus ,7,10.in cis and chromosome silencing of the chromosome bearing the Xist transgene [Gene knockout and transgenic experiments in mouse demonstrated that Xist is both necessary and sufficient for X inactivation ,12. NotaWith the discovery of Xist RNA in the early 1990s, the field turned to three important questions: (i) How is the Xist gene regulated so as to ensure that Xist expression occurs only on a single X chromosome in female cells? (ii) How does Xist RNA accumulate locally on the chromosome from which it is transcribed? (iii) How does Xist RNA bring about formation of repressive chromatin and gene silencing? In this perspective I focus on our progress towards understanding the second question, the molecular basis for localized accumulation of Xist RNA. Readers interested in advances in understanding Xist gene regulation and Xist-mediated chromatin modification are referred to other recently published reviews ,14.Understanding localized accumulation of Xist RNA presents several key challenges: How does Xist RNA evade transport from the nucleus to the cytoplasm? How does Xist RNA spread from the site of synthesis? Does Xist RNA spreading involve passive or active processes? Does Xist RNA have preferred binding sites? How is Xist RNA accumulation limited to a single chromosome territory? Although we currently have few definitive answers to these questions several interesting clues have begun to emerge. I will summarize this recent progress, setting out competing models and hypotheses, and will additionally provide suggestions for how these may be addressed experimentally in future studies.2.cis-acting locus on the X chromosome, referred to as the X inactivation centre (XIC), which must be present for that chromosome to undergo X inactivation. It was through efforts to pinpoint the XIC that Xist was discovered [in cis. This possibility was reinforced by cell fractionation experiments showing that Xist RNA is retained in the cell nucleus [in situ hybridization (FISH) analyses that demonstrated Xist RNA has the appearance of a discrete nuclear domain comprising clustered foci overlying the dense inactive X chromatin (Barr body) [in situ hybridization (RNA-FISH), using either conventional widefield or super-resolution three-dimensional structured illumination microscopy (3D-SIM) are shown in Classical genetic studies led to the proposal that there is a scovered ,6,15–17.rr body) ,8. This rr body) –20. Repret al. [Localized accumulation of Xist RNA over a single chromosome territory was substantiated by Duthie et al. , who obset al. . In lateet al. .Detailed analysis of Xist RNA on Xi in mitotic cells revealed that Xist transcripts are concentrated over gene-rich chromosome domains and excluded from gene-poor domains, including pericentromeric heterochromatin, resulting in a banded appearance . This obin cis have emerged. The lncRNA Rsx, which is evolutionarily unrelated to Xist, performs an equivalent function in X inactivation in marsupial mammals, accumulating over the Xi chromosome in cis and silencing underlying genes by inducing chromatin modification [in cis has evolved to coordinately repress underlying genes. Other lncRNAs thought to function similarly to Xist include Kcnqt1ot1 and Airn, which are required for imprinted repression of large chromosomal regions on mouse chromosomes 7 and 17, respectively [in cis with the site of transcription [Since the discovery of Xist RNA other examples of regulatory RNAs proposed to mediate their effects via localized accumulation fication . Similarfication . It is cectively –33. In bcription ,35. Furtin cis, Jeon & Lee [trans-localization of Xist RNA from the Xi chromosome to autosomes that bear an inducible Xist RNA transgene. Specifically, integration of the inducible Xist transgene in female mouse embryo fibroblasts (MEFs) resulted in reduced Xist RNA clouds on an endogenous Xi chromosome (referred to as squelching), and, following transgene induction, Xist RNA from the Xi was detected in association with the transgene-expressing chromosome, indicating that Xist transcripts can also localize to sites in trans. The latter finding was based on FISH analysis using a probe specific for Xist RNA produced on the endogenous Xi. Further analysis identified a short region of Xist RNA centred on repeat F, which binds the nuclear protein YY1 and which is required for transgenic Xist RNA accumulation and squelching. However, subsequent studies have found that the repeat F region is the major enhancer required for Xist RNA expression [While there is an overall consensus that Xist RNA accumulates on & Lee  reportedpression ; in lighpression ,39.in trans at expressed Xist transgene sites, as reported by Jeon & Lee [trans-located Xist transcripts at Xist transgene sites is low-level plasmid DNA contamination of the RNA-FISH probes. This is because Xist transgenes generally co-integrate with, and therefore potentially transcribe, associated plasmid sequences. Taking this uncertainty into consideration, in the context of this review I will focus on the generally held consensus that localized accumulation of Xist RNA over a defined chromosome territory occurs strictly in cis.The accumulation of endogenous Xist RNA on & Lee , is diff3.et al. [In an important early study Clemson et al.  reportedet al. . StudiesA further link between localized accumulation of Xist RNA and the nuclear matrix came through the discovery that the major nuclear matrix protein scaffold attachment factor A (SAF-A)/heterogeneous nuclear ribonucleoprotein U (hnRNPU) has, has a role in the formation of Xist RNA clouds . In thisSAF-A/hnRNPU has been reported to bind Xist RNA directly, and this was linked to the presence of an RGG domain at the C-terminal end of the protein ,47. WhetFurther evidence for co-association of Xist RNA and SAF-A/hnRNPU comes from an analysis using 3D-SIM super-resolution microscopy which found that Xist RNA co-localizes with SAF-A/hnRNPU within interchromatin spaces, more specifically at perichromatin boundaries . NotablyMore recently, a second nuclear matrix-associated factor, CDKN1A interacting zinc finger protein 1 (CIZ1), was reported to have a role in localized accumulation of Xist RNA ,52. CIZ1The essential requirement for CIZ1 in localized accumulation of Xist RNA is thought to be limited to somatic cell types. Specifically, CIZ1 null female mice are viable , indicatWhile CIZ1 null mice are viable, indicating that there is no major deficit in X inactivation, ageing animals do exhibit a female-specific lymphoproliferative disorder. A likely explanation for this finding is that CIZ1 has a critical role in localizing Xist RNA in activated lymphocytes. Specifically, resting female T- and B-cells from wild-type animals extinguish Xist RNA expression and then restore Xist clouds in response to antigen stimulation . Followia, is that SAF-A/hnRNPU undergoes a local ATP-dependent polymerization upon binding to Xist RNA, and that this provides a reticular substrate for anchoring Xist RNA molecules via interactions with CIZ1. To account for cell type-specific effects of CIZ1 we can speculate, as above, that other sequence-specific Xist RBPs can also function as anchoring factors, acting redundantly with CIZ1, notably in early embryogenesis when X inactivation is first established (figure 2b). This model is consistent with evidence indicating redundancy in the mechanism of Xist localization, at the level of both binding sites [The inter-relationship between CIZ1 and SAF-A/hnRNPU in localized accumulation of Xist RNA is not yet understood. In one study it was reported that SAF-A/hnRNPU knockdown in MEFs phenocopies loss of CIZ1 , and moreover that CIZ1 binding to the dispersed Xist RNA molecules is retained . The simfigure 2ng sites  and, as 4.Although it is clear that nuclear matrix proteins are important for anchoring Xist RNA, there remain many questions in relation to the nature of anchoring sites. An interesting observation is that the abundance of Xist RNA in dividing cells increases approximately twofold at S-phase , suggestin cis demonstrates that anchoring sites are not unique to the X chromosome.What then can we deduce about the nature of anchoring sites? As mentioned above, Xist RNA is concentrated over gene-rich domains and excluded from gene-poor regions and constitutive heterochromatin, indicating a link between active chromatin and Xist RNA anchoring. Interestingly, SAF-A/hnRNPU polymerization requires active transcription , and thiet al. [More recent progress has come from studies that applied RAP-seq  and CHARet al.  an inducet al. . Subsequet al. .et al. [et al. [The study by Engreitz et al.  further  [et al.  using CHTaken together the RAP-seq and CHART-seq studies indicate that chromosome topology/dynamics and the progression of Xist-mediated silencing are important in determining the location of Xist RNA-anchoring sites. Whether or not this is sufficient to explain the limited capacity of a sister chromatid for anchoring Xist RNA remains to be determined.5.Kon/Koff and dwell time), the concentration of Xist RNA (the product of the rate of synthesis and degradation) and the abundance of Xist RNA-anchoring sites . However, Xist RNPs are large, estimated to be around 40–50 nm3, while the interchromatin spaces in the Xi territory through which Xist RNA must diffuse appear to be relatively narrow [b). This model integrates the concept that preferred anchoring sites correlate with chromosomal regions that have relatively high contact frequencies with the site of Xist RNA synthesis.It should be noted that the aforementioned model makes a number of untested assumptions. One notable example is that Xist RNA molecules translocate away from the site of transcription by passive diffusion, being retarded by anchoring to the nuclear matrix. In one scenario individual Xist RNA molecules could undergo several cycles of anchor and release. Alternatively, each molecule may undergo only a single anchoring interaction with a duration that is of a similar order to the lifespan of individual Xist RNA molecules a. Howevey narrow . Thus, i6.Recent progress has allowed us to begin to formulate ideas for how Xist RNA accumulates locally over the chromosome from which it is transcribed. There are however several significant gaps in our understanding; moreover, there is a need to develop better tools and approaches to test emergent ideas. Here I highlight key issues for future work in this field.SAF-A/hnRNPU and CIZ1 are clearly not the only factors involved in Xist RNA anchoring, and similarly the Xist E-repeat, to which CIZ1 binds, is not the only important sequence element. Indeed, an early study that analysed the effect of multiple deletions in Xist sequence concluded that there is no single element in Xist RNA required for localized accumulation, but rather multiple elements that function redundantly . The worAn important caveat in considering the role of the A- and B-repeats and associated factors in localized accumulation of Xist RNA is that the mutations described also disrupt gene silencing and Xi chromatin modification. Thus, it is plausible that aberrant localized accumulation deficiencies are a consequence of deficiencies in silencing/chromatin modification, for example reflecting a relatively decompacted chromosome structure. In future studies it will be important to test this by attempting to define separation of function mutations in the key A- and B-repeat-associated factors.Identifying the factors and elements with a role in localized accumulation of Xist RNA will not in itself explain the underlying mechanisms. For this it will be necessary to accurately measure key parameters: localization, anchoring/diffusion dynamics and turnover of Xist RNA. Methods such as RAP-seq and CHART-seq allow high-resolution mapping of Xist localization and can also provide limited temporal information over long time courses, several hours to days. However, the data are an aggregate from many millions of cells and it is difficult to discern from them events at the single-cell level. It may be possible in the future to develop single-cell RAP-seq/CHART-seq, although this will require highly efficient capture of Xist–chromatin complexes. Also, a drawback with these methods is that they rely on efficient cross-linking of Xist RNA with neighbouring chromatin, which could introduce bias towards Xist RNA sites that are in closer proximity to chromatin. A complementary strategy to advance our understanding of local accumulation of Xist RNA is to further develop the application of super-resolution microscopy to define the organization of Xist RNA particles relative to other nuclear landmarks and structures, in either fixed or cryopreserved material. One option would be to apply correlative light and electron microscopy, which should provide details on the ultrastructural organization of Xist particles relative to chromatin and other nuclear landmarks.While methods based on analysing fixed/cryopreserved cells should be highly informative, analysis of Xist RNA dynamics ultimately requires the development of live-cell imaging approaches. In experiments to date, this has been achieved by labelling Xist RNA with green fluorescent protein using the MS2 aptamer system . In futuThe aforementioned approaches, using either fixed cells or live-cell imaging, will be more informative when coupled to perturbation experiments in which key factors/sequence elements are deleted/ablated. Here again there may be significant challenges, for example SAF-A/hnRNPU is required for viability in ES cells. Accordingly it will be important to make full use of systems for conditional knockout, using either inducible gene deletion or protein degradation-based strategies.A final and complementary approach to unravelling the mechanism for localized accumulation of Xist RNA will be to develop synthetic RNAs that can bind key factors, for example the anchoring protein CIZ1, using established RNA aptamer systems, such as MS2 ,71, BglG7.The unique and unusual property of Xist RNA to accumulate locally over the chromosome from which it is transcribed provides us with an intriguing and challenging mystery. From a position of near-complete ignorance we can now begin to identify the underlying mechanisms. The coming years are set to be exciting as we move towards completing the task of defining the key factors and of testing our developing models.


The discovery that the non-protein coding part of human genome, dismissed as “junk DNA,” is actively transcripted and carries out crucial functions is probably one of the most important discoveries of the past decades. These transcripts are becoming the rising stars of modern biology. In this review, we have casted a new light on RNAs. We have placed these molecules in the context of life origins, evolution with a big emphasize on the “RNA networks” concept. We discuss how this view can help us to understand the global role of RNA networks in modern cells, and can change our perception of the cell biology and therapy. Finally, although high-throughput methods as well as traditional case-to-case studies have laid the groundwork for our current knowledge of transcriptomes, we would like to discuss new strategies that are better suited to uncover and tackle these integrated and complex RNA networks. Caenorhabditis elegans (Drosophila melanogaster) with 14 000 protein-coding genes and only five fold more abundant compared to bacteria (Escherichia coli) with ≈ 4 500 protein-coding genes. The source of organism complexity and diversity might therefore rather rely on how these genes are used and regulated , since many of them have emerged as key players in the cell biology with important regulatory roles and, therefore, were associated to numerous diseases ranging from cancers to neurological disorders . Thus, n2+  are single-stranded and act by base pairing with other nucleic acids (RNAs or DNAs), it is very likely that a vast majority of non-coding transcripts adopts complex 3D structure(s) to achieve their biological functions. These “structured” RNAs act using very diverse mechanisms including RNA-RNA, RNA-ligand, RNA-protein, RNA-DNA, and RNA-substrate interactions . However2+ . Finallyin vitro (11-fold with reaction efficiencies (kcat/Km) up to 108 M-1 min-1, which is 103-fold less than what is observed for proteins catalyzing equivalent reactions  that complement(s), or partially overlap(s) with the functions carried out by other RNAs. This concept of prebiotic networks constituted of interacting RNA species that evolve and act co-operatively has been demonstrated experimentally  (E. coli (E. coli). The authors inserted two copies of this promoter in the genome of E. coli: one copy driving the expression of the Cyan Fluorescent Protein (CFP) and the second driving the expression the Yellow Fluorescent Protein (YFP). The authors reported high variability on the fluorescence type that was emitted by individual bacteria. The source of this variability relies on the stochasticity of genetic regulation that is explained by two different factors or noises . Secondly, “intrinsic” noises imply that, even if the concentrations and states of every cellular component would be identical in every cell, the rate of expression of particular genes would also vary from cell to cell due to stochastic microscopic events that are intrinsic to transcription/translation events and influence gene regulation  (A particularly important property of genetic regulatory networks is their intrinsic “sticity” . This ststicity”  and expl. coli…)  as well . coli…) . One of r noises . First, on rate) .E. coli , 80 min for Saccharomyces cerevisiae (yeast) to several decades for human neurons). Since random events can be quantified by a frequency, organisms with longer lifetime are more prone to stochasticity and chaotic drift. Therefore, it is more obvious that eukaryotic cells need to undergo a much tighter regulation compared to prokaryotes since it is more crucial for eukaryotes to compensate and regulate randomness and better control cell fate.As mentioned above, one of the main differences between prokaryotic and eukaryotic genomes is the proportion of non-protein coding DNA. Furthermore, this ncDNA portion increases with organism complexity: the ncDNA size follows a quadratic equation that is function of the total length of exonic DNA . Why do How do eukaryotes satisfy this required tighter regulation? The answer most likely relies on the complexity of their regulatory networks. Indeed, recent studies have shown that more complex networks are better at coping with both: intrinsic and extrinsic noises that are the sources of stochasticity. Intrinsic noise tends to decrease with network complexity, and extrinsic noise tends to have less impact in complex regulatory networks . Nonethetrans interactions) and generate specific structural motifs that could modulate their interaction to binding partners .The emerging question is: how can we tackle the huge complexity of regulatory RNA networks given that we have only scratched the surface of protein-based networks? Nowadays, the current trend to fill in the gap of knowledge in the biology, functions and structures of regulatory RNAs is undoubtedly the use of high-throughput methods . Next-geWhereas protein-based networks are highly specialized with delimited functions and tasks, RNA-based networks are much more complex systems to study. Indeed, RNAs are less specialized molecules that act as part of bigger networks where all individual RNAs can interact with many different other proteins and nucleic acids. RNAs also exhibit much more dynamic and plasticity in their structures, biochemical modifications (epitranscriptomic) and therefore binding partners and functions. In this regard, the recent progresses made in cryo-electronic microscopy (cryoEM) will certainly facilitate RNA structure determination and will also allow assessing the dynamic and plasticity of these structures.In addition, this high functional plasticity of RNAs might explain why inactivation of a regulatory RNA can be trickier to analyze and will most likely generate more subtle changes with only partial destabilization of the downstream cell pathway(s). This is why we need to adapt our experimental approaches to the complexity of RNA networks. Therefore, the idea is to detect differences in the dispersion but not necessary in the average of the affected pathway(s). This is why single-cell analysis constitutes an attractive approach to highlight subtle changes in the cell (sub)-populations. In this approach, single cell are sorted and isolated using several well-established methods  as wellFinally, we need to better understand how the different regulatory RNAs work as networks and how they interact and are connected to each other as well as with DNA and regulatory proteins to better understand their roles in the cell biology.How can we address this network complexity without considering the information theories? Indeed, a good analogy of complex biological regulatory network is the information network . In thisFrom this point of view, the analogy between RNA and information networks is striking. Since, as discussed in this review, we hypothesized that the major purpose of complex RNA networks such as the ones found in complex eukaryotic organisms is to “buffer” the stochasticity of biological systems. In other words, RNAs can protect living cells/organisms against chaotic drifts. With this view, RNA networks are analogous to “correction codes” found in the information theory. Therefore this involves that we could imagine to model RNA regulatory networks by extending information theories and thereby reach a wider and global comprehension of these molecules.In conclusion, tighter regulation of complex organisms doesn’t rely on their genome size (number of nodes/genes), but rather relies on the possibility of each molecule (nodes) to receive or exert more regulations (edges). In higher and complex organisms, spatio-temporal regulation is crucial and needs to be more tightly regulated with much more noise control. This noise control network is probably strongly intricate with the catalyzers and lower level regulators from the biotic era. Life has adopted two strategies: either it diminishes the impact of noises by shortening the lifetime using fast replication (thanks to the efficiency of proteins) or it increases the noise control in complex organisms. As demonstrated by MV wrote most of the manuscript. MD participated to the manuscript writing. MV and MD contributed the most to the ideas presented in the manuscript. MD and MG gave regular intellectual inputs and proofread the manuscript.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.


OBJECTIVES/SPECIFIC AIMS: Immune-based therapies hold great promise for treatment of refractory tumors. However, development is limited by a lack of identified immune correlates to vaccination. We recently showed that dendritic cells (DCs) prolong progression-free survival (PFS) and overall survival (OS) in patients with glioblastoma, and that DC migration to site draining lymph nodes robustly correlates with both PFS and OS. While this appears to be a reliable immune correlate, the complexity of routine labeling for PET and SPECT prohibits validation in a large clinical study. We therefore seek to develop a safe, translatable reporter that can be imaged with a widely available imaging modality. METHODS/STUDY POPULATION: The cationic liposome 1,2-doleoyl-3-trimethylammonium-propane (DOTAP) was loaded with MRI-imageable iron oxide nanoparticles (IONPs) with or without the neutral molecules PEG and cholesterol. The resulting nanoparticles were loaded with RNA to form RNA-NPs that were characterized with transmission electron microscopy (TEM) and used to transfect DCs in vitro; 4.7 T MRI was then used to image particles or cells in agarose gel phantoms. RESULTS/ANTICIPATED RESULTS: TEM images of RNA-NPs indicate the presence of IONP-loaded liposomes. In vitro transfection experiments demonstrate that iron oxide does not reduce RNA-NP-mediated transfection of DCs. Additionally, small amounts of either PEG or cholesterol within RNA-NPs increased transfection of DC2.4s and enhanced T-cell priming by bone marrow-derived dendritic cells. A series of 4.7 T MRI images of particles in cells, spleens, and LNs demonstrated quantifiable differences in particle density between groups. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that IONP-loaded RNA-NPs can be imaged with MRI and manipulated to augment DC function. Future work will include in vivo imaging in mice and safety studies to facilitate translation into first-in-human studies. Successful completion of this project would provide a powerful clinical tool to improve and track patient responses to immune therapy.


Finally, the target norovirous (NV) RNA was combined with the probe according to the principle of complementary base pairing, resulting in a decrease of the photocurrent. The results show that the absorbance spectrum of visible light is enhanced for CSZFRs compared with pure ZnO. Under visible light irradiation, the photocurrent of CSZFRs is up to 0.1 mA, which can improve the sensitivity of the photoelectrochemical (PEC) biosensor. In the measurement range of 0–5.10 nM, the measured concentrations (c) have a good linear relationship with the output photocurrent of the biosensor. The linear regression equation is expressed as I = 0.03256 − 0.0033c  with a detection limit of 0.50 nM. Therefore, this work realizes a rapid and sensitive method for the detection of NV RNA.In this work, the CdSe–ZnO flower-rod core-shell structure (CSZFRs) was prepared by ion-exchange method. The surface of CSZFRs was modified by 3-mercaptopropionic acid (MPA), and then the DNA probe was immobilized on the surface via chemical bond between -NH Norovirus (NV), one genus of the family of Caliciviridae, is one of the main pathogens causing nonbacterial gastroenteritis in humans. It is highly infectious and variable that can lead to serious public health problems especially in people with low resistance ,2. ThereNano ZnO has been widely used in the field of biosensors due to the advantages of high electron mobility, stable chemical properties, and good biocompatibility . BecauseHowever, nano ZnO has lager bandgap, which leads to an only response to the UV region of the solar spectrum and inhibit the utilization of solar energy. The semiconductor quantum dots (QDs) with narrow bandgap can effectively solve this problem ,18,19,20In this work, CdSe–ZnO flower-rod core-shell nanocable arrays (CSZFRs) were prepared via ion-exchange method and used as a new PEC biosensor for detection of norovirous RNA (NV RNA). The photocurrent is up to mA level. It shows a high sensitivity for the low concentration of NV RNA. In addition, it also has great advantages like convenient use, simple equipment, and fast response. The proposed PEC biosensor exhibits broad prospects and potential applications to be explored in biological detection and analysis respect.2·2H2O), zinc nitrate hexahydrate (Zn(NO3)2·6H2O), ammonium hydroxide (NH3·H2O) of 25–28%, hexamethy-lenetetramine (HMTA) and NaBH4 were purchased from Sino Chem. Reagent. Poly-ethylenimine (PEI) and N-hydroxysulfosuccinimide sodium salt (NHS) were obtained from Aladdin Reagent. Se powder was obtained from Shanghai Meixing Chemical Co., Ltd.  Cd(NO3)2 and 3-mercaptopropionic acid (MPA) purchased from Alfa Aesar. N-(3-Dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDAC) was obtained from Life Science Products & Services. Target complimentary NV RNA of genogroup II (G11) and DNA probe were purchased from Sangon Biotech .Indium doped tin oxide (ITO) substrate with 15 Ω/sq was obtained from Heptachroma Solar Tech. Zinc acetate dihydrate (Zn(Ac)2-/GCGACGAATTAGCTTGTATGATGTCGTCGC-3′DNA sequence: 5′-/NHRNA sequence: 5′-GACAUCAUACAAGCUAAUUC-3′2·2H2O in ethanol, followed by thermal decomposition at 300 °C for 20 min. Thirdly, the seeded substrates were placed in an aqueous solution containing 25 mM Zn(NO3)2·6H2O, 12.5 mM HMTA, 5 mM PEI, and 0.35 M NH3·H2O at 85 °C for 4 h. Finally, the as-prepared ZnO samples were dried in oven and then calcined at 450 °C for 30 min.CSZFRs were prepared by ion exchange method according to our previously reported method . ZnO flo2− ion solution (6.0 mM) prepared by reacting Se powder with NaBH4 and kept at 50 °C for 20 min under the condition of nitrogen gas. This process was repeated for two times to get a desirable thickness of ZnSe. The arrays of ZnO/ZnSe nanocables then immersed into Cd(NO3)2 aqueous solution (0.1 M) at 90 °C for 8 h, then ZnSe was converted to CdSe and CSZFRs were formed. At last, CSZFRs were dipped in MPA solution at 90 °C for 2 h.ZnO FRs were immerged in a SeEDAC and NHS solutions were used to activate the carboxylate groups on the surface of CSZFRs at 37 °C for 30 min. Then DNA probe  was added onto CdSe–ZnO/ITO and activated at 37 °C for 1 h (marked as CSZFR@DNA). The actual molar amount of DNA probes on the electrode surfaces is about 0.585 pmol, which was measured with the method of spectrophotometry. Different concentrations of 15 μL NV RNA, which were prepared using pH = 8 Tris-EDTA buffer, were hybridized with the DNA oligonucleotide sequence in the dark for 5 min (marked as CSZFR@RNA). Crystal structures of CSZFRs were characterized by powder X-ray diffraction  and field-emission scanning electron microscopy . The diffuse reflectance spectra were measured on a Shimadzu UV-2500 UV-Visible spectrophotometer . Fluorescence measurement was performed at room temperature on a Cary Eclipse fluorescence spectrometer  in a 300 μL quartz cuvette.The photocurrent measurement of the PEC biosensor was carried out using an electrochemical workstation . All electrochemical analyzes were performed using a conventional three-electrode system under the irradiation of xenon lamps , where UV light was cut with UV cut 400 filter. The saturated Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode as the counter electrode and the as-prepared CdSe–ZnO/ITO or ZnO/ITO as the working electrode. The electrolyte solution was 0.1 M phosphate buffer solution (PBS). The impedance spectrum of the PEC biosensor was measured using the same electrochemical workstation.n = 5).For the experiment of recovery, the human serum was diluted 1000 times, and then added different molars of NV RNA into the diluted serum. The operation was the same as above PEC measurement and repeated 5 times for each sample . The XRD pattern of CSZFRs has a broad peak between 20–30° and the diffraction peaks of 42° and 49.5° are not observed, which is due to the smaller size and lower amount of CdSe QDs. The result matches well with FESEM image of CdSe–ZnO.It can be seen from According to Rct. As 63−/4−] electrons. After loading the quantum dots, the impedance of CSZFRs increases. Additionally, the impedance value of CSZFRs after adding the probe DNA and the target RNA is much larger than that of the bare ZnO FRs and CSZFRs, which is mainly due to the DNA and RNA insulators. Moreover, the addition of DNA and RNA also makes its steric hindrance larger. And the charge transfer rate is inhibited after the hybrid of DNA probe and NV RNA. This result is consistent with Electrochemical impedance spectroscopy is an effective method to explain the information of impedance change and the characteristics of the electrodes in the modification process, in which the semicircle diameter represents the impedance of electron transfer at the interface of the dominant electrode I = 0.03256 − 0.0033c  and the detection limit is 0.50 nM. The value of limit of detection (LOD) is given by the equation LOD = −3 σ/s, where σ is the standard deviation of the blank measures, and s is the slope of linear equation. The PEC biosensor based on pure ZnO was also used for detecting RNA under visible light (As shown in le light . Howevern = 5). The results show that the developed PEC biosensor has good applicability.In order to verify the applicability of a PEC biosensor for NV RNA detection, we performed a standard recovery experiment in normal human serum. The results are shown in As the results shown, CSZFRs were successfully prepared by the ion-exchange method. UV-Visible diffuse reflectance spectra and fluorescent spectra show that CdSe can effectively enhance the visible light absorption of ZnO, promote the separation of a photogenerated electron-hole and improve the efficiency of photoelectric conversion. The CSZFRs based PEC biosensor has good detection performance for NV RNA. The photocurrent has a good linear relationship with the concentration of NV RNA in the range of 0–5.10 nM with a detection limit of 0.50 nM. The results reflect that the constructed PEC biosensor based on CSZFRs which is sensitive and fast for detecting NV RNA can be used for the detection of other biomolecules.