Caliciviruses are a leading agent of human and animal gastroenteritis and respiratory tract infections, which are growing concerns in immunocompromised individuals. However, no vaccines or therapeutics are yet available. Since the rapid rate of genetic evolution of caliciviruses is mainly due to the error-prone nature of RNA-dependent RNA polymerase (RdRp), this article focuses on recent studies of the structures and functions of RdRp from caliciviruses. It also provides recent advances in the interactions of RdRp with virion protein genome-linked (VPg) and RNA and the structural and functional features of its precursor. Caliciviridae is a large group of non-segmented, single stranded RNA viruses. Its genome is surrounded by a non-enveloped capsid with icosahedral symmetry, which forms a virion with a diameter of 27–40 nm ;,12;11,12Among the caliciviral proteins, the crystal structures of RdRp have been determined for RHDV , HuNoV gα atoms. They adopt an enclosed conformation, where the extended N-terminal domain bridged across the fingers and thumb domains. The tip of the thumb domain forms an extended loop and the bottom of the same domain comprises the C-terminal region, displaying significant conformational variation. In contrast, the palm domain, the most conserved domain of known polymerases [The crystal structures of the RHDV, HuNoV GII.4, NoV GI, and SaV GI RdRps show a remarkable degree of structural similarity, with 37–60% sequence identity, and resemble a right hand with fingers, palm, and thumb domains, with an N-terminal domain . A superymerases , and the4)2SO4 or PEG at pH 5.5–7.5, which does not seem to affect the oligomerization state of the protein molecules. Nevertheless, the MNV RdRp can form stable ball-like hexamers [These calicivirus RdRps contain one or two protein molecules in the asymmetric unit, which are not related by a noncrystallographic symmetry, displaying mainly a monomeric form, whereas the MNV RdRp structure contains three molecules per asymmetric unit. The MNV RdRp is found to be a monomer in solution, but with hexameric form in crystals . The molhexamers . When diThe central three-stranded β-sheet in the palm domain contains the DYTxxD and YGDD sequences , highly conserved in the RdRps of picornaviruses and caliciviruses, with which metal ions interact to be involved in the nucleotidyl transfer reaction . As the Structural studies of RdRp in complex with RNA and NTP revealed three key features; binding of metal ions and NTP substrate to the active site, rotation of the central helices in the thumb domain to be able to bind RNA template , and disThe error-prone nature of RdRps is a key feature in RNA synthesis, which alters the fidelity, leading to the rapid rate of genetic evolution during viral infection. A high-fidelity NoV RdRp mutant I391L displayed delayed replication kinetics in vivo, showing lower transmission rates between susceptible hosts than the wild-type virus . The NoVThe conformational variation among caliciviral RdRps is most significant at the C-terminal region, where the terminal helix and loop are located at the bottom of the thumb domain and near the active site cleft. In many RdRp structures, the C-terminal region (from residue 478 to the C-terminus in MNV) is unstructured or partly disordered . No visiCaliciviridae [Positive-strand RNA viruses are capable of initiating RNA synthesis either de novo or in a primer-dependent fashion: flaviviruses depend on de novo replication only, whereas picornaviruses and caliciviruses adopt de novo and primer-dependent initiation where VPg attached to the 5′ end of genomic and sgRNAs, acts as a primer ,36. VPg iviridae . It is kiviridae .Picornaviridae members [VPg nucleotidylylation is important for RNA replication in caliciviruses and picornaviruses, and genomic RNA purified from FCV and other vesivirus virions was not infectious after digestion of the VPg by proteinase K, suggesting that lack of VPg capping renders viruses non-infectious . A Tyr r members . Althoug members . The calReplication of many RNA viruses was proposed to facilitate the formation of higher-order protein complex structures, or large replication factories. For positive-strand RNA viruses, such as caliciviruses and picornaviruses, RNA replication complexes are membrane-anchored to be clustered in replication compartments. The functional significance of RdRp oligomerization in infected cells can be important for viral replication in RNA viruses . In picoIn addition, the GII.4 and MNV VP1 capsid protein were found to enhance cognate RdRp activities, coimmunoprecipitated with their cognate RdRps . The VP1The genome replication from RNA templates is essential in the evolution of RNA viruses, where the interactions between proteins and RNA play critical roles in the initiation of RNA synthesis . ReplicaLagovirus contains VP1 additionally. The polyprotein is cleaved by viral Pro, to release both the precursor ProPol and mature RdRp proteins. The ProPol precursors of FCV and NoV were first reported to be active in RNA synthesis [2+ [Lagovirus, the mature form of RdRp in RHDV and RCV in virus-infected hepatocytes is more active than the precursor [Vesivirus, in contrast, appears to utilize ProPol that is an active form for nucleotidylylation, due to a lack of proteolytic cleavage site in wild type virus [In NoV, ORF1 encodes a 200 kDa non-structural polyprotein, while the polyprotein in ynthesis ,57, showesis [2+ ,58. In Lrecursor . FCV in pe virus ,57,59,60pe virus ,62. AlthIt is also important to note that the HuNoV VPg binds NTPs, and its nucleotidylylation by the precursor is markedly greater than by the mature RdRp. The N-terminal His-tag of a recombinant HuNoV VPg has an inhibitory effect on nucleotidylylation by ProPol, but not RdRp, suggesting that the conformation of the N-terminal domain of VPg is crucial in the interaction with ProPol. The N-terminal domain of VPg, with a basic amino acid-rich region comprising the first 20 amino acids, is involved in the interaction with the Pro domain of the precursor . It has RdRp, that is the main target of antiviral drugs, plays a critical role in the replication of caliciviruses with rapid genetic evolution. With recent structures determined, structural analyses of calicivirus RdRps suggest that the rotation of the central helix in the thumb domain and displacement of the C-terminal tail at the active site are essential to bind RNA and substrates. The higher-order multimerization of RdRp may confer high efficiency in viral genome replication, and the ProPol precursor can nucleotidylylate VPg, with enhanced polymerization activity in some of caliciviruses.


Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The “psi” (Ψ) element within the 5′-untranslated region (5′UTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1) Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA) domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV) Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity. A hallmark of retroviruses is their ability to reverse transcribe single-stranded genomic RNA (gRNA) into double-stranded DNA for subsequent integration into the host cell genome. Following nuclear transcription and export into the cytoplasm, highly selective packaging of the full-length, unspliced gRNA into newly assembled virus particles is achieved, despite the presence of spliced viral RNA and a vast excess of cellular RNA ,2. HumanAlthough the minimal element that is both necessary and sufficient to confer selective HIV-1 gRNA packaging is not yet known, a gRNA packaging competition assay has defined a “core encapsidation signal” composed of the U5 region and stem-loops 1–4 (SL1–SL4) of Ψ . In the Several factors in addition to Gag/NC binding affinity for Ψ have recently been proposed to play a role in selective gRNA packaging. In vitro fluorescence-anisotropy (FA) salt-titration binding assays showed that HIV-1 Gag binding to RNA derived from the gRNA Ψ region is characterized by highly specific non-electrostatic binding and an effective charge that is consistent with an NC-only binding mode . In contIn the case of Rous sarcoma virus (RSV), nucleotides (nt) 156–315 in the 5′UTR of the gRNA (designated as MΨ) can direct packaging of heterologous RNAs with an efficiency similar to that of the full-length gRNA ,29. SubsKd(1M), which describes the non-electrostatic component of binding , and Zeff, the effective charge of the protein–nucleic acid interaction, which is the number of Na+ cations displaced upon nucleic acid binding . In. InKd(1M1 Gag∆p6  [27]. Wheff ~ 6) . HIV-1 MThe mechanism by which MA contributes to Ψ-binding specificity in the context of the Gag polyprotein is not known. To begin to address this question, two protein chimeras were investigated wherein MA domains of HIV-1 and RSV Gag were swapped. In H132R, the RSV MA domain is replaced by the 132-residue HIV-1 MA domain, whereas in R155H, HIV-1 MA is replaced by the 155-residue RSV MA domain B. Salt-tKd(1M) = 7.2 × 10−5 M, which was very similar to RSV Gag∆PR, whereas the Zeff was slightly higher at ~5 (versus ~4) (Kd(1M) ~ 1.9 M and a large Zeff of ~9. These data suggest that despite the presence of a heterologous HIV-1 MA domain, RSV Gag was still able to distinguish Ψ from non-Ψ RNA. In the reciprocal experiment, R155H maintained a specific Kd(1M) = 3.1 × 10−5 M with HIV-1 Ψ RNA and a low Zeff of ~4, while the chimera’s interaction with HIV-1 TARpolyA was characterized by a nonspecific Kd(1M) ~ 2.6 M and high Zeff of ~9 (The chimeric H132R Gag construct interacted with RSV MΨ with a highly specific rsus ~4) . Furtherff of ~9 . Thus, HKd(1M) value of 3.2 × 10−2 M and 4.0 × 10−2 M, respectively, and a Zeff ~ 6 for both RNAs, indicating that RSV Gag was unable to bind specifically to HIV-1 Ψ (Kd(1M) value of 1.9 × 10−2 M and 2.9 × 10−1 M, respectively, and a Zeff value of 7 and 8, respectively (Kd(1M) = 5.5 × 10−5 M and Zeff ~ 5, and bound 167 more electrostatically with a Kd(1M) = 1.0 × 10−2 M and Zeff ~ 8 (Kd(1M) = 6.7 × 10−5 M and Zeff ~ 5, whereas the RSV 167 interaction is characterized by a nonspecific Kd(1M) = 2.3 × 10−1 M and Zeff ~ 10  values of ~10−5 M and low Zeff, values (~3) that were essentially indistinguishable from binding to WT Ψ (Kd(1M) values, binding to these mutant RNAs was actually more specific (~1.5–6-fold) than binding to WT Ψ RNA.We next wanted to investigate the Ψ RNA elements required for specific recognition by Gag. In RSV, it was previously reported that mutations A197G and UGCG to GAGA (nt 217–220) in RSV µΨ   . Thus, wKd(1M) .To better understand how the mutations to RSV MΨ may affect recognition by Gag, the global conformation of the RNA variants was probed by native polyacrylamide gel electrophoresis (PAGE). Mfold analysis  of MΨ A1Kd(1M) = 3.6 × 10−5 M) and ~six fewer electrostatic interactions (Zeff ~ 5) than binding to TARpolyA (Kd(1M) = 5.6 × 10−1 M and Zeff ~ 11) (Kd(1M) ~ 6–8 × 10−5 M and relative specificity of 0.47–0.61 of Ψ-WT), and only a modest increase in the number of electrostatic charges mediating the interaction (Zeff ~ 5–6) (Kd(1M) ~ 3 × 10−4 M; relative specificity of 0.14), based on the variability in the data generated using this mutant, we concluded that it was not significantly different from Ψ-WT. In contrast, a significant loss in specificity was observed upon mutation of residues in the bulge regions of SL1 (Mut 1 and 2), as well as in the region downstream of SL3 (Mut 6) (relative specificity of 0.04–0.11). The final two HIV-1 Ψ mutants tested (Mut 7 and 8) bound HIV-1 Gag∆p6 with a relative specificity of 0.97–1.7 and interacted with a similar number of electrostatic contacts (Zeff ~ 4–5) as Ψ-WT  . HIV-1 Gf ~ 5–6) . Based o as Ψ-WT . AlthougTo determine whether the Ψ mutations disrupted the global RNA fold, all HIV-1 Ψ RNA variants were subjected to native-PAGE analysis. The majority of RNAs migrated as a single band corresponding to the RNA dimer, and there were no changes in the global conformation of the mutants relative to Ψ-WT . The onlIn the present work, we have shown that, as for HIV-1 Gag, specific recognition of Ψ RNA by RSV Gag involved a significantly greater nonelectrostatic binding component than interaction with non-Ψ RNA, and binding to Ψ was mediated primarily by the NC domain. In contrast, non-Ψ RNA interactions were characterized by increased electrostatic binding interactions, consistent with binding of both the NC and MA domains. A recent small-angle X-ray scattering (SAXS) and neutron reflectometry study showed that RSV Gag is likely sampling an ensemble of conformations in solution (elongated and bent), but was more compact when interacting with anionic membranes . This suThe lack of direct MA-Ψ RNA interactions in the case of HIV-1 and RSV Gag is consistent with recent CLIP-seq studies showing a lack of HIV-1 MA–gRNA interactions in cells ,65. HoweWe observed an RNA concentration-dependence in the capability of HIV-1 Gag to specifically interact with its cognate Ψ RNA element, with optimal discrimination between Ψ and non-Ψ RNA occurring at 20.5 nM RNA, which corresponds to a 1:20 RNA:Gag ratio . To dete2) for correct Gag PM localization . I. I61]. ILess is known about the specific RSV Ψ elements that are critical for gRNA packaging. However, two previous mutations (G214C/G215C and MΨ Triple) shown to reduce gRNA packaging efficiency by ~20- and ~50-fold, respectively , were teIn summary, in this work, we used an FA-based salt-titration assay to investigate binding of RSV Gag to MΨ and non-Ψ RNAs, revealing significant RNA-dependent differences in the binding mode. RSV Gag interacted with MΨ RNA preferentially using only the NC domain, while the increased electrostatic interactions observed with non-Ψ RNA are consistent with simultaneous binding of both NC and MA. Moreover, the presence of MA was required for optimal discrimination of Ψ and non-Ψ RNAs. Similar results were previously obtained in the case of HIV-1 Gag binding to its cognate Ψ RNA versus a non-Ψ RNA . Thus, t


Dendrobium huoshanense is an Orchidaceae perennial herb that is rich in polysaccharides and other secondary metabolites. By using D. huoshanense as the subject, we improved the method originated from CHAN and made it suitable for plants containing high amount of polysaccharides and polyphenols. The extracted total RNA was clear and non-dispersive, with good integrity and no obvious contamination with DNA and other impurities. And it was also evaluated by gel electrophoresis, nucleic acid quantitative detector and PCR assessment. Thus, as a simple approach, it is suitable and efficient in RNA isolation for plants rich in polysaccharides and polyphenols.Acquiring high quality RNA is the basis of plant molecular biology research, plant genetics and other physiological investigations. At present, a large number of nucleotide isolation methods have been exploited or modified, such as commercial kits, CTAB, SDS methods and so on. Due to the nature of different plants, extraction methods vary. Moreover, efficiency of certain approach cannot be guaranteed due to composition of different plants and extracting high quality RNA from plants rich in polysaccharides and polyphenols are often difficult. The physical and chemical properties of polysaccharides which are similar to nucleic acids and other secondary metabolites will be coprecipitated with RNA irreversibly. Therefore, how to remove polysaccharides and other secondary metabolites during RNA extraction is the primary challenge.  Dendrobium huoshanense C. Z. Tang et S. J. Cheng is a famous Orchidaceae perennial medicinal herb that has been used to remedy extensive symptoms, such as chronic superficial gastritis, throat phlegmonosis, to enhance immunity and so forth [D. huoshanense is a precious medicinal herb in China, and rare Daodi herb [D. huoshanense contains a variety of chemical components such as polysaccharides [D. huoshanense have the efficacy of enhancing immunity [D. huoshanense mainly focuses on germplasm resources, cultivation techniques and pharmacological effects.so forth . D. huosodi herb . Becauseodi herb . The govodi herb ,4–7. D. charides , alkaloicharides , amino acharides , flavonocharides  and so oimmunity , anti-oximmunity , anti-tuimmunity , cell apimmunity , lowerinimmunity , liver pimmunity  etc. At D. huoshanense is its high content of polysaccharides [D. huoshanense stem took up 36.23 per cent of its dry weight [D. huoshanense and other plants with similar traits. In this study, D. huoshanense was selected as the subject to extract total RNA from its stem, leaf and flower by applying modified CHAN method [Obtaining high quality RNA is the basis for many plant molecular biology researches . RT-PCR,charides –20. Studcharides  and totay weight . Howevery weight . TherefoN method –25. In aD. huoshanense C. Z. Tang et S. J. Cheng from Orchidaceae. Stems, leaves and flowers of D. huoshanense were washed with tap water, then sterilized with 75% ethanol for 45s, and then rinsed twice with sterile water. Subsequently, plant organs were placed on a sterile filter paper to remove water and finally ground rapidly in liquid nitrogen to obtain fine powder for RNA extraction.Subjects were collected from National Breeding Base of Seeds and Seedling of Medical Herbs for Essential Drugs (Anhui). Professor Nianjun Yu from Anhui University of Chinese Medicine identified the samples as Extraction buffer (EB) ; 20mM, EDTA; 1%(w/v) sodium dodecyl sulphate) was modified from the protocol proposed by CHAN  and PVP 75μL EB, 750μL CI and 30μL β-mercaptoethanol were mixed in a 2.0mL RNase-free microcentrifuge tube.D. huoshanense stem, leave and flower respectively in liquid nitrogen were added to tubes. Vortex vigorously. Place at 20°C for 5 minutes before centrifuging the samples at 4°C for 5 min at 12000×g. Note that the samples should not thaw when they were added to solutions prepared in step 1.50~100 mg of tissue powder made by grinding The supernatant (~700μL) was transferred to a new 2.0mL RNase-free microcentrifuge tube and 700μL of PCI was then added. Mix gently and centrifuge at 4°C for 5 min at 12000×g.The supernatant (~600μL) was transferred to a new 2.0mL RNase-free microcentrifuge tube. 600μL of PCI was added. Mix and centrifuge at 4°C for 5 min at 12000×g. This step resulted in ca 400 μL of supernatant.1/10 volume of 3M sodium acetate (pH = 5.2) and 2.5 volume of absolute ethanol were added to the supernatant. Mix gently and incubate at 4°C for 30 minutes.Centrifuge at 4°C for 10 min at 12000×g to recover the nucleic acids.200 μL of DEPC-treated water was added to dissolve the nucleic acids and 500μL of 10M LiCl was added to the solution. Mix gently and then place on ice for 60 minutes.Centrifuge at 4°C for 10 min at 12000×g. Discard the supernatant and wash the pellet with 800μL of 75% (V/V) ethanol. Air-dry in a ventilator for 5-10min.50 μL of DEPC-treated water was added to dissolve RNA. Incubate at 50°C for 5 minutes. RNA samples can be used immediately for downstream experiments or stored at -80°C for future use.D. huoshanense were collected and tested for integrity by 1.0% (w/v) agarose gel. A260/230 and A260/280 values for RNA samples were measured with an Analytic JENA Scandrop 200 to evaluate purity and yield.The total RNA extract of stem, leaf and flower of D. huoshanensis transcriptomic data. Primer 5.0 software was used to design primers for specific PCR amplification. Sangon Biotech synthesized the sequence. The forward primer of tubulin was 5’-TTC TGG GAG GTG ATC TGC GA-3’ and the reverse 5’-CGG GGG AAT GGA ATG AGG TT-3’. PCR reaction system: 33.5μL ddH2O, 5μL 10× Extaq buffer, 5μL 2.5mM dNTP Mix, 2μL cDNA, 2μL 10μM Forward Primer, 2μL 10μM Reverse Primer and 0.5μL Extaq enzyme. PCR reaction conditions: initial denaturation at 94°C, 2min; 30 cycles at 94°C for 30s, 52°C for 30s and 72°C for 2min. Later, 10μL of the PCR product was used for agarose electrophoresis using TBE buffer (1×) following the usual procedure. The remaining products were stored at -20°C.The first strand cDNA was synthesized using FastQuant RT kit  according to the manufacturer’s instructions. Through BLAST alignment, tubulin homologous sequences were searched in our D. huoshanense contains many polysaccharides, phenols, proteins and other secondary metabolites [D. huoshanense. By trying CHAN method [D. huoshanense RNA, the results were not ideal and the price for purchasing commercial kits was relatively high. Therefore, we set out to establish a simple, economical and efficient RNA extraction method in order to acquire high-quality RNA from plant rich in polysaccharides and polyphenols.Plant gene expression analysis, cloning, transcriptomic and other research often need to isolate and purify RNA from tissues and high purity, high concentration and good integrity RNA is always a must , 26. At abolites –11. ThesN method , Trizol,In this study, extracted total RNA was verified by gel electrophoresis, quantitative nucleic acid detection and PCR analysis. As a routine examination method –32, all 260 and A280 reveals seriousness of protein contamination. Likewise, A260/A230 provides clues for polysaccharides and other related metabolites contamination. Using the modified CHAN method, results from nucleic acid quantitative analyzer showed A260/A280 value of stem RNA at around 2.0, A260/A230 between 1.43 and 2.14 and the concentration ranged from 54.31 to 122.89 ng/μL Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as a reverse transcriptase, an activity that has never been demonstrated before for RNA. This activity is thought to have been crucial for the transition from RNA to DNA genomes during the early history of life on Earth, when it similarly could have arisen as a secondary function of an RNA-dependent RNA polymerase. The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate products containing up to 32 deoxynucleotides. It is likely that this activity could be improved through evolution, ultimately enabling the synthesis of complete DNA genomes. DNA is much more stable compared to RNA and thus provides a larger and more secure repository for genetic information. All known living things share the same genetic machinery, traditionally called the central dogma. According to this dogma, genes in DNA produce messages made from a similar molecule called RNA. These RNA messengers provide the instructions to make proteins, which then form structures and act as molecular machines inside cells. This process is found in all modern living things, but early life must have been much simpler.Many biologists believe that the earliest life only used RNA, which can both store information like DNA and perform tasks like a protein. Life evolved from this so-called ‘RNA world’ because DNA provides a more reliable long-term store of information, whilst proteins are more versatile and able to perform more tasks. This key step in evolution allowed life to move beyond basic chemistry and develop the size, complexity and diversity we see today. Yet, how this transition happened is not well understood. In particular, many believe an RNA molecule must have evolved the ability to make DNA from an RNA template, allowing early life to build the first genetic material made from DNA. This molecule would be referred to as a reverse transcriptase ribozyme.Modern living things do not contain such a molecule. Yet based on their previous work using RNA molecules to make copies of other RNAs, Samanta and Joyce attempted to develop an artificial reverse transcriptase ribozyme. The goal was to show that these ribozymes can be made and could theoretically have evolved naturally. The molecule Samanta and Joyce created was able to reliably produce short sections of DNA, with rare errors. This ribozyme is slower and makes more mistakes than molecular systems in modern biology, but it proves that reverse transcriptase ribozymes are possible.Using a process called test-tube evolution, which uses the same concepts as natural evolution to improve the qualities of biological molecules, Samanta and Joyce now plan to improve their ribozyme. The aim is to confirm that a reverse transcriptase ribozyme could have been a transformative early step in evolution of life on Earth that led to the first DNA genomes. This will be a critical addition to scientists’ understanding of how life became more complex and how the first cells formed. It is widely thought that RNA-based life preceded DNA- and protein-based life during the early history of life on Earth . PerhapsThe first reverse transcriptase may have been either an RNA or protein enzyme. The former seems plausible because such an enzyme could have derived from an RNA-dependent RNA polymerase, which would have been an essential component of RNA-based life. It has been argued that the translation machinery requires more heritable information than can be maintained by RNA genomes , thus plThere are several examples of RNA enzymes with RNA-dependent RNA polymerase activity, all of which were obtained by in vitro evolution . The mosa values of the 3´-hydroxyl in either RNA or DNA, this difference is ~100 fold  installation and evolutionary optimization of an accessory domain to increase catalytic efficiency ; (2) addThe 24–3 polymerase was tested for its ability to catalyze the RNA-templated addition of dNTPs to the 3´ end of an RNA or DNA primer . The enzFor all templates tested, the reaction proceeds similarly using either an all-RNA primer or an RNA primer that has a single 3´-terminal deoxynucleotides . When anTwo approaches were taken to confirm the identity of the reverse transcription products obtained using template 1. First, the presumed full-length materials, initiated by either an all-RNA or an all-DNA primer, were purified by denaturing polyacrylamide gel electrophoresis and subjected to partial digestion with DNase I. This enzyme degrades 3´,5´-phosphodiester linkages in DNA but not RNA. For the RNA-primed products, the extended portion was degraded by DNase and the primer portion remained intact, whereas for the DNA-primed products the entire molecule was degraded . AuthentThe second confirmatory approach involved analysis of the gel-purified, full-length materials by liquid chromatography/mass spectrometry. The RNA or DNA primer contained a 5´-fluorescein label to permit visualization in the gel and the reaction involved addition of deoxynucleotides residues having the sequence 5´-CGGGGGTG-3´. For the RNA-primed reaction the calculated mass was 8252.4 and the observed mass was 8252.4 ; for theTo further investigate the fidelity of reverse transcription, the reaction was carried out either in the presence of all four dNTPs or in a mixture lacking dGTP, dATP, TTP, or dCTP. Templates 3 and 4 were tested, both of which contain all four nucleotides and direct the synthesis of partial-length products containing up to 6 or 12 deoxynucleotides, respectively. For both templates, the size distribution of the products was the same when employing either all four dNTPs or a mixture that lacked dATP . For rea2, and were carried out at pH 8.3°C and 20°C. The reaction has a rapid initial burst phase, followed by a second slower phase that continues until >90% of the primer molecules are extended , which was incubated at 37°C for 2 hr. The DNA then was digested by adding 0.1 U/μL TURBO DNase I and continuing incubation for 1 hr. Ribozymes were transcribed from fully double-stranded DNA templates (20 µg/mL) that were obtained by PCR amplification of plasmid DNA encoding the 24–3 ribozyme (courtesy of David Horning).RNA templates were transcribed from 0.5 μM single-stranded DNA that had been annealed with 0.5 µM of a synthetic oligodeoxynucleotide encoding the second strand of the T7 RNA polymerase promoter. Transcription was carried out in a mixture containing 15 U/μL T7 RNA polymerase, 0.002 U/μL inorganic pyrophosphatase, 5 mM each NTP, 25 mM MgCl2, 0.05% TWEEN20, and 50 mM Tris (pH 8.3 or 9.0). Polymerization was carried out at 20°C and quenched by adding 250 mM EDTA. The biotinylated primers and extended products were captured on streptavidin C1 Dynabeads, washed twice with alkali  and once with TE-urea , and 8 M urea), then eluted with 98% formamide and 10 mM EDTA (pH 8.0) at 95°C for 15 min. The reaction products were analyzed by denaturing PAGE.RNA-templated polymerization of either RNA and DNA was performed using 100 nM ribozyme, 125 nM template, and 125 nM primer. The primer, which consisted of RNA, DNA, or RNA with a single 3´-terminal deoxynucleotide, contained both a fluorescein label and biotin moiety at its 5´ end. The ribozyme, template, and primer first were heated at 80°C for 2 min, then cooled to 17°C over 5 min and added to the reaction mixture, which also contained 2 mM each NTP or dNTP, 200 mM MgClDefined-length extension products for analysis by either DNase digestion or LC/MS were prepared using 1 µM ribozyme, 1 µM template, and 0.8 μM RNA or DNA primer. The reaction was carried out as described above at pH 8.3 for 21 hr. Presumed full-length materials were purified by electrophoresis in a denaturing 15% polyacrylamide gel, excised from the gel, eluted with 200 mM NaCl, 1 mM EDTA, and 10 mM Tris (pH 7.5), and ethanol precipitated.2, 0.5 mM CaCl2, and 20 mM Tris (pH 7.5), which was incubated at 37°C for 30 min, then quenched with 20 mM EDTA, followed by heat inactivation of the enzyme at 75°C for 10 min. The resulting products were analyzed by electrophoresis in a denaturing 15% polyacrylamide gel.The purified extension products were subjected to partial DNase digestion in a mixture containing 1 μM oligonucleotide, 0.1 U/μL TURBO DNase I, 10 mM MgClLiquid chromatography/mass spectrometry analysis was performed by Novatia LLC  using 50 pmol of purified extension products. Standard analyses were performed by electrospray ionization LC/MS on the Oligo HTCS platform, which achieves mass accuracy of 0.01–0.02%. Oligonucleotide sequence confirmation was performed by high-resolution ion trap tandem MS on an LTQ-Orbitrap ion mass spectrometer, which achieves mass resolution of 0.003% (FWHM). The parent ion was used to generate a fragment spectrum resulting from cleavage at phosphodiester linkages within the DNA portion of the molecule. ReSpect deconvolution software (Positive Probability Ltd.) was used to deisotope the MS/MS spectrum and to obtain a simplified fragment spectrum with exact masses. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably reviewed by Peter J Unrau (Reviewer #1) and Jack Szostak (Reviewer #2), and the evaluation has been overseen by a Reviewing Editor and John Kuriyan as the Senior Editor.Thank you for submitting your article "A Reverse Transcriptase Ribozyme" for consideration by The Reviewing Editor has drafted this decision to help you prepare a revised submission. We note that both reviewers recommend publishing the article essentially as is, but have offered some suggestions that you may wish to respond to in a revision.Review:This manuscript presents data demonstrating that the latest generation of the RNA-dependent RNA polymerase ribozyme originating from the Class I ligase of Bartel and Szostak is in fact capable of RNA-dependent DNA polymerization, in other words, the ribozyme has acquired reverse transcriptase activity. This new activity is not in itself highly surprising, given the overall chemical similarity between RNA and DNA and the corresponding nucleotides, although it is fair to note that previous, less optimized versions of this ribozyme had no detectable RT activity. Nevertheless, the emergence of RT activity in the absence of direct selection is highly significant, since it provides a proof of principle for the hypothesis that DNA genomes may have emerged from RNA genomes during the 'RNA World' phase of early life.The reviewers appreciated the use of MS to confirm the identity of the major full length product, coupled with drop-out reactions to show reasonable fidelity in the presence of all four substrates, along with the incorporation of G across from U in the absence of dATP. The mass spectrometry and gel analysis presented show quite clearly that dNTP extension of an RNA template occurs.The work is quite significant and demonstrates the potential for a transition from a RNA to DNA genome via RNA catalysis earlier in evolution.Minor points to consider:1) The inability of the polymerase to extend a DNA template is also quite interesting and should be expanded upon. With this in mind, in the third paragraph of the Results, what specifically does 'negligible' mean? No extension? A small amount? Please include the data behind this statement as any extension of a DNA template by a ribozyme polymerase would be quite interesting. Other than this mild concern the work should be published as submitted.2) The authors note in the fourth paragraph of the Introduction, that DNA synthesis is more difficult chemically due to the reduced nucleophilicity of the 3'-hydroxyl of deoxynucleotides vs. ribonucleotides. However, Wu and Orgel showed in 1992 (JACS 114:317) that nonenzymatic extension of a primer ending in a deoxynucleotide was only about 10-fold slower than from a primer ending in a ribonucleotide. Thus, decreased nucleophilicity may be responsible for perhaps a 10-fold effect on reaction rate, rather than the 100-fold effect noted by the authors, which seems to derive from a reference that actually measures a pKa change, not a change in nucleophilicity.3) On the other hand, the authors also suggest that the tendency of deoxynucleotides to adopt a 2'-endo sugar conformation could also make DNA synthesis more difficult. However they then go on to say that even deoxynucleotides adopt a 3'-endo conformation when bound to an RNA template. While this is true for duplex oligonucleotides, Zhang and Szostak showed in 2012 (JACS 134:3691) using NMR methods that rG but not dG tends to adopt a 3'-endo conformation upon binding to an RNA template. If the same is true for the dNTPs used in the present study, this conformational effect may indeed contribute to the difficulty of ribozyme catalyzed DNA synthesis. An intriguing possibility might be that in the course of evolutionary optimization for RNA synthesis, new interactions that constrain the sugar geometry have come into place, and that these new interactions increase DNA synthesis relative to RNA synthesis by constraining the sugar conformation of template bound dNTPs. Minor points to consider:1) The inability of the polymerase to extend a DNA template is also quite interesting and should be expanded upon. With this in mind, in the third paragraph of the Results, what specifically does 'negligible' mean? No extension? A small amount? Please include the data behind this statement as any extension of a DNA template by a ribozyme polymerase would be quite interesting. Other than this mild concern the work should be published as submitted.Results, third paragraph: We have added a figure  depictin2) The authors note in the fourth paragraph of the Introduction, that DNA synthesis is more difficult chemically due to the reduced nucleophilicity of the 3'-hydroxyl of deoxynucleotides vs. ribonucleotides. However, Wu and Orgel showed in 1992 (JACS 114:317) that nonenzymatic extension of a primer ending in a deoxynucleotide was only about 10-fold slower than from a primer ending in a ribonucleotide. Thus, decreased nucleophilicity may be responsible for perhaps a 10-fold effect on reaction rate, rather than the 100-fold effect noted by the authors, which seems to derive from a reference that actually measures a pKa change, not a change in nucleophilicity.a values  and non-enzymatic primer extension reactions . Prior to the original submission, I discussed this topic with Dan Herschlag, who felt the Åström et al. paper is the most relevant, but it is good to cite both papers. We also have toned down the opening sentence of the paragraph, changing “much more challenging” to “more challenging”.Introduction, third paragraph: We now discuss the reduced nucleophilicity of the 3´-hydroxyl of DNA compared to RNA based on consideration of both relative pK3) On the other hand, the authors also suggest that the tendency of deoxynucleotides to adopt a 2'-endo sugar conformation could also make DNA synthesis more difficult. However they then go on to say that even deoxynucleotides adopt a 3'-endo conformation when bound to an RNA template. While this is true for duplex oligonucleotides, Zhang and Szostak showed in 2012 (JACS 134:3691) using NMR methods that rG but not dG tends to adopt a 3'-endo conformation upon binding to an RNA template. If the same is true for the dNTPs used in the present study, this conformational effect may indeed contribute to the difficulty of ribozyme catalyzed DNA synthesis. An intriguing possibility might be that in the course of evolutionary optimization for RNA synthesis, new interactions that constrain the sugar geometry have come into place, and that these new interactions increase DNA synthesis relative to RNA synthesis by constraining the sugar conformation of template bound dNTPs.Introduction, last paragraph: We have clarified the point about sugar pucker, which refers to residues within the primer, not the incoming nucleotide. The former could potentially affect positioning of the reactive 3´-hydroxyl.


Reoviridae family, causes infection in children and infants, with high morbidity and mortality. To be viable, the virus particle must package a set of eleven RNA segments. In order to understand the packaging mechanism, here, we co-synthesized sets of RNA segments in vitro in different combinations and detected by two alternate methods: the electrophoretic mobility shift assay (EMSA) and the RNA-bead pull-down assay. We showed that viral positive-sense RNA segments interact with each other in a specific manner, forming RNA complexes, and that the RNA–RNA interactions followed a sequential order initiated by small RV segments. Further, we demonstrated that RNA complexes were perturbed by targeted specific antisense oligoribonucleotides (ORNs) complementary to short RNA sequences, indicating that the RNA–RNA interactions between different segments were sequence-specific. The same inhibitory ORNs also had the capability to inhibit virus replication. The combined in vitro and in vivo data inferred that RNA–RNA interactions and specific complex formation are essential for sorting different segments, possibly prior to, or during, genome packaging. As genome assembly is a universal requirement in the Reoviridae family, this work offers an approach towards a further understanding of the sorting and packaging mechanisms of RV and related dsRNA (double-stranded RNA) viruses.Rotavirus (RV), a member of the  Reoviridae family) with three layers of capsid proteins enclosing the viral polymerase complex and a double-stranded (ds)RNA genome of 11 segments (S1–S11) with different sequences and lengths  pCp  with T4 RNA ligase  in a T4 RNA ligase buffer, and incubating at 4 °C overnight. Unincorporated 32P was removed by exclusion chromatography . Unlabeled S10 and S11 RNAs were denatured at 80 °C for 1 min, immediately chilled, and then mixed with a folding buffer . Hybridization was performed with 1 pmol of pre-folded S10 RNA annealed with 32P-labelled ORNs  in the folding buffer in a 10 µL final volume [The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs  including scrambled (Scr) with 10 μCi [l volume . The comA series of eight antisense ORNs were designed to hybridize to the AUG initiating codon, stop codon and the 3′-UTRs of segments S1, S10 and S11. ORNs were modified at the 2′-OH ribose with a 2′-O-methyl group  to enable nuclease resistance and to avoid potential interferon responses ,28,29. TFor the ORN interference assay, the simultaneous transcription of two or more RNA segments (S8–S11) was performed in the presence of 20 pmol of S10 ORNs (S10.2 and S10.3) or S11 ORNs (S11.1 and S11.2), after determining the optimum inhibitory concentration of 20 pmol. The reactions were analyzed in 1% native gel electrophoresis, as described above, and the integrity of transcribed RNAs was verified by denaturing gel electrophoresis. For the RNA–RNA interaction specificity assay, co-transcription of S9–S11 was carried out in the presence of a 20-fold molar excess of transfer RNA (tRNA).5 cells) were transfected with 0.5, 1, 1.5 or 3 µM ORNs for 3 h prior to infection with RV (MOI: 0.1) for 1 h. After 16–18 h, cells were harvested and the virus titer was analyzed by a plaque assay. For the time course assay, the experiment was repeated using 1.5 µM ORNs, and transfected-infected cells were harvested at different time points. RV genome copy numbers were determined by qRT-PCR, detecting the negative-strand RNA as described below.For the inhibition of RV replication in the presence of ORNs, MA104 cells in 12-well plates  ORN by site-directed mutagenesis . For the2) for 30 min at 30 °C, followed by three washes. The pulled-down RV S1 or S5 were quantified by qRT-PCR.The Agarose beads-based RNA–RNA interaction assay was described previously . The bioFor the detection of ssRNA in RNA–RNA interactions and ORNs inhibition assays, ssRNAs were reverse-transcribed into cDNA using RevertAid Reverse Transcriptase  with specific reverse primers, and quantified with qPCR using the 7500 Fast Real-Time PCR system and SYBR select Master Mix . The primers were: for RRV S1: 2356F: 5′-AGAGTATGGGACAACTGACG-3′; RRV S1: 2570R: 5′-CACTTGAGAAAAGACGT GCGCAAATATCAGC-3′; RRV S5: q-rota S5F: 5′-CTGATCTCGGACTCTG-3′ and rota S5R: 5′-GGTCACAGTTTTTG-3′; RRV S8: q-rota S8F: 5′-GGAAAGTAATCCG-3′ and rota S8R: 5′-GGTCACATAAGCGC-3′; RRV S9: q-rota S9F: 5′-GTAGTCGACTATGTG-3′ and rota S9R: 5′-GGTCACATCATAC-3′; RRV S10: q-rota S10F: 5′-CATCGGACCTGATG-3′ and rota S10R: 5′-GGTCACAGGAAGAC-3′; and RRV S11: q-rota S11F: 5′-CTGTAAATATAAG-3′ and rota S11R: 5′-GGTCACAAAACGGAAGTGG-3′. The PCR conditions were 95 °C for 15 s and 55 °C for 30 s, for 40 cycles. The qPCR amplification was considered valid only when the standard curves had a correlation coefficient greater than 0.96 and a PCR efficiency within the range of 85–115%.Since the de novo positive-strand RNA segments must have been associated with the newly synthesised viral capsid, prior to synthesis of negative-strand RNA of genomic dsRNA segments, viral dsRNA quantification was performed by using primers specifically targeting the negative strands in the RT reaction.35S-methionine. Translation reactions were incubated at 30 °C for 90 min and treated with 1 µg of RNase A for 10 min at room temperature. Labelled proteins were visualized using a PhosphorImager screen .For the in vitro RV translation assay, various concentration of ORNs RV S1 AUG , S10.2, S11.2 (1 or 4 µM) or Scr (4 µM) were incubated with 300 ng of S1, S10 or S11 RV transcripts purified by phenol-chloroform-isoamyl alcohol, followed by exclusion chromatography  to remove other impurities. The mixtures of RNA and ORN were incubated for 20 min at 37 °C, and added to a reaction mix containing 7.5 µL of nuclease-treated rabbit reticulocyte lysate , 1 mM amino acid mix minus methionine, and 6 µCi To investigate if specific RNA–RNA interactions occurred among RV RNA segments, first, we synthesized exact copies of the small RNA segments (S8–S11) from the RV strain RRV by generating cDNA copies of genomic dsRNA segments from purified RV. Each cDNA copy was flanked by a T7 promoter at the 5′ terminus and a specific restriction enzyme site at the 3′ terminus, as described previously . For RNATo further investigate the interactions among all three segments , ssRNA of each segment was co-synthesized and analyzed by EMSA D. WhilstReoviridae family—the orbiviruses—where the 3′-UTRs of the RNA segments play an important role in RNA complex formation [The mechanism by which viruses with multi-segmented dsRNA genomes correctly package the complete set of genomic RNA segments is not fully understood. Our previous study on BTV demonstrated that the genomic RNA segments interact with each other in a coordinated manner that is related to the segment size and is initiated by the small segments, which leads to a sequential packaging mechanism . We predormation ,23,24,25ormation . The RNAOur data show that S11-coated beads captured S8 efficiently in the pull-down assay when S9 and S10 were present, but this was 100-fold less when only S10 was added in absence of S9 . The results obtained indicated that S11.2 inhibited the RNA complex formation or RNA–RNA interactions more efficiently than the S10.2 ORN in S8–S11 RNA combinations , suggestTo further confirm and substantiate the observed disruption on the RNA complex formation using S10.2 ORN, two mutations were designed on S10 RNA, one with a deletion (10.2del) and one with substitution mutation (10.2mut) spanning the same targeted area. The region of deletion mutation was the 27 nts (nt 570–596) after the NSP4 stop codon (5′-GAGGUUGAGCUGCCGUCGUCUGUCUGC-3′), complementary to S10.2 as shown in After demonstrating the capacity of the ORNs to inhibit the RNA complex formation in vitro, we investigated whether the inhibition of RNA–RNA interactions mediated by ORNs affected RV replication in cell culture. Since the ORNs were modified at the 2′-ribose to substitute the 2′-OH to 2′-O-methyl, these small ORNs should not have had any adverse cellular interferon effects during the virus replication ,28,29. TScr ORN and an S1 ORN  complemeTo further substantiate the inhibitory effects of the S10.2 and S11.2 ORNs on the virus growth, we measured the dsRNA synthesis in the presence or absence of these two ORNs at different time points of the viral replication. Since RV only produces dsRNA as genomic RNA segments, and each viral particle contains precisely one copy of each of the 11 genomic dsRNA segments, the copies of dsRNA segments represented the quantity of the viral genome present in the infected cells. Quantitative analysis of viral dsRNA using qRT-PCR confirmed that S10.2 and S11.2 significantly inhibited the virus replication B.The nucleotide sequences of the RRV 3′-UTR shared some similarity with those of human RV (HRV), and since the 3′-UTR of segment S10 was the longest among all segments, we assessed the effect of the S10.2 ORN (nt 570–596) on the replication of an HRV strain in MA104 cells. The S10.2 ORN corresponding region of the HRV S10 segment had only two mismatches in comparison to that of RRV. The inhibition of the virus growth was at least ~50% in the presence of S10.2  and Scr ORN (as a non-inhibitory control) were included. To further investigate the specificity of the ORNs to the target regions, hybridization assays of RNAs and ORNs were undertaken. The gel analysis showed that ORNs S10.2, S10.3 hybridized with S10 and S11.2 hybridized with S11 mRNA. No hybridization with the Scr control was detected when incubated with S11 RNA, suggesting specific annealing of the ORNs to their target sequences .The precise and specific mechanism of genome segment assembly during RV replication has yet to be elucidated. Previously, our laboratory has reported that BTV hierarchical intermolecular interactions between RNA segments are required for the efficient packaging of the viral genome ,18,19,23Since EMSA is not appropriate for the detection of very large complexes, an alternative pull-down assay using the immobilized RV segment S11 was established to evaluate the interactions between the larger and smaller genome segments. The smaller genome segments appeared only to interact with the larger genome segments (such as S1) via the medium-sized RNA segments. The medium-sized RNA segment S5 also required the presence of other intermediate-sized segments (S6–S10) in order to interact with S11. These data suggest that all 11 RV genome segments may form a networking RNA complex. We predict that the formation of an RNA complex comprised of the small RNA segments serves as the leading base for recruiting the remaining RNA segments by sequential interactions, and ultimately generating a complex composed of the complete set of 11 RV RNA segments, similarly to for BTV ,19.Based on previous studies of BTV, we proposed that the 3′-UTR of smaller segments might have also played an important role in RV genome assembly. Our data showed that intersegment interactions between the smaller segments could be disrupted in the presence of ORNs specifically targeting the 3′-UTR of segments S10 and S11, as demonstrated by altered RNA migration profiles on native agarose gels. The different effects of the S10- and S11-targeting ORNs on the complex formation were most likely due to the different secondary structures and contact points disrupted by each specific ORN. Deletion and substitution mutation in the S10 3′-UTR region targeted by the ORNs significantly disrupted the RNA complex formation, also suggesting that this region was likely to be the putative binding sites of S10 with other genome segments. In summary, our data suggest that both ORNs and specific mutations in the 3′-UTR of genome segments, are able to disrupt the specific RNA structures in the 3′-UTR, resulting in conformational changes that affect the correct sorting and/or assembly of RV genome RNA segments.This hypothesis was substantiated by demonstrating that the ORNs, which caused disruption of RNA–RNA interactions in a RRV strain in vitro, also had strong inhibitory effects on RV replication in cell culture, using a HRV strain. This suggests that ORNs targeting the 3′-UTR of RV RNA genome segments might cause the same inhibitory effect on RV replication of the two related RV species. The observed inhibition of RV replication was likely to be due to disruption in the genome sorting and/or packaging process, as the presence of these ORNs did not inhibit the virus protein expression. Some ORNs significantly inhibited the virus growth, but had mild effects on the RNA interaction. As the interactions may depend on multipoint contacts between RNA segments, blocking one region of an RNA segment will not necessarily fully diminish the complex formation in vitro, however, it is sufficient to prohibit the assembly of all RNA segments and consequently abolish the virus replication in vivo. Further RNA structural analysis is needed to confirm our hypothesis.Reoviridae family.Most current antiviral agents target specific viral enzymes, such as polymerases and proteases, and these often become ineffective after long-term use, as the target viruses develop a resistance to the drug. To minimize the possibility of antiviral drug resistance, it might be a useful strategy to target other stages in the virus replication process, such as viral genome packaging mechanisms possibly targeting specific sequences of the conserved 3′-UTRs ,38,39. O


Meeting report on the Cold Spring Harbor Asia conference on RNA Modifications and Epitranscriptomics, held in Suzhou, China, 13–17 November, 2017. Nature Methods—have recently revealed highly dynamic 'epitranscriptomes', in which RNA modifications regulate gene activities and cellular phenotypes in health and disease. The Cold Spring Harbor Asia (CSHA) conference on RNA Modifications and Epitranscriptomics highlighted cutting-edge research in this rapidly evolving field. The conference was organized by Michaela Frye , Chuan He , Tsutomu Suzuki , and Yungui Yang , and included more than 50 talks and hundreds of attendees.Methods for transcriptome-wide analyses of RNA modifications—highlighted as the 'Method of the Year 2016' by 6-methyladenosine (m6A) is the most prevalent type of reversible internal modification in eukaryotic mRNAs. Chuan He (University of Chicago) characterized m6A-modified transcripts as entering a separate track of fast decay and fast translation mediated through m6A reader proteins. One reader protein, YTHDF2, promotes m6A-mediated mRNA decay, while another reader protein, YTHDF1, promotes mRNA translation. The He group also discovered FTO and ALKBH5 as erasers of m6A modification, and found that these proteins have important functions. Misregulation of m6A-associated processes leads to stalled cell differentiation and human diseases, in particular cancer.More than 140 types of chemical modifications have been found in RNA. NAnother RNA modification, 5-hydroxymethylcytosine (5hmC), was characterized by Francois Fuks (Free University of Brussels (ULB), Brussels). The Fuks group developed hMeRIP-seq for transcriptome-wide mapping of 5hmC. Their studies of 5hmC characteristics in mammalian embryonic stem cells (ESCs) revealed its enrichment at the mRNA transcription start sites.RNA tailing is another prevalent type of RNA modifications. To study the regulation of RNA tailing, Narry Kim  and colleagues developed TAIL-seq, which determines transcriptome-wide poly-A tail length and 3’ end modifications simultaneously. Using TAIL-seq, they identified CNOT as the major deadenylase and elucidated the mechanistic interactions between the two CNOT subunits, CNOT6/6L and CNOT7/8, and the poly-A binding protein PABPC1. After PABPC1 and CNOT remove poly-A tails, the uridylation-catalyzing enzymes TUT4 and TUT7 add a U-tail to deadenylated mRNAs. More recently, the Kim group discovered that guanylation is catalyzed by a subset of TUTs and slows down deadenylation. The interplays of deadenylation, uridylation, and guanylation reflect a complex regulatory network of RNA modifications at the mRNA 3’ ends.Drosophila brains. Eddie Park  reported widespread population and allelic variation of A-to-I RNA editing in human cells and proposed a mechanism that is based on RNA secondary structure.RNA editing is an abundant RNA modification. Jian Lu  discovered that adenosine-to-inosine (A-to-I) RNA editing has provided an adaptive evolutionary force through systematic analyses of RNA editing sites in New types of RNA modifications are being discovered. Mark Helm  reported an LC-MS-based method to identify novel RNA modifications. A proof-of-concept study detected KDka427, a new RNA modification with a thioacetal structure. Further applications of this method may contribute to an expanding epitranscriptome vocabulary.Elucidating the functional significance of RNA modifications has been a major goal for the field. Numerous talks focused on the impacts of RNA modifications in various biological processes and diseases.6A regulates viral infection, as shown by Stacy Horner  in Flaviviridae, a family of RNA viruses. Specifically, m6A marks the positive-sense-stranded RNA genomes of viruses in this family and regulates the viral replicative life cycle. The presence of m6A on viral functional elements suggests an additional layer of gene regulation in virus-host interactions.In virology, m6A-modified antiviral transcripts in the nucleus. On the other hand, TET2 represses the expression of SOCS3, an inhibitor of the JAK3 pathway, by binding and oxidizing m5C in the SOCS3 3’-UTR, thus promoting infection-induced myelopoiesis. Jianlong Wang  also described critical roles of TET2 and RNA m5C oxidation in the post-transcriptional regulation of endogenous retrovirus (ERV) and ERV-associated genes in pluripotent stem cells.The immune response is also regulated by RNA modifications. Brenda Bass  presented findings on the roles of A-to-I RNA editing by ADARs (adenosine deaminases that act on RNA) and dsRNA termini-dependent cleavage by Dicer in immune response. ADARs mark cellular dsRNAs to preclude the antiviral response mediated by Dicer in invertebrates. Bass and colleagues elucidated the mechanisms of differential ATP requirement by invertebrate Dicer and its relationship with the recognition of self versus non-self dsRNAs. Xuetao Cao  discovered that in antiviral immune responses, DDX46 inhibits immune response by retaining mTaurine modification of uridine in mitochondrial transfer RNA plays a critical role in decoding during protein synthesis. Tsutomu Suzuki  and colleagues found that the lack of this modification is a primary cause of mitochondrial diseases, and proposed 'RNA modopathy' as a new class of human diseases. Kazuhito Tomizawa  demonstrated the essential role of taurine modification and its catalyzing enzyme Mto1 in mitochondrial translation and in control of the proteostasis network, which has therapeutic implications for mitochondrial diseases.6A eraser FTO acts as an oncogene in certain AML cells, and that inhibiting FTO activity can suppress leukemogenesis. Furthermore, an oncometabolite, R-2-hydroxyglutarate (R-2HG), exerts a broad anti-tumor activity by inhibiting the FTO/m6A/MYC/CEBPA signaling axis in FTO-high AML cells.RNA modifications have emerged as an important regulatory axis of tumorigenesis. In subtypes of acute myeloid leukemia (AML), Jianjun Chen  discovered that the mRNA modifications play a critical role in cell fate determination. Michaela Frye  demonstrated that several mechanisms of RNA modifications, in particular non-coding RNA modifications, regulate stem cell functions by providing a molecular rheostat for protein synthesis in fast response to environmental stimuli. For example, tRNA methylation responds to oxidative stress and regulates protein synthesis in stem cells.The mRNA cap is a collection of structures on the 5’ end of mRNA that recruit proteins mediating RNA processing, export, and translation. Victoria Cowling  described the identification of the mRNA cap methyltransferase RAM and its regulatory role in ESC differentiation, and reported how its methyltransferase-independent characteristics had been revealed by cross-linking and immunoprecipitation sequencing (CLIP-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) experiments.5C and m2G on small non-coding RNAs in mice. Mofang Liu  discussed the piwi/piRNA machinery in mammalian spermiogenesis and human male infertility, and revealed a novel mechanism for regulated packaging of genomic DNA into functional sperm. Ramesh Pillai  introduced a critical role of the m6A reader YTHDC2 in facilitating the meiotic gene expression program during mouse spermatogenesis. In oocytes, as shown by Donal O’Carroll , TUT4 and TUT7 catalyze uridylation and are required to sculpt certain transcripts for the maternal transcriptome, whereas the m6A reader YTHDF2 regulates transcript dosage during oocyte maturation and is a key determinant of egg quality.Several studies highlighted the functional roles of RNA modifications in germline cells. Qi Chen  illustrated the intergenerational transmission of paternally acquired traits via sperm. In particular, he described paternally acquired metabolic disorders that are induced by a high-fat diet and transmitted through the epitranscriptomic markers mTrm7 mutants grow slowly and have translation defects in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they are associated with intellectual disability in humans.Eric Phizicky  and colleagues characterized the highly conserved 2’-O-methyltransferase Trm7 in yeast. 6A modification. The mRNAs of several clock genes have high m6A levels, often in their 3’-UTRs, and inhibition of m6A stabilizes these transcripts and prolongs the circadian period.Jean-Michel Fustin  presented work demonstrating the control of circadian rhythm by m6A nuclear reader that specifically recognizes m6A-modified transcripts and regulates exon splicing by promoting or antagonizing splicing factor binding. In a re-analysis of previously published m6A-LAIC-seq data and related datasets, Li Yang  discovered a global downregulation of A-to-I editing in m6A-positive compared to m6A-negative RNA populations, suggesting a negative correlation between m6A modification and A-to-I editing.A recurring message at the conference was the interplay between multiple types of RNA modifications and other RNA regulatory processes. For example, an important molecular consequence of RNA modifications is differential affinity for RNA-binding proteins. Yungui Yang  and colleagues identified YTHDC1, an m6A writer METTL3 and the eIF complex that regulates protein expression. This looping mechanism ensures that ribosomes are more efficiently recycled in METTL3-bound mRNA transcripts.RNA modifications also mediate mRNA translational control. Richard Gregory  described a looping mechanism between stop-codon-associated mArabidopsis. The Chen group also identified HESO1 in catalyzing pre-miRNA uridylation. Uridylation of trimmed pre-miRNAs promotes their degradation.One session of the conference was dedicated to RNA modifications in plants. Xuemei Chen  presented a role for the TREXII complex in miRNA biogenesis, export, and loading onto AGO1 in 6A methylation complex in plants. The identified components include MTA (METTL3), MTB (METTL14), FIP37 (WTAP), Vir (KIAA1429), and Hakai, a novel m6A writer that may also be conserved in mammals. Guifang Jia  presented the identification of the first plant m6A eraser, AtALKBH10B. AtALKBH10B regulates the floral transition and vegetative growth in Arabidopsis.Rupert Fray  presented a study of the m1A-MAP, sequencing methods for detecting pseudouridine and N1-methyladenosine (m1A), respectively. Specifically, m1A-MAP leverages m1A induced mis-incorporation to achieve single-base resolution for m1A site identification, revealing the enrichment of m1A at the 5’-cap and 5’-UTR.Several speakers presented novel methods for epitranscriptome sequencing. Dan Dominissini  presented work on Nm sequencing. Leveraging the differential reactivity of 2’-O-methylated (Nm) and 2’-hydroxylated nucleosides, Nm-seq identifies Nm sites at base resolution and characterizes the unique features of Nm modification. Chengqi Yi  presented CeU-seq and m6A-CLIP to enable comprehensive, single-base-resolution mapping of m6A sites across the transcriptome. Yang Yu  presented a highly efficient and sensitive CLIP method called GoldCLIP that omits all gel purification steps to characterize transcriptome-wide protein-RNA interactions.Shengdong Ke  presented efforts in developing mOur understanding of the landscape, regulation, and functions of RNA modifications has advanced tremendously in the past few years. This CSHA conference brought together a multidisciplinary group of researchers studying RNA modifications in diverse biological systems using a broad range of approaches. With many questions still to be answered, we are excited about future scientific explorations at the new frontiers of RNA modifications and epitranscriptomics.


Infectious pancreatic necrosis (IPN) is an acute contagious systemic disease affecting several fish species and a critical disease in the salmonid fish farming industry. Here, we report the complete genome sequence of IPN virus (IPNV) RNA segments A and B, isolated from a farmed brook trout in Pennsylvania. Aquabirnavirus of the family Birnaviridae. This prototype virus of the Birnaviridae family is nonenveloped and measures approximately 65 nm in diameter with icosahedral symmetry. The IPNV genome consists of two linear double-stranded RNA segments which are designated segments A and B. Segment A is bicistronic and encodes a 106-kDa polyprotein (NH2-pVP2-VP4-VP3-COOH), whereas segment B is monocistronic and encodes the RNA-dependent RNA polymerase VP1 (Infectious pancreatic necrosis (IPN) is a highly contagious viral disease of salmonid fish caused by infectious pancreatic necrosis virus (IPNV). IPNV belongs to the genus Salvelinus fontinalis) and submitted to the Animal Diagnostic Laboratory, Pennsylvania State University. Kidney and spleen tissues from the brook trout were homogenized in viral transport medium (VTM) and inoculated in Chinook salmon embryo (CHSE-214) cells grown at 18°C in minimal essential medium supplemented with 2 mM glutamine, 1% nonessential amino acids, and 1× penicillin, streptomycin, and amphotericin B for virus isolation. After three freeze-thaw cycles, the infected viral supernatant and the lysed CHSE-214 cells were centrifuged at 1,500 × g for 10 min. The resulting supernatant was used for viral RNA extraction using a QIAamp viral RNA minikit .Along with several salmonid fish species, other types of fish, such as rainbow and brook trout, are also susceptible to IPNV. The IPNV infection manifests as swelling of the abdomen, bilateral exophthalmos, and pale gills. The infection is subclinical in fish older than 4 months, and the mortality rate ranges from 10 to 90% . Here, wde novo assembler to produce two RNA segments. The IPNV consensus sequences of segments A and B were annotated with Prokka (v1.12)  RNA enrichment and begins with RNA fragmentation and priming. An equimolar pool of libraries was made and sequenced on an Illumina MiSeq instrument using 150 × 150-bp paired-end sequencing according to the manufacturer’s protocol. Over 611,110 reads were generated for the IPNV genome. Low-quality bases were trimmed by Trimmomatic  using a  (v1.12) .MH010544 and MH010545, respectively.RNA segments A and B of isolate PA1 were submitted to GenBank and assigned the accession numbers


With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities.With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss techniques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of  Below, we discuss each technique’s usage, advantages, and limitations.RNA trafficking and localization are important processes that influence cellular physiology at the epigenetic, post-transcriptional, and post-translational levels. Conventional techniques such as qRT-PCR and DNA microarrays are cell lysate-based assays that provide ensemble averages of RNA expression levels. To improve our current understanding of the role of RNAs in health and disease, there is great interest among RNA biologists to visualize individual RNAs in cells. There are currently three popular approaches — fluorescence Single-molecule FISH (smFISH) is a powerful technique that uses multiple fluorophores to visualize specific RNA targets at the single-molecule level in cells The MS2 system takes advantage of the ability of the bacteriophage MS2 coat protein (MCP) to bind to an aptamer sequence (known as the MS2 aptamer) with high specificity and affinity  [4], [5]MBs are antisense stem-loop forming oligonucleotide probes labeled with a fluorophore at one end and a quencher at the other end In addition to the techniques described above, other techniques, including RNA-targeting CRISPR associated protein 9 (RCas9) Both the MS2 and MB systems are capable of imaging single RNA transcripts in living cells. Nonetheless, the MS2 system has been used more widely, despite the fact that MBs offer several advantages including smaller probe size, versatility in fluorophore/quencher selection, improved signal-to-background due to quenching, and the ability to image endogenous RNAs . One majO-methyl (2Me) MB backbone, we have recently developed an MB architecture called the 2Me/PSLOOP MB, based on the latter approach LOOP MB exhibits a marginal level of FPSs in various cell types and can be used for imaging single RNA transcripts in living cells LOOP MBs and explore their capabilities for single-molecule RNA imaging. We envision that the use of 2Me/PSLOOP MBs to study RNAs in living cells can further our knowledge of the role of RNAs in health and disease.To reduce nonspecific signals, MBs have been conjugated to macromolecules that are either too big to traverse the nuclear pores, such as quantum dots FULL MBs) and showed that these MBs enable detection of Coxsackie viral RNA replication for up to 12 h FULL MBs have longer intracellular stability and bioactivity than 2Me MBs FULL MBs still cause a detectable FPS FULL MBs could exhibit a punctate staining pattern that can be easily misinterpreted as RNA granules or even single RNA transcripts Conventional MBs, including those that are synthesized with backbones composed of DNA or 2Me RNA linked with phosphodiester bonds (DNA or 2Me MBs), can be highly sensitive to nuclease degradation. To confer nuclease resistance, a non-bridging oxygen of the phosphate may be replaced with a sulfur atom to form a chemically-modified internucleotide linkage known as the PS bond. Yeh et al. reported the first use of MBs that incorporate PS linkages throughout the probe backbone  of PS modifications. Consistent with this observation, 2Me/PSLOOP MBs, which have a fully-PS-modified loop domain and a phosphodiester stem, exhibited even lower FPSs than 2Me/PS10-LOOP MBs LOOP MBs opened nonspecifically within 10 h after delivery into several different cell types LOOP MBs were lower than those generated by 2Me/PSFULL MBs, suggesting that when the loop domain is highly PS-modified, stem domain modification is disadvantageous, as the additional PS groups can induce nonspecific binding while offering no additional increase in nuclease resistance. Overall, these findings demonstrate the feasibility of reducing the number of PS modifications in the MB backbone to reduce nonspecific binding while maintaining nuclease resistance.We hypothesized that partially-PS-modified probes may exhibit an optimal balance of nuclease resistance while avoiding excess nonspecific binding. To test this, MBs were synthesized with different numbers and distributions of PS linkages. In a variety of cell types including HEK293, HeLa, Jurkat, and primary BJ cells, we observed a general trend of MB performance relative to the degree of PS modification . For exaFULL than 2Me/PSLOOP MBs suggest that any nuclease resistance gained by PS modifications in the stem domain is offset by increased nonspecific binding due to greater number of PS modifications.Currently, debate over the primary causes of MB FPSs in living cells remains unresolved. Our findings that MBs with different PS modifications exhibit large differences in the degree of nonspecific opening can help explain why MBs open nonspecifically in cells. For example, single-stranded endonucleases appear to be the primary cause of MB nonspecific opening, as levels of FPSs are inversely correlated with the extent of PS modifications in the single-stranded loop domain LOOP MBs exhibit a marginal level of FPS raises the possibility of using MBs to image the dynamics and localization of single RNA transcripts in living cells with high accuracy. To determine whether 2Me/PSLOOP MBs can accurately detect single RNA transcripts, we developed a plasmid construct that encodes a transcript carrying an EGFP coding sequence followed by 32 tandem repeats of an MB target sequence (pEGFP-N1-32x) LOOP MBs and smFISH processing, bright MB and smFISH spots could be detected in the nucleus and the cytoplasm (LOOP MBs can detect engineered transcripts with high accuracy (FULL MBs and processed by smFISH, only 60% of the MB signals colocalized with smFISH signals. Thus, consistent with the analysis showing that 2Me/PSLOOP MBs generate lower FPS compared to 2Me/PSFULL MBs, 2Me/PSLOOP MBs can image single RNA transcripts more accurately than 2Me/PSFULL MBs in living cells.Our finding that 2Me/PSytoplasm A. Analysaccuracy B. By conLOOP MBs may be a promising tool to study the trafficking and localization of single RNA transcripts in real time. LOOP MBs at either 5′- or 3′-UTR. Furthermore, binding of the 2Me/PSLOOP MBs to 32 tandem repeats does not cause interference with EGFP translation as seen when 64 repeats are used (LOOP MBs do not affect cellular growth or physiology. Overall, these findings suggest that 2Me/PSLOOP MBs can be a noninvasive platform for imaging single RNA transcripts in living cells.Given their ability to detect single engineered RNA transcripts with high accuracy in live cells, 2Me/PSare used C, suggesare used D–E, suggLOOP MB, that elicits a marginal level of FPSs in cells as compared with conventional MBs LOOP MBs could accurately image single mRNA transcripts harboring 32 tandem repeats of an MB target sequence using conventional fluorescence microscopy. Currently, RNA dynamics at the single-molecule level has been studied primarily based on engineered RNA molecules that harbor large insertions of MB target or MS2 aptamer sites that potentially interfere with the activities of target RNAs. With further possible approaches for optimizing signal-to-background, such as fluorophore/quencher selection, and the use of more sophisticated imaging techniques, we anticipate that 2Me/PSLOOP MBs can be a promising platform for live-cell, single-molecule imaging of minimally-engineered RNA molecules, or even endogenous RNA molecules, providing researchers with opportunities to study RNAs with unprecedented spatial and temporal resolutions.Conventional MBs have been used to image RNAs in various cellular contexts, but their propensity for nonspecific opening in living cells limits their widespread applications in studies where more sensitive detection is necessary, such as imaging RNA localization and dynamics at the single-molecule level The authors declare no conflict of interest.


Programmed self-assembly of nucleic acids is a powerful approach for nano-constructions. The assembled nanostructures have been explored for various applications. However, nucleic acid assembly often requires chemical or in vitro enzymatical synthesis of DNA or RNA, which is not a cost-effective production method on a large scale. In addition, the difficulty of cellular delivery limits the in vivo applications. Herein we report a strategy that mimics protein production. Gene-encoded DNA duplexes are transcribed into single-stranded RNAs, which self-fold into well-defined RNA nanostructures in the same way as polypeptide chains fold into proteins. The resulting nanostructure contains only one component RNA molecule. This approach allows both in vitro and in vivo production of RNA nanostructures. In vivo synthesized RNA strands can fold into designed nanostructures inside cells. This work not only suggests a way to synthesize RNA nanostructures on a large scale and at a low cost but also facilitates the in vivo applications. RNA nanostructures have been demonstrated in a range of biological applications, but their assembly and delivery to cells is difficult. Here the authors demonstrate the in vivo assembly of a RNA nanostructure from a single transcript inside the cellular environment. Up to now, DNA/RNA self-assembly generally starts from chemically or enzymatically synthesized single-stranded DNA or RNA (ssDNA or ssRNA). This method is not desirable for large-scale production because of the excessive cost. A potential solution is to clone nucleic acids in bacteria, such as E. coli, in the same way as recombinant proteins.15 However, DNA molecules normally exist as duplexes in the cell. Though direct cellular production of ssDNAs is possible in some special cases such as by reverse transcriptase17 or M13 bacterial genomes5, there are limitations. For instance, the ssDNA length range is limited or additional enzymatic treatment after purification is needed.18 Comparatively, a more suitable choice is RNA. Cellular RNAs primarily exist as single-stranded, and their length can vary in a broad range. Recently, Geary et al. have demonstrated that ssRNAs, in test-tubes, can cotranscriptionally fold into designed nanomotifs, which can further assemble into 2D arrays.9 Delebecque et al. have cloned RNA nanostructures in E. coli to organize chemical reactions in vivo.12 However, the nanoscaled, structural details of the RNA complexes have not been thoroughly characterized under native conditions.Nucleic acids (DNA and RNA) have been extensively explored for molecular self-assembly and a wide range of nanostructures have been assembled from nucleic acids.E. coli. RNA nanostructures have been thoroughly characterized by gel electrophoresis, atomic force microscopy (AFM) imaging, and cryogenic electron microscopy (cryoEM).In this work, we have developed a versatile strategy to prepare well-defined nanostructures by folding individual long ssRNAs. Each nanostructure contains only one ssRNA molecule. The resulting nanostructures can be cloned, expressed, and self-folded in 19 Obviously, this thermal annealing process is not feasible for nucleic acid self-folding in vivo. A potential approach is to design the targeted nucleic acid nanostructures both thermodynamically stable and kinetically favorable. To achieve this goal, the ssRNA is designed to fold following a sequential and hierarchical pathway. Newly synthesized ssRNA would first fold into hairpins while transcription. Hairpin structures are not only thermodynamically stable but also topologically simple. They only involve local interactions, thus, fold quickly. If any alternative structure forms, it would readily rearrange into the target hairpin structure via local branch migration.20 Upon hairpin formation, which defines the RNA’s secondary structure, most of the RNA residues are inert as being in the content of duplexes, leaving a minimal amount of RNA residues as unpaired. The unpaired residues are able to further form long-range tertiary interactions, leading to the formation of fully folded, designed nanostructures. The overall folding pathway is similar to that of the naturally occurring complex RNA structures, such as hairpin ribozymes.21. Conceptually, the design concept resembles the principle that developed by Geary et al. However, a significant change is that the short dovetail seams (2–3 bps) are avoided. Such short helical domains are not very stable and are likely to deformation under mild stress.A key challenge of this approach is to design the folding pathway to avoid kinetic traps. For nucleic acid self-assembly, the target structures are designed to be thermodynamically stable, but often not kinetically favored. This problem is commonly solved by slowly cooling the samples from a high temperature  to a low temperature  over a long period of time.22, (iii) 3-way junctions in open conformation (o3WJ)23, (vi) 3-way junctions in stacked conformation (s3WJ) observed in the packaging RNA (pRNA) of phi29 bacteriophage,24 (v) coaxially stacked kissing loops (KLs) found in the dimerization initiation sites of HIV-1 RNA,25 (vi) a 3-way loop (3WL) interaction observed in phi29 pRNA,26 (vii) 4-way junctions in open conformation (o4WJ), and (viii) 90°–kink found in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA genome.27 According to the molecular designs, DNA templates coding for the ssRNAs have been synthesized so that RNAs can be produced by in vitro transcription. Alternatively, the DNA templates can be inserted into a plasmid and introduced into E. coli. Then cellular transcription machinery will transcribe the gene into the corresponding ssRNA, which spontaneously folds into the designed nanostructures inside the cells.The RNA nanostructures in this study are rationally designed based on natural RNA motifs and tertiary interactions Fig. , includiS) and a double-square (S2). Figure S contains four 90°-kinks and a KL interacting pair, while S2 contains four 90°-kinks and two 3WL pairs. After in vitro transcription, purified RNA molecules were thermally annealed  to promote the RNA folding. RNA molecules first folded into Mg2+–independent, intermediate, loose secondary structures, which then further folded into Mg2+–dependent, compact, tertiary structures via loop-loop (KL or 3WL) interactions. The resulting RNA square (S) samples were analyzed by native PAGE  is fully folded. To show the critical role of the KL interaction in the folding, a control molecule (S*) is prepared, in which one loop sequence is altered so that the KL interaction is interrupted  migrate identically to those annealed samples, indicating that the intramolecular folding of the designed RNA molecules is indeed a fast process. In addition, the comparison between S and S* molecules gives an estimation of the folding yield of S close to 100% as no RNA in the lane of S migrates like the partially folded S*. More strikingly, the RNA molecules could spontaneously fold into the designed structures cotranscriptionally. After transcription, the crude RNA samples were directly analyzed by PAGE without any purification or thermal treatment (the lane as indicated As Transcribed). The migration pattern was identical to those of purified and thermally treated samples, indicating that, as expected, the RNAs folded into the designed structures even in crude solutions without any annealing step. A similar result was also observed for S2  were designed  structure  is designed to fold from a 623-base-long ssRNA. It contains six edges and four vertices. All edges are four helical turns long. Three of them are standard A-form duplexes and each of the other three contains a KL interaction in the middle. Each of the vertexes is an o3WJ containing four unpaired uracils on each strand at the center to ensure sufficient out-of-plane flexibility to fold. Upon cooling, the ssRNA first folded into a three-branched structure  was prepared, in which a pair of KL interaction was disrupted by sequence mutation. It is expected to only fold into an open, 2D, double-triangular shape instead of the compact, fully folded, 3D tetrahedron  has a higher mobility than the open, 2D, double-triangle structure (T4*). The folding is fast, and no difference has been observed between quenching and annealing for each sample. In the lane of T4, no RNA migrates like the partially folded T4* molecule; suggesting that all T4 molecules are fully folded. In order to confirm the migration pattern and the folding ability, we have prepared two more RNA tetrahedrons with different sizes and their corresponding control molecules: a three-turn-edged tetrahedron  in E. coli . Then the cells were lysed by phenol or sonication. Without any further manipulation, the aqueous solution of the total cell lysates was directly examined by both PAGE and AFM imaging. On native PAGE, the desired RNA molecule from E. coli migrates the same as the in vitro prepared S2 sample  structure  nanostructures is a major challenging to the field of nucleic acid nanotechnology and has attracted significant efforts. The original idea was first proposed by Nadrian Seeman in 1997.In conclusion, we have developed a programmable ssRNA folding strategy for the preparation of complex nanostructures. Each nanostructure is primarily composed of RNA duplexes and is folded from one long ssRNA. Thus, the formation of the nanostructures is independent of stoichiometry and RNA concentration. This strategy allows cloning and expression of RNA nanostructures inside of cells in the same way as those of recombinant proteins; pointing to a cost-effective way for large-scale production of nucleic acid nanostructures. We expect this strategy would facilitate a wide range of in vivo biomedical applications of RNA nanotechnology, such as integrating multiple functional RNA moieties to regulate cellular processes.Double-stranded DNA templates were purchased from IDT, Inc. Polymerase chain reaction was applied to amplify DNA duplexes into large amount. The sequence of DNA templates can be found in Table Sequence of PCR primers:P1: 5′-TTCTAATACGACTCACTATA-3′P2: 5′-TAGATTCTTAGCACTGC-3′P3: 5′-CCCGTTTAGAGGCC-3′P4: 5′-CGTTATTGTACCCAACCCAGAAAAATCAC-3′S/S1*/S2/T2/T2*/T4/T4*: P1 + P2; FL/S2’/S4: P1 + P3; T3/T3*: P1 + P4.27 For KLs and pRNA loops, the exact sequence is not important, but sequence complementarities are ensured for the interacting pairs. (ii) The remaining duplex regions. They provide all the struts in the structures and do not involve long-range interactions. Their sequences are randomly generated by a computer software Tiamat.34 Roughly 10% of the Watson-Crick basepairs (A–T and G–C) are replaced by G–U pairs to facilitate PCR amplification of the DNA templates. Such G–U pairs are evenly distributed along the duplex regions. Finally, the RNA sequences are checked by a computer software Mfold35 to make sure that the RNA will fold into the designed secondary structures. If there are any undesired, strong secondary structures, the sequence will be manually altered to avoid such structures.When designing RNA sequences, we take two parts into consideration. (i) The single stranded loops in the RNA secondary structures, including the 90°-kink loop, KLs, pRNA interacting loops. They are critical regions and determine the long-range interactions and local conformations. For the 5-nt-long, 90°-kink loop, the sequence is strictly taken from the HCV IRES domain II.DNA double-stranded template was amplified by polymerase chain reaction using Taq DNA polymerase kit (New England Biolabs Inc.). 4–10 ng DNA template, 200 µM dNTPs, 200 nM of forward and reverse primers were dissolved in 100 µL standard Taq reaction buffer. Then 0.5 µL Taq DNA polymerase was added to the system. The solution was initially denatured for 30 s at 95 °C, and started 30 cycles at 95 °C for 30 s, 50 °C for 1 min, and 68 °C for 2–3 min. Then kept the final extension at 68 °C for another 5 min. PCR product was stored at 4 °C for short time storage and at −20 °C for long terms.RNA molecules were synthesized from in vitro transcription using T7 RNA polymerase . Corresponding DNA template was added, and the experiment was conducted by following the manufacturer-recommended protocol.2+ buffer and annealed by different processes: Quenched---65 °C/5 min, 0 °C/5 min; Regular anneal---65 °C/5 min, 50 °C/30 min, 37 °C/30 min, 22 °C/30 min, and 4 °C/30 min. TAE/Mg2+ buffer contained 40 mM Tris base (pH 8.0), 20 mM acetic acid, 2 mM EDTA, and 12.5 mM magnesium acetate.ssRNA was prepared in TAE/Mg5% denaturing PAGE gel was prepared with the 19:1 acrylamide/bisacrylamide solution, 8 M urea, and TBE buffer, containing 89 mM Tris base (pH 8.0), 89 mM boric acid, and 2 mM EDTA. The gel was run at 55 °C for around 1.5 h at 650 V on Hoefer SE 600 electrophoresis system and was stained with ethidium bromide (Sigma). The major band was cut under UV light and eluted out.2+ buffer. The gel was run at 4 °C. Then stained with Stains-All (Sigma) and scanned by an HP scanner (Scanjet 4070 Photosmart).4% native PAGE gel was prepared with 19:1 acrylamide//bisacrylamide gel and TAE/MgSamples were loaded into 1% agarose gel and run in TBE buffer on a FB-SB-710 electrophoresis unit (FisherBiotech) at room temperature under constant voltage of 80 V. The gel was stained in EB and illuminated under UV light. Images were taken by cell phone.l-lysine (5 µg/ml) was added onto freshly cleaved mica substrate and was incubated for 30 s. Then the surface was washed by 100 µL H2O and dried by compressed air. Annealed RNA solution was diluted to 50 nM and a drop of 5 µL was deposited onto pre-treated mica and incubated for 2 min. 20 µL TAE/Mg2+ buffer was further added. Imaging was performed in a fluid cell under tapping mode on a Multimode AFM  with SNL-10 probe .Mica surface  was pre-treated with poly-L-lysine  to increase the adsorption of small RNA particles. 20 µL poly-−/Ǻ2 dose condition at under-defocus 8 μm to enhance image contrast.3 μL RNA solution (50 nM) was deposited on the Lacey Carbon-only grid . The grid was blotted with two pieces of filter paper  and was immediately flash frozen in liquid nitrogen-cooled liquid ethane. Images were taken on FEI Titan Krios with accelerating voltage of 300 kV under 90 e36 Totally, 470 particles were selected to go through reconstruction process using EMAN2 reconstruction package, following the standard protocol. (Wiki at: http://blake.bcm.edu). A general summary of the steps includes: (1) Import and convert CCD micrographs; (2) Pick typical particles; (3) CTF determination; (4) Remove bad particles and build particle sets; (5) Make 2D reference-free class-averages; (6) Build an initial 3D model; (7) 3D high resolution refinement; (8) Evaluate the resolution of 3D map. About 470 randomly selected particles were used to build 12 class averages, which were used to generate initial models under different symmetries, such as C3 symmetry, TET symmetry or without symmetry  to get rid of proteins. Then the template and plasmid were ligated by T4 DNA ligase (New England BioLabs Inc.), and were incubated overnight at 16 °C.Double-stranded DNA template and pET23a plasmid (GenScript Inc.) were double-digested by XbaI and StyI-HF restriction enzymes (New England BioLabs Inc.) by following the manufacturer-recommended protocol. The reaction was performed in 100 µL CutSmartEscherichia coli (E. coli) cells for transformation. The mixture was kept on ice for 30 min, and then heat to 42 °C for 90 s and put on ice for 2 min. Spread the transformed bacteria on Lysogeny Broth  (RPI. Corp.) plates and incubate overnight at 37 °C. After the colonies growing into desired size, inoculate a colony into an individual aliquot of 6 ml LB/ampicillin liquid medium and shake overnight at 37 °C. 1.5 ml bacteria solution was taken out into another 6 ml fresh LB/ampicillin liquid medium and kept shaking at 37 °C until UV absorbance was around 0.5 OD at 600 nm, measuring by a UV/Vis spectrophotometer . Plasmid was extracted using plasmid isolation kit (Zymo Research Corp.). The purified plasmid was characterized by restriction enzyme digestion and gel analysis to confirm the correct insert size.Ligation product was combined with XL1-Blue competent TM (DE3) competent E.coli cells (Life Technologies Corp.) for RNA expression, following the manufacturer’s protocol. The cell culture process was the same as described above. When UV absorbance was around 0.5 OD at 600 nm, IPTG (1 mM) was added for induction and the solution was shaken at 37 °C for 3 h.The recombinant plasmid was transformed into BL21 Star2. Then destroy the bacterial membrane by adding 100 µL phenol solution (Sigma).38 Pipet out the aqueous layer and directly deposit it into native PAGE gel or scan under AFM. To prepare samples for denaturing PAGE gel or gel purification, 10 µL NaOAc  and 200 µL ethanol were added to 100 µL aqueous layer, followed by an ethanol participation in dry ice to get rid of salts. Alternatively, the cell pellet was re-suspended in 15 ml Buffer L and sit in an ice-water bath. Cells were lysed by sonication (without phenol) with Branson Digital Sonifier (10% amplitude). Sonicating for 5 s and stop 5 s; and repeat over until total of 10 min. 1 ml lysates were centrifuged at 16,000×g for 30 min to remove the cell debris and the upper layer was diluted 40 times with TAE/Mg2+ buffer for AFM imaging.After induction, centrifuge 1 ml bacteria culture solution in a 1.5 ml centrifuge tube and remove the suspension. Re-suspend the pellet in 100 µL Buffer L, containing 10 mM Tris-HCl (pH 7.4) and 10 mM Mg(OAc)Data supporting the findings of this study are available within the article and its Supplementary Information Files and from the corresponding author upon reasonable request.Supplementary Information