As a new kind of RNA, circular RNA (circRNA) is a endogenous non-coding RNA with circular structure, which has the characteristics of universality, stability, conservatism and specificity. CircRNA can specifically bind to microRNAs (miRNAs) in the form of competitive endogenous RNA, thus directly or indirectly regulating the expression of related genes. In addition to the role of sponge, circRNA also regulates parental gene expression, transcriptional translation and protein modification; and it can be used as a biomarker to develop potential diagnosis and treatment methods and evaluate prognosis. Due to changes in dietary habits and genetic factors, the morbidity and mortality of esophageal cancer (EC) in the world are still high, and are prone to early metastasis. Although the diagnosis and treatment techniques have been improved in recent years, the early diagnosis of EC is not common, and the 5-year survival rate of patients is still very low. This article reviews the function and significance of circRNA and discusses the research progress of circRNA as biomarkers in EC. Esophageal cancer (EC) is one of the most common malignant tumors in the world, with high morbidity and mortality in China. The histological types of esophageal cancer mainly include esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), 90% of which are ESCC . The incNon-coding RNA (ncRNA) is a kind of RNA which is transcribed from the genome, does not encode protein, and can perform various biological functions at the RNA level. There are many kinds of ncRNA. At present, there are mainly long-chain non-coding RNA (lncRNA), micro-RNA (miRNA), circular RNA (circRNA) and so on. LncRNA is a kind of RNA whose transcript length is more than 200 bp. MiRNA is a kind of small RNA with a length of about 18 to 25 nucleotides. LncRNA and miRNA both belong to linear ncRNAs. In previous studies, ncRNA studies are more focused on linear ncRNAs, which shown that these linear ncRNAs have a variety of functions in physiological and pathological processes. In recent years, circRNA has attracted much attention as a potential biomarker for the treatment of different cancers . HoweverAs a new type of RNA molecule, circRNA is a single-stranded ring molecule produced by the linear precursor mRNA (pre-mRNA) in a non-classical splicing square, which is transcribed by RNA polymerase II . Its traOne of the confirmed functions of circRNA is to regulate gene expression by acting as a miRNA “sponge” . CompareIn addition to the effect of circRNA on miRNA, another function of circRNA is its interaction with proteins. Previous studies have shown that circRNAs can regulate parental gene expression by affecting pre-mRNA. Ashwal-Fluss et al. found that there are conservative muscle-blind binding sites in circRNA (circMBL) and its flanking introns, which are firmly and specifically bound by MBL . The regThe regulation of circRNA at the transcriptional level may be the general function of the intron sequence circRNAs. Studies have shown that circRNA is abundant in the nucleus, in which ci-ankrd52 is mainly enriched in the transcriptional site of its parent genes, and is related to the extension mechanism of RNA pol II. circRNA acts as a positive regulator of RNA pol II transcription, indicating the cis-regulation of non-coding introns on the transcription of their parent genes . It is rCircRNAs, similar to linear mRNAs, it can be used as a template for protein synthesis. It has been reported that eukaryotic ribosomes can initiate translation on circRNA, but only if the RNA contains an internal ribosome entry site element (IRES) . A functThe expression pattern and characteristics of circRNA  make it an ideal biomarker. Patients with EC lack effective diagnostic markers and therapeutic targets, which is part of the reason for their poor prognosis. Therefore, there is an urgent need to find biomarkers or therapeutic targets to improve the clinical prognosis of EC. At present, it has been observed that some circRNAs can be used as diagnostic and prognostic markers in patients with EC. Table Accumulated evidence shows that circRNA is a potential biomarker of EC. The expression of circ_0004771 was up-regulated in plasma and tissues of patients with EC. QRT-PCR verification and ROC curve analysis showed that circ_0004771 had higher significance and better diagnostic value, and its expression level was significantly correlated with T grade (invasion of the primary tumor range) and vascular invasion, suggesting that circ_0004771 can be used as an index to judge the prognosis . At presIn order to understand the role of circRNAs in the progress of EC, there have been a large number of research reports on circRNAs in EC. More and more studies have shown that in EC, circRNAs can be used as an oncogene or tumor suppressor to regulate the proliferation, migration, invasion, apoptosis, cell cycle, epithelial–mesenchymal transition (EMT) and radioresistance of EC.The ability to regulate cell proliferation and apoptosis is very important in tumor therapy. Many literatures have shown that circRNAs mediates EC cell proliferation and apoptosis, or interaction, and regulates cell migration and invasion.As a new category of ncRNAs, the research on the role of circRNAs in the development of EC is still in its infancy. However, more and more evidences show that circRNAs plays an important role in the biological development of EC, such as regulating cell proliferation and apoptosis. For example, studies have found that the expression of ciRS-7 is up-regulated in ESCC, and ciRS-7 contains 19 miR-876-5p binding sites. Through the action of miRNA sponge, the tumor inhibitory activity of miR-876-5p is weakened and the expression of MAGE-A family of downstream targeted tumor antigens of miR-876-5p is enhanced. It is proved that the overexpression of ciRS-7 promotes the proliferation, migration and invasion of ESCC cells . The expIn the process of EMT, epithelial cells lose polarized tissue and gain the ability of migration and invasion . The abnIt has been found that the up-regulated expression of hsa_circ_0006948 in ESCC is related to lymph node metastasis and poor prognosis. In addition, further studies have shown that hsa_circ_0006948 promoted the proliferation, migration and invasion of through sponging miR-490-3p, and induced the formation of EMT in EC cells . SimilarAt present, in the treatment strategy of EC, chemotherapy, chemoradiotherapy and esophagectomy are the main treatment methods, but due to the emergence of drug resistance, some patients would have local recurrence and distant metastasis, leading low 5-year survival rate. Acquired radioresistance during radiotherapy is considered to be the most important cause of tumor local recurrence or treatment failure. Understanding the regulatory mechanism of circRNA involved in chemoradiotherapy resistance can identify new targets to optimize treatment.The evaluation of circRNAs found that among the 3752 candidate circRNA genes detected, 57 circRNAs were up-regulated and 17 circRNAs were down-regulated in the radioresistance EC cell line (KYSE-150R) compared with the radiation-sensitive ESCC cell line (KYSE-150). Studies have shown that radioresistance EC cells are related to the abnormal regulation of circRNA. Most of their target genes are enriched in the Wnt signal pathway, indicating that these circRNAs play a regulatory role in EC resistance through the Wnt signal pathway. The differential expression of circRNA in EC, especially circRNA_001059 and circRNA_000167, may be related to the radioresistance of EC cells and affects the treatment and prognosis of EC . The latWith the continuous progress in the field of RNA, circRNAs has become a new research hotspot. In recent years, our research has deepened our understanding of circRNA, from “transcriptional noise” to functional regulatory molecules that mediate different physiological and pathological processes. Its interaction with tumor has gradually attracted people’s attention. In this review, we mainly introduce the function of circRNAs and its as clinical biomarkers for the diagnosis, treatment and prognosis of EC, and further explore the role and significance of circRNAs in EC. Other unknown circRNA functions and potential biomarkers need to be studied in the future. Although circRNAs has made some progress in other human tumors, the research on the regulation of EC by circRNAs is still in its infancy, and some specific mechanisms of circRNAs-mediated EC are still unclear. Further study on the detailed mechanism of circRNAs regulating EC may provide new insights into how circRNAs can enhance or inhibit the occurrence and development of tumor, increase the research on the pharmacodynamics and safety of circRNA in the treatment of EC, and realize the clinical application of circRNAs as soon as possible.


An HTML vignette including documentation on the tool startup and usage, executable examples, and a video demonstration, are available at: http://bioconductor.org/packages/release/bioc/vignettes/INSPEcT/inst/doc/INSPEcT_GUI.html.The abundance of RNA species and their response to perturbations are set by the kinetics rates of RNA synthesis, processing, and degradation. However, the visualization, interpretation, and manipulation of these data require familiarity with mathematical modeling and command line tools. INSPEcT-GUI is an R-Shiny interface that allows researchers without specific training to effortlessly explore how the fine kinetic regulation of the RNA life cycle can shape gene expression programs. In particular, it allows to: (i) interactively visualize gene-level RNA dynamics; (ii) refine the model fit of experimental data; (iii) test alternative regulatory models; (iv) explore, independently from the availability of data, how the combined action of the RNA kinetic rates impacts on premature and mature RNA. INSPEcT-GUI is freely available within the R/Bioconductor package INSPEcT at  P and M, the concentrations of premature and mature RNA) and the RNA kinetic rates :The RNA life-cycle is composed by three main steps - the synthesis of premature RNA, its processing into the mature form, and the degradation of the latter. The dynamics of transcripts metabolism are set by the rates governing the kinetics of those steps, ultimately setting the abundance of premature and mature RNA species , and shaThis model is implemented, with various assumptions, by different tools [cDTA , DRiLL , INSPEcTde novo the role of the RNA kinetic rates.Despite the availability of these tools, anticipating the outcome of the joint contribution of various RNA life-cycle stages can be far from trivial. For example, how would a halved RNA degradation impact on the expression of a gene? How would the latter be affected if also the rate of RNA processing were increased? A tool that enable scientists to explore and manipulate these data, while not requiring specific training on mathematical modeling or familiarity with command line scripting, is missing. To lower the barrier to the field, we developed INSPEcT-GUI, an R-Shiny interface that fully complements the analytical framework implemented in INSPEcT . INSPEcTThe RNA kinetic rates  are modeled within INSPEcT-GUI with the following analytical functions: constant, sigmoid, or impulse. Thus, one of these functions, and the corresponding parameters, has to be set for each rate.At steady state, constant functions constrain the kinetic rates to fixed values , determiRather, when at least one of the three kinetic rates is set to variable, the abundance of premature and mature RNA species is determined through the numerical solution of the system of ordinary differential equations indicated in Eq. (1). Variable rates can be described by sigmoid or impulse functions. Sigmoids are S-shaped functions described by four parameters: starting and final levels, time of transition between those, and slope of the response . Impulsek1–3. In the “user defined” mode the parameters of k1–3 functions can be freely assigned to model RNA kinetic rates and explore their impact on premature and mature RNAs. When INSPEcT-GUI is applied to RNA expression data, it infers the parameters of the functional forms assigned to INSPEcT modeling results for both steady state or time-course experiments can be uploaded within INSPEcT-GUI for an interactive visualization and for further analyses . To facisynthesis, constant processing, and variable degradation rates, while “sp” stays for variable synthesis, variable processing, and constant degradation rates. Once a gene is selected, the temporal or steady state profiles of premature and mature RNA species are plotted, together with the corresponding profiles of the kinetic rates.INSPEcT-GUI allows controlling the INSPEcT analysis framework to reassess and potentially refine the selected regulatory model. Analyzed genes are divided according to their modeled RNA dynamics, grouping together those that have the same regulatory model. For example, “sd” stays for variable p-value and Akaike information criterion. These metrics are penalized for the model complexity, and can be used for the comparative evaluation of alternative regulatory models (p-value can be used to assess whether the model under consideration adequately explains the data. These metrics are evaluated in real-time, helping the user assessing if a change in the model parameters is lowering or increasing the ability to interpret the data.All plots include standard deviation and/or 95% confidence intervals, and experimental data can be smoothed. The functional forms assigned to each kinetic rate, and the corresponding parameter settings, are reported. Moreover, goodness of fit statistics are indicated, expressed as two-tailed chi-squared test y models . MoreoveThe model fit can be reassessed and potentially improved by providing additional minimization iterations, or by selecting a different minimization algorithm [choosing among Nelder-Mead  and BFGSFinally, alternative regulatory models can be tested by changing the functional forms assigned to one or more kinetic rates, and/or by modifying the corresponding parameters. Upon changes of functional forms or their parameters, the corresponding plots and statistics are updated on real-time. This allows the user to evaluate the impact and goodness of fit of alternative regulatory models, without the necessity of explicitly controlling the corresponding INSPEcT functionalities, which are automatically exploited under the hood.The previous section illustrated how INSPEcT-GUI can take advantage of the INSPEcT analytical framework to explore or manipulate RNA dynamics of individual genes. Alternatively, INSPEcT-GUI can be used to simulate how a given combination of constant or variable RNA kinetic rates impact on the temporal profiles of premature and mature RNAs. This approach allows exploring the effect of complex transcriptional and post-transcriptional regulations, without the need of experimental data.Using the same interface described in the previous section, and depicted in The recent development of experimental and computational methods able to dissect the dynamics of RNA metabolism is allowing the study of layers of regulation that were previously hard to characterize, which are revealing the detailed molecular mechanisms at the basis of complex gene expression programs. INSPEcT-GUI is seamlessly integrated with INSPEcT modeling functionalities, covering steady state or time course studies with or without the profiling of nascent RNA. Altogether, INSPEcT-GUI facilitates the interactive visualization and manipulation of gene level RNA dynamics, empowering scientists with no specific training with the possibility of exploring the effects of the combinatorial regulation of RNA kinetic rates.http://bioconductor.org/packages/INSPEcT/. An HTML vignette including documentation on the tool startup and usage, executable examples, and a video demonstration, are available at: http://bioconductor.org/packages/release/bioc/vignettes/INSPEcT/inst/doc/INSPEcT_GUI.html.INSPEcT-GUI is freely available within the R/Bioconductor package INSPEcT at SP and MP conceived the study. SP and MF developed the software. All authors contributed discussing and writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages. Coronaviridae family, is the etiological agent of the current COVID-19 pandemic that has generated an international public health emergency. As of May 3rd, 2020, the virus has infected more than 3.3 million individuals and killed over 238,000 people worldwide . Testing for the presence of the virus is of utmost importance for containment strategies aiming to reduce dissemination of the virus and prescription of appropriate clinical practices for affected patients. However, understanding and managing the full extent of the outbreak has remained a challenge for most countries due to significant bottlenecks imposed by diagnosis1.SARS-CoV2, a member of the 2, and novel approaches are constantly being reported10. All methods require an RNA extraction step to isolate the viral genetic material before its detection. Unfortunately, RNA extraction has become a serious bottleneck for COVID-19 diagnosis around the world due to shortages in RNA-extraction kits customarily used to process patients samples. This is particularly troublesome in developing countries lacking the infrastructure and capacities to produce these kits locally. Before the kit-era, which contributed to standardize and simplify molecular biology work, several RNA extraction methods were routinely used in research laboratories around the world. RNA isolation procedures typically involve three general steps: cell lysis, separation of RNA from other macromolecules such as DNA, proteins, and lipids, followed by RNA concentration. To prevent RNA degradation, cell lysis must be conducted under conditions that inhibit RNase activity, which is abundant in many cellular compartments12. RNA separation from other macromolecules is often achieved by a combination of pH and organic solvents, such as phenol/chloroform16. RNA concentration is most commonly achieved by high salt and isopropanol or ethanol precipitation20.Early detection of infection by SARS-CoV2 relies on the efficient detection of the viral genome using RT-qPCR. Several RT-qPCR-based tests are being used in clinical settingsWe reviewed the published literature to search for procedures of RNA extraction that could potentially be used to replace commercial kits. Many different protocols and variations have been published over the years that optimize or simplify the RNA extraction process from various types of samples. We tested five types of procedures to identify an efficient procedure for extracting RNA from clinical samples that is compatible with downstream RT-qPCR analysis. Of the procedures evaluated, a simple method based in acid pH separation of RNA was found the most suitable. It can be carried out in approximately 40 min for ten samples, and is not more laborious than current methods using commercial kits. This procedure requires reagents and equipment that can be found in any standard molecular biology laboratory, thus avoiding supply chain issues. The resulting RNA can be used to detect SARS-CoV2 by standard RT-qPCR testing protocols with robust results comparable to those obtained using commercial RNA-extraction kits.For validation of the RNA extraction procedures, the RNase P target was amplified in a one-step RT-qPCR reaction, as quality control for the extraction method. As shown in Fig. 21. The nucleocapsid viral proteins N1 and N2 were amplified as viral targets, and RNase P was also amplified as a control. We analyzed 50 clinical samples: 22 were positive, 11 were undetermined, and 17 were negative according to RT-qPCR recommended by CDC21, using RNA extracted with High Pure Viral RNA Kit (Roche). Undetermined samples are described as having a viral load around the limit of detection (LOD) of the RT-qPCR method that was reported as 100.5 RNA copies/µl21. This means that the RT-qPCR method can detect 16 RNA copies per PCR reaction. The PCR test used detects 2 targets of the virus: N1 and N2. The mean Cq value for N1 target reported for sets of dilutions that are ≥ 95% positive is around 3621. Therefore we analyzed the efficiency of both extraction methods in two different groups of samples: those with Cq N1 ≤ 36 and those with Cq N1 > 36. The results for the 50 samples are shown in Table To validate the acid pH method of RNA extraction, RT-qPCR using TaqMan probes and primers recommended by the CDC were usedImportantly, the processing time and laboriousness of the acid-pH method is similar or less than that of High Pure Viral RNA Kit (Roche) method. A detailed scheme of the method is shown in Fig. 22 are intended for RNA extraction from tissue, cultured cells, and cell-associated virus. Therefore, the first step of these protocols is centrifugation with subsequent lysis of the cell pellet. However, we need to recover free viral particles in solution, which do not sediment after routine centrifugation at 15,000 g. For this reason we used the uncentrifuged sample directly mixed with lysis buffer, with subsequent precipitation of viral RNA in the whole mix volume. Using uncentrifuged sample is the key step for efficient RNA recovery because when centrifuged sample was used in preliminar tests, Cq values were much higher than those obtained with High Pure Viral RNA Kit (Roche). Second, the acid pH method uses the anionic detergent Sodium dodecyl sulfate (SDS) that can lyse cells and viral coats through disruption of noncovalent bonds in proteins causing them to lose their native conformation12. Third, low pH and high concentration of salt make possible the selective recovery of RNA. Within the pH range of 5.5 to 6.0, RNA degradation is minimized22. RNA phosphodiester bond is more stable at acidic than alkaline pH, where it is susceptible to alkaline hydrolysis at pH greater than 623. Acid hydrolysis can only occur at pH lower than 224. Moreover, DNA and RNA have different solubility at different pH, mainly due to the 2′ hydroxyl group of RNA, which increases the polarity of this nucleic acid26. Therefore, it is essential to adjust the Lysis Buffer to pH 5, as described in materials and methodsHere we tested several kit-free RNA extraction methods compatible with RT-qPCR analysis and selected one simple procedure based on RNA extraction using acid pH. We validated this method using 50 clinical samples with results comparable to those obtained with commercial kits. There are three key aspects of this method that must be pointed out. First, the acid pH-based methods that we reviewedrd week of April 2020 for routine detection of SARS-CoV2 in clinical samples.It is worth mentioning that all of the samples that changed their report had Cq values that were around the cutoff value of 40. These changes occurred in both directions, meaning that some Cqs increased and some Cqs decreased. It would have been very clarifying to perform triplicated RNA extractions, in particular for undetermined samples, whose viral load is around the detection limit. Because of the above exposed information we consider the acid pH method robust and reliable. In fact, it is currently being used in our diagnostic laboratory since the 3The RNA extraction procedure with acid pH described here has many advantages over commercial kits to test for SARS-CoV-2 in the context of the current pandemic. This experimental procedure utilizes low cost reagents and equipment that can be found in standard molecular biology laboratories. The cost of extraction is a critical issue in most clinical laboratories, and the cost of our in-house method is around ten times lower than extraction kits. Moreover, DNase treatment is not necessary because SARS-CoV-2 detection is not altered in the presence of DNA. In fact, residual DNA may serve as the template for RNase P gene amplification. Because of current environmental concerns, we would also like to highlight the lower plastic contamination generated by this in-house method. Column-based extraction kits use several disposable tubes per sample, columns, bottles of buffer solutions, and plastic bags. Our in-house extraction method is by far, much more environmental friendly; it requires only two Eppendorf tubes per sample. Finally, our in-house method is comparable in hands-on time to commercial kits: it can be carried out in approximately 40 min for a set of 10 samples. However, it is important to mention that additional care must be taken in handling to avoid cross-contamination between samples.In conclusion, the RNA extraction procedure with acid pH described here is an excellent alternative to commercial systems to test for SARS-CoV2. Our results support a new method for RNA extraction from swab samples that can be used to detect SARS-CoV2 by standard RT-qPCR testing protocols. This procedure can be a helpful alternative for laboratories facing supply-chain disruption and commercial kit shortages.Two types of biological samples were used. For preliminary evaluation of the RNA extraction methods we used saliva samples obtained from two asymptomatic volunteers. Saliva is routinely collected for the initial assessment of viral infection. Two saliva samples were obtained from each volunteer and at least three independent RNA extractions were performed from each sample, obtaining a minimum of six RNA preparations to test each experimental procedure. For validation of the RNA extraction method selected, we used nasopharyngeal swabs in Universal Transport Medium (UTM). Swabs were obtained from 50 patients that attended the outpatient service of Red Salud UC-CHRISTUS  because of suspected coronavirus infection. Only one sample was obtained per patient: one portion of the sample was extracted using the High Pure Viral RNA Kit (Roche), and another portion of the same sample was extracted using the acid pH method. Informed consent was obtained from all participants and/or their legal guardians. Samples were processed in the Laboratory of Diagnostic Microbiology of the same institution. All methods were performed in accordance with the relevant guidelines and regulations. All procedures were approved by the Ethics Committee of the Pontificia Universidad Católica de Chile.18. First, 800 µL of TRIzol were added to 200 µL of sample and vortexed briefly. Then, 200 μL of chloroform were added, vortexed, and centrifuged at 12,000 g for 10 min at room temperature. The aqueous phase (600 μL) was recovered in a clean tube containing 600 μL of isopropanol. The tube was mixed by inversion and incubated at room temperature for 10 min. The tube was then centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was washed with 500 μL of 70% ethanol, centrifuged at 7,500 g for 5 min at 4 °C and the supernatant was discarded. The pellet was dried at room temperature for 10 min and resuspended in 25 μL of RNase-free water by incubating at 37 °C for 10 min.(1) TRIzol. The standard TRIzol-based method was evaluated28. Based on the procedure described by Plante et al. (2010)27 and Svec et al. (2013)28, a 200 μL aliquot sample was centrifuged at 12,000 g for 30 s at room temperature. Then, 2.5 μL of supernatant were added to 47.5 µL of a 1 mg/mL BSA solution (1:20 ratio), vortexed for 30 s and kept on ice or at − 80 °C until further use.(2) BSA-based method. Previous reports show that BSA has positive effects on RT-qPCR results when added to samples in the presence of inhibitors29. Based on the methods described by Heath (1999)22, Sambrook and Russell (2001)12, and Chomczynski and Sacchi (2006)14, 300 µL of pH 5 Lysis Buffer  were added to 200 µL of uncentrifuged sample and mixed by pipetting three times. Then, 150 µL of Precipitation Buffer  were added and mixed by inversion 10 times. Samples were incubated on ice for 5 min and centrifuged at 15,000 g for 3 min at room temperature. 600 μL of the supernatant were transferred to a clean tube containing 600 μL of isopropanol and incubated for 10 min at room temperature. A new centrifugation step was made at 15,000 g for 5 min at room temperature. The supernatant was removed, and the pellet washed with 300 μL of cold 70% ethanol and centrifuged at 15,000 g for 3 min at room temperature. Supernatant was discarded and tubes were inverted in paper towel. The pellet was dried, leaving the tubes open for 10 min. Finally, the pellet was resuspended in 50 μL of nuclease-free water pre-warmed at 70 °C. NOTE: If the buffer is stored for later use, it precipitates at 4 °C, so it needs to be heated for 5 min at 60 °C for its use.(3) Acid pH-based method. Under acidic pH, RNA can be separated from DNA and other molecules due to the differential polarity given by its hydroxyl groups, which maintains it in solution30, 50 µL of the sample were directly heated at 98 °C for 5 min and cooled at 4 °C. Then 19 µL of the sample were mixed with 1 µL of BSA (20 mg/mL) and kept on ice for immediate use or at -80 °C for later use.(4) High temperature-based method. Based on the method described by Fomsgaard and Rosenstierne (2020)31.(5) Direct use of the samples. An aliquot taken from the original sample was directly used to perform RT-qPCR analysisThe following experimental procedures were tested in this study. Saliva samples were centrifuged before taking an aliquot of supernatant for processing as described below.The 50 nasopharyngeal swabs used for the validation of the RNA extraction method selected, were extracted using High Pure Viral RNA Kit (Roche) according to instructions provided by the manufacturer. This RNA extraction method was considered as the gold standard for comparison purposes, and it is based in capture of RNA using columns with silica filters.32. RP1-F: AGATTTGGACCTGCGAGCG, RP1-R: GAGCGGCTGTCTCCACAAGT, and RP1-probe: TTCTGACCTGAAGGCTCTGCGCG. The RNase P gene is used as an internal control because many copies of it exist in the human genome, and it is readily detectable. The source of RNase P comes from the human cells that are present in every sample used. It is assumed that if human nucleic acids were extracted to detect the human gene RNase P, viral nucleic acids were also successfully extracted. The RNase P target is also amplified as a quality control for the extraction method and to corroborate the absence of PCR-inhibitors in the sample.For preliminary evaluation of RNA extraction procedures, we used RT-qPCR against the human RNAse P gene with primers and a Taqman probe previously describedFor RT-qPCR 5 μL of RNA from saliva samples, 2 μL of RNase-Free water, 1 μL of each RNase P primer, 1 μL of TaqMan RNase P probe and 10 μL of 2X TaqMan Fast Universal PCR Master Mix (Applied Biosystems), were used in a final reaction volume of 20 μL performed in a StepOnePlus Real-Time PCR System (Applied Biosystems).21. Two viral targets were amplified: the nucleocapsid viral proteins N1 and N2. Primers and probe for N1 were N1-F: GACCCCAAAATCAGCGAAAT, N1-R: TCTGGTTACTGCCAGTTGAATCTG, and N1-probe: FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1. Primers and probe for N2 were N2-F: TTACAAACATTGGCCGCAAA, N2-R: GCGCGACATTCCGAAGAA, and N2-probe: FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1. Primers and probe for RNase P were RP2-F: AGATTTGGACCTGCGAGCG, RP2-R: GAGCGGCTGTCTCCACAAGT, and RP2-probe: FAM–TTCTGACCTGAAGGCTCTGCGCG–BHQ1. A one-step RT-qPCR reaction was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems). Cutoff points for Cq values  required to decide whether a result is COVID-19 positive or negative were those specified by CDC as follows. To report a positive result, both viral targets N1 and N2 must be Cq < 40. To report a negative result both viral targets must be Cq ≥ 40. If one of the viral targets is Cq < 40 and the other is Cq ≥ 40, the result must be reported as undetermined. The RNase P target must be Cq ≤ 35.For validation of the selected RNA extraction procedure, RT-qPCR using Taqman probes and primers recommended by the CDC was usedMean Cq values obtained through both methods for each target gene were analyzed in pairwise comparisons using a paired Student’s t-test. The analysis was performed using GraphPad Prism 8 software.


RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA’s resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme  that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson–Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics. A fundamental feature of the proteins and nucleic acids is their homochirality. These natural biopolymers are composed of only one optically active ‘isomer’: L- amino acids or D-nucleotides. Although the origin of homochirality in biological macromolecules is not fully understood, it is assumed to be essential to ensure the high specificity and fidelity of the recognition mechanism between them. The stereospecificity of enzyme (protein) or ribozyme (nucleic acids) catalysed reactions is the foundation of the homochiral world.5dA and D-poly(rU) have been suggested, but no hybridization of L-dU oligomer and D-poly(dA) was observed [in vitro that selected L-DNA aptamers bind to a D-RNA target [Changing chirality is a strategy to overcome the low chemical stability of the phosphodiester bond of D-RNA in order to build more effective RNA-based tools. It is known that mirror-image ribonucleic acids consisting of L-ribose instead of D-ribose are resistant to hydrolysis with ribonucleases. Also L-DNA, where D-deoxyribose is substituted with L-deoxyribose, are resistant to nucleases ,2. Shortobserved . Also noobserved . A few yobserved . RecentlA target , but WatA target ,12.The intense interest in enantiomeric nucleic acids observed in the last 40 years has dampened recently because the finding that L- and D- oligonucleotides do not form a regular double helix with Watson–Crick base pairs .Recently, for the first time, some of us have shown that mirror image hammerhead ribozymes (L-ribozyme) and L-DNAzyme cleave the complementary D-RNA substrate of reciprocal chirality at the predetermined site ,15. On tChemically synthesized RNA oligonucleotides were from IBA (Germany) or ChemGenes (U.S.A.). The 14-nt synthetic D- and L-RNA oligomers with the sequence 5′CUUCAAGUCCGCCA3′ were labelled with fluorescein at the 5′ end. The D-HH (D-Rz), L-HH (L-Rz) hammerhead ribozymes were 33-nt long with the sequence 5′UGGCGCUGAUGAGGCCGAAAGGCCGAAACUUGA3′. Nucleotides of L33 at positions 1-6 and 29-33 for parallel complex were methylated at 2′OH. D-anti HH Ribozyme 5′UGUCAGCUGAUGAGGCCGAAAGGCCGAACCAGCGG3′ was synthesized and labelled with FAM.2 concentrations. Hydrolysis products were separated with 20% polyacrylamide gel electrophoresis (PAGE) in the presence of 7 M urea in 0.09 M Tris-borate buffer at pH 8.3. The gels were exposed to a Fuji Film FLA 5100 phosphoimager using the manufacturer’s (Fuji) BCIP/NBT Liquid Substrate System (Sigma), and bands were quantified using the ImageQuant software from Molecular Dynamics.The activity of the D- and the L-hammerhead ribozymes with labelled L- and D-RNA targets were measured in 10 μl reaction volumes containing 50 mM Tris-HCl at pH 7.5 at 37°C. The RNA substrate and L- or D-Rz ribozymes were denatured for 2 min at 73°C and cooled to 25°C at a rate of 1°C/min. The reactions were done at 0 and 25 mM MgClCD spectra were recorded on a JASCO J-815 spectropolarimeter. The L- and D-oligonucleotides were dissolved in 50 mM NaCl, 20 mM Tris-HCl pH 7.5 buffer to achieve a concentration of 3.0 μM. All samples were denatured at 90°C for 5 min and then slowly cooled to room temperature before data collection . The spe2. Equimolar amounts D-RNA and L-Rz (L33) was dissolved in 150 mM NaCl and 10 mM sodium phosphate pH 6.8. The solvent was H2O/D2O . The samples were annealed by heating at 90°C for 5 min and then slowly cooled to room temperature. The 3 mm thin wall tubes were used with a final sample volume of 200 μl. The water signal was suppressed by excitation sculpting with a gradient pulse. Spectra were processed and prepared with TopSpin 3.2 Bruker Software.All NMR spectra were acquired on a Bruker AVANCE III 700 MHz spectrometer, equipped with a QCI CryoProbe. D-RNA, L-Rz and their equimolar amounts (3 μM) complex 3 were dissolved in buffer 150 mM NaCl, 50 mM Tris-HCl pH 7.5 with 10 mM MgClhttp://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). We incorporated the three-way junction motif covering the catalytic center of the ribozyme (PDB: 3ZP8). RNAfold generates up to ten structure models and we selected only the model with the lowest total energy. Visualizations of the molecular models were done in PyMOL (http://www.pymol.org). Finally, the linker was removed, and the structure was stored. The heterochiral model L/D we got from D/D structure with the X-Plor program ver. 2.21 [The 3D structures of homochiral and heterochiral complexes of RNA hammerhead (Rz) ribozyme (33-mer) and RNA oligonucleotide (14-mer) were constructed based on the 3D crystal structure of the catalytic center of hammerhead ribozyme (3ZP8) with RNA Composer . The 14 er. 2.21 . The parer. 2.21 . FurtherGeometry of the D/D and D/L duplex models were of heterochiral complex. Correctness of the centre chirality was evaluated by a data processing validated with RCSB MAXIT (MAcromolecularEXchange Input Tool), covering geometry of covalent bond and chirality centres. The two other models L/L and L/D were obtained by changes of values of the Z axis from D/D and D/L, respectively.The fours starting structures (models) of DL, LD, LL and DD were built using SYBYL and UCSF Chimera ,22. In ttleap module of Amber 18 to prepare the topologies and coordinates for simulations. We utilized the AMBER ff99bsc0χOL3 force field for D-RNA and its modified version for L-RNA [OL3 to account for L-ribonucleotides involved corrections of dihedral angles (γ and χ). Each of the structures was solvated with TIP3P water molecules in a truncated octahedral box with minimal distance of 10 Å between solute and the box border [+ cations and NaCl excess salt was added to obtain the concentration of 150 mM by using Joung and Cheatham ion parameters [2 applied to solute atoms and finally without any restraints. Then, at the heating stage, the temperature was gradually raised to 300 K within 80 ps followed by 20 ps of equilibration while solute atoms restrained with harmonic potential of 15 kcal/(mol·Å2). Next, the restraints were gradually released in four rounds  of 100 ps each. After that, two sets of MD simulations were carried out: one set of entirely unrestrained simulations and a second set with small harmonic potential of 0.5 kcal/ mol·Å2 applied to base heavy atoms of residues involved in Watson–Crick hydrogen bonding between heterochiral strands, to preserve these bonds and to allow the sugar-phospate backbone to adjust to it. The long-range electrostatics interactions were calculated using the particle mesh Ewald method, periodic boundary conditions, and the non-bonded cut-off set to 9 Å. The covalent bonds were constrained using SHAKE, and the integration time step was set to 2 fs. The Langevin thermostat with collision frequency 2.0 ps −1 was used to control the temperature and the Berendsen barostat was used to enforce a constant pressure simulation. MD simulations were performed with the pmemd module of AMBER 18 on CUDA Device (Ge Force RTX2080). The data generated from MD were analysed using the CPPTRAJ module of Amber Tools [MD simulations were performed with the AMBER18 package . We usedor L-RNA ,25. The x border . The sysrameters . The soler Tools .Recently, we have shown that L-hammerhead ribozyme  hydrolysed a D-RNA target . The che2  and migrates slowly . One can2 . One shoTo get a deeper insight into the conformation of the heterochiral complexes, we measured the CD spectra of L/D-ribozymes with L/D-RNA substrates . The L- 1H NMR spectra. They show spectra of single-stranded D-RNA or L-RNA . For the complex with parallel stems new imino-proton signals in the region of 13–14.5 ppm were also observed: however, their pattern and intensity were different than those obtained for anti-parallel heterochiral catalytic complex . It suggests that the nature of the hydrogen bond interactions is different for the parallel and anti-parallel complex.To confirm the formation of heterochiral complexes and evaluate the nature of hydrogen bonding interactions in these complexes, we recorded the or L-RNA  without or L-RNA . This isHaving experimental data on the specific hydrolysis of RNA with catalytic nucleic acids (zymes) in a heterochiral manner  as well To understand how this complex is self-assembled, we developed a new approach to find the possible configuration of L-nucleosides. We started with the right-handed homochiral complex of D-RNA/D-ribozyme, called the D/D RNA model A. The moZ-coordinates of the right-handed (D-RNA/D-Rz) homochiral complex multiplied by -1 leads into the left-handed homochiral L-RNA/L-Rz complex , one can clearly see that Watson-Crick type base pairs within the heterochiral complex were very similar to those of the homochiral complex with some irregularities  and the chi angle values of the calculated heterochiral complex. The violin plot analysis of the molecular dynamics simulation of the D-RNA/L-Rz complex with positional restraints shows that all nucleotides of the L-Rz adopt C2′-endo sugar pucker (S-type) with pseudororation phase values (P) of approximately 1800 . This suormation . These rThe molecular structures can be chiral, and have mirror-image isomers of the opposite chirality. These isomers are called enantiomers or optical isomers, because they rotate plane-polarized light either to the right (D) or to the left (L). The building blocks of the life are homochiral, which means that naturally occurring amino acids, protein, sugars, and nucleic acids exist as only one chiral form. If these building blocks had mixed chirality, molecular chaos would ensue, and life would not be possible.In principle, mirror-image or looking-glass versions of chiral molecules should work in the same way as original ones, but they are resistant to attack by viruses or enzymes that have not evolved in a glass-looking world. That suggestion opens the door to a new dimension of mirror-image molecular biology with broad applications in diagnostics and therapeutics. During last 40 years there have been many studies showing the formation of complexes of two strands of opposite chriality; however, the conclusions drawn from their structure are not clear .In this work, we were interested in the structure of mirror image catalytic RNAs (L-RNA) and its interaction with RNA of reciprocal chirality (D-RNA). We concentrated on chemistry of the ribozyme because a specific RNA  hydrolysis is based on two important premises: (i) the tertiary structure of the active center should allow the substrate to adopt a precise conformation and formation of a transient active structure, and (ii) the active site of both the homochiral and heterochiral complexes should be very similar to perform the same specific catalysis.The D-hammerhead ribozyme activity against various D-RNAs (D-targets) has been studied recently ,15,30–32+2 ions, specific hydrolysis takes place. One can also see that L-HHRz binds to D-anti-HHRz . After binding to the substrate to L-Rz, no change of the intensity of the CD signals was detected indicating that in this case the heterochiral complex did not form C.Analysis of NMR spectra fully confirmed the formation of heterochiral L-Rz/D-RNA complex. New imino signals in the range of 13.5–14.5 ppm were observed in NMR spectrum when L-Rz and L-RNA/D-RNA was annealed .We have also consider parallel complex formation of D-RNA target with L-HH ribozyme. However NMR spectra of such complex  did not show down-field imine protons , and therefore did not support a parallel helices 1 and 3.Our 3D structure of the heterochiral models evidently shows that Watson-Crick base pairs between the ribozyme and substrate can be form although their geometry are slightly different C,D.It is interesting that using standard, well-known modeling tools and simple mathematical operation one can get antiparallel heterochiral Watson–Crick base pairs, that have geometry that is very similar with the canonical Watson–Crick base pairs C,D. As o5)A with D-poly (U), [To obtain deeper structural insight into the heterochiral complexes, we studied the molecular dynamics simulations of the complexes. These results suggest that the Watson-Crick like type pairing formed by strands of reversed chirality in helixes 1 and 3, are induced and stabilized by the homochiral helix 2 of the ribozyme. Such interactions maintain the active center of the catalytic RNA. Pairing between flanking L-nucleosides of ribozyme and D-RNA substrate is possible due to changes within sugar puckers and syn-anti conformation changes, which occur regularly every second nucleoside . Our resoly (U), . That oboly (U), .Click here for additional data file.


RNA, the transcriptional output of genomes, not only templates protein synthesis or directly engages in catalytic functions, but can feed back to the genome and serve as regulatory input for gene expression. Transcripts affecting the RNA abundance of other genes act by mechanisms similar to and in concert with protein factors that control transcription. Through recruitment or blocking of activating and silencing complexes to specific genomic loci, RNA and protein factors can favor transcription or lower the local gene expression potential. Most regulatory proteins enter nuclei from all directions to start the search for increased affinity to specific DNA sequences or to other proteins nearby genuine gene targets. In contrast, RNAs emerge from spatial point sources within nuclei, their encoding genes. A transcriptional burst can result in the local appearance of multiple nascent RNA copies at once, in turn increasing local nucleic acid density and RNA motif abundance before diffusion into the nuclear neighborhood. The confined initial localization of regulatory RNAs causing accumulation of protein co-factors raises the intriguing possibility that target specificity of non-coding, and probably coding, RNAs is achieved through gene/RNA positioning and spatial proximity to regulated genomic regions. Here we review examples of positional cis conservation of regulatory RNAs with respect to target genes, spatial proximity of enhancer RNAs to promoters through DNA looping and RNA-mediated formation of membrane-less structures to control chromatin structure and expression. We speculate that linear and spatial proximity between regulatory RNA-encoding genes and gene targets could possibly ease the evolutionary pressure on maintaining regulatory RNA sequence conservation. Many mechanisms for RNAs to regulate gene expression in the cell nucleus involve recruitment of regulatory protein factors, including chromatin modifiers and polymerase recruiters that affect the transcriptional output of genes . Non-codMost regulatory proteins, such as transcription factors, enter nuclei after their synthesis through pores to eventually interact specifically, or broadly, with chromatin regions. RNAs, on the other hand, emerge from their encoding gene at a defined genomic and spatial position. Therefore, the position of origin for a regulatory RNA and the spatial genome neighborhood are arguably critical in defining target gene specificity for such transcripts. Particularly RNAs with shorter half-lives might exert roles in gene expression regulation restricted to nuclear regions in immediate vicinity to their gene locus encompassing neighboring genes in cis or loci brought into close proximity in the spatial genome structure through chromatin looping or DNA contacts in trans.Once a polymerase engages in processive transcription, the synthesized, nascent transcript appears from the encoding gene locus. The RNA molecule grows in length with continuing transcription along the gene but stays tethered to chromatin by the polymerase until 3’ RNA cleavage followed by polyadenylation releases the RNA molecule . At a poNascent RNAs emerge as groups of multiple molecules in a short time window, so-called transcriptional bursts, which results in the amplification of available chromatin-tethered RNA binding sites and of protein recruitment to a given locus in that moment. Bursting, or discontinuous transcription, of active genes describes the temporal gating of transcription initiation into time windows of a few minutes and the interspersion of such “on” states with longer periods of promoter inactivity, or so-called “off” states . First iIntriguingly, a recent preprint applying RNA-FISH combined with expansion microscopy revealed that transcripts after completion of synthesis and chromatin dissociation remain locally restricted within sub-micron distances from gene loci for some time . The absOnce regulatory transcripts escape localization to the vicinity of their encoding gene locus, the target gene search is expected to rely primarily on differential affinities to for example different DNA sequences, chromatin modifications and other chromatin-associated factors, comparable to regulatory proteins entering the nucleus through pores.In summary, transcriptional bursts locally increase RNA concentration throughout the time of synthesis when RNA is tethered to chromatin and likely longer in an untethered state in the immediate vicinity of encoding genome loci. As a consequence, a high density of locally confined single-, double-stranded and structural RNA motifs presents itself at transcription units to concentrate and position nucleic acid-binding proteins with gene regulatory functions within the three-dimensional genome structure . Indeed,The information of a single allelic genome motif can therefore be locally amplified in the orders of 10–100-fold when converted into RNA during a single transcriptional burst. However, whether the regulatory potential of nascent RNA is realized might depend on factors that, besides transcription and degradation kinetics, include the sequence and structural features of the RNA, RNA modifications and, importantly, linear or spatial proximity to potential target loci. In the following paragraphs we revisit a few illustrative examples of regulatory RNAs and their effects on the transcriptional output of genes encoded in close distance on the same chromosome or in proximity either through chromatin looping or gene positioning to nuclear bodies.Xist, which forms RNA clouds covering exclusively the inactive X chromosome was the first regulatory lncRNA found to display genomic positional conservation across species (A case in point is illustrated by lncRNAs whose genomic position relative to neighboring genes is conserved (synteny) . The cla species , 1992. T species . Most pc species . Their e species .Evx1-as illustrates a possible sequence of steps that leads to the upregulation of Evx1 target transcription in cis. Evx1-as pcRNA and Evx1 coding transcripts are co-expressed in the primitive streak of mouse embryos . Estrogen treatment increases the number of cells, i.e., the burst frequency, that express eRNAs and the corresponding, estrogen-responsive target genes Foxc1 and P2ry2. Co-localization of eRNA and nascent target gene RNA spots increases from less than 5% in unstimulated cells around 5-fold to 25% after estrogen treatment  and its interactions with eRNAs serves as one mechanistic example of how regulatory RNAs contribute to target gene regulation. YY1 binds to active enhancers and promoters and forms dimers to stabilize DNA looping . DiffereFoxc1 upon co-localization with its enhancer and eRNA, genes close to large nuclear speckles containing the non-coding RNA Malat1 are subject to an increase in burst size and amplify their transcriptional output. After heat shock, Hsp transgenes and endogenous genes are induced at the same time irrespective of whether the gene is speckle-associated or not. However, cells with the Hsp gene positioned in the vicinity of nuclear speckles surpass cells whose heat-inducible gene is apart from speckles in signal intensity and size of nascent RNA spots. The boosted transcriptional response at speckles correlates with lower exosome activity and larger foci of elongating RNA polymerase II  The locations of these sites are biased toward the 5’ end of the Malat1 molecule, the RNA part transcribed first and therefore with the most long-lived chromatin association. (iii) Malat1 transcription is characterized by one of the largest transcriptional burst sizes  A Mst sizes . The temMalat1, non-coding RNA Neat1 triggers paraspeckle formation co-transcriptionally (Similarly to tionally . Increastionally .Parameters of single-cell and locus-specific transcription, transcript length, RNA decay and diffusion rates all influence the time of chromatin association for nascent, regulatory transcripts and are possibly of equal importance to the structural and motif content of the RNA in order to regulate proximal genes. We speculate that a large burst size combined with increased protein-binding motif occurrence toward the 5’ ends of RNAs is one solution in the parameter space to locally concentrate protein factors with RNA affinity for subsequent, site-specific regulation of nuclear processes. However, the act of transcription itself, independent of an increased abundance of specific RNA motifs, can affect the expression of proximal transcription units to some extent. Indication of regulatory RNA function with little sequence requirements is suggested by the correlation in expression of neighboring genes . FurtherH2b RNA was found capable to induce subnuclear structures resembling histone locus bodies and RNA from a β-globin minigene to assemble splicing speckle components into nuclear puncta (In a model in which co-transcriptional chromatin decoration with RNA overcomes barriers for transcription initiation or repression of proximal genes, protein-coding transcripts cannot be categorically excluded from regulatory roles commonly assigned to non-coding RNAs. Results from studies applying enhancer screening, followed by CRISPR-Cas9 manipulation, or analysis of gene expression levels associated with sequence variation in regulatory regions revealed protein-coding gene promoters as potent distal regulatory elements . The duar puncta . Chromosr puncta  in combir puncta  will proAll authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection. The function of RNA molecules is dependent on their sequence, but also on their structure. The great flexibility and dynamism of RNA folding underlies the versatile features of RNA elements. RNA structure is maintained by the widespread establishment of hydrogen bonds between different nucleotides, yielding a set of loops and stems that define the overall folding of the molecule. These help generate complex conformations, such as helices duplexes, triple-stranded structures and loop-loop connections. Ultimately, they allow for intricate networks of RNA-RNA interactions, the so-called interactome, which generates dynamic, high-order RNA structures . These nViral RNA genomes are compact entities with the capacity to perform different functions during the infective cycle, as well as carrying protein-coding information. Such genomic plasticity confers significant adaptive capacity, allowing new hosts and molecular contexts to be explored, thus, promoting the emergence of new diseases. Given their high mutation rates and population sizes, the evolutionary capacity of RNA viruses favors the appearance of variants that are resistant to the host immune system and to treatment. This is achieved by the viral genome, increasing its sequence variation without disturbing functions essential to the execution of the viral cycle . RNA virHepacivirus, family Flaviviridae. HCV was discovered three decades ago as the major causal agent of post-transfusion non-A, non-B hepatitis . Th. Th39]. The most widely accepted model of HCV IRES-mediated translation initiation can be divided into three well-defined steps ,41. In t2+ concentration . Th. Th83]. Different high resolution microscopy studies have provided a dynamic view of the IRES-80S complex ,62. The Translation initiation in HCV is, thus, a dynamic and highly controlled process dependent on structural units that operate in a coordinated fashion. Such dynamism may also favor the existence of different pathways for overcoming adverse environmental conditions.High-order RNA structures affect translation initiation, elongation and termination. In particular, mRNA circularization increases translational efficiency by encouraging ribosome recycling and providing protection against the action of exonucleases . In viraHCV RNA has acquired a sophisticated translational control system. The molecular mechanism involves three differently evolved functional regions A: (i) Sucis via the transfer of the translation machinery from the 3′ end of the genome to the IRES, especially during the termination stage of each translation round.The acquisition of a circular isoform by the HCV genome during translation initiation is enhanced by the likely oligomerization of the polypyrimidine tract-binding (PTB) protein, which binds to both the IRES and the 3′X tail ,90,91. FThe 3′X tail - a 98 nt-long region showing strong sequence and structure conservation  - was idA long-range RNA-RNA interaction that seems to be required for efficient HCV translation has also been reported  100]. T. T100]. HCV replicates in the cytoplasm once viral protein levels are adequate. Actively translating viral RNA molecules must be transferred to the replication complexes, which contain non-structural viral proteins embedded in the endoplasmic reticulum. This supramolecular complex generates a membranous web that provides a suitable microenvironment for replication ,109. In cis, both as binding platforms for cellular and viral proteins and as organizers of long-distance RNA-RNA contacts  . S. S116]. HCV RNA circularization during viral replication is also stabilized by the long-range interaction established between the apical loop of domain II in the IRES, and the DLS motif in the 3′X region B 21]. I. I21]. IThe control of HCV replication was associated with the 3′UTR for a long time. However, it is now known that the 5′UTR and the coding region contain important elements for viral RNA production C.In addition to the evident role of the 5′UTR in viral translation, it promotes a significant enhancement of replication efficiency. Domains I and II at the very 5′ terminus of the HCV genome  are indicis-acting replication domain , which improves the dimerization yield, and by the IRES at the 5′ end, which exerts an inhibitory effect on dimerization even in the presence of enhancer elements . The higVirion assembly in HCV is a complex process that relies strongly on the interaction of the RNA genome with the capsid - or core - protein. It also requires the coordinated action of viral proteins and host factors . To datThe HCV assembly process appears to be spatially associated with lipid droplets, to which the core protein is linked . InteracThe 3′X structural signal is not the only actor required for efficient HCV assembly. In contrast to the many RNA viruses that use large, stable RNA structural elements with high-affinity for the core protein as the encapsidation signal, HCV seems to takes advantage of multiple domains with low affinity for the core protein as a means of presenting the intact, full-length genome to the assembly complex D 140]. . 140]. Scis. It is especially important in viruses lacking the cap structure , the polyA tail  or both . In good agreement with the HCV model, the 5′-3′ end communications in other RNA viruses involve elements critical for translation and replication, making the closed-loop topology indispensable for viral fitness. Since translation and replication are mutually exclusive, it is also likely that circular isoforms operate as elements that manage the transitions between different stages of the viral cycle.Viral genome circularization is not exclusive to hepaciviruses. Indeed, circular RNAs are represented in all kingdoms of life. In RNA viruses, however, the ubiquitous circularization process acquires new importance, acting as a regulatory tool in In some cases, such as in polioviruses and reoviruses, RNA genome circularization occurs via a protein-protein bridge ,145. In gag gene at the 5′ end of the open reading frame with the 3′ terminal U3R region [Retroviral replication involves a reverse transcription step with two obligatory DNA strand transfers; these are needed to generate the complete full-length RNA genome, which is then packaged. To facilitate these strand transfer events, the virus uses RNA circularization dependent on RNA-RNA interactions ,148. In R region ,148. In In flaviviruses, end-to-end genome connections rely on at least three specific interactions involving complementary sequence motifs located at essential domains at both ends of the viral RNA B. The paThis brief overview reflects the universality of the circularization process in RNA viruses and its value in controlling the viral cycle. It is noteworthy that circular isoforms emerge under certain stimuli and may coexist with the linear form. Preserving the balance between different conformations helps ensure viral fitness and propagation.Protein coding-information represents only a small portion of the genetic information carried in the genome. Viral RNA genomes use an information coding system involving structural genomic domains that overlap the protein-coding system. These domains express their information by establishing a dynamic network of long-distance RNA-RNA interactions - the interactome - and the recruitment of cellular and viral factors. It is this network that governs the execution of the viral cycle. In the case of the HCV RNA genome, it seems clear that the 5BSL3.2 structural domain within the CRE, located at the 3′ end of the ORF, plays a central role in the management of the RNA-RNA interactome, and therefore, in the fine regulation of the viral cycle. During the early phase of infection, the RNA genome mainly functions as mRNA, and is therefore, dedicated to translation. However, the recruitment of viral RNA polymerase by the structural domains at the 3′ end of the genome, and the consequent modification of RNA-RNA interactions, leads 5BSL3.2 to repress IRES translation initiation. The 5BSL3.2 domain also promotes replication, increasing the number of viral genomes and favoring the formation of genomic RNA dimers. These replication competent-conformers remain, however, incompetent for packaging. The dissociation of the dimers yields competent forms for translation and consequent packaging.Exploring the interactome of other related RNA viruses and their functionality should provide further information on the regulation of the viral cycle and provide a means of identifying potential targets for the development of antiviral strategies. It should be anticipated that the lack of structural RNA domain conservation among different viruses means that different RNA viruses, even when closely related, will use different strategies involving different RNA domains to achieve the goal of regulating the essential steps of their infective cycle.


The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively. Enterovirus belongs to the family of Picornaviridae, and they are classified into 15 species octane (DABCO)  ref. D2, 780-2), and stored in the dark at 4 °C overnight.Before imaging the cells with the confocal microscope, we stained the nuclei of the cells with 4′,6-diamidino-2-phenylindole (DAPI)  diluted 1:40,000 in 1x PBS for 5 min at RT, mounted on microscopy slides (Thermo Scientific) with in-house-made Mowiol-Dabco  and Olympus Immoil-F30CC immersion oil. The lasers used were a 405 nm multiline diode laser, a 488 nm argon laser, and 543 nm and 633 nm HeNe lasers. The following software settings were used in image acquisition: unidirectional scan mode, dwell time—4.0 µs/pixel, image size—640 × 640 pixels, aspect ratio—1:1, sequential line capture, Kalman averaging with 3 scans.ISH-stained paraffin sections were imaged using an Olympus BX60 microscope fitted with an Olympus Colorview III camera.For the RT-qPCR and bDNA FISH experiments with the antiviral drugs, the drugs were added to the 10% DMEM, which was administered to the cells right before the start of the infection. Rac1-I  was used at 200 µM final concentration and Cal-I1  was used at 200 µM final concentration. Cell viability assays to determine the cytotoxicity of the drugs was performed with Cell Titer-Glo according to the manufacturer’s instructions .6 on well) as described in The RT-qPCR experiment was started with seeding 90,000 A549 cells per well on 4-well plates. After 24 h, the plates were infected with 1:10,000 diluted CVA9 virus . A total of 140 µL of the supernatant was used for the RNA extraction, and the rest was stored at −80 °C.After stopping the infection, the plates were thawed for 15 min at 37 °C and frozen for 15 min at −80 °C. This was repeated three times to lyse the cells and release the virus without destroying the viral RNA. Cell debris (400 µL) was transferred to a 1.5 mL LoBind Eppendorf tube  and centrifuged for 10 min at 16,700× Viral RNA was extracted with QIAamp Viral RNA Mini kit  according to the spin protocol provided in the kit’s handbook. Extracted RNA (60 µL) was stored at −80 °C.The primers for the RT reaction and the qPCR reaction were synthesized and acquired from Thermo Scientific. The primer for the −RNA had the following sequence: 5′-GAAACACGGACACCCAAAGTA-3′, and the primer for the +RNA had the following sequence: 5′-CGGCCCCTGAATGCGGCTAA-3′. The sequences for the primers were originally received from Matti Waris (University of Turku) and the sequences have been successfully used before to detect enterovirus RNAs.The RT reaction was performed by making a master mix  accordinControl reactions for the RT-qPCR experiment included negative controls for RNA extraction, negative controls for the RT reaction, and negative controls for the qPCR reaction. In negative controls, we used nuclease-free water  instead of the template RNA/cDNA.The qPCR was performed by making a master mix  accordinThe images acquired from the confocal microscope were refined and analyzed with Fiji v. 1.52s, a distribution of the ImageJ image processing software . First, Intensity calculations from bDNA FISH images were acquired by using the “Measure” tool with “Limit to threshold” in Fiji after thresholding the background as explained above. Approximately 100 cells per image was used for the calculations. The measurement calculates the area of all pixels that have intensity values above the set threshold, which is a quantitation of the amount of target transcripts in the sample image. According to the manufacturer, each dot in bDNA FISH images represents one target transcript, and the level of expression can be quantified by measuring the number of dots per cell, meaning that the amplification of the signal is a linear function. It is important to note that experimental conditions, such as the microscope and camera used, can affect the visibility of the RNAs, meaning that the results are comparable only with studies with similar settings.For the RT-qPCR data, average Cq values were calculated from the triplicate measurements for each sample and plotted as a bar chart with standard deviation error bars representing the amount of variance in the Cq values in the triplicates.During the optimization of the bDNA FISH method, we discovered that the −RNA was barely visible under the confocal microscope, possibly due to the +RNA and −RNA strands making double-stranded RNA intermediates, effectively inhibiting the binding of our RNA probes to the strands. To overcome this problem, we tested 100% methanol, up to 8 molar urea, and 95% formamide in 0.1x SSC buffer to separate the strands from each other. The results (not shown here) indicated that 100% methanol and up to 8 molar urea did not affect the visibility of either RNA strand, but that 95% formamide in 0.1· SSC buffer improved the visibility of the −RNA significantly. Using high temperature combined with 95% formamide to separate the RNA strands was critical for the success of our bDNA FISH experiments.None of the negative controls used for RT-qPCR crossed the Cq value threshold, proving that the samples were not contaminated by other RNA- or DNA molecules.In our first experiments, we used standard antibody-based IF to label the CVA9 capsid protein VP1 and bDNA FISH to label either the +RNA or the −RNA at timepoints of 1, 2, 3, 4, and 5 h post-infection (p.i.). The results indicated that the amount of viral capsid protein was negligible before 4 h p.i., and most of the capsid protein was located peripherally in the cell . The +RNIn the samples where we labelled −RNA instead of +RNA, the amount of −RNA was negligible at timepoints 1–3 h p.i., but some −RNA could be seen after 4 h p.i. . As withNext, we simultaneously labelled cell nuclei and viral dsRNA with IF, and −RNA and +RNA with bDNA FISH from cells infected with CVA9 at timepoints 0.5, 1, 2, 3, 4, and 5 h p.i. . The resAccording to our results, there was very little colocalization between the dsRNA and the −RNA , even though they seemed to mostly reside in the same cellular locations . The maj−4 ng, while that of −RNA was in the range of 1 × 10−6 ng.We used RT-qPCR to detect the amounts of enterovirus RNA molecules in CVA9-infected cells at timepoints 0, 1, 2, 3, 4, and 5 h p.i. . The resThe amounts of enterovirus RNAs were also calculated from intensity values of the bDNA FISH sample pictures using Fiji. The results are presented in pixels that have intensity values above the set threshold plotted against time . The resWe tested the effect of the antiviral drugs Rac1-I and Cal-I1 on the amount of enterovirus +RNA and −RNA molecules being produced in CVA9-infected cells at 5 h p.i. . The resThe cytotoxicity of the drugs Rac1-I and Cal-I1 was determined with a cell viability assay. After a 5 h incubation with the drugs, only a 10% drop in cell viability was detected with Cal-I1 and no noticeable drop in cell viability was detected with Rac1-I in comparison with a DMSO control . This prWe infected cells with CVA9 while the cells were under the effects of either Rac1-I or Cal-I1, and then simultaneously labelled the viral dsRNA, +RNA, and −RNA at timepoints 0.5 h p.i. and 5 h p.i. . The resCal-I1 seemed to be even more effective than Rac1-I, blocking the virus infection completely in almost all cells , panel BAssuming that the efficiency of our qPCR was close to 100%, the amount of cDNA  was doubled every PCR cycle. This can be presented with the equationThe amounts of −RNA and +RNA compared to the timepoint 0 h p.i. (background level) were calculated with Equation (3) from the Cq data in We applied ViewRNA ISH for formalin-fixed, paraffin-embedded (FFPE) samples to detect viral +RNA and −RNA in CVB1-infected and non-infected cells and mouse pancreas. In the first phase, we showed that the method detected both RNA strands in acutely and persistently CVB1-infected FFPE PANC-1 cells . Both +RNext, FFPE pancreas tissue samples from CVB1-infected and mock-infected mice were analyzed. A strong +RNA expression was seen throughout the exocrine part of the pancreas. The −RNA expression was also clear, but less strong compared to +RNA expression. Pancreas from mock-infected mice was negative for both + and −RNA .Picornaviridae family, are small, non-enveloped viruses with a single-stranded positive-sense RNA genome. While their life cycle is relatively well-known, the distribution and the amounts of the viral +RNA and −RNA strands during an infection has been clouded due to insufficient methods to study them. Here, we have shown that the amounts of both +RNA and −RNA are negligible before 3–4 h p.i.  is more effective than formamide in the separation of DNA strands , but unfSince the ViewRNA Cell Plus Assay Kit allows for the labelling of only three different molecules, we unfortunately could not label the capsid, +RNA, −RNA, and dsRNA all at once. However, our results suggest that the −RNA and dsRNA, which take part only in replication, are located quite centrally in the cell, indicating that the virus’ replication organelles (ROs) are located centrally in the cell as well. Along with capsid proteins, the +RNA is located peripherally  in the cell, indicating that the assembly of new virus particles takes place in the cell periphery. These findings are consistent with earlier literature describing ROs being usually located close to the perinuclear endoplasmic reticulum ,40 and vWe demonstrated that the antivirals Rac1-I and Cal-I1 effectively blocked the CVA9 infection in cells in vitro at 200 µM concentrations. It is noteworthy to mention that the images taken from the bDNA FISH experiment with the antivirals gave only a glimpse of the reality of the situation; even though the amount of RNAs in The results demonstrate that ViewRNA ISH with our custom probes is a suitable method to study the expression of +RNA and −RNA in acute and persistent enterovirus infection. The same probe sets detecting +RNA and −RNA of enteroviruses can be applied across ViewRNA platforms, and the method works well also for FFPE tissue samples. According to our results, the replication of the viral RNA starts 2–3 h after infection and the translation of RNA to capsid proteins starts shortly after at 3–4 h post-infection. The virus’ replication areas with newly emerging −RNA are located centrally in the cell, while the virion assembly most likely takes place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands is almost identical, contradictory to earlier literature. Considering these results, new antiviral drugs can be designed more efficiently to target the various steps of the enterovirus life cycle. Moreover, the bDNA FISH protocol we established can be easily and readily applied to study enterovirus RNAs in vitro more effectively.


Ziziphus jujuba Mill. var. spinosa was mapped and determined based on Illumina sequencing data. The complete cp genome is 161,606 bp and contains a pair of inverted repeat regions of 26,479 bp each, a large single-copy region of 89,292 bp, and a small single-copy region of 19,356 bp. It harbors 112 unique genes, including 78 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Phylogenetic analysis based on cp genomes indicates that the cp genome of wild Z. jujuba Mill. var. spinosa is similar to that of cultivated Z. jujuba and closely related to that of Z. incurva of the family Rhamnaceae.In this study, the complete chloroplast (cp) genome sequence of  Ziziphus species (family Rhamnaceae) are plants considered to have dual medicinal and food purposes, and are distributed mainly in warm and subtropical regions throughout the world  Hu ex H. F. Chou have traditionally been used as an ethnomedicine in Asian countries for thousands of years .The Z. jujuba Mill. var. spinosa tree growing in Qinglonghu town , Fangshan District, Beijing, China. Voucher specimens (accession number: ENC850490) were deposited at the herbarium of the Beijing Academy of Forestry and Pomology Sciences. DNA extraction was performed according to a modified CTAB protocol  software tools. Reference-guided assembly was then performed to reconstruct the cp genome with the BLAST program , a 161,606-bp cp genome was obtained for Z. jujuba Mill. var. spinosa. Annotation was performed using the Plann script (Huang and Cronk Fresh leaves were collected from a single Z. jujuba Mill. var. spinosa contains a pair of inverted repeat (IR) regions of 26,479 bp each, and large single-copy (LSC) and small single-copy (SSC) regions of 89,292 bp and 19,356 bp, respectively. The genome comprises 112 unique genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes . Among the annotated genes, 17 genes were found to contain introns, including 15 with a single intron each and two with two introns each (clpP and ycf3).The circular cp genome of Z. jujuba Mill. var. spinosa, a maximum likelihood analysis was performed based on 24 complete chloroplast genomes using the Randomized Axelerated Maximum Likelihood (RAxML) program (Stamatakis Z. jujuba Mill. var. spinosa was found to be similar to that of cultivated Z. jujuba and closely related to that of Z. incurva of the family Rhamnaceae (Z. jujuba Mill. var. spinosa in Ziziphus Mill.To identify the phylogenetic position of amnaceae . This co