Male gender was predominant in both groups, at 76% (23/30) in the HTGAP group vs. 54% (126/232) in non-HTGAP, p = 0.02; 53% (16/30) presented with DM vs. 18% (42/232), p < 0.0001. The patients with HTG presented higher CRP 48 h after admission: 207 mg/dL ± 3 mg/dL vs. non-HTGAP 103 mg/dL ± 107 mg/dL, p < 0.0001. Among the patients with HTGAP, there were 60% (18/30) with moderately severe forms vs. 30% (71/232), p = 0.001, and 16% (5/30) SAP vs. 11% (27/232) in non-HTGAP, p = 0.4 Among the predictive markers, only haematocrit (HT) and blood urea nitrogen (BUN) had AUCs > 0.8. According to a multiple regression analysis, only BUN 48 h was independently associated with the development of SAP (p = 0.05). Diabetes mellitus increased the risk of developing severe acute pancreatitis . In our cohort, HTGAP more frequently had local complications compared with non-HTGAP. A more severe inflammatory syndrome seemed to be associated with this aetiology; the best predictive markers for complicated forms of HTGAP were BUN 48 h and HT 48 h.Acute pancreatitis (AP) is the most common gastrointestinal indication requiring hospitalisation. Severe hypertriglyceridaemia (HTG) is the third most common aetiology of AP (HTGAP), with a complication rate and severity that are higher than those of other aetiologies (non-HTGAP). The aim of this study was to evaluate the supposedly higher complication rate of HTGAP compared to non-HTGAP. The secondary objectives were to find different biomarkers for predicting a severe form. This was a retrospective study that included patients admitted with AP in a tertiary department of gastroenterology and hepatology. The patients were divided into two groups: HTGAP and non-HTGAP. We searched for differences regarding age, gender, the presence of diabetes mellitus (DM), the severity of the disease, the types of complications and predictive biomarkers for severity, hospital stay and mortality. A total of 262 patients were included, and 11% (30/262) of the patients had HTGAP. The mean ages were 44.4 ± 9.2 in the HTGAP group and 58.2 ± 17.1 in the non-HTGAP group,  Acute pancreatitis (AP) is a common worldwide cause of morbidity and mortality, occurring in 40 out of 100,000 people in the Western population , and it Severe hypertriglyceridaemia (HTG), defined as triglyceride (TG) ≥1000 mg/dL, is a well-described risk factor for AP. It is the third most common aetiology after excessive alcohol intake and gallstone disease and is the most common aetiological factor up to 56%) for AP in pregnant women % for AP . HTG accThe most accepted theory of pathophysiological mechanisms is that excess TG is hydrolysed by pancreatic lipase, resulting in free fatty acids (FFAs). The excess FFAs consequently overwhelm the binding capacity of albumin and cause acinar and pancreatic capillary injury. In addition, hyperviscosity resulting from chylomicronaemia causes impaired pancreatic blood flow, leading to ischaemia and acidosis, ultimately resulting in further pancreatic injury ,10.Patients with HTGAP had higher rates of complications and more severe forms ,12; theyAs HTG is associated with diabetes mellitus (DM), some studies found that the risk of HTGAP was higher in diabetic patients  and thatThe main objective of this study was to compare the severity of HTGAP with that of AP of other aetiologies. The secondary objectives were to compare different severity-predicting markers and scores for severe forms of AP based upon aetiologies: HTGAP versus other aetiologies (non-HTGAP), such as the BUN, haematocrit (HT), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and bedside index for severity in AP (BISAP).We retrospectively reviewed the electronic medical records of patients who were admitted with AP in a tertiary department of gastroenterology from 1 January 2018 to 31 August 2019. We identified potential patients using the discharge diagnoses of AP  and ICD-10 code (K85.9)). We confirmed the diagnosis of AP based on the revised Atlanta criteria guidelines, which require at least two of the following three criteria to be fulfilled: (1) abdominal pain consistent with acute pancreatitis ; (2) serum lipase activity (or amylase activity) at least three times greater than the upper limit of normal and (3) characteristic findings of acute pancreatitis on contrast-enhanced computed tomography (CECT) and, less commonly, magnetic resonance imaging (MRI) or transabdominal ultrasonography . The excAccording to the revised Atlanta criteria, AP was divided into three degrees of severity: mild (MAP), moderately severe (MSAP) and severe (SAP). MAP was characterised by the absence of organ failure and the absence of local or systemic complications; MSAP included the presence of transient organ failure and/or local complications, whereas SAP was defined by the presence of persistent organ failure (≥48 h) . The locThe following information was collected and recorded from the patients’ charts: gender, age, blood pressure (mmHg), respiratory rate (breaths per minute), oxygen saturation (%), pulse rate (heart beats per minute), body mass index (BMI), BISAP score at admission, leucocyte number at admission (cells number/μL), thrombocyte number at admission (cells number/μL), haematocrit at admission and at 48 h (%), glucose level at admission (mg/dL), aspartate aminotransferase (AST) at admission (U/L), alanine aminotransferase (ALT) at admission (U/L), total bilirubin (TB) at admission (mg/dL), gamma-glutamyl transpeptidase (GGT) at admission (U/L), alkaline phosphatase (FAL) at admission (U/L), creatinine level at admission and at 48 h (mg/dL), BUN at admission and at 48 h (mg/dL), CRP at 48 h (mg/dL), NLR at admission and at 48 h, PLR at admission and at 48 h, the aetiology of AP, complications and mortality. HTGAP was diagnosed when TG levels were ≥1000 or ≥500 mg/dL and milky serum and no other aetiology was found. Based on the aetiology, the patients were split in two groups: HTGAP and non-HTGAP.2.BMI was calculated as weight in kg/(height in m)Based on the recorded data, NLR and PLR were calculated at admission and at 48 h, using the formulas: NLR = neutrophil count/lymphocyte count and PLR = platelet count/lymphocyte count.The BISAP score was evaluated at admission using the worst parameters available in the first 24 h. The five-point BISAP score system incorporates the following variables: BUN level >25 mg/dL, an impaired mental status, systemic inflammatory response syndrome (SIRS), age >60 years and the presence of pleural effusion. One point was assigned for each variable within 24 h of presentation and added for a composite score of 0–5 .The patients were followed up for 90 days after discharge, with visits to the outpatient clinic or by phone.The Emergency County Timisoara Hospital Committee for Ethics approved this study .p values and odds ratios (ORs). Multivariate logistic regression analyses were used to assess whether the inflammation markers were independent factors for predicting MSAP + SAP or SAP in patients with AP. The accuracy of each marker for predicting SAP was assessed using receiver operating characteristic (ROC) curves, and the analysis was carried out using the MedCalc 15.0 software. The positive likelihood ratio (+LR) and negative likelihood ratio (−LR) were also calculated using the following formulas: +LR = sensitivity/(100 − specificity); −LR = (100 − sensitivity)/specificity. A 2-tailed value of p < 0.05 was considered statistically significant.The variables are expressed as the mean ± SD or median (range) and categorical data, as percentages, as appropriate. Univariate analysis was used to calculate During the designated study period, a total of 282 patients with AP were identified in the records and retrospectively analysed. Meeting the exclusion criteria, 20 patients were excluded: 4 pregnant women, 2 patients under 18 years old, 8 patients with recurrent AP and 6 patients with missing data see . There wThe patients’ characteristics are presented in A total of 141 (53%) individuals were diagnosed as MAP, 89 (33%) presented as MSAP and 32 (12%) as SAP. The forms of AP of different severity of the two groups are also presented in We analysed the types of complications of the two groups, and there were significant differences between the rates of local complications in HTGAP versus non-HTGAP. p < 0.0001).Regarding comorbidities, a significant difference was only observed in the number of patients with DM, the number was higher in HTG patients (p = 1). No significant differences were observed regarding ICU admission (p = 0.8) or the need for surgery (p = 0.8).There were no significant differences regarding the length of hospitalisation between the two groups: the average lengths of hospital stay were 8 ± 6.7 days for the HTGAP group and 8 ± 9.3 days for the non-HTGAP group (p = 0.2).There was a single death in the HTGAP group, representing 3%, and 22 cases of death in the non-HTGAP group, representing 9%  underwent DFPP after admission; the rest of the patients were treated with oral fenofibrate. p values, +LR and −LR are summarised in We performed an ROC analysis for parameters that are known to reflect severity, to evaluate their strength in predicting SAP and MSAP + SAP in HTG and non-HTG patients. The obtained cut-off values, ROC, sensitivity (SE), specificity (SP), p = 0.7).In multiple regression analysis, out of the parameters with the best AUROC, only BUN 48 h was independently associated with SAP in HTGAP. In the HTGAP group, the presence of DM increased the risk of developing SAP , depending on the region of the globe where the study was performed and the ethnicity of the patients included . Given iAccording to our data, the patients in the HTG group were significantly younger; they were predominantly males, and DM was frequently associated (53%). Higher percentages of patients (70.9%) have been found in other studies with a previous diagnosis of DM . The preA history of DM and younger age were found to be independent risk factors for AP in patients with severe HTG, thereby increasing the ICU admission rate and prolonging hospital stays . In our Lipid mediators are widely appreciated for their important roles in proinflammatory processes. TG metabolism is associated with inflammation and insulin resistance; moreover, even if TGs are not inherently toxic to the pancreas, their breakdown into FFAs by pancreatic lipase causes lipotoxicity. The severity of pancreatitis is dependent on the severity of the inflammatory response and the injury caused by lipotoxicity. FFAs increase inflammatory mediators such as TNF-alpha, interleukin-6 and interleukin-10, and these inflammatory cytokines play an important role in HTGAP. In obese mice and humans, hypertrophied adipocytes have been found to secrete large amounts of monocyte chemoattractant protein-1 (MCP-1), a chemoattractant that enhances macrophage infiltration into adipose tissues and the production of cytokines . This map < 0.0001) and rarely had uncomplicated forms, and MAP was observed in only 23% of cases compared to 57% in AP due to other aetiologies. Most patients with HTGAP had MSAP according to the revised Atlanta classification (p = 0.0005). Similar observations were made in a French cohort [HTGAP patients had higher levels of CRP (h cohort  and in tAlthough complicated forms were frequent in the HTGAP group, the mortality rate was not higher than in the non-HTGAP group, probably due to the younger age in the HTGAP group and the equal share of severe forms, there were also no significant differences in the comorbidities, such as cardiovascular diseases or chronic renal failure, that were observed in the two studied groups, which may have contributed to more severe forms. There are literature data that show consistent findings regarding mortality .There has been a constant search for rapid biomarkers for adequate prognosis prediction for AP. The markers of systemic inflammation are determined using readily available laboratory tests, including the NLR, PLR, HT, BUN and CRP. Elevation of the NLR during the first 48 hours of admission was significantly associated with SAP and was an independent negative prognostic indicator in AP . Multiplp = 0.005 for predicting the forms with complications.Wang et al. concluded that NLR represents an inexpensive, readily available test with promise for predicting the disease severity in HTGAP with a cut-off value of 10 and AUROC of 0.8905 . HoweverThe main limitation of this study was that it was a single-centre retrospective study. The number of patients with uncomplicated forms of HTGAP was low, with no apparent causes; one reason might be that we are a referral centre for the western region of our country, and it was predominantly the more severe cases of HTGAP that were referred to our hospital. Due to the same situation, another possible source of bias was the high percentage of cases with local or general complications, MSAP and SAP, representing 46% of the total cases.In our cohort, HTGAP more frequently had local complications compared with non-HTGAP and, consecutively, more severe forms of AP, statistically significant only for MSAP. A more severe inflammatory syndrome seemed to be associated with this aetiology. The best predictive markers for complicated forms of HTGAP were BUN 48 h and HT 48 h.


The question of how RNA, as the principal carrier of genetic information evolved is fundamentally important for our understanding of the origin of life. The RNA molecule is far too complex to have formed in one evolutionary step, suggesting that ancestral proto‐RNAs (first ancestor of RNA) may have existed, which evolved over time into the RNA of today. Here we show that isoxazole nucleosides, which are quickly formed from hydroxylamine, cyanoacetylene, urea and ribose, are plausible precursors for RNA. The isoxazole nucleoside can rearrange within an RNA‐strand to give cytidine, which leads to an increase of pairing stability. If the proto‐RNA contains a canonical seed‐nucleoside with defined stereochemistry, the seed‐nucleoside can control the configuration of the anomeric center that forms during the in‐RNA transformation. The results demonstrate that RNA could have emerged from evolutionarily primitive precursor isoxazole ribosides after strand formation. Prebiotically plausible isoxazole nucleosides are shown to be excellent candidates for proto‐nucleosides and proto‐RNAs. The proto‐nucleoside forms 2 instructive H‐bonds with guanosine and can directly rearrange in RNA to give cytidine. The stereochemical outcome of the reaction is controlled by already present canonical seed‐nucleosides. All concepts that have been developed to explain how RNA oligonucleotides could have formed on the early Earth are based on the prebiotic formation of nucleosides, which oligomerize in an enzyme free reaction.5, 6 T1) with hydroxylamine (2) to give 3‐aminoisoxazole .3 was shown to react with urea (4) to give N‐isoxazolyl‐urea 5, which can next react with ribose to furnish the corresponding furanosides  and pyranosides . Subsequent reductive N−O bond cleavage with thiols in the presence of catalytic amounts of Fe2+ gives cytidines ‐proto‐RNA structures, can undergo the N−O cleavage induced rearrangement directly in RNA. This “in‐RNA” reaction creates a new stereocenter. Excitingly, we observe that the configuration of already present canonical seed‐nucleosides controls the stereochemical outcome of the “in‐RNA” cyclization reaction, which leads to an amplification of stereochemical information.A recently introduced concept Figure b of how 6α/β to C rearrangement directly in‐RNA, we prepared the IO3 phosphoramidites α‐8 and β‐8 incorporated both building blocks into the different RNA strands shown in 2SiCl2  to give 10. Subsequent protection of the 2′‐OH group with TBS‐Cl  provided 11. This was followed by a one‐pot reduction of the azide to the amine  and reaction of the amine with IO3 3 to generate the ribofuranoside 12 as the expected α/β‐mixtures. The two IO3 anomers α‐12 and β‐12 could be separated by flash column chromatography. The nucleosides were next independently 3′‐5′‐deprotected to 13  and 5′‐dimethoxytrityl‐protected  to give 14. The 5′‐DMT‐2′‐TBS‐protected IO3 ribosides 14 were finally converted into the phosphoramidites α‐8 and β‐8. While we were initially concerned about the stability of the isoxazole‐ribosides, we learned during the synthesis that all IO3 nucleosides are astonishingly stable. Similar to the previously reported urea nucleosides,To first investigate the possibility of performing the in Table . The synα‐8 and β‐8. The RNA synthesis was indeed possible without large adjustments of the RNA‐synthesis and deprotection protocol. We just had to extend the coupling time of α‐8 and β‐8 to 600 s. Deprotection was first achieved with ammonium hydroxide/methylamine (1 : 1) followed by a second deprotection step with TEA⋅3HF. Scheme S1 as an example containing a single β‐configured IO3 after purification. The MALDI‐TOF mass spectrum proves the integrity of the obtained RNA S1. With this experimental proof for the possible and efficient incorporation of the IO3 nucleoside into RNA, we next used the protocol to prepare the RNA strands S1–S8 containing multiple IO3 nucleosides in mixed sequence contexts.To investigate whether an incorporation of the IO3 nucleosides into RNA is possible, we used standard solid phase oligonucleotide chemistry and the phosphoramidites hylamine  : 1 follS2 . The obtained mixture of nucleosides was subsequently analyzed by LC‐HESI‐MS  and catalytic amounts of Fe2+ at 90 °C for 2 h. HPL‐chromatographic analysis of the transformation revealed a clean spot‐to‐spot conversion .With the purpose of proving that the presence of seed‐nucleosides is a prerequisite for the stereochemical amplification, we performed two control experiments. First, we used the strand S6) or α‐C (S7) at the 3′ position. LC‐HESI‐MS analysis of the digestion products after N−O bond cleavage and subsequent rearrangement showed us now mixtures of α/β‐C for both strands. However, a clear stereochemical bias is observed. For the β‐C carrying strand redS6 an α/β‐ratio of 1 : 3 is detected and for the α‐C carrying strand redS7 an α/β‐ratio of 2 : 1 was obtained. This shows that one stereochemically defined seed‐nucleotide is already sufficient to control the stereochemical outcome of the cyclization reaction.Based on this result, we next designed two 5mers with only one information carrying nucleoside in the form of a single β‐C (S1 (A/C/G/U‐R1) in which we varied the base opposite the IO3 and measured melting points. We also prepared four reference S1 strand (A/C/G/U‐S1), in which we replaced the IO3 by one of the other canonical bases  at the respective position features as expected, the highest melting point of 55 °C. The A : U base pair at this position created a duplex (A‐S1:U‐R1) which melted at Tm=48 °C. For the duplex G‐S1:U‐R1 with a G : U Wobble base pair at the respective position, we measured a melting point of 43 °C. When we investigated the IO3‐S1:R1 base pair, we noted that the IO3:G situation created a duplex IO3‐S1:G‐R1 with a melting point of 45 °C even higher than what was determined for the G : U Wobble base pair. More importantly, the pairing selectivity is remarkable. While the IO3:G containing duplex melts at 45 °C, all other base pairs created duplexes with melting points of 31 °C or even lower, establishing a ΔTm of minimum 14 °C. This is an unusual discrimination between the productive IO3:G situation and all other base pairing possibilities.For a chemical compound to function as an instructional proto‐nucleoside it is essential that the nucleoside can contribute to the stability of the RNA and that it can encode information.30, 31S8 and 8Can (see Supporting Information), which upon annealing result in formation of either G : U or IO3:G wobble base pairs at the central two positions in each duplex, respectively (GGUXGACC). These strands were prepared in mg‐quantities and the formed duplexes were analyzed by NMR spectroscopy . The high chemical shift similarity observed across the two samples, both in homo‐ and heteronuclear experiments, showed us that both palindromes form double‐strands with an overall A‐like conformation which is typical for RNA duplexes . 2D 1H–1H NOESY was next employed to elucidate the conformation of the IO3 base  should be addressed to the authors.Supporting InformationClick here for additional data file.


Formerly, we were able to inhibit hepatitis C virus propagation by applying a circular RNA decoy strategy against microRNA-122, which is essential for the viral life cycle. In another proof-of-principle study, we used circular RNAs to sequester microRNA-21, an oncogenic and pro-proliferative microRNA. This strategy slowed tumor growth in a 3D cell culture model system, as well as in xenograft mice upon systemic delivery. In the wake of the global use of an in vitro transcribed RNA in coronavirus disease 2019 (COVID-19) vaccines, the question arose whether therapeutic circular RNAs trigger cellular antiviral defense mechanisms when delivered systemically. In this study, we present data on the cellular innate immune response as a consequence of liposome-based transfection of the circular RNA sponges we previously used to inhibit microRNA function. We find that circular RNAs produced by the presented methodology do not trigger the antiviral response and do not activate innate immune-signaling pathways.Natural circular RNAs have been found to sequester microRNAs and suppress their function. We have used this principle as a molecular tool and produced artificial circular RNA sponges in a cell-free system by   in vitro transcription can be used to inhibit microRNAs that are involved in viral or cancerous human diseases. Here, we show that circular RNAs are not sensed by the cell-intrinsic innate immune system, a crucial precondition for their clinical application.Circular RNAs produced by  Harboring neither a 5′ cap nor a 3′ poly(A) tail, circRNA molecules belong to a class of mainly non-coding RNAs.,,,,,trans-acting molecule. Considering the latter, recent studies demonstrate that naturally occurring circRNAs have at least three major functions in eukaryotic cells: circRNAs can function as microRNA (miRNA) sponges, interact with RNA-binding proteins (RBPs), and act as nuclear transcriptional regulators—all together illustrating the impact of circRNAs on the regulatory networks governing gene expression.,,Even though naturally occurring circRNAs appear to be highly abundant, found in diverse species throughout the eukaryotic Tree of Life, and strictly regulated, the molecular function of most endogenous circRNAs is still unknown.cerebellar degeneration-related protein 1 transcript (CDR1as) gene, which expresses the cellular circRNA CDR1as/ciRS-7. CDR1as/ciRS-7 acts as a molecular sponge sequestering the cytoplasmic miRNA-7 via 70 highly conserved binding sites, hence its alternative name circRNA sponge for miRNA-7 (miR-7) (ciRS-7).,,When binding cytoplasmic miRNAs as molecular sponges, circRNAs are able to regulate and neutralize endogenous miRNA levels. In this context, two initial studies, published simultaneously in 2013 by the groups of Nikolaus Rajewsky and Jørgen Kjems, focused on the ,,,15,in vitro production, and stringent purification of artificial ciRSs as novel agents for miRNA inhibition.in vitro and in vivo, thereby reducing viral intracellular protein levels similar to Miravirsen.The described characteristics, functions, and importance of naturally occurring circRNAs imply the increasing relevance of artificially produced circRNAs as potential tool for molecular biology and medicine.MIR21 knockout resulted in a reduced tumor growth, ciRSs were designed to impair miR-21 activities in vivo.Moreover, the concept of artificial circRNA sponges was used to target miR-21. miR-21 is one of the earliest identified oncogenic miRNAs.,,,,Two fundamental parameters determine the fate of any pharmaceutical: (1) the efficacy and (2) the toxicity.in vitro transcription and ligation, and stringently purified by gel extraction, can bypass the cellular RNA sensors, and are not recognized by the innate immune system.In this study, we demonstrate that artificial circRNAs can be utilized as miRNA sponges, if produced in a cell-free system by ,,Encouraged by naturally occurring circRNAs containing binding sites that are able to sequester specific miRNAs, artificial ciRSs were designed to target the oncogenic miR-21.in vitro circRNAs production procedure, as described in detail elsewhere,in vitro transcription and transcript purification, (2) the transcript ligation based on the T4 RNA ligase, and (3) the gel purification of circular and linear constructs ), which mimic long double-stranded RNAs (dsRNAs), and single-stranded poly uridine (ssPolyU), we created an extensively double-stranded circRNA. In more detail, we engineered an artificial ciRS sharing the same basic structure as ciRS-21-bu and ciRS-21-rnd (stem loop and constant region) but containing a randomized self-complementary sequence of 50 bp, resulting in ciRS-21-double-stranded (ciRS-21-ds) A. The reisoforms C, and 4Digations ,8,9,20 pations ,,C. Notablations ,,C. Analytations ,,D and 4E.CXCL2, CXCL3, CXCL8, EGR1, IRAK2, and TRAF1 identified in RNA sequencing  and ssPolyU, or liposome transfected control (mock) cells , and 7. quencing  via qRT-quencing A, and 7AA levels B, and 7CA levels . In addiA levels . HoweverA levels .Figure 5CXCL2, CXCL3, CXCL8, EGR1, and TRAF1 mRNAs, as well as CXCL10 and INFB1  treatment , and 7. nd INFB1 , and 7. MDA5, PKR, OAS1, TLR3, TLR7, TLR8, and RIG-I after transfection (MDA5 and RIG-I mRNA levels upon HMW poly(I:C) and LMW poly(I:C) treatment, no transcriptional upregulation of RNA sensors could be detected after circRNA transfection  and purification method or purity of the final product, (2) RNA modifications, (3) secondary structure, and (4) the dose of the circRNA administered. However, in-depth analyses of cellular fate and immunogenicity of relatively short, highly purified artificial circRNAs produced in cell-free systems are still lacking.As a potential therapeutic tool, circRNAs have attracted the attention of many scientists in recent years. This has sparked a discussion about the immunogenicity of circRNAs.in vivo”) both by spliceosome-dependent exon circularization strategy,,in vitro”) using recombinant T7-phage RNA-polymerase-mediated transcription and circularization by either employing genetically engineered autocatalytic group I introns ,,,,,,To not only identify but also rather increase the functional understanding of circRNAs, a broad range of artificial circRNA expression systems was established. Relying on specifically designed overexpression vectors, circRNAs can be produced in cell culture  as marker of cellular circRNAs, which do not induce the known innate immune pathways, whereas unmodified circRNAs activated RIG-I and innate immune signaling. Interestingly, in a cell-free system for ciRS production by in vitro transcription, ligation, and stringent purification via gel extraction, and the given length of about 200 nt, our ciRSs (ciRS-21-bu and ciRS-21-rnd) were found to be non-immunogenic, even without any RNA modifications. This may be different with longer RNAs that are, e.g., constructed for translation purposes and not only miRNA sequestration. Nonetheless, when necessary for longer circRNAs, modified NTPs  can be used in in vitro transcription reactions, replacing the respective unmodified NTP, as it is common practice in mRNA vaccine production.In 2019, Chen et al.Surprisingly, the highly double-stranded construct ciRS-21-ds (with a 50-bp perfectly double-stranded region) did not only upregulate the analyzed chemokine-, interferon-, and other innate-immune-response-associated mRNAs but also PKR, a key RNA sensor in cellular immunity. The autophosphorylation of threonine at position 446 in PKR is an indicator of PKR dimerization and activation.In summary, the entirety of all properties of an artificial circRNA may be crucial regarding the recognition and RNA-sensor-mediated immune activation and thus the cellular fate of a potential therapeutic—from design, production strategy, and stringent purification to the extent of double-stranded regions in a ciRS. The latter provides opportunities and possibilities for immunomodulatory effects. Thus, it could be useful in certain applications to either specifically trigger or bypass the innate immune system with an artificial circRNA therapeutic.The circRNAs used here were designed as initially described in Jost et al.EcoRI restriction sites and gel purified via agarose gel electrophoresis. The purified template was used for in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit  with additional 10-fold molar excess of guanosine monophosphate (GMP) over NTPs . Transcripts were treated by RQ1 DNase  digestion to remove the DNA template. Next, excess NTPs were removed by size-exclusion chromatography . In vitro circularization was performed at 16°C overnight using the T4 RNA Ligase I . Ligation reaction was analyzed on analytic 6%, 7%, and 8% polyacrylamide-urea gels enabling distinguishment of linear and circular products. Using preparative 7% polyacrylamide-urea gels, circular and linear sponges (ciRS/liRS) were visualized and excised using UV shadowing and purified by gel extraction within PK Buffer at 50°C. Phenol and chloroform extraction was performed to purify RNA followed by ethanol precipitation. Purified sponges were analyzed on 6%, 7%, and 8% polyacrylamide-urea gels as described above. Find detailed information and protocol elsewhere.Template sequences were chemically synthesized and cloned into the multipurpose vector backbone containing the sequence elements described above, excised by flanking Circularity was proven by RNase R treatment. Therefore, 200 ng of the RNA was incubated with 2 U RNase R  for 30 min at 37°C. Afterward, 50% of the reaction was analyzed via polyacrylamide-urea gel electrophoresis and ethidium bromide staining.2. Twenty-four hours prior transfection 4.2 × 105 cells were seeded on 6-well plates. The transfection of cells was performed using Lipofectamine 2000  according to manufacturer’s instructions. Depending on the approach, 21 ng to 7 μg of the circular RNA sponges, LMW poly(I:C) , HMW poly(I:C) , or single-stranded (ss) PolyU  were added to the transfection reaction. If not harvested beforehand, cells were washed with PBS and medium was exchanged 4 h post-transfection.A549 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% COhttps://www.ncbi.nlm.nih.gov/tools/primer-blast/). Relative quantification of mRNA abundance was ascertained as described by Michael W. PfafflCells were lysed and total RNA was isolated using TRIzol  reagent. Two micrograms of total RNA served as template for reverse transcription using the qScript cDNA Synthesis Kit  according to manufacturer’s protocols. Two-step qRT-PCR with an annealing temperature of 58°C was performed utilizing Luna Universal qPCR Master Mix  based on SYBR green technology in both a QuantStudio 3 and StepOnePlus Real-Time PCR cycler (Thermo Fisher Scientific). Primer pairs were selected using the Primer Blast designing tool , which includes rRNA removal. Sequencing was performed using a NextSeq 500 High Output Kit (75 cycles).https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 22 June 2020) before and after trimming. Adapters were trimmed using Trim_Galore v.0.6.4 , which used cutadapt v.2.8,https://github.com/agordon/fastx_toolkit; accessed on 22 June 2020), also removing reads that became shorter than 90% of their original length (settings: -q 20 -p 90). The remaining reads were aligned to the human genome (GRCh38) with the associated annotation from EnsemblThe quality of the datasets was examined with fastqc v.0.11.9  with the generic GO subset from the Gene Ontology Resource.GSE192656).IDs of differentially expressed genes were converted from ensembl to entrez format via the package org.Hs.eg.db v.3.11.4,Cells were harvested by scraping, and total protein was extracted using PBS containing 0.5% Triton X-100. Proteins were separated via SDS-PAGE, containing 4% polyacrylamide stacking gel and 12% polyacrylamide resolving gel, and transferred to PVDF Western Blotting Membrane . Specific primary antibodies binding to p-PKR (Thr446) , PKR , and γ-tubulin  followed by horseradish-peroxidase-coupled secondary antibodies  were used to determine the protein abundance. Visualization was achieved utilizing luminol-based chemiluminescent substrates Lumi-Light Western Blotting Substrate  and Lumi-Light Plus Western Blotting Substrate  as well as INTAS ECL Chemocam Imager (Intas Science Imaging Instruments).


The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification. In SPAA (SPAAC) . As a re ethyl]-biotinamide, CAS: 717119-80-7, 5 mM, Amatek scientific, catalog: B-1328) under the catalysis of ADPRC  in 100 μl of ADPRC reaction buffer   was reacted with HEEB (4HCO3 in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 2 μl. The gradient was as follows: 0 min 0% B, 2 min 0% B, 4 min 20% B, 17 min 40% B, 19 min 100% B, 24 min 100% B, and 30 min 0% B. The retention time of NAD and ADPR-Biotin (product) was 9.393 min and 9.929 min, respectively. The corresponding compounds were purified from HPLC and then analyzed by LC–MS by Agilent Technologies 6120 Quadrupole with a C18 column . The mobile phase was composed of (A) 0.1% formic acid in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 5 μl. The gradient was as follows: 0 min 10% B, 4 min 100% B, and 7 min 100% B.Samples were analyzed by an Agilent Technologies 1260 Infinity with a C18 column  monitoring at 260 nm. The mobile phase was composed of (A) 100 mM NHTAATACGACTCACTATTACTGTGTCGTCGTCGTCTGCTGTCTCTCTCTCGCGGGC-3′; boldface letters denote the sequence of T7 class II promotor (ϕ2.5)) and (anti-sense: 5′-GCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3′) ) and (anti-sense: 5′-GGCAGCAAGCCGGAGGAGCAGAAGAACGACAGGGCCAGCGAGCCGCAGGCGGCAGCGCGCACGCCCACGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3′). To prepare spike-in ppp-RNA of 62 nt, oligonucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template ) and (anti-sense: 5′-TGGCTGGCCGCATGCCCGCGGCAGAAGTGACATTATGGCTAAAGTTGTGGTAGCACTTCCCTAATAGTGAGTCGTATTA-3′). To prepare spike-in NAD-RNA (500 nt) and m7Gppp-RNA (500 nt) with identical sequence, oligonucleotide without adenine was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5′-AAGAAGAACACCAAGAAGCCGAACAACAGCAAGACGGCCAAGAAAAAGACGAAGAGGCAGAAGAAGAAGAACACCAGCAAGAGCCCCAGGAAGAAGCCGACCACCAAGAAGACGAAGCCCAACAGCACGAAGCGGAACACCAGGGAGACGCCCACGAACAACACCACGGCGCGGGACAAGAAGAAGCCGACGACCAAGAAGAAGAAGGAGCGCACCAGGACGAAGCCAACGGGCAAGGCGGACAAGAAGAAGACGAGCAGCAACAAGAGGACGGGGAAGCGGCAGAAGCACAGCACGCCGAAGGACAGGGAGGACACGAGGGAGGGCCAGGGCACGGGCAGCAAGCCGGAGGAGCAGAAGAACAACAGGGACAGCAAGCCGAAGGAGGCAACGCCCACGCCCACGCCGGACACGCAGAACAAGAGGCCGAAAACGACGCCGACCAGCACGACCAGGAAGGGCACCACCCCGGAGAACAGCACCACGCCCAAGCACACCATAATAGTGAGTCGTATTA-3′). For in vitro transcription, 10 μM of double-stranded DNA (dsDNA) template in 100 μl transcription buffer , along with 1 mM of each of GTP, CTP and UTP, with 1mM ATP , 4 mM NAD (for NAD-RNA) or 4 mM m7GpppA  (for m7G-RNA), 10 μl of T7 RNA polymerase , 5% DMSO, 5 mM DTT and 2.5-unit RNase inhibitor were added and the transcription mixture was incubated at 37°C for 4 h. The reaction was incubated with 11-unit DNase I  at 37°C for 30 min to remove the DNA template. RNA was then extracted using acid phenol/chloroform and precipitated with isopropanol  at -80°C overnight. The RNA pellet was washed twice with 75% ethanol, air-dried, redissolved in DEPC-treated H2O, and stored at –80°C. For spike-in RNAs used as internal controls for gene-specific qRT-PCR assessment, NAD-RNA is 106 nt as described above. To prepare ppp-RNA, 88 nt oligo nucleotide was synthesized  and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 106 nt RNA ) and (anti-sense: 5′-CAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGACCTCTCACACACAGTTCGCCTCTTTGATTAACGCCCAGCGTTTTCCCGGTATCCAATAGTGAGTCGTATTAGTGATC-3′). To prepare 45 nt m7Gppp-RNA, oligo nucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 45 nt RNA ) and (anti-sense: 5′-CGACAGGGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3′).To prepare short RNA of 38 nucleotides (nt), 10 μM template oligo DNA (template sequence: 5′-GATCACGATC-3′)  were com7G-RNA (500 nt) and ppp-RNA (500 nt) spike-ins with different sequences from that of NAD-RNA . The synthesis of m7G-RNA (500 nt) was as described in  and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5′- TGGCCTTGTAGGTGGTCTTGACCTCAGCGTCGTAGTGGCCGCCGTCCTTCAGCTTCAGCCTCTGCTTGATCTCGCCCTTCAGGGCGCCGTCCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATGGTCTTCTTCTGCATTACGGGGCCGTCGGAGGGGAAGTTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGTCCTGCAGGGAGGAGTCCTGGGTCACGGTCACCACGCCGCCGTCCTCGAAGTTCATCACGCGCTCCCACTTGAAGCCCTCGGGGAAGGACAGCTTCAAGTAGTCGGGGATGTCGGCGGGGTGCTTCACGTAGGCCTTGGAGCCGTACATGAACTGAGGGGACAGGATGTCCCAGGCGAAGGGCAGGGGGCCACCCTTGGTCACCTTCAGCTTGGCGGTCTGGGTGCCCTCGTAGGGGCGGCCCTCGCCCTCGCCCTCGATCTCGAACTCGTGGCCGTTCACGGAGCCCTCCATGTAATAGTGAGTCGTATTAGTGATC-3′). To prepare ppp-RNA, oligonucleotide was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5′-TCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATAATAGTGAGTCGTATTA-3′).For boronate affinity experiment, we synthesized mribed in . To prep2) at 37°C for 1 h. Product was purified with Zymo column  according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).NAD-RNA (1 μg) was reacted with 40 mM and 100 mM HEEB (1 M stock in DMSO) under the catalysis of ADPRC (25 μg/ml) with 0.4 U/μl of RNase Inhibitor  in 100 μl of ADPRC reaction buffer  were washed 3 times with immobilization buffer  , and the254nm (0.18 J/cm2) twice. Membrane was blocked in 5% BSA in PBST (0.1% Tween20 in PBS) for 1 h, followed by incubation with Alexa Fluor® 790 Streptavidin  at room temperature for 2 h in the dark. Membrane was washed with PBST and PBS for 3 times, respectively, and imaged on the Odyssey LiCor CLx scanner (Li-Cor Biosciences) with the software set to auto-detect the signal intensity at 800 nm channel. Finally, the membrane was stained with methylene blue solution (0.3% w/v methylene blue + 30% v/v ethanol + 70% v/v H2O)  which was then cross-linked by UVv/v H2O)  to visua7Gppp-RNA (45 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  at 37°C for 1 h. 100 μl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction  and 0.4 U/μl of RNase Inhibitor  at 25°C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris–HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H2O. The biotinylated RNA on the beads was extracted with Trizol LS  and chloroform. Input (see above) RNAs and the biotinylated RNA on the beads were analyzed by an 8% polyacrylamide TBE urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of mreaction . RNAs we7Gppp-RNA (38 nt) was performed with 1 μl of NudC  in 25 μl of NudC reaction buffer  at 37°C for 30 min. Product was purified with Trizol LS  and analyzed by an 8% polyacrylamide TBE–urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) or m7Gppp-RNA (45 nt) were mixed together and incubated with 500 nM NudC  in 25 μl of NudC reaction buffer  at 37°C for 30 min. Product was purified with Trizol LS  and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of m2O was then added and acid phenol/ether extraction was performed to stop the reaction . Total RNAs (100 μg) was incubated with 100 mM HEEB (1 M stock in DMSO), ADPRC  and 0.4 U/μl of RNase Inhibitor  in 100 μl of ADPRC reaction buffer at 37°C for 1 h. 100 μl of DEPC-treated Hreaction . RNAs we2O. To ensure complete elution, biotin-conjugated RNAs were replaced from streptavidin beads by incubating with 1 mM biotin buffer  at 94°C for 8 min, followed by incubation with 500 nM NudC  in 25 μl of NudC reaction buffer  at 37°C for 30 min. After NudC treatment, biotinylated-RNAs that are resistant to NudC catalysis, potentially derived from contaminating m7G-RNAs, were retained on beads by incubation with high-capacity streptavidin particle  at 25°C for 30 min. Eluted RNAs in the supernatant were used for next step.After HEEB reaction, biotinylated RNAs were incubated with streptavidin bead particles  and 0.4 U/μl of RNase Inhibitor  at 25°C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris–HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H7Gppp-RNA (38 nt) was performed with or without 1 μl of yDcpS  in 1X yDcpS reaction buffer  in 20 μl total volume at 37°C for 1 h. Product was purified with Zymo column  according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) and m7Gppp-RNA (106 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with or without ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  at 37°C for 1 h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates were used for qRT-PCR. Reverse transcription was performed with gene specific primers using SuperScript III SuperMix . qPCR was performed using SYBR Green master mix  to detect NAD-RNA, ppp-RNA or m7Gppp-RNA from three biological replicates using specific primers described in t test.Total RNAs (100 μg) and 100 ng of spike-in NAD-RNA (106 nt), ppp-RNA (106 nt) or mInput (see above) and NudC-eluted RNAs from four biological replicates of livers from 2- and 18-month old C57BL/6 mice were used for NGS library construction, in accordance with manufacturer's instructions . Library quality was assessed by Bioanalyzer 2100 , and quantification was performed by qRT-PCR with a reference to a standard library. Libraries were pooled together in equimolar amounts to a final 2 nM concentration and denatured with 0.1 M NaOH . Libraries were sequenced on the Illumina NovaSeq 6000 system .7Gppp-RNA, 1% NAD-RNA/99% m7Gppp-RNA, 5% NAD-RNA/95% m7Gppp-RNA or 10% NAD-RNA/90% m7Gppp-RNA, respectively. The mixture of total RNAs and spike-in RNA (500 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  at 37°C for 1 h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates of livers from 18-month old C57BL/6 mice were subjected to polyA-selected RNA sequencing as mentioned above.Total RNAs (100 μg) were mixed with 1 ng of 3′-end polyadenylated spike-in RNA (500 nt) that had 0% NAD-RNA/100% m7Gppp-RNA (38 nt) was ligated with 5 μM 3′ adaptor oligo listed in 1 μg of NAD-RNA (38 nt) or mBoronic acid beads  were washed 3 times with binding buffer , and then incubated with RNA oligonucleotide in binding buffer in a thermomixer at 37°C for 2 h. Beads were then washed 3 times with wash buffer ; RNA oligonucleotide bound by the beads was extracted with Trizol LS ). Purified RNA product was analyzed by an 8% polyacrylamide TBE–urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).7Gppp-RNA, and 1 ng of ppp-RNA). RNAs were incubated with yDcpS  in 50 μl of 1X yDcpS reaction buffer at 37°C for 1 h, to remove m7G cap. After purification with Trizol LS , yDcpS-treated RNAs were combined with 100 μmol oligo d(T)30VN in 100 μl 0.5× SSC buffer , and incubated at 80°C for 5 min followed by 50°C for 60 min. Then, 1 μl RNase H  and 5.6 μl 10× Reaction buffer  were added. The mixture was incubated at 37°C for 20 min, and purified with Trizol LS . This step aims to promote 3′-end adaptor ligation by removing polyA sequence tract from the endogenous transcripts. Prior to 3′-end ligation, polyA-depleted RNAs were treated with 2 U CIAP  in the presence of 40 U RNaseOUT at 37°C for 1 h to remove 5′-terminal phosphate. RNA was extracted with Trizol LS . 5 μg of CIAP-treated RNA products were ligated with 100 μM 3′ adaptor oligo listed in 2O. Reverse transcription was performed with adaptor-based primer listed in 7G-RNA (500 nt) was used as a negative control. Primers were listed in t-test.Total RNAs were extracted from three biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, total RNAs (50 μg) were mixed with polyadenylated spike-in RNAs   with theDESeq2 (v1.30.0) . NAD-RNAs were defined as fold change of normalized transcript counts ≥2 and FDR <0.05 in NudC-treated samples compared to those in input samples. Gene annotation information, such as chromosome, gene-types, gene-lengths and intron coordinates were retrieved from Gencode (M23) annotations. The violin plot, bar plot, line chart and scatter plot were generated by R package ggplot2 (v3.3.2) (Genes that had zero count in more than 25% (3 out of 12) sequencing libraries were removed before performing differential analysis. Principle component analysis (PCA) was performed with R function ‘prcomp’ using a transformed counts matrix by function ‘vst’. To identify NAD-RNA from total RNA-seq data, we used R package v1.30.0)  to perfo(v3.3.2) .gprofiler2 (v0.2.1)  in Cytoscape (v3.8.2)  . Pathway(v0.2.1)  and the (v0.2.1)  without (v3.8.2) . Network(v3.8.2) .2) at 37°C for 1 h. 100 μl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction  following the manufacturer's protocol with primers listed in Total RNA was extracted from four biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, 100 μg total RNA, mixed with 1 ng of NAD-RNA (106 nt) and 1 ng of ppp-RNA (106 nt), was incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  ethyl]-biotinamide) as a reactant, allowing only one reaction to biotinylate NAD-capped RNAs. To avoid contaminating signals, we designed a NudC-based post-treatment to elute biotin-conjugated RNAs specifically derived from NAD, but not m7G-capped transcripts from streptavidin beads. Therefore, our method circumvented laborious steps to purify mRNAs followed by clearance of m7G-capped RNAs. With significantly simplified procedures, our method enabled NAD-RNA profiling directly from total RNA. Based on the same platform, qRT-PCR analysis on specific NAD-RNAs could be readily applied.Compared to previous methods that require multiple reactions, we introduced HEEB  and NAD-RNA (38 nt) with 100 mM HEEB to the reaction. While NAD-RNA reacted with HEEB, as indicated by the presence of a biotinylated form, m7G-RNA had no such a product, reflecting specificity  but not m7Gppp-RNA (38 nt) as shown by a lower-sized band corresponding to the de-capped product  to the same reaction. The result clearly showed that NudC was able to selectively de-cap NAD-capped (38 nt) but not m7G-capped RNA (45 nt)  but not m7Gppp-capped forms (38 nt) from streptavidin beads , but not NAD-RNA (38 nt) was sensitive to yDcpS treatment, a decapping enzyme that hydrolyzes the triphosphate linkage of m7G-capped RNA  Figure . We tests Figure  and F. Apped RNA .7G-capped RNA (m7Gppp-RNA), respectively). HEEB reacted with NAD-RNA (106 nt) and m7Gppp-RNA (106 nt), but not ppp-RNA (106 nt), resulting in a band retained by the streptavidin beads (7Gppp-RNA (106 nt) could be detected on streptavidin beads; treatment of NudC, however, found no evidence to elute m7Gppp-RNA (106 nt)  Figure .7G-cap, followed by polyA tails. Presumably, endogenous transcripts may contain both NAD and m7G-capped forms, though the percentage may differ for particular genes. The rationale of this design is to mimic endogenous genes that have either low (0% or 1%) or relatively high (5% or 10%) NAD modification. Total RNAs were mixed with spike-ins that had different ratios of NAD-RNA and m7G-RNA. The sample that contained 100% m7G-RNA spike-in represented a gene with no NAD capping, which allowed the assessment of the specificity of ONE-seq platform. Other samples that contained 1%, 5%, and 10% of NAD- relative to m7G-RNA spike-ins were used to determine the capture sensitivity. We subjected 100 μg total RNA from mouse livers mixed with 1 ng spike-in RNA to ONE-seq experiment, followed by polyA-selected RNA sequencing. In the sample mixed with 100% m7G-RNA spike-in, we found no enrichment  but had either NAD or mt Figure . As demot Figure , represee Figure , suggeste Figure , reflect7G-capped RNAs.Fourth, we determined the noise-cancelling effect of NudC by comparative analysis of RNA-seq datasets. Total RNAs, after HEEB reaction, were captured by streptavidin beads, followed by either NudC-catalyzed elution (NudC+) or mock treatment (NudC-) . polyA-s7G and NAD-capping.We tested the utility of ONE-seq. We profiled NAD-RNAs from mouse liver tissues of young (2-month) and aged (18-month) cohorts. After quality control, we obtained in average ∼12.3 million high-quality and uniquely mapped sequencing read pairs from each library . Assessmcis diols, occurring naturally at the 3′-end of RNA, as well as in the 7-methylguanosine of m7G-cap and the nicotinamide riboside of NAD-cap  at the terminal is ligated to the 3′-end of RNA, such that the vicinal-diol moiety of the ribose at the 3′-end of RNA no longer exists. At this step, affinity binding can only occur between the boronyl group from boronic acid and 1,2-cis diols from the nicotinamide riboside of NAD-cap. Importantly, we perform reverse transcription using an adaptor-specific primer to harvest RNAs with 3′end blocker, thereby abolishing false signals from boronate binding of RNAs that are not properly ligated with adaptor. Consequently, only RNA transcripts that contain NAD-cap can be selectively enriched by boronate affinity.We validated newly identified NAD-RNAs by an ADPRC-independent method. To do this, we applied boronic acid beads. Our rationale lies at the fact that boronyl groups of boronic acid can form relatively stable complexes with 1,2- NAD-cap . Inspire NAD-cap ,38, we d NAD-cap . First, 7G, but not NAD, from RNA oligos (38 nt). In the presence of RNA ligase, 3′adaptor was efficiently ligated to the spike-in RNAs (38 nt), yielding an upper band in the gel  that were designed with different sequences. This experiment clearly demonstrated that NAD-RNA (500 nt) spike-ins, but not ppp-RNA (500 nt) and m7Gppp-RNA (500 nt), were selectively and significantly enriched by boronic acid beads  and transcription (e.g. RNA polymerase II) were found to contain NAD-cap. Also, transcripts with 5′-NAD modification were enriched in cell cycle (e.g. centromere protein and CDK) and DNA damage responses (e.g. ATM and TP53). Not just housekeeping genes, transcriptional factors (e.g. BMP5 and Interferon), epigenetic pathways , and histone-related genes were also subject to NAD modification Figure . In mitoIn line with an age-modulated decrease of NAD , both thAbove evidence supported the notion that ONE-seq platform permits epi-transcriptome-wide profiling directly from total RNA, prompting us to extend its application for gene-specific assessment by qRT-PCR. To do this, we included non-capped ppp-RNA (106 nt) as a baseline negative control, and NAD-RNA (106 nt) as a positive control. Total RNA and internal spike-in controls were subjected to ONE-seq experiment, followed by qRT-PCR. The relative abundance was calculated between NudC + and input samples Figure . Our datN-acetylglucosamine (UDP-GlcNAc), can be incorporated at the 5′-end of RNA during transcription initiation in both prokaryotes and eukaryotes , flavin adenine dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc) and uridine diphosphate karyotes . CapZymeNCIN cap . As a re7G) has been found to contaminate the NAD-RNA profile. As such, SPAAC-NAD-seq , Mup13 (http://genome.ucsc.edu/s/lda97/Mup13_ONE-seq), Mup19 (http://genome.ucsc.edu/s/lda97/Mup19_ONE-seq) and Mettl7b (http://genome.ucsc.edu/s/lda97/Mettl7b_ONE-seq) can be accessed at UCSC Genome Browser online sessions. All source codes for data analysis are available at Zenodo https://doi.org/10.5281/zenodo.7293917.All high-throughput RNA sequencing data as well as transcript quantifications have been deposited at the Gene Expression Omnibus under accession number GSE194271. Genome browser views for Cyp2c70 (gkac1136_Supplemental_FilesClick here for additional data file.


The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure–function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields. This massive research effort is inspired by the immense potential that RNA offers to basic research and in clinical settings. RNA is a natural biopolymer that is highly integrated in a plethora of mechanisms essential for life and thus, understanding RNA’s folding, structure, and functions would allow for the rational design of RNA-based therapies and nanodevices suitable for biomedicines In order to advance the applications of RNA in the fields of nanotechnology and nanomedicine, an interdisciplinary effort must be undertaken. To further this goal, the ISRNN has hosted a biennial meeting series, entitled “Biomotors, Viral Assembly, and RNA Nanobiotechnology”. At these events, researchers gather from across the globe to share and discuss their findings in each of these areas, with the most recent boasting presentations from over 15 countries by 68 independent researchers. This critical review aims to cover recent research highlights and perspectives in selected topics; namely, we discuss advancements in the fields of biomotors and biomachines, nanopores, nanozymes, viral frameshifting elements, RNA structure prediction and assessment, and RNA nanotechnology.22.12.1.1The 2021 Nobel Prize in Physiology and Medicine was given to Professors David Julis and Aderm Patapoutian for their discoveries of receptors that sense heat and pain. These breakthroughs launched intense research activities that lead to a rapid increase in our understanding of how our nervous system senses changes in temperature and mechanical stimuli. NompC is a TRP  channel that is in the same family as the capsaicin receptor TRPV1. NompC is also a special mechanosensitive ion channel responsible for the sensation of touch and balance in Drosophila melanogaster in vitro methods. These findings were further validated by Prof. Michael Krieg’s research group via an in vivo C. elegans behavior experiment C. elegans was controlled by two ion channels. Straight-forward movement was controlled by the TREK2 channel, a mechanosensitive potassium channel that can sense membrane stretching and transmit electric signals. The omega turn movement of C. elegans was found to be controlled by the NompC channel, which is stimulated by cellular compression C. elegans can only go straight while the TREK2-knocked out C elegans can only perform the omega turn movement. Therefore, they summarize an interesting physical model of C. elegans. When the C. elegans moves straight, the TREK2 channel opens due to the stretch of nerve cells while NompC remain closed. On the contrary, when s C. elegans performs an omega turn, the sensory cells are compressed, opening the NompC channel while TREK2 is closed. These findings showed that NompC plays key roles in sensing touch and proprioception in C. elegans, which will provide promising logic element for designing smart C. elegans.The Shu group’s experimental collaborators, Prof. Zhiqiang Yan’s group, found, using the cell-attached patch clamp method that positive pressure acted on the NompC expressed by Drosophila melanogaster S2 neuro cells, which simulated the compression of the gating spring on NompC The push-to-open gating mechanism of NompC represents a special mechanism for sensing the compression of sensory cells, which is different from other traditional mechano-sensitive ion channels that mainly rely on membrane surface extension and respond to cell expansion. This model provides a promising area for designing smart biosensors in response to mechanical stimuli. Although the sequence and structure of other TRP ion channel family members, such as TRPV1 and TRPV4, is similar to NompC, whether the gating mechanism of these TRP channels obeys the push to open model remains an open challenge for structural biology. In the near future, the humans will begin to expand their explorations of space. The low gravity conditions in space pose a large challenge to the health of astronauts. Astronauts will face quite different health challenges from those living on earth. The relative studies on the gating of TRP channels under extreme conditions such as low gravity will give new insight to solve these problems.2.1.2A key characteristic of “living beings” is movement. These motion activities are usually performed by ATPase motors 2.22.2.1Nanopore technology has demonstrated its ability to detect fine molecular details, allowing the study of subtle changes in protein or RNA conformation While recent efforts to sequence proteins at the single-molecule level using nanopores have shown promise 2.2.22O2, which is abundant in tumor cells, to abstract electrons from the surroundings and thus induces apoptosis or necrosis of tumor cells. The catalase-like activity converts H2O2 into O2, which is useful to attenuate tumor hypoxia. The superoxide dismutase-like activity is useful for scavenging O2•−, providing the nanomaterials with anti-oxidant activity. Owing to their enzyme-like activities, the nanomaterials have potentials in tumor chemodynamic, anti-hypoxia, and antioxidant protection therapies  mechanism to direct synthesis of their replicase proteins, a critical switch in their replication program that can be therapeutically targeted The −1 PRF is a kinetically driven process. Elongating ribosomes are forced to pause over heptameric “slippery sequences” by metastable frameshift stimulating elements (FSEs) on mRNAs, typically pseudoknots e. g., 3–4 orders of magnitude, decreases in viral copy numbers First demonstrated using the yeast “Killer” virus system Although numerous academic and private entities are currently targeting the −1 PRF signal of SARS-CoV-2, the challenge of drugging a highly dynamic RNA structural element situates these efforts at the cutting edge of technology. Further, while −1 PRF is an essential regulatory element for many viruses, each viral −1 PRF signal is unique, likely necessitating developing −1 PRF signal-specific drugs for every virus. Additionally, as it is becoming clear that −1 PRF is used to regulate human gene expression 2.3.2Despite the ongoing vaccination campaigns across the world, we are entering the third year of the COVID-19 pandemic with new waves of infection by SARS-CoV-2 and witnessing mutations that continue to evade our immune system while producing alarming symptoms in increasing cohorts and parts of the world. It has become clear that simple solutions are difficult and that more fundamental scientific knowledge, as well as clinical evidence, is required to address current and future viral infections that inevitably will arise.Fortunately, the level of scientific cooperation and advances we have witnessed since early 2020 offers hope. Besides successful vaccine development efforts, progress on unraveling the complex and multifarious biophysical aspects of the virus life cycle and infection trajectory has helped us describe how the virus hijacks our own protein-synthesis machinery into making viral proteins efficiently and propose new lines of defense against the deadly disease it carries. These insights into the life cycle of the virus and mode of action are invaluable for further development of drugs and other strategies to combat current and future viral epidemics.Investigations of the RNA viral agent itself are crucial for understanding how the RNA invader replicates itself, is translated by the human ribosomal machinery, assembles, and synthesizes a suite of viral proteins that enable the continuation of its invasive trajectory. Importantly, RNA-targeting therapeutics and vaccines can disarm the origin of the infection rather than its products and potentially be more effective in the long term. However, the complexity of the RNA molecule and the lagging science about its modeling, imaging, and drug screening, as compared to proteins, poses several challenges. With technological improvements in RNA delivery systems, the rise of CRISPR-based gene editing systems Of particular interest by many groups is the RNA frameshifting element (FSE), a small region in the open reading frame ORF1a,b region A of the When encountering ORF1b, out of register with respect to ORF1a, the ribosome backs up one nucleotide in the 5′ direction to define a different sequence of codons . BecauseThe FSE’s crucial role in viral protein synthesis makes it an excellent target for therapeutic intervention . Already, several groups have explored FSE structure by modeling The Schlick group focuses on better understanding the conformational landscape of the FSE using a combination of complementary graph-based modeling and chemical reactivity and other experimentsMany have characterized the FSE of SARS-CoV-2 as a 3-stem hairpin (H-type) pseudoknot with colinear Stems 1 and 2 intertwined via a pseudoknot and perpendicular Stem 3. This association has persisted from the SARS-CoV FSE characterization three viable structures of the FSE: two 3-stem pseudoknotted RNAs , and one unknotted, 3-way junction RNA (3_5) (Developing further and applying the RNA-As-Graphs (RAG) graph-theory tools for handling pseudoknots NA (3_5) B. The fl2.42.4.1Predicting RNA secondary structure has long been dominated by folding-based algorithms, which require a scoring function coupled with a minimum-searching algorithm. These predictions, however, are often limited to nested and stacked canonical base pairs , while ignoring other hydrogen-bonded bases stabilized by tertiary interactions. The ignorance reflects the difficulty to predict “tertiary” base pairs without knowing three-dimensional structures. Structure-stabilized base pairs include un-nested (pseudoknot), noncanonical and isolated lone base pairs as well as base triplets. Thus, a gold-standard dataset for these “tertiary” base pairs requires experimentally determined three-dimensional structures. However, there are only a few thousand RNA structures that have been solved so far. Such a small number of RNA structures makes it challenging to apply deep learning to RNA structure prediction., without information of homologous sequences) can provide a significant improvement over the stagnant accuracy of secondary structure prediction by folding-based algorithms in recent years. The most significant improvement is in “tertiary” base pairs such as pseudoknots, noncanonical and lone base pairs.Recently, Singh et al. showed that the above problem can be addressed by using a highly accurate but approximate database of RNA secondary structures for initial training https://sparks-lab.org.To further improve the above prediction, it is necessary to incorporate sequence conservation and co-variation information as in proteins. However, unlike proteins, 4-letter coded RNA can easily lose their sequence identity in evolution. Thus, it is necessary to incorporate structural information in homology search as has been done in INFERNAL Despite the above progress, many challenges remain. For example, RNA homologies remain difficult to find, for long RNA sequences (>1000 nucleotides) in particular 2.4.2Development of new nanotechnologies based on engineered RNA structures depends on knowledge of the fundamental properties of RNA folding and its conformational dynamics Viruses ubiquitously use structured RNA elements as part of their overall infection strategy, and for regulating the formation of the capsid Tyr). TLSTyr are found at the 3′ end of specific single-stranded positive-sense RNA viral genomes Tyr are tRNA mimics that undergo programmed conformational changes, but their structure had remained elusive for decades.Examples of structured, multifunctional, and perhaps conformationally dynamic RNA elements are the tyrosine-accepting tRNA-like structures (TLSTyr RNA (∼55 kDa) in both its unbound form and bound to Tyr-AARS  that can compute the SHAPE profile from a 3D RNA structure. Combined with computer simulations that can generate a pool of candidate 3D structures, SHAPER would provide a novel, SHAPE data-driven, method to efficiently identify the native structure or near-native structures by removing SHAPE-incompatible structures from the pool of candidate structures.The talk from Shi-Jie Chen’s research group from the University of Missouri, Columbia, focused on a novel method for data-assisted RNA structure prediction. As a readily available alternative to X-ray footprinting of RNA for assessing secondary and tertiary structures which does not require the use of a synchrotron, selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) provides insights into the flexibility of nucleotides The key product of the model is a new estimator of SHAPE reactivity for each nucleotide in a given 3D structure Overall, this new model displays substantial promise in the assessment tested. For a structure, the Pearson correlation is calculated between the predicted SHAPE profile and the experimental profile. The result  of the SThere are two major challenges to the SHAPER model. First, the SHAPE data used to train the model are from various systems and may rely on different data processing of the raw data. Therefore, the model may suffer from the quality of the training data set. This has been shown by a previous attempt of using machine learning to predict the SHAPE profile from a 3D RNA structure 2.4.4Small angle X-ray scattering (SAXS) experiments are valuable in providing information about size and shape of RNA molecules and thus optimally complement other biophysical techniques Molecular dynamics simulations allow for RNA dynamics to be investigated at atomistic resolution and are currently a standard tool for structural biology studies In a recent work Overall, these results show that both conformational dynamics and solvent effects play an important role in SAXS spectra of RNA, and indeed, neither of the two effects can separately account for the experimental results. The importance of the solvent might be ascribed to the highly charged nature of RNA. At the same time, the observation that rich and heterogeneous conformational ensembles are required to match experiments underlines the importance of RNA dynamics in analyzing data obtained in solution.Given the relative simplicity of small (and wide) angle X-ray scattering experiments (SAXS and WAXS) when compared to other biophysical techniques, as well as their capability to capture dynamical effects, the application of these techniques to RNA systems is gaining momentum. At the same time, several methods are emerging to combine them with molecular modeling techniques so as to extract the maximum amount of information. We believe that this field will see other methodological developments in the next few years. In addition to methods based on maximizing entropy, such as the one discussed above, other techniques based on the maximum parsimony principle, such as sample-and-select, have been recently applied to RNA 2.52.5.1RNA folds into thermostable structures and nanocomplexes with defined stoichiometry, size, and shape from one or multiple RNA oligo strands Methods for examining the motion and movement of RNA utilize nuclear magnetic resonance (NMR) RNA moves to change conformations in the presence of a ligand during the binding of proteins, or when undergoing co-transcriptional dynamic folding and restructuring, demonstrating movement during enzymatic behaviors in accordance with the induced fit model of substrate ligand interactions 2.5.2Strand displacement is a reaction between DNA or RNA strands that is essential to dynamic nucleic acid nanotechnology, and has been used in nucleic acid circuits designed for molecular computing, as well as in synthetic biology and molecular sensing The Sulc group has studied toehold mediated strand displacement reaction (TMSD) While the kinetics and thermodynamics of the TMSD reaction between DNA strands has been studied in detail for DNA nanotechnology via computational and experimental studies, there has not been a systematic study of such systems experimentally carried out when RNA strands are involved. Experiments have also not been carried out for hybrid reactions, where either RNA invades a DNA duplex, thus creating RNA-DNA hybrid, or when DNA strands invades an RNA duplex. The Sulc group has performed an experimental study where they varied the length of the toehold for RNA TMSD, as well as introduced mismatches between the invader and the substrate. Through this work, it was found that increasing toehold length speeds up the reaction exponentially, as was previously observed for DNA. Introducing mismatches slows the reaction, and the closer the mismatch is to the interface of the toehold / incumbent strand, the larger the slow-down. For a six-nucleotide toehold, it was observed that RNA invading an RNA duplex is faster than RNA invading a DNA duplex. For a toehold of length 3 nucleotides, they found RNA invading DNA to be faster than RNA invading RNA.It should be noted that these studies are just a first step towards understanding the strand displacement kinetics that involves hybrid DNA-RNA systems. Such systems are still not integrated into available simulations and the design tools that our community uses to design and test strand displacement circuits. The studies which involve invasion (of DNA or RNA) into DNA-RNA hybrid circuits have not been performed yet, and testing of effects of different sequences and positioning on the 5′ or 3′ end of substrate is also needed for better understanding of RNA TMSD reaction. Finally, while the thermodynamics of DNA and RNA duplexes has been carefully studied over the years, and is currently incorporated into widely used dynamic programming tools for secondary structure prediction such as NUPACK or RNAStructure, the free energies of fully complementary DNA-RNA hybrids were measured in the above work. The thermodynamics of mismatches in DNA-RNA hybrids has still not been measured.2.5.3As was revealed by the COVID-19 pandemic, there are numerous biological challenges that we are not prepared for With traditional small molecule drugs, there are two main components -- the dianophore that defines drug’s biodistribution and pharmacokinetics, and the pharmacophore that determines its targeted function and biological responses. Therefore, even minor changes to a chemical composition of a small molecule drug can have drastic effects on its function and distribution and consequently, would require its complete re-evaluation On top of being used as therapeutics, nucleic acids can now be employed as nanoscaffolds to coordinate simultaneous delivery of multiple different TNAs that are intended for synchronized action in the same diseased cell Due to the intended applications, it becomes important to understand how NANPs interact with the human innate immune system, evolutionary equipped with the diverse set of pattern recognition receptors (PRRs) that act specifically against non-self nucleic acids. During the extensive course of systematic studies in clinically relevant model systems, several important factors responsible for NANPs’ immune recognition have been discovered Mechanistic studies helped to disseminate how NANPs can be recognized by the immune cell machinery to trigger the observed immune responses. For example, endosomal TLR7 and TLR9 and a cytosolic RIG-I have been identified to be important regulators of NANPs’ immune recognition by human immune cells Overall, NANPs technology introduces a unique class of therapeutics that bring new possibilities for immunotherapies and targeted drug delivery with their structural versatilities. These particles have the potential to interact with human immune system in unique ways and, if designed correctly, can become a basis for a new class of safe and effective therapeutics. The knowledge and experience we gained by now are testimony to NANPs' potential. Further studies of how NANPs produce the observed immunological effects will pave the way for novel applications.33.1via the Zoom webinar platform. The first day of the conference presentations were focused on viral nanomachines and vaccines. Throughout these sessions, speakers presented their research on molecular machines and other platforms to understand viral mechanisms to produce better antiviral therapeutics including SARS- CoV-2. This day included a variety of computational and experimental results, highlighting the synergy between these two approaches to solving biochemical problems.The conference was successfully delivered over a period of four days, December 12–16, 2021, The second day of the conference was themed around RNA nanotechnology and therapeutics. Much of RNA’s unique structural and functional properties were exhibited here. There is a tremendous effort to understand and predict the structure and function of RNA in therapeutic applications using computational methods to model RNA platforms. One of the highest barriers to overcome in RNA therapeutics is delivery into the cytoplasm. Researchers presented current carrier options and the about the search for alternative methods. Applications of RNA nanotechnology were also discussed, in particular their uses in gene therapies, diagnostics, and targeted delivery were highlighted.Day three looked into modeling nanostructures for health and life and engineering molecular machines to mimic motor proteins. There was also significant interest in nanozymes to perform enzyme like functions for therapeutics against cancers and viral infections. The design and function of anti-cancer RNA therapies were discussed at length, including methods for effective delivery and the uses of small-interfering RNAs. It was also shown how computational modeling helps to inform the understanding and design of RNA based platforms by improving our ability to predict RNA structures and interactions.The final day of the conference focused more heavily on biomotor imaging, nanopore sensing and DNA packaging systems. The design of nanopores for various molecular functionalities, including sequencing, was discussed, with talks highlighting the ability of nanopores to function with both proteins and nucleic acids. Methods for using nanopores to monitor reaction kinetics were also shown. DNA delivery methods into cells were discussed in depth and attendees were shown how, by utilizing molecular machines, DNA can be packaged and excreted from viruses and virus-like particles. There was also substantial attention given to the structure, functions, and mechanisms of ATPase Motors.Lewis Rolband, Damian Beasock, Yelixza Avila, and Leyla Danai (University of North Carolina at Charlotte) moderated the conference through the assignment of presentation permissions to speakers, enforcing time requirements, transitioning between sessions, introducing session moderators and keynote speakers, and assisting with any minor technical difficulties that arose during the event.The core mission of the International Society of RNA Nanotechnology and Nanomedicine (ISRNN) is to promote the most recent headway in RNA nanotechnology while facilitating the exchange of information and formation of interdisciplinary collaborations among researchers around the world. Founded in 2016, the society focuses heavily on the applications of RNA nanotechnologies as RNA therapeutics, biomedicines, diagnostics, and imaging. The leadership team of the ISRNN is comprised of more than 30 distinguished researchers from seven countries, with members across the globe. In order to further their primary objectives of disseminating the latest research and connecting the researchers behind these advancements, the ISRNN hosts regular conferences. In addition to the Biomotors, Viral Assembly, and RNA Nanobiotechnology conference series, the ISRNN hosts other meetings, such as their 2020 Webinar Series, which had speakers and attendees from at least 10 countries. Members of the ISRNN are given priority to organize meeting programs, speak at all meetings hosted by the society, receive discount for publications at ISRNN sponsored journals, and have access to a variety of benefits such as free RNA Nanotechnology books and privileged publications.The 3rd Biomotors, Viral Assembly, and RNA Nanobiotechnology conference was a resounding success that brought researchers together from across the globe through a convenient online only format. This format allowed for the conference to have a much greater reach than an in-person event would have, and, as such, it brought together as many researchers as possible in these and related fields to share highlights of their most recent work and catalyzed the discussion and development of new ideas. RNA has powerful therapeutic potential; by highlighting recent advances and connecting researchers, the ISRNN hopes that RNA nanotechnology and nanomedicines can reach their full potential as quickly as possible.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


BLVRB depletion accelerated epithelial-mesenchymal transition (EMT), cell migration and invasion. In contrast, BLVRB overexpression was associated with reduced EMT and cell migration and invasion in CCA. BLVRB suppression activated Notch signaling, and activated c-Notch enhanced EMT by upregulating Snail expression levels, thereby increasing cell migration and invasion in CCA. Our results identified an unexpected function of BLVRB in CCA migration and invasion through the regulation of Notch/Snail signaling.Cholangiocarcinoma (CCA) is one of the most lethal types of solid tumors worldwide. Lymph node metastasis is common in the early stage, which is associated with recurrence and reduced survival time after CCA resection. The molecular pathogenesis of CCA is complex and requires extensive investigation. It involves multiple genomic alterations and the dysregulation of signaling pathways. Biliverdin reductase B (BLVRB) is a non-redundant NAD(P)H-dependent biliverdin reductase that regulates cellular redox status by reducing biliverdin to bilirubin. This study aimed at describing the biological functions and molecular mechanisms of BLVRB in human CCA. Prognostic clinical data showed that low expression BLVRB was associated with poor prognosis and lymph node metastasis.  It is the most common biliary tract malignancy and the second most common primary hepatic malignancy Notch gene can lead to increased Notch signaling. In healthy tissues, the Notch cascade transmits information from the outer to the inner part of the cell, integrating this information into appropriate developmental or physiological responses, such as those involving Wnt/β-catenin signaling and transforming growth factor-β Accumulating evidence has indicated that the Notch cascade, a highly conserved pathway in most multicellular organisms, is a crucial signaling cascade in many cancer types, and aberrant Notch signaling has been implicated in CCA In this study, we investigated the biological functions and molecular mechanisms of BLVRB in human CCA. We found that BLVRB was downregulated in CCA. Genetic studies have shown that the knockdown of BLVRB-mediated Notch signaling affects tumor cell migration and invasion in CCA. In addition, clinical research shows a strong association between low BLVRB expression levels and a poor prognosis in CCA. We conclude that BLVRB suppression promotes Notch activity, which is important for tumor cell dissemination. BLVRB represents a potential therapeutic target for this rare disease.2 at 37 °C.Clinical samples from 188 CCA patients who underwent radical surgery at Zhongshan Hospital  between 2012 and 2015 were analyzed in this study. Patients were included in the study according to the following criteria: (1) CCA was diagnosed by the department of pathology, (2) the patient did not receive preoperative chemotherapy or radiotherapy, and (3) the patient underwent radical surgery. Patient data were excluded if (1) the clinical records were incomplete or (2) follow-up information was missing. The tumor specimens were identified by biliary specialists and resected by pathologists. Patient characteristics and clinical parameters, including sex, age, tumor size, tumor number, lymph node metastasis status, liver cirrhosis, and tumor differentiation status were obtained from medical records and re-estimated by two pathologists and surgeons. The use of human tissue samples and clinical data was approved by the Ethics Committee of Fudan University . All patients involved provided their informed consent. The CCA cell lines, RBE and HCCC9810, were purchased from the Shanghai Branch Cell Bank of the Chinese Academy of Sciences . Human 293T, RBE, and HCCC9810 cells were cultured in RPMI-1640 . All media were supplemented with 10% fetal bovine serum  and cells were cultured under a humidified atmosphere with 5% COAll biopsy samples taken from the core of CCA tumoral tissues and peritumoral normal tissues were formalin-fixed, paraffin-embedded, and placed on glass slides. The slides were baked, deparaffinized, rehydrated, and endogenous peroxidase activity was blocked. Next, 5% bovine serum albumin was used to block nonspecific antigens. After antigen retrieval, the tissue sections were incubated overnight at 4 °C with anti-BLVRB , anti-cleaved Notch1 , or anti-Snail  antibodies. After washing with phosphate-buffered saline, the slides were incubated with an anti-rabbit IgG antibody conjugated with horseradish peroxidase. Finally, the sections were stained with hematoxylin, dehydrated, and covered with coverslips. To evaluate the immunohistochemistry (IHC) data, we used a composite expression score ranging from 0 to 12 that was calculated as the product of the percentage of stained cells and the intensity of staining. The percentage of staining was graded as: <5% (score 0), 5-25% (score 1), 26-50% (score 2), 51-75% (score 3), and 76-100% (score 4). The intensity of staining was graded as negative (score 0), weak (score 1), moderate (score 2), or strong (score 3). Multiplication of the two scores was regarded as the final staining score BLVRB shRNA1 (GCGGTGCAAGCAGGTTACGAA) and BLVRB shRNA2 (GAAGGCTCATGGTGTGGACAA), which were cloned into a pLKO.1-puro vector. Lentiviruses were generated by cotransfection of one of the above recombinant plasmids with packaging plasmids (psPAX2 and pMD2G) into 293T cells using polyethyleneimine . To establish stable cells, the transfected cells were treated with 4 mg/mL puromycin for 48 h after lentiviral infection.To express BLVRB proteins, the corresponding open reading frame cDNA was cloned into a lentiviral recombinant pCDH-puro vector. The shRNAs used in the present study included Cells were lysed on ice for 20 min in radioimmunoprecipitation assay lysis buffer  supplemented with a protease inhibitor cocktail  and a phosphatase inhibitor cocktail . The protein concentration of each sample was measured using a bicinchoninic acid assay kit . The proteins were subsequently separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with a primary antibody and then with a secondary antibody. The blots were visualized using an ImageQuant LAS 4000  with an enhanced chemiluminescence assay. The following antibodies were used: anti-BLVRB , anti-BLVRA , anti-E-cadherin , anti-Claudin-1 , anti-N-cadherin , anti-Snail , anti-ZEB1 , anti-cleaved Notch1 , anti-non-phospho (active) β-catenin , anti-non-phospho (active) β-catenin , anti-β-actin , and horseradish peroxidase-conjugated secondary antibody .Total RNA was isolated using TRIzol reagent  and cDNA was synthesized using a PrimeScript RT Reagent Kit , with a mixture of oligo dT and random primers, after genomic DNA elimination. mRNA expression levels were measured by real-time PCR using an ABI-7500 system  and a 2×SYBR Green qPCR Master kit  according to the manufacturer's instructions. Relative expression levels were calculated by determining the threshold cycle values of the samples. All data were normalized to β-actin as an internal control.BLVRB forward, 5ʹ- CTCATGGTGTGGACAAGGTCGT-3ʹ and reverse, 5ʹ-CATCACAGCCACGTACTTCAGG-3ʹ; β-actin forward, 5ʹ-GCGGGAAATCGTGCGTGACATT-3ʹ and reverse, 5ʹ-GATGGAGTTGAAGGTAGTTTCG-3ʹ; E-cadherin forward, 5ʹ-CGAGAGCTACACGTTCACGG-3ʹ and reverse, 5ʹ-GGGTGTCGAGGGAAAAATAGG-3ʹ; N-cadherin forward, 5ʹ-TGCGGTACAGTGTAACTGGG-3ʹ and reverse, 5ʹ-GAAACCGGGCTATCTGCTCG-3ʹ; Snail forward, 5ʹ-TCGGAAGCCTAACTACAGCGA-3ʹ and reverse, 5ʹ-AGATGAGCATTGGCAGCGAG-3ʹ; and Slug forward, 5ʹ-TGTGACAAGGAATATGTGAGCC-3ʹ and reverse, 5ʹ-TGAGCCCTCAGATTTGACCTG-3ʹ.The primers used for real-time PCR analysis were as follows: After total RNA was extracted, mRNA was isolated using oligo (dT) magnetic beads and cut into small fragments for cDNA synthesis. Libraries were generated using a NEBNext Ultra RNA Library Prep Kit  for an Illumina system, following the manufacturer's instructions. Sequencing was performed using a HiSeq XTEN platform .4) were suspended in 200 μL of serum-free Dulbecco's modified Eagle's medium (DMEM) and seeded on the upper chamber. The lower chamber was filled with DMEM supplemented with 10% FBS. After incubation at 37 °C for 20 h, the cells that migrated to the lower chamber were fixed with methanol for 15 min at room temperature and stained with 0.1% crystal violet for another 15 min. The medium surface in the upper chamber was carefully wiped with a cotton-tipped applicator. Cells that migrated to the lower chamber were counted in five non-overlapping fields and photographed. For invasion assays, the bottom chamber was coated with Matrigel basement membrane matrix .Transwell assays of tumor cells were performed in 24-well transwell plates with an 8-μm pore size, according to the manufacturer's instructions . For tumor cell migration/invasion assays, cells , as previously described CBF1-luciferase reporter construct and a Renilla luciferase reporter plasmid. Luciferase activity was examined 24 h after transfection using a dual-luciferase reporter assay system  according to the manufacturer's instructions. Firefly luciferase activity from the CBF1 reporter construct was normalized to the control Renilla luciferase included in the kit. Luciferase activity was expressed as the fold increase relative to unstimulated conditions.To assess the effect of BLVRB on Notch-mediated transcriptional activity, we used a traditional dual-luciferase assay consisting of four t-test or analysis of variance (ANOVA) was used to assess the differences between the control and treatment groups. Survival curves were plotted using the Kaplan-Meier method and compared with a log-rank test. All assays were repeated at least three times. The differences were considered to be statistically significant at P < 0.05.Statistical analyses were performed using SPSS 13.0 statistical software . A chi-squared or rank-sum test was used to investigate the relationship between BLVRB expression and clinicopathological factors. A two-tailed Student's BLVRB levels were lower in tumor tissues than in peritumor tissues . According to IHC staining, BLVRB was localized in the cytoplasm and nucleus of bile duct tissues. In normal peritumor biliary tissues, we observed a strong expression level in 42.3% (80/189), moderate expression level in 28.0% (53/189), weak expression level in 25.9% (49/189), and negative expression in 3.7% (7/189) . The expression level of BLVRB in CCA tissues was significantly lower than that in normal peritumor biliary tissues. None of the CCA tissue samples showed a strong BLVRB expression level, 8.7% (16/184) showed a moderate expression level, 84.2% (155/184) showed a weak expression level, and 7.1% (13/184) of samples were negative for BLVRB expression . To illustrate the clinical relevance of BLVRB expression in CCA, patients were grouped according to the expression level of BLVRB . According to the Kaplan-Meier analysis, the overall survival (OS) rate of the group with low BLVRB expression levels was lower than that of the group with high BLVRB expression levels . This suggests that the downregulation of BLVRB is associated with poor prognosis in patients with CCA. A low BLVRB expression level was strongly associated with lymph node metastasis (Table P = 0.016). No association was observed between BLVRB expression level and gender, age, tumor size, tumor number, vascular invasion, differentiation, or liver cirrhosis. Next, we divided patients with CCA into two groups according to lymph node metastasis and assessed the BLVRB expression pattern by IHC. The IHC scores were lower in patients with lymph node metastasis than in patients without . Therefore, BLVRB levels seem to be negatively associated with lymph node metastasis.We compared the transcriptomes of 11 paired tumor and peritumor tissues and found differentially expressed genes in CCA. BLVRB in HCCC9810 and RBE cells, and the efficiency of BLVRB knockdown was verified by western blotting . Low ays Fig. A and 4 BSnail is a typical downstream Notch target gene BLVRB depletion on Snail expression  produces BV by heme degradation, while BLVRs generate BR by conversion of BV. Two BLVRs, BLVRB and BLVRA, are encoded in humans. These enzymes share little sequence similarity, though their structures are typical of the short-chain dehydrogenase/reductase superfamily Heme is one of the most abundant molecules in the body and its core function is the synthesis of hemoglobin/myoglobin, which is involved in the transport of OCompared to BLVRA, few studies have considered the function of BLVRB. In the present study, BLVRB impaired CCA migration and invasion via Notch signaling. However, the physiological functions of BLVRB remain unclear. Recent evidence in humans has implicated redox-defective BLVRB in a thrombopoietic mechanism determining hematopoietic lineage fate. BLVRB is highly expressed in multiple organisms. It is vital for normal organ function, except for hematopoietic and erythrocyte/platelet functions. In this study, we found that low levels of BLVRB expression in clinical specimens were associated with poor prognosis and that BLVRB overexpression inhibited the migration and invasion of CCA cells. Moreover, the suppression of CCA cell migration and invasion by BLVRB may be dependent on catalytic activity (data not shown). Future research should attempt a more detailed characterization of BLVRB-regulated metabolites and description of the molecular events in BLVRB-regulated tumorigenesis.In the human liver, the Notch signaling pathway is the key regulator of normal cholangiocyte differentiation Homo sapiens CBF1, Drosophila melanogaster suppressor of hairless, and Caenorhabditis elegans Lag-1) protein family. In the absence of a Notch signal, RBPJ remains bound to Notch target genes, where it represses transcriptional output The Notch signal transduction cascade results in the proteolytic processing of the Notch receptor and the subsequent assembly of a transcriptional coactivator complex containing the NICD and the transcription factor, RBPJ. RBPJ is also known as CBF1 (C promoter binding factor 1), KBF2 (H-2K binding factor-2), and RBPJκ . RBPJ belongs to the CSL (BLVRB participated in the activation of the Notch pathway. As in many other cancer types, the activation of Notch signaling led to EMT induction in CCA cell lines, thus conferring tumor progression. In summary, we found that BLVRB depletion promoted CCA cell migration and invasion by activating Notch signaling, which is a novel function of BLVRB. However, this mechanism requires further elucidation, as it is a potential target for the prevention and treatment of CCA.In conclusion, the knockdown of Supplementary figure.Click here for additional data file.