N-terminal truncated sequence (TwCDA-S) had chitin deacetylase and chitinolytic activities after expression in Escherichia coli. High-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC-MS) indicated that TwCDA and TwCDA-S could catalyze the deacetylation of oligosaccharide (GlcNAc)5. TwCDA had higher deacetylase activity, and also catalyzed the deacetylation of the β-chitin polymer. A dinitrosalicylic acid (DNS) assay showed that TwCDA-S had high chitinolytic activity for (GlcNAc)5, and the optimal reaction temperature was 35 °C. Liquid chromatography combined with time-of-flight mass spectrometry (LC-coTOF-MS) detected the formation of a N-acetylglucosamine monomer (C8H15NO6) in the reaction mixture. Altogether, we isolated a chitin deacetylase from a marine diatom, which can catalyze the deacetylation and degradation of chitin and chitin oligosaccharides. The relevant results lay a foundation for the internal regulation mechanism of chitin metabolism in diatoms and provide a candidate enzyme for the green industrial preparation of chitosan and chitin oligosaccharides.β-Chitin is an important carbon fixation product of diatoms, and is the most abundant nitrogen-containing polysaccharide in the ocean. It has potential for widespread application, but the characterization of chitin-related enzymes from β-chitin producers has rarely been reported. In this study, a chitin deacetylase (TwCDA) was retrieved from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database and was heterologously expressed in vitro for functional analysis. The results showed that both the full-length sequence (TwCDA) and the  N-acetylglucosamine (GlcNAc) is the most abundant renewable polymer in the ocean [Thalassiosira and Cyclotella in centric diatoms are the most important producers of marine chitin [Thalassiosira , the chitin content in the cell wall of T. weissflogii was the highest, indicating that T. weissflogii might have a strong ability to synthesize chitin and is an excellent material for the exploration of chitin-related enzyme resources and chitin derivatives.Chitin, β-1,4)-linked ,4-linkedhe ocean . It is ahe ocean . Its nathe ocean . The chahe ocean ,7. Previhe ocean ,9. Diatohe ocean ,11,12. Che ocean . Thalasse chitin ,15. Throe chitin  found thChitin and its derivatives are used in medicine, pharmaceuticals, cosmetics, the food industry, agriculture, environmental protection, etc. However, due to the strong hydrophobicity of chitin, its application is greatly limited, and its economic value has not been fully appreciated . In contSogatella furcifera and found that three of them were associated with molting defects and high mortality with nymph–adult molting. Li et al. [EScDA-l gene in Eriocheir sinensis, which resulted in an abnormal ultrastructure of the cuticle, suggesting that EsCDA-l is indispensable for molting. However, present research is still largely restricted to bacteria, fungi, and crustaceans, and there have been few reports of chitin deacetylase in eukaryotic microalgae. To our knowledge, the only report on functional CDA in diatoms noted the overexpression of two diatom CDA genes (PtCDA and TpCDA) in Phaeodactylum tricornutum to address the ambiguous characteristics of CDAs from P. tricornutum and T. pseudonana [Chitin deacetylase  belongs to the carbohydrate esterase 4 (CE4) superfamily. It participates in the degradation pathway of chitin and has a wide range of substrate specificity. It can catalyze the deacetylation of both chitin oligosaccharides and insoluble chitin . The inii et al.  knocked eudonana .CDA (TwCDA) was identified from T. weissflogii, and its sequence structure, evolutionary relationships, and enzymatic activities were integrally characterized. The results verified the role of TwCDA in the degradation of chitin in vitro, which lays a foundation for determining the underlying regulation mechanism of chitin metabolism in diatoms, and they provide a new candidate enzyme for the preparation of chitosan and chitooligosaccharides.In this study, a putative T. weissflogii (9021) was acquired from the Microalgae Collection Center at Ningbo University, Ningbo, China, and grown in an optimized f/2 liquid medium provided by Shanghai Guangyu Biological Technology Co., Ltd.  (−2 s−1) with shaking at 100× rpm. Cells at the exponential phase were collected by centrifugation, flash-frozen in liquid nitrogen, and stored at −80 °C for further experiments., China) . Cells wT. weissflogii by a method optimized from the report by Noishiki et al. [g. The supernatant and pellets were decolorized by adding methanol , deproteinized with 5% KOH , demineralized with 0.34% NaClO2  and 0.1 N HCl , and purified with 1% HF . At the end of each step, the sample was centrifuged at 19,000× g to remove the supernatant, and finally it was dehydrated at 80 °C and stored at −80 °C [Thus far, there is no pure beta-chitin standard for sale on the market. β-Chitin was extracted from i et al. . The celt −80 °C . An α-chT. weissflflogii frozen sample in TRIzol reagent . Then, 1.5 mL TRIzol reagent was added to the frozen algal pellet; it was placed at room temperature for 5 min and centrifuged at 10,000× rpm, at 4 °C. The supernatant was added to a new 2 mL tube with 300 μL chloroform and incubated at room temperature for 10 min prior to centrifuging at 10,000× rpm for 5 min. The supernatant was added with 750 μL isopropyl alcohol and centrifuged at 10,000× rpm, 4 °C, for 10 min. Finally, 500 μL 75% ethanol was added and the supernatant was removed by centrifugation at 8500× rpm at 4 °C to obtain RNA. The extracted RNA was reverse-transcribed into cDNA using a reverse transcription kit , with 200 μL of a reaction mixture of RNA, gDNA eraser, and 2 × SPARK scriptII RT Plus Master Mix. The mixture was incubated in a thermal cycler with the following protocol: 42 °C for 5 min, 50 °C for 30 min, and 80 °C for 5 min. The synthesized cDNA can be directly used for subsequent PCR amplification.Total RNA was extracted by grinding the CDA homologous sequence  was retrieved from the T. weissflogii transcriptome dataset, using a CDA from T. pseudonana (TpCDA) [TwCDA sequence was synthesized with codons optimized for E. coli . In order to improve the subsequent heterologous expression, we also removed the transmembrane helices to amplify the N-terminal truncated TwCDA-S. TwCDA-S-F (5′-CGGGATCCATGGAAGATACCATGGCAGC-3′) and TwCDA-S-R (5′-CCAAGCTTCTCGAGCAGGATCATAGTCG-3′), containing a BamHI and a HindIII restriction site, respectively, were designed for PCR amplification. A total of 20 μL PCR reaction mixture was used, with 1 μL of each primer, 10 μL of 2 × Phanta Master Mix , 2 μL of cDNA template, and 6 μL of ddH2O. The PCR amplification procedure was as follows: 95 °C for 30 s; 35 cycles of 95 °C for 10 s, 55 °C for 5 s, and 72 °C for 10 s; and 72 °C for 1 min. The products were sequenced and analyzed using BLASTN.A full-length  (TpCDA)  to query (TpCDA) . The fulT. pseudonana and Cyclotella cryptica by CLUSTALW . ProtParam was used to predict the physical and chemical parameters (molecular weight and isoelectric point) of TwCDA [http://www.genome.jp/tools/motif/, accessed on 27 February 2022) was applied to analyze the motifs in the Pfam and NCBI-CDD databases. EUK-MPLOC and PSORT were jointly used to predict the TwCDA subcellular localization [http://www.cbs.dtu.dk/services/SignalP-4.1/, accessed on 27 February 2022) for signal peptide and signal anchoring sequence prediction [http://www.cbs.dtu.dk/services/TMHMM/, accessed on 27 February 2022) was used to predict transmembrane helices. The secondary structures were illustrated by ESPript [https://www.imicrobe.us/#/projects/104, accessed on 10 July 2022), PhycoCosm , and PLAZA . Homology alignment was conducted using BLASTP. MEGA X was used to construct the neighbor-joining tree based on the Jones–Taylor–Thornton (JTT) model and the Gamma-distributed matrix-based model with 1000 bootstrap replicates [The TwCDA amino acid sequence was aligned with CDA proteins from of TwCDA . The Motlization ,41, withediction . TMHMM S ESPript , and the ESPript . For phyplicates .TwCDA was synthesized into pET-32a vectors with a His-tag . At the same time, TwCDA-S with a reduced transmembrane helix structure, which was amplified as an BamHI/HindIII fragment by PCR, was cloned into the pET-32a vectors with His-tag, resulting in the fusion plasmid pET-TwCDA-S.The pET system was used to express TwCDA and TwCDA-S in vitro. The codon-optimized full-length sequence of E. coli cells (BL21 (DE3) pLysS and Transetta (DE3)) were transformed with the recombinant expression plasmids (pET-TwCDA and pET-TwCDA-S). The E. coli cells were then incubated in 1 L of Luria–Bertani (LB) medium with 100 µg/mL of ampicillin and 20 µg/mL of chloramphenicol at 37 °C. When OD600 reached 0.6, 0.1 mM IPTG was supplemented, and the induction conditions were changed to 120× rpm and 15 °C. After 24 h of incubation, the transformed cells were harvested by centrifugation at 4500× rpm at 4 °C for 30 min. Then, the cells were disrupted by sonication for 25 min (3 s on and 5 s off cycle) . One EDTA-free protease inhibitor tablet  was added to the 35 mL extracts prior to the centrifugation at 12,000× rpm at 4 °C for 45 min. The supernatant was then filtered through a 0.45 μm filter membrane to obtain a crude enzyme solution. The crude protein concentration was measured using a BCA protein quantification kit .The 2O solution three times, CDA target bands were visualized by incubating the membranes in ECL reagents with the ChemiDoc XRS Imaging System and the Image Lab 6.0 software .The protein concentration in crude protein was determined with a BCA kit  according to the manufacturer’s instructions. We then boiled the samples for 10 min in loading dye , and loaded 60 μg of the crude extract onto 12% acrylamide gels for separation by SDS-PAGE. Primary  and secondary  antibodies were used for the Western blotting test via the iBind™Flex Western System  at room temperature for 4 h, following the manufacturer’s protocol. After washing with ddHc. 3 mg) crude proteins. The reaction was carried out at 250× g, 37 °C for 48 h, and terminated by freezing the mixtures in liquid nitrogen [5 and β-chitin. Each experiment was performed with two replicates. Deacetylation activity was determined by measuring the released acetic acid via high-performance liquid chromatography (HPLC) with an acetic acid content detection kit . The experimental conditions were as follows: an Ultimate AQ-C18  HPLC column  was used for detection at 30 °C with a 210 nm UV filter, and the flow rate was set to 0.4 mL/min. Each acetic acid assay was performed with three replicates. A standard curve generated from 175, 87.5, 17.5, 8.75, 1.75, and 0.875 μmol/mL acetic acid was used to calculate the acetic acid concentration in the sample, using Equation (1). Gas chromatography-mass spectrometry (GC-MS) was used to further detect whether acetic acid was produced in the product.Crude protein extracts of TwCDA and TwCDA-S were prepared with a phosphate buffer containing 20 mM sodium phosphate pH 7.5, 20 mM imidazole, 500 mM sodium chloride, 5% glycerol, and a protease inhibitor. The reaction volume was set to 10 mL, containing 50 mM triethanolamine (TEA) pH 7.0, 2 mg/mL substrate, and 1 mL . To obtain the optimal reaction conditions for the TwCDA, we measured the enzyme activity at various temperatures . Each experiment was performed in triplicate, and a statistical analysis was conducted with SPSS 26.0 . The chemical formula prediction of the reaction products was detected by liquid chromatography combined with time-of-flight mass spectrometry (LC-coTOF-MS), with the inactivated crude enzyme group and blank group 5 aqueous solution) as the control groups.Some chitin deacetylases also have the ability to degrade chitin sugar chains . The dinp < 0.05 was considered a significant difference; p < 0.01 was considered an extremely significant difference.The SPSS version 26 software was used to analyze the experimental data statistically, and one-way analysis of variance was used for the data. The results were expressed as mean ± standard deviation (SD). T. weissflogii was used to extract chitin. The content of chitin obtained from the supernatant and cell pellets was 0.047 g and 0.0762 g, respectively. The total yield of chitin was calculated to be 20.53% of the total cell mass (fresh weight). Infrared spectrum detection showed that the chitin extracted from T. weissflogii had a single peak at 1625 cm−1, while α-chitin standard forms a double peak at 1620 cm−1 and 1652 cm−1  encoded 658 amino acids, which had a predicted molecular weight (MW) of 71.15 kDa and an isoelectric point (pI) of 6.21. TwCDA-S (885 bp) encoded 295 amino acids, with a predicted MW of 31.89 kDa and a pI of 5.11. TwCDA has a signal anchor, whereas TwCDA-S does not. Secondary structure prediction showed that both CDAs had a similar composition, with a predominance of the α-helix structure (~45%). The TwCDA tertiary structure reveals a single domain (residues 403–651) adopting a fold reminiscent of an (α/β)8 topology  and C. cryptica (PLAZA Reference Sequence: CC00G25130). Multiple-sequence alignments indicated that TwCDA has 95% and 96% query coverage and 38.61% and 39.31% identity with T. pseudonana CDA and C. cryptica CDA, respectively 5 group were detected by GC-MS, and it was verified from the molecular weight of the characteristic peak that acetic acid  was indeed produced in the mixture (The deacetylation activities of TwCDA and TwCDA-S on (GlcNAc) by HPLC . The con by HPLC . The HPL HPLC 5 was used as the substrate, TwCDA showed significantly higher chitinolytic activity than the control groups, with activity 1.48-fold (against pentamer) and 1.46-fold (against β-chitin) higher than the control groups (p < 0.05), respectively , respectively  . Taking the amount of enzyme required to produce 0.1 μmol NAG in 1 h as the unit of enzyme activity, we drew the specific activity bar chart with the number of units of enzyme activity per milligram of enzyme . We compectively A. Comparectively A. This ias 35 °C B. It was8H15NO6) .CDA gene from T. weissflogii, a high producer of β-chitin, and explored its structural and enzymatic properties.Diatoms represent an important source of β-chitin, which has been reported to be a better material for application compared with α-chitin . β-Chiti8 barrel fold with six or seven strands and encompass a mononuclear metalloenzyme that employs a conserved His–His–Asp zinc-binding triad to carry out acid/base catalysis [8 topology, which is consistent with CDA structure studies. The full-length sequence contains 658 amino acids, but there are 403 amino acid residues in its N-terminal region, whose tertiary structure is not predicted, denoting the unstructured nature of this region [5 and the partially acetylated chitosan polymer [Most CDAs have a conserved (α/β)atalysis ,51,52. Is region  reported polymer . TogetheSynedra acus, which expanded the taxonomic distribution of CDA in diatoms and provided new sources of novel CDA enzymes. A phylogenetic tree found that TwCDA was closely related to T. pseudonana CDA and C. cryptica CDA. The evolutionary position of C. cryptica is adjacent to T. pseudonana. This was consistent with the finding that T. pseudonana likely descended from a freshwater ancestor in Cyclotella [The putative CDA sequences from the MMETSP, PhycoSM, and PLAZA databases show that the CDAs are dominated by sequences from marine centric diatoms and the Thalassiosirales contain the most abundant CDA homologues. Sequence homology alignment found that CDAs were retrieved from two independent divisions: centric diatoms and pennate diatoms. In pennate diatoms, we first reported the presence of CDA homologous sequences in clotella .T. pseudonana CDA–GFP fusion proteins is in the ER. These transmembrane helix structures may cause difficulties for our protein expression in vitro. However, producing a prokaryotic membrane protein in the simple, flexible, and inexpensive E. coli system has already proven successful in many cases [E. coli [E. coli through the rational design engineering of strains or by modifying target proteins. In addition, Brosson et al. [Encephalitozoon cuniculi, produced a recombinant form of the EcCDA protein that was slightly truncated to exclude transmembrane helices, and obtained a high yield of purified protein. Therefore, we removed as many transmembrane helices as possible on the basis of preserving the complete functional domain and expressed it in E. coli. We simultaneously expressed TwCDA with the full length of ORF and TwCDA-S with the transmembrane structure removed, and carried out enzyme activity detection. It was found that TwCDA-S had a higher expression level and higher chitinolytic activity. Although TwCDA retains its transmembrane helical structure, its expression level is acceptable and it has higher deacetylase activity.TwCDA was predicted to have a structure of eight transmembrane helices. The prediction of subcellular localization showed that TwCDA was potentially located in the endoplasmic reticulum. This was in accordance with the results of Shao et al. , who repny cases ,56,57. U[E. coli . To addr[E. coli  found thn et al.  studied Francisella tularensis (35 °C) and Coprinellus congregatus (35 °C) [We found that TwCDA-S had optimum chitinolytic enzyme activity at 35 °C. At present, the optimal conditions for the enzyme activity of chitinase have been studied in animals, plants, bacteria, and fungi. According to the previous results, the optimal temperature is 25–65 °C ,62,63. T (35 °C) ,65.Pestalotiopsis sp., and revealed that this enzyme is inactive against insoluble α-chitin and β-chitin, slightly active against colloidal chitin, and active against soluble chitosan polymers. In our study, HPLC results revealed that TwCDA could catalyze both (GlcNAc)5 and β-chitin, but TwCDA-S can only catalyze (GlcNAc)5. We speculate that the N-terminal TMH or signal-anchoring sequence is essential for the catalysis of the polysaccharide. This speculation was supported by the previous report that a signal anchor is important for fungal CDAs to function efficiently at the cell surface [Previous results have shown that most CDAs had high activity on chitooligosaccharides, water-soluble chitosan, and colloidal chitin, but low or no activity toward chitin polymer ,66. Rese surface . The eff2 or GlcNAc [Most of the reports on CDAs are about its deacetylation activity; there have been few reports on its chitinolytic activity . Chitinar GlcNAc . Thus, tr GlcNAc . Since wr GlcNAc ,67,71. HIn summary, a putative TwCDA gene was identified and characterized, and the catalytic products of TwCDA recombinant protein were explored. To our knowledge, this is the first analysis of chitin deacetylase with both chitin deacetylase and chitinase activities from diatoms, and the first report of a CDA that could catalyze β-chitin polymer. Although we used a crude TwCDA preparation instead of a pure enzyme in this study, it is worth mentioning that crude enzymes are often used in industry . TherefoThalassiosira weissflogii was heterologously expressed in vitro for functional analysis. The results showed that both the full-length sequence (TwCDA) and the N-terminal truncated sequence (TwCDA-S) had chitin deacetylase and chitinolytic activities after expression in Escherichia coli. TwCDA and TwCDA-S could catalyze the deacetylation of oligosaccharide (GlcNAc)5. TwCDA had higher deacetylase activity, and also catalyzed the deacetylation of the β-chitin polymer. TwCDA-S had high chitinolytic activity for (GlcNAc)5, and the optimal reaction temperature was 35 °C. Altogether, we isolated a chitin deacetylase from a marine diatom, which can catalyze the deacetylation and degradation of chitin and chitin oligosaccharides. The relevant results lay a foundation for the internal regulation mechanism of chitin metabolism in diatoms, and provide a candidate enzyme for the green industrial preparation of chitosan and chitin oligosaccharides. However, our enzyme activity reaction was carried out on the basis of a crude enzyme solution, and a sufficient quantity of active recombinant enzyme was not obtained after purification. We need to find solutions regarding how to retain the enzymatic activity during the purification process and further analyze the enzyme kinetic parameters for a deeper understanding of TwCDA.In this study, a chitin deacetylase (TwCDA) from


RNA Plasticity: Novel Structures, Shapes, Modifications, and Functions” was constituted with a focus on various aspects of plant RNA biology including novel structures, modifications, and its biological roles as regulatory molecules. In applied biotechnology, novel designs drive technological innovations and advancements that help resolve contemporary society’s significant challenges.This Research Topic on “The diversified roles played by the different forms of RNA molecules in the past few decades exemplify its significant role in cellular regulation, as it acts as a messenger of information from genomic DNA to the production of distinct proteins as well as regulates unique biological processes via flexibility of RNA structure and functions. The diversity of design and roles of RNA types has recently increased rapidly . In addiBragg and Rieske investigated hydroponically applied exogenous dsRNA translocation and retention in a commercially and ecologically important hardwood tree, white oak . dsRNA recovery in treated seedling material was confirmed by gel electrophoresis and Sanger sequencing. Both approaches demonstrated that white oak tissues absorbed and translocated exogenous dsRNAs. According to the findings, such root uptake of dsRNAs in hardwood seedlings may protect single trees against certain tree-feeding pests or illnesses.For instance, the double-stranded RNA (dsRNA) produces host-specific gene silencing in many insects; such dsRNAs are being utilized to reduce pests in agricultural and horticultural crops. Although many forest pests are susceptible to RNAi, their use in forests has gotten little attention. Before forest pest suppression, dsRNA specificity, efficacy, and behavior must be determined. In their article Huang et al. report that m6As in the transcriptome of hexaploid wheat spikelets during the flowering stage. Authors discovered they are evenly distributed across the A, B, and D subgenomes, while their extents and placements fluctuate across homologous genes. Homeologous genes with similar expression levels are enriched in m6A-methylated genes. The effects of m6A methylation on mRNA translation are negatively related to mRNA expression levels. m6As in coding sequences and 3’-UTRs inhibit mRNA translation, but those at start codons and 5’-UTRs enhance it. The presence of m6A-containing mRNAs in “translation” and “RNA transport” processes and pathways suggest that they may influence the translation of genes involved in translation regulation. Small RNAs (miRNA and phasiRNA) inhibit translation more than m6A methylation, and no synergistic effect was seen. It is proposed that m6A methylation status triggers translation regulation amplification. These data suggest that m6As controls translation in hexaploid wheat.RNA N6-methyladenosine (m6A) is a modification in eukaryotic mRNAs . m6A wasMarin-Sanz and Barro present their investigation of the transcriptome of E82 grain and leaf tissues during grain filling in order to better understand regulatory pathways in response to gliadin silencing. Gluten proteins provide wheat dough with viscoelastic properties ((Marin-Sanz and Barro). Proline and lysine-histidine transporters were down- and up-regulated in the leaf. Lysine-rich globulins compensate for gliadin silencing in the RNAi line, indicating that these proteins are regulated independently of the other SSPs. These findings help to interpret the complex TF network during grain loading by explaining protein compensatory mechanisms.operties  but can Shinde et al. highlight the importance of RNA methylation in plant development in their review. Plants primarily methylate RNA with N6-methyladenosine (m6A). Mutations influence Arabidopsis phenotypic changes in writers, erasers, and RNA methylation readers. Unmethylated TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1-messenger RNA does not pass from shoot to root, whereas methylated TCTP1-mRNA does , growth, and development. Moreover, the increasing evidence of RNA plasticity in various forms and modifications in higher organisms indicates that the highly evolved regulatory mechanisms are established, which control and integrate with other complex processes like epigenetic modifications via RNA editing, splicing, and structure. We hope this special Research Topic will provide you with significant insights into the versatile roles of RNA in plant biology.UK: Conceptualization, Formal Analysis, Funding acquisition, Validation, Writing – original draft, Writing – review & editing. RD: Formal Analysis, Resources, Writing – review & editing. LT: Formal Analysis, Investigation, Methodology, Writing – review & editing.


The European Society of Human Genetics—Young Committee (ESHG-Y) aims to support young human geneticists by developing strategies and programs for better education and creating a strong network in all European countries. In this report, we present the ESHG-Y projects conducted since its conception. We organized the educational sessions at the ESHG Annual Conference, the European Dysmorphology Meetings, and a virtual session in collaboration with the European Board of Medical Genetics (EBMG). Also, the ESHG-Y regularly promotes relevant activities and succeeded in creating an active network of young geneticists. Our representatives have a supportive role in well-known organizations such as: ESHG Board, ESHG Scientific Program Committee, ESHG Education Committee, EBMG, ERN-Ithaca, Unique - Rare Chromosome Disorder Support group, Orphanet, EuroGEMS, MOOC BIG and more. Taking into consideration all activities and ongoing projects, we can state that the ESHG-Y successfully achieves its objectives and brings young professionals together. The European Society of Human Genetics—Young (ESHG-Y) was launched with great enthusiasm as a committee, in June 2019 by human genetic trainees with common goals and a shared vision regarding human genetics [“Active members” are elected by all trainee members of the ESHG for a period of 2 years  and are responsible for all decisions taken by the committee;“Consultants” are elected based on the request of the committee from former ESHG-Y Committee members; additionally they are not allowed to vote and their role is to supervise ongoing projects;“Associate members” are invited directly by the ESHG-Y due to the individual’s outstanding Curriculum Vitae and achievements to fill different skill gaps and are not allowed to vote.In the last year, significant adjustments have been made and currently, the structure of the committee is composed of different types of members Fig. :“Active In addition, the committee grants the position of “Officer” to any volunteer that is involved in our projects.ESHG-Y’s initiative allows trainees to have a significant impact by creating a strong network between all European countries and sharing professional experiences. Our mission is to represent and support the young European geneticists by developing strategies and programs that aim for a better education. We intend to create and implement new projects that will enhance the performance of young geneticists and sustain them to be heard, understood, and empowered. The efforts of our committee are centered on four main objectives: organize scientific events, ensure equal access to educational opportunities, create a professional network, and supporting junior geneticists to become leaders Fig. .Fig. 2ESSince our commencement in 2019, we have engaged with different entities and developed multiple projects that are in accordance with our mission and objectives. In the last 4 years, partnerships with highly regarded organizations have continued to grow, maintaining their positive long-term trend.Our main focus is to invest energy and resources into creating compelling educational opportunities for the young generation of human geneticists. Relevant scientific events to which we contributed and had a decisive role were: the ESHG Annual Conference and the European Dysmorphology Meeting.The ESHG-Y organized a workshop focusing on professional development during the 2020 ESHG Annual Conference. Ever since then, we have organized an annual scientific session addressed to the young generation of geneticists. We participated with three posters at this conference between 2020–2022 Table . AdditioStarting in 2021, two ESHG-Y members have been involved in organizing the annual European Dysmorphology Conference which is held by ERN-ITHACA.All junior geneticists have the right to equal access to education. However, due to limited access to infrastructure, knowledge, or human resources, becoming a well-trained geneticist can be challenging in a developing country. ESHG-Y tries to fill this gap by developing collaborations with important entities that offer educational opportunities in the field of human genetics.From 2020 ERN-ITHACA has offered the ESHG-Y the opportunity to contribute with presentations at their biannual ITHACA Board Meeting; subsequently, collaborations with the EuroDysmorpho Meeting and UNIQUE were successfully established.UNIQUE is a registered charity that offers support and knowledge to families and caregivers of patients affected by a rare chromosome disorder; more than that it provides a network for patients and professionals. UNIQUE and ERN-ITHACA aim to offer accurate and simplified information on rare genetic disorders for non-native English-speaking families. For this purpose, the ESHG-Y has coordinated since March 2021 the recruitment of native speakers of different languages on a voluntary basis as proofreaders for the UNIQUE automated translated guides. The volunteers were recruited from the European Young Geneticist Network (YGN). We have since succeeded in enrolling 32 volunteers that contributed with translations in French, German, Italian, Lithuanian, Polish, Portuguese and Spanish.ESHG-Y also collaborated with the European Joint Program on Rare Disease (EJP RD) by providing support with the development of the “Diagnosing Rare Diseases” MOOC for which we recruited six beta-testers and five mentors in 2021.st of December 2021, together with Dr. Jonathan Berg (Chair of the ECMGG Exam Committee) and Dr. Laura Pölsler (Secretary of the ECMGG Exam Committee), we organized a special virtual session called “Becoming European Board Certified in Medical Genetics and Genomics”. The discussion aimed to highlight the importance of holding the European Certification in Medical Genetics and Genomics (ECMGG) degree and to provide essential information about this exam.Also in 2021, our committee was offered the opportunity to have a liaison member in the European Board of Medical Genetics. On the 1DNA Day, April 25th, is commemorated internationally as a celebration of genetics. The DNA Day Essay and Video contest, supervised by Dr. Christophe Cordier, intends to challenge European high school students to reflect on the importance of human genetics. The ESHG-Y committee became involved as judges for the first time in this event in 2021, providing support with the evaluation of the essays and videos. The topic suggested by us, “How can DNA help us to discover ancient human history?” was elected for 2022.The EuroGEMS website provides impressive digital educational resources related to genetics and genomics. The online material is addressed to genetic specialists, non-genetics professionals, students and the public , 4. The Since 2021, we have had two representatives in the ESHG Education Committee (ESHG EduComm) to support their projects. The ESHG International Mentorship Programme annually provides five young professionals the opportunity to undertake a 1 week educational and observational placement within a European genetic institution. The Programme was launched in 2022 and received applications from different continents. Members from the ESHG-Y were involved in the election process of the awardees and had a supportive role.In 2022, the ESHG’s Podcast “Genetic Sounds”, coordinated by Dr. Sofia Douzgou, was launched with great excitement. This podcast discusses different topics in genetics that involve us all and is addressed to both professionals and non-professionals. Current and former ESHG-Y representatives were invited to make significant contributions in the 2nd episode “Let’s talk about sex in genetics” and the 3rd episode “Let’s talk about access to genetic services”.In addition to these partnerships, in the future the committee will also seek to produce and disseminate biannual newsletters, to keep young members of the ESHG updated on upcoming training courses, events, and development opportunities.Having the opportunity to share experiences and opportunities between developed and developing countries in Europe will surely lead to important progress in the field of human genetics and will inspire and engage young geneticists to reshape the future of human genetics.The ESHG-Y committee is present on social media through an active Facebook page and a Twitter channel (@eshg_young), both used for disseminating information regarding our projects. Furthermore, since 2019, the ESHG-Y manages the Young Geneticist Network (YGN) which aims to allow its members to share and discuss information related to human genetics [To further promote our activities a joint manuscript 2022) from the YGN and ESHG-Y Committee was published in the European Journal of Human Genetics, a journal with broad international readership 022 from .In 2022, the ESHG-Y was invited to summarize its accomplishments in the ESHG Newsletter, this way highlighting the importance of involving the young generation in such organizations.For 2023 our Committee has been invited to offer support for the Young Investigator Forum (ICHG2023 Meeting) affiliated to the African Society of Human Genetics (AfSHG).Having good leadership that promotes the ESHG-Y’s mission, provides direction and is crucial for engaging the young generation in projects that aim to equally develop the genetic field in Europe and beyond.In order to represent the young human geneticists’ interests representatives from the ESHG-Y are involved in different committees like the ESHG Board, ESHG Scientific Program Committee (SPC), ESHG Education Committee (EduCom), ESHG Social Media Committee and European Board of Medical Genetics (EBMG). All these committees have the purpose to organize and promote different scientific events and facilitate access to a diverse portfolio of genetic courses and educational material.In 2022, the ESHG Journal offered section editor and editorial board positions for ESHG-Y representatives, while the ESHG Public and Professional Policy Committee (PPPC) offered us a permanent position as an observer.Former, current, and future members of the ESHG-Y gain experience and notability by being involved in the ESHG-Y Committee, whilst also developing friendships and collaborations with renowned geneticists from Europe. This allows them to become leaders and forces of change in their own national scientific communities and to augment the development of the human genetics field within their own countries.The “ESHG Observership for Young Geneticists” program (first edition in 2023) offers annual financial support (2000 euro) for five young human geneticists who wish to observe specific areas of clinical care or laboratory techniques at highly regarded European human genetics departments for a maximum period of 1 month;The “ESHG-Young European Committees Group” encourages young geneticist to organize in formal committees associated to European human genetic professional organizations, and aims to connect them and develop a strong network;The “ESHG-Young International Leadership Group” has the objective to build a strong partnership with all young international professional organizations and work together on projects; furthermore one of its main activities is to co-create virtual sessions regarding common topics periodically.For the near future, we will continue to promote ESHG-Y’s activities and attract new members and we would like to create a network of young geneticist committees in all European countries. We will be seeking to develop further activities:Moreover, we will continue our established collaborations and partnerships and we aim to engage many more young geneticists focusing on the opportunities and advancements in European genetic education.In the last 4 years, the ESHG-Y has succeeded in achieving its objectives by developing ongoing projects and partnerships. With the growth and expansion of the genetic field comes the need to inspire, interconnect and encourage the younger generation to have a major professional impact on the equitable development of this speciality in Europe.


Quantification of the dynamics of RNA metabolism is essential for understanding gene regulation in health and disease. Existing methods rely on metabolic labeling of nascent RNAs and physical separation or inference of labeling through PCR-generated mutations, followed by short-read sequencing. However, these methods are limited in their ability to identify transient decay intermediates or co-analyze RNA decay with cis-regulatory elements of RNA stability such as poly(A) tail length and modification status, at single molecule resolution. Here we use 5-ethynyl uridine (5EU) to label nascent RNA followed by direct RNA sequencing with nanopores. We developed RNAkinet, a deep convolutional and recurrent neural network that processes the electrical signal produced by nanopore sequencing to identify 5EU-labeled nascent RNA molecules. RNAkinet demonstrates generalizability to distinct cell types and organisms and reproducibly quantifies RNA kinetic parameters allowing the combined interrogation of RNA metabolism and cis-acting RNA regulatory elements. SU) or 5′-bromo-uridine (BrU) that get incorporated during transcription into newly synthesized RNAs 8. Subsequent steps involve either the chemical isolation of metabolically labeled RNAs before sequencing or the bioinformatic inference of labeling through PCR-generated mutations after sequencing 10. These methods have substantially simplified the methodology, but all share common limitations. Alternative splicing is a fundamental cellular process with biological and clinical relevance in which exons from the same gene are joined in different combinations, leading to different, but related, mRNA isoforms 11. However due to the limitation of using short-read sequencing, established methods cannot make high-confidence assignment of isoforms or collect information on individual RNA molecules. This prevents association of RNA kinetics with cis-acting transcriptional regulators such as alternative transcription start site and poly(A) site usage, post-transcriptional RNA modifications, and poly(A) tails, known regulators of RNA metabolism 12.The life cycle of RNA from transcription to decay is tightly regulated in cells. Unraveling of the kinetic parameters of RNA metabolism is critical for understanding gene regulation and cellular response to environmental cues. Existing methods for studying RNA dynamics in mammalian cells are based on short-read RNA sequencing (RNA-Seq) and involve the exposure of cells (pulse) to nucleoside analogs such as 4-thiouridine  that identifies individual 5EU-modified RNA molecules following direct RNA-Seq (dRNA-Seq). RNAkinet can analyze entire experiments in hours, instead of days that nano-ID does, and predicts the modification status of RNA molecules directly from the raw nanopore signal without using basecalling or reference sequence alignment. We show that RNAkinet generalizes to sequences from unique experimental settings, cell types, and species and accurately quantifies RNA kinetic parameters, from single time point experiments. Being able to interrogate whole RNA molecules, RNAkinet allows, for the first time, the combined interrogation of RNA metabolism of single isoforms and cis-acting RNA regulatory elements, such as alternative splicing, poly(A) tail length and post-transcriptional RNA modifications.24. Given that the half-life of the majority of RNAs ranges in the order of a few hours, it is expected that the majority of RNA molecules would incorporate a 5EU 26. We defined this as our positive dataset while RNA from HeLa cells cultured without addition of 5EU served as our negative dataset  as previously described  dataset . RNA froing data .27. Nano-ID comprises a fully connected neural network with 2,590,493 trainable parameters that include information from the raw electrical signal, basecalling and alignment that was trained on fewer than 700,000 examples  are subject to overfitting particularly when over-parameterized examples , bottom.n a read . We expln a read , while tbackbone . Transfe labeled , C.To ensure proper training, testing, and validation of RNAkinet, we followed a rigorous approach for data preparation. First, we performed basecalling and alignment to the human genome and subsequently separated the raw reads by chromosome. We isolated reads on chromosome 1 and 20, accounting for 8–9% and 2% of the total reads, to be used for testing and validation, respectively . This en32, padding with zeros or any value would interfere with actual values in the electrical current that have a physical meaning in the signal generated from the sequencer. To avoid this, other tools have used fixed length windows of the input signal 33. However, this is not ideal for the purpose of detecting 5EU modification, since the 5EU labeling efficiency per nucleotide is low (2–3%) 24 raising the possibility of a labeled sequence not containing the modification in a given window. To address this, we trained the network on single whole sequences without padding and designed our network to accept sequences of any length and share learned features across the entire sequence.Raw electrical signal from the sequencing device is encoded in a one-dimensional array with varying size dependent on read length. Varying length input can be challenging for NNs and is typically addressed by padding to the length of the longest input. Given that in a typical dRNA-Seq experiment read lengths vary dramatically from a few bases to several kilobases in length , most seOur training process involved randomly sampling from our positive and negative sets uniformly to ensure a balanced ratio for training. Since we used multiple datasets as negatives, those were sampled proportionally based on the number of reads they contained. Due to the large size of our dataset, we adopted a multi-read fast5 file format for quick loading, and we only loaded files when required to reduce the memory footprint. The same approach was used for inference, which ensured the network used the minimum amount of computational resources. Finally, we limited the input of the model to raw nanopore signal, not utilizing any additional information about the sequence, such as basecalling, alignment, or other metadata. This makes RNAkinet adaptable for future applications since it is independent of external basecaller or alignment software.To test the performance of RNAkinet, we first compared the distribution of predicted modification probabilities on the training data. Our results showed that both RNAkinet and RNAkinet-TL could robustly distinguish labeled RNAs and that they were learning the training data . We subsTo further explore the training process and validate that RNAkinet does not overfit to reads in the training chromosomes, we calculated the ROC for individual chromosomes and combined –E. We fo24. Since this is also a human cell line, we again tested on reads that aligned to chromosome 1 to ensure that RNAkinet had not previously seen the corresponding sequence space and thus performance would not be confounded by read sequence. Our results show that RNAkinet could successfully distinguish labeled RNA molecules along all replicates of K562 cells (24 were prepared with an earlier iteration of the ONT RNA sequencing kit (SQK-RNA001) the intricacies of which have not been modeled during training. Similarly, we found that RNAkinet had comparable precision, recall and accuracy on both datasets  or control (negative) 62 cells . Quantif62 cells . A minordatasets . Again, datasets –E.24 in which cells were subjected to heat shock at 42°C for 60 min at the presence of 5EU and performed differential expression analysis using DESeq2 34  site usage, poly(A) tail length and post-transcriptional RNA modifications simultaneously A prevalent limitation of machine learning tools is their potential inability to generalize and extrapolate findings to different systems. To address this, we adopted a thorough data preparation protocol aimed at achieving broad domain representation. Furthermore, we subjected RNAkinet to rigorous validation across a variety of distinct and completely independent experimental conditions, confirming its capability to generalize across various RNA sequencing kits, cell types, and organisms. This also highlights the robustness of the training process which coupled with lack of external dependencies introduces a path to future developments and updates as nanopore sequencing technology advances. We also placed particularly emphasis on model architecture to avoid overparameterization. By incorporating convolution and recurrent layers, RNAkinet captures long-range interactions inherent in the electrical signal while it avoids overfitting to training data thus dramatically reducing the parameter space and achieving high computational efficiency being able to process ~600,000 reads/h .Finally, we show that RNAkinet can quantify isoform-level RNA kinetic parameters that significantly correlate with measurements from methods that lack isoform-level resolution. This feature enables the study of RNA kinetics at unprecedented resolution allowing associations with post-transcriptional regulatory cues at single molecule resolution. We anticipate that RNAkinet will enable a novel set of study designs that will involve a holistic interrogation of RNA and gene regulation in cells.™) were cultured at 37°C, 5% CO2, 90% humidity in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (GeminiBio), 1% MEM-nonessential amino acids (Invitrogen), 2 mM L-Glutamine. iPSC-derived i3Neurons were cultured and differentiated as previously described 36. For metabolic labeling, cells were cultured in media containing 400 or 500 μM 5-Ethynyl-uridine (ThermoFisher) for 2 to 24 hours as indicated. Total RNA was extracted using TRIzol reagent (Invitrogen) according to manufacturer’s instructions followed by DNase I treatment (MilliporeSigma). RNA concentration and integrity were determined using a Nanodrop ND-1000 (ThermoFisher) and Qubit™ RNA IQ assay (ThermoFisher), respectively. Library preparation for direct RNA sequencing was performed as previously described 37 with modifications. Briefly, poly(A) RNAs were purified from 50 μg of total RNA using Oligo d(T)25 Magnetic Beads (New England Biolabs). 500 ng of poly(A) mRNA was used for library preparation using SQK-RNA002 sequencing kit (Oxford Nanopore Technologies). The final library was quantified using Qubit dsDNA High Sensitivity assay kit (ThermoFisher) and sequenced on a MinION device using FLO-MIN106 flow cells (Oxford Nanopore Technologies).NIH3T3 cells (ATCC CRL-1658) were cultured at 37°C, 5% CO2, 90% humidity in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% bovine calf serum (GeminiBio), 1% MEM-nonessential amino acids (Invitrogen), 2 mM L-Glutamine. Cells were passed weekly by gentle dissociation with trypsin-EDTA 0.25%. HeLa cells (ATCC CCL-2) and HEK-293T , and the rest were used for training. The first 5000 raw signal values from each read were cropped to avoid sequencing artifacts and any reads shorter than 5000 raw signal values or longer than 400000 raw signal values were discarded. The filtered reads were then normalized by median absolute deviation before being passed through the neural network.In order to prevent data leakage and overoptimistic results that could emerge from using k-fold cross-validation with random shuffling, the reads were split into training, validation, and testing sets based on a chromosome they originated from. For evaluating precision, recall, and F1 metrics, which are sensitive to imbalanced data, the splits were up-sampled to have a balanced ratio of positives and negatives. The reads were basecalled with Guppy 6.4.8, aligned to the Ensembl human genome (GRCh38) and transcriptome with Minimap2 To create a detection tool for 5EU modification, a convolutional neural network classifier was designed. It was trained with data from the training split described above and was optimized to perform a binary classification of raw nanopore signals into either modified or unmodified categories. The network was made up of multiple convolutional blocks extracting local patterns from the input signal, followed by a bidirectional recurrent layer with GRU units that aggregated the extracted information across the whole length of the sequence in both directions. These features were then pooled with max pooling, average pooling, and concatenated with hidden states from the last hidden states of the recurrent layers. To allow the network to accept signals of varying lengths without padding as an input, this pooling layer at the end of the network was utilized to aggregate information across the length dimension into a fixed-size vector. This vector was then fed into a small dense feed-forward network to predict the final 5EU modification score.39 and PyTorch Lightning frameworks. Snakemake 40 workflows were developed for the entire process of data splitting, model creation, and evaluation to be reproducible, scalable to large computational clusters, and adaptable to be used on newly sequenced datasets.For training the network on sequences of variable length, a batch size of size 1 was used. Additionally, to simulate minibatch learning, gradients were accumulated over 64 sequences. The network was trained with a learning rate of 0.001 and weight decay of 0.01 using the AdamW optimizer. The model was trained for 1000 epochs with early stopping on the validation set AUROC metric with threshold 0 and patience of 50 evaluation steps. The first 1000 learning steps were used as warm up steps where the learning rate was scheduled to linearly increase from 0 to the final learning rate of 0.001. To accelerate the network training, a single A100 Nvidia GPU was utilized. The implementation and training of the network were done using PyTorch To accurately capture the change in gene expression between control and condition experiments using 5EU detection, a metric called relative modification increase was introduced. This metric used the percentage of modified reads of a given transcript from both the condition and control experiments and calculated a relative increase in the condition experiment compared to the control. A positive relative modification increase indicated a higher prevalence of modified reads under the experimental condition, while a negative value indicated a decrease.Supplement 1


E. coli. We showed that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We found that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We proposed a physical barrier model in which bulky structures in 5’UTR prevent ribosome dislodging and thereby increase the translation output. This study provides biophysical insights into the regulatory role of 5’UTR structures in bacterial translation, highlighting their potential applications in tuning gene expression.Translation initiation in bacteria is frequently regulated by various structures in the 5’ untranslated region (5’UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aimed to explore how G4 formation in mRNA (RG4) at 5’UTR impacts translation using a T7-based in vitro translation system and in  In bacteria, the absence of a nuclear membrane necessitates rapid post-transcriptional regulations to enable quick responses to the environmental stimuli11. Untranslated regions (UTR) of RNA have emerged as key players in regulating translation initiation by presenting noncanonical structures to translational machinery or by recruiting proteins and enzymes that recognize RNA sequences, modifications, or structures13.Gene expression is a tightly regulated process to ensure efficient utilization of resources and adaptation to changing environments. This regulation occurs at various levels, including transcription, translation, and the level of mRNA and protein14. Second, bacterial 5’UTRs often harbor cis-acting regulatory elements, such as upstream open reading frames (uORFs), which stall the ribosome and control the access to downstream TSS. Additionally, bacterial 5’UTRs can serve as a platform for RNA-binding proteins and small RNAs that regulate translation efficiency. For example, small RNA coupled with an RNA binding protein Hfq can bind a 5’UTR to stimulate translation initiation or trigger mRNA degradation16.The 5’ untranslated region (5’UTR) of bacterial mRNA serves multiple functions that are critical for gene regulation and protein synthesis. First, it typically contains a conserved AG-rich Shine-Dalgarno (SD) sequence, located a few nucleotides upstream of the translation start site (TSS). The SD sequence base-pairs with the 16S ribosomal RNA (rRNA) to guide the binding of the small ribosomal subunit, providing a well-defined mechanism for initiating translation17. Several studies showed that secondary structures, such as pseudoknots and hairpin stem-loop that form across the SD sequence can inhibit translation by preventing ribosome binding19. Notably, temperature sensitive hairpins in the 5’UTR of E. coli can regulate translation by masking or unmasking RBS or start codon (AUG) to turn on or off translation initiation, respectively20. Similarly, riboswitches control translation through changes in mRNA conformation upon ligand binding, enabling rapid responses to environmental cues22. Specific sequence elements, such as purine-rich regions or G-quadruplexes, can also affect translation in a context-dependent manner. Previous studies demonstrated that an RNA G-quadruplex (RG4) structure located near the SD sequence inhibits the base pairing between 16S rRNA and the mRNA24. However, it remains unclear how and to what extent the 5’UTR structure impacts bacterial gene regulation.Furthermore, secondary structures within 5’UTR play a critical role in regulating RNA stability and translation efficiency. Specifically, co-transcriptionally folded structures can influence translation initiation and the rate of translation. Previous research on bacterial 5’UTR primarily focused on the ribosome binding site (RBS), which includes the SD sequence and a short range (10–20 nt) upstream and downstream of the SD region25. In this context, the transcribed mRNA bears a G4 structure at the 5’ end, which prompted us to investigate whether such G4 structures in RNA modulates translation outcome. G-quadruplexes form in single stranded DNA or RNA that harbors repetitive runs of guanines interspersed with non-guanine, loop sequences. Four guanine bases come together in a coplanar arrangement to form a tetrad, which stacks in multiple layers. The size and stability of the structure depend on the composition and the length of the loops26. Increasing evidence suggests that RG4 structures are involved in translation regulation in eukaryotes, often blocking translation initiation when present in the 5’UTR31. Some RG4 structures have also been shown to function as Internal Ribosome Entry Sites (IRES), stimulating translation independent of a start site34. Furthermore, computational studies have identified numerous PQS across prokaryotic species positioned non-randomly in non-coding RNA segment that precedes mRNA, indicating an evolutionarily conserved function of 5’UTR RG4 in bacterial genome37. While some studies demonstrate reduced translation by the 5’UTR RG4, it is not clear if different sequence composition and hence structure of RG4 would drive a different type and level of translational regulation.Our previous research has uncovered the role of potential G-quadruplex sequence (PQS) in non-template DNA in promoting transcription through co-transcriptional formation of R-loop and G4 structureE. coli translation. We inserted a series of PQSs in non-template DNA, upstream of a GFP reporter gene to allow for the formation of RG4 at 5’UTR. Using the T7 expression system, we measured in vitro transcription and subsequent translation in real-time, which were used to calculate the translation efficiency. We found that the presence of RG4 in the 5’UTR led to enhanced translation both in vitro and in E. coli. Longer loops within RG4 resulted in higher translation yield. Moreover, insertion of a hairpin upstream of an RG4 further increased translation. Taken together, we demonstrate that the translation enhancement scales with the size of the 5’UTR structures. We propose a mechanism by which the 5’UTR structures act as a physical barrier that may prevent dissociation of ribosome from the mRNA and thereby promote translation.In this study, we investigated the role of 5’UTR RG4 structures in in vitro translation assay which contains reagents for the T7 RNAP for transcription and E. coli translation system (38. We prepared DNA construct with T7 promoter followed by the ribosome binding site (RBS), a fluorescent reporter which encodes superfold GFP (sfGFP), and a transcription terminator sequence. We used our previously established protocol to measure the real-time transcription; DNA molecular beacon becomes fluorescent when annealed to a transcribed RNA which bears a complementary sequence  by the transcription signal (molecular beacon).To quantify the translation efficiency, we set up an n system 25, 38. Wsequence 39. In paread-out . Hence, 25. The initiation rate of transcription was quantified by taking the linear increase of fluorescence intensity in the initial phase of each curve  on translation, we inserted a potential G-quadruplex forming sequences (PQS) in between the T7 promoter and the RBS such that the PQS is 44 bp downstream of T7 promoter and 43 bp upstream from the RBS. The PQS was inserted into either a template (T) or a non-template (NT) strand for comparison. Hence, the PQS insertion in NT is expected to produce the G4-bearing transcript which can fold into RG4 while the PQS in T, and the scrambled control (C) sequence will not fold into RG4 . In agrech curve . Surpris control . The trandition. . The 4-f T and C , indicat T and C . This fu43. Based on the result obtained above, we asked if different PQS sequences produce various levels of translational enhancement. To focus on the effect of G4, we removed the sequence upstream of PQS and inserted a series of PQS with varying loop length without changing the guanine triplets . Despite varying levels of NMM signal acquired for different RG4s, single molecule FRET assay displayed that both small (short looped) and large (long looped) RG4 form a stable G4 structure without structural dynamics . All PQS-NT which result in RG4 containing RNA consistently led to higher translation product than its counterpart PQS-T construct  to calculate the folding energies of all positions except position 2, because RG4 folding cannot be accurately predicted by currently available tools. The folding energy, ΔG estimated for the positions 3, 4, and 3 + 4 were −5.6, 0.9, and −11.8 kcal/mol, respectively, indicating weakly folded state of the downstream sequence (Supplementary table 1). To avoid interference with the ribosome binding, we decided to vary sequence located upstream of PQS. We note that the position 1 sequence used in By comparing the results presented in What is the mechanism that enables 5’UTR structures to elevate translation in a size dependent manner? We reasoned that the 5’UTR structure may have an impact on either the ribosome or the mRNA. To define the mechanism, we set out to test four hypotheses: i) RG4 increases the ribosome binding affinity ; ii) RG446. This hypothesis posits that the mRNA containing RG4 structure (based on PQS-NT) will recruit more ribosomes than the mRNA without RG4   in viral translation, PQS-T) , resulti, PQS-T) –1, an RG, PQS-T) –2, a sin, PQS-T) –3, an RG, PQS-T) –4, a ssR, PQS-T) –6. If ou, PQS-T) . The tra, PQS-T) , suggestd was 367 (± 25), 364 (± 23), 370 (± 21) nM for NT, C and T respectively, indicating negligible difference in RBS accessibility among the three constructs . Strikingly, when applied to the translation reaction, DHX36 reduced translation in a dose-dependent manner  which were recorded simultaneously by the plate reader. The GFP intensity displayed the same orientation dependence of NT > C > T as we observed in vitro . We cloned six sets of plasmids with varying PQS sequences inserted either in NT or T and quantified the translation efficiency. The result reflects the same pattern as before; PQS-NT produces higher GFP signal than PQS-T and the translation further increases when bulkier PQS is inserted in NT  and the longer loop (199) RG4 via smFRET assay. Surprisingly, both RNAs showed a steady high FRET state, indicating a stably folded G4 structure . This evidence suggests that the G4 folding in RNA is inherently more stable than the G4 in DNA. To further weaken the RG4, we mutated the middle guanine to adenine to destabilize the core of RG4. Strikingly, the mutation constructs still showed a similar enhancement effect as the original RG4 , reflecting that the mutated RNA can still fold into a structure that can increase translational output. Taken the data together with the dual-hairpin result , translational enhancement up to 4–6 folds increase occurs regardless of the structure, but the strongest effect requires both stable folding and large size of the structures, for example, 177, 555 and 199 with a hairpin with a long stem.We find that RG4 with longer loops induces higher translational enhancement . While l24. Therefore, we inserted G4 at 46 bp upstream to SD to prevent such structural inhibition. Whether the level of enhancement relies on the distance between RG4 and RBS, or RG4 and other structures warrants future study.The effect of RG4 depends on its location. We found that translation was abolished when PQS was positioned at 10 bp upstream of the SD sequence (data not shown), likely by blocking ribosome subunit association to the RBS. This agrees with Holder and Hartig’s previous work which demonstrated that inserting G-quadruplex 20 bp upstream of the start codon decreased translation efficiency without changing transcription. Again, G4 likely inhibits the interaction of the 16S ribosomal RNA with the SD region56, in which an upstream hairpin provides a temporary position for the ribosomal 30S subunit to stay, waiting for the unfolding of the SD sequence. According to this model, the rate-determining step becomes the recruitment of ribosome subunit, which is slower than the waiting time for unfolding. In our case, however the extended sequence adjacent to SD 58. However, in our system, CTT is unlikely because T7 RNAP transcription rate (220 ~ 230 nt/s)60 is significantly higher than E. coli ribosome translation rate (42–51 nt/s) 61. That is, ribosomes will be loaded onto a nascent RNA post RNA synthesis rather than being coupled to transcription. In order to examine the effect in the presence of CTT, we also cloned the same 5’UTR sequence and GFP gene to an E. coli promoter PL–LacO system. Surprisingly, we still observed a huge increase of GFP signal in non-template construct than in template , suggesting the structural effect may still function in regular E. coli gene under CTT. However, the mechanism in a pure E. coli system should be studied more systematically in the future.We also considered that the translation enhancement may result from the coupled transcription and translation in E. coli system. We focused primarily on RNA G-quadruplex and hairpin structures. Although we examined varying sizes of both the RG4 and hairpin, our study opens a wide window of opportunity for future studies, for example, investigating the role of a pseudoknot structure positioned either upstream or downstream of RG4 or in between RG4 and RBS. Furthermore, we want to note that in all our experiments, PQS insertion into template (PQS-T) which produces C-rich transcript, strongly suppressed the translation efficiency both in vitro and in vivo experiments. This suggests an opposite function of C-rich RNA in down-regulating the gene expression. In conclusion, our study provides a new mechanism and function of 5’UTR mRNA in bacterial translation and provides a novel cloning scheme for tunable gene expression system.To summarize, we have demonstrated a size-dependent 5’UTR structure effect in translation enhancement in T7 and GFP reporter. The details were described in an earlier publication25. DNA sequences were listed in Supplementary Table 2. The recombinant plasmids were transformed into NEB-5α for DNA extraction and BL21-DE3 for E. coli expression assay. For dual-color reporter system, a mCherry gene was cloned from pET mCherry vector  into the GFP plasmid by NEB HiFi DNA Assembly kit. Linear DNA samples for in vitro translation were PCR-amplified from GFP plasmid. The T7 promoter forward primer and T7 terminator reverse primers (see Supplementary Table 2) were designed and ordered from Integrated DNA Technologies (IDT) for amplification. The amplified linear DNA held the T7 promoter, 5’UTR with PQS, GFP gene, and T7 terminator and was purified by gel electrophoresis and Gel Extraction Kit (QIAquick).All the DNA samples were started from the plasmid construction with a single 2, 0.5 mM CaCl2) at 37°C for 30 minutes. Later, the reaction was quenched by adding 1 μL 0.5 M EDTA, followed by inactivating at 75°C for 10 minutes. RNA was purified by Monarch RNA CleanUp kit (NEB).RNA samples were prepared by HiScribe T7 Quick High Yield RNA Synthesis kit (NEB) at 37°C overnight. Each reaction (20 μL) had 1 μg of DNA, NTP mix, T7 RNA Polymerase, and RNase free water. The overnight product was firstly digested by DNase I (0.1 U/μL) in DNase reaction buffer  and performed by TECAN Spart plate reader at 37°C. Each reaction (25 μL) was premixed with 55 ng linear DNA (4 nM), 400 nM molecular beacon (see Supplementary Table 2), RNase inhibitor murine (0.8 unit/μL), and 10 μL Solution A. To measure the RG4 formation, NMM was added in premixed solution at final concentration of 1 μM. The reaction was initiated by adding 7.5 μL Solution B and loaded on a 384-well plate . The Cy3, GFP, and NMM were excited at λex 545, 485, and 393 nm and detected at λem 570, 510, and 610 nm, respectively. Both excitation and emission were assigned with 10 nm slit size. The initiation transcription rate was quantified from the linear part (10 to 25 minutes) of the Cy3 intensity curve. The translation rate was quantified from the linear part (25 to 50 minutes) of the GFP intensity curve. Each rate was normalized to the transcription rate and translation rate of 10 hp control DNA construct, respectively. The normalized translation efficiency was calculated by dividing the normalized translation rate to the normalized transcription rate. The half-life was defined by the time that the intensity reached 50 percent of the plateau.Ensemble 2, 2 mM spermidine, 1 mM dithiothreitol), RNase inhibitor murine (0.4 unit/μL), T7 RNA polymerase (1.25 unit/μL), and 1 mM NMM. The reaction was initiated by adding NTP mix for a final concentration of 1 mM. Each reaction (100 μL) was loaded on 96-well transparent plate (Thermo Scientific). The data was collected at λex 393 nm (slit size 10 nm) and λem 610 nm (slit size 10 nm). For emission spectrum, the data was collected at λex 393 nm (slit size 10 nm) and λem 580–650 nm (slit size 10 nm).Ensemble in vitro transcription for real-time NMM measurement was performed by TECAN Spark plate reader at 37°C. Each sample was prepared with 1 nM linear DNA template in transcription buffer . RNA purification protocol was described in RNA Preparation. The reaction was performed with 55 ng of 10 hp cMyc-NT DNA and 5 μM of competitor RNA by plate reader at 37°C.RNA competition assay was modified from ex 640nm and λem 665nm with 10 nm slit size. The binding assay was conducted by incubating 400 nM beacon and RNAs of 10hp control, T, and NT at 37°C. RNA purification protocol was described in RNA Preparation. The data points were collected by titrating RNA concentration in a 2-fold series dilution from 1.75 μM to 24 nM. The Kd was fitted to the binding curve by OriginPro. For accessibility assay, each sample (10 μg of RNA) was mixed with 400 nM of the RBS beacon and incubated with or without heating treatment. The heated samples were incubated at 80°C for 5 minutes and cooled by 10°C intervals for 5 minutes until 37°C. After incubation, intensities of all the samples were measured by TECAN plate reader at 37°C.A molecular beacon complementary to the ribosome binding site (RBS) was ordered from IDT and labeled by Cy5 and quencher at each end (see Supplementary Table 2). The Cy5 intensity of beacon was measured at λin vitro translation, post-translation samples (20 μL) were treated with DNase I (0.1 U/μL) in DNase reaction buffer  at 37°C for 30 minutes. The reaction was quenched by 1 μL of 0.5 M EDTA, followed by heat inactivation at 75°C for 10 minutes. The DNA-free samples were diluted 10-fold, and 2 μL of each diluted sample was used to synthesize cDNA by ProtoScript II cDNA Synthesis Kit (NEB). The reaction was incubated at 25°C for 5 minutes, followed by 42°C for 1 hour and 80°C for 5 minutes. The cDNA was diluted 100-fold before qPCR measurement. The qPCR sample (20 μL) contained 1 μL 100-fold diluted cDNA, 10 μL SYBR Green Supermix (NEB), and 250 nM primers (see Supplementary Table 2). The data points of cycles were plotted in For E. coli strand with DHX36 plasmid was made in the lab and described in a previous publication52. The E. coli was inoculated in TB medium and grew overnight. Next day, the culture was diluted to OD600 of 0.01 and grew till OD600 of 0.6 at 37°C and induced protein expression with 1 mM IPTG at 14°C for overnight. The purification protocol followed previous publications52. Protein concentration was quantified by standard BSA (NEB) calibration curve by SDS-PAGE, and the aliquots of protein samples were stored in −80°C. For DHX36 titration assay, the protein was diluted by TNM buffer  to avoid the change of reaction volume and buffer condition. 1 μL of each titrated DHX36 and additional 1 mM ATP were premixed with solution A, and the reaction was initiated by adding solution B. The experimental protocol is described in the In Vitro Translation Assay.The DNA Preparation, were transformed into BL21(DE3) E. coli, and grew in LB medium at 37°C overnight. The cultures were diluted to OD600 of 0.01 and grew at 37°C by TECAN plate reader with a 24-well transparent plate (1 mL for each culture). OD600 measurements were performed every 10 min, followed by an orbital shaking mode (215 rpm) for a 10 min interval. The gene expression was induced by 1 mM IPTG at OD600 of 0.4. After the induction, the protein expression was monitored by an auto-loop measurement of 10 min shaking, OD600, GFP , and mCherry  for 10 hours. The translation efficiency was defined by the ratio of GFP to mCherry and normalized to real-time OD600 . The ratio of 210 min after induction represented the maximal efficiency of each strain. The cultures after 210-min induction were collected and extracted mRNA for RT-qPCR. The pellet was treated with 20 mg/mL lysozyme, and the mRNA was extracted by using Qiagen RNeasy Kits. The primers of qPCR were listed in Supplementary Table 2.PQS-contained dual fluorescence plasmids, described in 62. RNA sample (10 nM) was diluted to 25 pM and immobilized on a PEG-coated quartz slide by neutravidin (0.05 mg/mL). The reaction buffer  with an oxygen scavenging system . The smFRET experiment was performed by two solid-state lasers, 532 nm and 640 nm lasers. Each measurement was recorded with a 100 ms time resolution by smCamera software and analyzed with Interactive Data Language (IDL). The outputs were processed with custom MATLAB script to generate trajectories and FRET histograms. The details of smFRET data process were described in previous publications63. For DHX36 assay, protein (1 nM) or ATP (1 mM) was premixed into reaction buffer, and a flow system was applied to study real-time binding and unwinding events63.The PQS RNA oligonucleotides (see Supplementary Table 2) for smFRET were purchased from IDT with terminal amine modification for Cy3 labeling. The 18-mer RNA primer for immobilizing PQS RNA on slides was purchased from IDT and later labeled Cy5. The labeling protocol was described in a previous publication. RNA samples were annealed in TE buffer  at the ratio 1:1. The mixtures were heated at 80°C for 5 min and slowly cooled to room temperature (1°C per min). The single molecule assays were performed by using a home-built prism-type total internal reflection fluorescence microscope (TIRFM) at room temperature (23.0 ± 1.0°C)P-values were reported in Supplementary Table 3.Data shown in


Supramolecular self-assembly is a powerful tool for the development of polymolecular assemblies that can form the basis of useful nanomaterials. Given the increasing popularity of RNA therapy, the extension of this concept of self-assembly to RNA is limited. Herein, a simple method for the creation of nanosized particles through the supramolecular self-assembly of RNA with a three-dimensional macrocycle from the calixarene family was reported for the first time. This self-assembly into nanoparticles was realized using cooperative supramolecular interactions under mild conditions. The obtained nanoparticles are able to bind various hydrophobic  and hydrophilic (doxorubicin) drugs, as a result of which their cytotoxic properties are enhanced. This work demonstrates that intermolecular interactions between flexible RNA and rigid calixarene is a promising route to bottom-up assembly of novel supramolecular soft matter, expanding the design possibilities of nanoscale drug carriers. Despite the intensive development of anticancer therapy, cancer is one of the leading causes of death. The main reason for failure of therapy is a large number of genetic changes in cancer cells and their rapid growth, which leads to the emergence of drug resistance ,2. Due tOne of the most widely studied classes of substances for gene transfection are surfactants. A special type of surfactant is macrocyclic amphiphiles based on three-dimensional cyclodextrins and calixarenes. Such macrocycles can have a hydrophilic head fragment formed by ionic groups located on one of the edges of the macrocyclic skeleton and a hydrophobic part formed by n-alkyl substituents on the opposite rim, which is a prerequisite for their aggregation into various nanostructures. Due to their low toxicity and lower aggregation threshold compared to conventional non-macrocyclic surfactants, macrocyclic amphiphiles are promising components for transfection . In 38]38], poroIn addition to cyclodextrins, calix-like macrocycles can also be used to bind RNA. Undoubtedly, the cationic nature of these macrocycles also plays a key role in binding to nucleic acids, and as a result of this electrostatic interaction, the stability of RNA against thermal denaturation is increased . The polMost of the studies devoted to the binding of nucleic acids using macrocycles are focused on both non-covalent complexes with DNA and covalent conjugates with deoxyribonucleotides. In the work , calix4 and its The first step in the supramolecular design of non-covalent conjugates based on VR and RNA began with the study of their co-aggregation using turbidimetry by measuring the optical density at 450 nm. Due to the rather high absorption of both pure components in the region of 220–300 nm, it was impossible to follow the changes in their characteristic absorption bands in the mixed system; therefore, the turbidimetric titration method was used to monitor the formation of joint VR–RNA aggregates in solution. There is no turbidity in pure RNA solutions a, but thThe dynamic light scattering (DLS) method was used to determine the sizes of the formed joint aggregates. This method showed that the particle size in pure RNA solutions in the concentration range of 0.005–2 mg/mL does not exceed 8 nm a. The prThe effect of various concentrations of VR on RNA solutions with constant biopolymer concentrations of 0.1 and 0.5 mg/mL was studied further using DLS . The minThe ordering of the RNA structure in the presence of VR was confirmed by TEM. The TEM image for pure RNA a shows fSince the binding of VR to RNA is mainly due to strong electrostatic interactions between the negatively charged phosphate groups of the biopolymer and the viologen fragments on the upper rim of the macrocycle, the changes in the specific conductivity of solutions and the zeta potential of particles were further studied. The specific conductivity in a solution of pure RNA increases with an increase in concentration with an inflection at 1.2 mg/mL a. The prWhen VR is added, the specific conductivity of mixed solutions becomes lower, and the resulting dependence has a greater slope compared to the dependence for pure RNA b, which Due to the presence of a large number of phosphate groups, an increase in the proportion of RNA in a mixture with a constant VR concentration of 0.02 mg/mL leads to a decrease in the zeta potential of the particles towards negative values a. In an Morphological changes in RNA molecules under the action of VR can be traced using circular dichroism spectra, which are recorded to confirm the change in polynucleotide conformation as a result of VR binding. The spectrum containing negative bands at 209 and 239 nm, as well as a positive maximum at 268 nm, indicates that the RNA in the pure solution is in the A-conformation a. The ad1H NMR spectroscopy has been used to confirm intermolecular interactions between VR and RNA in an aqueous medium. Since the equimass complex VR:RNA = 1:1 precipitated in aqueous solution due to the high concentrations required to obtain 1H NMR spectra, a mixture with a slight 1.5-fold excess of VR was examined using 1H NMR  and N–H bond (~3100 cm−1 and ~3300 cm−1); the band at 2949 cm−1 corresponds to the stretching vibrations of the C–H bond; bands in the range of 1400–1700 cm−1 cover various stretching and bending vibrations of the C=C, C–N, C=O bonds included in the structure of nitrogenous bases; the antisymmetric and symmetric PO2 stretching bands correspond to the bands at 1237 and 1072 cm−1; bands in the region of 800–900 cm−1 correspond to vibrations of the sugar–phosphate backbone. A pure VR is characterized by bands of bending vibrations of the C–H bond at 3400 cm−1 and stretching vibrations of the same bond at 3117, 3038, 2923 and 2853 cm−1 resorcinol were studied. Due to the cooperative effect of electrostatic and hydrophobic interactions between them, the spontaneous formation of nanoparticles in an aqueous medium at room temperature and without the use of ultrasonic treatment is realized, which meets the criteria of green chemistry. The results of a set of physicochemical methods provided information about an interesting structural-ordering property of calix[4]resorcinol in solution of larger open-chain molecules, which leads to the formation of joint nanoparticles. The obtained nanoparticles can increase the water solubility of hydrophobic antioxidants (quercetin and oleic acid), which allows increasing their biocompatibility. With an excess of RNA relative to calix[4]resorcinol, nanoparticles are formed that are capable of binding not only lipophilic substances, but hydrophilic doxorubicin. The obtained drug-bound forms exhibit enhanced cytotoxicity and penetrating ability into tumor cells. Despite the fact that these forms also demonstrated cytotoxic activity against normal cells, it can be said that the use of RNA and an oppositely charged macrocycle leads to joint aggregation, initiating the formation of promising nanoparticles for delivering both a chemotherapeutic agent and an antioxidant. The use of such nanoparticles in anticancer therapy will allow for the combined therapy of tumors by suppressing the development of tumors with doxorubicin and leveling the side effects with antioxidant.