The roles of these processes in coping with various stimuli, biological development, disease, or tumor development in vivo and in vitro are self-evident and may become a new direction for cancer in the future. In this review, we summarize the category, characteristics, and therapeutic precis of RNA modification, m6A in particular, with the purpose of seeking the systematic regulation axis related to RNA modification to provide a better solution for the treatment of EC.RNA modifications are mostly dynamically reversible post-transcriptional modifications, of which m6A is the most prevalent in eukaryotic mRNAs. A growing number of studies indicate that RNA modification can finely tune gene expression and modulate RNA metabolic homeostasis, which in turn affects the self-renewal, proliferation, apoptosis, migration, and invasion of tumor cells. Endometrial carcinoma (EC) is the most common gynecologic tumor in developed countries. Although it can be diagnosed early in the onset and have a preferable prognosis, some cases might develop and become metastatic or recurrent, with a worse prognosis. Fortunately, immunotherapy and targeted therapy are promising methods of treating endometrial cancer patients. Gene modifications may also contribute to these treatments, as is especially the case with recent developments of new targeted therapeutic genes and diagnostic biomarkers for EC, even though current findings on the relationship between RNA modification and EC are still very limited, especially m6A. For example, what is the elaborate mechanism by which RNA modification affects EC progression? Taking m6A modification as an example, what is the conversion mode of methylation and demethylation for RNAs, and how to achieve selective recognition of specific RNA? Understanding how they cope with various stimuli as part of  It is commonly believed that RNA is the vector that transmits genetic information from DNA to the machinery that synthesizes proteins. Aside from translation, RNA has a hand in many other essential biological processes such as genetic controlling, protein synthesis, degradation, and even catalysis of chemical reactions, which largely rely on intricate RNA modification and structure . In non-per se (Ψ) was the most abundantly modification in RNA , and were viewed as irreversible decorations that contributed to the structural stability of RNA previously. However, many studies have confirmed that RNA modifications are reversible and that in recent years this has been executed with dynamic modulation of RNA transcription, splicing, localization, decay, and RNA-RBP pattern . We collper se , 5-methyper se . The abuper se , inosineper se . Pseudoun in RNA . N7-methn in RNA . N4-acetn in RNA  and N6,2n in RNA .The disruption of gene expression patterns controlled by epigenetics can lead to autoimmune diseases, infection, cancers , inflammCollectively, there have been extensive studies about the modifications of RNA during recent years, with modified nucleotides detected in abundant cellular RNAs and the specifically modified bases might exert various effects in RNA metabolism consisting of structure formation, dynamic stability, splicing, transportation, cellular localization, and translatability . In the The dynamic and elaborate manipulation of reversible RNA modification such as m6A, was previously predominantly dependent on methyltransferases and demethylases. The regulators associated with RNA modification were divided into “Writer ”, “Erasers (remove the modification in RNA)”, and “Readers/binders (accurately recognize and bind to RNA modification site)”, which could widely influence the steady state and fate of RNAs . What RNCancer conceptualization aims to distill complex phenotypic and genotypic diversity into a minimally structured set of principles, with the hallmarks continually updated. The latest hallmarks of cancer include avoiding immune destruction, tumor-promoting inflammation, deregulating cellular energetics, sustained proliferative signaling, genome instability, and mutation, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, resisting cell death . Even thFirst of all, m6A has been well-studied for its role in cancer, and the deposition in RNA are mainly mediated by a methyltransferase  complex, including METTL3, METTL14, WTAP, KIAA1459/VIRMA, ZC3H13, HAKAI and RBM15/15B, or METTL16 under existence of SAM. The overwhelming majority of m6A sites occurred at a consensus motif “DRACH”  . DemethyWhen it comes to m5C modification, NSUN1-7 and DNMT2 are ‘Writers’ that have been used to catalyze the methylation that occurred at cytosine, conversely, TETs and ALKBH1 were responsible for the removal of m5C . ALYREF WBSCR22, WDR4, RNMT, and METTL1 have been shown to be “Writers”, while TGS1 and H29K are “Erasers” for m7G modification . StudiesTreatment for tumors generally comprises surgery, hormonal therapy, Chemo/Target therapy, immunotherapy, radiotherapy, viruses/bacteria , and mor1 m6A modification, which could be inhibited by neplanocin A (NPC), Cycloleucine, and 3-Deazaadenosine ;2 METTL3, which was effectively repressed by STM2457  and UZH13 S-Adenosylhomocysteine (SAH), Antiviral agent 23/24 and STM2120  was iden4 FB23 , Rhein, 5 ALKBH5, whose potential inhibitor was IOX1 ;6 DC-Y3 and DC-Y13-27 were for YTHDF2 ;7 SND1 was corresponding to Thymidine 3′,5′-disphosphate ;8 IGF2BP2, which was restrained by CWI1-2 ;9 MSI2, whose inhibitor was Romidepsin (FK228) .Apart from surgery, hormonal therapy, and radiotherapy, we introduced Chemo/Target therapy and immunotherapy associated with m6A. Above all, we collected the small molecule inhibitors that targeted m6A related proteins, which included :1 m6A moAs for immunotherapy, ablation of METTL3 could effectively impair the YTHDF1 mediated translation of SPRED2 and increase M1/M2 like macrophage, as well as regulatory T cell infiltration into tumours . Further46, DNMT1, DNMT2 (TRD⁃MT1), DNMT3A and DNMT3B and m5C methylation-binding proteins (Readers) ALYREF, YBX1 and RAD52. Proof-of-concept studies have shown that dysregulated m5C regulators targeted by small molecule inhibitors have the potential for cancer treatment  and tumor microenvironment plasticity.We systematically summarized the landscape of m6A modification associated regulation pathway in EC . In term+ T cells in the coculture system using EC and CD8+ T cells, mechanistically, downregulated METTL3 protects NLRC5 from degradation through m6A modification and YTHDF2 dependent inhibition  through modulating FZD10 and EIF3C, etc . It was hibition . The conHOXB13 mRNA, the Wnt signaling pathway was activated, resulting in EC transfer /estrogen could remarkably induce FTO expression and transfer to nuclei, activate PI3K/AKT and MAPK signal pathways, and participate in enhancing proliferation and invasion of EC through modulating CyclinD1 and MMP2/9 level  divides endometrial cancer into four different types, including the: 1) DNA polymerase ε (POLE) super mutant type, which had a very high mutation load and a good prognosis; 2) the MicroSatellite Instability (MSI) type , with a py of EC . Over thAlthough the role of m6A in cancer has gradually been revealed, many challenges remain. Firstly, the mechanism of m6A regulators in tumors is largely unknown, such as the role and mechanism of “Readers” in cancer in m6A methylation modification is still a big gap; Secondly, although many studies have shown that m6A-related regulators and pathways could be used as new targets in cancer treatment, there is a lack of certain clinical practice, and m6A can affect the expression of genes in many aspects. That is to say, its side effects cannot be ignored. The coming mission will aim to deeply explore the molecular mechanism of “Writers”, “Erasers” and “Readers” in m6A modification involved in the regulation of tumorigenesis, as well as evaluate the correlation between m6A and cancer in combination with clinical data. Therefore, strengthening our understanding of tumor malignant transformation, ultimately, will be conducive to seeking and designing novel prospective targets for cancer therapy soon.


RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free-electron laser sources to reveal the formation and ready identification of angstrom-scale features in structured and unstructured RNAs. Previously unrecognized structural signatures of RNA secondary and tertiary structures are identified through wide-angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base-paired intermediate to assume a triple-helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. This method may help to rapidly characterize and identify structural elements in nucleic acids in both equilibrium and time-resolved experiments. Dynamically changing RNA structures are visualized in time-resolved, high-resolution solution scattering experiments. Most genomic DNA is transcribed into RNA, yet only a small fraction is translated into proteins  has been frequently applied to measure RNA structure  than reported in previous studies  sources present a unique opportunity to illuminate micrometer-scale, tens of femtoliter sample volumes with high-flux beams. At the Linac Coherent Light Source , beam intensities at the sample are ~1000 times higher than at state-of-the-art synchrotron beamlines, enabling measurement of previously impossible-to-detect signals using time-resolved WAXS  is information rich for nucleic acid samples. q < 0.25 Å−1) of rU30 and rA30 (q portions of these data (for q < 1.0 Å−1) were previously published , the small but distinct peak  scattering profiles of RNA structures, only one of which has been previously identified . In particular, when unstructured single strands combine to form a duplex, a peak appears near q = 0.4 Å−1. According to machine learning models , shown in q peak of q = 0.8−1) is absent from this measured profile, consistent with third strand filling the major groove. The cartoon schematically indicates the conformation of the folded triplex.Last, the ending state of the experiment  and a sample-absent  exposure, a total of 4 min of data acquisition yields one time point. Each profile is scaled to account for the varying intensity of the XFEL pulse that created it. Details about profile acquisition, computation, normalization, selection, and background subtraction are provided in Materials and Methods and figs. S4 to S7.To capture WAXS profiles with sensitivity to millisecond-scale conformational changes, time-resolved data were collected from micrometer-sized liquid jets at the Coherent X-ray Imaging (CXI) beamline at LCLS. Mixing injectors . t = 6, 10, 60, and 1000 ms. The curves are shown along with the t = 0 state (acquired at [RNA] = 0.5 mM). Four features are visible at the earliest time point acquired, 6 ms after the initiation of folding (orange). A shoulder appears near q = 0.4 Å−1 and the major groove–associated peak appears near q = 0.8 Å−1. Two higher q peaks also appear, though they are not well resolved. On this short timescale, the lower q data indicate that some fraction of the sample is in the duplex state. The appearance of triplex-associated peaks also suggests partial, but not yet full, structuring of the triple-helical backbone. It is unclear whether all molecules fold through a mandatory duplex intermediate or whether some fold directly to triplex. This distinction will require measurements at shorter time points. Nonetheless, it is clear that the RNA shows some duplex and some weak triplex features within 6 ms of folding initiation.Time-resolved experiments were performed at RNA concentrations of 1 and 0.5 mM. Different amounts of Mgq, base stacking peak. Sixty milliseconds after the initiation of folding (purple curve), the triplex peaks, as well as the base stacking peak, are firmly established. These trends are further underscored at 1000 ms after folding (green). Here, the lower q shoulder near q = 0.4 Å−1 has settled into a form that resembles that of the static triplex (fig. S2). At this time point, the scattering profile displays all of the SAXS/WAXS peaks seen in an equilibrium, folded curve, acquired on our laboratory source (fig. S8). Deviations in the baseline levels can be explained by temperature variations . The signal-to-noise ratio of each curve depends on the flow rate of the sample, which determines the total amount of RNA in the beam-illuminated sample volume, and more critically on the beam power. In some cases, ice crystals formed in the jet (or at the nozzle), creating some very intense diffraction spots on the detector. We attenuated the beam to protect against damage. The fraction of beam power used to collect data for each time point is provided in tables S3 and S4.In the 10-ms profile (yellow), diminished duplex features are observed: The major-groove peak is reduced. The triplex peaks are better articulated, and a small, but not quite resolvable, perturbation in intensity appears near the highest-Overall, the RNA’s folding strategy can be established directly from the curves shown in The order of appearance of all of the peaks is recapitulated by the measured time series at [RNA] = 0.5 mM, where data points were acquired at 0, 6, 100, and 500 ms . Notably, the transient duplex intermediate again appears at 6 ms and vanishes at all later times. Slight deviations were seen in the amount of triplex present at 6 ms, likely a result of mixing conditions.This distinctive view of dynamic structural changes from single strand to triplex underscores the highly dynamic nature of the RNA backbone, even in the duplex state, until it captures a third strand in the proper triplex geometry. Once the proper backbone arrangement is assumed, the structure is locked into place by base stacking. This experiment provides direct evidence that the RNA backbone drives folding, a conclusion that was previously hypothesized by comparison with molecular dynamics simulations  states, as well as from transient states during a folding reaction. When coupled with lower-angle data that reveal the relative spatial arrangement of these structural elements, this work highlights the potential of solution scattering to uniquely and directly characterize both static and dynamic RNA structures.Presently, less than 2000 RNA structures are deposited in the Protein Data Bank. If a substantial database of structures can be built, either directly or by solving the folding problem, new deep learning techniques  features in solution scattering. Once these upgrades are realized, atomic resolution in solution structures, at room temperature, and on biologically relevant timescales is imminent.In addition to enhancing structure solving methods, this work highlights the unique ability of highly brilliant, XFEL x-rays to provide the exceptional sensitivity needed to observe subtle structural changes in macromolecular systems. Although there is already much to learn from this now demonstrated technique, XFEL technology is rapidly advancing. The increasing pulse repetition rates at XFELs worldwide [from hundreds to millions of pulses per second  as single strands. Molecular reconstitution, including buffers and annealing protocols, are distinct for each sample and are described in detail in the “Sample Preparation” section in the Supplementary Materials.Mixing injectors [as described in  and wide-angle (q = 0.3 to 3.2 Å−1) regimes were read simultaneously using two Pilatus 1M detectors  arranged in series. The transmitted x-ray beam intensity was also recorded during each measurement. Centering and calibration of the beam on both detectors were performed using a silver behenate standard in BioXTAS RAW, as well as masking, radial averaging, and buffer subtraction  of Brookhaven National Laboratory  using the setting WAXS_STD. Data collected from the BioXolver are analyzed using BioXTAS RAW, as described above. The quoted sample temperatures, when noted, were achieved by temperature controlling the sample capillary.Time-resolved data were acquired from ~16,000 frames for each sample present and sample absent (buffer) profile. The number of frames for each data point, for all data shown in


TM D microarrays. The mean signal values of the two groups were compared using a one-way ANOVA. A total of 26,639 different RNA transcripts were identified, comprising both mRNA and ncRNA subtypes. Approximately 6% of transcripts showed statistically significant differential abundance between the two groups. The mRNA sodium channel modifier 1 (SCNM1) was detected at a level 3.8 times lower, and the immature microRNA-130b was detected at a level 1.5 times higher in the group with TBUT 2 s compared to the group with TBUT 10 s. This study demonstrates the potential for using tear fluid EV-RNA in DED diagnostics.Currently, diagnosing and stratifying dry eye disease (DED) require multiple tests, motivating interest in a single definitive test. The purpose of this study was to investigate the potential for using tear fluid extracellular vesicle (EV)-RNA in DED diagnostics. With a role in intercellular communication, nanosized EVs facilitate the protected transport of diverse bioactive molecules in biofluids, including tears. Schirmer strips were used to collect tears from 10 patients presenting with dry eye-related symptoms at the Norwegian Dry Eye Clinic. The samples comprised two groups, five from patients with a tear film break-up time (TBUT) of 2 s and five from patients with a TBUT of 10 s. Tear fluid EV-RNA was isolated using a Qiagen exoRNeasy Midi Kit, and the RNA was characterized using Affymetrix Clariom Dry eye disease (DED) is a chronic condition characterized by both the loss of tear film homeostasis and ocular surface inflammation . SymptomThe tear film forms an interface between the cornea and the environment, and its integrity is essential for both the health of the eye and for visual acuity . LacrimaAs DED encompasses diverse etiologies, there is a significant degree of heterogeneity between subjects; thus, the diagnosis and stratification of patients can be challenging. There is presently no single gold standard symptom or test ,9. InsteAlthough the pathophysiology of DED is incompletely understood, inflammation is considered integral to both the development and propagation of the condition . A disruTear fluid collection is both non-invasive and relatively easy. The study of tear fluid has the potential to expand our understanding of DED and unveil new diagnostic opportunities. To date, minimal research has focused on the extracellular vesicle (EV) content of tear fluid. EVs are a heterogeneous group of nano-sized particles with a phospholipid membrane enclosing a cargo of nucleic acids, proteins, and lipids . They arEVs are present in all biological fluids and function in intercellular communication . They maThe primary aim of this study was, therefore, to characterize the RNA content of EVs isolated from the tear fluid of patients with symptoms of DED using microarray technology. Additionally, the secondary aim was to identify any potential differences in the abundance of specific EV-RNA transcripts between two groups of patients with dry eye-related symptoms, one with extremely low tear film stability (TBUT 2 s) and one with near normal tear film stability (TBUT 10 s). 8 particles/mL. The size distribution was between ~100 and 430 nm, and aggregates were seen. The concentration of EVs, as determined by nanoparticle tracking analysis (NTA), was ~10Transmission electron microscopy (TEM) allowed for the direct visualization of EVs, as evidenced by cup-shaped particle morphology and a size range between 100 and 350 nm. TEM provided confirmation of the presence of EVs in the pooled tear fluid sample A. Flow cytometry analysis, using anti-CD9-coated magnetic beads and phycoerythrin (PE)-coupled anti-CD9 detection antibodies, indicated an increased level of the membrane-associated protein marker, tetraspanin CD9, in a pooled tear fluid EV sample relative to the isotype control B. This cWestern blotting analysis verified the presence of EV marker proteins in a pooled tear fluid sample following EV isolation using a modified Qiagen exoRNeasy column approach C. The me® Fluorometer, Nanodrop™, and Agilent Bioanalyzer analysis. The fluorometric analysis showed that group 1 (patients with TBUT 2 s) had an average EV-RNA concentration of 2.3 ng/µL, with a range of 1.9 ng/µL to 3.0 ng/µL. For group 2 (TBUT 10 s), the average EV-RNA concentration was 1.9 ng/µL, with a range of 0.6 ng/µL to 2.7 ng/µL. Based on the Agilent Bioanalyzer analysis, most of the RNA in all samples showed lengths of approximately 200 nucleotides or shorter . The (mean) signal values ranged from 3 to 24,568, corresponding to the relative abundance of each transcript in the samples. Most RNA subtypes so far defined were identified in the samples, including messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), circular RNA (circRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Y-RNA (yRNA), small Cajal body-associated RNA (scaRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). The RNA transcripts were categorized according to their RNA subtype, as defined by Affymetrix, and the comparative abundance of the different RNA subtypes was determined A. The cap < 0.05). ncRNAs represented a slightly higher percentage of these transcripts compared to mRNA  was compared with group 2 (TBUT 10 s), 1598 transcripts were seen to have statistically significant differential abundance  and beta-2-microglobulin (B2M)) in three samples and one ncRNA transcript (yRNA1) in four samples. The results showed transcript levels consistent with the microarray signal values. q-values > 0.05), the study demonstrated the potential for using tear fluid EV-RNA in diagnostics, with further investigation recommended.The Affymetrix microarray analysis of tear fluid EV-RNA performed in this study produced a large dataset of transcripts describing a diverse selection of RNA subtypes in all samples. A comparison of the RNA profiles between the two categories TBUT 2 s (group 1) and TBUT 10 s (group 2) identified several mRNA and miRNA transcripts with statistically significant differential abundance between the groups. While no clear RNA patterns or biomarker candidates were revealed  and non-coding RNA (ncRNA). Together, the different RNA subtypes form a complex, intricate network of interactions controlling a multitude of cellular processes . It has There is general agreement that EV-RNA is involved in intercellular communication ,28. WhilThere remain many facets relating to EV-RNA intercellular communication that still need to be resolved. Little is known about the selectivity of RNA into EVs or the fate of the RNA after it has been delivered into the target cell . Adding While all subjects included in the current study showed clinical signs of DED, their clinical presentation varied considerably. Unfortunately, present diagnostic tests have a relatively low reproducibility  and cannAlthough no transcripts were identified with clear RNA biomarker potential for tear film instability, 6% of the transcripts were seen to have differential abundance between the EVs of group 1 and group 2. The comparison of these profiles  revealedTear fluid is composed of secretions from lacrimal glands (main and accessory), meibomian glands, conjunctival goblet cells, and epithelial cells of the ocular surface. As such, the cellular origins of tear fluid EVs are presumably quite diverse. To date, only a minor number of publications have focused on EVs in tear fluid ,52,53,548). As aggregates were seen, increasing the vortex time prior to NTA may be of benefit for future studies. This current study employed three novel methods of biomolecular analysis for studying tear fluid EVs and tear fluid EV-RNA. Firstly, a Qiagen exoRNeasy EV-RNA isolation kit was used to isolate EV-RNA from tear fluid. Secondly, Affymetrix microarrays were used to analyze the EV-RNA. Additionally, a Qiagen exoRNeasy affinity column was used with RIPA lysis buffer, in a modified approach, to isolate tear fluid EV proteins for use in Western blotting. Ideally, the EVs used for characterization should be isolated by the same method used for EV-RNA analysis; however, the Qiagen exoRNeasy kit combines both EV and RNA isolation. This did not enable the extraction of EVs for characterization. SEC was originally used to isolate EVs from the tear fluid; however, the yield was too low for use in characterization, even with pooled samples. This was evident based on the results from the NTA and initial pilot projects for flow cytometry and Western blotting. Therefore, the characterization of EVs was performed on pooled whole tear fluid samples (TEM and flow cytometry) and together with a modified Qiagen exoRNeasy column approach for Western blotting. Of further note, the particle concentration following SEC EV isolation as measured by NTA was below the manufacturer’s recommended range for result reliability  and underwent a battery of diagnostic tests, including measurement of tear fluid production (Schirmer 1 test without anaesthesia), fluorescein tear film break-up time (TBUT), and assessment of ocular surface staining (OSS). Two groups of tear fluid samples were then selected according to the subjects’ TBUT; samples from patients with a TBUT of 2 s (considered extremely reduced tear film stability) were placed in group 1, and samples from patients with a TBUT of 10 s  were placed in group 2. An overview of the clinical data from each subject is presented in Schirmer tear test strips  were used for tear fluid collection, for a period of five minutes. The strips were then placed in individual 1.5 mL collection tubes containing 500 µL phosphate-buffered saline (PBS). The samples were then frozen at −80 °C until use.g for 10 min, RT, with the Schirmer tear test strips still in place. The strips were then removed, and the samples were again centrifuged at 3000× g for 10 min, RT, to remove cellular waste and debris. The supernatant was transferred to new tubes. A portion of each sample was directly used for EV-RNA isolation, and the remaining portion was frozen at −80 °C until further use in EV characterization. Thawed samples were pooled prior to EV characterization. Samples were thawed and individually centrifuged in a two-step differential centrifugation procedure. The samples were first centrifuged at 300× EVs were isolated from a pooled tear fluid sample using size-exclusion chromatography (SEC) . Fractions 7–10 were combined, and the size distribution and concentration of the EVs were determined by nanoparticle tracking analysis (NTA), using a NanoSight NS500 . The sample was diluted 1:1 with filtered PBS , vortexed, and then analyzed in triplicate. The camera level was set to 13, and the detection threshold was set to 3. Videos 60 s in length of a continuous flow of sample were recorded. Data analysis was performed using NTA 3.1 software. 2O. The grids were then incubated with 2% methylcellulose containing 0.3% uranyl acetate on ice for 10 min. The grids were then air-dried before being viewed with a Tecnai G2 Spirit transmission electron microscope . A Morada digital camera and RADIUS imaging software (http://emsisasia.com/radius/) were used to create images, which were further processed using Adobe Photoshop. Transmission electron microscopy (TEM) was performed on a pooled tear fluid sample. Formvar carbon-coated copper grids were placed on top of 5 µL drops and followingly incubated for 5 min at RT, before being washed 3 times with distilled H®  were added to 100 μL of the tear fluid sample and to each of three controls . Overnight incubation was performed in a HulaMixer Sample Mixer at 2–8 °C. Following wash steps, 150 μL of assay buffer was used to redistribute the beads, and 100 μL from each sample was then combined with 25 μL of PE-conjugated detection antibodies  or 25 μL of isotype-matched control . An orbital shaker  was then used for 45 min at 1000 rpm. Following two wash steps, a final 100 μL of assay buffer was added to each tube. All samples and controls were then placed on ice, ready for flow cytometry analysis. A BD Accuri™ C6 Cytometer  was used for flow cytometry EV detection. Flow cytometry was performed on a pooled tear fluid sample. Aliquots of 20 μL Dynabeadsg. The lysate and controls, SW480 cell lysate and recombinant Exosome standard , were heated to 70 °C for 10 min and then loaded on a 4–12% Bis-Tris gel . CD9 and CD63 were examined under non-reducing conditions and Hsc70/Hsp and calnexin under reducing conditions. Proteins were blotted onto a PVDF membrane , which was then blocked with casein before overnight incubation with primary antibodies. The antibodies used were anti-CD9, 1:750 , anti-CD63 1:600 , anti-Hsc70/Hsp 1:1000 , and anti-Calnexin 1:1000 . Secondary antibodies used were Rabbit TrueBlot and Mouse TrueBlot . The blots were imaged using SuperSignal™ West Dura Extended Duration Substrate  with an Amersham™ Imager 600 .Western blotting was performed on a pooled tear fluid EV-isolated sample. EV isolation was conducted using a modified Qiagen exoRNeasy EV isolation method see . BrieflyEV-RNA was isolated from each tear fluid sample using a Qiagen exoRNeasy Midi Kit , according to the manufacturer’s guidelines. A sample input volume of 300 µL was used. Following the QIAzol and chloroform steps, 200 µL of the aqueous phase, containing the RNA, was transferred to new tubes. A DNase step was included following the 100% ethanol-centrifugation step. RNAse-free water was used to recover the RNA, producing samples of approximately 12 µL. The RNA elutes were immediately placed on ice, prior to RNA quantification and characterization, and then frozen at −80 °C until further use.® 2.0 Fluorometer  combined with a Qubit® microRNA Assay Kit , according to the manufacturer’s guidelines. Sample volumes of 1 µL were used. The measurements obtained were then used to calculate the Affymetrix microarray sample input volumes. Additionally, contaminants (guanidine or phenol) were assessed using a Nanodrop™ One Spectrophotometer , using sample volumes of 1 µL.Sample RNA concentrations were determined using a QubitQuality analysis was performed on 1 µL of each RNA sample using a Bioanalyzer  together with an Agilent RNA 6000 Pico Assay Kit  in accordance with the manufacturer’s guidebook. TM WT Pico Reagent Kit (Cat no 902623) was used to prepare the RNA samples prior to their application to Clariom™ D microarray chips . This was performed according to the manufacturer’s guidelines and using 2 ng of total EV-RNA per sample. A HeLa cell sample was used as a positive control with a total RNA input of 0.5 ng. Nuclease-free water was used as a negative control. Poly-A RNA was used as an exogenous positive internal control in all samples. A Veriti® 96-Well Thermal Cycler  was used for the pre-IVT PCR amplification with 9 PCR cycles applied. The prepared samples of fragmented and labeled ss-cDNA were then injected into individual ClariomTM D microarray chips, which were incubated for 17 h at 45 °C in an Affymetrix GeneChip 645 hybridization oven with a rotation of 60 rpm. Following the hybridization process, the microarray chips were immediately washed and stained in a GeneChip™ Fluidics Station 450 using a GeneChip™ Hybridization, Wash, and Stain Kit . The microarray chips were then scanned with a GeneChipTM Scanner 3000 System and analyzed with GeneChipTM Command ConsoleTM software, version 4.0. All Affymetrix instruments and software were from Affymetrix, Santa Clara, CA, USA.A GeneChipReverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was used to validate the Affymetrix detection of selected RNA transcripts.® 96-Well Thermal Cycler and qPCR with a ViiA™ 7 Real-Time PCR Instrument . The reagents used were SuperScript™ Enzyme Mix (Cat no 11754-050) and VILO™ Reaction Mix (Cat no 11754-250) for RT, and Taqman™ Fast Advanced Master Mix (Cat no 4444557) together with Gene Expression Assays  for qPCR, all provided by Thermo Fisher Scientific, Oslo, Norway.RT-qPCR was performed on three RNA samples to validate the presence of two mRNA transcripts (RPL9 and B2M). The transcripts were selected based on the relatively high signal values detected in the samples and the availability of the assays in the laboratory. Total cellular RNA was used as a positive control, and RNA-free water was used as a negative control. Reverse transcription (RT) was performed using a VeritiAdditionally, to substantiate the presence of an ncRNA transcript, the validation of yRNA1 was performed externally, at the Department of Microbiology, Oslo University Hospital, on four RNA samples.® Genomics Suite® software (https://www.partek.com/) was used for the statistical analysis. A one-way ANOVA was applied to determine any statistically significant abundance between the groups, with p-values less than 0.05 considered to be indicative of statistical significance. Additionally, q-values and fold changes were determined. Furthermore, a principal component analysis (PCA) was performed on the data, and a cluster heatmap was produced using the 100 transcripts with the most statistically significant difference in signal values between the groups.The fluorescent signal values of the RNA transcripts identified by the Affymetrix microarray analysis were recorded, and mean values were calculated for each group: TBUT 2 s (group 1) and TBUT 10 s (group 2). PartekThis study provides the novel characterization of the RNA content of tear fluid EVs from patients with dry eye-related symptoms, using microarray technology. Thousands of RNA transcripts, both mRNA and ncRNA, were successfully detected, supporting the potential for the use of tear fluid EV-RNA in DED diagnostics. Approximately half of the transcripts identified were coding RNAs, and approximately 6% of the transcripts showed a significant differential level of abundance between patient groups with TBUT 2 s and TBUT 10 s. While the comparison of RNA profiles revealed no conclusive results, the protein-coding gene SCNM1 and the immature mir-130b stood out as genes of interest. These findings may be useful in guiding and supplementing further research into tear fluid EV-RNA. Additionally, future knowledge concerning the roles of the different ncRNA subtypes may reveal information yet undisclosed.


Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized   Circular RNAs (circRNAs) are a class of covalently closed RNA molecules produced from precursor mRNA back splicing and are widely found in nature . Due to in vivo, which leads to a longer half-life for their effects as a therapeutic drug. CircRNAs can also be designed and optimized for effective expression in specific tissues and cells , the RNAs to be circularized, and a 45-nt antisense region were chemically synthesized by Beijing Genomics Institute  and cloned into a double-enzyme-digested linearized pCDH vector.The active ribozyme contained the sequences from 28 to 414 nt of the The overexpression plasmids of specific circRNAs based on flanking ICSs and paired control plasmids were obtained from the Public Protein/Plasmid Library , which contains a general front and back circular frame. The indicated RNA sequences were cloned into the construct by EcoRI/BamHI restriction sites.Tetrahymena ribozyme. Specifically, the exon was divided into two parts, and the split GFP gene fragments were inserted into the vector in reverse order. The CVB3 IRES was then inserted between the split GFP gene start and stop codons, resulting in a large exon downstream of the ribozyme. Similarly, we used Gibson Assembly to obtain the full-length sequence. The primers and the sequences of these constructs are listed in For the GFP reporter gene vector, a synthesized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) fragment and an exon that encoded GFP were inserted into the abovementioned circRNA overexpression vector using the 2 to a density of 70%-80% and were transfected with Lipo8000 Transfection Reagent  or Lipofectamine 3000 reagent .Human cervical cancer (HeLa), breast cancer (MCF7), HEK293T, colon cancer , and prostate cancer (DU145 and PC-3) cell lines were purchased from the American Type Culture Collection . HeLa, MCF7, HEK293T and HCT-116 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) , while HCT-8, PC-3 and DU145 cells were maintained in RPMI 1640 medium with 10% FBS. All cells were cultured at 37 °C in a humidified incubator containing 5% COin vitro transcription were amplified, and an upstream T7 promoter was added by PCR using Phanta Flash Master Mix . The product was identified with 1.2% agarose gel electrophoresis and purified with a TIANgel purification kit . T7 RNA polymerase  was used for in vitro transcription reactions that lasted 2 h at 37°C. After in vitro transcription, the RNA products were treated with DNase I  at 37°C for 30 min to remove DNA templates. Each circularization reaction was supplemented with 2 mM GTP and incubated at 55°C for 15 min to induce RNA circularization, followed by quick cooling to 4°C. The acetate/ethanol precipitation method was used to purify the RNA . The error bar represents the standard error of the mean (SEM) of three independent experiments. The primers used for the PCR are listed in Total RNA was extracted using TRIzol reagent  according to the manufacturer's instructions and subsequently treated with DNase I  at 37°C for 15 min, followed by treatment at 65°C for 10 min to inactivate DNase I. The OD value 260/280 of the extracted RNA was between 1.9 and 2.1 and the OD value 260/230 was greater than 2.0. One microgram of RNA was reverse-transcribed using Evo M-MLV RT Premix  according to the manufacturer's instructions. The RT product was used for RT-PCR amplification with Phanta Flash Master Mix . The RT-PCR product was then separated on a 1.2% agarose gel and scanned with a scanner . The same cDNA template was diluted 4-fold for quantitative real-time PCR (qPCR) with a SYBR Green Premix Pro Taq HS qPCR kit . The reaction procedure for qPCR was incubation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 58°C for 30 s on an iQ5 Real-Time PCR Thermal Cycler . The 2In brief, RNA was resolved on formaldehyde denatured agarose gel, followed by transfer to a nylon membrane. Digoxigenin (Dig)-labelled cDNA probes were made using a DIG DNA Labelling kit . The membrane was then hybridized with specific Dig-labelled probes at 42°C. The NB probe is listed in The cells were washed twice with phosphate-buffered saline (PBS), and total protein was extracted with a Whole Protein Extraction Kit . Protein samples were separated by SDS−PAGE and then transferred to PVDF membranes. Subsequently, the membranes were blocked in 1 × casein buffer for 1 h at room temperature and then incubated with anti-cleaved-parp1 antibody , anti-cleaved-caspase 3 antibody , anti-GAPDH antibody  and anti-β-actin antibody  at 4°C overnight. The membranes were washed three times with TBS-T, incubated with secondary antibodies  for 1 h, and detected with a gel imaging analysis system .3 transfected cells were harvested and plated in 100 μl of medium containing 10% FBS per well in 96-well plates. Then, 100 μl of medium containing 10% Cell Counting Kit-8 (CCK-8) reagent  was added to each well at different time points . Then, the cells were incubated at 37°C for 1 h. The absorbance at 450 nm was measured by a multimode microplate reader .A total of 3  ×  103 transfected cells were seeded in each well of a 6-well plate and cultured at 37°C for 14 days. On the final day in culture, the cells were fixed for 15 min with methanol and then washed twice with PBS. Then, the cells were stained for 30 min with a 0.5% crystal violet solution. The colonies were photographed and counted with ImageJ software. Alternatively, acetic acid was used to wash off the crystal violet, and was taken the eluent to measure the OD value at 570 nm on a multimode microplate reader .A total of 1.0  × 105 transfected cells in 200 μl of serum-free medium were seeded in the upper chamber, and 600 μl of medium containing 10% FBS was added to the lower chamber. After incubation at 37°C for 24 h, the cells on the upper membrane surface were removed with a cotton swab, and the cells that had migrated to the bottom surface of the upper membrane were fixed in methanol for 30 min and then stained with 0.5% crystal violet for 30 min. The number of migrating cells was counted with ImageJ software. Alternatively, acetic acid was used to wash off the crystal violet, and the eluent was taken to measure the OD value at 570 nm on a multimode microplate reader .A 24-well transwell insert with an upper chamber was used. Briefly, 1.0 × 10The transfected cells were plated in 12-well plates and grown to confluence. The cell monolayer was wounded using a 200-μl pipette tip. The wounds were imaged at 0, 24 and 48 h after wounding with a phase-contrast microscope .t test or one-way ANOVA. P values <0.05 were considered statistically significant.GraphPad Prism version 8.0 was used to analyze all the experimental data. Differences between experimental groups were evaluated by a two-tailed Student's The desired RNA sequence to be circularized was inserted downstream of the optimized ribozyme and a complementary antisense region was added to the pCDH backbone upstream of the ribozyme to facilitate the folding and stabilization of the ribozyme. The catalytic domain in the 5' portion of the ribozyme was fused with an adjustable 13-nt-long IGS. The first 9 nt of the 3' terminus of the IGS sequence was complemented by pairing with the 3' terminus of the circRNA and a U residue was inserted in the 3' terminus of the circRNA. In contrast, the last 6 nt of the 5' terminus of the IGS was designed for incomplete complementary pairing with the 5' terminus of the target circRNA sequence to form helix P10. The secondary structure of RNA precursor was predicted by RNAFold .T. thermophila ribozyme-based RNA circularization system   was chosen as a target circRNA to evaluate the established m Figure . As a re) Figure . The cir) Figure . qRT-PCR) Figure , and the) Figure . Furthero Figure . Similars Figure . CircDNMs Figure .T. thermophila ribozyme-based RNA circularization system was suitable for general circRNA overexpression. To avoid adding an extra uridine residue (‘U’) at the 5' splice site, we adjusted the IGS sequence to correctly hybridize the 5' and 3' terminal sequences of the rearranged circRNA sequence to form the P1 and P10 helices , circFOXO3 (hsa_circ_0006404), and circHIPK3 (hsa_circ_0000284) were used to determine whether the s Figure –C, rights Figure –C, left T. thermophila ribozyme-based RNA circularization system had overwhelming advantages over the commonly used flanking ICS method.Divergent primers were designed for detecting circRNA. Convergent primers were designed for detecting total RNA including the forms of precursor RNA and circRNA. Linear primers were designed for detecting linear RNA that represented the RNA form that is not circularized in the precursor RNA Figure . The resT. thermophila ribozyme-based circularization system . A . A T. ths Figure . We perf studies ,31. Simim Figure . GFP flum Figure . Moreovem Figure .T. thermophila ribozyme-based circularization system achieved a circularization efficiency of 80% in vitro and in mammalian cells, and the circRNA produced by this system was completely consistent with the designed sequence without introducing any additional nucleotides. Due to the complexity of the intracellular environment in mammalian cells, the splicing efficiency of ribozyme varies with gene sequence and cell type. In recent years, the ribozyme derived from the T. thermophila group I intron has been widely applied in gene delivery, mRNA repair, and mRNA detection (Tetrahymena ribozyme to synthesize long circular RNA in E. coli based on the PIE strategy, and this circular transcript could be translated into an active β-glucosidase (Efficient and precise preparation of circRNAs is important for the functional study of circular mRNA pharmaceutics in the future. Previous strategies for circRNA synthesis did not meet the requirements due to their inefficiency and the introduction of additional nucleotides. Therefore, it is necessary to further simplify the procedure and improve production efficiency to achieve industrial-scale production. In our current study, the etection ,33. Liescosidase . Howeverin vitro to produce a large number of precise circRNAs as protein overexpression vectors through simplified reaction and purification processes. We used the GFP reporter system to further verify the possibility of our RNA circularization system acting as an mRNA for translation. The coding region of GFP was divided into two parts: the first part containing the initiation codon was preceded by an IRES sequence, which could act as an mRNA cap structure in mammalian cells. The second part of GFP was located in front of the IRES and behind the self-splicing ribozyme. Without the introduction of additional nucleotides, the coding sequence of GFP will not frameshift and green fluorescence will be expressed under the mediation of IRES. For any circRNA synthesized using the ribozyme described here, it is necessary to prevent the end-joining sequence from becoming a stop codon to ensure the translation of the full-length circRNA. Recently, it has been reported that exogenous circRNA was prepared by the PIE strategy in vitro to produce neutralizing antibodies against SARS-CoV-2 (in vivo.Because of its covalently closed circular structure, circular mRNA is more resistant to endonuclease degradation and more stable than linear RNA , thus prRS-CoV-2 . In contet al. developed a systematic approach to rapidly assemble and test elements engineering circular RNA for enhanced protein production. CircRNA protein production was increased several hundred-fold by optimizing vector topology, 5' and 3' untranslated regions, IRES, and synthetic aptamers recruiting translation initiation machinery (Recent research has indicated that the nucleotide modification of circRNAs attenuates the innate immune response and may enhance the future applicability of circRNAs in treatment . Therefoachinery , which pT. thermophila ribozyme-based circularization system can mimic the natural noncoding circRNA and participate in various intracellular functions, and may also be used in the preparation of circular mRNAs to express therapeutic proteins. Because of its unique characteristics, circRNA may become a safe and effective choice for therapeutic use as well as vaccination against viral infection, including new SARS-CoV-2 variants. We believe this novel RNA circularization system will soon have broad industrial, scientific, and clinical applications.In conclusion, the circRNA produced by our gkad554_Supplemental_FilesClick here for additional data file.


Trypanosoma brucei RESC1–RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5′-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5′-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs.Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of  Trypanosome brucei, Trypanosoma cruzi and Leishmania spp., all causative agents of neglected tropical diseases. Their distinguishing feature is the presence of the kinetoplast, a DNA-containing granule located within the large mitochondrion. Kinetoplast DNA (kDNA) comprises circular chromosomes, so-called maxicircles, which harbour protein-coding genes  (The protein platform that coordinates the complex editing cycle within the editosome is the x (RESC) . RESC cox (RESC) ,16,17, tx (RESC)  and the x (RESC) . For thex (RESC) ,23. Howex (RESC) .The proteins RESC1  and RESC2  have been identified as the gRNA binding core of the RESC1-7 module ,24. RESCTrypanosoma brucei RESC1–RESC2 heterodimer and show that the RESC2 subunit binds the 5′-triphosphate-nucleoside of RNA in a phosphate binding tunnel that resembles the phosphate binding site in a group of RNA capping enzymes. Since only gRNAs, but not other mitochondrial RNA species of kinetoplastids carry a 5′-triphosphate, we propose that the RESC1–RESC2 module discriminates gRNAs from the RNA pool, protects them from exonucleolytic cleavage and targets them to the editing reaction.Here, we report the cryo-EM structure of the 2; 20 mM Imidazole; 2% Glycerol; supplemented with 1 mM of PMSF, 2 μg/ml of DNase and 25 μg/ml of RNase) and lysed by sonication . The lysate was clarified by centrifugation  and loaded into a 5 ml HisTrap HP affinity chromatography column (Cytiva), that was washed with 5 column volumes of lysis buffer, 10 volumes of high salt buffer  and 5 column volumes of low salt buffer . The sample was eluted with an imidazole gradient and dialysed to a low salt ion exchange buffer , when needed, his-tag was cleaved during dialysis with 3C protease. After dialysis, the sample was injected in a HiTrap Q HP anion exchange chromatography column (Cytiva), and eluted with a NaCl gradient. The protein fractions were pooled, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C. As a final purification step, frozen aliquots were thawed and injected in a Superdex 200 size exclusion column (Cytiva), pre-equilibrated with the desired buffer for downstream applications . All steps were performed at 4°C.N-terminally tagged 6xHis-3C-RESC1 Tb927.7.2570) and untagged RESC2 (Tb927.2.3800), without their mitochondrial targeting sequences , coupled to MALS detector miniDAWN™ TREOS (WYATT). The sample  was injected into the column with a 0.5 ml/min flow. The data were processed using the ASTRA V software (Wyatt Technology). For the complex of RESC1–RESC2 and gRNA, RESC1–RESC2 and gRNA were mixed to final concentrations of 15 and 60 μM, respectively, and injected in the column. The mass was calculated by two-component analysis.The size exclusion chromatography-multiple angle light scattering (SEC-MALS) experiments were performed with a Superdex 200 Increase 10/300 GL column, in SEC buffer . Exclusively for the experiments with RESC1wt/RESC2K311S and related controls, the buffer contained 400 mM of NaCl. Different combinations of RESC1–RESC2 and gRNA  connected to an AKTA Micro system (Cytiva) and pre-equilibrated with SEC buffer , at finThe mass photometry experiment was performed in the Refeyn OneMP, with RESC1–RESC2 in a final concentration of 100 nM. The contrast-to-mass conversion was achieved by calibration using a native protein ladder, following the manufacturer's guidelines.in vitro transcribed using the MEGAshortscript™ T7 Transcription Kit (Invitrogen) . Oligonucleotides containing the sequence of the gRNA gND7(506) (GGGAUAUAAAUACGAUGUAAAUAACCUGUAGUAUAGUUAGUGUAUAUAGUGAAA) and a T7 promoter were annealed to make partially single-stranded templates. Following the manufacturer guidelines, the templates were added to the reaction to a final concentration of 200 nM, followed by nuclease-free water, nucleotide solutions, reaction buffer and T7 Enzyme mix, and incubated at 37°C for 4 h. After transcription, the reaction was mixed with a gel loading buffer and loaded in a 7 M urea–PAGE. The band of RNA was visualised in a TCL place using the U.V. shadowing method, cut from the gel and eluted in gel elution buffer  at 37°C, overnight. After elution, the RNA was precipitated with isopropanol and resuspended in nuclease-free water.The gRNAs used in this work were either synthesised by Biomers   or 2; 0.5 mM TCEP). UltrAuFoil R 1.2/1.3 300 Au mesh (Quantifoil) grids were glow discharged with residual air for 30 s, on each side in a PELCO easiGlow device operated at 30 mA. Peak fractions were diluted 1:5 to 1:10, and 2 μl of the sample were applied in each side of the grid before blotting for 3 s (blot force 0) and vitrified in liquid ethane using a Vitrobot MARK IV (Thermo Fisher Scientific) operated at 4°C and 100% humidity.For Cryo-EM sample preparation, RESC1–RESC2 heterodimer  was injected in a Superdex 200 Increase 3.2/300 size exclusion column (Cytiva) pre-equilibrated with SEC buffer  was added, and the sample was incubated for another 30 min before being injected in a Superdex 200 Increase 3.2/300 size exclusion column (Cytiva) pre-equilibrated with SEC buffer . Sample vitrification was performed the same way as for the apo sample.For the gRNA bound RESC1–RESC2 sample preparation, RESC1–RESC2 heterodimer (2 mg/ml) was incubated with 2 at the sample level, fractionated into 100 movie frames. Three movies were acquired per hole, for a total of 6145 movies, with a defocus range from −1.2 to −2.5 μm.For the apo RESC1–RESC2 complex, micrographs were collected with a 300 kV Titan Krios (Thermo Fisher Scientific) electron microscope at EMBL Heidelberg, equipped with a K2 Summit direct electron detector and a GIF Quantum energy filter (Gatan). Data were acquired using serialEM  at a nom2 at the sample level, fractionated into 60 movie frames. One movie was acquired per hole, for a total of 4420 movies, with a defocus range from −1.2 to −3.5 μm.For the gRNA bound RESC1–RESC2, micrographs were collected with a 300kV Titan Krios (Thermo Fisher Scientific) electron microscope at ESRF Grenoble, equipped with a K3 direct electron detector and a Quantum-LS energy filter (Gatan). Data were acquired using EPU at a nominal magnification of 105 000, resulting in a pixel size of 0.84 Å .The model of RESC1–RESC2 was generated by submitting the full-length sequences of RESC1 and RESC2 to the Alphafold ColabFold notebook using the multimer mode, and the best-ranked of the 5 resulting models was selected ,36 . Th. ThTrypay Figure  and D. Fy Figure .2 and involves about 28% of the resolved domain, as calculated by PDBePISA. In each subunit, a hydrophobic patch binds the β-harpin of the other subunit, mediated by interactions between β-2 and β-3 of the hairpin with β-4, β-5 and helix B of the barrel . . 48). Fuin vitro transcribed RNA that harbours a 5’-triphosphate and a chemically synthesised RNA that harbours a 5’-hydroxyl of identical sequence, we observe complex formation exclusively with the 5′-triphosphate-containing RNA , for example, has been described to be associated with RESC1–RESC2, but in co-immunoprecipitation experiments with tagged KREH2, a RESC-associated helicase, only RESC1–RESC2 and gRNA, but not RESC6 were detected . This fiGiven the lack of larger RESC complex structures, we can only speculate how RESC1–RESC2 engages with other components of the RESC complex. Both RNA-dependent and RNA-independent interactions have been identified within various RESC sub-assemblies (reviewed in ). The adBidirectional transcription of the gRNA genes from minicircles generates long sense and antisense gRNA precursors with 5’ overlapping regions, which are polyuridylated by RET1 and trimmed at the 3’-end by the MPsome complex. These processing steps result in symmetric duplexes of 30–60 nucleotides containing both the sense gRNA transcript and the antisense non-guide RNA (ngRNA) with uridylated 3’-tails and preserved 5’-triphosphates . It is cTrypanosoma cruzi and Trypanosoma brucei  and EMD-16593 (gRNA bound) and the apo model is deposited at PDB: 8CDP.gkad217_Supplemental_FileClick here for additional data file.


Automatically determined band widths in simulated backscatter Kikuchi patterns exhibit differences from the double Bragg angles that correlate with the scatterer density. Corrections are proposed to compensate for this. a/a of the mean lattice parameters derived from as many Kikuchi band widths as possible. For dynamically simulated Kikuchi patterns, Δa/a can reach up to 8% for phases with a high mean atomic number Z, whereas for much more common low-Z materials the offset decreases linearly. A predicted offset Δa/a = f(Z) is therefore proposed, which also includes the unit-cell volume and thus takes into account the packing density of the scatterers in the material. Since Z is not always available for unknown phases, its substitution by Zmax, i.e. the atomic number of the heaviest element in the compound, is still acceptable for an approximate correction. For simulated Kikuchi patterns the offset-corrected lattice parameter deviation is Δa/a < 1.5%. The lattice parameter ratios, and the angles α, β and γ between the basis vectors, are not affected at all.A band width determination using the first derivative of the band profile systematically underestimates the true Bragg angle. Corrections are proposed to compensate for the resulting offset Δ Although the first derivative works automatically and gives reproducible results, it yields slightly different lattice parameters ahkl from Kikuchi band widths indicating the (inverse) distance to the reciprocal-lattice point hkl. The resulting distribution of ahkl as calculated using the software CALM  patterns in Part I of this series (Nolze a/a = (aCALM − a0)/a0 can easily be displayed as a function of the mean atomic number a/a < 8%). To a first approximation it scales with et al.  or from excess deficiency effects. It is also conceivable that the differences are due to a deviating electron energy, E0 but rather an energy distribution is likely to be relevant for experimental BKD patterns, whose governing maximum is slightly below E0  the relative offset Δa/a is shown as hollow symbols for as many elements as possible of different structure types; for some elements more than a single modification was analysed, e.g. for Fe all three are shown. The light-grey error bars refer to σhkl which also increases with Z. The distribution of hollow symbols can be roughly described by a linear approach, a) as a dark-grey line. However, clearly visible hump-shaped deviations from this line occur for elements with Z = 25–30, Z = 40–50 and Z = 70–80.In Fig. 1b) as blue filled circles. The undulating curve is due to the varying binding forces and the resulting similarly varying packing density of the atoms, which can be described in a similar way to the mass density. Therefore, for a better description of the offset curve, the mass density is taken into account, which is as much of an unknown for an unknown phase as the lattice parameters to be determined. However, aCALM and the unit-cell volume Vuc derived from it are completely sufficient for a suitable estimate.The positions of these humps are reminiscent of the mass density ρ, shown for comparison in Fig. 1muc and volume Vuc of the unit cell. muc is equivalent to the number of formula units (n) per unit cell multiplied by the atomic mass M. Since for elements M ≃ 2Z we can formulate a relationship between density ρ and atomic number Z,n/Vuc)Z represents the proton density, which is assumed to be crucial for backscattering since the interaction happens between the primary electrons and the core of the atom. If there is an anomaly in the proton density, a deviation from the linear lattice parameter offset follows.The mass density ρ is the quotient of the mass b) proves that, apart from a proportionality factor of 1.25, the density ρ (light-blue circles) for elements is almost congruent with 3.321nZ/Vuc (red filled circles). [The factor 3.321 results from 2 ( ≃ M/Z) divided by Avogadro’s number 6.02214076 × 1023 and multiplied by cm3/Å3 = 1024.]Fig. 1a) can be satisfactorily predicted by a quadratic equation with nZ/Vuc as an additional correction term in . By inserting Z and Vuc into , 12 with B2 (CsCl), 16 with B3 (zincblende) and eight with B4 (wurtzite). Compounds of light, heavy, or both light and heavy elements were selected since we expected the largest deviation in the fit for such phases.To start with the simplest possible phases, the master patterns of 96 binary phases of type In addition, BKD patterns were analysed for a further 136 common phases that consist of up to five elements and are of greater technical or mineralogical interest, such as borides, carbides, oxides, sulfides, arsenides, selenides, carbonates, sulfates, silicates and various intermetallic phases.CALM and a fit using the quadratic correction in . The different colours of the circles distinguish between the different structure types. For a better comparison with Fig. 1Direct comparisons of the a/a = ±1%. The distribution of the filled circles confirms that for many phases the difference between linear and quadratic fits is small. Nevertheless, especially for i.e. one really only sees the outliers. Comparing the frequency distributions of the relative deviations of the predicted offset (fit) and the offset from CALM for elements and compounds, it turns out that they are quite similar .The analysis of all studied compounds showed that the element with the highest atomic number on Fig. 7: the comZmax and assuming n = 1, the following parameter set can be fitted: p = 0.117, q = 0.055 and r = 0.526. The diagram in Fig. 8a) displays the predicted offset (black dots) but this time plotted as function of Zmax. Since CALM are shifted to the right, which results in a lower linear slope of the distribution (light-grey dotted line). The remaining offset difference ΔCALMfit− between the offset predicted with Z = Zmax and n = 1 and the one resulting from CALM is shown in Fig. 8b). Although the deviation is about one-third larger than in Fig. 2n, it is still often less than 1%, which in our view represents a real alternative for phases whose chemical composition is not well known except for the heaviest element of the compound.Replacing Zmax will not be as good as for a phase with known composition, but we expect that the supposed error increases only for phases with heavy elements and amounts to a maximum of 2% [Fig. 8b)].Thus, the resulting correction of the absolute lattice parameters by the predicted offset via 3.aCALM is systematically too high. The offset approximately correlates with Zmax and n = 1 is, to a first approximation, also an acceptable approach to estimate the offset Δa/a, a0 used during signal simulation of the master pattern is presented in Fig. 9For simulated Kikuchi patterns Zmax tend to generate higher deviations. For the 249 compounds studied, the relative deviation is given by Higher ight 2009. Howeveri.e.a/b, c/b, α, β and γ, remain unchanged, the missing basis vector lengths can be determined: Since all other lattice parameters,


A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low- and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits. The lysis buffer contains a mixture of detergent and chaotropic salt to lyse the sample and inactivate nucleases4. Upon addition of nanoparticles dispersed in isopropanol, the chaotropic agents from the lysis buffer promote binding of released NAs to the silica coating5. When a magnetic field is applied, the superparamagnetic properties of the nanoparticles allow isolation of adsorbed NAs from the solution6. Following a series of alcohol wash steps, the purified NAs are eluted in nuclease-free water and ready for use in downstream applications. In fact, NAxtra has been shown to rival the performance of commonly used viral NA extraction kits at considerably reduced cost1. Thus, the possibility of applying this technology to isolate NA from other biological sources is currently being explored.During the COVID-19 pandemic, a shortage of commercial kits for SARS-CoV-2 RNA isolation became a limiting factor for PCR-based testing of infection status in many countries, including Norway. This prompted the development of a nucleic acid (NA) extraction technology at the Norwegian University of Science and Technology (NTNU), a technology that has since been commercialized as NAxtra by Lybe Scientific AS. NAxtra is comprised of two components, namely a customized lysis buffer and silica-coated superparamagnetic iron oxide nanoparticles7, the most widely used extraction procedures include the standard phenol–chloroform method8, and column- or bead-based commercial kits7. Regarding the former, phenol and chloroform are hazardous chemicals that may contaminate the purified sample and affect downstream analyses9. Meanwhile, commercial kits are costly, with those relying on spin-columns being low throughput and difficult or impossible to automate. Cost-effective, high-throughput alternatives exhibiting comparable or improved performance to the current commercial kits would lower expenses for molecular research and help to accelerate advancements in these fields. For this purpose, we developed an automatable, simple, and efficient method for NA isolation from mammalian cells and organoids based on the low-cost NAxtra technology. We show that the NAxtra-based method is successful in isolating high-integrity DNA and RNA from various cell and organoid types. It has a flexible input range from one hundred to one million cells and produces similar results to commonly used commercial kits while being less costly, less time-consuming and more user friendly. We have implemented the method on the robot systems KingFisher Flex and KingFisher Duo Prime for automated extraction of 96 or 12 samples, respectively. This enables rapid and consistent total NA isolation in 14 min, and DNA or RNA purification in 22 min (+ nuclease incubation time). Finally, the extracted NA is shown to be suitable for (RT-)qPCR and has been sequenced by NGS.DNA or RNA purification from mammalian sources is frequently required in molecular research, e.g., for disease-related studies. Although numerous methods have been developed over the years10. In many cases, DNA and/or RNA analyses are fundamental to reveal key molecular mechanisms. Therefore, robust and reliable extraction of high-quality DNA/RNA is essential. To illustrate the potential of the NAxtra-based method for NA extraction from different cell types, isolation of total NA, DNA and RNA was conducted for 100,000 cells of an adherent cell line (HAP1), a suspension cell line (JJN-3) and primary cells . Extraction yields . The AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN) was used for comparison.The NAxtra total NA procedure has a cell input limit of 100,000 cells, while the DNA and RNA protocols can be performed for at least 1,000,000 cells. To demonstrate the flexible cell input range of the NAxtra-based method, NA extraction was conducted for 100 to 1,000,000 cells of HAP1. Extraction was evaluated in terms of DNA/RNA yield for 500,000 and 1,000,000 cells, and in terms of (RT-)qPCR cycle threshold (Ct) values, for 100 to 100,000 cells. Relationships between cell numbers  and Ct values were in all cases highly linear for the NAxtra-based procedures  Fig. B, with hFor RNA extraction, the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN) produced the highest total RNA yields for 500,000 and 1,000,000 cells of HAP1 Fig. B, followmirVana Total RNA isolation kit (Applied Biosystems), respectively  for further NA extraction. Hence, the total NA yield obtained using the MagMAX kit is expected to correspond to approximately half of the NA yield obtained using NAxtra. This was observed for RNA yields in total NA samples. However, the average reduction in DNA yield for MagMAX total NA samples compared to NAxtra was more than sixfold, indicating that the NAxtra method is superior for DNA extraction from HAP1 cells  are widely used in molecular research, bead-based methods have become increasingly popular due to their practicality. To compare the efficiency of the NAxtra method to bead-based alternatives, extraction of total NA, DNA and RNA from 100,000 cells of HAP1 was performed using the MagMAX Total Nucleic Acid Isolation Kit (Applied Biosystems), MagMAX DNA Multi-Sample Kit (Invitrogen) and MagMAX 11. Compared to cell culture, the increased structural complexity of organoids may impede homogenous cell lysis during NA extraction, thereby requiring additional homogenization steps. To investigate the potential of the NAxtra procedure in extracting NAs from higher complexity samples, DNA and RNA were extracted from different organoid types . The resulting DNA and RNA yields from single retinal organoids were shown to be similar for NAxtra and MagMAX  and MagMAX MAX Fig. . Lower p12. During NA extraction, the presence of nuclease activity or factors intrinsic to the extraction procedure, e.g., shear forces for column-based methods, may result in degradation and reduced NA integrity. To assess NA integrity, capillary or automated electrophoresis are commonly used, after which an RNA Integrity Number (RIN) or DNA Integrity Number (DIN) is determined, ranging from 1 (lowest integrity) to 10 (highest integrity)14. The integrity of DNA and RNA extracted from different cells and organoid types using NAxtra was assessed by these methods genetic alterations or gene expression changes, requires NA of high integrity1 recently showed that eluates obtained using NAxtra on nasopharyngeal swabs are compatible with various RT-qPCR master mixes for detection of SARS-CoV-2 RNA. In Fig. NA extraction methods vary in their ability to remove impurities that may inhibit downstream reactions such as PCR or sequencing. Impurities can derive from solutions used in extraction, such as residual salts or organic solvents, or from insufficient removal of cellular components. Ravlo et al.Another typical downstream application for extracted NAs is NGS, which requires high quality NA material. Whole genome 5-mC and 5-hmC sequencing with the BGI platform (GEO accession ID: GSE240177) and RNA sequencing with the Illumina platform (GEO accession ID: GSE240564) have been successfully performed on NAs extracted from mammalian cells using NAxtra.16 or as biomarkers for various diseases20. To assess the ability of the NAxtra-based method to extract small RNAs, the method was compared to a bead-based commercial kit designed to effectively purify such molecules, namely the MagMAX mirVana Total RNA isolation kit (Applied Biosystems). Total RNA was extracted from 1,000,000 cells, and the presence of five miRNA targets  and one mRNA target (ACTB) was detected using RT-qPCR , are extensively studied due to their role in RNA silencing, and potential applications in disease therapyPCR Fig. . The resThe NAxtra-based method for isolation of RNA and DNA from mammalian cells and organoids is shown to be highly versatile in terms of sample type and input range. When compared to alternative commercial kits, it produces similar yields while being both less costly and faster, with a processing capability of 96 samples in 14 min when automated. The extracted NAs are shown to be of high integrity and compatible with various kits for (RT-)qPCR, and NGS. Altogether, our data shows that the NAxtra method for isolation of RNA and DNA derived from mammalian cells and organoids is straightforward, flexible, rapid, and cost-effective, being an attractive alternative to commercially available alternatives.l-glutamine  and 1% Penicillin  Streptomycin  (Gibco). Cultures were incubated at 37 °C in a Steri-Cycle CO2 Incubator (Thermo Scientific) containing 5% CO2. Cells were harvested at 70–80% confluency. For comparison of the NAxtra-method with the MagMAX mirVana Total RNA isolation kit (Applied Biosystems) for 1 million cells, the cell pellets were used directly in NA extraction. For other comparisons, cell pellets were flash frozen in liquid nitrogen and stored at − 80 °C until NA isolation.JJN-3, HAP1 and primary fibroblasts, provided by the Department of Clinical and Molecular Medicine (IKOM) at NTNU, were cultured in RPMI-1640 (Sigma-Aldrich), IMDM (Gibco) and DMEM (Sigma-Aldrich) medium, respectively. All media were supplemented with 5% fetal bovine serum (Sigma-Aldrich), 1% .21, Sun et al.22, and Hofbauer et al.23, respectively, with minor modifications, such as changes in medium components and/or differentiation mediators. Retinal organoids derived from two individuals were collected at day 10 of differentiation, with average diameters of 630 µm and 570 µm. The smaller retinal organoids were used for NAxtra-based NA extraction from single organoids . Vascular organoids made with three variations of the final culture stage, i.e., embedded in Matrigel with MV2 medium, embedded in Matrigel with neural differentiation medium , and in neural differentiation medium without Matrigel, were collected at day 12 of differentiation with respective average diameters of 1.5 mm, 1 mm and 800 µm. The 1 mm organoids were used for extraction from single vascular organoids, while combinations of the three sizes were used for isolation from four to six vascular organoids. Cardiac organoids were collected at day 11 of differentiation with an average diameter of 620 µm. Organoids were imaged with the Olympus UC90 attached to an Olympus CKX53 Cell Culture Microscope (Holger Hartmann AS). Collected organoids were stored at − 80 °C until NA extraction.Retinal, cardiac, and vascular organoids were generated following procedures described by Yamasaki et alids Fig. . Single mirVana Total RNA isolation kit (Applied Biosystems), elution volumes were 100 µl for 1 million cells and 50 µl for 100,000 cells. For comparison to MagMAX DNA Multi-Sample Kit (Invitrogen) and MagMAX Total Nucleic Acid Isolation Kit, the elution volumes were 100 µl and 50 µl, respectively.Samples of selected cell or organoid inputs, resuspended in 100 µl PBS, were mixed with 200 µl NAxtra LYSIS BUFFER (Lybe Scientific). Each sample was added a 400 µl mixture of either 20 µl NAxtra MAGNETIC BEADS (Lybe Scientific) in 380 µl isopropanol , or 60 µl in 340 µl . Lysis, binding, washing, and elution was performed on the KingFisher Flex Purification System with a 96 Deep-Well Head (Thermo Scientific) or KingFisher Duo Prime Magnetic Particle Processor with a 12 Deep-Well Head (Thermo Scientific). For DNA samples, RNase treatment was conducted by adding 40 µg PureLink RNase A (Invitrogen) and nuclease-free water to a final reaction volume of 100 µl  or 200 µl , followed by a 2 min incubation at room temperature. For RNA samples, DNase treatment for cell inputs ≤ 100,000 was performed using 2.5 µl RNase-Free DNase (QIAGEN), 10 µl Buffer RDD (QIAGEN) and nuclease-free water to a final volume of 100 µl for each sample, with room temperature incubation for 10 min. For cell inputs > 100,000, a mixture of 2.5 µl RNase-Free DNase (QIAGEN), 20 µl Buffer RDD (QIAGEN) and 27.5 µl nuclease-free water was prepared for each sample. For 5 min at room temperature eluates were incubated with 20 µl of the mixture before adding the remaining 30 µl with another 10 min of incubation. Nuclease-treated eluates were combined with 200 µl NAxtra LYSIS BUFFER (Lybe Scientific) and 400 µl  or 600 µl  isopropanol before another round of mixing, washing and elution was performed on the KingFisher Flex Purification System or KingFisher Duo Prime Magnetic Particle Processor. Final elution volumes for comparison to the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN) were 50 µl in all cases except for DNA from cell inputs exceeding 100,000 cells, where 100 µl was used. When comparing to the MagMAX The AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN) is a widely used spin column-based method that allows simultaneous purification of DNA and RNA from cells or tissue samples. DNA and RNA extraction was performed according to manufacturer’s instructions. Elution volumes for the DNA fraction were two volumes of 100 µl if the eluate was to be used for assessment of yield, and 50 µl if the eluate was to be used for qPCR. Elution volumes for the RNA fraction were 50 µl.mirVana Total RNA isolation kit (Applied Biosystems) is a leading product on the market, which enables isolation of total RNA, including microRNA, from multiple sample types using magnetic beads. Extraction of total RNA was conducted on the KingFisher Duo Prime Magnetic Particle Processor with a 12 Deep-Well Head (Thermo Scientific) according to kit instructions. Elution volumes were 100 µl for 1 million cells and 50 µl for 100,000 cells.The MagMAX The MagMAX DNA Multi-Sample Kit (Invitrogen) is designed for rapid DNA isolation from multiple sample types, including cells and organoids, using magnetic beads. DNA extraction was executed on the KingFisher Duo Prime Magnetic Particle Processor with a 12 Deep-Well Head (Thermo Scientific) according to kit instructions, including a two-step elution with final elution volumes of 130 µl.MagMAX Total Nucleic Acid Isolation Kit (Applied Biosystems) is used for isolation of total NA from a wide range of sample types. Isolation of total NA was performed on the KingFisher Duo Prime Magnetic Particle Processor with a 12 Deep-Well Head (Thermo Scientific) according to manufacturer’s instructions. The method involved a bead-beating step that allowed only 56.1% of the lysate to be used as input for extraction. For each cell sample, isolation was conducted by two parallel runs of 115 µl lysate with a combined elution volume of 50 µl.DNA and RNA quantity was assessed for duplicates of each sample and five standards using Quant-iT dsDNA HS Assay Kit (Invitrogen) and Quant-iT RNA HS Assay Kit (Invitrogen), according to manufacturer's instructions.All qPCR reactions were performed by thermal cycling on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with reaction volumes of 10 µl and duplicate no template controls (NTCs) for each master mix. The primers are listed in Supplementary Table The 305 bp, single-locus genomic DNA amplicon of the KAPA Human Genomic DNA Quantification and QC Kit was amplified according to manufacturer’s instructions.MYC was amplified in reactions of 2.5 µl eluate, 1X MYC TaqMAN Copy Number Assay Hs00834648_cn (Applied Biosystems), and either 1X RealQ Plus 2 × Master Mix for Probe (Ampliqon) or 1X Luna Universal Probe qPCR Master Mix (NEB), with annealing at 62°C and remaining reaction conditions according to master mix instructions.ACTB mRNA was reverse transcribed and amplified in reactions of 2.5 µl template RNA, 1X ACTB PrimeTime XL PCR Assay Hs.PT.39a.22214747 (Integrated DNA Technologies), and either 1X qScript XLT 1-Step RT-qPCR ToughMix (Quantabio), 1X One-Step Takyon Ultra Probe 4X MasterMix (Eurogentec), or 1X 1Step RT qPCR Probe 4X Kit (highQu) with annealing at 62 °C and remaining reaction conditions according to master mix specifications.Power SYBR Green PCR Master Mix (Applied Biosystems). Reactions were performed with enzyme activation at 95 °C for 10 min, and amplification in 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s.Reverse transcription of mature microRNA (miRNA) was performed using the miScript II RT Kit (QIAGEN) with HighSpec Buffer and 12 µl RNA template input. Duplicate dilutions of the resulting cDNA were prepared according to kit recommendations, and subsequent detection of miRNA targets by qPCR was conducted with 1 µl diluted cDNA, 300 nM target-specific primer (Supplementary Table NAs were separated on 1.2% or 0.4% agarose gels in Tris–acetate-EDTA (TAE) buffer, with 0.5 µg GeneRuler 1 kb plus DNA ladder (Thermo Scientific) or 0.1 µg GeneRuler High Range DNA ladder (Thermo Scientific), respectively. Visualization was performed by post-staining with GelRed Nucleic Acid Gel Stain (Biotium) according to manufacturer’s instructions. Gel images were acquired using the Kodak Gel Logic 200 Imaging System. Gel images presented in the results section have been inverted and cropped. Original gel images are provided in Supplementary Figs. RNA integrity was assessed with the Agilent RNA 6000 Pico kit on the Agilent Bioanalyzer 2100 (Agilent Technologies) according to kit instructions. The Agilent 4200 TapeStation System with GenomicDNA ScreenTapes (Agilent Technologies) was used for DNA integrity analyses according to manufacturer’s instructions.P ≤ 0.05 as the probability level determining significance.Statistical analyses were performed in GraphPad Prism (version 9.5.1) by two-sided, unpaired t-tests, using Supplementary Information.