The flexible strain sensor was fabricated using MoS2 and showed high performance of low detection limit (0.09%), high gauge factor , and high stability . Besides, we demonstrated its applications in real-time monitoring of health-related physiological signals such as radial artery pressure, respiratory rate, and vocal cord vibration. Our findings suggest that the laser-assisted method is effective and capable of synthesizing wafer-scale 2D TMDs, which opens new opportunities for the next flexible electronic devices and wearable health monitoring.The two-dimensional (2D) transition metal dichalcogenides (TMDs) are promising flexible electronic materials for strategic flexible information devices. Large-area and high-quality patterned materials were usually required by flexible electronics due to the limitation from the process of manufacturing and integration. However, the synthesis of large-area patterned 2D TMDs with high quality is difficult. Here, an efficient and powerful pulsed laser has been developed to synthesize wafer-scale MoS  •2 film has been synthesized by pulsed laserWafer-scale patterned MoS•2 film strain sensor shows low limit detection, high GF, and stabilityThe MoS•2 film strain sensorThe healthy-related singles have been monitored by the MoS Physics; Sensor system; Nanomaterials Flexible electronic sensors are the essential components of a new generation of strategic flexible information devices. As the basic and widely used devices, flexible strain sensors have attracted increasing attention . High-quControllable synthesis of wafer-scale TMDs is the pursuit of scientific researchers . Recentl2. Semitransparent flexible strain sensors were fabricated using the laser-written MoS2 film. The electromechanical behavior of the MoS2 film strain sensor was then investigated, and applications of the sensor were explored. Health-related signals have been monitored, such as radial artery pressure, respiratory rate, and vocal cord vibration.In this study, the laser-assisted method was used to synthesize programmable wafer-scale MoS2 film process is illustrated in 2 film. Before laser writing, the SiO2/Si wafer with an oxidation layer of 285 nm was cleaned with plasma (4)2MoS4) precursor solutions were prepared and spin coated on the SiO2/Si wafer. Then, the SiO2/Si wafer was baked to evaporate residual solvents 2MoS4, and the following chemical reactions occurred (The programmable patterned MoSh plasma B. The amsolvents C. The laoccurred .(Equatio2/Si substrate was rinsed in dimethylformamide solution (DMF) to remove any residual precursor film, and a programmable MoS2 pattern remained on the SiO2/Si substrate. As shown in 2 film with a pattern of “2D Materials,” matrix electrodes, wrist and pulse waves, and the logo can be effectively and efficiently realized.After laser writing, the SiO2 film after laser writing was shown in in situ optical and scanning electron microscopic (SEM) images of the MoS2 films have been added in 2 uniformity films had been successfully synthesized by laser. The high-magnification SEM image of the interface with and without laser writing is shown in 2 films and precursor films are uniforms. The Raman spectrum was used to identify the materials, as shown in −1, was consistent with the Raman peak of Si and raised from SiO2/Si substrate. After laser writing, apart from the Si peaks, two typical Raman peaks at 381.6 cm−1 and 407.1 cm−1 were observed, indicating that the 2D 2H phase MoS2 crystal film was successfully synthesized. The peak at 381.6 cm−1−1d, Mo 3p, S 2p was increased, which indicated that the surface element concentration of the Mo and S increased during laser writing. The high-resolution XPS spectra of Mo 3d and S 2p is shown in d spectrum of the samples with and without laser writing  into 2p1/2 (163.8 eV) and 2p3/2 (162.6 eV) with laser writing, which signified the formation of 2H-MoS2  of 0.78 nm, indicating that five layers of MoS2 were successfully synthesized.Optical image of the MoSnal mode . X-ray p writing E, four p writing . With laectively . The bin to Mo4+ . Compare 2H-MoS2 . The unr2, have unique advantages in applications of flexible stress sensors. The MoS2 film strain sensor was fabricated as shown in 2 film was transferred from the SiO2/Si substrate using the polymethyl methacrylate (PMMA)-assisted transfer method. A 50-nm Au interdigital electrode that had an electrode spacing of 100 μm was fabricated using laser etching technology on the polyethylene terephthalate (PET) substrate. The MoS2 film covered the PET substrate, and PMMA was removed by acetone. The electromechanical behavior of the MoS2 film strain sensor is shown in 2 film strain sensor. The initial resistance (R0) of the MoS2 film strain sensor is 8.0 MΩ. Under the forward bending, the resistance of the MoS2 film strain sensor is increased. The sensor response can be calculated as 0.09, 0.39, 1.45, 2.20, and 6.82 under different compressive lengths (ΔL) of 0.05, 0.1, 0.5, 1, and 2 mm shown in 2 film strain sensor shows the opposite evolution result indicating that our MoS2 film strain sensor is the piezoresistive sensor (2 film on PET. As shown in C) and arc length (L) using the following formula .The r can be calculated to be 53.0, 37.5, 16.7, 11.7, and 8.2 mm with ΔL increases from 0.05 mm to 2 mm. Thus, the bending strain (ε) can be calculated by the following formula : of the MoS follows : and GF are shown in terature , 2019b. l strain . On the tability .2 film strain sensor, which has a low limit of detection and high GF, can be used for small-signal detection. Radial artery pressure (RAP) is used as an indicator in personal health and clinical diagnoses, and the shape of the RAP wave can assist doctors in diagnosing diseases such as arterial stiffness and high blood pressure  and reflected wave (P2). Heart rate with time delay (ΔtDVP = tP1-tP2) and radial artery augmentation index (Alr = P2/P1) could be extracted to be 277 ms and 42%, respectively, which were in the normal range for healthy males. Furthermore, the MoS2 film strain sensor was connected to a respirator to monitor breathing rate in real time, as shown in 2 film strain sensor was also attached to a human throat 2MoS4 film could be effectively decomposed to form high-quality MoS2 film by a pulsed laser. Furthermore, the strain sensor was fabricated using the MoS2 film and showed a low detection limit (0.09%), high GF , and stability. Moreover, health-related signals, such as radial artery pressure, respiratory rate, and vocal cord vibrations, were monitored using the sensitive strain sensor. Our work contributes to the research on the development of large-area, high-quality 2D materials and adds to the diversification of flexible electronic materials by providing a new way to apply 2D TMD materials.In conclusion, we demonstrated a simple and efficient direct laser writing method to synthesize patterned, wafer-scale, and layered MoS2 film on a SiO2/Si substrate. However, given the laser processing range, preparing a larger MoS2 film with continuous fabrication needs further exploration. In addition, direct synthesis of a high-quality, large-area MoS2 film on a flexible substrate using a laser will greatly promote printed and flexible electronics development.This work focuses on synthesizing wafer-scale MoSiamxwwang@nwpu.edu.cn).Further information and requests for resources and materials should be directed to and will be fulfilled by the lead contact, Prof. Xuewen Wang (This study did not generate new unique reagents.2 film synthesis process is shown in 4)2MoS4 in 20 mL DMF, 8 mL ethanolamine, and 8 mL n-butylamine mixed solution. The precursor solutions were magnetically stirred at 1000 rpm for 60 min and then stirred by ultrasound for an additional 60 min. A 285 nm SiO2/Si substrate was cleaned under air plasma at 100 W for 2 min. Then, the precursor solution was spin-coated on the SiO2/Si substrate with a gradient procedure of 500, 1,000, 1,500, and 2000 rpm for 10 s and 2500 rpm for 30 s. After, the SiO2/Si substrate was placed on a hot plate and baked at 150°C for 3min.The MoS2 film synthesis, the commercial laser marking machine  used a fiber laser with a wavelength of 1.06 μm (spot size of ∼22 μm). The prepared SiO2/Si substrate was placed under the field lens (110 mm2), and a programmable pattern was marked using EZCAD laser marking software. Different laser states can be obtained by adjusting the laser parameters, such as working distance (the distance between sample surface to the lens), laser power, pulse frequency, laser spacing, and writing speed. For a typical 5 mm × 10 mm area, the laser with a power density of 5.26 × 109 W/m2 (laser fluence of 2.63 J/cm2) have been adopted under the optimized laser parameters as follows: working distance (170 mm), laser power (2 W), pulse frequency (200 kHz), laser spacing (2 μm), and writing speed (50 cm/s). After laser writing, the SiO2/Si substrate was washed in DMF at 130°C for 60 s to remove any residual precursor film. Lastly, the substrate was dried at 150°C, and the programmable MoS2 pattern remained on the SiO2/Si substrate.For the MoS2 film were characterized using optical microscopy (Nexcope NMM910), Raman (WITEC Alpha 300R), AFM (Asylum Research Cypher S), XPS (Thermo SCIENTIFIC ESCALAB 250Xi) and SEM (FEI Apreo S). The Raman characterization was conducted using a 532 nm laser calibrated with a Raman peak of Si at 520 cm−1. The binding energy of the XPS results was corrected by assigning a value of 284.8 eV to the adventitious C 1s line.The morphology and microstructures of the MoS2 film  was synthesized on an SiO2/Si substrate. Then, PMMA (950 A5) was spin-coated on the SiO2/Si substrate at a speed of 500 rpm for 5 s and 2000 rpm for 60 s. After baking at 120°C for 5 min, the SiO2/Si substrate was immersed in a hydroxide (KOH) solution at 90°C for 10 min. Then, PMMA was cleaned using deionized water and ready for device fabrication.The device fabrication process is shown in 2. The laser parameters are as follows: working distance (115 mm), laser power (2 W), pulse frequency (200 kHz), laser spacing (10 μm), and writing speed (1 m/s). PMMA with the MoS2 film was put on the PET to ensure the MoS2 film came into close contact with the electrode. After natural drying, PMMA was removed using acetone and cleaned using isopropyl alcohol, deionized water, and ethanol, respectively. After drying, the conductive carbon oil served as a bridge between the Au electrode and the test wire. The carbon oil was cured for 60 minutes at 60°C for further tests.A 50 nm Au film was deposited on a 100 μm thickness PET substrate using the thermal evaporation system . An interdigital electrode with an electrode spacing of 100 μm was prepared using laser etching technology that had the same laser under the field lens of 70 mmA stepper motor was used to control the strain applied to the sensor, and a source meter  was used to measure the resistance of the sensor. The LabVIEW program was used for data acquisition. In addition, a multimeter  was used to test the health-related signals.Each value of sensor response (with standard deviation) and gauge factor shown in


Pregabalin and gabapentin improve neuropathic pain symptoms but there are emerging concerns regarding their misuse. This is more pronounced among patients with substance use disorder, particularly involving opioids. Co-ingestion of gabapentinoids with opioids is increasingly identified in opioid related deaths, however, the molecular mechanism behind this is still unclear. We have sought to determine whether pregabalin or gabapentin directly modulates acute μ receptor signaling, or μ receptor activation by morphine.The effects of pregabalin and gabapentin were assessed in HEK 293 cells stably transfected with the human μ receptor. Their effect on morphine induced hyperpolarization, cAMP production and ERK phosphorylation were studied using fluorescent-based membrane potential assay, bioluminescence based CAMYEL assay and ELISA assay, respectively. Pregabalin/gabapentin effects on morphine-induced hyperpolarization were also investigated in AtT20 cells.Pregabalin or gabapentin  did not activate the µ receptor or affect K channel activation or ERK phosphorylation produced by morphine. Neither drug affected the desensitization of K channel activation produced by prolonged (30 min) application of morphine. Gabapentin  and pregabalin (1 µM) did not affect inhibition of forskolin-stimulated cAMP production by morphine. However, pregabalin (100 µM) potentiated forskolin mediated cAMP production, although morphine still inhibited cAMP levels with a similar potency to control.Pregabalin or gabapentin did not activate or modulate µ receptor signaling in three different assays. Our data do not support the hypothesis that gabapentin or pregabalin augment opioid effects through direct or allosteric modulation of the µ receptor. Pregabalin at a high concentration increases cAMP production independent of morphine. The mechanism of enhanced opioid-related harms from co-ingestion of pregabalin or gabapentin with opioids needs further investigation. Gabapentin and pregabalin improve analgesia in acute perioperative pain and chronic neuropathic pain . They arWhile both gabapentin and pregabalin are GABA analogs, no direct action at GABA receptors has been identified . The anaThe µ receptor (previously known as µ opioid receptor) and its endogenous ligands are crucial regulators of cells and circuits that detect, integrate, and respond to painful sensations. Clinically used opioids exert their analgesic action through activation of the µ receptor , howeverAnimal studies suggest gabapentinoid activity in modulating neuropathic pain is opioid independent . HoweverTo date, there has been no investigation of the actions of gabapentin or pregabalin at the µ receptor. We, therefore, hypothesized that gabapentin and pregabalin enhance opioid effect through direct modulation of the µ receptor. The current study investigated recombinant human µ receptor modulation of three distinct effector systems: activation of G protein-coupled inwardly rectifying potassium channels (GIRK), inhibition of adenylyl cyclase, and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation.+ . Selection was performed using 150 µg/mL hygromycin gold and resistant cells are considered isogenic due to integration of the plasmid into the same genomic locus in every clone (Flp-In™ system).The study was performed in Human Embryonic Kidney Flp-In™-293 (HEK) cells from Life Technologies  and mouse pituitary (AtT20) cells from ATCC (CRL-1795). HEK cells were stably transfected with human GIRK 4 construct subcloned into pcDNA3.1+  and 3-ha+  using Fu2.Cells were cultured and maintained in growth media composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine saline (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 80 µg/mL hygromycin gold. HEK cells additionally had 400 µg/mL geneticin added to the culture media. All cells were stored in a humidified incubator at 37 °C in an atmosphere with 5% CO2.The effect of pregabalin or gabapentin on morphine induced hyperpolarization was evaluated using a fluorescence-based membrane potential assay . One day2HPO4 0.338, NaHCO3 4.17, HEPES 22, KH2PO4 0.441, MgSO4 0.407, MgCl2 0.493, CaCl2 1.26, glucose 5.56 . A total of 90 μL of dye was added to each well and incubated for 60 min at 37 °C in ambient CO2 prior to fluorescence reading. All drugs were diluted in HBSS and loaded on the compound plate. Drug concentrations were ten times that of the final concentration to accommodate for dilution after addition to the well. Fluorescence was measured using FlexStation 3 plate reader (Molecular Devices) with an excitation wavelength of 530 nm and emission of 565 nm at 2 s intervals.FLIPR membrane potential blue dye was reconstituted to 50% of the manufacturer’s recommended concentration in modified Hank’s buffered salt solution (HBSS). This was composed of (in mM) NaCl 145, NaBaseline fluorescence was recorded for 2 min after which 20 µL of vehicle or drug was added. Data were calculated as a percentage change from baseline fluorescence after correction for vehicle addition.2. A total of 24 h after transfection, the cells were detached from the plate and resuspended in L-15 (no phenol red), supplemented with 1% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 15 mM glucose. Cells were then plated in 96 well white-walled clear bottom plates pre-coated with poly-D-lysine and incubated overnight at 37 °C in ambient CO2.Bioluminescence based cAMP assay was performed in HEK-µ cells to investigate the effect of pregabalin or gabapentin on morphine mediated inhibition of cAMP production . The celOn the day of the assay, all drugs were prepared at ten times the final concentration in HBSS containing 30 µM forskolin to reach the desired final concentration upon adding to the cell suspension. Cellular cAMP levels were measured using kinetics settings in the FlexStation 3 and luminescence detection was set at an emission spectrum of 461 nm (Rluc) and 542 nm (YFP). A total of 10 µL coelenterazine-h substrate was added, and the baseline luminescence was measured for 5 min before adding the drugs. Inverse BRET ratio was calculated at the emission of 461/542 so that the increase in ratio corresponds to increase cAMP production. The area under the curve was calculated for each concentration of the drug after correcting for vehicle and data were expressed as a percentage change in forskolin response after drug addition.Opioid-induced ERK phosphorylation was measured using whole cell ELISA in HEK-µ cells. The cells were resuspended in L-15 and plated in a 96 well clear microplate pre-coated with poly-D-lysine and incubated overnight at 37 °C. The next day, cells were serum starved for an hour in serum free L-15 before adding the drugs. Cells were then treated for 5 min with different concentrations of drugs prepared in serum free L-15. Some wells were separated for treatment with the positive control  and negative control (10 µM U0126). After 5 min of incubation, drug solution was removed, and the plate was immediately placed on ice. Cells were fixed using 4% paraformaldehyde for 15 min at room temperature. The cells were washed 3 times with PBS then permeabilized with 0.1% Triton-X in PBS. After 30 min, the permeabilizing agent was removed and the plate was incubated at room temperature for 2 h with blocking solution  in PBS with 0.01% Tween-20). After 2 h of blocking, the solution was removed, and cells were incubated overnight at 4 °C with rabbit α-phospho-p44/42 MAPK (Thr202/Tyr204) antibody diluted at a ratio of 1:500 with 1% BSA in PBS supplemented with 0.1% Tween-20 (PBS-T). Next day the cells were washed 3 times with washing buffer (PBS-T) and treated with secondary antibody  for 2 h. Cells were washed three times and incubated in the dark with ELISA peroxidase substrate  for 45 min. Finally, the reaction was stopped using 1N HCl. Absorbance was measured at 450 nm using the BMG PheraStar FS plate reader. Data were expressed as mean ± SEM for five individual experiments performed in triplicates and the concentration-response curve was plotted after normalizing to the positive control.50 and Emax for CAMYEL assay in measuring the effect of 100 µM pregabalin and isoproterenol in forskolin response. P values < 0.05 were considered significant for all the analysis.All data were expressed as mean ± SEM of at least five independent experiments performed in duplicates unless otherwise stated. Concentration-response curves were plotted using the four-parameter non-linear regression equation in GraphPad PRISM 8 . Data were statistically tested using one way ANOVA corrected using Bonferroni method within each set of comparisons. Two tailed unpaired student t-test was used to compare the mean of ECMorphine was a kind gift from the University of Sydney, Australia. Pregabalin and gabapentin were purchased from Tocris, Australia. Forskolin was provided by Ascent Scientific Ltd. Culture reagents and buffer salts were supplied by Sigma Aldrich  or Thermo Fisher Scientific . The CAMYEL plasmid encoding the cAMP sensor YFP-EPac-RLuc was originally from ATCC (MBA-277). Phospho-ERK1/2 antibody and anti-rabbit IgG HRP linked antibody were from Cell Signaling Technologies . Antibiotics were from Invivogen  and coelenterazine h was from Promega . The membrane potential dye was purchased from Molecular Devices, CA.pEC50 of 6.6 ± 0.5. Incubation of pregabalin or gabapentin at 1 µM or 100 µM for 5 min did not change the morphine-induced GIRK activation in HEK-µ cells  or HEK-µ cells . In HEK-µ cells, membrane hyperpolarization in response to morphine, although lower in amplitude, persisted with minimal abatement over the 30 min it was measured  and 16 ± 3% (p = >0.99) in the presence of 100 µM pregabalin and gabapentin respectively, with a constant potency value of 6.7 ± 0.3 in all three conditions , 47 ± 5% (p = >0.99) and 35 ± 4% (p = 0.95) respectively with corresponding pEC50 values as 6.9 ± 0.1, 6.8 ± 0.2 and 7.0 ± 0.2  in the presence of 100 µM pregabalin in HEK GIRK4 wild type cells were plotted. Pregabalin did not affect the maximum response of forskolin but caused a significant increase in potency of forskolin (p = 0.015), however, it did not affect the response of isoproterenol (To test the hypothesis that this increase may be due to activation of Gαoterenol .p > 0.99) and 100 µM gabapentin (p > 0.99) did not affect the phosphorylation of ERK on HEK µ cells. We found that morphine Emax for ERK1/2 phosphorylation was 28 ± 2% of the PMA response with pEC50 value of 6.9 ± 0.2. There was no significant change in the morphine-induced phosphorylation in the presence of 1 µM or 100 µM of either pregabalin or gabapentin (p = >0.99) and 22 ± 4% (p = >0.99) with 1 µM or 100 µM pregabalin with pEC50 of 6.6 ± 0.4 (p = 0.31) and 6.5 ± 0.4 (p = 0.23), respectively. Co-administration of 1 µM or 100 µM gabapentin with morphine resulted in maximum phosphorylation of 30 ± 4% (p = 0.73) and 27 ± 4% (p = 0.92) respectively with a pEC50 value of 6.7 ± 0.3 (p = >0.99)Finally, the effect of gabapentin or pregabalin on the ligand interaction of µ receptor on ERK phosphorylation pathway was investigated using ELISA. Acute application of 100 µM pregabalin  concentrations of approximately 0.4 μg/mL (2.5 µM) . A totals and µ receptor being activated by Gi/Go, highlight the need of more studies to better understand this unknown mechanism and if it has any link to the synergistic toxicity seen with opioid and pregabalin.The finding that higher dose pregabalin augmented forskolin-stimulated cAMP activity with no effect from gabapentin also identifies a previously unrecognized difference in the drugs. Pregabalin and gabapentin are often considered interchangeable clinically. There are some differences in pharmacokinetic parameters, such as dose-dependent (non-linear) absorption and lower bioavailability of gabapentin . LookingAs the lethality of pregabalin/gabapentin combined with opioids is increasingly recognized, so has the need to understand the molecular biology of this toxicity. Both positive and negative allosteric modulators of the µ receptor have been identified . Recent Performing the experiments in different cell lines strengthens the robustness of the findings, hence we used two different cell lines. Although HEK cells showed a lower maximum response and minimal acute signal desensitization in response to prolonged exposure to morphine as compared to AtT20 cell, pregabalin or gabapentin did not alter morphine response in either HEK or AtT20 cells, which supports our findings.In conclusion, this is the first study to look at the impact of pregabalin and gabapentin on functional assays of human µ receptor signaling. Gabapentin and pregabalin do not mediate their analgesic or toxic effects by a direct effect on the µ receptor. No allosteric modulation of µ receptor activity was identified in assays of GIRK, cAMP or ERK. High dose pregabalin caused increase in cAMP production, and the mechanism behind this warrants further investigation.10.7717/peerj.11175/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11175/supp-2Supplemental Information 2Click here for additional data file.


E. coli serotype O157. To better understand host recognition by both phages, here we focused on characterizing the structures and functions of their RBPs. We identified two distinct tail fibers in the genome of the podovirus EP335: gp12 and gp13. Using fluorescence microscopy, we reveal how gp13 recognizes strains of E. coli serotypes O157 and O26. Phage EP75 belongs to the Kuttervirus genus within the Ackermannviridae family and features a four TSP complex (TSPs 1–4) that is universal among such phages. We demonstrate enzymatic activity of TSP1 (gp167) and TSP2 (gp168) toward the O18A and O157 O-antigens of E. coli, respectively, as well as TSP3 activity (gp169.1) against O4, O7, and O9 Salmonella O-antigens. TSPs of EP75 present high similarity to TSPs from E. coli phages CBA120 (TSP2) and HK620 (TSP1) and Salmonella myovirus Det7 (TSP3), which helps explain the cross-genus infectivity observed for EP75.Bacteriophages (phages) are widely used as biocontrol agents in food and as antibacterial agents for treatment of food production plant surfaces. An important feature of such phages is broad infectivity towards a given pathogenic species. Phages attach to the surfaces of bacterial cells using receptor binding proteins (RBPs), namely tail fibers or tailspikes (TSPs). The binding range of RBPs is the primary determinant of phage host range and infectivity, and therefore dictates a phage’s suitability as an antibacterial agent. Phages EP75 and EP335 broadly infect strains of  Besides O157, over 50 other STEC serogroups are associated with human illness  making the range of pathogenic E. coli that require targeting by the food industry highly diverse Bacteriophages (phages) are viruses that infect and kill specific host bacteria, while leaving human cells and non-target bacteria unaffected Listeria[20], Salmonella[22], Pseudomonas[23], and YersiniaE. coli[25], Klebsiella pneumoniaeAcinetobacter baumannii[28], has led to their investigation as anti-virulence agents to target multidrug resistant bacteria and re-sensitize them to antibiotics.The principal determinant of a phage’s infectivity is the binding specificity of its receptor binding proteins (RBPs), i.e., tail fibers and tailspikes (TSPs) E. coli, of which O157 is best known owing to its association with foodborne illnesses E. coli contain a backbone of four residues with a single residue side-branch, although the backbone can consist of two to six residues and be linear or branched. The O-antigens of the seven major STECs consist of three , four , or five (O103) residue repeats, can be branched (O111), and even sialylated (O145), demonstrating the wide variety of O-antigen structures across these important serogroups E. coli phage T4 bind to the LPS core during host adsorption E. coli phages G7C E. coli O-antigens means that a single TSP will recognize one substrate or possibly a few closely related O-antigen structures E. coli phage CBA120 features four TSPs that confer specificity towards different E. coli and Salmonella O-antigens The Gram-negative cell’s outer leaflet is composed almost exclusively of LPS. LPS consists of a membrane-bound lipid A, a core oligosaccharide, and a repetitive glycan polymer called the O-antigen. Variations in the O-polysaccharide repeat has given rise to over 170 unique O-antigens in E. coli O157 strains E. coli O157 on beef when compared to chemical preservatives  Recently, we described the isolation and genomic characterization of two phages EP75 and EP335, which have broad and complementary specificity to 22.1E. coli and Salmonella strains were obtained from various culture collections as indicated in 2.2600 of 0.6), when expression was induced with 0.5 mM isopropyl-D-thiogalactopyranoside  for 16 h at 20 °C with agitation. Cells were harvested by centrifugation . Cells were suspended in phosphate buffered saline + 0.1% Tween-20, pH 8.0 (PBS-T), containing 5 mM imidazole, cooled to 4 °C, and lysed using a Stansted pressure cell homogenizer . Purification was performed by gravity flow immobilized metal affinity chromatography (IMAC) with low-density Ni-NTA resin . A solution of 0.1% PBS-T + 5 mM imidazole was used as a wash buffer after which 0.1% PBS-T, pH 8.0 + 250 mM imidazole was used for elution. Proteins were dialyzed into 25 mM Tris, pH 7.4 and stored at 4 °C. Proteins were analyzed by SDS-PAGE (Criterion TGX stain-free gel) using PageRuler Unstained Protein Ladder (Thermo Scientific™) as a marker and imaged by UV or stained with InstantBlue® (Expedeon) using a Gel Doc XR + Imaging system (BioRad).A pQE30-based plasmid featuring N-terminal His- and GFP-tags 2.3600 of 1.0 in PBS-T, and transferred in 500 µl aliquots into 2 ml microcentrifuge tubes. 50 µg of GFP-tagged RBP was added to the cells and incubated for 90 min on an overhead rotator. Cells were spun down and washed once with 1 ml PBS-T and resuspended in 200 µl PBS-T. 150 µl of the cell suspension was pipetted into a well of a black, flat bottom 96-well microplate  and the fluorescence intensity of bound GFP-RBP was measured at ambient temperature using a POLARStar Omega spectrophotometer  at 485 nm excitation, 520 nm emission with (1000 x) fixed gain. Fluorescence binding was performed in triplicate with mean (raw fluorescence) ± standard deviation. For fluorescence microscopy, 4 μl of the cell suspension was imaged using a confocal inverted microscope (Leica TCS SPE) equipped with an ACS APO 63×/1.30 oil CS lens objective with excitation at 488 nm and emissions collected with a PMT detector in the detection range of 510 to 550 nm. Transmitted-light microscopy images were obtained with the differential interference contrast mode. Images were acquired with a Leica DFC 365 FX digital camera controlled with the LAS AF software. Fiji v2.0.0 (ImageJ software) was used to generate final images.Overnight cultures of individual strains were spun down , resuspended to an OD2.4Bacterial lawns were prepared for each test strain by mixing 200 µl of overnight bacterial cultures with 5 ml soft agar (4 mg/ml agar) poured onto a LB agar plate (12 mg/ml agar) and left to solidify for 30 min. 10 µl of EP75 TSPs 1–4, GFP or BSA controls at 2 mg/ml were spotted onto the lawn and incubated for 16 h at room temperature, 30 °C, or 37 °C. Active TSPs produced translucent and circular haloes on target bacterial lawns. Plates were imaged with an iPhone XR 12-megapixel camera under a lightbox with contrast and brightness adjustments applied to whole images to improve halo visualization.2.54), after which an appropriate volume was added to 4 ml Yeast Glucose agar  containing 100 µl of an E. coli NCTC13128 overnight culture. Subsequently, the 4 ml LB top agar was poured over an LB agar bottom plate (1.5% agar) and allowed to solidify at room temperature. The titration plates were then incubated overnight at 30 °C followed by plaque enumeration.The double layer agar method 2.6E. coli or Salmonella cell surfaces on phage adsorption, overnight cultures of E. coli or Salmonella were diluted to an OD600 of 0.5 in 200 µl LB medium. TSPs or GFP  or no protein were then added to the cells and incubated for 10 min at room temperature. The cells were collected by centrifugation  and resuspended in 200 µl LB medium. Approximately 300 plaque forming units (PFU) of EP75 was added to the resuspended pellets followed by an incubation of 10 min at room temperature. The solution was centrifuged  and 100 µl of supernatant (containing non-bound phages) was mixed with 4 ml Yeast Glucose agar containing 100 µl E. coli NCTC13128 and poured on top of an LB agar plate. Plates were incubated at 30 °C overnight followed by PFU enumeration . A sample containing 300 PFU of EP75 and no bacteria or protein was treated the same and used as input control (PFUinput). Relative phage adsorption was calculated as (PFUinput - PFUsupernatant)/PFUinput.To determine the inhibitory effect of EP75 TSP enzymatic activity of the 2.7E. coli or Salmonella cells on phage efficiency of plating a phage infection inhibition assay was performed. For E. coli strains, a single colony was inoculated in LB broth and incubated at 37 °C at 150 rpm. After reaching an OD600 of 0.3, aliquots of 100 µl of the culture were immediately incubated on ice to stop growth. TSPs or GFP  or no protein was added to the aliquots and incubated for 20 min at 37 °C. For Salmonella strains, an overnight culture was diluted to OD600 of 0.3 in LB medium and divided in 100 µl aliquots at room temperature. TSPs or GFP  or no protein was added to the aliquots and incubated for 20 min at room temperature. Approximately 100 PFU of EP75 were added to individual aliquots , mixed with 4 ml Yeast Glucose agar and poured on top of an LB agar plate. Plates were incubated at 30 °C overnight followed by PFU enumeration and determination of the efficiency of plating (EOP): PFUtreatment/PFUno protein.To determine the inhibitory effect of EP75 TSP enzymatic activity on 2.8www.FGCZ.ch) using standard protocols. In brief, samples were diluted 2-fold with 1% trifluoroacetic acid and transferred to autosampler vials for LC/MS. 10 μl of sample was injected into an ACQUITY UPLC@ BioResolve-RP-mAb 2.7μ 2.1x150 450 A  column. For separation and elution on an Acquity UPLC station , a gradient buffer A (0.1% formic acid in water)/ buffer B (0.1% formic acid in acetonitrile) at a flow rate of 200 µl/min at 500C over 25 min was applied. Analysis was performed on a Synapt G2 mass spectrometer  directly coupled with the UPLC station. Mass spectra were acquired in the positive-ion mode by scanning an m/z range from 100 to 4000 Da with a scan duration of 1 s and an interscan delay of 0.1 s. The spray voltage was set to 3 kV, the cone voltage to 50 V, and source temperature 80 °C. The data were recorded with the MassLynx 4.2 Software . For single peaks, the recorded m/z data were then deconvoluted into mass spectra by applying the maximum entropy algorithm MaxEnt1 (MaxLynx) with a resolution of the output mass 0.5 Da/channel and Uniform Gaussian Damage Model at the half height of 0.5 Da.Protein masses were identified using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) at the Functional Genomics Center Zürich, Switzerland (2.9post hoc Tukey’s multiple comparison test using GraphPad Prism (version 8.2.0).Data presented in all graphs were obtained from three independent experiments and shown as the mean ± standard deviation. Statistical significance was calculated by two-way ANOVA with 2.10BLASTp sequence comparisons were performed using the NCBI website platform with default parameters. BLASTn sequence alignments were performed with 30% minimal identity on 100 base pair (bp) minimum alignments using in-house, python-based software.33.1Kuravirus genus (previously Phieco32virus) within the Podoviridae family E. coli phages KBNP1711 , NJ01  and the type phage phiEco32 . To explain the broad and specific binding range of EP335 towards O157 strains . The central segment of gp13 (Phe236-Ala422) was predicted to resemble the homotrimeric tip of the E. coli phage T4 short tail fiber . In addition, the C-terminal 114 residues of gp13 were predicted to form an intramolecular chaperone domain (IMC), which assist with the folding and maturation of phage RBPs before autoproteolytic removal, as described for the long tail fiber of phage S16 (PDB ID: 6F45) Bacillus phage GA-1 neck appendage (PDB ID: 3GUD) E. coli siphoviruses DT57C and DT571/2 (LtfA and LtfB) and podovirus phiEco32 (gp14 and gp15) have been determined to form a branched fiber structure whereby LtfB/gp15 connects to LtfA/gp14, which then attaches the dual fiber complex via its N-terminus to the phage tail apparatus Whole genome sequencing previously identified phage EP335 (GenBank: MG748548) as a member of the B). As shown in Fig. 1C, LC-ESI-MS analysis of GFP-gp13 shows a reduction in the molecular weight by 11.8 kDa, corresponding to cleavage between Ser621 and Asp622, which confirmed our previous identification of a self-cleaved C-terminal IMC domain. Similar to other IMC-containing homotrimers, cleavage occurs after a highly conserved serine residue (Ser621) E. coli hosts was assessed. GFP-gp13 bound strongly and evenly to the surfaces of E. coli O157 strains susceptible to phage EP335 infection, i.e., enabled plaque formation . GFP-gp13 also bound to phage-susceptible O26 strains TW04584 and TW04588; however, the level of binding was visibly weaker than that against the O157 strains as observed by fluorescence microscopy , with the fluorescent signal too weak to detect by fluorescence spectroscopy (E. coli strain 777/1 (O157), which was prone to EP335 activity (turbid zones produced by phage EP335 spots) but lacked discernable plaque formation. However, E. coli strains 264 (O157) and the C600 rough strain (lacking O-antigen) that are prone to phage activity but not plaque formation, were not bound by GFP-gp13. Finally, E. coli ECOR34, which features a branched O88 antigen, was the only strain susceptible to phage infection that was not bound by GFP-gp13. Unfortunately, no binding was observed for GFP-gp12 against any strains tested ; however, no binding was observed for either protein. While gp13 functions as an RBP binding to O157 and O26 E. coli strains, there are various hypotheses that could explain the lack of binding by GFP-gp12: (i) attachment of GFP to gp12 renders the protein non-functional; (ii) gp12 requires an additional intermolecular chaperone, similar to other phage RBPs Is common for phage RBPs Fig. 1troscopy . Bindings tested . Based o3.2Kuttervirus genus within the Ackermannviridae family E. coli phages PhaxI  and type phage CBA120 , as well as Salmonella phages such as Det7 . We aimed to understand the broad and cross-genus host range  indicates conservation of this RBP network.Whole genome sequencing previously identified phage EP75 as a member of the B) to explore their individual receptor binding and enzymatic properties against different E. coli and Salmonella hosts , including conservation of all active site residues E. coli O157 strains tested. Interestingly, the EP75 TSP2 was not active against phage-resistant O157 strains (D). There is no similarity between the C-terminal domain of EP75 TSP3 and the TSP3 of CBA120, which instead presented 97.2% sequence similarity (100% coverage) with the O-antigen degrading TSP (gp207) of Salmonella phage Det7 (D), including conservation of all active site residues Salmonella serovars that feature O4 or O9 antigens and are susceptible to EP75 infection such as S. Enteritidis (O9), S. Panama (O9), S. Typhimurium (O4), and S. Derby (O4) (S. Typhimurium O-antigen S. Braenderup (O7), which is also susceptible to EP75 infection E. coli O157 strains with TSP2 significantly reduced the number of phages that could adsorb to the cells (<25% average pulled down compared to > 93% pulldown with no protein) due to binding and degrading of its O-antigen receptor (A). Similarly, the EOP of EP75  was significantly reduced when O157 strains were incubated with TSP2, again due to the enzymatic removal of the O-antigen receptor required for infection of these strains (C). No significant difference in EOP or phage adsorption could be observed when other TSPs or GFP were added as they all produced similar results as the protein-free control. Treatment of Salmonella strains with TSP3 followed a similar trend and produced a significant reduction in the adsorption and infectivity of phage EP75 towards the Salmonella strains tested (B&D). Again, no significant difference in EP75 adsorption or infectivity was observed when other TSPs or GFP control were added to Salmonella.Given the 99.5% sequence identity (99% coverage) of the EP75 TSP2 and its CBA120 counterpart E, includ strains , suggest strains D. There age Det7 D, includrby (O4) . The O4 receptor A. Simila strains C. No sigs tested B&D. Aga3.4Salmonella podovirus HK620 (NC_002730.1) endo-N-acetylglucosaminidase TSP that specifically targets and degrades E. coli O18A1 LPS EP75/E372HK620 and D384EP75/D339HK620) Salmonella O18 serogroup LPS. Phage EP75 could infect the O18A strain DSM10809, with TSP1 also producing clear haloes against this strain; however, no activity could be observed for phage EP75 or TSP1 against the O18A1, O18B, or O18B1 strains tested (E. coli DSM10809 with TSP1 significantly reduced the ability of phage EP75 to bind to the bacteria with no difference in adsorption observed when treated with other TSPs or a GFP control (D). Interestingly, the only difference between the O18A1 and O18A LPS is the former has a branched glucose (Glu) linked to the terminal N-Acetylglucosamine (GlcNAc) of the repeating unit (E) Fig. 4C Inset), which potentially explains the lack of activity against the branched O18A1 LPS.TSP1 shares high similarity with only the N-terminal region of its CBA120 TSP1 counterpart . HHpred analysis revealed the C-terminus of TSP1 (Ser158 to end) shares high structural similarity and 45% sequence identity with the well-characterized TSP (gp9) of s tested . As conf control D. Intereing unit E [62] su3.5A). BLASTp analysis demonstrated > 95% sequence identity (100% coverage) with other Salmonella phage TSPs from the Kuttervirus genus, e.g., phages ST-W77 (NC_049378.1), moki (NC_049506.1), and SJ_3 (NC_024122.1), which has been used to treat Salmonella infections in pigs -41) as having the same structure as CBA120 TSP4 . Nevertheless, while the CBA120 TSP4 targets and degrades the O78 antigen of E. coliE. coli ECOR-71). Given that all E. coli and Salmonella serotypes within the current host range of EP75 could be accounted for by corresponding activity of TSP1, TSP2, or TSP3, we expect TSP4 to target a different Salmonella or E. coli serotype or even another species, which requires further investigation.As no halo formation or phage inhibition could be observed for TSP4 against any of the strains, we attempted to determine its putative enzymatic activity based on sequence and structural similarity to other characterized phage TSPs. The first half of TSP4 shares 95.2% and 93.7% sequence identity with its CBA120 and Det7 counterparts, respectively, suggesting conservation of the N-terminal attachment domains necessary for construction of the three TSP networks for connecting to the phage baseplate A. BLASTp4E. coli and Salmonella serotypes can now be explained by the individual activities of TSP1 (E. coli O18A), TSP2 (E. coli O157), and TSP3 . Overall, the TSPs of EP75 provide excellent examples of the modular nature of phage genomes and the exchange observed between functional modules, i.e., between C-terminal enzymatic domains of phage TSPs during evolution E. coli siphoviruses DT57C and DT571/2 and podovirus phiEco32 Fig. 1). Given the infectivity of EP335 towards these two serotypes, their unbranched O-antigen structures are expected to be the primary receptor used by the phage. However, this does not remove the possibility that other surface structures are also used by EP335 for adsorption or infection, especially as the two RBPs could still recognize different surface structures.By combining structure and sequence analyses with enzymatic and cell binding experimentation we gained a deeper understanding of the host range determining characteristics of the EP75 and EP335 RBPs. While the target of TSP4 remains elusive, the cross-genus infectivity of EP75 toward certain Finally, it is interesting to note that O157 strains 264 and TW04583, which are not infected by either phage, also are not recognized by their respective RBPs (TSP2 of EP75 and gp13 of EP335). Similarly, non-infected O26 strain NCTC08960 is also not recognized by EP335 gp13. Potentially, modification to the O-antigens (not identified by serotyping) or other extracellular barriers exist for these and other non-infected O157 or O26 strains and requires further investigation. Overall, this study demonstrates how structure- and sequence-based characterization of phage RBPs can be used to provide important information on the different receptors used by phages, which could aid the selection of phages with broader infectivity for implementation in phage-based antibacterial and therapeutic products in the future.Conceptualization MD and JTvM; Investigation MD, SW, JTvM, LZ, RGS, CM; Project administration MD; Resources MJL; Supervision MD, JTvM; Validation MD; Visualization MD; Roles/Writing - original draft MD; Writing - review & editing MD, JvTM, MJL, SW, RGS, LZ.Sander Witte: Investigation, Writing - review & editing. Léa V. Zinsli: Investigation, Writing - review & editing. Rafael Gonzalez-Serrano: Investigation, Writing - review & editing. Cassandra I. Matter: Investigation. Martin J. Loessner: Resources, Writing - review & editing. Joël T. van Mierlo: Conceptualization, Investigation, Supervision, Writing - review & editing. Matthew Dunne: Conceptualization, Investigation, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.N6-methyladenosine (m It instenosines . While menosines . Additiopre-mRNA . In mRNAanscript . Howevere 3′ UTR ,16.6A, it is essential to precisely locate the modification. Although m6A had been identified over four decades ago, only recent technological breakthroughs allowed a transcriptome-wide mapping of m6A  enabled mapping of m6A within a ∼100 nucleotide (nt) window and paved the way to further understand and dissect the cellular and physiological functions of m6A (6A iCLIP (miCLIP), which allows the transcriptome-wide mapping of individual m6A residues at single-nucleotide resolution  cells to identify peaks that are significantly depleted upon Mettl3 KO and validated selected m6A sites by an orthogonal method. The resulting high-confidence m6A sites within DRACH and non-DRACH motifs were used to train a machine learning model, named m6Aboost, which recognizes the specific characteristics of m6A sites in miCLIP2 data. We applied m6Aboost to multiple miCLIP2 datasets from human and mouse. Thus, our new miCLIP2 protocol in combination with our m6Aboost machine learning model allow to globally predict m6A sites in miCLIP2 datasets independently of a Mettl3 KO.Here, we present the optimized miCLIP2 protocol, along with the machine learning-based analysis tool m6Aboost to overcome these limitations. Experimental improvements comprise two separately ligated adapters, two independent cDNA amplification steps and a bead-based size selection . These a6A) standards, ammonium acetate, and LC/MS grade acetonitrile were purchased from Sigma‐Aldrich. 13C9‐A was purchased from Silantes, GmbH . 2H3‐m6A was obtained from TRC, Inc. . All solutions were prepared using ultrapure water . 0.1–1 μg of poly(A)+ RNA was degraded to nucleosides with 0.003 U nuclease P1 (Roche), 0.01 U snake venom phosphodiesterase (Worthington), and 0.1 U alkaline phosphatase (Fermentas). Separation of the nucleosides from the digested RNA samples was performed with an Agilent 1290 UHPLC system equipped with RRHD Eclipse Plus C18  with a gradient of 5 mM ammonium acetate  and acetonitrile (solvent B). Separations started at a flow rate of 0.4 ml/min and linearly increased to 0.5 ml/min during first 7 min. Then, washing and re‐conditioning was done at 0.5 ml/min for an additional 3 min and linearly decrease to 0.4 ml/min during the last minute. The gradients were as follows: solvent B linear increase from 0 to 7% for first 3 min, followed by isocratic elution at 7% solvent B for another 4 min; then switching to 0% solvent B for last 4 min, to recondition the column. Quantitative MS/MS analysis was performed with an Agilent 6490 triple quadrupole mass spectrometer in positive ion mode. Details of the method and instrument settings are described in (13C9‐A), 282.1→150.1 (m6A) and 285.1→153.1 (2H3‐N6-mrA). Quantification of all samples utilized biological triplicates, and averaged values of m6A normalized to A, with the respective standard deviation are shown.The experiments were performed as described in . Ribonucribed in . MRM tral-glutamine (Life Technologies) and 1% penicillin-streptomycin (Life Technologies) at 37°C with 5% CO2. All cell lines were monitored for mycoplasma contamination. Anaplastic thyroid carcinoma-derived C643 cells  were cultured on 15 cm dishes in DMEM (Thermo Fisher Scientific) supplemented with GlutaMAX (Thermo Fisher Scientific) and 10% FBS at 37°C and 5% CO2.The HEK293T cell line was cultured in Dulbecco's Modified Eagle Medium  containing 10% fetal bovine serum , 1% Mettl3 KO genotype were taken from a previous publication (Mouse embryonic stem cells (mESC) with wild-type and lication  and cult6A validation in HEK293T cells using SELECT, m6A was depleted by using the METTL3 inhibitor STM2457 (STORM Therapeutics) (6A depletion quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To this end, HEK293T cells were treated with 2–20 μM STM2457 in DMSO 0.05–0.2% (v/v) or DMSO alone 0.2% (v/v) as a negative control. After 16 h of treatment, the cells were washed with ice-cold PBS and collected on ice.For mpeutics) . STM2457For RNA extraction from HEK293T and mESC cells, cells were washed in ice-cold PBS and collected on ice for the isolation of total RNA using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's recommended protocol. For C643 and RAW 264.7 cells, cells were washed with PBS, and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Prior to isolation of poly(A)+ RNA, total RNA samples were treated with DNase I (New England Biolabs) according to the manufacturer's protocol, and subsequently cleaned up again by using TRIzol LS (Thermo Fisher Scientific).25 Magnetic Beads using the manufacturer's recommended protocol . Poly(A)+ concentration was measured using Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific). For RAW 264.7 cells, poly(A)+ RNA was extracted by incubating 100 μg total RNA with 200 μl Dynabeads solution  and purified following the manufacturer's protocols.For HEK293T and C643 cells, poly(A)+ RNA was extracted using Oligo d(T)The quality of poly(A)+ RNA was ensured using High Sensitivity RNA ScreenTapes for the 2200 TapeStation system (Agilent). If a predominant peak for ribosomal RNA was still detectable, an additional round of poly(A) selection was performed, resulting in one round of selection for mESC and RAW 264.7 cells, and two rounds for HEK293T and C643 cells.2O for each condition. 1 μl of 0.1–0.4× diluted fragmentation buffer was added . The mixture was incubated for 7–12 min at 70°C in thermomixer at 1,100 rpm and put immediately on ice. 1 μl of 0.1–0.4× diluted STOP solution was added. The solution was mixed and placed back on ice until use. Time of fragmentation and dilution of fragmentation reaction solutions were optimized prior to miCLIP2 experiments for each new batch of RNA.Poly(A)+ RNA was fragmented using RNA fragmentation reagents from Thermo Fisher Scientific. 1 μg of poly(A)+ RNA was filled up to 22 μl with H6A antibody purchased from Synaptic Systems (order number 202 003).All miCLIP2 experiments were performed with rabbit anti-m6A antibody was added to the 24 μl of fragmented RNA and rotated for 2 h at 4°C. The IP mixture was placed on a parafilm-coated dish and UV irradiated with 2 × 150 mJ/cm² of UV 254 nm. The mixture was placed back into the tube, another 500 μl of IP buffer and 50 μl of washed protein A beads were added. The mixture was rotated at 4°C for 1 h. The beads were magnetically separated and the supernatant was discarded. The beads were washed two times with high-salt wash . The second wash was rotated for at least 1 min at 4°C. Subsequently, the beads were washed two times with PNK buffer  and resuspended in 1 ml PNK buffer (the samples can be left at 4°C until ready to proceed).50 μl of protein A Dynabeads  were magnetically separated, washed two times in 900 μl IP buffer  and then resuspended in 50 μl IP buffer and put at 4°C until use. 6 μg of mThe beads were magnetically separated and resuspended in 20 μl of 3′ end RNA dephosphorylation mixture . The mixture was incubated for 20 min at 37°C in a thermomixer at 1,100 rpm. The beads were washed once with PNK buffer, once with high-salt wash (rotate wash for at least 1 min at 4°C) and again washed two times with PNK buffer.The supernatant was magnetically removed and the beads were resuspended in 20 μl of L3 DNA linker ligation mixture . The mixture was incubated overnight at 16°C at 1,100 rpm in a thermomixer. Subsequently, 500 μl of PNK buffer was added. The beads were washed two times with 1 ml high-salt buffer and two times with 1 ml PNK buffer. After the first wash, the mixture was transferred to a new tube.32P-γ-ATP, 0.4 μl 10× PNK buffer [New England Biolabs], 3 μl H2O) was added and incubated for 5 min at 37°C in a thermomixer at 1,100 rpm. Next, the supernatant was removed and 20 μl of 1× NuPAGE loading buffer  was added to the beads and incubated at 70°C for 5 min.The beads were magnetically separated and 4 μl of hot PNK mix . 0.5 l of 1× MOPS running buffer (Invitrogen) was used. Additionally, 5 μl of a pre-stained protein size marker was loaded. The gel was run for 50 min at 180 V. The dye front was cut and discarded as solid radioactive waste. For transferring the protein–RNA complexes to a Protan BA85 Nitrocellulose Membrane, a Novex wet transfer apparatus was used according to the manufacturer's instructions. The transfer was performed for 1 h at 30 V in 1× transfer buffer with 10% methanol. After the transfer, the membrane was rinsed in 1× PBS buffer. Afterwards, it was wrapped in saran wrap and exposed to a Fuji film at 4°C for 30 min, 1 h, or overnight. The film was exposed to a Typhoon phosphoimager.2O and transferred to a PCR tube.The protein-RNA complexes were isolated by using the autoradiograph as a mask by cutting the respective regions out of the nitrocellulose membrane. The fragments were placed in a 1.5 ml tube and 10 μl proteinase K  in 200 μl PK buffer  was added and incubated at 37°C for 20 min at 1,100 rpm. 200 μl of PK buffer + 7 M urea  was added and incubated at 37°C for 20 min at 1,100 rpm. The solution was collected and added together with 400 μl phenol/chloroform (Sigma P3803) to a 2 ml Phase Lock Gel Heavy tube . The mixture was incubated for 5 min at 30°C at 1,100 rpm. The phases were separated by spinning for 5 min at 13,000 rpm at room temperature. Next, the aqueous layer was transferred into a new tube. Precipitation was performed by addition of 0.75 μl glycoblue , 40 μl 3 M sodium acetate pH 5.5 and addition of 1 ml 100% ethanol. After mixing, the mixture was placed at –20°C overnight. The mixture was spun for 20 min at 15,000 rpm at 4°C. After removing the supernatant, the pellet was washed with 0.9 ml 80% ethanol and spun again for 5 min. After removing the supernatant, the pellet was resuspended in 5 μl H2O, 4 μl 5× RT buffer [Invitrogen], 1 μl 0.1 M DTT, 0.5 μl RNasin, 0.5 μl Superscript III) the mixture was incubated in a thermocycler . 1.65 μl of 1 M NaOH was added and incubated at 98°C for 20 min. Subsequently, 20 μl of 1 M HEPES-NaOH pH 7.3 was added. This will eliminate radioactivity from strongly labelled samples after the next step and prevent RNA from interfering with subsequent reactions.RT primers and dNTPs  were added to the resuspended pellet and incubated in a thermocycler . After adding the RT mix  was pipetted on ice. To ensure homogeneity, the ligation-master-mix was mixed by flicking and spinning it down and was subsequently added with the linker-sample-mix. After vigorous stirring, another 1 μl RNA ligase was added to each sample and mixed by stirring. The mixture was incubated at RT at 1,100 rpm overnight.The linker was prepared by heating the linker mix (2 μl L##clip2.0 (10 μM stock) 1 μl 100% DMSO) for 2 min at 75°C and keeping it on ice afterwards for > 1 min. The DNA linker L##clip2.0 has the sequence 5′-/5Phos/NNNNXXXXXXNNNNNAGATCGGAAGAGCGTCGTG/3ddC/-3′, where N’s are the 4-nt and 5-nt random nucleotides from the unique molecular identifier (UMI) and X’s are the 6-nt the sample-specific experimental barcode given in 2O.Per sample, 5 μl MyONE Silane beads were prepared. The MyONE Silane clean-up was performed as described in the previous Silane clean-up step with following modification: After washing the beads in 500 μl RLT, the beads were resuspended in 60 μl RLT buffer and added to the already bead-containing sample. After the precipitation was performed as previously described, the dried beads were resuspended in 22.5 μl HThe PCR mixture .2O, 30 μl Phusion PCR mastermix [New England Biolabs] and the samples were mixed with 147.5 μl ProNex chemistry. This is a 1:2.95 (v/v) ratio of sample:beads. This was optimized in previous experiments  will be size selected in parallel to monitor ProNex size selection efficiency. ProNex chemistry was adjusted to RT by keeping it for 30 min at RT. 50 μl of ULR-Phusion mix  was added to 1 μl of the pre-amplified library. The PCR reaction was performed in a thermocycler ; 72°C, 3 min; 16°C, hold). 2 μl of the amplified library was run on a High Sensitivity D1000 Kit in a TapeStation system. Repeat this step until libraries are seen without over-amplification.2O, 2 μl primer mix P5Solexa/P3Solexa, 10 μM each, 20 μl Phusion HF Mix [New England Biolabs]) was added to 10 μl cDNA. The PCR was performed in a thermocycler using the same program as in the optimization PCR with the optimized cycle number. 2 μl of the amplified library was run on a High Sensitivity D1000 Kit in a TapeStation system. If the results looked fine, the second half of the library was also amplified and combined with the first half. Finally, the concentration under the peak was determined using TapeStation software, and replicates were combined either in equal molarities or equal volumes.From previous results of the PCR cycle optimization, the minimum of PCR cycles was used to amplify 2O. Again, for comparison the selected and unselected ULR Phusion mix was analyzed as described previously. The 100-nt/75-nt ladder fragment ratio should be around 4.5.Before submitting the samples for sequencing, another round of bead-based size selection was performed to remove residual primers. This ProNex size selection was performed as described above with the following modifications: After ULR preparation, the samples and beads were mixed in a 1:2.4 (v/v) ratio of sample:beads. This was optimized in previous experiments in . After t6A modifications at several putative m6A sites identified from our miCLIP2 data. Experiments for mESC cells were performed with RNA from mESC WT cells and compared to RNA from mESC Mettl3 KO cells. Experiments for HEK293T cells were performed with RNA from cells treated with 20 μM METTL3 inhibitor STM2457 (STORM Therapeutics) (We used the elongation and ligation-based qPCR amplification method SELECT  to indeppeutics)  or DMSO Mettl3 KO or METTL3 inhibitor-treated cell lines, the amount of m6A is greatly reduced. Due to m6A-mediated RNA degradation or stabilization processes, absence of m6A may influence the abundance of specific transcripts. To ensure usage of same RNA amounts, Qubit (Thermo Fisher Scientific) with Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific) was used to precisely measure RNA concentrations. To ensure usage of equal amounts of transcripts, qPCR experiments were performed for normalization of input RNA amounts in WT versus m6A-depleted cell lines.For 6A site (termed X site), two primers (Up and Down primer) were designed flanking the site of interest. To precisely measure RNA concentrations before each experiment, Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific) was used. An influence of m6A on transcript stability may lead to a difference in transcript abundance upon Mettl3 KO. Therefore, qPCR for the respective transcript was performed and the amount of total RNA for each SELECT experiment was normalized. To further monitor usage of equal amounts of input material, an Up and Down primer were designed flanking an adjacent nucleotide (termed N site). N sites between X-8 and X+4 were used as input control. According to the previously published SELECT method, 20 ng of poly(A)+ RNA was used per experiment. The RNA was mixed in a total volume of 17 μl in 1xCutSmart buffer containing 40 nM Up primer, 40 nM Down primer and 5 μM dNTPs. The RNA and primers were annealed by incubation in a thermocycler . 0.02 U Bst 2.0 DNA polymerase, 0.5 U SplintR ligase and 10 nmol ATP in a volume of 3 μl in 1× CutSmart buffer was added and incubated at 40°C for 20 min. After denaturation at 80°C for 20 min, the mixture was kept at 4°C. Using the Applied Biosystems ViiA7 Real-Time PCR system, qPCR was performed. The 20 μl qPCR reaction mixture contained 2 μl of the final reaction mixture after denaturation, 0.2 nM per qPCR primer, 2x Luminaris HiGreen Lox Rox (Thermo Fisher Scientific) and ddH2O. The quantitative qPCR reaction condition was run as follows: 95°C, 5 min;  x 40 cycles; 95°C, 15 s; 60°C, 1 min; 95°C, 15 s (collect fluorescence at a ramping rate of 0.05°C/s); 4°C hold. qPCR data analysis was performed using QuantStudio Real-Time PCR Software v1.3. All experiments were performed in three technical replicates (separate SELECT reactions). Oligonucleotides used for SELECT are listed in For the quantitative real-time PCR (qPCR)-based validation of a presumed mYthdc1, Mif4gd) comparing Mettl3 KO and WT mESCs. Cells were grown on irradiated CF1 mouse embryonic fibroblasts  under normal FCS/LIF conditions, as described before (Taq DNA Polymerase (New England Biolabs) in a 25 μl reaction, according to the recommended protocol, using 0.5 μl cDNA as template, a shared forward primer located in the upstream exon and two isoform-specific reverse primers in the intron (IR) and the downstream exon (spliced), respectively. All three primers were used in a final concentration of 200 nM each, rendering the shared primer as a rate-limiting factor in the reaction. Primer sequences were: Ythdc1_shared (5′-CCATCCCGTCGAGAACCAG-3′), Ythdc1_IR (5′-CCAACGTGACCATGTGAAATCC-3′), Ythdc1_exonic (5′-TGGTCTCTGGTGAAACTCAGG-3′), Mif4gd_shared (5′-CCTGAGAGTCTGAGCAGGGA-3′), Mif4gd_IR (5′-AAGCCTTGGCCTCTATGTGC-3′) and Mif4gd_exonic (5′-AGCCGTCCCGGATTAGGATA-3′). The PCR reaction was carried out in a thermocycler at 94 °C for 30 s, 30 cycles of  and final extension at 68°C for 5 min. PCR products were analyzed by capillary gel electrophoresis on the TapeStation 2200 system using D1000 ScreenTapes (Agilent) according to the manufacturer's recommendations. Band intensities were quantified using the TapeStation Analysis Software and frequency was calculated as the relative proportion of IR and spliced transcript abundance.Reverse transcription followed by polymerase chain reaction (RT-PCR) was performed to validate changes in isoform frequencies of selected transcripts .https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and filtering reads based on sequencing qualities (Phred score) of the barcode region (FASTX-Toolkit v0.0.14) (http://hannonlab.cshl.edu/fastx_toolkit/), seqtk v1.3 (https://github.com/lh3/seqtk/), reads were de-multiplexed based on the experimental barcode (positions 6 to 11 of the reads) and adapter sequences were removed from the read ends (Flexbar v3.4.0)  . UMIs we v3.4.0)  using ST v3.4.0) . When ru v3.4.0)  and dupl v3.4.0) . Reads w).After removing duplicates, all mutations found in reads were extracted using the Perl script parseAlignment.pl of the CLIP Tool Kit  . The lis39The code for miCLIP2 data processing as described here is available from two recent data analysis publications ,37.BAM files with reads without C-to-T mutation were used for peak calling with PureCLIP (v1.3.1)  individuIn order to calculate the relative signal strengths of all peaks within a transcript, we calculated the mean number of truncation events for all peaks in the same transcript. Then, we divided the individual truncation read number of each peak by the mean of the peak strength in the corresponding transcript, leading to a value representing the relative peak strength.one-run), any change of transcript abundance will lead to incorrect fold change and FDR estimations, resulting in false positive calls in down-regulated genes. We tested four different approaches to overcome this, namely separately running DESeq2 on peaks of individual genes (gene-wise) or groups of genes with similar abundance change (bin-based), by building a combined DESeq2 model on peak signals and transcript counts using interaction terms (2-factor) as well as by using DEXSeq (dexseq-run)   instead 2-transformed fold change (log2FC) and the false discovery rate (FDR) from the bin-based differential methylation analysis between WT and Mettl3 KO cells, we used peaks at A to compile a positive  and negative  set. Both were combined and then randomly split into a training set (80%) and an independent test set (20%). We then extracted 27 features, including the nucleotide sequence in a 21-nt window around the central A, the transcript region as well as the relative signal strength (log2) and the number of associated C-to-T transitions (log2). We initially tested three different machine learning algorithms  and evaluated their performance based on precision-recall curves and area under the curve (AUC) as well as by comparing F1-score, Matthews correlation coefficient (MCC), precision, accuracy, sensitivity and specificity on the independent test set. Based on these measures, we selected the AdaBoost-based predictor, which we named m6Aboost .Based on the loghttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to the mouse genome (assembly version GRCm38.p6) and its annotation based on GENCODE release M23 using STAR (v2.6.1b) (RNA sequencing (RNA-seq) libraries were sequenced on an Illumina NextSeq500 as 84-nt single-end reads, yielding 31–35 million reads per sample. Basic sequencing quality checks were applied to all reads using FastQC (v0.11.8) (v2.6.1b) . When ruv2.6.1b)  using RP2-transformed fold changes.For differential gene expression analysis, mapped reads were counted with htseq-count   into genIntron retention (IR) analysis was done with IRFinder (v1.3.0)  using bumy $ok = ($pA[8] > 0 || $pB[8] > 0) && ($pA[19] > 0 || $pB[19] > 0) && separatedAB;and line 186 into:if  > 0) &&  > 0)) {P values were corrected using Benjamini-Hochberg adjustment.For downstream analysis, IR events were filtered for IRratio ≥ 0.03 in at least one condition and mean IntronDepth ≥ 3. 6A sites therein were filtered for a difference in MazF cleavage efficiency > 0.1 between WT and Mettl3 KO, yielding a total of 580 reliably identified m6A sites from mESC cells. Two hundred of these (34.5%) overlapped at single-nucleotide resolution with the 4,464 predicted m6A sites at ACA from our mESC miCLIP2 data.Processed MAZTER-seq data from  were dowet al. , which was previously shown to yield highest truncation efficiency in miCLIP experiments .,24.6A pr2 Figure  and opti2 Figure . The RNAs Figure  (17). Afs Figure , crosslis Figure . Transferagments . The resragments .n = 3 replicates) from wild-type (WT) as well as Mettl3 knockout (KO) mESCs. The latter lack the primary m6A methyltransferases Mettl3 and hence, lost most of m6A mRNA methylation .,50.n = 3n Figure . The remn Figure . Peak can Figure . The numn Figure . To allon Figure .Mettl3 KO, indicating that they are part of the unspecific background signal of the employed antibody or m6A sites independent of Mettl3  which is known to reside at 5′ cap structures and is also recognized by the m6A-specific antibody . Best performance was achieved with the bin-based approach, in which genes were stratified according to their expression change upon Mettl3 KO , which served as ‘positive set’ of true m6A sites in the following analyses . As previously described, the positive sites accumulated nearby stop codons and in 3′ UTRs, and the underlying sequences resembled the DRACH motif  and hence represented the nonspecific background in the data.In order to learn about the features of genuine mO Figure . All miCO Figure   at non-DRACH motifs (non-DRACH m6A). While most of these non-DRACH motifs still contained the AC dinucleotide . Indeed, we detected Mettl3-dependent methylation at A in the GGATT motif, reflected in a reduced efficiency of the qPCR amplification when the m6A mark is present  and Hic2  , Ccnt2  and Phb2   Figure . For com) Figure .Mettl3 KO, indicating that the antibody may show a residual background activity against the DRACH motif itself. SELECT experiments for two out of two selected sites in the genes Nanog (chr6:122711605:+) and Zfp710 (chr7:8008671:+) confirmed that the respective A indeed did not carry an N6-methyl modification  in the negative set. The miCLIP2 signal at these peaks did not decrease in the n Figure .6A sites, that are modified in a Mettl3-dependent manner. In addition to canonical DRACH motifs, we identified a fraction of m6A modifications at non-DRACH motifs which show the same characteristics and Mettl3 dependency as m6A sites at DRACH motifs.All together, we defined a positive set of > 10,000 m6A detection independently of an accompanying KO dataset, we built a predictive machine learning model to discriminate true m6A sites from background signal in the miCLIP2 data  and negative  sets identified in the differential methylation analysis upon Mettl3 KO. The unbalanced setup was chosen to reflect the predominance of nonspecific background in the miCLIP2 data . In total, m6Aboost extracted 25,456 putative m6A sites in 9,363 genes  predicted ms Figure . These nO Figure  and D, st Figure , suggested genes , possibled genes . Altoget6A sites to those detected by the antibody-independent method MAZTER-seq in the same cell line (6A sites from MAZTER-seq (200 out of 580 sites) were also present in our data, further supporting the validity of our approach.As an orthogonal support, we compared our predicted mell line . MAZTER-6A sites + RNA from RAW 264.7 cells, a mouse macrophage cell line . Out of 462,073 miCLIP2 peaks, m6Aboost identified a total of 19,301 m6A sites . Overlay6A sites were located in exons. However, we also detected a number of m6A sites in retained introns. Interestingly, the intronic m6A sites showed a strong accumulation towards the 5′ splice sites -selected RNA, most identified ms Figure , suggests Figure , we coul1 Figure , indicats Figure . This trs Figure . Generals Figure , indicatn = 4 replicates with 30 million truncation events on average, 6A sites in 7,552 genes, corresponding to 21% of all peaks at A + RNA from human HEK293T cells   and RHOB (chr2:20448702:+) .We used SELECT to validate the presence of mpeutics) , which pn to 22% . We then) Figure . As a co6A sites with binding sites of the cytoplasmic m6A reader protein YTHDF1 from published iCLIP data  were not detected by miCLIP2 + RNA from C643 cells, a human thyroid cancer cell line + mRNA (Most current protocols for antibody-based mA)+ mRNA ,56. In oA)+ mRNA . HoweverA)+ mRNA .6A sites from all RNA input concentrations were consistently enriched at DRACH motifs and nearby stop codons . Of note, we find that m6A modifications occur outside of DRACH motifs  and validate selected m6A sites at non-DRACH motifs using an orthogonal antibody-independent method. Similar motifs were previously reported and recently confirmed in direct RNA sequencing data (Oxford Nanopore Technologies)  in m5C-miCLIP  ,60. Impos Figure , captureC-miCLIP ,61.6A sites from any miCLIP2 dataset. Several machine learning approaches have been developed to predict m6A sites from the primary RNA sequences . It is known that certain biochemical features such as the truncation rate at the crosslinked antibody and the distribution of C-to-T transitions vary with each antibody .The computational code is available in the GitHub repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE163500).All miCLIP2 and RNA-seq data sets generated in this study were submitted to the Gene Expression Omnibus (GEO) under the SuperSeries accession number GSE163500 (gkab485_Supplemental_FilesClick here for additional data file.


We created a mutation of lips-6 to study its effects on lifespan and the ascaroside profile. We found that lips-6 had a wild-type lifespan and a wild-type ascaroside profile. These results suggest that the lips-6 gene plays a minimal role in C. elegans lifespan biology and does not affect the ascaroside profile until it is specifically activated by starvation in adult worms.The  Caenorhabditis elegans has made the creation of null mutants easier than ever.The putative lipid hydrolase lips-6 is known to function in the DAF-12/DIN-1 pathway to promote fat mobilization during periods of starvation in adult worms . Similarly, it was found to be strongly downregulated in response to dauer ascarosides given during the L1 to L2d decision in larval worms . Here, we have created a mutant of the putative lipase lips-6 and studied its lifespan as well as its ascaroside profile.The efficient CRISPR/Cas9 genome editing toolkit in the nematode lips-6 that has a 348 base pair deletion, spanning one-quarter of the entire gene (lips-6(sy1325) and found that it has a wild-type lifespan phenotype (lips-6(sy1325) and found that, when compared to a series of unrelated strains, the ascaroside profile was also wild-type. Our results indicate that until the lips-6 gene is activated by starvation during adulthood, it plays no role in lifespan or the worms’ changing ascaroside profile.First, we made a mutation of ire gene a. We perhenotype b. We alsStrain Creation: We used the CRISPR/Cas9 method essentially as described in Köhler et al. (2017). We used the guide sequence AATACTTATTCGCAACAACA. To detect a deletion mutation of lips-6, we used forward primer TACGGTAGCCGTTATGCTTG and reverse primer GCTGATGTGACTGCTGTTCG. In sy1325, there is an in-frame deletion of 348 base pairs traversing one-quarter of the gene; the deletion flanking sequences are ACGACAATACTTATTCGCAA_deletion_GGAGCTCAGAAAATTGACGT.Longevity Assays: The longevity assays were done using the method described in Larsen et al. (1995). Ten adult worms were kept on each 6cm plate in order to prevent food scarcity or overcrowding – 100 worms were assayed for each strain. The first day of the survival curve is the first day of adulthood. Worms were scored every day as alive, dead, or missing. Worms were deemed dead if they neither moved, pumped, nor responded to stimulation using the end of a platinum wire pick.Nematode Cultures: Cultures were started by picking 20 C. elegans hermaphrodites onto 10cm NGM agar platescontaining OP50 E. coli and incubated at 22°C. After 96 h, each plate was washed with 25 mL of S-complete medium into a 125 mL Erlenmeyer flask, 1 mL of concentrated E. coli OP50 was added (see above), and cultures were shaken at 220 RPM at 22°C. After 72 h, cultures were centrifuged at 1000 G for 1 min. After discarding the supernatant, worm pellets were washed with 30 mL of ddH2O and centrifuged at 1,000 G for 1 min, and the supernatant was discarded. 24 mL ddH2O was added, along with 6 mL regular bleach  and 900 uL 10 M NaOH, and the mixture was shaken for 2.5 min to prepare eggs. Eggs were centrifuged at 1,000 G and the supernatant was removed. Eggs were then washed with 30 mL M9 buffer and suspended in a final volume of 5 mL M9 buffer in a 50 mL centrifuge tube. Eggs were placed on a rocker and allowed to hatch for 24 h at 22°C. L1 larvae were counted and seeded at 70,000 L1s per 25 mL S-complete culture, and 1 mL of OP50 was added per 25 mL culture in 125 mL Erlenmeyer flasks. Cultures were incubated at 220 RPM at 22°C for 72 h.Metabolite Extraction: Lyophilized culture media were crushed and homogenized by shaking with 2.5 mm steel balls at 1300 RPM for 3 min in 30 s pulses while chilled with liquid nitrogen (SPEX sample prep miniG 1600). Dried material was then extracted with 15 mL methanol in 50 mL centrifuge tubes, rocking overnight at 22°C. Extractions were pelleted at 5000 G for 10 min at 4°C, and supernatants were transferred to 20 mL glass scintillation vials. Samples were then dried in a SpeedVac (Thermo Fisher Scientific) vacuum concentrator. Dried materials were resuspended in 1 mL methanol and vortexed for 1 min. Samples were pelleted at 5000 G for 5 min and 22°C, and supernatants were transferred to 2 mL HPLC vials and dried in a SpeedVac vacuum concentrator. Samples were then resuspended in 200 uL of methanol, transferred into 1.7 mL Eppendorf tubes, and centrifuged at 18,000 G for 20 min at 4°C. Clarified extracts were transferred to fresh HPLC vials and stored at −20°C until analysis.Mass Spectrometric Analysis: High resolution LC−MS analysis was performed on a ThermoFisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid quadrupole-orbitrap high resolution mass spectrometer equipped with a HESI ion source. Metabolites were separated using a water-acetonitrile gradient on an Agilent Zorbax Eclipse XDB-C18 column  maintained at 40°C. Solvent A: 0.1% formic acid in water; Solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 1% B for 5 min after injection and increased linearly to 100% B at 20 min, using a flow rate 0.5 mL/min. Mass spectrometer parameters: spray voltage 3.0 kV, capillary temperature 380°C, probe heater temperature 300°C; sheath, auxiliary, and spare gas 60, 20, and 2, respectively; S-lens RF level 50, resolution 240,000 at m/z 200, AGC target 3×106. The instrument was calibrated with negative ion calibration solutions (ThermoFisher).All CRISPR/Cas9 system reagents were ordered from IDT except for the Cas9 protein, which was kindly provided by Tsui-Fen Chou. Sequences were downloaded from WormBase.Strain Generated:lips-6(sy1325) IVPS7427 List of Strains.


E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using  E. coli — CRISPR classifications can be found in [Prokaryotes have evolved multiple defence strategies against incursion from mobile genetic elements , including activation of their CRISPR-Cas systems. These comprise a DNA locus called ‘CRISPR’  and genes encoding ‘Cas’ (CRISPR associated) proteins, and sometimes additional non-Cas proteins. Comprehensive recent perspectives of CRISPR systems can be found in ,2,3, andfound in —are catafound in . Cascadefound in ,9,10,11—found in . The comfound in ,14,15. Rfound in —the impofound in . In CRISfound in . Therefofound in , by theiE. coli ygcB (encoding Cas3) is repressed by H-NS, reviewed in [5N, 10N-methylene tetrahydrofolate, a product of bacterial glycine metabolism catalysed by enzymes of the Glycine Cleavage System [E. coli for CRISPR interference [E. coli, removal of H-NS (Δhns) gives constitutive expression of Cas3 and Cascade that protects against λ phage plaques at 30 °C, but protection is lost at 37 °C [Control of the Cas3 nuclease-helicase activity is exerted at several junctures. Transcription of iewed in , and CRPiewed in , but de-iewed in ,23, and e System . The prerference , and intrference , althougat 37 °C . The losat 37 °C . It was at 37 °C , in whicE. coli, we previously reported that the efficacy of CRISPR defence against phage λ is restricted by temperature [hns E. coli cells expressing Cas3, Cascade and λ-targeting crRNA when compared with plaques at 30 °C (cas3 (E. coli wild-type Cas3 was purified fused to tandem (His)6- maltose binding protein (MBP) affinity tags, the latter to improve protein solubility and stability when expressed in E. coli [E. coli is more susceptible to λ plaque formation at 37 °C because Cas3 is required for protection against them.In CRISPR systems Cas3 nuclease is targeted to destruction of MGEs, and potentially provides MGE DNA as protospacers for updating the CRISPR immunity locus. In perature ,28. Thisat 30 °C A. This e°C  spectra were generated to detect changes in Cas3 secondary structure composition and thermodynamics when heated from 20 °C to 55 °C. This yielded thermograms of CD spectra changes at 222 nm, for alpha-helix and beta-strand composition , that, fThese data show temperature-dependent changes in Cas3 structure that correlate well with changes in nuclease activity and the function of Cas3 in CRISPR defence at 30 °C, which becomes inactivated at 37 °C A. We nexE. coli Cas3 is not available, so we turned to the I-E subtype Cas3-DNA co-structures from Thermobifida fusca (PDB 4QQW-Z) [E. coli Cas3—30% amino acid identity, E-value 7e−54—to identify areas for mutagenesis. Many Cas3 proteins comprise a fusion of an HD-nuclease domain with superfamily 2 helicase (RecA-like) domains and an additional predicted C-terminal domain of unknown function (E. coli Cas3 numbering) and the other (Trp-230) located close by   as a clofunction A, reviewfunction A,B. The function C, one loclose by A,B, and W230A, Cas3W406A, Cas3W149A and Cas3W152A for assays measuring their nuclease activity compared to the wild type protein. Using the DNA fork (20 nM) substrate and measured as a function of time, wild type Cas3 (56 nM) converted maximally 92% of DNA into nuclease product at 30 °C, compared to 40% at 37 °C  were significantly less active than wild type Cas3 at both temperatures. However, Cas3W406A (56 nM) was active similarly to wild type Cas3 at 30 °C and hyperactive compared to wild type Cas3 at 37 °C (mean of 73% fork converted to nuclease product). Therefore, the Cas3 Trp-406 residue seemed to modulate Cas3 nuclease activity because changing it to alanine substantially overcame the inhibitory effect of elevated temperature on nuclease activity. There was no evidence in Electrophoretic Mobility Shift Assays (EMSAs) for Cas3W406A binding to DNA more stably or with greater efficacy than wild type Cas3  formed less pronounced protein-fork DNA complex in the same EMSAs  [E. coli Cas3 Trp-406 is abutted to another tryptophan residue (Trp-230 in E. coli). This Trp-230/406 pair is close to the ssDNA binding channel in the iHDA1 (Thermobaculum terrenum (PDB 4Q2C) [E. coli Cas3 to begin molecular dynamics simulations of these tryptophan residues at 30 °C and at 37 °C. In molecular dynamics simulations, we identified temperature dependence for interaction of the highly conserved residues Trp-406 and Trp-230. Increasing temperature resulted in increased distance, from a mean of approximately 6.1 Å at 30 °C to 11.6 Å at 37 °C (. When in close proximity (30 °C), Trp-230 and Trp-406 from a stable π–π interaction that was broken in the first phase of MD simulations at 37 °C and remained broken through the rest of simulations  , the tryhe iHDA1 B,C. We uDB 4Q2C)  to generDB 4Q2C)  a predicat 37 °C . When inulations B. The inulations A comprisulations . At 30 °ulations C panel iW406A was also a hyperactive ATPase that, based on the structural models, may explain hyperactive nuclease activity. Cas3 ATP hydrolysis was measured using the malachite green reporter assay [. ATPase activity of each Cas3 mutant was similar whether measured at 30 °C or 37 °C, also contrasting with the much-reduced nuclease activity from three of the mutants at the higher temperature. Cas3W406A was not hyperactive as an ATPase at 37 °C. Therefore, the effect of temperature and the Trp-406 residue are exerted only on Cas3 nuclease activity.CRISPR interference requires Cas3 nuclease and ATP-dependent DNA translocase activities ,37. Struer assay  at 30 °CE. coli CRISPR interference, Cas3 is recruited to MGE DNA at an R-loop formed by the Cascade ribonucleoprotein complex [W406A nuclease activities against a plasmid targeted by Cascade in vitro (W406A instead of wild type Cas3 (lane 6). An interesting result was observed when Cascade R-loops were pre-formed on M13 DNA—in these reactions, wild type Cas3 nuclease activity was inhibited by Cascade, observed as the absence of linearized M13 and re-appearance of supercoiled M13 (W406A in this respect was unaffected by Cascade (lane 8).In  complex . Cas3 th complex ,40,41,42 complex ,40, and  complex ,43. The in vitro B. The Cailed M13 B lane 7,W406A nuclease activity in the presence of Cascade were repeated with the addition of ATP, to observe for processive nucleolytic degradation of the M13 DNA , uncoupled from the constraint of Cascade. This intriguing observation indicates that the nuclease hyperactivity of Cas3W406A can also overcome the modulating effect of Cascade in vitro.Assays comparing Cas3 and Cas3 M13 DNA C. Consisin vitro , reactioW406A nuclease hyperactivity impacted CRISPR catalyzed protection against phage lambda (λvir). The assay requires expression of Cas3 and Cascade in E. coli to determine if these cells are resistant to lambda plaque formation [vir spacers for targeting of Cascade to λvir DNA. In addition, the native ygcB gene encoding wild type Cas3 within the CRISPR-cas locus was replaced by the ygcBW406A allele, encoding Cas3W406A. Cells were lacking H-NS (Δhns) to de-repress transcription of Cascade and Cas3 encoding genes. At 30 °C, wild type Cas3 cells were fully protected from plaque formation when challenged by λvir, but this protection was lost at 37 °C  reside in a region of Cas3 we refer to as iHDA1, a domain interface needed to channel ssDNA between the RecA1 helicase domain and the HD nuclease active site. In a structural analysis of Cas3 , the DNAive site , and thafunction .Cas3 structure therefore exerts intrinsic control over nuclease activity, via a gate formed of Trp-406 opening and closing access of ssDNA to the nuclease active site .This tryptophan residue, and its binding partner at lower temperature, Trp-230, is well positioned for this, being close to the HD active site—a 12 Å mean distance from the centre of mass of the Trp-406/Trp-230 pair and the HD active sites in MD simulations at 30 °C. Allosteric regulation of nucleases in biological systems ensures that they are targeted to destroy only appropriate substrate nucleic acids at an appropriate time. For Cas3, the interaction with Cascade during CRISPR interference provides targeting, and functional levels of Cas3 in cells rely on the presence of the chaperone HtpG , anotherW406A may be of use in this respect, particularly if it can be successfully targeted by Cascade but then has nuclease activity that is not modulated by Cascade.Bacterial Cas3 and Cascade have been deployed for genetic insertions or deletions in human cells ,48,49. TA concluding summary of the work presented here is shown in E. coli strains and their derivation are listed in ygcB,) was amplified from E. coli MG1655 genomic DNA and cloned into pBAD-HisA  via XhoI and EcoRI sites to construct plasmid pAH4 for overexpression and purification of Cas3 with an N-terminal histidine tag. The plasmid for purification of (His)6-MBP-Cas3 protein was described in [ribed in . Point mygcB with either a (His)6-MBP tag (pCas3) or (His)6-tag only (pAH4) was transformed into BL21 A.I. (Invitrogen) and selected on agar containing 100 µg/mL ampicillin. Cells were grown in LB broth with 100 μg/mL ampicillin at 37 °C until the absorbance at 600 nm (OD600) reached 0.3 and L-arabinose 0.2% (w/v) was added at this point to induce protein overexpression. Growth continued for another 3 h at 37 °C before cells were resuspended in lysis buffer  glycerol and 0.5 mM phenylmethylsulphonyl fluoride (PMSF)) and stored at −80 °C. Cells were lysed and centrifuged at a relative centrifugal force of 48,000 g for 1 h at 4 °C.Plasmid encoding 3PO4 buffer pH 8.0, 300 mM NaCl, 20 mM imidazole). Proteins were eluted from the resin via a linear gradient of increasing imidazole (50 mM to 125 mM) in 50 mM Na3PO4 buffer pH 8.0 and 300 mM NaCl. Samples were analyzed by 10% (w/v) SDS-PAGE and Western blotting with anti-histidine antibody . Proteins were concentrated with ultrafiltration in an Amicon Ultra 3K device (Merck) in 50 mM Na3PO4 buffer pH 8.0 and stored at −80 °C.Cell lysates containing over-expressed Histidine-tagged Cas3 were loaded onto Ni-NTA resin pre-equilibrated with lysis buffer, and bound proteins washed with a further buffer  pre-equilibrated with buffer A  glycerol). The column was then washed with a further buffer  glycerol) and re-equilibrated with buffer A. (His)6-MBP-Cas3 was directly eluted from Ni-NTA resin with buffer B  glycerol), and recovered proteins were loaded into 1 mL heparin column  and then 1 mL MBPTrap HP column  both pre-equilibrated with buffer MA . Cas3 did not bind to heparin column and therefore the column flow through was loaded directly into the MBPTrap HP column. Proteins were eluted from the MBPTrap HP column with buffer MB  and dialysed into buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 30% (v/v) glycerol for storage at −80 °C.Cell lysates containing over-expressed (His)The DNA fork substrate for evaluating Cas3 nuclease activity shown in 2, 100 mM NaCl, 0.1 mg/mL BSA, 20 mM DTT), at 30 or 37 °C. Reactions were stopped by adding 50 mM Tris pH 8.0, 100 mM EDTA, 5 mg/mL proteinase K and 1% (w/v) SDS, followed by incubation at 37 °C for 15 min. Reactions were mixed with Orange G loading dye 1 (80% (v/v) glycerol, Orange G) for electrophoresis in 10% acrylamide 1 x TBE gels, or Orange G loading dye 2  for electrophoresis through 10% acrylamide denaturing gels.Cas3 proteins were incubated with 20 nM of fork DNA in buffer O  for electrophoresis in 10% acrylamide 1 x TB gel containing 0.13 M Tris-HCl (pH 7.6), 45 mM boric acid, 8% acrylamide and 5 mM DTT. Running buffer containing 0.13 M Tris-HCl (pH 7.6), 45 mM boric acid, 5 mM DTT, and 0.1 mg/mL BSA was used in EMSA.For EMSAs, Cas3 proteins were incubated with 20 nM of fork DNA in buffer E , at room temperature for 1 h. Reactions were mixed with 8 μL Orange G loading dye 1  at 30 or 37 °C for 30 min, to form an R-loop complex. Cas3 proteins were added and the mixture incubated for a further 120 min before directly mixing with Orange G loading dye (80% v/v glycerol and Orange G) and loading for electrophoresis through a 1% TAE agarose gel. If ATP was to be included, it was added to a final concentration of 2 mM in the Cas3 protein sample. All reactions were stopped using 50 mM Tris pH 8.0, 100 mM EDTA, 5 mg/mL proteinase K and 1% SDS, before adding Orange G dye for electrophoresis in a 1% TAE agarose gel.For Cas3 nuclease reactions in the presence of Cascade, the Cascade complex was prepared as described in . The crR2, 100 mM NaCl, 0.1 mg/mL BSA, 20 mM DTT) supplemented with 20 nM of MW14 ssDNA and 2.5 mM ATP. Reactions were incubated at either 30 °C or 37 °C for 60 min before adding eight reaction volumes of pre-mixed colour reagent, incubating at room temperature for 2 min and stopping the reaction by adding one volume of 3% (w/v) sodium citrate. Colour reagent was prepared using six volumes of 0.045% (w/v) malachite green hydrochloride mixed with one volume of 4.2% (w/v) ammonium molybdate in 4 M HCl. Samples were transferred to 96-well flat bottom plate (Life Technologies) to measure phosphate production by dye absorbance at 660 nM. The total amount of phosphate product (nmol) was calculated from a phosphate standard curve plotted using NaH2PO4 at 2–16 nmol in 100 μL reactions developed as described above.Cas3 ATPase assays followed the method described in  that usew/v) maltose. LB plates were overlaid with 3 mL of 0.6% (w/v) LB top agar containing 0.2 mL of cells. Then, 10 μL aliquots of serially diluted λvir phage in 10 mM MgSO4 were spotted on the surface of set agar and allowed to soak in. Plates were incubated overnight at 30 °C or 37 °C. The sensitivity of the cells to infection was represented as the plaque-forming units (PFUs) by plaques from several dilutions being counted and their number per ml calculated.This assay was done according to reference . Cells wcas3W406A allele was made in pAH4 by site-directed mutagenesis to create plasmid pIIB39—details of the primers and plasmids are in the E. coli strain IIB1309 (T3 λc Δcas1::FRT Δhns::kan) using the gene replacement method described in [ygcB) containing the mutation for W406A was cloned from plasmid pIIB39 into the pKOV plasmid using the NEBuilder® HiFi DNA Assembly kit and primers listed in E. coli cells and incubated over night at 30 °C. Transformed colonies were screened using PCR and sequencing for the presence of the correct cas3 gene insert. Correct plasmid with the insert was purified and transformed into the strain IIB1309 (λT3 λc Δcas1::FRT Δhns::kan) and plated onto chloramphenicol agar to induce plasmid integration at 42 °C. From the 42 ° C plate, 1–2 colonies were picked into 3 mL of LB broth with 5% w/v sucrose, grown over night at 37 °C and plated next day onto LB plate with 5% w/v sucrose to induce loss of the replacement vector. Individual colonies were then tested by phage infection assay and loss of antibiotic resistance. Colonies that showed plaques at 30 °C and sensitivity to antibiotic chloramphenicol were selected. The cas3 gene was amplified and sequenced in the Macrogen Europe service to confirm the point mutation.The ribed in . The ful6-Cas3 used a Jasco J-815 CD spectrophotometer connected to a Peltier temperature control system. Protein samples were diluted in 50 mM sodium phosphate (pH 8.0) to a concentration of 0.1–0.2 mg/mL in a total volume of 300 µL, for analysis in a 1 mm path length thermo-cuvette. Spectra were measured from 190 nm to 260 nm, and at 222 nm, at 20 °C with continuous scanning (50 nm/min) using a response time of 1 s and bandwidth of 1 nm. Delay was set at 30 s, sensitivity as standard and data pitch as 0.2 nm. For an assessment of the thermal denaturation of Cas3 proteins, the CD ellipticity (mdeg) was recorded at 222 nm in the temperature range from 20 °C to 55 °C for Cas3 and from 30 °C to 90 °C for archaeal Cas3 with a temperature slope of 1 °C/min and data pitch set as 1 °C or 5 °C, with delay of 30 s for each temperature. The buffer CD signal was subtracted from all measurements. Data were collected considering the HT voltage applied to detector was at the maximum of 600 V. Data were processed and fitted to a Boltzmann sigmoidal curve using the Origin software. Changes in the secondary structures of the Cas3 and Cas3W406A proteins were calculated at two temperatures using the CD experimental data at Dichroweb [Circular dichroism (CD) measurements of purified (His)ichroweb , BeStSelichroweb  and algoichroweb ,34.Escherichia coli Cas3 were prepared from structures of Cas3 from Thermobaculum terrenum (PDB ID: 4Q2C) [Thermobifida fusca (PDB ID: 4QQW) [tleap module of Amber16 program package [2+ ions and DNA atoms were obtained from the Amber ff14SB and the Amber OL15 force fields. Parameters for ATP were obtained from literature. Before molecular dynamics (MD) simulations, systems were energy minimised in several cycles using different constraints (the detailed procedure is described in [2). For the next 200 ps, the system was equilibrated at a constant temperature under constant pressure using the weak coupling Berendsen thermostat algorithm with the time constant for heat bath coupling set to 2 ps. During the production phase, systems were simulated at constant pressure with a Berendsen thermostat [+ ions. Simulations were carried out using the Amber 16 and simulation packages. The following systems were subjected to computational simulations: (i) protein complexes with two Mg2+ ions and ATP, (ii) protein (wild type and mutants) complexes with two Mg2+ ions and ssDNA. Each system was simulated at lower (301 K and/or 303 K) and higher (310 K and/or 317) temperature (Starting models of D: 4Q2C)  and TherD: 4QQW)  using thD: 4QQW)    and WHATD: 4QQW)  in order package . Proteinribed in . During ermostat  and the perature . The tra