Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded  Subsequctively) .Sulfolobus islandicus required transcription of DNA target genes and depended on the presence of an uncharacterised Cas protein known as Csx1  to target and degrade cognate RNA molecules . In a fo as Csx1 . Similar protein . Csx1 an protein , suggestargeting .Thermococcus onnurineus, which demonstrated that the HD nuclease domain has ssDNA endonuclease activity in vitro  domains, resulting in activation of the HEPN ribonuclease domain at the C-terminus  molecules once activated by target RNA binding ,23. cOA,terminus  and exteterminus . Subsequterminus . All of terminus ).Vibrio metoecus, a type III-B system comprising subunits Cmr1-6 where the Cmr2/Cas10 subunit lacks an HD-nuclease domain, together with a Cas6 enzyme for crRNA generation   . NucC isVibrio type III-B system generates cA3 on specific target RNA binding, resulting in the activation of the NucC effector nuclease for non-specific dsDNA degradation. By coupling these activities to a fluorescent readout, we obtain a limit of detection of 2 fM for SARS-CoV-2 RNA targets.In the past few years, the collateral cleavage activities of Cas12 and Cas13 have been repurposed for the development of rapid and specific assays capable of detecting specific RNA or DNA sequences . Two recEscherichia coli and restriction sites for cloning incorporated where necessary. All final constructs were verified by sequencing . Vibrio metoecus nucC (NucC) was obtained as a G-Block with flanking restriction sites for cloning. After digestion with NcoI and SalI, nucC was ligated into NcoI and XhoI-digested pEV5HisTEV . Synthetic genes were codon-optimized for V5HisTEV  to allowV. metoecus Cmr interference complex (VmeCmr) was assembled using the NEBuilder® HiFi DNA Assembly kit (New England Biolabs) following the Gibson assembly strategy (cmr1–3 and cmr4-6 was driven by T7 promoters. The cmr2 (cas10) gene included a sequence encoding a TEV-cleavable, N-terminal His8-tag.The expression construct pACE-Cmr for the strategy . All frastrategy  containeV. metoecus CRISPR array was designed as two repeat sequences flanking two oppositely directed BpiI recognition sites (5′-gtgtcttcgtaccttgaagacca) to allow later insertion of the target/spacer sequence of choice. The synthetic cassette also contained flanking NcoI and SalI sites that were used to clone the pre-array into MCS-1 of pCDFDuet™-1 . Vibrio metoecus cas6f, obtained as G-Block with flanking NdeI and XhoI sites, was subsequently cloned into MCS-2 of the latter construct to give pCDF-notarget_CRISPR  from the commercially available plasmid 2019-nCoV_N_Positive Control (IDT) and cloned by restriction digest / ligation into MCS-1 of pRSFDuet™-1 (Novagen) to give pSG220. The N gene plus the upstream T7 promoter region was PCR-amplified from pSG220 using Phusion™ High-Fidelity DNA polymerase (Thermo Scientific) and standard primers ACYCDuetUP1 and DuetDOWN1. The agarose gel purified PCR product served as template for in vitro transcription using the MEGAscript® T7 kit (Invitrogen) according to the manufacturer's protocol. After DNase I digest the transcript was purified by phenol:chloroform extraction / iso-propanol precipitation. The integrity of the transcript was verified by agarose gel electrophoresis. RNA species were quantified by spectrophotometry using a dilution series for each sample; for RNA oligomers the molar concentration was obtained from OD260 measurement and the calculated extinctions coefficients (IDT); N gene transcript concentration was estimated using the generic ϵ260 of 0.025 (ng μl–1)–1 cm–1 and a molecular weight of 464,564 Da (from the formula: MW = 1449 nt * 320.5 Da + 159 Da). All RNA species were dispensed into single use aliquots and stored at −70°C.RNA oligonucleotides were obtained from IDT. The mRNA for the SARS-CoV-2 N gene was obtained by The NucC reporter substrate was obtained by heating and slow cooling of the 5′-FAM- and 3′-Iowa Black®-labelled strands (25 μM each) in 10 mM Tris–HCl, pH 8, 50 mM NaCl. The dsDNA substrate was stored in the dark at −20°C.2 for 72 h. Cells were then fixed with 5% (v/v) paraformaldehyde/phosphate buffered saline (PBS) and plaques visualized by crystal violet staining (0.2% w/v). The virus titer was determined to be 6 × 106 plaque forming units (PFU)/ml. Ten-fold serial dilution of the SARS-CoV-2 stock were prepared using PBS and viral genomic RNA extracted using the QIAamp viral RNA mini extraction kit (Qiagen) according to the manufacturers spin protocol and the recommended 5.6 μg polyA carrier RNA/140 μl virus sample. The QIAamp viral RNA extraction kit procedure inactivates SARS-CoV-2  was conducted using a class II Microbiology safety cabinet inside a Biosafety Level 3 (BSL3) biocontainment facility. Vero E6 cells  were routinely cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and used to generate a high-titer SARS-CoV-2 working stock. The virus stock was generated by infecting Vero E6 cells at a multiplicity of infection (MOI) of 0.01 in DMEM supplemented with 1% (v/v) FBS. Cell supernatant was harvested 72 hours post-infection, clarified by centrifugation for 5 min at 276 × g, aliquoted, flash frozen in liquid nitrogen and stored at −80°C. Virus stock titer was determined by plaque assay using a confluent monolayer of Vero E6 cells in 12-well plates, infected with 10-fold serial dilutions of virus stock (200 μl/well) and incubated for 1 h with shaking at 37°C. The virus inoculum was removed, and cells overlaid with 1% (v/v) avicel (Dupont) in DMEM supplemented with 2% FBS and incubated at 37°C/5% CORS-CoV-2  thereforEscherichia coli BL21 Star™ (DE3) cells (Invitrogen) were co-transformed with pACE-Cmr and pCDF-target_CRISPR. The wild type VmeCmr construct and a Cmr4-D26A variant designed to abolish target RNA cleavage were expressed and purified in the same way. Overnight cultures were diluted 100-fold into LB containing 100 μg ml–1 ampicillin and 50 μg ml–1 spectinomycin, incubated with shaking at 37°C, 180 rpm until the OD600 reached 0.8. After induction with 200 μM IPTG, incubation was continued at 27°C overnight. Cells were harvested by centrifugation and pellets stored at −20°C. Cells were resuspended in lysis buffer  glycerol, pH 7.5) and lysed by sonication. The cleared lysate was loaded onto a pre-equilibrated HisTrap Crude FF  column, washed with lysis buffer and eluted in a gradient with increasing imidazole concentration (to 0.5 M). VmeCmr complex-containing fractions were pooled, dialysed at 4°C overnight in the presence TEV protease against lysis buffer without imidazole. The protein solution was passed through the HisTrap Crude FF column a second time, the VmeCmr complex-containing flow-through was concentrated using an Amicon Ultracentrifugal filter  and further purified by size exclusion chromatography  using 20 mM Tris–HCl, 500 mM NaCl, 10% (v/v) glycerol, pH 7.5 as mobile phase. Fractions from the main peak (VolRet 53 ml) containing the VmeCmr complex were combined, concentrated as above and the NaCl concentration adjusted to 50 mM by dilution with gel filtration buffer lacking NaCl before further purification over a HiTrap Heparin column (Cytiva). The sample was loaded in 20 mM Tris–HCl, 500 mM NaCl, 10% (v/v) glycerol, pH 7.5 and eluted with a NaCl gradient. Fractions containing the VmeCmr complex, eluting at ∼200 mM NaCl, were combined and concentrated as before to 3–4 mg ml–1, dispensed into single-use aliquots and flash-frozen in liquid nitrogen. Aliquots were stored at −70°C. Protein concentrations were determined by UV quantitation  using calculated extinction coefficients . The concentration of VmeCmr complex was estimated using an extinction coefficient of 610 240 M–1 cm–1, which was obtained by adding the values for the protein component in Cmr11 Cmr21 Cmr31 Cmr44 Cmr53 Cmr61 stoichiometry and an estimated value for the crRNA .E. coli C43(DE3) was used as the expression host, size exclusion chromatography was carried out on a HiLoad® 16/60 Superdex® pg 200 column  using 20 mM Tris–HCl, 250 mM NaCl, 10% (v/v) glycerol, pH 7.5 as mobile phase, and no heparin purification was performed. The NucC peaks eluting at approximately 160 ml and 180 ml during gel filtration were collected separately, and likely correspond to hexameric and trimeric species, respectively, as observed previously  glycerol, 500 μM ATP for 90 min. Enzyme was removed by spin filtration , and the filtrate was analysed by LC–MS. Compounds were separated on a Kinetex® EVO C18 column  using a Dionex UltiMate 3000 chromatography system with the following gradient of acetonitrile (B) against 10 mM ammonium bicarbonate (A): 0–2 min 2% B, 2–10 min 2–8% B, 10–11 min 8–95% B, 11–13 min 95% B, 13–14 min 95–2% B, 14–18 min 2% B at a flow rate of 300 μl min–1 and column temperature of 40°C. Elution was monitored by UV at 254 nm and by mass spectrometry . Data were analysed using Xcalibur™ (Thermo Scientific) and visualised in Prism (GraphPad).To determine which cOAs are produced by VmeCmr, 500 nM VmeCmr [3 by NucC, 500 nM NucC and 100 μM cA3 were incubated at 37°C for 1 h under the same conditions as for cOA production. Samples were processed and analysed as described above, however, elution was monitored by UV spectrophotometry alone.To test for degradation of cAlex/em 485/520 nm) in black, non-binding half-area 96-well plates (Corning). Key experiments were performed in at least two independent experiments; representative examples are shown, and data are provided in Supporting Information. Screens for selected assay conditions for both NucC alone and in combination with VmeCmr are also found in Supporting Information. The standard nuclease assay contained 25–250 nM wild-type or Cmr4-D26A variant VmeCmr complex in 12.5 mM Tris–HCl, pH 8.0, 10–20 mM NaCl, 10 mM MgCl2, 10% (v/v) glycerol, 500 μM ATP, 125 nM FAM : Iowa Black® double-stranded DNA substrate .All assays were performed in duplicate unless stated otherwise on a FluoStar Omega plate reader (BMG Labtech) using fluorescence detection . Each 20 μl reaction contained 10 μl 2X iTaq universal SYBR® Green reaction mix, 0.25 μl iScript reverse transcriptase, 2 μl premixed N2 primer/probe mix, and 2 μl sample. The reverse transcriptase was omitted to test for DNA contamination. Samples were either a 10-fold serial dilution of SARS-CoV-2 N gene transcript  or SARS-CoV-2 extracts. Each condition, including a non-template control, was run in triplicate. The RT-qPCR reaction was performed in a Stratagene Mx5000P instrument using the 2-step protocol for SYBR Green with Dissociation Curves settings and monitoring ROX as reference dye. Cycle conditions were: 10 min at 50°C; 1 min at 95°C; 40 × 10 s at 95°C, 30 s at 60°C; the dissociation curve was measured at 55–95°C. Amplification plots and dissociation curves are shown in RT-qPCR was performed using the 2019-nCoV RUO Kit  in combination with the iTaq™ Universal SYBRtT) when the fluorescence intensity crossed a threshold value. The threshold value was set to 1/8th of the maximal measured fluorescence signal of the reference substrate  were used for data analysis. Statistical analyses were performed with Prism 8 (GraphPad). Target specificity was analyzed by extracting the time point  plus 10 standard deviations were deemed detected. The lowest measured concentration detected was taken as the LoD. The quoted LoDs represent the average LoD from at least two independent experiments.V. metoecus, NucC (NucC hereafter) is the only effector protein encoded by the CRISPR locus . This system can be programmed to detect any RNA sequence by changing the spacer sequence in the CRISPR array. The VmeCas6 enzyme processes the pre-crRNA transcript to generate mature guide RNAs with the 5′-handle CUUAGAAA  system by using target RNA complementary to the crRNA spacer sequence of the ribonucleoprotein complex  of the fluorescent signal generated by 250 nM NucC (trimer concentration) using synthetic cA3 in the absence of VmeCmr. The signal reached saturation in less than 5 min for cA3 concentrations of ≥10 nM, with cA3 concentrations of ≥100 nM achieving maximal activation of the system  , which is immediately 3′ of the RNA duplex formed between the target RNA and the crRNA, corresponding to the repeat-derived 5′-handle of the crRNA. When an anti-sense CRISPR RNA binds, it base-pairs along the length of the PFS, preventing activation of the HD nuclease or cyclase activities of Cas10 ,23,26,49(TmaCmr)  revealed(TmaCmr)  and furt(TmaCmr) .3 present, as a convenient readout for the cyclase activity of VmeCmr. At position −1, all four nucleotides were well tolerated but a guanine at position −1 resulted in a slightly higher level of activation of VmeCmr. Introduction of a U:A base pair at position −2 reduced activity significantly, but equivalent base pairs introduced singly at positions −3 to −5 had only a modest impact on activity  and compared their activity when presented with either the N gene transcript or an RNA oligonucleotide containing the target sequence plus flanking region  to 6 (lowest concentration). The extracts were diluted 20-fold in triplicate into our coupled VmeCmrN209 / NucC assay. Both the wild type and the Cmr4 D26A (cas7 mutant) complexes were activated by SARS-CoV-2 RNA extracted from 6 × 103 to 6 × 106 PFU ml–1 stocks (Extracts 4–1) in the presence of polyA carrier RNA. As it is likely that many more RNA molecules than viable viral particles existed in the viral stock, we sought to estimate the N gene concentration in the extracts by comparison to a standard curve of known N gene transcript concentrations using RT-qPCR . IndeedA260/A280 ratio of the purified complexes. One possibility is that E. coli mRNA with partial matches to the crRNA sequence can co-purify with the VmeCmr complex and are sufficient to provide partial activation of the cyclase.One unexpected finding was that the background cyclase activity of VmeCmr in the absence of any target RNA varies between constructs and is proportional to the maximal activity when target RNA is present . Thus, t5 copies of the viral RNA (8 fM) can be readily detected  system of 1 × 107 copies (100 pM) (8 copies (1 nM) detected by the TtCmr system   and 1.2 r system . The >10rget RNA , with borget RNA ,26. The rget RNA . Possibl3 generating type III CRISPR systems coupled to the NucC effector nuclease as the basis for a rapid, sensitive and specific assay for any desired RNA sequence. The sensitivity of our best performing complex is on a par with first generation Cas13 assays; by way of comparison, Lbu-Cas13a programmed with a single guide RNA has a LoD of 10 fM of target RNA (Our work provides a proof of concept for the use of cArget RNA ,67. Therrget RNA . ApproacAll relevant data are included in the manuscript and the gkab1190_Supplemental_FileClick here for additional data file.


Bursaphelenchus xylophilus that spread by stem-borers Monochamus alternatus feeding on pine leaves, which has brought inestimable economic losses to the world's forestry due to lack of effective prevention and control measures. In this paper, we put forward a proposal for utilizing nanoHKUST-1 to encapusulate the Pyrethrins II that a nerve agent extracted from plant to control M. alternatus, including toxicity mechanism research, traceable biopesticide monitoring, and environment assessment for the first time. The highly biocompatible nanoHKUST-1 can solve the problems of poor water solubility, easy degradation and low control efficiency of Pyrethrins II.Pine wilt disease as a devastating forest disaster result from The results illustrated the biopesticide loading efficiency of PthII@HKUST-1 reached 85% and the cumulative release of pH-dependent PthII@HKUST-1 was up to 15 days (90%), and also effectively avoid photodegradation . 50 nm PthII@HKUST-1 made it easily penetrate the body wall of MA larvae and transmit to tissue cells through contact and diffusion. Moreover, PthII@HKUST-1 can effectively enhance the cytotoxicity and utilization of Pyrethrins II, which will provide valuable research value for the application of typical plant-derived nerve agents in the prevention and control of forestry pests. PthII@HKUST-1 as an environmentally friendly nano-pesticide can efficiently deliver Pyrethrins II to the larval intestines and absorbed by the larvae. PthII@HKUST-1 could also be transmitted to the epidemic wood and dead wood at a low concentration (10 mg/L).Here we speculate that nanoHKUST-1 will bring new opportunity to research biopesticide inhibition mechanism of different agricultural and forestry pests, which will break through the existing research limitations on development, utilization and traceable monitoring of biopesticide, especially for the study of targeting specific proteins. Monochamus alternatus Hope (MA) is not only an important stem-borer of pine, but also an important vector insect of pine wilt disease [Bursaphelenchus xylophilus to invade the host [B. xylophilus and MA because of low adhesion and transmission efficiency in the stem and branches of pine. The dosage of pesticides is multiplied to achieve the desired control effect, which not only increases the production cost but also causes the resistance of target organism to pesticides, and posing a serious impact on the safety of soil and groundwater [Stem-borers can seriously hinder the transportation of plant nutrients and water by drilling into branches and trunks, and harm a variety of economic and ecological tree species, leading to withering and even death , 2. Mono disease , 4. Pine disease –8. In ththe host . Therefoundwater –8. ThereIn recently years, nanotechnology has gradually been applied in the agriculture and forestry pest control , 10. EspPyrethrum cinerifoliun [MOFs  are a kind of organic–inorganic hybrid materials, also called coordination polymers, which are different from inorganic porous materials and general organic complexes , 20. It rifoliun . It is irifoliun . HKUST-13BTC, 98%), copper acetate (Cu(OAc)2⋅H2O, 98%) and acetic acid  were provided by Shanghai Macklin Biochemical Co., Ltd. ; Fluorescein isothiocyanate  and trypan blue were supplied by Coolaber Science & Technology Co., Ltd ; DAPI Staining Solution and 4%Paraformaldehyde Fixative Solution were supplied by Shanghai Beyotime Bio-Technology Co., Ltd ; DMEM medium and Fetal bovine serum (FBS) were obtained from ThermoFisher scientific Co., Ltd ; Insect cell culture medium sf9 was purchased from Thermo Fisher Scientific . Nematode growth medium (NGM) was obtained by Shijiazhuang cmore Technology Co., Ltd ; CCK-8 kit was got from New Cell & Molecular Bio-Technology Co., Ltd; The C. elegans, the Bristol strain N2 and escherichia coli OP50 (E. coli strain OP50) were obtained from Sunybiotech Science & Technology Co., Ltd ; DF-1 cells were C/E chicken embryo fibroblasts which were resistant to endogenous ALV in the E subgroup, and were a gift from the Institute of Poultry Diseases and Tumors of the United States Department of Agriculture that was preserved by our laboratory for generations. Other reagents were from Beijing Aitemon Co., Ltd .Pyrethrins II  was purchased from Shanghai Acmec Biochemical Co., Ltd ; Trimesic acid (HX-ray diffraction (XRD) patterns were recorded with a Bruker D8 Discover 25 Advance diffractometer using nickel-filtered Cu Kα radiation (λ = 1.5406 Å). UV-visible spectra were recorded on a DaoJin UV-2550 spectrophotometer. Thermogravimetric analysis (TGA) was performed on a TG-DTA6300. Transmission electron microscopy (TEM) images was taken by a JEOL JEM 2100F at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) images were obtained by a JEOL JSM-6700F field-emission SEM with an accelerating voltage of 10 kV. Nitrogen sorption were performed in a MicroActive ASAP 2460 (2.02 version) adsorption apparatus at 77 K up to 1 bar to obtain pore volume and pore size by analyzing nitrogen adsorption and desorption isotherms. Confocal laser scanning microscope (CLSM) images were performed on a Leica TCS SP8 microscope. Fluorescent inverted microscope (Nikon TE2000), flow cytometer (BD LSRFortessa 4), stereo light microscope , biological microscope , high performance liquid chromatography (HPLC) is configured with Waters 1525 column oven and Waters 2998 UV detector. Cell viability was measured on a ThermoFisher Varioskan Lux Multifunctional microplate reader.2·H2O was dispersed in 10% aqueous acetic acid, and 0.42 g H3BTC/20 mL ethanol was added drop by drop under high speed stirring for 60 min. The blue precipitate was collected by centrifugation of 14,000 rpm for 30 min, and washed with ethanol several times, then the precipitation was replaced with deionized water twice, stored at  − 80 °C for 2 h before freeze-drying. The obtained lyophilized powder of nanoHKUST-1 was redispersed in 20 mg/mL PthII/ethanol and stirred for 48 h. The mixture was centrifuged to collect the precipitate and washed with ethanol several times to harvest PthII@HKUST-1.HKUST-1 as a member of the MOFs is widely used in the fields of medicine, catalysis, membrane engineering, etc., which is chosen to load biopesticide into HKUST-1, hoping to avoid the application problems of small-molecule biopesticide on the basis of reducing environmental pollution, and realize the high-efficiency application of pesticides. However, the size of HKUST-1 is usually large, and although it is speculated that it can effectively kill the insect of MA, it may not be transmitted efficiently on the diseased pine trees. So, this will prompt us to consider this harsh problem. In this paper, nanoHKUST-1 was chosen for encapsulating PthII to research the toxicity mechanism, traceable biopesticide monitoring and environment assessment. Simply, 0.6 g Cu (OAc)1 mg FITC was ultrasonically dissolved in 40 mL deionized water, and 50 mg nanoHKUST-1 powder was added to the FITC solution to stir at 300 rpm for 4 h in the dark. The product was centrifuged at 14,000 rpm for 20 min, and the supernatant was removed. The obtained precipitate was washed with deionized water several times to harvest FITC@HKUST-1.5 mg nanoHKUST-1 powder was dispersed in different concentrations of PthII/ethanol solution, and stirred at 300 rpm for 48 h. After the reaction, the supernatant was collected at 14,000 rpm for 20 min to measure the content of free PthII using an UV spectrophotometer. The loading efficiency of PthII@HKUST-1 was calculated according to the following formula .\documenThe release curve of PthII@HKUST-1 was determined according to the method described in literature . PthII@H1 mg PthII@HKUST-1 powder was ultrasonically dispersed in 10 mL of PBS with pH of 5.0, 7.0 and 9.0, and spread in a 9.0 cm petri dish, then natural dried in the dark. The petri dishes containing PthII@HKUST-1 were exposed to ultraviolet light for different time, Free PthII and PthII water emulsion (PthII/WE) were as a control. Free PthII was dissolved in ethanol and spread in 9.0 cm a petri dish, and PthII/WE was directly spread in 9.0 cm a petri dish. The anti-photolysis performance of PthII@HKUST-1 was measured by UV spectrophotometer at 228 nm, that is, the difference between the mass of PthII before irradiation and the remaining amount after irradiation.Firstly, ten of 5th instar MA larvae were placed in 6 cm petri dishes, respectively. The dispersed FITC@HKUST-1 (10 μg/mL) was sprayed on the larvae by small pressure spray can (soLo-408). After incubating for 5 min, the larvae are repeatedly rinsed with deionized water by small pressure spray can to remove non-adhered FITC@HKUST-1. The treated larvae were paralyzed with ethyl acetate and placed under a stereo light microscope  to observe the fluorescence distribution on body wall and stomata. The main respiratory system of most insect larvae is through larval stomata, and a small amount spread through the body wall. Therefore, larval stomata and body wall gully area were focused on detection during the shooting. Secondly, the evenly dispersed FITC@HKUST-1 (10 μg/mL) solution was mixed with the artificial diet to feed MA larvae after starving for 24 h. After the larvae had fully fed for 1 h, the larvae were stored in 4% paraformaldehyde fixative solution for 48 h in the dark, and observed the fluorescence distribution of gut through dissecting intestines and making paraffin section. The fluorescence distribution of the larvae’s intestines was observed with a fluorescence microscope. Finally, 50 of 5th instar MA larvae were placed in 6 cm petri dishes, respectively. PthII@HKUST-1 (10 g/mL) was sprayed on the larvae by small pressure spray can and extend the crawling time of the larvae  according to the previous operation. The epidermis of the larvae was dissected to observe the distribution of PthII@HKUST-1 on the epidermis through SEM.4 cells/well, and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin–streptomycin and 5% CO2 for 24 h at 37 °C. After incubating, the original medium of DF-1 cells were removed and added fresh medium containing FITC@HKUST-1 (10 μg/mL). After 4 h of incubation, DF-1 cells were washed with ice PBS for three times, and digest with 0.25% trypsin for 5 min, and then the reaction was terminated with ice PBS. DF-1 cells suspension was centrifuged at 2000 rpm for 5 min with the temperature of 4 °C to remove supernatant, and resuspend the cells in ice PBS. The cellular uptake of FITC@HKUST-1 was observed through CLSM.In order to further study the cellular uptake of PthII@HKUST-1, DF-1 cells (C/E chicken embryo fibroblasts) were obtained as a model cell line, FITC@HKUST-1 was used to indirectly characterize the distribution of PthII@HKUST-1 on the surface of DF-1 cells. Firstly, DF-1 cells were inoculated into a 24-well plate at a concentration of 5 × 10Primary cell culture medium including 80% insect cell culture medium, 10% fetal bovine serum FBS and 10% phenylthiourea saturated solution, and subculture medium including 80% insect cell culture medium and 20% fetal bovine serum FBS, were ready for the following experiment. Under sterile conditions, the well-developed MA larvae were soaked in 70% ethanol for 20 min and absorbed the ethanol through the sterile absorbent paper. Then the insect needle was pierced into larval pronotum, and the insect blood cells were taken with a dot capillary. The blood cells were put into the primary cell culture medium and cultured under aseptic conditions at 27 °C for 7 days. After cultivation, the cell culture medium was transferred to the subculture medium and continued to incubate, and half of the subculture medium was taken out and replaced with fresh medium every week. PthII@HKUST-1, PthII/WE, Free-PthII and nanoHKUST-1 were added to the subculture medium with same concentration of PthII under aseptic conditions at 27 °C for cultivation. After 3 days of cultivation, the cell suspension was diluted with PBS and observed the cell morphology with a biological microscope.3 cells/well, and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin–streptomycin and 5% CO2 for 24 h at 37 °C. After incubating, the original medium of DF-1 cells were removed and added fresh medium containing PthII@HKUST-1, and then PBS, PthII/WE and Free PthII were as control groups. Except for the PBS group, PthII/WE and PthII@HKUST-1 had the same amount of Free PthII. After 4 h of incubation, the medium of DF-1 cells were changed to the original medium to continue cultivate for 96 h. The medium was removed and added 20 µL, 5 mg/mL of CCK-8 to incubate for 4 h in the dark. The supernatant was removed and added 100 µL DMSO to detect the absorbance at 450 nm by a microplate reader. The calculation formula of cell viability is as follows:DF-1 cells were inoculated into 96-well plates at a density of 5 × 10Sample is the absorbance value of the sample group, ABlank is the absorbance value of the blank group, and AControl is the absorbance value of the PBS group.Among them, A3. There were 30 larvae under each concentration gradient, and 10 larvae are a set of repeated group, and the control group was treated with water.The contact toxicity and stomachtoxicity of PthII@HKUST-1 were determined by the biopesticide membrane contact method and the feed mixing biopesticide method , 25. FirFeed mixing biopesticide method: 3 g of artificial feed were put into the breeding box, and 1 mL PthII@HKUST-1 solution was added. The well-developed 3rd-instar larvae were put into the breeding box. There were 30 larvae under each concentration gradient, and 10 larvae are a set of repeated group, and the control group was treated with water. The breeding boxes were placed in a constant temperature incubator with a temperature of 26 ± 1 ℃ and a relative humidity (RH) of 65 ± 10%. The survival rate of MA larvae was counted at 24 h and 48 h after feeding. If the larva did not move when touched with a brush. HKUST-1, PthII/WE and Free PthII were as control.2HPO4·12H2O (12.1 g), KH2PO4 (2.4 g), NaCl (4.0 g) and MgSO4·7H2O was dissolved in 1 L deionized water. Lysate solution: NaClO: 2 mol/L NaOH = 2:1 (v/v). The Caenorhabditis elegans, the Bristol strain N2 was cultured in inoculated Escherichia coli OP50 at 20 ℃. Adult nematodes in the egg-laying stage were washed with M9 buffer solution into the EP (Eppendorf) tube, then centrifuged to discard the supernatant, and M9 buffer solution was added to wash twice. The lysate solution was added and performed intermittent shaking fully lyse the nematodes. Then the M9 buffer solution was added and washed 3 times repeatedly to remove the lysate components. Finally, the M9 buffer solution was used to transfer the eggs that settle at the bottom of the centrifuge tube to an empty petri dish without medium, and incubated in an incubator at 20 °C for 12 h to obtain L1 instar larvae.Preparation of M9 buffer solution: NaE. coli OP50 solution at ratio 1:1 to obtain a culture solution with final concentrations of 0, 0.001, 0.01, 0.1, 1, 10, 100 and 1000 μg/mL. The prepared culture medium was spread evenly into the petri dish just poured with NGM medium and placed in a 37 °C incubator for 1 h. After the solution was dry, the petri dish was placed upside down and incubated for 11 h. The nematodes that reached L4 instar after being cultured for 2 days were transferred to a medium containing different concentrations of reagent. After 24 h of feeding, LC50, body length and width, and head swing frequency were measured.Before conducting the environmental safety assessment of nanomedicines, we firstly studied the stability of HKUST-1 in M9 buffer and also in nematode serum considering the stable delivery of HKUST-1 to nematodes, fetal bovine serum (FBS) was chosen for mimicing the in vivo serum environment of nematodes. Simply, HKUST-1 were dispersed in M9 buffer and 10% FBS for 7 days to observe their size changes by DLS detection. Then, HKUST-1, Free PthII and PthII@HKUST-1 were dispersed in M9 buffer solution, and mixed with 50: different concentrations of reagent (200 μL) were added to the 96-well plate, and then 30 of L4 nematodes were picked into each well. Each concentration treatment was repeated three times. After contacting with the agent in the incubator at 20 °C for 24 h, counted dead nematodes under the microscope to calculate the LC50.Determination of LCC. elegans. In one independent experiment, three L4 nematodes with the same activity were selected, one infected with Free PthII for 24 h, one infected with HKUST-1 for 24 h and one infected with PtII@HKUST-1 for 24 h, and then place them on three slides dripping with M9 buffer solution (60 μL). Each reagent was independently replicated 3 times, and total 90 nematodes were determined. After 1 min of stable adaptation, the head swing and body bending frequency of nematode were observed and recorded under a microscope within 1 min. The head of the nematode swings from one side of the body to the other, and the angle > 90° is defined as one head swing. A sine wave movement of the nematode relative to the long axis of the body is defined as a one body bending. In addition, the nematode to be tested was anesthetized, then observed and photographed with a microscope, and the body length and width was measured using Image J software.Determination of head swing frequency, body bending frequency and body length and width: the conventional application concentration 20 μg/mL of free PthII, HKUST-1 and PthII@HKUST-1 are configured to study the effect of three reagent on the body length, width and mobility of Although it has been clarified that PthII@HKUST-1 could effectively kill MA larvae in the direct application process, it is still a big problem that cannot be ignored whether it can effectively penetrate the bark of the diseased pine into the trunk and spread through the water transport. After all, realizing the practical application of PthII@HKUST-1 is our core value of preparing nano-pesticides. As an important evaluation standard, transmission of PthII@HKUST-1 in tree trunks were studied in this paper. Experiment base on studying transmission of PthII@HKUST-1 was is located at Taishan Forest Area in the city of Tai'an, Shandong Province, China . An electric drill was used to drill a hole with a diameter of 5 mm and a depth of 4 cm in the trunk at a 45-degree angle downward from the ground of 30 cm. First, FITC@HKUST-1 was used to inject trees and the diseased pine was sawn off after different processing time. The diseased pine was cut into small wooden pieces along the direction of the pine pith to observe the transmission of FITC@HKUST-1 through the small animal image. Second, PthII@HKUST-1 was injected with the concentration 1.0 mg/L, 10 mg/L and 50 mg/L, respectively, and PthII/WE with the same concentration as a control. After 3 days, sawdust was taken 5 cm above the punch at different depths. The method of subcritical water was used to extract PthII from wood chips. Simply, the sawdust was mixed with deionized water and put into a small polytetrafluoroethylene tank, then tanked into the reaction kettle and placed in a high-temperature furnace at 120 ℃ for 30 min. The reaction kettle was taken out, and quickly cooled, centrifuged to remove the aqueous solution. Sawdust was added methanol and mechanically shaken for 2 min, then centrifuged to retain the supernatant, continued to repeat the above operation 2 times. The methanol solution was collected to measure the content of PthII by an ultraviolet spectrophotometer. Third, the dead wood was sprayed with the concentration 1.0 mg/L, 10 mg/L and 50 mg/L, respectively, and PthII/WE with the same concentration as a control. After 3 days, sawdust was taken 5 cm above the punch at different depths. The method of subcritical water was also used to extract PthII from wood chips with the same operation method.p < 0.05.All experiments in this work were repeated three times, and statistical analysis of the data was performed by analysis of variance (ANOVA), and all the data were subjected to normality and homogeneity tests using DPS v 7.05. All graphical data are reported as the mean ± standard deviation (SD). Significance levels were set at * 2/g. HKUST-1 has excellent adsorption properties because of it’s open unsaturated copper metal sites, which brings opportunities for the successful adsorption of PthII. The changes in thermal weight loss of PthII@HKUST-1 with the temperature range of 0–800 ℃ were observed through the thermal weight loss curve , makes it easily penetrate the body wall of MA larvae and transmit to tissue cells. This phenomenon can be further proved from Fig. 0 nm, makFurthermore, the process of PthII@HKUST-1 passing through the epidermis into the larval body was observed by SEM Fig. . The untDue to effective cell uptake of PthII@HKUST-1, it could increase the cytotoxicity and exert the bioactivity , 30. TheIn order to verified the above conclusions, the cytotoxicity of PthII@HKUST-1, PthII/WE, Free PthII and HKUST-1 was measured the in vitro Fig. F. With tContact toxicity refers to the pesticide entering the pest body by contacting the epidermis of the insects to exert its activity . Due to Caenorhabditis elegans has short life cycle, complete genome, easy reproduction, etc., it was often used to assess the acute toxicity and lethal effects of heavy metals, organics and pesticide, and was widely used in environmental assessments such as soil and wastewater [C. elegans was as the object to initially assess the impact of carrier on the environment. It can be seen from Fig. 50 of PthII@HKUST-1 to C. elegans was 806.82 μg/mL, which implied very low in acute toxicity. The effect on the body length, width and mobility of C. elegans under the conventional application concentration of 20 μg/mL were explored. As shown in Fig. C. elegans after PthII@HKUST-1 treatment, and there was no significant difference in body length and width from the PthII@HKUST-1 treatment.Benefiting from the advantages of stewater , 35. TheC. elegans, and it could be considered that PthII@HKUST-1 was an environmentally safe carrier material. There are reports on the application of HKUST-1 to the development of medical drugs [C. elegans at regular concentrations, and was safe for soil microorganisms. It could be considered for the development of chemical pesticide carriers.At the same time, the body bending frequency and head swing frequency of the HKUST-1 treatment group were slightly higher than that of the PthII@HKUST-1 treatment group, but the difference was not significant. The above results showed that PthII@HKUST-1 treatment has no effect on the body shape and activity ability of al drugs , but theBecause the outer epidermis of the tree is dead tissue, it is composed of keratinized cells, produced by cork cambium. Cork is the main component of the outer layer of the bark, which can insulate moisture and gas from passing through, protect the tree, and is a natural barrier for chemical control. Therefore, we deliver nano-biopesticide into the tree by injection. PthII@HKUST-1 was designed to control BX and its vector insect MA, and the transportation efficiency in the pine tree trunk would directly affect its application prospects. As shown in Fig. In summary, we put forward a proposal for utilizing PthII@HKUST-1 on prevention and control pine wood nematode and vector insect MA, including toxicity mechanism research, traceable biopesticide monitoring, and environment assessment for the first time. In order to solve the problems of poor water solubility, easy degradation and low control efficiency of PthII, the highly biocompatible material nanoHKUST-1 was used as a carrier for embedding PthII (biopesticide loading efficiency reached 85%) to prepare controlled release nano-pesticides. The particle size advantage of nanoHKUST-1 (50 nm) made it easily penetrate the body wall of MA larvae and transmit to tissue cells through contact and diffusion. In addition, PthII@HKUST-1 can effectively enhance the cytotoxicity and utilization of PthII, which will provide valuable research value for the application of typical plant-derived nerve agents in the prevention and control of forestry pests. Moreover, PthII@HKUST-1 could be transmitted to the epidemic wood and dead wood at a low concentration. We speculate that the multifunctional designed PthII@HKUST-1 will initiate new curiosity and enthusiasm in studying the uptake/inhibition mechanism of pine wood nematode and vector insects, and guide specific target genes. This will break through the limitations of existing research on pine wilt disease and related forestry pests.


Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo. The spatio-temporal control of gene expression is essential to many fundamental biological processes from development and differentiation to synaptic plasticity and memory. This is achieved by coordinating maturation, distribution, stability, and decay of RNAs ,2. All tThe aim of this review is, after a brief summary of RNA and RBP affinity purification methods, to introduce the rapidly evolving field of in vivo proximity labeling techniques. The review will give an in-depth overview of the application of these techniques in identifying proteins in individual mRNP complexes and characterizing local transcriptomes in cells.The classic approach to study RNA–protein interaction is RNA-co-immunoprecipitation (RIP) ) or formaldehyde have facilitated the characterization of less stably associated binding partners  (for an The (standard) CLIP protocol has been modified and optimized over the last decades  to oWhile RIP and CLIP focus on the identification of RNAs associated with specific proteins, other affinity-based methods have been developed to characterize the proteins associated with a specific RNA. The common theme of RNA affinity purification is the capturing or immobilization of an RNA of interest (expressed either in vitro or in vivo) followed by identification of its bound proteins via immunoblotting or mass spectrometry. Two general strategies are used for capturing an RNA of interest: antisense oligonucleotides (ASOs) and RNA aptamer tags.O-methylated RNA chimeric probes can be used, which also allows to reduce the input material required for capturing [ASOs can be considered as an equivalent to antibodies for isolating RBPs. They can be designed to hybridize to either a single RNA-specific sequence, a sequence shared by many RNAs like the poly(A) tract in mRNAs or to several regions tiling the RNA of interest. For capturing RNA–protein complexes, biotinylated ASOs are used for the retrieval of the RNA of interest through hybridization, followed by capturing with streptavidin-beads, elution, and identification of the binding proteins. To preserve RNA–protein interactions, crosslinking by UV, formaldehyde or both is usually performed before cell lysis (RNA interactome capture (RIC), ,23,24,25apturing ,26. The apturing ,27. The apturing ), but alapturing . Anotherapturing .An alternative way to capture RNAs is to use aptamers encoded in their sequence. Such aptamers are recognized by specific RNA-binding proteins or domains that—when fused to a protein tag, for example—allow pull-down of the RNA for identification and analysis of its interactors. Several different aptamer sequences are currently in use. The MS2 and R17 stem loops, and the BoxB hairpin originate from different bacteriophages and are recognized by the MS2 coat protein (MCP), R17 coat protein, or the phage λ N-peptide, respectively (reviewed in ). FurtheA general consideration is the number of aptamers to be introduced in the RNA, which can range between 1 and 24 ,40. StudS transferase  or withse  have bese  is a mutated version of the ase BirA , a DNA-base BirA ,56,57). ase BirA  took advase BirA ,58,59 cease BirA , or the a gondii .The main drawback of BirA* is the long labeling time required to achieve sufficient biotinylation (6–24 h), which prevents its use for capturing interactome snapshots ,62. Sinc2O2 . In iewed in ), which A complementary proximity labeling approach that has been developed takes advantage of the PafA proximity ligase from bacteria. PafA mediates ligation of a small protein PupE to lysine residues on the surrounding proteins . A caveaA more recent development of proximity labeling enzymes are the split versions like Split-BioID, Split-APEX2, and Split-TurboID ,78,79,80Following the great success of proximity biotinylation in mapping protein localization and protein–protein interaction, APEX2 was applied to transcriptome mapping . In the Drosophila S2 cells and neurons. The comparison of the RNA targets between neuronal subtypes allowed the identification of cell-type specific RBP-RNA interactions [C. elegans poly(U) polymerase (PUP-2) that adds a uridine tail to the 3′-end of proximal RNAs. After lysis, RNAs are reverse-transcribed using a primer designed to enrich uridylated RNAs. The resulting cDNA libraries are analyzed using paired-end sequencing for the identity, the number of reads and the number of Us added to the RNA. By fusing PUP-2 to Puf3p in S. cerevisiae the authors identified mostly target mRNAs with known Puf3 binding sites [S. cerevisiae [Proximity labeling methods have also been applied to the characterization of RNAs bound by individual RBPs. This includes methods that are not based on the biotin-streptavidin system but on modifying the sequence of the RNA at or close to the site of protein–RNA interaction . In TRIBractions ,89. Anotractions ,91 . In vivo proximity labeling can serve as a good entry point to this problem. By recruiting the labeling enzyme to a specific RNA of interest, its associated proteins will be biotinylated. Two different strategies have been introduced for tethering the labeling enzyme to an RNA: aptamer tagging and CRISPR/gRNA guidance. Aptamer tags like MS2 or BoxB RNA loops have long been used for mRNA localization studies and to follow the dynamics of transcription and translation (reviewed in ). FurtheIn RaPID, a short RNA of interest is flanked by two BoxB aptamers (see below for more details). Its co-expression with a fusion of the BoxB recognizing λ N-peptide and BirA* or BASU allows the biotinylation of proteins bound to or associated with the flanked RNA sequences. RaPID was used to identify proteins that bind to known RNA motifs  andTethering a biotinylating enzyme to an RNA using the CRISPR system has the advantage of targeting native RNAs at their endogenous expression levels without the need for aptamer fusion b. The teThe application of tethering proximity labeling enzymes to RNA via dCas13/gRNA requires, however, a thorough optimization. For example, the target sequence of the gRNA has to be single stranded and accessible . WithoutdCas13-based and MS2-based tethering of a labeling enzyme to the same RNA does not necessarily identify the same set of protein interactors. By comparing the hTR RNA interactome identified by MCP-APEX  with that of dCas13d-dsRBD-APEX2, Han et al. surprisingly found only partially overlapping datasets, with MCP-APEX identifying more potential interactions in total . The difSimilarly to the aptamer methods, the dCas13 based approaches have only been tested for highly abundant or overexpressed RNAs. Therefore, further optimization of RNA-centric proximity labeling is needed to achieve the interactome detection of endogenously, low expressed RNAs.Proximity labeling has already proven to be a valuable complement to other methods for the analysis of RNA–protein interactions. In the near future, one can expect more and better variations of the approach to address questions in RNP composition and function. The strength of proximity labeling approaches is that they allow the identification of more binding partners, including those interacting transiently, which would not be detected by other methods. However, they do not distinguish between direct binders and proximally located partners. Thus, other techniques like CLIP and RIP still provide valuable information that is complementing results obtained by proximity labeling. This can be exemplified with Xist, a long non-coding RNA that interacts with multiple protein partners. When applied to Xist, proximity-labeling based CARPID identified 73 XIST-interacting proteins, among them 19 that had been previously found as functionally significant binders . However


Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions  Hydros cancer ,4. Semi-s cancer . Therefos cancer , cartilas cancer  and in vodelling –10. In pin vitro –13.The biological response of cells to a particular stimulus, such as a treatment, the presence of other cells and cell types, or alterations to microenvironment, can be reflected by changes in gene expression. Such differences can be observed by quantitative polymerase chain reaction (qPCR), northern blotting, high-throughput next-generation RNA sequencing (RNASeq) or whole-transcriptome sequencing. These methods require RNA preparations of high quality. The presence of hydrogel residues  can impact the use of RNA in these downstream applications . Large pFor RNA isolation, the cell lysis protocol is crucial, as to achieve cell disruption the hydrogel must also be disrupted. In addition, handling of cells can change their expression profile within minutes, requiring disruption of the gels along with cell lysis to occur in parallel to limit any effects on expression profiles. Therefore, the standard approach of digesting gels with collagenase  followedHere, we compared two protocols which combined chemical and mechanical disruption with either flash-freezing or prolonged storage at −80°C. Subsequently, we tested three different RNA purification techniques based on a column, enzyme or a combination approach. We evaluated RNA yield and quality as well as the performance of cDNA synthesized from the various RNA preparations in real-time qPCR (RT-qPCR).. 2. 2.1−1 FGF-2 , 100 U penicillin and 100 µg ml−1 streptomycin . All cells were maintained at 37°C in a humidified incubator containing 5% CO2, with media changes every 2–3 days.HUVECs were isolated as previously described  and expa. 2.26 and 6 × 105 cells ml−1 hydrogel, respectively. Vascular endothelial growth factor (VEGF) , human fibroblast growth factor 2 (FGF-2) and stromal cell-derived factor 1 (SDF-1)  were included into the gel at a concentration of 5 µg ml−1 each. Additionally, 2 mol of RGD-SP (H2N-GCWGGRGDSP-CONH2) was added to the gel. A molar ratio of starPEG to heparin-maleimide of 1 : 0.75 was used to obtain a stiffness of approximately 500 Pa (storage modulus). The starPEG–heparin hydrogels were maintained in EGM-2 for 7 days at 37°C in a humidified incubator containing 5% CO2.Three-dimensional co-cultures were obtained using hydrogels comprising synthetic starPEG and maleimide-functionalized heparin, as described previously ,11. Brie. 2.33 cells cm−2 and CAFs at 5.5 × 103 cells cm−2. The cells were maintained at 37°C in a humidified incubator containing 5% CO2, with media changes every 2–3 days for 7 days.HUVECs and CAFs were cultured separately on tissue culture plastic. HUVECs were seeded at 6.8 × 10The RNA from both cell types was isolated separately using the Zymo Direct-zol RNA MiniPrep kit  following the manufacturer's protocol. A mixture of the RNA from the 2D mono-cultures was used representing the cell ratio after 7 days in co-culture .. 2.4Zymo Direct-zol RNA MiniPrep kit (Integrated Sciences) includes a lysis step based on TriZOL. When using the Norgen Total RNA Purification Plus kit , cell lysis was achieved by shock freezing the samples in liquid N2, followed by using the lysis buffer present in the kit. Lysates from both preparations were additionally mechanically disrupted by passing the suspension through first a 19-gauge and then a 21-gauge needle. Both RNA isolation kits are based on the principle of using nucleic acid binding columns. All steps in the manufacturers' protocols were followed. DNA removal was performed by an on-column digestion with DNAse I (Zymo kit) or by passing the cell lysate through a DNA binding column prior to RNA isolation (Norgen kit). A detailed step-by-step protocol of the procedures is outlined in For the isolation of RNA from cells cultured in starPEG–heparin hydrogels, two different isolation kits were tested which use specific techniques for cell lysis. The In the following text, preparations from the Zymo Direct-zol RNA MiniPrep kit are labelled with ‘Zymo kit’ and preparations from the Norgen Total RNA Purification Plus kit are labelled with ‘Norgen kit’.. 2.5Bacteroides eggerthii  in 1× Heparinase Reaction Buffer was added to the RNA and the reaction incubated at 30°C for 1 h. Additionally, a combination of both procedures was tested. Following Heparinase digestion, the OneStep™ PCR Inhibitor Removal kit (Integrated Sciences) was used to remove residues of the enzyme and reaction buffer from the RNA preparation. A detailed step-by-step protocol is described in Following extraction, three purification methods were tested for the RNA preparations. Using the OneStep PCR Inhibitor Removal kit (Integrated Sciences), the RNA was passed through a purification column to remove organic compounds from the RNA preparation. The work was carried out according to the manufacturer's instructions. The use of heparinase digestion to purify RNA has been described previously . Based o. 2.6The quality of the RNA was assessed by UV–Vis spectrophotometry (NanoDrop) as well as by chip-based capillary electrophoresis .For the spectrophotometrical measurements, 2 µl of undiluted RNA was pipetted onto a NanoDrop 1000 instrument (ThermoFisherScientific). Measurements were carried out using the NanoDrop 1000 software, v. 3.8.1. Bioanalyser measurements were performed on an Agilent Bioanalyzer 2100  using an RNA Pico Kit (Agilent Technologies) according to the manufacturer's protocol. Additionally, qPCR was used to assess the downstream performance of the various RNA preparations. For each RNA sample, 1 μg was reverse transcribed to cDNA using SuperScript IV VILO  according to the manufacturer's instructions. All incubation steps were carried using a PTC-200 DNA Engine thermal cycler .The reference genes ribosomal protein L32 (RPL32), TATA-box binding protein (TBP) and Ubiquitin C (UBC) were then used for the qPCR assay. The reaction set up contained 1× PowerUp SYBR Green Master Mix , 200 nM of each, forward and reverse primer and 4 µl of cDNA. The primer sequences are presented in . 2.7t-test was performed comparing each extraction/purification combination to the 2D control using R v. 3.6 [p ≤ 0.05.For the statistical analysis of Ct values, Student's R v. 3.6 . Statist. 3z-stack confocal image depicting cells cultured within the starPEG–heparin hydrogel is presented in figure 1a. Figure 1b shows a macroscopic photograph of the hydrogel.A representative 2D projection of a c(i), arrowhead). Upon centrifugation of the lysate at 10 000g for 2 min at 4°C, this observation was confirmed by the presence of a pellet, presumably consisting of hydrogel material (figure 1c(ii), arrowhead). Small hydrogel pieces present within the sample may clog the column and result in low RNA yield and quality. Therefore, an additional centrifugation step was implemented to remove any residual hydrogel material (Step 9).Following the lysis step , the celfigure 1a). Upon the use of the additional centrifugation step, the 230 nm peak was no longer present (figure 2b). In addition, the RNA yield was identified to be slightly reduced (approx. 19%) from 470 to 384.7 ng µl−1 when the extra centrifugation step was included.The results of the UV–Vis spectrophotometry of RNA samples isolated with and without an additional centrifugation step are presented in figure 2a–d with figure 2b).When the tested purification methods of isolated RNA were examined, a similar general appearance of the UV–Vis spectrophotometry graphs , in the Norgen kit preparations, a higher 28S peak was observed (figure 4e–h). This was also reflected by the rRNA ratio which was found to be below 2.0 for the RNA isolated with the Zymo kit but above 2.0, for the samples isolated with the Norgen kit (figure 4e–h).A Bioanalyser was used in conjunction with Agilent RNA 6000 PICO chips to further evaluate the quality of the prepared RNA. This identified clear 5S, 18S and 28S peaks as well as clear bands on the virtual electrophoretogram gel in all samples . While tfigure 4rgen kit . AdditioRNA integrity number (RIN) is commonly used as an indicator of RNA quality assessment. The algorithm computes a number between 1 and 10 based on various parameters of RNA integrity , where the higher the number is, the better the RNA quality . The RINAs an additional measurement of RNA integrity and quality, qPCR was also used to evaluate the downstream performance of the different RNA preparations. We selected three genes that are commonly used as endogenous controls, ribosomal protein L32 (RPL32), TATA-box binding protein (TBP) and Ubiquitin C (UBC). The cycle threshold (Ct) represents the cycle number at which the fluorescence generated by the reaction crosses a predetermined threshold. Commonly, the Ct value is used as a means to study the relative expression levels of genes of interest between a range of samples. For the current assay, the expression level of the genes examined was not of interest. In this study, changes in Ct values between samples were used as a measure of the presence of inhibitory molecules in the reaction. The Ct values obtained for the 2D control were 23.79 ± 0.25, 21.24 ±0.11 and 14.51 ± 0.06 (mean ± s.d.), for UBC, TBP and RPL32, respectively. For UBC, slightly increased values were found for the samples isolated with the Zymo kit but not the Norgen kit. However, for both TBP and RPL32, the 3D samples showed slightly increased Ct values compared to the 2D controls. The magnitude of the change was dependent on the extraction kit and the purification method. Samples isolated with the Norgen kit showed generally a smaller increase in Ct values . Overall. 4Bioengineered 3D models are widely used to study cell–cell and cell–matrix interactions within a physiologically relevant microenvironment . The isoWe found that the implementation of an additional centrifugation step prior to transferring of the sample to the RNA-binding column significantly increased the quality of RNA obtained. By removing residual hydrogel fragments, contamination of the RNA was reduced and an improvement in RNA quality was observed that surpassed the slight drop in RNA yield observed.A260/280 and A260/230 ratios obtained by UV-spectrophotometry indicated the absence of contaminants in the RNA preparations from both kits. The rRNA ratios for the isolated RNA samples ranged between 1.3 and 2.8. RNA isolated with the Zymo kit had a lower rRNA ratio overall when compared with the Norgen kit. While preparations with an rRNA ratio of 2.0 are typically considered as high-quality RNA, it has been reported that a low rRNA ratio does not necessarily denote poor mRNA quality [Only minor differences in RNA yield, quality and integrity were identified between the two tested RNA isolation kits. The  quality ,28. The Minor differences were observed in the amplification of the three selected endogenous controls used in the study between the 2D control and 3D samples. A slight increase in Ct values was observed in the 3D samples independent of the RNA isolation kit used with the extent being slightly less in the samples isolated with the Zymo kit. However, purification of the RNA did not result in significant changes to the observed Ct values. The slight variations in Ct values are likely due to the starting material rather than the RNA preparations. The samples for the 3D RNA extraction were prepared from the same batch of cells and hydrogels. For the controls, the equivalent type and density of cells were cultured on a 2D tissue culture plastic surface.The cell density is a crucial factor for RNA yield and the success of subsequent applications. The used cell density resulted in an adequate amount of RNA, between 4 and 20 µg in total. Depending on the application, a significant lower cell number can become a limiting factor for RNA extraction. However, in our hands, the SuperScript IV VILO reverse transcriptase provided successful amplification with efficiency between 90 and 107% with an RNA amount as low as 1 ng (data not shown).To remove hydrogel residues and heparin contamination, we tested also heparinase digestion, a PCR inhibitor removal column as well as a combination of these steps. No significant improvements in RNA quality were observed with these variations in purification techniques. However, the purification techniques might be of interest for other 3D cell culture techniques. They can be adapted for models with higher matrix density or different glycosaminoglycans, for example, by using other enzymes to purify the RNA.2 with the Norgen kit was found to be of little benefit. By contrast, small RNAs were found to be more abundant in RNA extracted with the Norgen kit which may be of relevance to specific research questions. However, the difference in small RNA composition may be redundant when considering total RNA isolation as there are specific commercial kits for the extraction of small RNA molecules that deliver higher throughput and reproducibility for these specific research questions.Both, the Norgen Total RNA Purification Plus kit and the Zymo Direct-zol RNA MiniPrep kit, yielded a good quantity of high-quality RNA suitable for cDNA synthesis and RT-qPCR. Here, we have identified that it is essential to centrifuge the cell–hydrogel suspension prior to RNA isolation, to remove any hydrogel residue. No obvious contamination of the RNA was present after isolation and a subsequent RNA purification step was found to be unnecessary for extracting RNA from cells cultured in starPEG–heparin hydrogels. While the Zymo kit needed a longer handling time due to the additional freezing step, handling of liquid NHere, we present two protocols to extract high-quality RNA from glycosaminoglycan-containing hydrogels suitable for gene expression analyses. Both methods and the purification techniques can easily be adapted for other 3D cell culture models based on natural or semi-synthetic materials, for example, hyaluronic acid hydrogels, to produce a robust template for subsequent interrogation.


Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low‐input direct RNA‐seq, improving pathogen detection and transcript identification in a microbial community standard with spike‐in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.The requirement of a large input amount (500 ng) for Nanopore direct RNA‐seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid‐phase reversible immobilization (SPRI) beads. A novel solid‐phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non‐polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen High input requirement in Nanopore direct RNA‐seq is attributed to significant sample loss associated with library purification using solid‐phase reversible immobilization (SPRI) beads. This investigation employs immobilization of poly(A) polymerase and T4 DNA ligase to magnetic microbeads for RNA library preparation to circumvent SPRI bead purification. This approach enables low input transcriptomic analysis and improves sensitivity of pathogen detection. Nanopore direct RNA‐seq avoids the artifacts and biases associated without amplification process. Enabling the interrogation of native transcripts has inspired new ideas in RNA research, as evidenced by recent studies that elucidate transcriptome complexity from transcript isoforms and strand‐specific information that includes RNA base modifications. In addition, Nanopore sequencing using the portable MinION device allows for point of care, real‐time identification, and characterization of RNA viruses such as Ebola virus, Venezuelan equine encephalitis virus, Zika virus, influenza A virus, and most recently, SARS‐CoV‐2. Furthermore, it allows for a single sequencing reaction to detect multiple, single‐stranded RNA viruses.High‐throughput RNA sequencing (RNA‐seq) techniques have transformed biological and biomedical research facilitating the understanding of transcriptional regulatory networks and identifying diagnostic and prognostic biomarkers for numerous diseases.1 The  NGS library construction, however, can cause specific biases. It is of great interest to identify the technical and methodological artifacts that may introduce biases and affect enzymatic efficiency and offer viable solutions to improve the Nanopore RNA‐seq outcomes.Nanopore direct RNA‐seq will continue to grow in popularity with further improvement to sequencing chemistry, library construction methodology, and data processing pipelines. For instance, lowering the RNA input requirement can improve the robustness of transcript profiling and identification, provide increased sensitivity to identify single nucleotide polymorphisms , as well as facilitate early and sensitive detection of pathogenic bacteria and RNA viruses. In addition, quantification of gene expression and characterization of RNA biomarkers require unbiased coverage across the transcriptome. Hence, RNA‐seq based research relies on a high‐fidelity library preparation that can represent the entire transcriptome  so that the RNA‐seq data can reflect genuine biological effects. It is well known that sequencing chemistry and bioinformatics tools can alter the final outcomes in next‐generation sequencing (NGS).10 NGS including Listeria monocytogenes, the etiological agent of listeriosis, which has one of the highest case fatality rates among all foodborne illnesses. The current protocol demands high RNA input (500 ng), which hinders the implementation of this technique for biomedical research using low input amounts and sensitive pathogen surveillance as a point‐of‐care platform. The protocol for polyadenylated transcripts includes enzymatic addition of adaptor(s) and multiple purification steps with solid‐phase reversible immobilization (SPRI) beads  in the presence of T4 DNA ligase. The RNA–RTA hybrid products are then reverse transcribed in the presence of reverse transcriptase. The synthesis of a cDNA strand can improve throughput, presumably by destabilizing RNA secondary structures. Next, the reverse‐transcribed RNA–RTA complex is ligated to a second adaptor (RMX) preloaded with a motor protein required to guide the RNA strand through a nanopore channel in a flow cell. Additionally, 3′ polyadenylation is required prior to adaptor ligation  before RNA‐seq. Furthermore, the current library preparation method comprises several distinct enzymatic reactions, resulting in a laborious and time‐consuming workflow. Therefore, it is highly desirable to improve the efficiency in library preparation and reduce sample loss inherent with SPRI bead‐based purification to enable Nanopore direct RNA‐seq viable for small input quantities or rare, difficult‐to‐obtain samples.Over the past decade, SPRI paramagnetic bead‐based chemistry has been widely used for nucleic acid handling procedures such as NGS library construction. While providing convenience in the removal of enzymes and other components, SPRI bead‐based purification can result in a fairly substantial sample loss, and thus researchers are required to begin their library preparation with large amounts of RNA input.2, 9L. monocytogenes as a model. We exploited the immobilization of poly(A) polymerase (PAP) and T4 DNA ligase on magnetic microbeads as a means to achieve enzyme removal  RNA library for Nanopore direct RNA‐seq using l Figure , as oppo22.1L. monocytogenes RNA for Nanopore direct RNA‐seq ‐coated magnetic microbeads (Im‐Ligase) can be utilized to catalyze a variety of ligation reactions. In this work, we demonstrate that this immobilized T4 DNA ligase catalyzes efficient ligation of two ONT adaptor‐derived substrates and can be easily removed from the reaction mixture  showed a significant sample loss with an average recovery rate of 34%  and a substantial loss (recovery rate of 37%) using the Sol‐RT 100 protocol with a bead purification step. The Im‐cpl protocol generated an average of 1.22 M reads from the replicates , we further conducted a series of library preparations from lower inputs of s Figure . By conts Figure . It is a2.5Escherichia coli O157:H7, Salmonella Enteritidis, and L. monocytogenes transcriptome  and displayed higher fidelity with a greater mean read length (537 nt), compared to the Sol‐RT method with a higher failed reads rate (30.8%) and a much shorter mean read length (129 nt). MG‐RAST also successfully identified all three pathogens. The results shown in Table E. coli, S. Enteritidis, and L. monocytogenes, respectively, considerably higher than the Sol‐RT results, recovering 158, 32, 10 reads, respectively. The RNA‐seq data analysis of the spike‐in transcript standards is described later.The Im‐cpl protocol for low‐input RNA was further validated using a cocktail community standard composed of me Table 2. The c2.6Figure To validate the effect of enzymatic processing strategies on 3′ poly(A) tail length, we analyzed the 500 ng input libraries of four different protocols Figure 3. The d2.7L. monocytogenes EGD‐e. All protocols displayed satisfactory mapping results, generating a mapped rate of ≈99% before rRNA sequences were removed. The results further demonstrated a satisfactory RNA mapping rate of 84% from the Im‐cpl protocol, a significant improvement from 79% from the Sol‐RT protocol, after rRNA sequences were removed  was first performed to evaluate the variability of each library preparation protocol. The average genes identified by the two replicates from each of the Im‐cpl 500, Sol‐RT 500, Im‐cpl 100, and Sol‐RT 100 protocols are 1309, 1504, 954, and 442 genes, respectively. While the replicates of the Im‐cpl methods produced consistent results in both gene content and transcript quantities, there is a larger variation between the Sol‐RT replicates with 100 ng of RNA input  identified twice more genes (1081 genes) than the Sol‐RT protocol (Sol‐RT 100), which identified only 336 genes Figure . The datFigure Table We also examined and compared the sequencing coverage of captured genes by the two different methods Figure 5. The pL. monocytogenes transcriptome also showed that both Im‐cpl and Sol‐RT libraries produced similar gene coverage distribution and resemble L. monocytogenes transcriptome when 500 ng of RNA input amount was used for library preparation. On the other hand, the Im‐cpl method offered better transcriptome coverage than the Sol‐RT protocol for 100 ng low RNA input compared with the reference  of input RNA. Our solid‐phase catalysis strategy, for the first time, allows for both enzymatic processing and enzyme removal from the final RNA library and thus enables the preparation of sequencing‐ready libraries without bead‐based purification, a widely adopted process in many NGS workflows. The coupled reaction protocol using immobilized enzymes avoided bead‐based clean‐up, resulting in substantial reduction of sample loss and identification of more transcripts for L. monocytogenes, with both 100 and 500 ng input amounts and 1 ng L. monocytogenes input in the community sample. The higher transcript identification rate is consistent with better recovery of the spike‐in RNA controls. The data suggest that our new methodology may help to capture a more complete transcriptome that may offer insight into the nature and expression of the transcriptome. This finding is important for the choice of reliable library preparation for RNA‐seq studies because accurate measurement of RNA transcript sequences and their copy numbers is a foundation for understanding dynamic transcript expression . Therefore, the method presented may potentially reduce false interpretation of transcriptomic data, as unbiased RNA‐seq data are critical for accurate quantification of gene expression, differential gene expression, and post‐transcription modifications. Further study is required to determine whether this method is applicable to a low‐input RNA‐seq analysis of various sources of rare and difficult‐to‐purify samples, including clinical human specimens. It is also of interest to examine if our new method can facilitate RNA‐seq analysis of other non‐polyadenylated RNAs such as long noncoding RNAs that play major roles in cancer.The new strategy was designed based on solid‐phase enzyme catalysis by covalent conjugation of two relevant enzymes to magnetic microbeads. In conjunction with a coupled enzymatic processing scheme, we created the one‐pot formulation for fast and robust RNA library preparation workflow, potentially enabling ONT's long‐read sequencing for native RNA molecules from rare and difficult‐to‐obtain, such as clinical specimens or environmental samples. Our strategy has consistently resulted in higher sequencing yields with satisfactory qualities and more complete transcriptome profiles of xpression18  tailing, RTA ligation for RT, if any, and RMX ligation) Figure . The new The Sol‐RT protocol, however, demands longer library preparation time than the Im‐cpl protocol significantly. The Im‐cpl method utilizes a much shorter yet highly efficient workflow. It generates a comparable throughput as the Sol‐RT protocol with 500 ng input but significantly higher yields and more accurate transcriptome profiling for low RNA inputs. The Sol‐RT library generated similar read output with a slightly higher number of genes identified compared to a flow cell run from one‐third of a typical Im‐cpl library; however, the latter can be entirely sequenced by multiple flow cell runs, resulting in both higher read output and identification of higher number of expressed genes. Furthermore, this highly streamlined Im‐cpl protocol is better suitable for automated library preparation and sequencing workflow.Our data confirmed that the Sol‐RT protocol with the incorporation of reverse transcription step can drastically increase the throughput of direct RNA‐seq compared to the Sol‐seq and Sol‐cpl protocols, as previously described.2 The E. coli poly(A) polymerase has low processivity and incorporates ≈1–2 nucleotide(s) per substrate binding event. Following the coupled reaction scheme, both PAP and ligase can potentially function during the entire 30 min incubation time, increasing the overall efficiency of adaptor ligation to the RNAs in a library. An RNA molecule with a sufficient length of 3′ poly(A) tail can anneal to an RTA possessing a single‐stranded sequence of 10 bases of dT at 3′ end. Consequently, adaptor ligation would deplete the pool of the unligated RNA molecules over time, thereby increasing the effective concentration of poly(A) polymerase to process the remaining RNA substrates. Indeed, our poly(A) tail length analysis revealed significantly shorter poly(A) tail length profiles when the couple reaction scheme was performed with either soluble or immobilized enzymes. Slightly longer poly(A) length associated with soluble enzyme treatment is likely due to faster kinetic property and/or higher soluble enzyme activity than the immobilized form. It has yet to be determined whether poly(A) length can influence Nanopore sequencing performance. On the other hand, the fact that the Sol‐RT method produced the longest mean 3′ poly(A) tail and the high read output indicates that 3′ poly(A) tail length is unlikely a major factor. Moreover, the coupled reaction protocol allows for ligase to function for the entire 30 min period compared to 10 min ligation with the sequential reaction protocol.It is noteworthy that both coupled enzymatic reaction protocols, Sol‐cpl and Im‐cpl, yielded significantly more reads than the sequential reaction protocol (Sol‐seq), albeit they have the same total 30 min reaction time. Our preliminary study indicates that  This approach has significantly enhanced the enzymatic activity of E. coli poly(A) polymerase. In addition, immobilized DNA ligase displayed proficient efficiency in adaptor ligation despite the fact that fewer enzyme units were used in place of highly concentrated soluble T4 DNA Ligase. Therefore, our work presented has demonstrated the utility of immobilized enzymes and can potentially contribute to the establishment of a highly efficient and less biased RNA library method. With high‐throughput sequencing already being applied in clinical contexts, there is a pressing need for the automation of multireaction workflows. With this new tool, we can explore new possibilities for a highly integrated system for fully automated library preparation, sequencing, and analysis in various research and clinical applications.Solid‐phase catalysis based on immobilized enzymes can bring forth many unique benefits to the molecular workflows for Nanopore RNA‐seq, including enzyme removal without heat inactivation and bead‐based purification, thereby facilitating many enzyme‐based applications. Successful implementation of enzyme immobilization technology requires innovative solutions to overcome potential challenges such as lower specific activity, higher costs, and additional equipment needs. Our previous study indicates that immobilization of enzymes can result in lower enzymatic activity in comparison to their soluble counterpart, and higher activity of the immobilized form can be achieved by surface modification of the magnetic microbeads.21 Thi4E. coli O157:H7 (ATCC 43895), Salmonella enterica serovar Enteritidis (ATCC 13076), and Listeria monocytogenes (ATCC 19115) were acquired from the American Type Culture Collection . Monarch Total RNA Miniprep Kit from New England Biolabs, Inc.  was used for total RNA extraction. Magnetic beads were purchased from General Electric and modified for enzyme immobilization. SuperScript III reverse transcriptase (Ref #. 18080–093) was purchased from Invitrogen by Thermo Fisher Scientific. Spike‐in RNA variant controls (SIRV‐Set 4) were purchased from Lexogen. All other soluble enzymes were obtained from NEB. 20× phosphate buffer saline (#9808) was purchased from Cell Signaling Technologies, MA, USA. The chemical reagents are in analytical grade.Ni‐NTA Agarose was purchased from Qiagen (Cat #. 30230) for protein purification. RNA Clean XP beads were obtained from Beckman Coulter, Inc. (Cat #. A63987) for bead‐based purification for RNA library preparation. lization.21 SupE. coli PAP was subcloned into pSNAPf‐tag (T7) (NEB) using NEBuilder (NEB). The resulting plasmid pPASH was used to express and purify a fusion protein (termed PASH) comprised of E. coli PAP, SNAP‐tag, and six‐histidine tag following the protocol described previously.E. coli T7 Express  transformed with pPASH was inoculated to 5 mL of LB/ampicillin medium and cultured at 37 °C for 4 h. 1 mL of subculture was further inoculated to 1 L of LB/ampicillin medium and incubated at 37 °C for about 3–4 h until OD600 reached 0.6–0.7. Then 3 mL of 100 × 10−3m isopropyl β‐d‐1‐thiogalactopyranoside was added to the medium (with a working concentration of 0.3 × 10−3m) to allow the expression of SNAP‐tagged poly(A) polymerase recombinant protein (PASH). The expression was carried out at 16 °C overnight. The culture was then harvested and centrifuged to discard the supernatant. The pellets were then lysed using a sonicator on ice and centrifuged for crude extract. The clarified cell lysate was loaded onto Ni‐NTA resin, followed by extensive washing with wash buffer . The fusion protein was then eluted using elution buffer . The eluted protein was dialyzed against Diluent C buffer No BSA (NEB) overnight and stored for future experiments. The concentration of enzymes was determined using the Bradford assay. The kinetic studies were carried out as described for NEB poly(A) polymerase.The coding sequence of eviously.21E. 6‐benzylguanine (BG) modified magnetic beads were characterized to catalyze ligation of various substrates , BG‐PEG4‐PASH (PEG750)  five times. Next, 25 µg PASH protein was dissolved to 125 µL of the buffer solution and further loaded to the washed beads. The mixture of enzyme and beads was incubated overnight at 4 °C. The beads immobilized with PASH were washed using the same buffer 8 times to remove the excessive enzymes. Diluent C buffer with no BSA (NEB) was used to resuspend the beads and for storage at −20 °C. Immobilized poly(A) polymerase with different modification strategies , and BG‐PEG4‐PASH (PEG750)) was evaluated using capillary electrophoresis, and BG‐PASH (PEG750) proved to be the immobilized form with the highest activity, which was stored and used for the following experiments presented  broth at 37 °C for 22 h. Total RNA extract of L. monocytogenes was performed according to the previous report. Particularly, 1 mL of the culture was centrifuged at 16 000 × g for 2 min to harvest the cell pellet. 250 µL of 3 mg mL−1 lysozyme diluted in TE buffer was added to suspend the cell pellet followed by incubation at 37 °C for 1 h. Total RNA was extracted using the Monarch Total RNA Miniprep Kit . Specifically, 40 µL of RNA with DNA contamination sample was eluted in nuclease‐free water. Then, 20 µL of DNase I , and 20 µL of DNase I reaction buffer were added to the eluted sample and incubated at 37 °C for 30 min for DNA digestion. Finally, RNA Priming Buffer and Wash Buffer were used for RNA purification. The RNA extract was quantified by high sensitivity Qubit Assay and Nanodrop, respectively, and stored at −80 °C. A similar protocol was used for preparing the community standard, which consists of E. coli O157:H7, S. Enteritidis, and L. monocytogenes. Particularly, a single colony of each strain was inoculated in 10 mL of BHI medium, respectively, followed by incubation at 37 °C for 24 h. 2 mL of each overnight culture was used for total RNA extraction using NEB Monarch Total RNA Miniprep Kit.Sequential reaction protocols start RNA library preparation with poly(A) tailing using poly(A) tailing, followed by ligation of poly(A) tailed RNA with adaptors using T4 DNA ligase. These two steps can be performed simultaneously in one‐pot (coupled reaction protocols) using soluble or immobilized enzymes.L. monocytogenes total RNA were prepared using various protocols  polymerase , and 500 ng of L. monocytogenes total RNA. The mixture was supplemented with nuclease‐free water to 30 µL in a 0.2 mL thin‐walled PCR tube. The mixture was incubated at 37 °C for 20 min for poly(A) tailing. 1 µL of RTA, 6 µL of RMX, and 3 µL of T4 DNA ligase were then added to the poly(A) tailed RNA sample to make a final volume of 40 µL. The mixture was incubated at 25°C for 10 min to allow the ligation of the adaptors to the poly(A) tailed RNA samples. For the coupled reaction protocol using soluble enzymes (Sol‐cpl), all components used for the Sol‐seq reaction protocols were mixed at once, as a one‐pot reaction. 40 µL of the mixture was subjected to incubation at 37 °C for 20 min, followed by 25 °C for 10 min. The libraries prepared by Sol‐seq and Sol‐cpl were purified with RNAClean XP beads as follows: 40 µL of resuspended RNAClean XP Beads was added to the 40 µL of adapter‐ligated RNA and mixed by pipetting. The reaction mixture was incubated on a Hula mixer (rotator mixer) at room temperature for 5 min, and the RNA sample was pelleted on a magnet. The supernatant was pipetted off, and 150 µL of the Wash Buffer was added to the beads and resuspended for washing. The beads were pelleted, and the supernatant was pipetted off. The previous step was repeated. The tube was removed from the magnetic rack, and the pellet was resuspended in 21 µL of Elution Buffer. The mixture was incubated at room temperature for 10 min to allow the elution of RNA. Beads were pelleted on a magnet until the eluate was clear and colorless. 21 µL of the eluate was collected and retained into a clean 1.5 mL Eppendorf DNA LoBind tube. RNA concentration was measured using high sensitivity Qubit Assay Kit, and the final yield and recovery rate were determined. In addition, a pair of libraries were prepared according to the Sol‐seq protocol using soluble enzymes without bead‐based purification [Sol (‐BP)]. For the L. monocytogenes RNA libraries of 500 ng input prepared by the coupled method using immobilized enzymes (Im‐cpl protocol), 2.5 µL of immobilized poly(A) polymerase (BG‐PASH (PEG750)) and 3 µL of immobilized T4 DNA ligase was applied to replace their soluble counterparts. Both immobilized poly(A) polymerase and immobilized T4 DNA ligase were removed after the one‐pot reaction from Im‐cpl mixture. Due to the usage of immobilized enzymes, bead‐based purification was not applied for the Im‐cpl method.All libraries with 500 ng of s Figure . For theL. monocytogenes RNA libraries with low‐inputs of 100, 50, 20, and 10 ng were prepared using the Im‐cpl protocol by mixing the following components in a 0.2 mL thin‐walled PCR tube: 3 µL of quick ligation buffer, 0.45 µL of 5 m NaCl solution, 1.5 µL of immobilized poly(A) polymerase, and L. monocytogenes total RNA . 0.5 µL of RTA, 3.0 µL of RNA RMX and 1.5 µL of Im‐ligase and nuclease‐free water were added to make a final volume of 15 µL. The reaction was incubated at 37 °C for 20 min and 25 °C for 10 min. The immobilized poly(A) polymerase and immobilized T4 DNA ligase beads were removed from the reaction mixture by placing the tube on the magnetic rack.L. monocytogenes RNA libraries were also performed using soluble enzymes with a reverse transcription step (Sol‐RT) as recommended by ONT  polymerase (NEB), and different inputs of RNA, 500 ng (Sol‐RT 500) and 100 ng (Sol‐RT 100), respectively. The mixture was supplemented with nuclease‐free water to 12.5 µL and incubated at 37 °C for 20 min for poly(A) tailing. Next, 1 µL RTA and 1.5 µL T4 DNA ligase were added to a final volume of 15 µL. The mixture was then incubated at 25 °C for 10 min to allow RTA ligation. Then, 9 µL nuclease‐free water, 2 µL 10 × 10−3m dNTP, 8 µL first strand buffer, 4 µL 0.1 m DTT, and 2 µL SuperScript III reverse transcriptase were added to a final volume of 40 µL. The reverse transcription reaction was performed at 50 °C for 50 min followed by 70 °C for 10 min. RNA samples were purified with RNAClean XP beads after treatment with soluble enzymes. 72 µL of resuspended RNAClean XP Beads was mixed with the 40 µL of adapter‐ligated RNA by pipetting, followed by incubation on a Hula mixer (rotator mixer) at room temperature for 5 min. The bead fraction, pelleted on a magnet, was washed with 150 µL of fresh prepared 70% ethanol. RNA was eluted in 20 µL of nuclease‐free water by incubation at room temperature for 5 min. The eluted RNA was then mixed with 8 µL quick ligation buffer, 6 µL RMX adaptor, 3 µL nuclease‐free water, and 3 µL T4 DNA ligase, and the mixture was incubated at 25 °C for 10 min for RMX ligation. The final bead‐based purification was carried out as described above for Sol‐seq and Sol‐cpl. RNA yields were determined by high sensitivity RNA Qubit assays.T Figure  for bothE. coli O157:H7, 10 ng of S. Enteritidis, 1 ng of L. monocytogenes RNAs, and 1 ng of Spike‐in RNA variant control (SIRV‐Set 4) mixture provided by Lexogen.A pair of community standard libraries were prepared by the low‐input Im‐cpl protocol and Sol‐RT protocol, respectively. Each library is composed of 39 ng of Direct RNA sequencing of the prepared libraries was performed on ONTs GridION using R9.4 flowcells. Each sequencing run was performed until less than ten active nanopores were available for RNA seq, or 72 h of sequencing running was achieved, which comes earlier. For the RNA libraries prepared using soluble enzymes (Sol‐seq and Sol‐cpl) and the protocols with reverse transcription (Sol‐RT 500 and Sol‐RT 100), 20 µL of each sample recovered from bead purification was mixed with 17.5 µL of nuclease‐free water to 37.5 µL. The mixture was further mixed with 37.5 µL of RNA running buffer (RRB) to a final volume of 75 µL for loading into a flow cell. For the libraries prepared by a coupled reaction approach using immobilized enzymes with one‐third of the library being loaded [Im‐cpl 500 (1/3)], or by soluble enzymes without bead purification [Sol (‐BP)], an equivalent amount of RNA comparable to that of the sequential approach using a Sol‐seq protocol was used for a sequencing run; the volume was first brought up to 37.5 µL by addition of nuclease‐free water followed by mixing with 37.5 µL of RRB to a final volume of 75 µL. The entire Im‐cpl library with 500 ng input (Im‐cpl 500) was subjected to three flow cell runs with each flow cell loaded with 13 µL of the library. For low‐input RNA libraries , 13 µL of each 15 µL prepared sample was mixed with 24.5 µL of nuclease‐free water to a final volume of 37.5 µL before the addition of 37.5 µL RRB for a sequencing run.L. monocytogenes reference genome using Minimap2. All FastQ files of passed base‐called reads were collected combined for future analysis, and only reads present in the “pass” folder were used in subsequent analysis. Comparison across experiments of the read length and distribution, number of reads, and reference identity were performed using NanoComp.Raw sequencing reads were collected via a local base‐calling algorithm with MinKNOW, base‐called using Guppy, and aligned to the  The passed reads were also aligned to the ribosomal DNA sequences of L. monocytogenes, S. enterica, and E. coli using minimap2. Subsequently, the unmapped non‐RNA reads were mapped to the composite reference genome of the three aforementioned organisms, and reads were differentiated from organisms using Samtools v1.9.The FastQ files of passed reads of the community sample libraries were first extracted from the base‐called fast5 files and were analyzed via MG‐RAST for metatranscriptomic analysis and taxonomic identification.23 TheSIRV‐Set 4  contains the long SIRV with 15 long SIRV RNAs of 4–12 k and ERCC standard RNAs. The non‐RNA reads from the community sample libraries were mapped to the long SIRV standard sequences (SIRV_longSIRVs_multi‐fasta_200709a.fasta) and the ERCC standard RNA sequences (SIRVome_isoforms_ERCCs_longSIRVs_200709a.fasta). The mapped read counts were calculated using Samtools v1.9. Read counts and mapped read length were plotted in R studio 1.3.1073. Statistical analysis of poly(A) tail size of different samples was conducted in R studio 1.3.1073.For poly(A) tailing analysis, Oxford Nanopore raw reads were base called using guppy base caller . Each of the large datasets was divided into 20 smaller subsets and used the tailfindr v1.2 program to deduce poly(A) tail lengths of all of the base‐called reads.25 StaL. monocytogenes genome using minimap2 and unmapped output reads. The non‐rRNA reads were then mapped to the L. monocytogenes reference genome (NC_003210), and only primary alignments were used for downstream analysis. The resulting alignment bam files were converted to bed files by bedtools v2.29.2, and the alignment sizes were computed from the bed files. Plotting of alignment size distributions of different samples was carried out in R studio 1.3.1073.The FastQ files were first extracted from the base‐called fast5 files and then aligned to the ribosomal DNA sequence of the ut reads.24 TheL. monocytogenes reference genome from the minimap2 alignments. Gene counts were normalized by gene lengths and the total number of aligned reads. Normalized read counts TPM were used for generating scatter plots and measuring linear correlation by Pearson correlation coefficiency between the datasets in R studio v1.3.1073. The raw gene count matrices of two technical replicates of the Im‐cpl and the Sol‐seq datasets were used for differential gene expression analysis using the Bioconductor package DESeq2 v3.12 in R Studio v1.3.1073.The featureCount v2.0.1 was used to quantify reads that map to individual genes in the ignments.28 GenL. monocytogenes reference genome (NC_003210) downloaded from NCBI. Gene body sequencing coverage was analyzed and visualized using the RSeQC package.The bedCoverage command of the bedtools (v2.29.2) was used to calculate sequencing coverage of individual genes based on the minimap2 alignments and the gene annotation of the  package.30L. monocytogenes reference genome from the minimap2 alignments. The gene counts were normalized by gene lengths, and the total number of aligned reads. The normalized read counts TPM were used for pairwise comparison and correlation plots between different methods. Pearson correlation was used to measure the linear correlation between two datasets. 1097 common genes between Im‐cpl 500 (1/3) and Sol‐RT 500, 1327 common genes between Im‐cpl 500  and Sol‐RT 500, and 274 common genes between Im‐cpl 100 and Sol‐RT 100 were used respectively for pairwise comparison and Pearson correlation coefficiency calculation. The raw gene count matrices of two technical replicates of the Im‐cpl and the Sol‐seq datasets were used for differential gene expression analysis using the Bioconductor package DESeq2 v3.12 in R Studio v1.3.1073. DESeq2 uses negative binomial distribution to make estimates and perform statistical inferences on differences. There were 1083 genes compared for differential expression, and none of the genes was found differentially expressed at a p‐value of 0.05. For per‐gene sequencing coverage analysis, sample sizes of gene sequencing coverage analysis are Im‐cpl 500: 1319; Im‐cpl 100: 1081; Sol‐RT 500: 1528, and Sol‐RT 100: 336  with 500 ng RNA input were repeated twice. Sol‐RT with 500 ng input was repeated three times, and Im‐cpl with 500 ng input was repeated four times. For low input RNA‐seq, Im‐cpl with 50, 20, 10 ng input and Sol‐RT with 100 ng input were repeated twice. Im‐cpl with 100 ng input was repeated four times. Reads and bases generated were obtained from Oxford Nanopore sequencing report. Mean and standard deviation were calculated using Excel. For Poly(A) tail length analysis, reads from two replicates were pooled for poly(A) tail length analysis. Sample sizes of the four protocols used for poly(A) tail length analysis are: Sol‐seq 253987 reads, Sol‐cpl 1334900 reads, Im‐cpl 2331366 reads, and Sol‐RT 747482 reads. The mean and median poly(A) tail lengths were calculated for each protocol and were used for comparison between different protocols. Plotting of poly(A) tail length distributions was done using R studio 1.3.1073. For Alignment length analysis, sample sizes of alignment length analysis are: Im‐cpl 500: 250249 primary alignments, Im‐cpl 100: 205448 primary alignments, Sol‐RT 500 116680 primary alignments, and Sol‐RT 100 41892 primary alignments. The mean and median alignment lengths were calculated for each dataset. Plotting of alignment length distributions was done using R studio 1.3.1073. For Gene expression analysis: The featureCount v2.0.1 was used to quantify reads that map to individual genes in the ignments.28 TheThe authors declare the following conflict of interest(s): Y.F., L.S., A. Z., Z. S., and M. X. are employees of New England Biolabs, Inc., a commercial supplier of reagents for molecular biology and inventors of a US patent related to this work .Supporting InformationClick here for additional data file.