Gastric cancer (GC) is one of the most pernicious tumors that seriously harm human healthcare. GC metastasis is one of the prime cause of failed cancer treatment, but correlation between N6-methyladenosine (m6A) and GC metastasis was less reported.Methylated RNA immunoprecipitation sequencing (MeRIP-seq) of GC tissues was conducted. Quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) were taken to determine the expression of ALKBH5 in GC tissues and cell lines. RNA-seq together with MeRIP-qRT-PCR was used to screen the target gene of ALKBH5. RNA pulldown, mass spectrometry and RNA immunoprecipitation (RIP) were used to search the “reader” protein of target gene. The mechanism was also validated via a tail vein injection method for lung metastasis model.Decreased expression of ALKBH5 was detected in GC samples, and it was correlated with clinical tumor distal metastasis and lymph node metastasis. ALKBH5 interference promoted metastasis of GC cells and this effect was closely related to the demethylase activity of ALKBH5. PKMYT1, as a downstream target of ALKBH5, promoted invasion and migration in GC. Caused by ALKBH5 knockdown or its demethylase activity mutation, upregulated expression of PKMYT1 indicated that ALKBH5 modulates expression of PKMYT1 in an m6A-dependent manner. IGF2BP3 helped stabilize the mRNA stability of PKMYT1 via its m6A modification site.This study established an ALKBH5-PKMYT1-IGF2BP3 regulation system in metastasis, representing a new therapeutic target for GC metastasis.The online version contains supplementary material available at 10.1186/s12943-022-01522-y. Gastric cancer (GC) is the fifth most universal cancer type and ranks third among causes of cancer-related death in the world , 2. MetaRNA modification, a form of epigenetic regulation, has been found to exist widely at the transcriptome level. As the most frequently modified form of eukaryotic mRNA, N6-methyladenosine (m6A) has been found to participate in a variety of biological processes . M6A is The role of m6A in diverse types of cancer has been reported recently , 16. PubIn this research, we identified the inhibitory effect of alkB homolog 5 (ALKBH5), one of the key demethylases, on the metastatic ability of GC cells. We further screened the key downstream molecules and modification sites and revealed the potential mechanism by which ALKBH5 regulates the invasion of GC cells.GC tissues and adjacent normal tissues from 49 GC patients were acquired from the Department of Surgery of Xinqiao Hospital and patients were diagnosed as GC by the departments of Pathology in hospital. Samples were quickly placed in liquid nitrogen for refrigeration. Informed consent for this procedure was gained from each participant.The human GC cell lines used were purchased from ATCC. HGC-27, BGC-823 and SGC-7901 GC cells were grown in DMEM medium with 10% FBS (Gibco) and 1% Penicillin–Streptomycin . Cells were identified with STR profiles. Mycoplasma decontamination was conducted once every two months.Cell pellets were charged at specific times and washed twice with PBS. Cell extracts were cleavage in RIPA . After 30 min of centrifugation at 12,000 × g, the supernatant was quantified using bicinchoninic acid (BCA) method . After quantification, sample was boiled at 100℃ with SDS buffer for 5 min. Protein extractions were separated using 10% or 12% SDS/PAGE gels. After incubated with primary and indicated HRP-conjugated secondary antibodies , membrane signals were exposed by chemiluminescence system . Antibodies used were as follows: Anti-ALKBH5 , Anti-GAPDH , Anti-PKMYT1 , Anti-IGF2BP3 .Stable interference and overexpression of ALKBH5 lentiviruses were generated by GenePharma . PcDNA3.1 vector were used for the construction of the full-length PKMYT1-CDS, CDS-mut1, CDS-mut2, ALKBH5, and H204A plasmids, The ALKBH5-H204A plasmid and its lentiviruses were obtained from Genechem . Small interfering RNAs (siRNAs) targeting PKMYT1 and IGF2BP3 were directly synthesized . Transfection of plasmids or siRNAs was conducted using Lipofectamine 8000 . Cells were collected for further experiments two days after transfection. Sequences are listed in the Additional file 6 cells suspended in 100 μl PBS into the tail vein (n = 5 per group). A Bruker Molecular Imaging Software  was used for monitoring metastatic progression 10 min after intraperitoneal injection (150 mg/kg) of D-Luciferin, Potassium Salt D  dissolved in DMSO. The luciferase signal intensity was kept on the same scale. Mice were executed after 6 weeks and lungs were removed for immunohistochemical analysis and hematoxylin–eosin (H&E) staining. All operations were in line with laboratory animal management norms.After randomly assignment and anesthetization, nude mice were injected with 5 × 10https://www.omicstudio.cn/tool.TRIzol reagent  was used for RNA extraction. RNA was quantified using a Nanodrop 2000. PrimeScriptTM RT reagent kit  and TB GREEN SuperMix  were used for reverse transcribed and the qRT-PCR reactions to analyze mRNA expression. GAPDH was regarded as internal standard control to normalize the data. Experiment was repeated three times. For RNA-seq, RNA samples were sequenced by Lianchuan  and analyzed using the OmicStudio tools at MeRIP was performed as formerly described  with minAfter the samples qualified, FastQC software was used to perform basic quality statistics on the raw sequencing data (raw reads) and obtain high quality reads. BWA (Burrows Wheeler Aligner) was used for more accurate alignment of reads to the reference genome . The reaThe Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit  was used. Briefly, protein A/G magnetic beads conjugated with rabbit immunoglobulin G , ALKBH5  or IGF2BP3  antibody were incubated with cell lysates supplemented with RNase inhibitor at 4 °C overnight. After washed for 6 times, RNA–protein complexes were added into proteinase K buffer. Finally, RNA was extracted using phenol–chloroform method. QPCR was performed to determine relative interaction between ALKBH5 or IGF2BP3 protein and PKMYT1 transcripts.Total RNA of samples was extracted. EpiQuik m6A RNA Methylation Quantification Kit  was taken to evaluate the global m6A levels of the mRNA. Briefly, 200 ng poly-A-purified RNAs were added to each assay well, and the relevant antibody were added to each well in a suitable diluted concentration, respectively. The OD450 was measured of each well. Quantification was performed according to the standard curve to calculate m6A levels.http://david.abcc.ncifcrf.gov/), the OmicStudio tools at https://www.omicstudio.cn/tool, and Cytoscape 3.7.0. P < 0.05 was considered statistically significant.GO analysis and GSEA were performed using DAVID . TMAs were deparaffinized, subjected to antigen retrieval, and incubated with antibodies against ALKBH5 (1:500), PKMYT1 (1:500), and IGF2BP3 (1:400). Upright microscope system  was used for images capturing. The stained slides were assessed with integrated optical density (IOD) using Image-Pro Plus software.4 cells in 200 ul serum-free medium were added into the upper layer. After 24 h, migrated cells in the lower layer were fixed, stained with crystal violet, and imaged for quantification. Pre-colded matrigel  were added into the upper layer for the invasion assay.For the transwell assay, chambers were present in a 24-well culture table with 800 ul DMEM containing 10% FBS prepared at the bottom. Then, 5 × 10GC cells were plated into 24-well plates and incubated for 24–48 h until a 100% growth. Then, 200 ul micropipette tips were used to generate wounds. Next, cells were washed twice with 500 ul PBS. Then, 500 ul serum-free medium was added to the well. Photos were captured every 6 h for 24 h. Images were analyzed using ImageJ software.GC cells were grown into 12-well plates. Actinomycin D  was adopted to the plates. Cells were collected at the constant times for RNA extraction. The remaining PKMYT1 was analyzed by qRT-PCR. MRNA half-life was calculated using linear regression analysis. GAPDH was used for normalization.Biotin-labeled PKMYT1 ssRNA probes were synthesized in vitro by Sangon Biotin , while the PKMYT1-CDS, CDS-mut1 and CDS-mut2 mRNA were first transcribed using the MEGAscript T7 Transcription Kit , and then biotin-labelled using Pierce RNA 3′ End Desthiobiotinylation Kit . Next, 20 pmol of biotinylated RNA together with cell lysate was mixed with streptavidin agarose beads  at 4℃ overnight. After six washes, streptavidin beads were collected for western blot or mass spectrometry.Statistics were analyzed using GraphPad Prism 9 . Two-tailed unpaired student’s t-test or one-way ANOVA analysis was adopted. The Spearman/Pearson correlation analysis was used to evaluate relationships between ALKBH5 expression, PKMYT1 and IGF2BP3. Survival curves of GC patients were performed using Kaplan–Meier analysis. Receiver operating characteristic (ROC) curves were generated by GraphPad Prism 9. Each experiment has a minimum of three replications.To assess overall levels of m6A modification in GC, 5 pairs of clinical tumor tissues with adjacent normal samples were randomly selected. Using the EpiQuik m6A RNA methylation quantification kit, we observed significantly higher levels of m6A in GC tissues than in adjacent normal tissues Fig. A, indicaM6A levels are primarily balanced by methyltransferases  and demethylases (FTO and ALKBH5), while methyltransferases METTL3 and METTL14 have been reported to regulate GC progression , 26. NevTo excavate the potential feature of ALKBH5 in GC progression, we first assessed the expression of ALKBH5 among GC cell lines Fig. A-B. AfteIt had been reported that ALKBH5 H204A, which mutates amino acid 204, may result in a deficiency of ALKBH5 demethylase activity . To clarTo explore the potential target of ALKBH5 in GC metastasis, RNA-seq in control and ALKBH5 overexpressing BGC-823 cells was conducted Fig. A. RNA-seTo identify the primary target gene that plays a key downstream of ALKBH5, 816 genes, whose m6A levels were notably higher in GC than that in adjacent tissue, were selected according to the MeRIP-seq result. Combined with 3715 genes that were significantly altered after overexpression of ALKBH5 in the RNA-seq results, we obtained 237 common genes. To further identify the downstream target genes essential in GC, we identified 2596 genes that had significantly high expression (log2 FC > 1.2) in GC using the GEPIA database, and 36 genes were ultimately obtained. A literature review was used to screen four genes  that have been reported in GC Fig. E. The m6Both tissue microarray and TCGA database results indicated highly expressed PKMYT1 in GC, and its expression was strongly correlated with poor prognosis Fig. I-M. ROC http://www.cuilab.cn/sramp/). The prediction results exhibited five potential m6A modification sites with very high confidence in the mRNA sequence of PKMYT1  , IGF2BP1Both the literature and our findings indicate that m6A modification is crucial for the metastasis of GC, therefore, targeting dysfunction of m6A regulators represents a promising strategy for cancer therapy. Several studies have shown that small molecule inhibitors targeting dysfunction of m6A regulators have therapeutic potential for cancer . FurtherOverall, we have identified ALKBH5 as a tumor suppressor in GC metastasis, and this role is dependent on the demethylase activity of ALKBH5. PKMYT1, a downstream target gene of ALKBH5, can be recognized and bound by the “reader” protein IGF2BP3 after m6A modification. The mRNA stability of PKMYT1 gets enhanced, resulting in higher expression level and ultimately a significant promotion on GC metastasis.Additional file 1: Figure S1. ALKBH5 was correlated with tumor stage and lymph node metastasis in database analysis.Additional file 2: Figure S2. ALKBH5 inhibited invasion and migration in BGC-823 cell.Additional file 3: Figure S3. PKMYT1 promoted invasion and migration in GC cell.Additional file 4: Figure S4. Mutation of m6A modification site suppressed invasion and migration ability of PKMYT1.Additional file 5: Figure S5. ALKBH5/PKMYT1/IGF2BP3 regulation system may exist among digestive system.Additional file 6: Table S1. RT-PCR Primer Sequences.Additional file 7. The specific sequence of CDS and mut plasmids in PKMYT1.Additional file 8. PKMYT1 mRNA-SRAMP prediction Results.Additional file 9. 823-RNA-seq Gene_differential_expression.Additional file 10. 823-RNA-seq GSEA gene list.


Cryptosporidium, a protozoan pathogen, is a leading threat to public health and the economy. Herein, we report the development of a portable, colorimetric biosensing platform for the sensitive, selective and label/PCR-free detection of Cryptosporidium RNA using oligonucleotides modified gold nanoparticles (AuNPs). A pair of specific thiolated oligonucleotides, complementary to adjacent sequences on Cryptosporidium RNA, were attached to AuNPs. The need for expensive laboratory-based equipment was eliminated by performing the colorimetric assay on a micro-fabricated chip in a 3D-printed holder assembly. A smartphone camera was used to capture an image of the color change for quantitative analysis. The detection was based on the aggregation of the gold nanoparticles due to the hybridization between the complementary Cryptosporidium RNA and the oligonucleotides immobilized on the AuNPs surface. In the complementary RNA’s presence, a distinctive color change of the AuNPs (from red to blue) was observed by the naked eye. However, in the presence of non-complementary RNA, no color change was observed. The sensing platform showed wide linear responses between 5 and 100 µM with a low detection limit of 5 µM of Cryptosporidium RNA. Additionally, the sensor developed here can provide information about different Cryptosporidium species present in water resources. This cost-effective, easy-to-use, portable and smartphone integrated on-chip colorimetric biosensor has great potential to be used for real-time and portable POC pathogen monitoring and molecular diagnostics. Cryptosporidium spp. is a major intestinal protozoan pathogen in adults, children and numerous animal species worldwide. Recently, cryptosporidiosis, an infection caused by Cryptosporidium, has become a global threat to public health and the economy1. Moreover, Cryptosporidium contamination has increased the challenge to deliver safe drinking water in developing and developed countries4. The pathogen can cause death in children and immuno-compromised individuals and severe cryptosporidiosis in healthy adults6.Cryptosporidium oocysts in water samples, including fluorescence microscopy-based methods  1623)7, immunoassays9 and molecular techniques11. These methods are time-consuming, insufficient, expensive and require well-trained personnel. In particular, immunological and molecular techniques require extensive sample preparation, including labelling with expensive fluorescence labels, amplification and sophisticated lab-based equipment for detection and data analysis12. These challenges make this method unsuitable for on-site detection and the fast-decision-making process14. Therefore, it is necessary to develop a simple, user-friendly, rapid, cost-effective, label/PCR-free and sensitive method for on-site detection of Cryptosporidium in water samples.Several techniques have been used to detect 17. Moreover, colorimetric assays can be visualized with the naked eye without the need for sophisticated instruments or tedious training18. Gold nanoparticles (AuNP) colorimetric assays are of particular interest because of their unique features, including their catalytic properties and controllable sizes19. More importantly, they are easily synthesizable and modified with several thiolated molecular recognition elements20. Furthermore, the unique localized surface plasmon resonance (LSPR) properties of AuNPs allow them to have different colors based on their size21. For example, AuNPs with 20 nm have a red color, and their LSPR can be measured at a wavelength of 520 nm22. Colloidal AuNPs are negatively charged and hence repel each other. The repulsion between the particles allows them to stay colloidal and maintain their red color. However, when salt such as NaCl is added, these negative charges are neutralized (masked), resulting in the aggregation of AuNPs. Aggregation results in a decrease in the distance between the particles and an increase in their size. This results in a change in color from red to blue due to a large shift of the LSPR peak (absorption at a longer wavelength)23.Recently, colorimetric detection based on inorganic nanomaterials has attracted great attention due to their simplicity, cost-effectiveness, and distinct color variation associated with their morphology or size change24. Due to these features, AuNPs have led to a new generation of biosensors with high stability, selectivity and sensitivity.In DNA biosensing, gold nanoparticles are usually modified with a biological recognition element such as a complementary oligonucleotide specific and selective to the target DNA or RNA. The hybridization between the modified AuNPs and the target DNA or RNA in the sample results in a change in color from red to blue. The color change is proportional to the analyte concentration in the sample and can be qualitatively detected by the naked eyes25. On-chip biosensors are one of the most exploited techniques used to produce cost-effective and portable miniaturized sensing platforms26. A typical on-chip-based biosensor consists of a recognition element , transducer, and detector33. The recognition element dictates the biosensing platform specificity and selectivity34, while the transducer transforms the response produced from a biorecognition event to a measurable signal35. These platforms can be advantageous in a wide range of applications, including food safety36, diagnostics37, security and defense39, and environmental monitoring40. This is due to their cost-effectiveness, disposability, simplicity, low power requirements and sample-reagent usage40. However, most colorimetric detection methods still need advanced and expensive equipment to read and analyze the results42. Therefore, the applicability of using these techniques for on-site detection and point-of-care (POC) is still limited44.Recently, portability has become an increasingly important goal for developing sensing platforms for many applications45. Given the iniquitousness of smartphone access, smartphone-integrated biosensing platforms can serve as a accessible and low-cost solutions for routine (daily) testing without requiring highly-trained personnel and bulky, expensive and complicated laboratory instrumentations such as spectrophotometers and microscopes, especially in resource-limited countries and rural areas47. As an example, Breslauer et al.48 developed a mobile phone-mounted light microscope with 350×magnification and demonstrated its potential for global health applications. Smartphone-based image capture and analysis systems have also been applied for monitoring immunoassays such as colorimetric and fluorescence assays50, DNA diagnostics51, label-free detection of pathogens52, quantum-dots labelling54 and fluorescence microscopy55. Smartphones have also been used as a spectrophotometer with a wavelength resolution of 5 nm56. These advantages have led to increasing research and industrial interest in using smartphones to detect analytes of environmental59, food safety61, personal health62, and biomedical64 applications. This has led to integrating smartphones as smart detectors, signal inducers, and data processors with the on-chip-based biosensing technology for rapid, cost-effective, easy-to-use, real-time and POC diagnostics68. An additional attachment or cradle is used with these integrations to mount the smartphone and other components72. These integrated systems have allowed many analyses to be performed remotely outside the laboratory (at the point of need)74.In the last decade, smartphones with internal memory storage, touchscreen displays, internet connectivity (enabling data exchange wirelessly over the internet), advanced compute power, and high-resolution cameras have gained significant market acceptance for POC applicationsCryptosporidium RNA. Compared to DNA, RNA is shorter and allows for the direct detection of a single mismatch or mutation without amplification (PCR). Hence, RNA was used as the target analyte in this project75. The colorimetric sensing mechanism is based on two sets of modified AuNPs with specific oligonucleotides complementary to adjacent sequences on Cryptosporidium RNA. Upon adding Cryptosporidium RNA into the modified AuNPs solution, hybridization between two complementary sets of oligonucleotides immobilized on the AuNPs and the RNA occurs, forming a network of oligonucleotide-AuNPs. The aggregation of AuNPs results in a colorimetric shift due to the change in LSPR due to a reduction in the distance between the AuNPs and hence an increase in the size of the AuNPs. The outcome is a visible color change of the AuNPs from red to blue, observed and recorded within 5 min . A smartphone was chosen based on its lightweight, portability, on-site image analysis, and data transmission capability. All these advantages are suitable for in-field detection and POC applications. The sensing platform in this research has provided an exclusive sensitivity, selectivity and portability towards the detection of Cryptosporidium RNA: a 5 µM of Cryptosporidium RNA spiked in water samples can be detected via the developed on-chip colorimetric assay in 30 min, reducing the turnaround detection time following RNA extraction from contaminated sites. The results generated by the developed sensing platform were in good agreement with those generated by the conventional spectroscopic techniques. All these confirm the developed sensing platform combined with the 3D-printed holder assembly integrated with a smartphone could directly detect different Cryptosporidium RNA concentrations in a spiked sample without DNA amplification. Furthermore, the on-chip sensing platform developed in this research showed the sensor's capability to detect Cryptosporidium and distinguish between its different species. This capability is crucial to allow appropriate risk assessments.This research describes the development of a simple, cost-effective, easy to fabricate, sensitive and portable on-chip-based colorimetric biosensor for the direct and label/PCR-free detection of min Fig. . The colCryptosporidium RNA were designed and purchased from Integrated DNA Technologies  (Table 3), dextran sulfate, formamide, sodium chloride and sodium dodecyl sulfate were purchased from Sigma-Aldrich (Canada). Illustra NAP 5 columns were purchased from VWR (Canada). All other reagents and solvents were of the analytical grade and purchased from Sigma Aldrich. Ultrapure water was used throughout the experiments.Gold nanoparticles (AuNPs) with a diameter of 20 nm were purchased from Cytodiagnostics (Canada). According to the manufacturer, AuNPs were synthesized by citrate reduction, and thus they have negatively charged citrate ions adsorbed on their surface, which enhance their stabilization via electrostatic repulsion. Two sets of thiolated-oligonucleotides capturing probes and a) Table . Sodium All ultraviolet–visible (UV–vis) spectral measurements were carried out by double beam spectrophotometer, Cary 3500 UV–vis. A 1-cm path length quartz cuvettes were used for all measurements. An Oakton pH meter  was calibrated against three standard buffer solutions with three different pH values  and used for the pH determination.76. All glass slides were cleaned by using piranha solution followed by an oxygen plasma treatment for 10 min. Chromium and gold layers with thicknesses of 50 nm and 250 nm, respectively, were sputtered (Angstrom Engineering) on the cleaned glass slides in an argon atmosphere. Sensing transparent circular zones with a diameter of 3 mm were patterned using the standard lithography process, as described in3. A lift-off procedure was followed to create hydrophobic circular barriers around the detection zone. This process has led to circular hydrophobic barriers around the detection spot to control the sample in the sensing area.The fabrication of the chip-based colorimetric biosensor was performed as previously described in43. The smartphone touch screen is used to control the detection process. Furthermore, the smartphone's built-in Wi-Fi chip allows a 2.4 GHz wireless network to transfer data for further analysis on an external computer or external storage when required. A three-dimensional-printed holder assembly was designed and fabricated to house all the chips and the smartphone inside a compact unit.The 3D portable holder assembly integrated with a smartphone is shown in Fig. The conjugation of the two sets of thiolated oligonucleotides Table  to gold 3.A 1 mL solution of unmodified gold nanoparticles was transferred to a 1.5 mL Eppendorf tube in the second part. Then, 24 µL of the reduced thiolated oligonucleotides  was added to the gold nanoparticles and incubated for 1 h at room temperature. Following this step, 0.1 M sodium phosphate buffer (pH 7.0) was added to the mixture in a stepwise manner (in 10 μL increment with 5 min incubation time in between) until a final concentration of 10 mM sodium phosphate (pH 7.0) was achieved. Subsequently, 1 M NaCl was added in a similar stepwise manner until a final concentration of 100 mM was reached. The mixture was then incubated at 4 °C for 16 h. The above steps minimize the aggregation of the gold nanoparticles by immobilizing the thiolated oligonucleotides properly onto the gold nanoparticles' surface, which improves the overall stability and quality of the conjugation process. The conjugated gold nanoparticles were then centrifuged at 17,000×g for 30 min to pellet the oligonucleotide gold conjugate. Lastly, the supernatant was removed, and the oligonucleotide gold conjugate was resuspended in 200 μL of 10 mM sodium phosphate buffer, 100 mM NaCl and 0.01% (w/v) NaNCryptosporidium RNA using the developed sensing platform, 3 µL of AuNPs modified with probe A (25 μM), and 3 µL of AuNPs modified with probe B (25 μM) were first added to the sensing zone on the fabricated chip. Next, 5 µL of different concentrations of Cryptosporidium RNA  and 4 µL of hybridization buffer (a stock solution of 16% dextran sulfate and 20% formamide) were added to the modified AuNPs. The chip was then incubated at 70 °C for 15 min, followed by incubation at 37 °C for 20 min. Lastly, the chip was inserted in the holder assembly at room temperature. The presence of Cryptosporidium RNA in the sample results in a detectable color change after 5 min in the detection zone patterned on a chip, which can be visualized by the naked eye.For the colorimetric detection of HSV) color space. This previous step was performed to achieve a linear relationship between the color components and the color change. Image analysis was performed by calculating the Saturation value of the recorded images' HSV coordinates and correlated to the sample's analyte concentration.For quantitative analysis, the color change is captured by a smartphone camera  integrated with a 3D-printed holder assembly under controlled light conditions. A region of interest (ROI) in the captured image was selected and fixed for consistency measurements. An open-source application  from the android application store was installed on the smartphone and used for converting the ROI from the red, green, blue (RGB) color space to the Hue, Saturation, Value  was calculated using the following expression: LOD = 3SDb/m, where SDb is the standard deviation of the blank and m is the slope of the calibration curve78.To assess the developed on-chip colorimetric sensor's applicability, a calibration curve was obtained by plotting ATo evaluate the developed sensing platform's selectivity, aqueous solutions spiked with a non-complementary RNA consisting of 42 nucleotides (random sequence RNA) were tested and used as the control  before and after conjugation Fig. . Gold naCryptosporidium RNA concentrations, as described in “630/A523 ratio was used in this study to investigate the aggregation state of the modified AuNPs. Figure 630/A523 ratio, and a noticeable color change of AuNPs from red to blue was observed.The colorimetric assay was first tested with different Furthermore, the color change can be visualized at high concentrations of the target analyte (> 25 µM)  of the HSV coordinates of the recorded images and a distinct color change . Furthermore, small volumes result in a shorter analysis time so that the total analysis time can be reduced from hours to 30 min. Moreover, multiple samples from different contaminated sites can be analyzed at once, which will further shorten the analysis time and result in a large-scale screening process.79. However, unlike our sensing platform, in79, DNA amplification was conducted to enhance the sensitivity. This has resulted in increasing the cost, analysis time and hindered portability and thus on-site detection. In our sensing platform, DNA oligonucleotides were used and the capture probe for the detection of RNA. This has increased the number of base-pairs mismatch between DNA and RNA and thus incomplete hybridization, which results in less sensitivity and detection limit. The use of RNA oligoprobes (capture probe) is expected to enhance the detection limit and sensitivity by 10 orders of magnitude. This is due to the high degree of stability of RNA/RNA hybrid duplexes compared to RNA/DNA ones80.The detection limit achieved was high (5 µM) compared to those reported inCryptosporidium RNA and the random sequence RNA was chosen to investigate the effect of other random bacterial sequences RNA in high-contaminated samples. The results show that a high concentration of a random sequence RNA does not induce any noticeable color change  to 53 (Cryptosporidium RNA). The results confirm the high selectivity of the developed colorimetric assay and the sensing platform to detect Cryptosporidium RNA in complex samples.To investigate the selectivity of the developed colorimetric assay, the degree of the color change of the modified AuNPs towards the analyte of interest and a non-complementary sequence RNA consists of 42 nucleotides (random sequence RNA) were recorded by both UV–vis spectroscopy Fig. a) and thAdditional experiments were performed to investigate the repeatability, practicality, accuracy and reliability of the developed sensing platform. Five samples with different concentrations  were prepared as described in “In general, the obtained results are in good agreement, with maximum errors occurring at the lowest concentration (5 µM).Cryptosporidium with the developed sensing platform in terms of detection principle, advantages, and disadvantages.Table Cryptosporidium specific RNA. This sensing platform eliminated the need for PCR amplification, well-trained personnel and bulky instruments. Two sets of AuNPs modified with oligonucleotides capturing probes specific and complementary to adjacent sequences on Cryptosporidium rRNA were used to enhance the specificity and selectivity towards the analyte of interest to reduce the inference from undesired substances in the sample. After assessing the colorimetric assay using UV–vis spectroscopy, the assay was tested using the developed sensing platform to confirm the applicability, reproducibility and reliability of the sensing platform for the quantitative detection of Cryptosporidium.A simple and cost-effective colorimetric assay combining the chemical and photoelectric properties of AuNPs on a chip with 3D-printed holder assembly integrated with a smartphone was developed for the sensitive, label-free and on-site detection of Cryptosporidium RNA. Furthermore, the sample volume used for measurements using the developed sensing platform was 15 µL, which reduces the sample volume and cost required for a single measurement required for analysis compared to the conventional method. In addition to the direct measurement of Cryptosporidium RNA without amplification, this sensing platform enables a simple way to screen multiple samples for Cryptosporidium from different contaminated sites and quantitatively determine the concentration of Cryptosporidium in the sample.Under optimized conditions, results obtained from the UV–vis spectroscopy and the developed sensing platform showed a linear detection range between 5 and 100 µM with a detection limit of 5 µM. The detection limit obtained by the developed sensing platform was similar to the detection limit obtained by UV–vis spectroscopy without using bulky and advanced laboratory equipment. The total turnaround analysis time using the developed sensing platform was reduced from hours to 30 min. Even when a high concentration of random sequences RNA in spiked samples were tested with the developed sensing assay, the developed sensing platform showed high selectivity and specificity towards Cryptosporidium RNA. The developed sensing platform shows maximum errors at low RNA concentrations. Future research efforts are required to characterize the effect of AuNPs size and RNA-induced aggregation in order to enhance the detection limit. Future studies are also required to study the replacement of the DNA oligonucleotides with RNA oligoprobes to increase the degree of stability of RNA/RNA hybrid duplexes compared to the RNA/DNA ones80 as well as testing the sensor with other potential interferants. Multidisciplinary research efforts are required to design and test real-time and high-throughput systems for detecting Cryptosporidium spp. in the water supply chains with high sensitivity and reproducibility in order to harmonize global efforts to monitor and prevent waterborne infectious diseases using such technologies.This smartphone-integrated sensing platform is a steppingstone towards creating ubiquitous and accessible pathogen detection technologies that could potentially be used in resource-limited settings, however, there are some outstanding challenges that need to be met before a field-ready technology is accomplished. One limitation of this technology is that it suffers from limited environmental control when used in open environments where ambient light or vibrations can negatively impact the sensing performance. Overcoming this challenge requires constructing the 3D attachment or enclosure with all the necessary hardware to perform measurements and then connect the attachment through Bluetooth or wire to the smartphone. This will reduce the interference from ambient light and vibrations and enable better control and stability of the attachment. Another challenge is lowering the detection limit below 5 µM of


RNAs play essential roles in various biological processes. Mounting evidence has demonstrated that RNA subcellular localization and intercellular/systemic trafficking govern their functions in coordinating plant growth at the organismal level. While numerous types of RNAs  have been found to traffic in a non-cell-autonomous fashion within plants, the underlying regulatory mechanism remains unclear. Viroids are single-stranded circular noncoding RNAs, which entirely rely on their RNA motifs to exploit cellular machinery for organelle entry and exit, cell-to-cell movement through plasmodesmata, and systemic trafficking. Viroids represent an excellent model to dissect the role of RNA three-dimensional (3D) structural motifs in regulating RNA movement. Nearly two decades of studies have found multiple RNA 3D motifs responsible for viroid nuclear import as well as trafficking across diverse cellular boundaries in plants. These RNA 3D motifs function as “keys” to unlock cellular and subcellular barriers and guide RNA movement within a cell or between cells. Here, we summarize the key findings along this line of research with implications for future studies on RNA trafficking in plants. The vascular system, including xylem and phloem, mediates the systemic transportation of molecules. The xylem system is mainly responsible for the transportation of water and minerals, while the phloem system transports photosynthates and macromolecules. Various proteins, RNAs, as well as viruses and viroids can be found in the translocation stream of phloem  of a potexviral RNA mediates cell-to-cell movement of the fused GFP reporter RNA . A recent report showed that mle mRNAs . This firter RNA . In addifficking . The detvia phloem  as a model, specific RNA three-dimensional (3D) motifs responsible for crossing multiple cellular boundaries have been identified, providing solid genetic evidence that those RNA motifs guide specific trafficking in plants , 2021. IRNA molecules form various helices and loops in their secondary structures. Helices are composed of contiguous Watson-Crick (WC) base pairs . In contrast, loop regions are composed of diverse non-WC base pairs that are highly arranged . Many locis as the same orientation and trans as the opposite orientation) of glycosidic bonds for each base pair. Therefore, there are 12 base-pairing geometries considering the combinations of interacting edges and glycosidic bond orientations  that can participate in interaction with other base edges to form non-WC-WC base pairs in loop motifs . Adding ntations . Two bason atoms . Isosteron atoms . Effortson atoms . In addion atoms . TherefoPospiviroidae all enter the nucleus for replication. How RNA nuclear import is regulated remains as an interesting question. Most RNAs are made in the nucleus, and the prevailing view is that those RNAs either traffic to the cytoplasm or remain in the nucleus for function. In recent years, more and more RNAs, besides viroids, have been found to enter the nucleus (Viroids of the family  nucleus . Using P nucleus .cis-WC-sugar and trans-WC-Hoogsteen base-pairings); (C) two triads are formed through base-pairings from two strands; and (D) this motif is often found in hairpin stem-loop structure  during vegetative propagation through cuttings from PSTVdNT-infected tobacco plants  were present in epidermis, mesophyll, and bundle sheath cells but could not be loaded into phloem in inoculated leaves. It is unclear whether this regulation is unidirectional or bidirectional because transgenic expression of loop 7 mutants in companion cells all converted to wild-type sequences, rendering it difficult to assess the regulation of trafficking direction.An early attempt to screen for PSTVd loss-of-function trafficking mutants identified two nucleotides (U43 and C318) that did not affect replication but abolished systemic trafficking . Interescis-WC–WC base pair with a water molecule insertion  revealed that U43 and C318 form a nsertion . All thensertion . Indeed,nsertion .N. benthamiana plants. A total of 11 loop mutants were found to impair systemic trafficking  of PSTVd is critical for RNA moving from epidermis to palisade mesophyll . Mutagenfficking .Using the JAR3D program , the terin situ hybridization analysis was performed using loop 6 mutants to determine the role of loop 6 in regulating viroid movements, those mutants, if replicable, were trapped in palisade mesophyll cells without entering spongy mesophyll  pair, and A325-G36 trans-Hoogsteen-Sugar (tHS) pair  pair . The C32ressures . Loop 6 partners . Loop 19partners . This mo-pairing .via the IMPa-4 based nuclear import pathway  that have been identified in many endogenous mobile transcripts . However5C methylation as a regulatory mark for mRNA translocation crossing graft junctions is a significant advancement in understanding the trafficking of endogenous RNAs  and the US National Institutes of Health (R15GM135893). Funders had no role in the study design, in the collection, analysis, and interpretation of the data, in the writing of the report, and in the decision to submit the article for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Novel coronaviruses emerged as zoonotic outbreaks in humans in 2003 (SARS), 2012 (MERS), and notably in 2019 (SARS2), which resulted in the COVID-19 pandemic, causing worldwide health and economic disaster. Vaccines provide the best protection against disease but cannot be developed and engineered quickly enough to prevent emerging viruses, zoonotic outbreaks, and pandemics. Antivirals are the best first line of therapeutic defense against novel emerging viruses. Coronaviruses are plus sense, single stranded, RNA genome viruses that undergo frequent genetic mutation and recombination, allowing for the emergence of novel coronavirus strains and variants. The molecular life cycle of the coronavirus family offers many conserved activities to be exploited as targets for antivirals. Here, we review the molecular life cycle of coronaviruses and consider antiviral therapies, approved and under development, that target the conserved activities of coronaviruses. To identify additional targets to inhibit emerging coronaviruses, we carried out in silico sequence and structure analysis of coronavirus proteins isolated from bat and human hosts. We highlight conserved and accessible viral protein domains and residues as possible targets for the development of viral inhibitors. Devising multiple antiviral therapies that target conserved viral features to be used in combination is the best first line of therapeutic defense to prevent emerging viruses from developing into outbreaks and pandemics. Coronaviruses are ubiquitous in bats, and spillover events into humans occurred in 2003 with SARS, again in 2012 with MERS, and most recently in 2019 with SARS-CoV-2. The initial coronavirus outbreaks were warnings for the COVID-19 pandemic, which to date has claimed over 5.97 million lives worldwide and over 954,500 in the United States (as of 2 March 2022) [Yearly vaccination provides excellent protection against targeted influenza A and B circulating strains, but there are years where the circulating influenza viruses change too much, and the vaccine protection loses efficacy. As reported by the CDC, influenza vaccine efficacy over the past decade ranged from a high of 60% to a low of 19% . The mRNAs demonstrated by the decreased vaccine efficacy as new variants emerge, vaccines may not protect as well against emerging novel coronaviruses. While next generation vaccine platforms, including mRNA vaccines, will improve the response time against emerging viruses, vaccine production may still take months to develop, manufacture to scale, and administer to populations during an emerging outbreak. Further, while vaccines are safe and effective, vaccine hesitancy is a major threat to world health , and ensThe first therapeutic line of defense against emerging viruses is antiviral medications. Antivirals are essential to treat infected individuals, especially in the absence of a vaccine. Our current arsenal of antivirals did little to limit the destruction caused by SARS-CoV-2, emphasizing the need to develop robust antiviral therapies to employ during novel viral outbreaks. While the pandemic has enhanced research into coronavirus antivirals, much work remains to prepare for the next coronavirus outbreak. Antivirals that target the conserved activities or interactions of viral molecules are likely to provide efficacy against multiple variants and strains, especially when employed in combination. The identification of unique antiviral targets conserved among coronaviruses is the first step to prevent future pandemics caused by emerging coronaviruses. Here, we review the coronavirus molecular life cycle, consider approved and promising antiviral inhibitors, and identify novel conserved antiviral targets.To identify valuable protein targets within coronaviruses, we performed polymorphism analysis of essential viral proteins to define the most conserved residues across human and bat β-coronaviruses. Using the sequence variation analysis tool from the National Institute of Allergy and Infectious Disease-sponsored Virus Pathogen Database and Analysis Resource , ViPR, wUsing Mol* Viewer , we thenCoronaviruses are enveloped viruses, with one positive-sense, single-stranded RNA genome segment ranging from 27 to 32 kb. The genome is bound by viral nucleocapsid phosphoprotein (N), which is surrounded by the viral envelope, composed of viral envelope protein (E), membrane protein (M), and the spike glycoprotein (S) . The envThe interaction between the host receptor and the RBD of the spike allows host cell proteases to cleave the spike into the S1 and S2 subunits. Some coronavirus spike proteins contain an additional furin cleavage site to facilitate host cleavage and viral entry into host cells. The SARS-CoV-2 spike protein contains a furin cleavage site, unlike SARS-CoV, that is not required for infectivity, but contributes to SARS-CoV-2 increased transmission . Once S1The spike protein binds to the host cell surface receptor through the receptor binding domain (RBD). The RBD of the spike protein is variable among different coronavirus strains, which, expectedly, use different host receptors . Yet, thTM, a fusion inhibitor approved by the FDA for clinical use against HIV-1. Another class of antivirals, called anchor inhibitors, target the conserved FP domain of Gp41 and are under development [The development of pan-CoV fusion inhibitors is an active research area and important goal to stave off emerging coronavirus outbreaks . The S2 elopment . OverallTranslation occurs first as the coronavirus RNA genome is modified with a 5′ cap and 3′ poly A tail, similar to eukaryotic mRNA. Upon the release of the RNA genome, the viral RNA is immediately recognized by the host ribosome and translated into a large polyprotein, pp1a. Viral proteases are among the first to be translated within pp1a and serve to cleave the large polypeptide into functional proteins . During TM for the hepatitis C virus. There are numerous FDA-approved protease inhibitors to target HIV-1 protease activity. Unfortunately, these protease inhibitors did not demonstrate clinical efficacy against SARS-CoV-2 [Protease inhibitors are successful antiviral therapies for other viruses that rely on polyprotein cleavage as a mechanism of viral protein expression, such as OlysioRS-CoV-2 . HoweverRS-CoV-2 . OptimizRS-CoV-2 . Indeed,RS-CoV-2 . Proteas2 were omitted to highlight the two most exposed amino acids. We found both G23 and W213 highly conserved, with a polymorphic score of 0. G23 is somewhat accessible with an ASA of 53 Å2 for both the monomer  binds full-length genomic RNA and plays a role in the regulation of viral transcription and replication. The phosphorylation of the viral N protein by host glycogen synthase kinase-3 (GSK-3) is required for the production of longer subgenomic mRNA transcription and full-length RNA genome replication. Phosphorylated N recruits host RNA helicase DDX1 to facilitate readthrough for the synthesis of longer subgenomic mRNA and genomic RNA . As enou2 in the monomer , membrane (M), and spike (S) proteins are translated by ribosomes in the endoplasmic reticulum (ER) and travel to the ER Golgi intermediate compartment (ERGIC). When in the ER/ERGIC, virus proteins traffic to the Golgi apparatus and trans-Golgi network (TGN) for glycosylation and other post-translational modifications ,61. The 2  drugs from at least two different HIV drug classes, for example, targeting the protease, integrase, and replicase at the same time, have proved effective . SuccessThe molecular life cycle of coronaviruses present opportunities to develop novel inhibitors to block essential and conserved viral molecular activities and interactions. Conservation indicates importance. Conserved activities and interactions across the coronavirus family are high-priority targets to stop emerging coronaviruses, as conserved targets are less likely to tolerate change that leads to resistance. We examined several coronavirus proteins because it is imperative to discover multiple targets and employ combinatorial antiviral therapies to prevent emerging coronaviruses. Nsp5 and the viral replicase are targeted by the approved antiviral therapies Paxlovid and Molnupiravir, respectively. Antiviral resistance has yet to emerge, but studies examining the safety of combinatorial therapy in these two antivirals may demonstrate this approach to be more effective and less likely to result in the emergence of resistant viruses. Nsp13, encoding the viral helicase, is a conserved target being actively pursued . While aHere, we reviewed the molecular life cycle of coronaviruses, discussed the current state of antiviral therapies to target SARS-CoV-2, and identified additional conserved and accessible residues of essential viral proteins that should be further studied to serve as targets for the development of novel antivirals. Targeting multiple activities and interactions conserved among bat and human coronaviruses with antivirals will provide protection against even emerging coronaviruses, leading to better global outcomes during the inevitable next coronavirus emergence.


When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β- Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. RdRp is an important target for the development of antiviral drugs against coronaviruses10. Structures of RdRp have been reported for SARS-CoV-111 and SARS-CoV-216 and provide insights into the mechanisms of RNA-dependent RNA synthesis17. The structures also enable mechanistic studies that can rationalize the molecular processes underlying the antiviral activity of compounds targeting RdRp.Coronaviruses use an RNA-dependent RNA polymerase (RdRp) for the replication and transcription of their RNA genome20. The nucleoside analog remdesivir can circumvent proofreading because its incorporation does not terminate elongation but only stalls RdRp after the addition of three more nucleotides24. Remdesivir was the first FDA-approved drug for the treatment of patients with COVID-1928, but its effectiveness is disputed29, emphasizing the need to develop new antiviral drugs.Antiviral drugs often target viral polymerases and function as nucleoside analogs that terminate RNA chain elongation. However, such chain-terminating antivirals are generally not effective against SARS-CoV-2 because coronaviruses carry an exonucleolytic proofreading activity that can remove misincorporated nucleotides from the nascent RNA 3′ endd-N4-hydroxycytidine (NHC or EIDD-1931)31. Molnupiravir interferes with the replication of various viruses36, including SARS-CoV-238. It inhibits SARS-CoV-2 replication in human lung tissue39, blocks SARS-CoV-2 transmission in ferrets40 and reduces SARS-CoV-2 RNA in patients41. In contrast to approved drugs such as remdesivir that are administered by infusion, molnupiravir is orally available. Molnupiravir has been tested in phase I trials42 for safety, tolerability and pharmacokinetics, and phase II/III studies are currently ongoing41 . Available data suggest that molnupiravir acts as a mutagenizing agent that causes an ‘error catastrophe’ during viral replication43. Indeed, NHC can introduce mutations into viral RNA, as shown for Venezuelan equine encephalitis virus44. Also, the sequencing of influenza virus populations has indicated that NHC causes G-to-A and C-to-U transitions in viral RNA30, and the same transitions have been found for SARS-CoV-237.Another promising drug candidate for the treatment of patients with COVID-19 is molnupiravir (or EIDD-2801), which also targets the RdRp of SARS-CoV-2. Molnupiravir is an isopropylester prodrug of the nucleoside analog β-Despite this progress, a systematic biochemical and structural analysis of molnupiravir- or NHC-induced RNA mutagenesis by viral RNA polymerases is lacking. In this Article, we quantify the effects of molnupiravir or NHC on the RNA synthesis activity of SARS-CoV-2 RdRp using a purified biochemical system and defined synthetic RNAs. Together with structural analysis, we establish the molecular mechanism of molnupiravir-induced RNA mutagenesis. Our results provide detailed insights into the mechanism of action of molnupiravir, which is entirely distinct from that of remdesivir or chain-terminating nucleoside analogs.We first tested whether purified SARS-CoV-2 RdRp can use the active form of molnupiravir, NHC triphosphate (‘MTP’) Fig. , as a su45 that have been predicted to allow for base pairing with either G or A46.When nucleotides G or A were present at the RNA template position +1, NHC monophosphate (‘M’) was readily incorporated instead of C or U, respectively Fig. . Time-deWe next tested whether the incorporation of NHC monophosphate (M) into nascent RNA interferes with further RNA extension. We first conducted RNA elongation assays with a scaffold that allowed for RNA extension by four nucleotides (nt) Fig. . We obseO4-chlorophenyluridine  in Hi5 insect cells. Production of the bacmid, V0 and V1 viruses was carried out as described in ref. 15. Sixty hours after transfection with the corresponding V1 virus, the cells producing nsp12 with a 6xHis-MBP N-terminal tag were collected by centrifugation  and lysed by sonication in lysis buffer A1  glycerol, 30 mM imidazole pH 8.0, 5 mM β-mercaptoethanol, 0.284 μg ml−1 leupeptin, 1.37 μg ml−1 pepstatin, 0.17 mg ml−1 PMSF and 0.33 mg ml−1 benzamidine). The lysate was clarified by centrifugation at 74,766g for 60 min and ultracentrifugation at 100,000g at 4 °C for 60 min, followed by filtration through a 0.45-μm filter . The protein was bound to HisTrap HP prepacked columns  pre-equilibrated in lysis buffer, washed with high salt buffer B2  glycerol, 5 mM β-mercaptoethanol) followed by washing with buffer A1, and eluted with a gradient of 0–80% A1-B1 buffers over 30 column volumes (CV)  glycerol, 400 mM imidazole pH 8.0, 3 mM MgCl2 and 5 mM β-mercaptoethanol). Fractions containing nsp12 were pooled and the tag was cleaved with His-tagged tobacco etch virus (TEV) protease overnight during dialysis against buffer D  glycerol, 5 mM β-mercaptoethanol). The protein solution was applied to a HisTrap HP prepacked column to remove uncleaved protein, tag and TEV protease. The flow-through containing nsp12 was further purified by ion exchange chromatography using Hi TRAP Q HP and SP HP prepacked columns  equilibrated with buffer D. The unbound protein was concentrated and further purified via size exclusion chromatography on a HiLoad S200 16/60 column  in buffer A2  glycerol, 1 mM MgCl2, 1 mM Tris(2-carboxyethyl)phosphine (TCEP)). The peak contained monomeric fractions of nsp12 that were pooled, concentrated to 55 μM, aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C.Preparation of SARS-CoV-2 RdRp, composed of nsp12, nsp7 and two copies of the nsp8 subunits, was carried out as described in ref. 15. Briefly, both proteins were expressed in Escherichia coli BL21(DE3) RIL from the pET-derived vector 14-B  in LB medium individually. Cells were grown to an optical density at 600 nm of 0.4 at 30 °C and protein expression was induced with 0.5 mM IPTG at 18 °C for 16 h. After collection, cells were resuspended in lysis buffer A1  glycerol, 30 mM imidazole pH 8.0, 5 mM β-mercaptoethanol, 0.284 μg ml−1 leupeptin, 1.37 μg ml−1 pepstatin, 0.17 mg ml−1 PMSF and 0.33 mg ml−1 benzamidine). Nsp8 and nsp7 were purified separately using the same purification procedure. The cells were lysed using a French press and three cycles. Lysates were subsequently cleared by centrifugation . The supernatant was applied to a HisTrap HP column , pre-equilibrated in lysis buffer. The column was washed with high-salt buffer B2  glycerol, 5 mM β-mercaptoethanol) and with buffer A1  glycerol, 30 mM imidazole pH 8.0 and 5 mM β-mercaptoethanol). The protein sample was then eluted using a gradient of 0–80% B1 over 30 CV  glycerol, 400 mM imidazole pH 8.0, 3 mM MgCl2, 5 mM β-mercaptoethanol). The protein sample was dialysed in buffer D  glycerol, 5 mM β-mercaptoethanol) in the presence of His-tagged TEV protease at 4 °C. The dialysed sample was subsequently applied to a HisTrap HP column , pre-equilibrated in A1 buffer. The flow-through that contained the untagged protein of interest was applied to an ion exchange chromatography HiTrap Q column . The unbound protein sample, containing nsp8 or nsp7, was concentrated using a 10,000 molecular weight cutoff Amicon Ultra Centrifugal Filter (Merck) and applied to a HiLoad S200 16/600 system  equilibrated in buffer A2  glycerol, 1 mM TCEP). Peak fractions containing proteins were pooled, concentrated to 430 μM (nsp7) and 400 μM (nsp8), aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C.Nsp7 and nsp8 were prepared as described in ref. 23. All assays except those shown in Fig. RNA scaffolds were designed according to published SARS-CoV-2 RNA extension assays15, except for the following changes. The final concentrations of nsp12, nsp8, nsp7 and RNA were 3 μM, 9 μM, 9 μM and 3 μM, respectively, except that 1 μM of RNA was used in the assays in Figs. 2, 5 mM β-mercaptoethanol) for 10 min at 30 °C and the reactions were started by addition of NTPs. After 20 min of incubation at 30 °C, the reactions were stopped with 2× stop buffer . RNA products were resolved on 20% denaturing polyacrylamide-urea gels in 0.5× TBE running buffer and visualized with a Typhoon 95000 FLA Imager . Bands were quantified with Image Lab (Bio-Rad). The obtained data were plotted and analyzed with GraphPad Prism version 9.All unmodified and 5′ FAM-labeled RNA oligonucleotides (Supplementary Table O-DMT-2′-O-TOM-O4-chlorophenyluridine was prepared as described in ref. 55 and converted to the corresponding N4-hydroxy-N4-benzoylcytidine 3′-(2-cyanoethyl) diisopropyl phosphoramidite (M-PA) in three steps. Details of the synthetic procedures are provided in the following, and NMR spectra of the isolated compounds are provided in the O-TOM-protected ribonucleoside phosphoramidites (70 mM in CH3CN) and ethylthiotetrazol  as activator, with 4-min coupling time, as previously described55. The oligonucleotides were deprotected with 25% NH4OH/EtOH 3/1 at 55 °C for 6 h, followed by 1 M TBAF in THF for 12 h, and carefully purified by denaturing polyacrylamide gel electrophoresis to remove a minor fraction, in which NHC was converted to U during deprotection.5′-4; gradient: linear, 0–48% solvent B, 4% solvent B per 1 CV) and HR-ESI-MS .The purity and identity of the RNA oligonucleotides were analyzed by anion-exchange HPLC , 6 M urea; solvent B: 25 mM Tris-HCl (pH 8.0), 6 M urea, 0.5 M NaClO−1, for two heating and two cooling ramps between 10 and 90 °C. Melting curves were normalized to the absorbance at 95 °C, fitted to a two-state transition model with linearly sloping lower and upper baselines, and the melting temperatures were determined at the inflection point of the curves.Thermal melting experiments were performed in 10 mM sodium phosphate buffer pH 7.0, 100 mM NaCl, at an RNA duplex concentration of 20 µM. Absorbance versus temperature profiles were recorded at 260 nm on a Varian Cary 100 spectrometer equipped with a Peltier temperature controller, at a heating rate of 0.5 °C min2Cl2, CH3CN). For workup and purification, distilled solvents  were used. Column chromatography was performed on silica gel  with a particle size of 0.040–0.063. Thin-layer chromatography was performed on Alugram aluminium sheets . NMR spectra were recorded using Bruker Avance III (400 MHz) spectrometers. Chemical shifts (δ) are given in ppm, relative to the residual solvent signals as internal standards . Data are reported as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Coupling constants (J) are given in Hz. HR-ESI-MS results were recorded on a Bruker micrOTOF-Q III spectrometer. The detected mass-to-charge ratio (m/z) is given, as well as the calculated monoisotopic mass -N4-hydroxy-2′-O-(triisopropyl-silyloxymethylcytidine), 5′-O-DMT-2′-O-TOM-O4-chlorophenyluridine  was dissolved in anhydrous CH3CN (4 ml) under a nitrogen atmosphere. 4-Dimethylaminopyridine  and NEt3  were added, followed by hydroxyl amine hydrochloride . After stirring for 21 h at ambient temperature, the reaction mixture was diluted with CH2Cl2 and washed with saturated aqueous NaHCO3 (2×). The organic phase was dried over Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (n-hexane:EtOAc + 1% NEt3 1:2 to 2:1) to yield the product  as a colorless foam.To synthesize compound 2 -N4-hydroxy-2′-O-(tri-isopropylsilyloxy)methylcytidine), a solution of compound 1  in anhydrous CH2Cl2 (4 ml) was treated with DMAP  and NEt3  under a nitrogen atmosphere. Benzoic anhydride  was added in three portions within 3 h and the resulting reaction mixture was stirred for one more hour at ambient temperature. Volatiles were removed under reduced pressure. The crude residue was purified by column chromatography (n-hexane:EtOAc + 1% NEt3 1:2) to yield compound 2  as a colorless foam.To synthesize compound 3, -N4-hydroxy-2′-O-(tri-isopropylsilyloxy)methylcytidine 3′-cyanoethyl-N,N-diisopropyl phosphoramidite), compound 2  was dissolved in anhydrous CH2Cl2 (2 ml) and cooled to 4 °C. 2-Cyanoethyl N,N,N′,N′-tetraisopropyl phosphoramidite  and 4,5-dicyanoimidazol  were added in two portions within 1 h. After one additional hour at 4 °C, the reaction mixture was allowed to warm to ambient temperature, then stirred for one more hour. The solvent was evaporated and the crude residue was purified by column chromatography (n-hexane:EtOAc + 1% NEt3 2:1 to 1:1) to yield compound 3  as a colorless foam.To synthesize compound 2, 1 mM TCEP) at 4 °C. Peak fractions corresponding to RdRp–RNA complex were pooled and diluted to 1.5 mg ml−1. An additional 0.2 nmol of the annealed RNA scaffold were spiked into each sample before grid preparation. A 3 µl volume of the concentrated RdRp–RNA complex was mixed with 0.5 µl of octyl β-d-glucopyranoside  and applied to freshly glow-discharged R 2/1 holey carbon grids (Quantifoil). The grids were blotted for 7 s with blot force 5 using a Vitrobot Mark IV instrument (Thermo Fischer Scientific) at 4 °C and 95% humidity and plunge-frozen in liquid ethane.RNA scaffolds for structural studies were prepared by annealing two RNA oligonucleotides, because the length of the NHC-containing RNA was limited for technical reasons. The first RNA oligo was designed to form a template–product hybrid with a hairpin leaving a 10 nt product overhang at the 3′ end. A second oligo containing 8 nt complementary to the overhang was annealed to obtain an RNA template–product scaffold with a single nick at position −10 in the template RNA  operated at 300 keV. Inelastically scattered electrons were filtered out with a GIF Quantum energy filter (Gatan) using a slit width of 20 eV. Images were acquired using a K3 direct electron detector in counting mode (non-super resolution) at a nominal magnification of ×105,000, resulting in a calibrated pixel size of 0.834 Å px−1. Images were exposed for a total of 2.0 s with a dose rate of 20.5 e− px−1 s−1, resulting in a total dose of 59.6 e−/Å2 that was fractionated into 80 frames. Our previous cryo-EM analysis of SARS-CoV-2 RdRp–RNA complexes showed strong preferred particle orientation in ice15. To obtain more particle orientations, all data were collected with 30° stage tilt. Motion correction, CTF estimation and particle picking and extraction were performed using Warp57. A total of 11,060 and 10,230 videos were collected for M-A- and M-G-containing structures, respectively.Cryo-EM data were collected with SerialEM57 1.0.9. Particles were imported to cryoSPARC58 2.15 and subjected to two-dimensional (2D) classification. 2D classes representing contamination or broken particles were selected and used for calculating two ab initio structures. All particles were then used for supervised 3D classification against five references, where four were originating from the ab initio reconstruction of contamination and broken particles and one was a previous RdRp–RNA complex structure (EMD-1199523). The class containing RNA-bound RdRp yielded ~800,000 particles, which were subjected to homogeneous 3D refinement. The refined particles were then exported to RELION 3.159 and focus-refined in 3D with an initial local angular sampling of 3.7° and a mask around RdRp that omitted the nsp8 sliding poles and the upstream, second RNA turn. To improve the quality of the density in the active site, particles were 3D-classified without image alignment  using the same mask. The best class was focus-refined to an overall resolution of 3.3 Å. Local resolution was estimated with Relion 3.1 using a kernel size of 10 Å. For the RdRp–RNA complex containing the M-G base pair, 2.2 million particles were extracted using Warp57 1.0.9. Further processing was as for the first complex, except that the class containing RNA-bound RdRp yielded 850,000 particles and the refinement resulted in a reconstruction at an overall resolution of 3.2 Å.For the RdRp–RNA complex containing the M-A base pair, 2.5 million particles were extracted using Warp7B3D23) as starting model. The model was first rigid-body-fitted into the density and then manually adjusted in Coot60. The protruding second RNA turn and the nsp8 extensions were removed from the model due to discontinuous cryo-EM density for these regions, indicating mobility. Restraints for molnupiravir monophosphate (M) were generated in phenix.elbow61 and the structures were refined using phenix.real_space_refine62 with restraints imposed on secondary structure and the backbone phosphate geometry of M. Model quality was assessed using MolProbity within Phenix63, which revealed excellent stereochemistry for both structural models  in the template strand with either A or G at the 3′ end of the product strand. Stable M-A or M-G base pairs are formed and accommodated in the RdRp active center. The overview structure is shown as a ribbon model: gray, nsp12; blue, nsp7; green, nsp8; red, product RNA; blue, template RNA; M highlighted in yellow. The predicted metal ion in the active site of RdRp is indicated with a magenta sphere. Close-up views are depicted as stick models with the same color code. Predicted hydrogen bonds for M-A and M-G base pairs are shown as black dashed lines.Supplementary Video


Hevea brasiliensis Muell. Arg.) is a tropical tree species that produce natural rubber. Self-rooted juvenile clones (SRJCs) are novel rubber tree planting materials developed through primary somatic embryogenesis. SRJCs have a higher rubber yield compared with donor clones (DCs). The molecular basis underlying increased rubber yield in SRJCs remains largely unknown. Here, the latex from SRJCs and DCs were collected for strand-specific and small RNA-seq methods. A total of 196 differentially expressed long noncoding RNAs (DELs), and 11 differentially expressed microRNAs were identified in latex between SRJCs and DCs. Targeted genes of DELs were markedly enriched for various biological pathways related to plant hormone signal transduction, photosynthesis, glutathione metabolism, and amino acids biosynthesis. DELs probably acted as cis-acting regulation was calculated, and these DELs relevant to potentially regulate rubber biosynthesis, reactive oxygen species metabolism, and epigenetic modification. Furthermore, the DELs acting as microRNA targets were studied. The interaction of microRNA and DELs might involve in the regulation of natural rubber biosynthesis.The rubber tree ( Hevea brasiliensis Muell. Arg.; Natural rubber, an important industrial raw material, is widely used to produce tires and medical gloves . NaturalNovel rubber tree planting materials, called self-rooted juvenile clones (SRJCs), are developed through the somatic embryogenesis pathway , 2002. SLong noncoding RNAs (lncRNAs) belong to noncoding RNAs that are longer than 200 nucleotides (nt) in length. LncRNAs can regulate gene expression through sequence complementarity with RNAs or DNAs  and takeIn a rubber tree, natural rubber is synthesized on the surface of rubber particles suspended in the latex (the cytoplasm of laticifers). The laticifers are specialized vessels located adjacent to the phloem of the rubber tree . Rubber H. brasiliensis cultivar CATAS7-33-97 SRJCs and CATAS7-33-97 DCs were cultivated in the experimental field of Guangdong NongKen Tropical Crop Science Institute. Three SRJC groups (10 trees in each group) and three DC groups (10 trees in each group). Latex (10 ml from each plant) was collected from SRJCs or DCs. The latex was allowed to drop directly into liquid nitrogen in an ice kettle. After the latex drops in liquid nitrogen, small spherical particles are formed immediately. About 2–3 min, 10 ml of latex can be collected. Then the frozen latex was transferred to an RNAase-free tube and stored in liquid nitrogen. After all, samples were collected, they were returned to the laboratory and stored at −70°C or used immediately. Before RNA extraction, the samples in each group were mixed in equal quantities, and then RNA extraction buffer was added to start RNA extraction.Eight-year-old −1 Tris–HCl (pH 8.0), 100 mmol.L−1 EDTA, 1.4 mol.L−1 LiCl, 20% (v/v) sodium dodecyl sulfate and 5% (v/v) mercaptoethanol, and the suspension was incubated at room temperate for 20 min, and was centrifuged at 12,000 × g for 10 min at 4°C. The lower liquid was transferred to a fresh tube. Then an equal volume of phenol/chloroform/isoamyl alcohol  was added and mixed evenly. The mixture was centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was transferred to a fresh tube, an equal volume of phenol/chloroform/isoamyl alcohol  was added again and mixed evenly. The mixture was centrifuged at 112,000 × g for 15 min at 4°C. The supernatant was transferred to a fresh tube, and 1/3 vol of 8 mol.L−1 LiCl was added, and mixed well, and stored at 4°C overnight. The reaction mixture was centrifuged at 12,000 × g for 15 min at 4°C. The final pellet was washed with 70% (v/v) ethanol, air-dried, and dissolved in RNase-free water. Total RNA was monitored using agarose gel electrophoresis. The concentration and integrity of total latex RNA were measured using Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer  and the RNA Nano 6,000 Assay Kit of the Bioanalyzer 2,100 system .The latex total RNA was isolated by the described method  with somFor lncRNA sequencing, the ribosomal RNA was depleted from the latex total RNA according to the Epicenter Ribo-Zero Gold Kit . Then, six ssRNA-seq libraries (3 replicates in each sample) were constructed by the protocol of the Illumina TruSeq™ RNA sample prep Kit  and were sequenced on the Illumina Hiseq 2,500 platform . For sRNA sequencing, six sequencing libraries (3 replicates in each sample) were constructed following the protocol of the NEB Next Ultra-small RNA Sample Library Prep Kit for Illumina , and sequencing was carried out on the Illumina Hiseq 2,500 platform. Library construction and sequencing were carried out by Novogene Co., LTD .To generate clean data from ssRNA-seq, raw reads of FastQ format were firstly processed through in-house Perl scripts using the quality control software FastQC.2fold-change| > 1 and p < 0.05  between SRJCs and DCs was determined using the DESeq R package (1.8.3). The p-values were adjusted using the Benjamini and Hochberg method  and Rfam (Release 11.0) databases, and the reads annotated as small nucleolar RNA, small nuclear RNA, small cytoplasmic RNA, ribosomal RNA, and translocation RNA were removed. The remaining clean reads were mapped to the rubber tree reference genome  by usingg method . Correctcis- or trans regulation was implemented according to a previously described method  termsHbACT7 was used as the internal reference gene . qRT-PCR was performed using SYBR Premix Ex Taq Kit  on the Mx3005P Real-Time PCR System . The nce gene . The priT method .The systematic identification of lncRNAs in rubber trees was performed in latex from SRJCs and DCs using ssRNA-seq. In total, 642 million clean reads were obtained by all ssRNA-seq data . A totalIn total, 3,013 and 2,982 lncRNAs were identified in SRJCs and DCs, respectively. A total of 196 DELs, including 57 downregulated and 139 upregulated lncRNAs, were identified between SRJCs and DCs . Among DGO enrichment and KEGG pathway analyses were performed on the 1796 target genes of DELs. Under three GO categories, the term with the largest proportion in biological process was cellular nitrogen compound metabolic process . The anaA total of 122 lncRNA–mRNA pairs related to cis-acting regulation were obtained . Among tA total of 8 DELs and 4 genes were randomly selected to examine the expression by qRT-PCR. qPCR results showed the expression of selected DELs and genes were highly consistent with ssRNA-seq data .A total of 15,778,815 and 11,396,302 raw reads were obtained in SRJCs and DCs, respectively. After removing reads less than 18 nt, adapter sequences, and low-complexity sequences, averages of 3,354,892 and 3,867,160 unique reads were obtained . More thIn total, 136 miRNAs, including 108 novel and 28 known miRNAs, were found in DCs and SRJCs. About 41.96, 29.23, and 14.61% of these miRNAs had lengths of 21, 24, and 22 nt, respectively . Among 1The construction of the lncRNA-miRNA-mRNA network is helpful to understand the regulatory role of DELs in rubber trees. We constructed the lncRNA–miRNA–mRNA network based on the co-expression of mRNA and DELs, and DELs–miRNA interaction pairs. As shown in HbSRPP, encoding the SRPP participating in natural rubber biosynthesis , National Natural Science Foundation of China (32171827), and National Key Research and Development Program of China (2018YFD1000502).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.