A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection. Among four genera of coronaviruses, humans can be only infected with the members of alpha and beta coronaviruses. Many of these human coronaviruses such as HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, leading to common colds, are not regarded as severe pathogens [Real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most widely used laboratory method for RNA virus detection owing to its sensitivity and selectivity ,4. HowevTo overcome the limitation, nucleic acid-based on-site detection methods that combine isothermal nucleic acid amplification have been suggested recently ,9,10. OnIn order to enhance the system’s sensitivity, nucleic acid sequence-based amplification (NASBA) method as an isothermal amplification method has been coupled to the toehold reaction . HoweverIn this study, we introduced reverse transcription loop-mediated amplification (RT-LAMP) for RNA target to the toehold reaction as an alternative to NASBA. To obtain RNA as an amplification product after the LAMP DNA amplification, we strategically modified a LAMP primer to include a T7 promoter upstream of the amplicon sequence. This modified LAMP amplification strategy was coupled with the paper-based toehold reaction. To test the system’s usability and adaptability, we applied this method to detect RNA coronaviruses, MERS-CoV and SARS-CoV-2. Finally, we demonstrated that the modified RT-LAMP-coupled toehold reaction could detect MERS-CoV and SARS-CoV-2 with high sensitivity and specificity.Due to the flexibility to design a pair of toehold switch and its cognate trigger RNA, the toehold reaction has been widely used as an RNA recognition-based detection system for pathogen identification and microbiome profiling ,13,14. TThe toehold switches and trigger sequences were designed via NUPACK, a software that designs the sequence of interacting nucleic acids to have a designated secondary structure . The toelacZ gene encoding β-galactosidase that hydrolyzes chlorophenol red-D-galactopyranoside  into chlorophenol red . In this way, only when the target RNA sequence exists, the translation of the lacZ gene can be triggered. The resulting LacZ enzyme converts CPRG to CPR and the color change on a paper can be monitored through naked eyes  or without (off-state) trigger RNA. Toehold switch sensors that show high on/off fold-change, that is, high absorbance at the on-state but low absorbance at the off-state, were selected for further experiments. Among the sensors tested, sensor M6 for MERS-CoV and sensor S1 for SARS-CoV-2 were selected for downstream tests as they showed the low absorbance in the off-state b and theNext, we evaluated the sensitivity of sensors, M6 and S1. After testing a series of target RNA concentrations, we found that the toehold reaction was initiated with as little as 250 nM of target RNA for both sensor M6 and S1 d. HoweveMERS-CoV  and 7.99RS-CoV-2 .To enhance the detection limit, we introduced nucleic acid amplification technology. One of the isothermal amplification methods, NASBA, was commonly used to amplify RNA and the compatibility with the toehold reaction was already demonstrated . HoweverReverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid, one-step and straightforward isothermal amplification method . DespiteIn order to generate transcripts from the amplified DNA, we used T7 RNA polymerase and its promoter, which have been often used for such conversion from DNA to RNA ,12. We fThe RT-LAMP reaction consists of a starting-structure-producing step and a cycling amplification step a,b. The When the T7 promoter and T7 terminator are inserted at the 5′ end of outer primers (F3 and B3), the T7 sequences will be excluded from the dumbbell structure. When the T7 promoter and T7 terminator are inserted at the 3′ end of the outer primers, strand displacement cannot occur. Also, the addition of the T7 promoter and T7 terminator at each the 3′ or 5′ ends of the inner primers (FIP and BIP) will prevent self-primed elongation as illustrated in step 4 to 5 or step 6 to 7 when added at the 3′ or 5′ end of FIP, respectively and in step 1 to 2 or step 5 to 6 when added at the 3′ or 5′ end of BIP, respectively a,b.Another option for the T7 promoter and T7 terminator is between F1c and F2 in the FIP primer and B1c and B2 in the BIP primer, respectively. First, the T7 promoter sequence was added to the FIP primer with direct or complementary sequence c,d. T7 pLoop primers that hybridize to the stem-loop regions of RT-LAMP intermediate products are known to accelerate the amplification reaction further . We intrThe detection limit of the modified RT-LAMP-coupled toehold reaction was tested by using in vitro transcribed target RNA spiked in either water unless mentioned otherwise or saliva a.For sensor M6, when the Loop primer B was used, the detection limit was dramatically enhanced compared to the assay with no loop primers, reaching 1200 copies per reaction  b. When bFor sensor S1, when the RT-LAMP resultant using the Loop B and F primers were employed for the toehold reaction, 120 copies per reaction  induced the toehold reaction c, implyiTo determine this assay is compatible with possible inhibitors of human samples, synthetic RNA spiked into human saliva was used as proxy clinical samples. Nasopharyngeal swab (NP swab) is a standard collection sample for diagnosis of respiratory infection. However, the NP swab is relatively invasive . As an aIt is known that 10% of human saliva samples are acidic, which can affect the pH in LAMP reaction . The toeSaliva samples from healthy humans were spiked with MERS-CoV or SARS-CoV-2 RNA at a 1:5 ratio and they were mixed with the previous 2X optimized saliva stabilization solution at a 1:1 ratio . Then, tWe then investigated the feasibility of the modified RT-LAMP-coupled toehold reaction as a rapid diagnostic test. To reduce the overall turnaround time, we first cut down the RT-LAMP reaction time from 60 min to 20 min. Using the RT-LAMP amplicons, we monitored the absorbance change of the toehold reactions a. We obsSince the coronaviruses share substantial sequence similarities, we tested the MERS-CoV or SARS-CoV-2 sensors for possible cross-reactivity against other coronaviruses, including SARS-CoV, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and HCoV-229E. To do so, we first aligned each viral sequence homologous to the target region of MERS-CoV  or SARS-RNA toehold switch sensors are robust molecular switches that can be used to detect RNA molecules in a sequence-specific manner. This flexible switch had been combined with a paper-based cell-free molecular diagnostic platform to detect various target nucleic acids ,13,14. HInterestingly, the addition of a T7 terminator, which is typically known to improve the termination efficiency, was not helpful in this case. Possible explanation is due to the failure of RT-LAMP. When we analyzed the RT-LAMP product, a typical ladder-like amplicon pattern observed in the agarose gel was not seen with the T7 terminator-inserted case, indicating the failure of RT-LAMP . The RT-The modified RT-LAMP-coupled toehold reaction developed in this study achieved high sensitivity. Notably, for both MERS-CoV and SARS-CoV-2 target RNA, we detected as few as 120 copies per reaction , the highest sensitivity reported using a toehold switch sensor. When NASBA was used as an amplification step for the toehold reaction, the detection limit was 3 fM of Zika viral RNA . The senMoreover, the modified RT-LAMP that we demonstrated in this study can amplify target RNA in a relatively short period of time. Usually, LAMP is a fast isothermal amplification method that requires 20–60 min . NASBA rIn summary, we developed the modified RT-LAMP that can amplify target RNA and convert the resulting amplicon into RNA molecules. This new amplification method connected to the toehold reaction based on a paper-based cell-free expression system, which enabled colorimetric detection of MERS-CoV and SARS-CoV-2. This modified RT-LAMP-coupled toehold reaction showed high sensitivity (120 copies per reaction), high selectivity against 7 other coronaviruses and short turnaround time (70 min). We believe the modified RT-LAMP toehold reaction developed in this study would be useful to detect not only coronaviruses but also any organisms that contains RNA in a fast, sensitive, selective and colorimetric manner.orf1ab and upE regions, which are commonly used targets for conventional RT-PCR probes [orf1ab and N gene regions for S1 and S2, respectively. Unwanted in-frame stop codons at the N terminus of the lacZ gene were discarded. DNA oligonucleotides  containing toehold switch sequences were assembled into a vector that contains the lacZ reporter gene  using Gibson assembly .The genome sequences of MERS-CoV and SARS-CoV-2 were obtained from GenBank (Accession number: JX869059 for MERS-CoV and NC_045512 for SARS-CoV-2). For MERS-CoV, we first divided the genome sequence into 30 sections and ran NUPACK to choose target sequences and toehold switches . The toeWe followed the previously described paper preparation protocol . BrieflyCell-free reaction contains components necessary for in vitro transcription and translation . Cell-frTemplate DNAs that include target RNA regions with 100 bp-extra bases at each end and T7 promoter sequence were synthesized (Integrated DNA Technologies). The template DNAs were in vitro transcribed into RNA. For the in vitro transcription, 200~1000 ng of target DNA, 2 μL 10X reaction buffer, 1 μL DTT , 0.8 μL NTPs , 0.5 μL RNase Inhibitor , 2 μL T7 RNA polymerase  and RNase-free water up-to 20 μL were mixed. The mixture was incubated at 37 °C for 16 h, then treated with 1 μL of DNase I  at 37 °C for 30 min. The resulting RNA was purified with the Riboclear™(plus!) RNA kit  and used as target RNA. The target RNA was quantified  and diluted in nuclease-free water as needed.lacZ in the cell-free reaction. Meanwhile, sensors for SARS-CoV-2 were directly reacted with transcripts of the RT-LAMP amplicon yielded from 200 aM (sensor S1) or 1 fM (sensor S2) of initial target RNA in the cell-free reaction. To obtain the fold change, we first subtracted background absorbance that does not contain sensor DNA and trigger RNA from each absorbance value of the cell-free reaction with or without trigger RNA. At this stage, any negative value was adjusted to zero. Then, the fold change was calculated as the adjusted absorbance of the sensor with trigger RNA divided by that without the trigger RNA.For the initial screening of toehold switch sensors, M1 to M10, for MERS-CoV, a high concentration of trigger RNA (2500 nM) that was obtained via in vitro transcription was directly used to induce the toehold-regulated expression of https://primerexplorer.jp/e/, accessed on 20 April 2020). The parameter of F1-B1c distance was modified from 0–100 to 36–100 to locate a target region of the toehold switch sensor between F1 and B1c. For the RT-LAMP reaction, WarmStart LAMP Kit 2X Master Mix , which contains a strand displacement DNA polymerase and reverse transcriptase in an optimized buffer, 10X Primer Mix , target RNA (40%) were assembled at room temperature according to the manufacturer’s recommendation. The reaction mix was incubated at 65 °C for 60 min in a heat block, then heat-inactivated at 80 °C for 5 min. The reaction products were directly used in the subsequent cell-free reaction.The RT-LAMP primers were designed using Primer Explorer v.5 software , EDTA , NaOH , Proteinase K  and Nuclease free water. The final concentration of 2X stabilization solution was 5 mM for TCEP-HCl, 2 mM for EDTA, 29 mM for NaOH and 100 μg/mL for Proteinase K. 6 μL of stabilization solution was added to 1 μL Saliva  mixed with 5 μL synthetic RNA in nuclease-free water. It was heated at 95 °C for 10-min and 10 μL of it was put into LAMP reaction.We investigated the cross-reactivity of the toehold sensors against other closely related coronaviruses. Each homologous region of the other coronaviruses to the target region for sensor M6 or S1 was first synthesized and a T7 promoter was included in the upstream of each homologous region . The syn


Adolescent health has become a priority on the global health agenda. Parent-adolescent communication regarding sexual and reproductive health (SRH) issues can help to reduce adolescent risk-taking sexual behaviours.This study was to describe the situation of SRH communication, and to determine the factors associated with SRH communication between high school students and their parents in Vientiane Capital prefecture, Lao PDR.A multistage sampling technique was applied. A self-administered questionnaire was implemented among a sample of 384 high school students aged 14–17 in Vientiane. SRH communication in this study was recorded as the frequency with which adolescents discussed with their parents at least four topics on SRH issues during a six-month period prior to the interview. Data were entered and analysed using Epi Data software version 6.0 and STATA software version 14.2.Slightly more than one-fifth of the students (21.3%) communicated with parents on SRH issues. The multivariate logistic regression model showed that being a male adolescent , urban school locations , a mature father , positive attitudes towards general communication with parents  and accessibility to multiple SRH information sources  were significantly associated with adolescent-parent communication on SRH issues.This study showed that student-parent communication on SRH issues was low, so policymakers should develop programs to improve SRH communication skills in all schools and encourage open discussion among family members, especially with respect to the participation of adolescent girls. The positive attitudes of students and multiple sources of SRH information were also important factors in improving SRH communication. Adolescent health has become a priority on the global agenda with an added focus on addressing adolescent health in low- and middle-income countries if the Sustainable Development Goals (SDGs) are to be reached . AdolescParent-adolescent sexual communication can positively affect safer sexual behaviour among adolescents, including utilisation of modern contraceptive methods. Through discussion of adolescent sexual activity, parents can influence on adolescents’ sexual attitudes, values and beliefs that help reduce sexually risky behaviour ,12. For SRH communication may protect adolescent children from SRH issues, but there is little research on what helps adolescents initiate the conversation about SRH matters with their parents . The sigThis study employed a cross-sectional design with a multi-stage sampling technique. First, it selected one school from 14 public high schools in four urban districts  and one school from five public high schools in one rural district (Sangthong) by random sampling due to limited time and budget, with one urban-based High School and one rural-based High School selected. Next, proportional sampling was used to select the number of classes in each grade. Finally, all students in the selected classes were invited to participate in the study.The population size was 950 adolescent students in grades 9 to 12 at one High School in urban and the one High School in rural area. The sample size was 384 which was determined using the single population proportion formula when considering the following assumptions p = 0.5 (it was hypothesised that the percentage frequency of outcome in the population was 50% for the estimated proportion of students communicating on SRH issues with parents due to unknown the proportion of the proportion of SRH communication between adolescents and parents in Lao PDR); d = 0.05 (a confidence level of 95% and a margin of error of 5%); Z (∝/2) = 1.96 (a significance level of 5% (α = 0.05)). To this was added a 10% non-response rate and design effect of 1.3.The independent variables included four sections: (1) socio-demographic factors , (2) the attitudes of students, (3) influence person  and (4) SRH information sources .The questionnaire about attitudes towards general and SRH communication was validated according to the Parent–Adolescent Communication Scale ,23 and tThe attitudes towards general communication with parents constituted 15 questions. The scale of reliability coefficient for the attitudes towards general communication with parents was 0.816. A summated composite score was calculated with a minimum score of 15 and a maximum of 60. The actual scores were classified base on Bloom’s criteria  and modiThe dependent variable for the questionnaire was measured by the frequency communication between adolescents and parents on SRH issues by incorporating the eight questions related to SRH communication with the father and eight questions related to SRH communication with the mother. This was assessed using a Likert Scale of eight items/topics with responses ranging from 0) never = never per six months, 1) rarely = 1 time per six months, 2) sometimes = 2 times per six months to 3) often more than 2 times per six months which was derived from the weighted Topics Measure of Family Sexual Communication Scale ,26. The The data collection team included eight people who were trained on the objectives of the study, the content of the questionnaire and how to conduct the study in a high school setting. After that, the team visited each selected school and identified eligible students. Two research assistants were allocated per class and explained the purpose of the research to the students and the content of each section of the questionnaire. The students completed the self-administered questionnaire, and the research assistants checked the completeness of all answers. If there were missing answers, the research assistants returned the questionnaire to complete the missing questions. After checking all questionnaires, the students were allowed to leave the classrooms.The data were entered and cleaned using Epi Data software version 6.0. The data were analysed using STATA version 14.2. Descriptive statistics was used to describe the numbers and percentages of the dependent and independent variables. Then, the bivariate analysis was performed between the independent variables and the dependent variable. The variables in the bivariate analysis found to be significant at p < 0.05 were entered into the multivariable logistic regression model. In the multivariate analysis, standard data analysis techniques were applied. Variables having p < 0.05 in the multivariate analysis were taken as significant predictors. Crude and adjusted odds ratios with their 95% confidence intervals were calculated and presented in texts and tables.In total, 384 students aged 14–17 in grades 9 to 12 were enrolled into the study. About 63% of the respondents were from an urban district and slightly higher than half were females, in addition the mean age of respondents was 15.7. The reported mean age of their fathers was 47.2 and 40.6% of their fathers had graduated from the tertiary education level. Their fathers were mostly employed as government staff or private businesses. In addition, the mean age of their mothers was 42.6 and 41.9% of mothers had graduated from high school or vocational college. About 27.1% of mothers were housewives .Table 1.About 21.3% of students in this study had discussed at least four topics in eight topics for SRH issues with their parents during the six months prior to the survey. Of these students, male adolescents discussed the topics more frequently with their parents than female adolescents (29.2% versus 16.1%). Moreover, male participants also discussed SRH issues with their father and their mothers more often than female participants. These results highlight that boys talk more than girls to both their parents about SRH issues. However, more adolescent boys and girls talk with their mothers than with their fathers .Table 2.Outside of the family, adolescents wanted to discuss with health staff on SRH issues comprised about 59.4%, followed by with peers (47.9%) and with their teachers (32.8%). The main reason that adolescents wanted to discuss SRH issues with health staff was that health staff had more knowledge of sexual and reproductive health than them, but the highest percentage of the reason for why adolescents chose someone for discussion was that they were always a good listener for them in SRH issues.About 70.8% of both male and female students had positive attitudes towards and accepted the importance of maintaining discussion about general matters with their parents and more than half of adolescents agreed that their parents were good listeners. Moreover, 94.1% of students had positive attitudes towards and accepted the importance of discussing SRH issues with their parents, meaning that almost all adolescents were concerned about SRH problems and wanted to consult their parents. However, the frequency of SRH communication between parents-adolescents was low .Table 3.The main sources of SRH information for adolescents were health facilities because they received good information from them. In addition, the internet was cited as the second most important source of SRH information due to the fact that the internet was easy to access for SRH information .Table 4.The multivariate logistic regression model showed that being a male adolescent , urban location , having a mature father , positive attitudes towards general communication with parents  and accessibility to many sources of SRH information  were significantly associated with adolescent-parent communication on SRH issues (p < 0.05) .Table 5.Parents are important role models in adolescents’ lives. They can directly or indirectly transmit values, traditions and lifestyles to their children. Positive family communication helps adolescents develop the values, security, and sense of worth that can lead to healthy decision-making including around sexual health . Issues In this study, less than 1 in 4 adolescents discussed SRH issues regularly with their parents, which indicates that SRH issues may be difficult for parents and adolescents to discuss. The reasons for this in this study are not clear but are likely to include embarrassment, lack of confidence or socio-cultural norms that make sexual activity a taboo topic ,29. ThisThis study indicated that both male and female adolescents were more comfortable discussing SRH issues with mothers than with fathers. This might be mothers were also perceived to be better at listening than fathers. Fathers may find it harder to discuss potentially embarrassing relational topics and have an open conversation about sexual health with their adolescent children and may need further support in initiating such conversations .Unlike a study in southern Ethiopia , the preAdolescents with older fathers were more likely to discuss SRH issues as found in a previous study in Unguja-Zanzibar, Tanzania . This maAdolescents who have positive attitudes about communication with their parents were more likely to discuss SRH issues than those who had negative attitudes towards such communication. More than half the respondents identified that it was important to discuss SRH issues with their parents, particularly if they are good listeners. Thus, developing good listening skills is likely to motivate sons and daughters to talk with their parents ,35, and The study also showed that adolescents who received information about SRH from multiple sources were more likely to discuss these issues with their parents. Similarly, a study in northwest Ethiopia mentioned that students who had obtained SRH information were more likely to communicate about SRH issues with their parents than those who did not have SRH information. This could be explained by the fact that the respondents have some awareness and might be more eager to communicate on SRH issues and the information they received might prepare them to begin that communication ,27. AdolAs with all studies, this study has limitations. Firstly, the sample selected only one rural and one urban school so it may not represent Lao PDR population overall. Secondly, adolescents in this study were limited to middle adolescents, aged 14 to 17. The other issue was that the responses from adolescents might be different from what their parents might really have perceived. Thirdly, our measure of sexual communication focused on several sexual risk and protective behaviours but did not assess many other topics that theorists and scholars have described as being part of sexual health such as intimacy and sexual pleasure. In addition, recall bias might have occurred as the SRH communication might or might not have happened: remembering their experiences of SRH communication with their parents during the previous six months may be inaccurate, so the adolescents’ responses might be over reported or under reported. Fourthly, the study did not capture the parents’ perspectives; however, anecdotal evidence suggested that adolescents’ perspectives are valid in parent-adolescent sexual communication.Parent-adolescent sexual and reproductive health communication was very limited and associated with sex of adolescent students, school location, attitudes of adolescents, younger age of fathers and information sources. Our findings emphasised the need to improve SRH communication for adolescents and parents in Lao PDR. Therefore, sex education should improve SRH communication skills in all schools, especially in student populations that have a high percentage of younger fathers. In addition, the programmes should involve multiple sectors like health, education and youth services to provide many sources of information to change the negative attitudes of students towards SRH communication with parents to positive ones. Moreover, parents should focus on both male and female students equally in discussing SRH issues. In particular, parents should discuss SRH issues with girls because they will bear the burden of unplanned pregnancies. Further studies should be carried out from the parents’ perspective to identify factors that affect the discussion of SRH issues between students and parents in other provinces of Laos. Qualitative research should be more in-depth in relation to the SRH topics discussed between parents and adolescents and the barriers to SRH communication between parents and adolescents.


The development of RNA self-assemblies offers a powerful platform for a wide range of biomedical applications. The fabrication process has become more elaborate in order to achieve functional structures with maximized potential. As a facile means to control the structure, here, we report a new approach to manipulate the polymerization rate and subsequent self-assembly process through regulation of the reaction viscosity. As the RNA polymerization rate has a dependence on solution viscosity, the resulting assembly, crystallization, and overall sizes of the product could be manipulated. The simple and precise control of RNA polymerization and self-assembly by reaction viscosity will provide a way to widen the utility of RNA-based materials. RNA nanotechnology has advanced extensively for its potential biomedical applications by leveraging a wide range of biological functions of RNA. Additionally, the versatility of RNA for bottom-up self-assembly to achieve the defined size and structures gives a further advantage to fully exploit the therapeutic potential of RNA ,2,3,4. I4−) as side products. Meanwhile, the Mg2+ cation in the reaction added as a co-factor for the enzyme forms a complex with pyrophosphate, resulting in magnesium pyrophosphate  [2PPi, is a biocompatible compound and a naturally occurring biological molecule [2PPi precipitate out from the solution and form crystalline structures with the adsorption of produced RNA and finally assemble into a unique sponge-like structure [2PPi crystallization and RNA hybridization. RCT is a sophisticated process that generates RNA strands and pyrophosphate (PPiMg2P2O7) . The inomolecule . Mg2PPi tructure ,12,13. SIn a previous study, RNAi microsponges were fabricated by RCT to deliver siRNA . HoweverIn this context, we controlled the enzymatic RNA polymerization by regulating viscosity to manipulate external reaction conditions and studied how viscosity affects the RNA microstructures fabrication by altering the enzyme activities and molecular interactions between reaction components . FurtherAll oligonucleotides were purchased from Integrated DNA Technologies . T7 RNA polymerase, RNA polymerase reaction buffer , and ribonucleotide triphosphates (rNTPs) were purchased from New England BioLabs . Eighty percent glycerol solution was purchased from Intron Biotechnology . Sodium pyrophosphate and magnesium chloride solutions were purchased from Sigma Aldrich .1 is the viscosity value introduced into software, R1 is the measured value of particle size, η2 is the actual solution viscosity, R2 is the known radius of the beads. The measured particle sizes were introduced into the equation, and the viscosity of the solution was calculated.The viscosity of the solution with different glycerol contents was calculated by a modified equation derived from the Stokes-Einstein equation  . The 100g for 10 min. To synthesize larger particles, the initial circular DNA concentration was decreased from 10 μM to 3 μM, and the final rNTP concentration was increased from 1 mM to 2 mM. Sense and anti-sense circular DNAs were synthesized from linear and primer DNA, as previously reported  Table 1Table 1. v/v)). Then, the mixture was incubated for 30 min at room temperature.Sodium pyrophosphate was solubilized at a concentration of 20 mM into nuclease-free water. Magnesium chloride was diluted to 16 mM with nuclease-free water. Next, sodium pyrophosphate and magnesium chloride solution were simply mixed by 1:1 volume ratio, and 80% glycerol solution was added at the desired concentration  was added to the final concentration of 10 mM to destroy the magnesium pyrophosphate crystalline structure. For the agarose gel electrophoresis analysis, each sample was loaded with 4X Gel loading dye  and 1X GelRed  and loaded to 1% agarose gel in Tris-acetate-EDTA buffer  and carried out at 100 V for 60 min and analyzed with GelDoc EZ imager . For the polyacrylamide gel electrophoresis (PAGE) analysis, 10% polyacrylamide gel was prepared and carried out at 100 V for 60 min in 1X tris-borate-EDTA buffer . Then, the gels were stained with 1X GelRed in 1X tris-borated-EDTA buffer for 20 min and analyzed as mentioned above. To obtain RNA yields from the gel image, the signal intensity for each lane was analyzed and compared to that of the single-strand RNA ladder . The PAGE analysis was performed three times, and the RNA yields were averaged.Scanning electron microscopy (SEM) images were captured using SNE-3000M , operating at an accelerating voltage of 15 kV. Samples were prepared by dropping a diluted particle solution into a silicon wafer. The number averages of RNA microstructures fabricated at each viscosity was measured by DLS measurement, operated at 25 °C, measurement angle at 90°, three times with 20 runs. For the manipulation of solution viscosity, we chose glycerol as an additive. Glycerol is a viscous liquid due to the presence of three hydroxyl groups in the molecular structure, which is usually added in the enzymatic reaction medium to confer long-term stability of the enzyme. As the glycerol contents in the reaction increase, the viscosity of the reaction medium increases A. In addNext, we investigated the effect of manipulating the solution viscosity in the RNA polymerization reaction B–D. To aTo evaluate the effect of an altered reaction rate on the final products, the size of the RNA microstructures was assessed. As glycerol contents increase from 5% to 50%, the size was reduced gradually from 782.5 (±7.85) to 301.6 (±15.29) nm E,F. For 2PPi by viscosity is a factor that affects the RNA microstructure fabrication. Of note, the discrepancy between the exact sizes of RNA microstructures and crystal structures of Mg2PPi here comes from the continuous and gradual feeding of the RNA and pyrophosphates in the enzymatic fabrication.To further assess the dependence of inorganic crystallization on reaction viscosity, the magnesium pyrophosphate crystals were synthesized without RNA. The tendency to decrease in size was consistent as glycerol contents increased . As the −3 to 1.5 × 10−3 at different viscosities, variously sized pools of RNA microstructures were achieved (In addition to the previous study, which reported that the increased rNTP concentration is known to reduce the size of RNA particles , the effachieved A,B. At aachieved C. Of notachieved D. Of notThus far, the enzymatic reaction was controlled rationally here by adjusting a single factor that does not participate in the reaction. At a suboptimal viscosity achieved by 5 to 30% glycerol contents in enzymatic reaction, the increased viscosity resulted in an increased reaction rate with reduced sizes of the final products. In comparison, higher viscosities led to impeded enzyme processing owing to the limited molecular movements. Notably, the rationale was consistent with changes in the active components of the reaction. Furthermore, the various size pools with different RNA contents were also achieved. Although it is not provided in a manuscript, the total costs of 300 nm RNA structure fabrication were reduced by about 25 folds relative to regulation through enzyme concentration.In this study, the size of the RNA structure was precisely regulated by a single non-reactive factor, viscosity. The increased viscosity at 30% by the addition of glycerol affected the reaction rate, reducing the sizes of hybrids. On the other hand, the controllability of RNA microstructure fabrication became minimal at higher viscosities owing to the interference in polymerase activity. Furthermore, manipulating reaction components along with viscosity achieves variously sized pools of RNA microstructures, ranging from 300 nm to 1.3 μm. Finally, the required size of the structures is different depending on the biomedical application, and therefore, we suggest new approaches to manipulate the size of RNA microstructures to satisfy diverse biomedical demands precisely and economically. In a follow-up study, the biological response and therapeutic efficiencies of the synthesized RNA microstructures with various sizes should be tested. Moreover, the limitations of noncationic, nucleic acid-based microstructures should be overcome through functionalization of the surface with various engineering tools.


Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1–7 complex initiates mRNA decay, while the nuclear Lsm2–8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2–8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1–7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1–7 loads onto RNA from the 3′ end and removal of the Lsm1 carboxy-terminal region allows Lsm1–7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism. The Sm-like archaeal proteins (SmAPs) are homologous to the eukaryotic Sm proteins, but their biological roles are less well understood  RNAs (The Lsm1–7 complex is cytoplasmic and mediates messenger-RNA (mRNA) decay, a major posttranscriptional mechanism for regulating gene expression . Binding complex , and the of Pat1 . However of Pat1 , protect of Pat1 , stabili of Pat1 , suppres of Pat1 , and pro of Pat1 . Human aoU) RNAs . In contoU) RNAs . The molatac snRNAs . For Lsm2–8, we demonstrated that the complex can bind to U6 snRNA and further associate with protein Prp24 to form the complete U6 snRNP, a known interaction previously observed for the S. cerevisiae U6 snRNP .U6 snRNP . We wishKd = 26 and 100 nM, respectively)  B. This fSupplemental Fig. 2a). We confirmed that the RNA remains completely intact after prolonged incubation with Lsm2–8, further supporting the idea that the last uridine is covalently attached but disordered in the structure .We crystallized the Lsm2–8 complex bound to UUUUU-3′-OH and UUUUU > p and determined their structures by X-ray diffraction to resolutions of 1.9 and 2.3 Å, respectively . In the Supplemental Fig. 2c,d). The cyclic phosphate causes the RNA chain to make a sharp turn of nearly 180°, which positions the terminal nucleobase to stack over the last histidine at the carboxyl terminus of Lsm8. The terminal uracil base adopts an unusual syn conformation and forms a hydrogen bond to the Lsm3–Asp83 side chain  , which is comparable to the binding observed for longer oligoU tracts . The oligo ACCCAUUUUU binds less tightly than U15, indicating that the length of the oligoU tract plays a role in binding affinity.Since  of Pat1 , we firs of Pat1 C. Owing (Δ56C–7) D. Lsm1Δ5d to A15 E. Upon oΔ56C–7 construct has higher affinity for RNAs than Lsm1–7 . For example, the oligonucleotide UUUUUACCCCC binds poorly to Lsm1–7 (Kd = 189 nM) and tightly to Lsm1Δ56C–7 (Kd = 34 nM) (Δ56C–7) complex can bind tightly to the UUUUA sequence irrespective of its position in the RNA. Thus, the Lsm1 carboxy-terminal extension functions to prevent high affinity binding to RNA, except for 3′ terminal sites. We further tested the effect of 3′-end nucleotide identity on binding and found that tetrauridylate followed by a single adenosine binds more tightly than an RNA with a pentauridylate tract  E. These te tract E.Interestingly, we observe that the carboxy-terminal 12 residues of Lsm1 strongly discriminates against cyclic phosphate RNAs. Comparison of a monoadenylated RNA and a cyclic phosphate RNA shows an approximate 14-fold reduction in binding affinity for the cyclic phosphate RNA, which is substantially attenuated by deleting only a small region from the carboxyl terminus of Lsm1 D,F. We cΔ56C–7 variant of the Lsm1–7 complex bound to the U-tract RNAs UUUUUA and AUUUUG and determined their structures to resolutions of 1.8 and 2.1 Å, respectively . The next four uridines occupy pockets in Lsm4, Lsm1, Lsm2, and Lsm3, respectively. The terminal adenosine reaches across Lsm6 to form a hydrogen bond to Asn66 of Lsm5 .We were unable to obtain crystals of wild-type Lsm1–7 in complex with RNA. Since deletion of the carboxyl terminus of Lsm1 generally enhances binding affinity E, we theectively ; Fig. 4. of Lsm5 . In placΔ56C–7 bound to AUUUUG, all nucleotides are visible within the electron density map. The path of the RNA around the interior of the Lsm1–7 torus is a right-handed helix, with the phosphodiester backbone in the center and the nucleobases splayed out into binding pockets. The six nucleotides of the RNA make an almost complete turn with the last nucleotides 6 Å below the first  . We also tested the binding specificity of the double mutant by comparing its ability to bind an RNA terminating with a cyclic phosphate versus the terminal adenosine. While Lsm1–7 can discriminate between these RNAs with a 14-fold difference in affinity, the double mutant shows only a sevenfold difference, corresponding to a twofold loss in binding specificity .Since the terminal purines are both within hydrogen bonding distance of Lsm5 Asn66 and are in van der Waals contact with Lsm5 Asn68 E,F, we tΔ56C–7 can only bind tightly to the RNA with a 5′ stem–loop and single stranded 3′ end  (Kd > 1 µM). This loss in binding affinity cannot be attributed to the 5′ cytidines, which do not affect binding to single stranded RNA , which may facilitate initial contact with RNA. Furthermore, comparison to the human Sm ring bound to U4 snRNA .In the crystal structures, the 5′ end of the RNA hexamer is nearest the “proximal face” of Lsm1–7 while the 3′ end is nearest the “distal face” A, the sactively) C. When tnded RNA E. With hU4 snRNA  shows hoS. pombe Lsm2–8 complex is highly similar to human and other organisms , so we expect the structural and functional data presented here to be broadly general in eukaryotic biology.Phylogenetic analyses suggest that the Lsm/Sm family of proteins arose approximately 2.5 billion years ago through two waves of gene duplication, the first wave resulting in the Lsm genes from which the Sm proteins subsequently diversified . As Lsm syn conformation of the terminal uracil base. Quantum chemical calculations of 2′,3′ cyclic UMP indicate that the cyclic phosphate stabilizes the syn conformation of uridine to be significantly more favored over the anti conformation . It is therefore clear that the carboxyl terminus of Lsm8 and nearby residues are important for specifically recognizing posttranscriptionally modified U6 snRNA , which has a stretch of 3–6 uridines ending with a 3′-OH. Consistent with this, Lsm2–8 still binds to oligoU RNA ending in 3′-OH, with a Kd of 100 nM. We find that Lsm2–8 also binds tightly to 3′ monoadenylated RNA . Interestingly, we note that 3′ monoadenylated U6 is present in human cells and is the preferred substrate for Usb1 . This may be to avoid certain RNAs in the cytoplasm; for example, tRNA splicing proceeds through a cyclic phosphate intermediate  bind well to Lsm1Δ56C–7 but not Lsm1–7. This is likely due to steric clash involving the Lsm1 carboxy-terminal region .Lsm1–7 preferentially loads onto single-stranded RNA from the 3′ end. The trajectory of the RNA in Lsm1l region B. The Lsl region . AccordiS. pombe Lsm1–7  for n system . In the n system  to yieldS. pombe Lsm2–8 expression plasmid was transformed into E. coli BL21(DE3) STAR pLysS cells (Invitrogen # C602003). Cells were grown in terrific broth (RPI # T15100-5000.0) containing ampicillin with shaking at 37°C until the OD600 was approximately 2, at which point isopropyl β-d-1-thiogalactopyranoside was added to a final concentration of 1 mM and the cells were allowed to continue growing with shaking at 37°C for another 24 h, at which point the OD600 was approximately 16. Cells were harvested by centrifugation and the resulting cell pellet was resuspended in 0.025 volumes of buffer A  per original volume of cell culture . Protease inhibitors (EMD Millipore # 539137-10VL) were added, as were DNase I (Sigma # DN25-100MG) and lysozyme (Sigma # 62971-10G-F) at a final concentration of 0.02 and 0.5 mg/mL, respectively. The resuspended cells were subjected to a single freeze-thaw cycle prior to sonication and clearance of cell debris by centrifugation.The resulting −1 cm−1 at 280 nm  using gravity flow at room temperature. Buffer A was used for washing and buffer A supplemented with 500 mM imidazole pH ∼ 7 was used for step elution. The pooled eluate was then dialyzed overnight at 4°C with 20 kDa MWCO membranes (Pierce # 66012) into buffer B . Centrifugation was used to remove any precipitation that formed during dialysis. Soluble protein was further purified via glutathione agarose chromatography (GenScript # L00206) with gravity flow at room temperature in fresh buffer B. Step elution was performed in buffer B supplemented with 50 mM HEPES acid, 50 mM sodium HEPES, and 10 mM reduced glutathione. One milligram of TEV protease was added to the eluate, which was then dialyzed overnight as above, but at room temperature against 1 L of buffer C . Dialyzed protein was then purified using gravity flow chromatography at room temperature on Strep-Tactin agarose (IBA GmBH # 2-1208-025) in fresh buffer C, with step elution in buffer C supplemented with 2.5 mM biotin. The resulting eluate was manually applied at room temperature to a 1 mL HiTrapQ anion exchange column  that had been preequilibrated in buffer B. The column was then attached to an AKTA chromatography system at 4°C and elution accomplished by applying a linear gradient of NaCl up to 2 M in buffer B. The Lsm2–8 complex desorbed from the column at ∼250 mM NaCl. Peak fractions were collected, pooled, and the concentration of the purified complex was determined by UV absorbance and the estimated molar extinction coefficient of 46,300 Mt 280 nm . Final pThe Lsm1–7 complex was produced as described for Lsm2–8, with the following exceptions. The multi-ORF expression system was assembled into a single plasmid through ligation independent cloning as described for Lsm2–8, with the ORFs assembled in order Lsm6, Lsm3, Lsm2, Lsm1, Lsm4, Lsm7, and Lsm5. Lsm7 lacked a purification tag.Protein expression, purification and storage were performed as with the above Lsm2–8 complex, with the exception that Strep-Tactin chromatography was not used and instead the eluate from glutathione agarose chromatography was loaded directly onto a HiTrapQ column after overnight incubation with TEV protease at room temperature. Peak fractions were collected, pooled, and the concentration of the purified complex was determined by UV absorbance and the estimated molar extinction coefficient at 280 nm . ElectroIn order to prepare the Lsm5–N66A/N68A or Lsm1 carboxy-terminal truncation mutants of Lsm1–7, a modified inverse PCR mutagenesis protocol was used on the multi-ORF Lsm1–7 expression plasmid. PCR amplification was performed in “GC” optimized Pfusion buffer (NEB # M0532S) using a gradient of annealing temperatures and oligonucleotides that anneal only within the unique open reading frame regions of each Lsm locus. Amplicons of correct length were gel purified prior to phosphorylation and ligation. All mutant clones were confirmed to be of correct length and sequence by analytical restriction enzyme digestion and Sanger sequencing.S. pombe Prp24 was cloned into a modified variant of plasmid pET3a, encoding an octahistidine tag, biotin acceptor peptide sequence (A codon optimized open reading frame (Genscript) for sequence , and tobS. pombe Prp24 was produced as described above for the Lsm proteins with the following exceptions. Cells were harvested by centrifugation and the resulting cell pellet was resuspended in 0.1 volumes of buffer A  per original volume of cell culture .The −1 cm−1 at 280 nm  that had been preequilibrated in buffer B. The column was then attached to an AKTA chromatography system at 4°C and elution accomplished by applying a linear gradient of NaCl up to 2 M in buffer B. The peak fractions were collected, pooled, diluted twofold against fresh buffer B and then manually applied at room temperature to a 5 mL HiTrap Heparin cation exchange column  that had been preequilibrated in buffer B. The column was then attached to an AKTA chromatography system at 4°C and elution accomplished by applying a linear gradient of NaCl up to 2 M in buffer B. The peak fractions were pooled and the final concentration of protein was determined by UV absorbance and the estimated molar extinction coefficient of 114,600 Mt 280 nm . ProteinS. pombe U6 nucleotides 1–100  Triton X-100. Synthesis of RNA was performed at 37°C for 2 h. Trisodium EDTA pH 8.0 was then added to a final concentration of 100 mM to halt transcription and solubilize accumulated magnesium pyrophosphate. The transcription was then concentrated to ∼400 µL with 10 kDa MWCO spin filters (Amicon # UFC901008) prior to addition of 6 mL of 100% formamide and subsequent electrophoresis on a denaturing 10% (19:1) polyacrylamide gel containing containing 8 M urea, 100 mM tris base, 100 mM boric acid, and 1 mM EDTA acid. Full-length U6 RNA was identified by UV shadowing, extracted by scalpel, and removed from the gel matrix by passive diffusion overnight at room temperature with gently shaking into a solution containing 300 mM sodium acetate, 50 mM HCl, 1 mM EDTA, 1 mM sodium azide, pH ∼ 5.6. Soluble RNA was separated from solid acrylamide by filtration through 0.22 micron filters and then manually applied to a 5 mL HiTrap Q column  that had been equilibrated in buffer D . Bound RNA was washed with 20 mL of buffer D and then eluted in a single step with buffer D adjusted to 2 M NaCl. Eluted RNA was pooled and concentration and buffer exchange were accomplished by three iterations of 10-fold concentration (Amicon # UFC501008) and redilution against buffer E . The final RNA concentration was estimated using UV absorption and an anticipated extinction coefficient of 1,223,990 M−1 cm−1 at 260 nm  and treated with human Usb1, which rapidly removes 3′ adenosine residues from oligoU tracts and leaves a 3′ cyclic phosphate that is not subjected to ring opening as with S. cerevisiae Usb1  were purchased from Integrated DNA Technologies and purified by urea PAGE and ion exchange as above for full-length U6, with the following exceptions: UV shadowing was not used (or required) to visualize the RNA after denaturing electrophoresis, concentration of RNA after anion-exchange chromatography was performed with 3 kDa MWCO spin filters (Amicon # UFC500308), and the final RNA concentration was estimated using UV absorption and an anticipated extinction coefficient of 75,000 M−1 cm−1 at 495 nm  in black 96 well microplates (Greiner Bio-One # 655209) and imaged on a Tecan Infinite M1000Pro using an excitation wavelength of 470 nm and emission wavelength of 519 nm. For each sample, 100 µL of RNA at 2 nM was added to 100 µL of protein at a defined concentration between 0.4 nM and 5 µM. Fluorescence polarization was measured in duplicate from two independent titrations using different protein concentrations. Binding curves were fit using nonlinear regression in GraphPad Prism 4 to the following four parameter equation: FP = FPmin + (FPmax − FPmin)/(1 + 10^((logKd − log[protein])*H)), where FPmin and FPmax are the minimum and maximum polarizations, Kd is the binding dissociation constant, and H is the Hill coefficient. H was constrained to be 1 during nonlinear regression. Depicted binding curves are normalized to FPmin and FPmax. All raw binding data are provided in Supplemental Data File 1.All fluorescence polarization binding assays were performed in buffer H  using 20 kDa MWCO membranes (Pierce # 66012). The dialyzed complexes were concentrated to ∼10 mg/mL with 50 kDa MWCO spin filters (Amicon # UFC505008) prior to high-throughput crystallization screening on a Mosquito crystallization robot (TTP Labtech). Initial crystallization hits were obtained exclusively using the precipitant pentaerythritol propoxylate (5/4 PO/OH) in a MIDAS screen (Molecular Dimensions # MD1-59). Crystals were optimized by hanging drop vapor diffusion at 16°C, using 2 µL of Lsm2–8/RNA complexes mixed with 2 µL of crystallization reagent containing 100–200 mM KCl, 50–100 mM HEPES pH 7.4, 35% pentaerythritol propoxylate (5/4 PO/OH). Crystals were vitrified by direct immersion into liquid nitrogen.The Lsm2–8/RNA complexes were reconstituted by adding crude RNA from IDT to protein in an approximately twofold stoichiometric excess. The complexes were then dialyzed overnight at 4°C against 1 L of buffer I  (S. pombe Lsm4) (S. pombe Lsm5/6/7)  (S. cerevisiae Lsm rings (Phenix.refine (Phenix.refine.For the 3′ diol terminated structure (PDB 6PPN), three diffraction data sets were collected from two isomorphous crystals at 100 K on beamline 24-ID-E at the Advanced Photon Source. Molecular replacement was used to obtain initial phases with initial search templates PDB 4EMG (be Lsm3) , PDB 4EMbe Lsm4) , PDB 4EMsm5/6/7) , and homsm5/6/7)  of S. poctively) . Moleculsm rings . Structux.refine  using sex.refine  and addiS. pombe Lsm2–8 structure for molecular replacement. Twelve diffraction data sets were collected from two isomorphous crystals at 100 K on beamline 21-ID-D at the Advanced Photon Source. Structure refinement was performed in Phenix.refine  was determined as above, but using addition of a different RNA prior to dialysis and using the above x.refine  using seSchizosaccharomyces pombe Lsm1–7 complexes lacking the carboxy-terminal 56 residues of Lsm1 were reconstituted with RNA as above, adding 5′-UUUUUA-3′ for PDB 6PPQ or 5′-AUUUUG-3′ for PDB 6PPV. Crystals were obtained by mixing 0.2 µL of complex with 0.2 µL of the following mixture: 20 mM sodium formate, 20 mM ammonium acetate, 20 mM trisodium citrate, 20 mM sodium potassium tartrate, 20 mM sodium oxamate, 100 mM sodium HEPES base, 100 mM MOPS acid, 10% PEG 8000, and 20% ethylene glycol. Crystals were vitrified by direct immersion into liquid nitrogen.Phenix.refine (The 3′ adenosine terminated structure (PDB 6PPQ) was determined by merging three diffraction data sets collected from a single crystal at 100 K on beamline 21-ID-D at the Advanced Photon Source. PDB 6PPN was used for molecular replacement. Structure refinement was performed in x.refine  using seThe 3′ guanosine terminated structure (PDB 6PPV) was determined from a single diffraction data set collected from a single crystal at 100 K on beamline 21-ID-D at the Advanced Photon Source. PDB 6PPQ was used for molecular replacement and refinement was conducted as above. The 3′ guanosine binding pocket did not exhibit residual Fo–Fc density as above for adenosine.http://www.pymol.org). Structural biology applications used in this project were compiled and configured by SBGrid (Supplemental Data File 2. A PyMol session with annotation matching that used throughout the manuscript is provided as Supplemental Data File 3.For all structures presented here, simulated annealing omit maps were prepared to confirm the presence of bound RNA in the final models deposited into the Protein Data Bank. All figures were generated with PyMOL (y SBGrid . Electroy SBGrid  as impleSupplemental material is available for this article.


Drawing on the reflections and discussions from a special session at the 2021 Global Health Bioethics Network summer school, this paper has summarised the key challenges faced by Frontline Workers (FWs) across research sites in Africa and Asia in performing the everyday ‘body work’ entailed in operationalising global health research. Using a ‘body work’ lens, we specifically explore and map key challenges that FWs face in Africa and Asia and the physical, social, ethical, emotional, and political labour involved in operationalising global health in these settings. The research encounter links with wider social and economic structures, and spatial dimensions and impacts on the FWs' performance and well-being. Yet, FWs' ‘body-work’ and the embedded emotions during the research encounter remain hidden and undervalued. This reStudies show that the research context shapes the research encounter, and that FWs often seek to appease participants’ demands and their own conscience to make Global Health research viable . Recogni1.2In this paper, we draw on the reflections and field experiences of FWs, community engagement practitioners, bioethicists, and health researchers based at the five Wellcome Trust Africa and Asia Programmes (AAP) sites, as well as participants across the sites, and from other organisations see . All sitTo promote and support ethical reflection, and to build capacity across the AAPs to address ethical issues in research, the Global Health Bioethics Network (GHBN) was established in 2011 as a collaborative partnership between the five AAPs, and the Ethox Centre at the University of Oxford. This paper is based on a special session at the annual GHBN Summer School of 2021, organised by the authors and focusing on challenges and interventions to support fieldworkers in the AAPs. Fifty-five GHBN members joined the virtual session chaired by BN, MLS, PCC, DN and JV, with the aim of mapping challenges faced by FWs across sites and co-producing solutions. All members were briefed about the aims of the session and at the start of the session, permission was obtained from attendees to record the plenary session and use detailed written notes to write a publication.1.3The session began with a presentation by BN and a guest presenter, PK, to situate the topic within the wider literature and to offer practical reflections. GHBN members were then split into five breakout rooms based on their geographical region in Asia and Africa to discuss key challenges experienced by FWs, existing interventions, and proposed solutions to address these. Breakout room discussions were summarised in a plenary session, offering opportunities for further joint deliberation. Based on detailed notes taken during breakout and plenary discussions, we collated, consolidated, and categorised the cross-site discussions into six main themes. In addition, we reflected upon these themes considering the contributions they make in furthering our understanding of the ‘body work’ lens over a series of subsequent virtual meetings.22.1Below, we highlight six interconnected themes identified from the GHBN special session relating to the challenges faced by FWs in conducting Global Health research. During the session, those who worked as FWs relayed experiences that they and/or their colleagues had faced during fieldwork, while the PIs and those in managerial roles, explained experiences that had been shared to them by FWs during de-brief sessions at the respective sites. We conceptualise these experiences as the challenges resulting from the ‘body work’ conducted by FWs during research encounters with the participants. Although each theme is grouped into a main category, challenges were found to be complex, and to intersect and reinforce one another. 2.2Performance of ‘body work’ threatens integrity of FWs’ own bodies, due to the associated risks to their physical safety. GHBN members across sites highlighted some of the physical risks commonly facing FWs in the daily performance of their ‘body work’. While many communities appreciated the work of FWs, FWs tended to bear the brunt of the blame from communities when their expectations were not met. Some FWs reported being at risk or subjected to verbal, and physical abuse by communities. For instance, in South Africa, there were reports of FWs being chased away from participants’ homes, while in high crime locations, female FWs felt particularly at risk of or were threatened with sexual assault. Attacks by stray dogs, exposing FWs to risk of rabies disease were also reported risks of being in ‘the field’.In the context of COVID-19, it was reported that visiting participants’ homes in cases where FWs lacked appropriate PPE exposed FWs to risk of infection across all sites. Additionally, some communities were wary of FWs bringing COVID-19 into their homes during research visits, which could potentially expose FWs to further physical harm. The physical risks and labour undertaken during fieldwork often overlapped with emotional labour, which will be discussed in more detail later.2.3The research encounter involved significant relational and emotional labour required from FWs in negotiating access to participants’ bodies-presence of mind and body to carry out the research. Members further discussed that building and maintaining relationships with research communities often brought multiple challenges to FWs. Being accepted into a community was said to take hours of relational labour to gain trust to implement data collection and maintain trust throughout the study and beyond. Relationship building may not always be immediately successful or long-lasting.FWs sometimes faced resistance for a variety of reasons, including the disjuncture between communities’ expectations of the research, and the remit of research institutions. For example, in South Africa, participants perceived research as a service to meet their basic needs including housing, food, water and electricity, which were not provided by the government. In some instances, communities’ misconceptions of study reimbursement as payment created conflicts when ‘payments’ were perceived to be insufficient, and FWs were thus blamed for ‘stealing’ money. This not only resulted in broken trust that had been built over extended time, but also took an emotional toll on FWs.Further challenges were emphasised for FWs doing ‘body work’ within their own communities. FWs felt they had to work particularly hard to fulfil expectations to bring resources back to their communities, even if this was beyond the scope of their role. In other instances, study methods were perceived to place participants in uncomfortable situations, for example when FWs were asked to visit a participant’s home for a research interview leaving participants feeling embarrassed by their impoverished living conditions. In one site, a FW spoke about a study with home visit interviews and while many participants were okay with researchers coming to their homes, the FW felt that a few participants were not comfortable when the FW arrived at their home. This in turn created significant emotional discomfort among FWs who felt obligated to do more after spending time listening to the participants who had spent an hour or over detailing their struggles.COVID-19 was said to create additional burdens for FWs trying to build new research relationships with communities, due to restrictions around visiting communities to start conversations and set up collaborations. In Indonesia, this process took several weeks using remote channels, whereas before COVID-19, it would have taken considerably less time.2.42.4.1The complexity of conducting a truly informed consent process was a recurring theme across sites. Balancing institutional obligations, like reaching recruitment targets and ensuring that participants make informed choices about study participation created pressure and anxieties for FWs across the sites. Discussions among members suggested that FWs often rush through the informed consent forms and/or enrol participants even when they lacked comprehension about the study. In some instances, FWs’ own lack of understanding about clinical concepts undermined informed consent as they were unable to provide accurate information. This was further complicated by time pressure, and where scientific terms and ethical concepts needed to be translated, especially when equivalent expressions did not exist in the local language, thereby affecting the meaning and the concept of consent.Impacted by social and health disparities, participants often viewed the study resources as a means for improving their lives, thereby undermining their choice to decline participation. Participants’ socio-economic status and power relationships were also seen to shape the informed consent process in important ways. For example, participants consented to performance of ‘work’ on their own bodies as a way to ensure or gain access to services and resources. For example, in South Africa, study incentives in the form of food vouchers, and phone credits were said to take precedence over informed consent. In hospitals-based studies, participants who valued hierarchical relationship structures were perceived to ‘blindly’ trust healthcare workers, making the informed consent process peripheral.Challenges relating to maintaining participants’ confidentiality and autonomy were also highlighted. Narratives suggested that culture rather than ethical guidelines carried greater weight during fieldwork, evidenced by the importance participants placed on consulting with the immediate family about their research participation, and expecting family members to disclose information about their participation. The location of where ‘body work’ is carried out influenced FWs’ performance and well-being. When conducted in the space of a home or community, rather than a designated research institution or healthcare setting, the performance of ‘body work’ often become more complex, … as FWs negotiated and managed the participant but also other family and community members. In South Africa, enrolment of adolescents was perceived to be especially challenging for FWs, as some caregivers or parents expected FWs to advise young people about risky behaviours . Additionally, a lack of privacy at participants’ homes, such as when family or community members wished to join the interview, commonly compromised confidentiality, with FWs often struggling to ask them to leave. Failing to ensure participants’ confidentiality and autonomy weighed heavily on FWs, particularly when the research explored sensitive topics .2.4.2Aside from the ethical issues arising around procedural aspects of research, FWs also frequently faced ethical challenges and moral dilemmas in the everyday practice of Global Health research, which were closely linked to FWs’ physical and social labour described earlier. For example, many FWs found themselves emotionally distressed when coming face-to-face with participants’ unmet basic needs (e.g. for food), or when asking sensitive questions as part of a study , knowing that the study did not provide direct support to address participants’ hardships.The heavy socio-emotional and physical labour required from FWs in trying to live up to the high expectations placed on them by research communities – who often held them in high regard and expecting them to solve their problems – was said to leave FWs feeling exhausted and ‘burned out’. Furthermore, emotional, and ethical tensions also arose for FWs whose own views, values and beliefs conflicted with the requirements of a particular study protocol. For example, drawing blood samples when a fieldworker believed this to be unethical, or promoting COVID-19 vaccination when a fieldworker was themselves vaccine hesitant among others. The moral distress caused by ethical dilemmas during fieldwork was said to be heightened among FWs who had close social and emotional ties with their research communities.2.5In the context of research in the Global South, ‘body work’ involves providing psychological and emotional support for the study participants, for which most FWs have not receive adequate training. While the Standard Operating Guidelines for referrals exist, there is widespread ambiguity and lack of institutional guidance about the limits of FWs’ roles and responsibilities. This ambivalence compounded many of the ethical and emotional challenges they faced. As described above, in contexts of high poverty, this lack of clarity often leads to community expectations for FWs to meet basic needs, as well as moral distress among FWs faced with deprived communities and competing community interests. The lack of support for FWs facing such recurrent structural challenges (e.g. FWs interviewing participants who are hungry) was seen to be a key area requiring further institutional support.2.6Underpinned by the institutional and political contexts in which they operated, FWs experienced important structural challenges, ranging from increasing workloads, inadequate institutional support, and seasons of isolation in the discharge of their work. FWs were commonly reported to work on multiple studies for different PIs, with high demands to meet specific recruitment targets. Moreover, due to their lower hierarchical positions, FWs often felt hesitant to inform senior researchers of their workload and time constraints for fear of reprisals. Participants noted that the senior researchers are distant from the ‘bodies’ on which their work is focused. This was seen as an expression of power relations. Consequently, FWs frequently lagged in achieving their recruitment targets across different studies. FWs experience the tensions between ‘body time’ and the demands of ‘clock time’ attached to the performance of their work; the dependence of FWs’ ‘body work’ labour process on the unique and specific emotional needs of each research participants are in direct tension with the rationed and streamlined demands of ‘clock time’, which FWs find themselves bound by  and lessons learnt during their work. This was further compounded by the institutional requirements and good research practice to maintain participants’ confidentiality in the process of data collection. These prevailing institutional challenges were said to further exacerbate the precariousness of FWs’ work environments by compounding the social, emotional, and ethical challenges they faced.3The ‘body work’ nexus provides an opportunity for examining how the social and economic contexts, and power relations impact the FWs’ performance during the research encounter. FWs experience enact hidden burdens and various forms of labour while working on the ‘frontline’ of Global Health research . These ‘Thirdly, the challenges related to FWs’ role in safeguarding the procedural aspects of ethical research  as well as the challenges related to high workloads highlight the important temporal dimensions of FWs’ ‘body work’ in Global South contexts. For example, FWs experienced tensions between ‘body time’ of their participants and the demands of ‘clock time’ in their everyday work, such as when ascertaining informed consent, or when needing to attend to the unique and specific emotional needs of their participants. Such labour appears in direct conflict with the rationed and streamlined demands of ‘clock time’, which FWs find themselves bound by under the demands of ambitious research timelines and recruitment targets. Similarly, others have argued that ‘the dependence of the body work labour process on bodily needs makes it difficult to rationalise or speed up’ .Additionally, the reflections emphasise the importance of the spatial dimension of conducting ‘body work’ in Global South contexts, in that they highlight the significance of the location where such work is carried out. For example, when conducted in a home or community setting, rather than a designated research institution or healthcare setting, the performance of ‘body work’ often becomes more complex, as negotiating and managing the research process not only involves the FW and participant, but also the wider family and/or community members. Finally, the spatial separation and physical distance between FWs conducting ‘body work’ on the frontlines and PIs and research institutions who solicit such research but are distant from the ‘bodies’ on which their work is focused on, can be seen to be expression and representation of the underlying hierarchies and power relations underpinning the research encounter itself .Our reflections expand on previous work by illustrating how FWs are largely left unsupported while navigating everyday ethical challenges in their work . As we pWhile the discussions focused mainly on mapping challenges experienced by FWs, there was consensus that potential solutions must be multifaceted to address the structural problems underlying the challenges faced by FWs in operationalising global health. Furthermore, members emphasised the importance of strengthening the institutional systems already in place, for example by adapting the debrief sessions to address and support the embodied experiences facing FWs instead of mainly focusing on the research process. In Kenya, an institutionalised model ‘ethics reflections’, has been developed to support and build FWs’ capacities to respond to the ethical dilemmas .Finally, despite the increased attention and consideration being paid to the challenges faced by FWs across the AAPs research sites, many issues still surfaced in our members’ discussions. As illustrated, these issues are complex and require multi-faceted approaches and solutions to strengthen and support the work of FWs.4We summarised the key challenges faced by Frontline Workers (FWs) across research sites in Africa and Asia in performing the everyday ‘body work’ entailed in operationalising global health research. Our findings show that FWs’ ‘body work’ and the embedded emotions during the research encounter remain hidden and undervalued. More research is therefore needed to explore solutions to the challenges facing FWs operating in Global South contexts.


Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses. Picornaviruses are important pathogens but their assembly is incompletely understood, preventing development of anti-viral drugs and vaccines. We report identification of multiple RNA stem–loops, with purine trinucleotide sequence motifs in each loop, distributed across the genomes of two enteroviruses, enterovirus-E and poliovirus. Each motif appears to have affinity for its cognate capsid protein. Cryo-EM reconstruction of enterovirus-E without imposed symmetry identifies many repeated CP-RNA contacts. These include stem-loops adjacent to the particle two-fold axes, which appear conserved between viruses, as are the amino acid side-chains that contact all the genomic RNA segments. Mutagenesis shows that these amino acids are essential for virion formation, consistent with an RNA-dependent assembly mechanism. These results suggest that all enteroviruses utilise a common assembly mechanism that requires defined CP-RNA contacts. Picornaviridae family cause major disease in a wide range of vertebrate species and include >30 genera -Met/Cys (Perkin Elmer) was added. For EC optimisation, GuHCl  was added to media at 3, 3.5, or 4 hpi.2 hours post-infection (hpi) the media was replaced with Met/Cys-free media (Gibco). At 2.5 hpi 10 μCi/mL [35S]-Met/Cys/ GuHCl-containing media was replaced, the cells washed with PBS and the media exchanged for serum-free DMEM.EV-E was propagated and radiolabelled as described above. GuHCl (2 mM) was added at 4 hpi to inhibit RNA synthesis and trigger EC production. At the chase time-points of 1h, 1h 15 min, 1h 30 min and 1h 45 min following GuHCl addition  the [260 and A280. For EC experiments fractions were also scintillation counted. Sucrose was removed by dialysis into PBS.EC purification was performed below 10°C to maintain native capsid antigenicity. Post-infection, lysate was freeze-thawed, transferred to a Falcon tube and 0.5%(v/v) Nonidet-P40 (NP40) added. Cellular debris was collected by low-speed centrifugation  and the supernatant transferred to a fresh tube. The pellet was resuspended in 0.5 mL ice-cold phosphate buffered saline, pH 7.2 (PBS) containing 0.5%(v/v) NP40, and freeze-thawed again. The solution was re-clarified by low-speed centrifugation and added to the initial supernatant. Viral particles and ECs were pelleted through a 30%(w/v) sucrose cushion at 154,000g for 5 h. The pellet was resuspended in PBS/ 0.5%(v/v) NP40 on a rocker overnight at 4°C. Virus and ECs were separated on a 15–45%(w/v) linear sucrose gradient at 354,000g for 1 h. The gradient was fractionated measuring the absorbances at ACarbon-coated copper grids  were glow-discharged before application of 5 μL samples which were blotted after 30 s. Grids were stained twice with 2%(w/v) uranyl acetate and blotted dry, before viewing in a Jeol JEM-1400 TEM. Images were acquired at 40,000x magnification on a Gatan US1000 CCD camera.SELEX selection was carried out largely as previously described .2, 1%(v/v) NP40, pH 8.5) at 4°C for 30 min. Dissociation under these conditions was checked by nsTEM of equivalent non-immobilised samples (not shown). The beads were partitioned via magnetic separation (2 min) and washed three times in PBS to remove unbound protein. The beads were resuspended in 300 μL PBS. The naïve RNA selection library was transcribed from an N40 DNA library (TriLink Biotechnologies) (Purified EV-E ECs in PBS (~180 μg) were biotinylated at a 1:20 molar ratio of capsid lysines: Biotin (NHS-LC-LC-biotin). The modification was confirmed by western blotting before the ECs were immobilised on streptavidin-coated magnetic beads by rolling at 4°C overnight. Biotinylated ECs were dissociated to pentamer  in dologies)  using Hi2O. Bound RNA was eluted from the beads by heating to 95°C for 5 min in a PCR thermocycler, followed by magnetic removal of the beads. The released RNA was reverse transcribed and amplified using a OneTaq RT-PCR kit. The enriched library was transcribed to RNA with a T7 transcription kit, the DNA template removed with TURBO DNase and purified with RNAClean XP resin (Agencourt). The concentration of RNA was determined after each round and 1 μM RNA was added to the next round of SELEX.The purified N40 RNA library (50 μL at 10 μM),  was addeCP binding stringency was increased across the ten rounds by: decreasing the positive bead incubation time over the first five rounds from 25 to 5 min; and increasing the number of washes from 10 to 15 over rounds 6–10. For rounds 5 and 10, a competition was carried out to remove RNA sequences that had a higher affinity for the EC than for the inner pentamer surface. The SELEX round was performed as above but after the washes, the positive beads were incubated with either 0.1 mg/mL of biotinylated capsid (round 5) or EC (round 10). A further three washes were carried out to remove the capsids and any capsid-bound RNA aptamers. Elution, etc, was then performed as above. The amplified DNA of round 10 was then subjected to Next-Generation sequencing using Illumina MiSeq.Polio pentamer SELEX followed a similar protocol to the EV-E SELEX, with differences in protocol described below. The N30 library  was usedE.coli supercompetent cells (Stratagene). 10 individual clones were randomly selected via blue/white screening, and then Sanger sequenced.The 10th round RNA pool was reverse-transcribed, PCR-amplified and ligated to the pGEM-T vector followed by transformation to B = L − log4x where The Bernoulli score of a given aptamer was calculated by sliding its sequence along the genome in increments of 1 nt to generate genome sequence frames for comparison with the selected region of the aptamers. In order to treat information at the 5′ and 3′-ends of each selected region on an equal footing to the middle sections in the histogram plot, we also considered partial overlaps, obtained from alignments of the 3′ region of the aptamers to the 5′ regions of the genome and vice versa, gradually increasing in steps of 1 nt until full overlap was reached. For each such alignment, we calculated the maximum Bernoulli score, defined as 0N(Li)3i . This isSince typically the fragment contributing to the score was smaller than the length of the aptamer and contained some mismatches, we identified the largest fragment of the aptamer that had the highest Bernoulli score, and therefore, the lowest probability of having aligned to the genome fragment by chance. For each comparison frame, the fragment of the aptamer that aligned to the genome with the maximum Bernoulli score was identified via computation of the contributions of different mismatches to the scores of any contiguous subset of the frame. If this maximum score was larger or equal to 12, we logged it into the data file (using the frequency of the aptamer read) that was subsequently used to compute the histograms used.The library selected against EV-E capsid protein contains 2,563,876 aptamer reads, of which 147,489 are unique. 55,088 (37.35%) of the unique reads match to the genome with a matching probability equivalent to 10 nucleotides, which is ~1 in a million. In order to facilitate the identification of the PSs in the genome we used an even stricter criterion for the data shown in The highest peak from the Bernoulli analysis , peak 9 We therefore analysed the matches with the aptamer pool in more detail. Firstly, we note that the library was preferentially enriched for A (from 26.1% to 34.5%) and G (from 24.7% to 41.7%), whilst the genome has only a mild bias towards purines  . This suWe used RNAcomposer to render candidate PS SLs in 3D and compare with the cryo-EM density. The 3D structure of the lowest energy fold for Peak 9 is shown in - Å-2 per frame, with a total exposure time of 1 s and an accumulated exposure of 49.5 e- Å-2. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher) at -0.75 μm to -3.5 μm defocus.Purified EV-E samples in PBS (3 μL) were applied onto 400-mesh lacey grids coated in a 3 nm carbon film . The sample was left to adsorb for 30 s before most of the sample was blotted away manually and this process was repeated 4 times. The grids were vitrified using a Leica EM GP freezing device (Leica Microsystems). Chamber conditions were set at 4 °C and 95% relative humidity. Grids were glow discharged for 30 seconds prior to application of the samples. Data were collected on a FEI Titan Krios electron microscope operated at 300 kV and images recorded on a FEI Falcon III detector operating in linear mode. A total of 8,785 movies were recorded at a calibrated magnification of 75,000x, yielding a pixel size of 1.065 Å on the specimen. Each movie comprises 39 frames with an exposure rate of 1.27 eMovies were motion-corrected and dose-weighted with MOTIONCOR2 . AlignedFollowing 3D refinement with icosahedral symmetry, the symmetry of the dataset was expanded such that each particle was assigned 60 orientations that corresponded to its icosahedrally redundant views. A cylindrical mask was created to exclude all of the virion structure except the area of interest, using SPIDER . We sepaThe crystal structure of EV-E  (PDB:1BEhttps://deposit-pdbe.wwpdb.org/deposition). Graphics were produced by UCSF Chimera [The FSC curve between the final model and map after post-processing in RELION (Model vs Map), is shown in  Chimera .E. coli (NEB), then extracted using a maxiprep kit (Qiagen) and purified via the Monarch DNA clean-up kit (NEB). The wild-type and mutant plasmids were digested with NotI  was supplied by Genscript Biotech (Netherlands) in a pUC57 plasmid vector, which encompasses ampicillin-resistance. The gRNA was flanked with a 5′ T7 promoter and hammerhead ribozyme and a poly-A40 tail terminating in a unique NotI restriction site at the 3′ end . Mutatioith NotI , the rea2O. The RNA was DNase treated again to ensure no plasmid DNA carryover. A 1 μL aliquot was taken from each RNA for a no-RT control in the qPCR reaction. The rest of the RNA was reverse-transcribed to cDNA using a sequence-specific primer (EV-E_R_7100–7081: ACGCACGAACTTCCTCTTCA) and Superscript IV (Invitrogen).BSR-T7 cells were grown to 80% confluency in 6-well plates. Linear plasmid DNA (1 μg of each) was transfected into wells using Viafect transfection reagent (Promega). Plates were incubated for 18 h at 37°C. Post-transfection the plates were freeze-thawed to lyse cells and the media removed. The cell lysate was treated with DNase and RNase A (10 μg/mL) at 37°C for 30 min to remove plasmid DNA and non-encapsidated RNA. Encapsidated RNA was extracted using Trizol; the precipitated RNA was pelleted, washed x3 with 70% (v/v) ethanol, air dried and resuspended in 12 μL H2O. The concentration of the experimental samples was quantified using a standard curve of EV-E RNA in the same qPCR run that followed the same RT and qPCR steps as the experimental samples.The cDNA and the no-RT controls were added to 5 μL qPCR mastermix , 400 pM each of the EV-E-specific primers , and made up to 10 μL with HBHK cells were grown 80% confluency in 6-well plates (Corning). Cells were washed with PBS and viral lysate from the above transfection added, made up to 1.5 mL with serum-free media (Sigma). Plates were incubated at 37°C for 2 h, then lysate was removed and the cells overlayed with 2 mL 1%(w/v) SeaPlaque agarose in DMEM with 2%(v/v) Horse Serum (Gibco) for 48 h. Cells were fixed with 2%(v/v) formaldehyde (Sigma) for 30 min, and stained with crystal violet solution (Sigma) before visualisation.EV-E plasmid, either wild-type (WT) or packaging signal mutation at Y9 or W38 in VP2 or R50 in VP1 was transfected into BSR-T7 cells. Following an 18h incubation, transfection product was purified by freeze-thaw and passaged three times on BHK cells. The lysate was harvested and viral titre was quantified by plaque assay and qPCR (as previously described).WT and W38A transfection product lysates were probed for EV-E VP3 by Western blot. 40 μL of each was run on an SDS-PAGE gel and transferred to nitrocellulose membrane. The primary antibody was mouse anti-VP3(EV-E) 8B1  diluted 1:500 in TBS-T, secondary antibody was goat anti-mouse IgG biotin . The bands were visualised using Western Blue Stabilized Substrate for Alkaline Phosphatase .260 and A280 absorbancies, as well as light-scattering were measured. The latter are shown in WT and W38A lysates were pelleted through a 30%(w/v) sucrose cushion (as described above in EC purification). Lysates were loaded onto a TSK 6000 column in PBS. The AS1 Fig(A) Optimisation of EV-E EC production. Analysis of viral CP radiolabelling, post-treatment with GuHCl, of an EV-E infection fractionated on a linear sucrose density gradient. Inset: nsEM images of peak fractions showing ECs and virions. EM scale bars, here and in (B), = 100 nm. (B) Pulse-chase of ECs to virions. Bars represent peak fraction scintillation counts of ECs and virions from sucrose density gradients, as in (A), following removal of GuHCl inhibition at different time points. Inset: nsEM images from peak fractions of ECs (1 h) and virions (1.75 h). (C) Confirmation of biotinylation of EV-E for SELEX target by western blot. (D) N40 library sequence information and primers for the EV-E selection. The T7 sequence is underlined in red. (E) Poliovirus EC pentamer SELEX target preparation following the same protocol as for EV-E. Sucrose density gradient fractions showing scintillation counts for each fraction. Inset are nsEM images of the peak PV virion and EC fractions and an autoradiography gel of these peak fractions. (F) N30 library sequence information and primers for the PV selection with T7 sequence underlined in red.(TIF)Click here for additional data file.S2 Fig(A) Frequency plot of the EV-E aptamer sequences in the selected and naïve libraries. The selected library contains 147,489 total unique reads within a total of 2,563,876 sequences (24% with a frequency >2). The most frequent aptamer read was 212,070. (B) EV-E RNA library base composition before and after selection. (C) List of peaks above background in the anti-EV-E Bernoulli Plot  and thei(TIF)Click here for additional data file.S3 Fig(A-F) Resolution and model validation of EV-E structure. (A) Cryo-EM image of EV-E. Bar = 500 Å. (B) Fourier Shell Correlation (FSC) resolution curve for the icosahedrally averaged 3DR of EV-E. Resolution based on the gold standard 0.143 criterion is 2.23 Å. (C) Cross-validation against overfitting of the model, FSC curve for the final atomic model refined against the post-processed map , and FSC curves for the randomly shifted and refined atomic model against the half map used in the refinement  and against the half map not used in the refinement . (D-F) Surface (top) and slab (bottom) viewed along a two-fold axis coloured and low-pass filtered based on local resolution as indicated in the colour key  for the (D) icosahedrally averaged density map shown at 1 σ, (E) density map obtained after symmetry expansion and focused classification on two-fold axis shown at 2 σ, and (F) density map obtained after symmetry expansion and focused classification on genome density shown at 1.2 σ. (G-H) Quality of the cryoEM density map. G) Atomic model of the asymmetric unit of EV-E shown as ribbon diagrams  colour-coded as in (TIF)Click here for additional data file.S4 FigNot I; cut and uncut plasmid were run on a 1%(w/v) agarose gel to confirm digestion.(A) Schematic of EV-E infectious clone; the EV-E genome was inserted into a pUC57 vector, the structural proteins of P1 region are filled in yellow-green and the sites of interest are highlighted, green boxes denote the chosen mutagenesis sites. (B) Mutations introduced individually into the infectious clone. (C) Plasmid digestion with (TIF)Click here for additional data file.S1 MovieMovie showing the reconstructed EV-E virion, together with the focused classifications at its symmetry axes. The cryo-EM density maps are shown as colour-coded surface, grey semi-transparent surface and grey mesh, with the density corresponding to RNA facing the bottom part. The atomic model is shown as ribbon diagrams and sticks coloured in orange for the RNA, and colour-coded as previously for the CP. Residues involved in the contact are indicated and coloured by heteroatom.(MP4)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.