Oyster reefs have the potential as eco-engineers to improve coastal protection. A field experiment was undertaken to assess the benefit of oyster breakwater reefs to mitigate shoreline erosion in a monsoon-dominated subtropical system. Three breakwater reefs with recruited oysters were deployed on an eroding intertidal mudflat at Kutubdia Island, the southeast Bangladesh coast. Data were collected on wave dissipation by the reef structures, changes in shoreline profile, erosion-accretion patterns, and lateral saltmarsh movement and related growth. This was done over four seasons, including the rainy monsoon period. The observed wave heights in the study area ranged 0.1–0.5 m. The reefs were able to dissipate wave energy and act as breakwaters for tidal water levels between 0.5–1.0 m. Waves were totally blocked by the vertical relief of the reefs at water levels <0.5 m. On the lee side of the reefs, there was accretion of 29 cm clayey sediments with erosion reduction of 54% as compared to control sites. The changes caused by the deployed reefs also facilitated seaward expansion of the salt marsh. This study showed that breakwater oyster reefs can reduce erosion, trap suspended sediment, and support seaward saltmarsh expansion demonstrating the potential as a nature-based solution for protecting the subtropical coastlines. These habitats include coral reefs5, reef-forming bivalves9, dense vegetation of kelps and seagrasses11, salt marsh vegetation14 and mangroves18. They have the capacity to reduce flow and dampen wave energy through their physical structures and by doing so, they trap and stabilize sediments, allowing to keep pace with sea-level rise by natural accretion and growth23. Moreover, they offer additional ecosystem services including: (1) water quality regulation25; (2) ecosystem succession27; and (3) fisheries production30. The use/design of sustainable ecosystems that integrate human society with related natural habitats for the benefit of both is called ecological engineering33. It provides opportunities to combine engineering principles with ecological processes to reduce environmental impacts of man-made infrastructure34.Coastal habitats play a critical role in coastal adaptation strategies as they can reduce the vulnerability of coastal communities to natural hazards like flooding, eroding shorelines and sea level rise36. Until 2015, a total of 1,576 km2 area was lost due to shoreline erosion at an annual rate of 6.3 km2 in 1985–1995 and 11.4 km2 in 2005–2015, respectively35. Shoreline erosion is increasingly threatening coastal communities and their livelihoods37, forcing thousands of people to migrate to the mainland38. This is particularly severe in offshore (island) areas, such as in the islands of Kutubdia and Sandwip that are frequently impacted by storm surges, increasing astronomical tides and erosive waves associated with southwest monsoon winds35.The coastline of Bangladesh has changed rapidly over the last few decades39. However, only 60 km out of a total of 957 km sea facing embankments are protected by a forest belt, which is gradually degrading due to ever increasing cyclones40. Moreover, mangroves were being deforested due to construction of  aquaculture ponds and salt pans in many intertidal areas41. The succession and growth of other vegetation types, such as salt marshes varies with season and is becoming less efficient in trapping sediments due to incoming erosive waves during high energy periods in the monsoon season  oyster breakwater reefs on an eroding intertidal mudflat of the Kutubdia Island at southeast Bangladesh. We evaluated the hypothesis that wave attenuation by these oyster breakwaters could reduce sediment erosion, promote mudflat stability, and enhance lateral saltmarsh expansion and growth. Oysters occur abundantly in the study area and the intertidal rock oyster, The reefs, being ~0.6 m in height after settling above the mud, dissipated wave energy, acting as wave breakwaters. Dissipation of wave energy depended on tidal height (water level) and wave height Fig. . Wave hep < 0.001) higher sedimentation occured at the landward mudflat of reef sites than the control sites  less as compared to control mudflats areas  lower at reef sites. Sediment deposition occurred during the post-monsoon (i.e. November) predominantly in the vegetated areas at both control and reef sites  higher at the saltmarsh areas of reef sites than at the salt marsh areas of the control sites during this period. In the mudflat area (i.e. seaward and landward mudflat) no distinct changes were observed in the post-monsoon season and elevation remained identical at the end of the monsoon period  occurred up to 35 m landward of the reefs with a maximum accretion of 29 ± 1 cm  and October 2016 . On the other hand, erosion occurred at distance class between 0–5 m and 35–100 m along the assessed cross-sections for both reefs and control sites (p = 0.32) over a 12 months period. Cumulative changes in terms of either the erosion or accretion rates along the whole transect (0–100 m) demonstrated that erosion on average are dominant at both reef and control sites, but at different rates. Average net erosion rates are almost 2x higher over the entire transect at control sites (0.051 m3 sediment m−2) vs. at the reef sites (0.023 m3 sediment m−2).Average sediment accretion or losses along cross sections of the difference map indicated an average accumulation of 0.11 mtes Fig. , and thip > 0.05). The difference in clay percentage seaward of the reefs vs. the control sites was minimal (p = 0.32).Sediment at the study site mainly consists of clay particles (>84%)  at control sites as compared to the reef sites  showed a seasonal pattern Fig. . During tes Fig. . During −2 area) were about 36% higher at reef sites than that of the control sites . Saltmarsh started to expand again in the post-monsoon season, while the steam density increased, reaching maximum during the winter months  and hot (~4 °C above the average) air from the north western India moves into the northern Bay of Bengal during the pre-monsoon (March-May) season causing a pre-monsoonal depression in the region51. At that time the coast receives strong longshore currents and waves. As a result, deposited sediments begin to erode from intertidal flats. Sediment erosion reaches a maximum rate during the monsoon (June-September) season, when southwest monsoonal winds result in huge rains and much more turbulent waves52. Subsequently, new sediments deposited in the late post-monsoon season (November), that continues throughout the dry winter season. The tidal flat seems unchanged at the end of the winter as compared to the previous winter period  period due to low energy incoming waveset al.53 observed that reefs were more effective in reducing shoreline erosion in an exposed area compared to a sheltered area. The interaction of a reef with larger waves could explain for this difference. Waves and wave dissipation are the primary reasons for the observed local morphological changes. Our study clearly indicates that breakwater reefs can successfully reduce the wave heights (i.e. wave energy). It provided better stability in sediment movement from intertidal bed and reduces erosion, which was not the case for control site without reefs. A 50 cm vertical relief of breakwater reefs resulted in long-periods of reduced wave height, resulting in an area of influence over 30 m behind the reef favoring sediment accumulation. Moreover, this accumulated sediment, which normally erodes during the monsoon months, was successfully trapped behind (lees) the reefs, thus reducing erosion and stabilizing the tidal flat locally. However, these effects might vary, depending on the intensity of monsoon. The net sediment budget at the end of monsoon (i.e. October) after 2016, but before 2015 also indicated that the oyster breakwater reef could reduce erosion rate by ~50% (i.e. 0.014 m3 sediment/m2 area) along the entire reef cross-sectional tidal flat area than the adjacent control areas . They suggested that longer extended reefs were more likely to generate larger impacts on tidal flat morphology. The experimental reef units used in this study were only 20 m in overall length resulting in an impact on the mudflat morphology up to 30 m area at the landward side. Beyond this distance, erosion was unchanged for reef and control sites. This entire zone was severely affected continuously by wave actions particularly during the monsoon period. The effect of the reef could be enhanced by extending the length and height of the reefs depending on immersion time in the investigated site. Moreover, the extent of the impact also depends on tidal range, wave condition, bed slope and flow directions at the reef construction site54. Studies55 along US coast have indicated that the effect of reefs in controlling shoreline erosion is quite variable over time, with their efficacy only viewed as significant during storm induced erosion. However, this study showed that oyster breakwater reefs were equally effective in trapping sediment for all seasons including stormy periods .If we focus on the effect of the monsoon season alone, sites reach their highest erosion state, oyster breakwater reefs appear to be effective locally at reducing tidal flat erosion Figs  and 4. T57. As particulate-laden water moves over an oyster reef, eddies slow water flow to the point where deposition may occur, and the amount of deposition dependent upon suspended sediment concentration in ambient water and their particle size and flow speed. Suspended sediment concentration were higher in ebb than the flood tidal phase, but the tidal current was relatively stronger in ebb tide compared to flood tide, suggesting that sediment deposition rate varies with the tidal phase. Longshore flow and current velocity showed some variation in and around the reef areas. Depth-averaged mean current velocity was less strong in the landward side of the reef than the seaward side58. Whitman and Reidenbach59 conducted a study of flows along mudflats and found that flow velocity was reduced by a factor of 2 compared with flows over an adjacent oyster reefs. In this study, high sediment deposition behind the reefs suggest these low flow areas tended to trap fine sediments. However, drag exerted on current flow by an object like oyster reefs and its influence on sediment deposition and particle flux are a function of the length and orientation of that object in the direction of the flow60. Perpendicular reefs were found more effective than parallel or circular reefs at producing hydrodynamic conditions that maintain higher deposition rate56. In this study, the reefs were placed parallel to coast as breakwater for attenuating wave energy that also effective in trapping sediment. We did not see any possibility that the placement of the parallel breakwater reefs accelerates erosion in adjacent areas including the control sites.Not only the waves, regular flow and current dynamics around oyster reefs have also been shown to be crucial in driving local sedimentation61. As indicated above, reefs can influence the tidal flat morphology during the monsoon period, impacting nearby salt marsh vegetation in terms of increased plant survival and lateral marsh expansion  in control site. Alteration of the physical conditions by the deployed reef structures protected the salt marsh during the monsoon season, resulting in the seaward expansion and fast regeneration of the marsh after the monsoon season. Previous studies have only recorded a reduction of marsh edge erosion after deployment of the oyster reefs62. This is the first study from subtropical region showing seaward expansion of salt marshes after deployment of reef structures. Furthermore, a much larger area can be influenced and protected using longer reef structures and increasing the vertical relief that, in turn, can elevate the intertidal bed and create other important shallow habitats, like mangroves64. Breakwater structures have often found to play a successful role to rehabilitating mangrove forests in subtropical regions65. Therefore, oyster breakwater reefs not only have the potential for stabilizing the tidal flats but also useful for restoring or enhancing other ecologically important habitats such as salt marsh, offering favorable conditions for many organisms to dwell and grow 66.In monsoon season, water flows were strong and erosion of other intertidal ecosystem like salt marshes occur. Short-distance interactions between the reef structures and adjacent salt marshes could accelerate the process toward more local tidal flat stabilityion Figs  and 6. A58. For example, the central and southwestern coasts of Bangladesh may not be ideal for deploying artificial oyster reefs as these areas are prone to large amount of sediment-laden freshwater discharge through the Ganges-Brahmaputra-Meghna (GBM) river system. While ~397 km coastline and offshore islands of the southeastern coast, which receives much influence from the Bay of Bengal than other parts and blessed with plentiful oyster spat supply, are deemed suitable for year round growth of oysters58. The development of oyster reefs is thus feasible in those areas to improve salt marsh and mangroves ecosystems for generating multiple ecosystem services and reducing the vulnerability of coastal islands from erosion.Apart from the investigated Kutubdia Island, breakwater reefs can also be implemented in other coastal areas of Bangladesh where natural habitat of oysters is present. Oyster settlement and growth, in general, are characterized by relatively high levels of salinity, Chlorophyll-a, dissolved oxygen and pH. Therefore, estuaries dominated by freshwater plume and coastal areas with high suspended sediments are less suitable for oystersIn conclusion, the use of oyster breakwater reefs at lower intertidal zone can protect the tidal flat in front of primary embankments by changing the shoreline eco-morphology. The results of this study demonstrated that oyster breakwater reefs are particularly useful in reducing erosion at lower intertidal areas as the reefs successfully stabilized sediments in both high (monsoon) and low energy periods. Additionally, they enhanced the growth of adjacent salt marsh vegetation, which expanded their seaward edge thereby further stabilizing the adjacent unconsolidated sediments. This effect can be improved by widening the reef length and height. Moreover, the reef structures provide space for new oysters to grow and develop as biogenic habitat overtime leading to a self-sustained oyster reefs. Therefore, along the coast of Bangladesh, where larval supply of oysters are abundant, the eco-engineered breakwater structures hold promise for a more sustainable shoreline protection against erosion.−1 67. About 40 km of earthen embankments, including 4 km of concrete blocks, have been constructed since the 1990s to protect the island, although a large area still unprotected and exposed to tidal flooding and erosion. The earthen embankments often collapse and cannot prevent flooding during the monsoon period, and require maintenance every year with new alignments as there is a constant loss of the foreshore. The east part of the island, which is characterized by wide tidal mudflats, is a suitable habitat for saltmarsh and mangroves68. Currently, 290 ha area are vegetated by mangroves/salt marsh covers, which is only 4.2% of the total island area69. Oysters occur along the shoreline where substrates are available.A manipulative field experiment was carried out at Kutubdia island Fig. , located71. In the study area, the climatic conditions are mild and dry during winter, with air temperature ranging from 6.2–22.4 °C. Winds are north-easterly at the beginning of winter but become north-westerly by the end. Air temperature is maximum (~38.5 °C) during the pre-monsoon season. Heavy southwest monsoon rains begin in early June, continuing in to mid-October. During the monsoon season, floodwaters from rainfall lowers the salinity to estuarine conditions (10–15 ppt). Salinity levels in other seasons, including the post-monsoon season remain steady, >22 ppt. Annual average rainfall varies from 2,300–3,200 mm52. Suspended sediment concentrations in the water are quite high, varying among seasons from 100–700 mg l−1 72. Tides along the coast are semi-diurnal, and the tidal range is approximately 4 m with a seasonal variation of mean tide level (MTL) 50–80 cm. Water current direction is from the north during peak flood tides and from the south during ebb tides. During the pre-monsoon and monsoon seasons strong longshore currents prevail50 with high and variable waves because of summer storms, reaching a height of 0.75 m or more depending on wind conditions. Average annual wind speed ranges from 0.8–2.2 m sec−1.A field experiment was carried out at the Boroghop jetty site at the eastern side of the island Fig. . Here th74. In a dynamic and high energy coast, like the study site, more robust reefs are needed with high vertical relief to avoid smothering by sediments and less physical damage to the deployed structure during the monsoon season6. Three oyster reefs were constructed on a tidal mudflat of Kutubdia Island using precast concrete rings. Each of concrete ring was 0.8 m in diameter, 0.8 m high, and 0.05 m thick with four holes in them50, a structure similar to reef balls75. Each reef contained 41 concrete rings, each placed in two rows next to each other, resulting in 20 m long reefs . However, successful spat fall (>300 spat m−2) was observed in year two, when rings covered with high densities of oysters S. cucullata  and other marine organisms such as barnacles, sea anemones, gastropods and polychaetes. The overgrown rings were transported to the experimental site in March 2016 and termed as “oyster breakwater reefs”. A terminology OysterBreaktm was also used for a similar experimental setup in Vermilion Cove, Louisiana, USA27 . Top 50 cm of the reef substrates were covered with as thick as ~10 cm layer of live and dead oysters, while the dynamic bottom part (30 cm) was occupied by various benthic epifauna, mostly reef forming polychaetes, Sabellaria sp.  along a transect crossing each of the three reefs  and along two transects crossing two adjacent control sites (CS) , before (2015) and after (2016) the construction of the reefs. These measurements were conducted in October at the end of the monsoon season after which the maximum erosion was observed. A digital elevation model (DEM) was made in R software (version 3.3.0) using the packages grDevices and graphics. Based on the measured relative elevations, net sediment accretion or erosion could be calculated by subtracting the DEM for 2016 from 2015. To calculate net sediment accretion or erosion in terms of sediment volume (m3) for either the reef and control sites, five-cross sections, three crossing the middle of the reef area and two crossing the control area from baseline till the edge of the mangrove forest, were made from the DEMs of 2015 and 2016. In this regard, a 1 m wide grid pattern was used to calculate sediment accretion/erosion in terms of volume (m3) by multiplying the sediment height increase/decrease (in meters) and the area of corresponding grid (1 m2) in each cross section. Cross sections for RS and CS were averaged for each year, with changes in sediment accretion/erosion (m3 sediment m−2 area) along the cross-sections measured by comparing the year 2016 with 2015.Additionally, elevation was measured on a 1 m by 1 m grid, covering the entire study area , while particle sizes <75 µm retained on the sieve (No. 200 mesh) were determined by a sedimentation process, using a soil hydrometer . The soil was classified by plotting the particle-size fraction  in the USDA textural triangle system77.To investigate sediment granulometry changes, monthly sediment samples were taken 5 m seaward and 10 m landward of each reef and at the same height at the control sites Fig. . Measure−2) was counted monthly in a fixed quadrat (1 m2) 15 m from the leeside of the reefs, which was initially 2–3 m inside the salt marsh edge.Reference points were set at the lee side of the reefs and at the same height at the control sites by placing benchmark sticks in the middle of the reef to measure lateral (i.e. seaward expansion or landward retreat) salt marsh movement Fig. . To dete3), mean clay percentages, salt marsh stem density at reefs and at the control sites were verified, using a simple t-Test. Before statistical analysis, the normality of response variables was tested using the Kolmogorov-Smirnov Test, and a homogeneity of variances using Levene’s Test. All analyses were performed using IBM SPSS statistics software (Version 2015) by setting statistical significance at p ≤ 0.05.The statistical difference in net sediment accumulation or loss (mSupplimentary information


Cells frequently simultaneously express RNAs and cognate antisense transcripts without necessarily leading to the formation of RNA duplexes. Here, we present a novel transcriptome-wide experimental approach to ascertain the presence of accessible double-stranded RNA structures based on sequencing of RNA fragments longer than 18 nucleotides that were not degraded by single-strand cutting nucleases. We applied this approach to four different cell lines with respect to three different treatments . We found that long accessible RNA duplexes were largely absent in native cell lysates, while the number of RNA duplexes was dramatically higher when proteins were removed. The majority of RNA duplexes involved ribosomal transcripts. The duplex formation between different non-ribosomal transcripts appears to be largely of a stochastic nature. These results suggest that cells are—via RNA-binding proteins—mostly devoid of long RNA duplexes, leading to low “noise” in the molecular patterns that are utilized by the innate immune system. These findings have implications for the design of RNA interference (RNAi)-based therapeutics by imposing structural constraints on designed RNA complexes that are intended to have specific properties with respect to Dicer cleavage and target gene downregulation. RNA–RNA interactions play structural and functional roles in nearly every step of the gene expression pathway, such as RNA interference/microRNA pathways, RNA editing, RNA splicing, and interferon response. A variety of approaches were developed to map RNA sequencing (RNA-Seq) data to the genome, such as Hierarchical Indexing for Spliced Alignment of Transcripts (HISAT)  and ShapIn addition, RNA antisense purification (RAP) can be used to determine RNA–DNA interactions . An RNA-The length of potential RNA–RNA duplexes influences their role in the formation of ribonucleoprotein (RNP) granules. Recently, a phase-transition model was proposed where, depending on the local concentrations of RNAs and proteins, RNP granule formation is either protein-driven, RNA-driven, or driven by both RNA–RNA and protein–protein interactions . The empIt is also known that a variety of cellular surveillance mechanisms are sensitive to the length of RNA duplexes. In addition to the aforementioned RNA interference (RNAi) pathway that is triggered by ~21-bp helices, the RNA-binding protein, protein kinase R (PKR), is activated by interactions with long RNA helices consisting of at least 30 base pairs, which then triggers downstream effects such as downregulation of protein synthesis or even apoptosis ,17. ExogFinally, the formation of long duplexes via RNA–RNA interactions has potential therapeutic applications. For example, self-assembling RNA complexes that contain functional Dicer-substrate RNA helices were utilized for target gene downregulation. Such designed RNA complexes may correspond to quasi-symmetric nanostructures resembling rings ,20, but Here, we present a novel method for enriching and cloning cellular double-stranded RNAs (dsRNAs) in their physiological condition. The idea of this approach is to utilize RNA-Seq combined with RNA single-strand cutting enzymes in order to obtain sequencing information pertaining to long RNA duplexes. Furthermore, a variety of bioinformatics analysis approaches were utilized in order to computationally characterize RNA–RNA interactions. Applying such methods, we investigated whether or not long RNA–RNA base pairs with more than 18 base pairs are formed in human cells.To enrich RNA duplexes, we sought to digest single-stranded RNAs (ssRNAs) using endoribonucleases. Given that the combined formulation of RNase A and RNase T1 is well characterized for specific digestion of single-stranded RNAs, a cocktail containing A and T1 (RNase A/T1), which is a standard, and a readily commercially available mixture, was chosen for the following experiments. A 35-bp-long dsRNA helix with five-nucleotide ssRNA as an overhang at both ends and two variants embedding a bulge or a mismatch in the middle was used as a control to probe the activity of RNase. The concentration of RNase A/T1, digestion time, and digestion temperature were optimized  removal, were heated up and reannealed before being subjected to the same RNase A/T1 digestion. In addition, total RNAs from HEK293T cells and HeLa cells were digested by RNAase A/T1 directly without the depletion of rRNA in order to capture RNA duplexes involving rRNAs. Samples with rRNA removal are labeled as “ribo-removed” while the ones without are labeled as “protein-removed” in We created two additional sequencing datasets corresponding to HeLa and 293T cells, focusing on reads with molecular weight corresponding to a consistently exhibited band (indicated by an arrow in z-score was computed by comparing the interaction energy with respect to the native read sequence (the dominant read) to 20 interaction energies corresponding to shuffled versions of the same sequence. The resulting z-scores corresponding to each transcript are plotted in z-scores for the interaction energy of the native rRNA sequence, respectively). The median of the z-scores was positive for most RNA types; in other words, on average, most RNAs seem to avoid interaction with this particular ribosomal RNA region. However, a functional analysis of the RNAs that have an overrepresentation of sites complementary to the 28S expansion segment  showed an enrichment for genes that are associated with cellular compartments including the cell membrane, nuclear membrane, or the cytoskeleton.A bioinformatics analysis of potential interactions of this sequence fragment with the transcriptome suggests that many cellular transcripts are subject to positive or negative selection for complementarity to this one ribosomal sequence region. We utilized the RNAduplex program to compute potential RNA–RNA interactions . The sum of the interaction energies of the minimum free energy structure, as well as suboptimal structures within 2 kcal/mol, was used as a score to measure the RNA–RNA interaction quantitatively. For each RNA of a certain class, a We computed, for each gene, the differential expression  with respect to a particular treatment using .For many transcripts, one can identify regions that overlap with We searched for reads that partially matched two different transcripts. We required that the 5′ end of a read matched for at least 18 nucleotides (nt) with respect to one transcript, while the 3′ end of the same read matched another transcript (again for at least 18 nt). Also, reads that matched a transcript in their entirety or that could be explained by intra-transcript interactions were removed. Using this approach, we found very few cases of sufficient evidence for RNA ligation events.We mapped the per-nucleotide coverage of the obtained reads onto the 28S rRNA sequence. Shown in We searched computationally in an all-versus-all manner for candidate read pairs of RNA–RNA duplex formation as described in In this paper we obtained transcriptome-wide information about long RNA duplexes by utilizing RNA-Seq and RNA single-strand processing enzymes on the experimental side followed by a variety of bioinformatics analyses. We arrived at interesting conclusions that are discussed below.cis and trans sense/antisense regions. It was found that cells frequently express sense and antisense transcripts simultaneously [The “pan-transcriptome”  contains many aneously . The lacaneously . In ordeWe found, in the present experimental results, orders of magnitude higher occurrence of duplexes in cell extracts where proteins were removed compared to cell lysates. This suggests that RNA-binding proteins prevent a large portion of RNA–RNA interactions. Moreover, the difference with respect to the presence of ribosomal RNA in observed evidence for RNA–RNA interactions for datasets where proteins were removed suggests that one important function of RNA-binding proteins is the prevention of interactions between ribosomal and non-ribosomal RNAs.Also, the found difference in medium helix length in an all-versus-all search of all read pairs in native lysates (with shuffled sequences leading to longer predicted duplexes compared to unshuffled read sequences) suggests that there is a pervasive cellular constraint on preventing the formation of long duplexes. As described, we computed a duplex structure for all reads with respect to all other reads of the same length in the same dataset. One can, in this way, for any read, identify the length of the longest predicted helix. We then computed, for each dataset, the median of the longest predicted helices. We found that the median of the helix length with the longest predicted interaction duplex was only 12 base pairs . What would explain this striking discrepancy between expectation and obtained experimental results? As possible explanations, we propose  the following: (a) reads corresponding to mismatch-containing helices were found in our datasets because they were protected from degradation by proteins at the stage of enzyme digestion of single-stranded regions; (b) reads corresponding to mismatch-containing helices were found in our datasets because the degradation by single-strand cleaving enzymes was incomplete. The fact that we found an increase in predicted contiguous helix lengths in the case of the protein-removed datasets, combined with the fact that, for all datasets, we found a large fraction of reads (at least 50%) for which the longest predicted helix interaction could not account for the entire read length, suggests that both scenarios (a) and (b) contribute to the observed findings.Furthermore, duplexes longer than about 18 base pairs can be processed by Dicer, leading to the shortening of duplexes and the degradation of the constituent transcripts. Dicer-processed duplexes were reported, in some cases, to result in endo small interfering (RNA) that then can lead to post-transcriptional downregulation of mRNAs .Long RNA duplexes correspond to a danger- or pathogen-associated molecular pattern (DAMP or PAMP), that may trigger a response from the innate immune system via several pathways. Duplexes with lengths greater than 30 base pairs can trigger a global cellular response of downregulation of translation or even apoptosis via binding to protein kinase R (PKR). The 2,5-AS/RNA L pathway is triggered by RNA helices with lengths of about 70 base pairs or longer. This pathway—thought to be an innate immune response to invading viral RNA—leads to the degradation of the recognized RNA. It should, thus, be no surprise that long duplexes are uncommon in cells because long duplexes are, as indicated, targeted for degradation by several different kinds of endo-nucleases. Also, the DAMP/PAMP “danger” pattern would be less informative and more difficult to interpret by the cell if there were a substantial amount of “beneficial” long RNA duplexes. In other words, our experimental results fit very well into the current thought regarding the workings of the innate immune system.Additionally, we searched for chimeric reads that mapped to two distinct transcript sequences. Those reads could be evidence for endo-ligase events. With the exception of ribosomal RNA, we did not find chimeric reads that would consistently support particular RNA–RNA interactions. The lack of perfectly complementary regions and endo-ligation events suggests that the majority of the observed sequence data are a result of partial digestion by the administered nuclease cocktail or protein-covered single-stranded regions. This allows, nonetheless, the firm conclusion that long perfectly complementary RNA duplexes are virtually non-existent in the examined cell lines.We found that a large portion of reads mapped to ribosomal RNA. Also, a substantial fraction of reads mapped to ribosomal expansion segments. These are regions that are likely to be flexible and located near the surface of the ribosome structure or are protruding outward. This suggests that flexible regions at distal ribosomal regions are important for RNA–RNA interactions. One may note an analogy between weakly structured or unstructured RNA regions and unstructured regions of proteins that were frequently found to be important for inter-molecular binding and recognition events .An extreme case of this tendency toward surface-accessible regions is the high coverage of 28S ribosomal expansion segments 7 (ES7L) and 27 (ES27L) in the analysis of the gel-extraction experiments see . InteresIt seems plausible that ribosomal segments whose role it is to be highly interacting with RNAs and proteins are both weakly folded and yet protected from single-strand cutting nucleases via unusual structural features such as triple helices. More research will be needed to elucidate the structural biology of ribosomal expansion segments.We consistently found that the number of contiguous RNA duplexes was dramatically higher if proteins were removed. This suggests, that RNA-binding proteins—to a large extent—prevent RNA base-pairing. In other words, RNAs are more prone to be involved in inter-strand interactions in vitro compared to in vivo environments. In the case of intra-strand interactions, Rouskin and coworkers stated that there “are vastly fewer structured mRNA regions in vivo than in vitro” for the case of intra-strand interactions . This suA variety of strategies for utilizing RNA for therapeutic or diagnostic purposes were reported in the literature. These approaches are typically based on one of a handful of foundational technologies such as RNA interference (RNAi), clustered regularly interspaced short palindromic repeats (CRISPR), ribozymes, antimirs, or antisense RNAs. In all cases, the exogenously delivered RNAs are potentially subject to human cellular surveillance. This work highlights that an exogenous long RNA duplex can potentially be unambiguously recognized because intra-cellular occurrence of long RNA duplexes is apparently rare. A fitting metaphor might be that a “coin dropped into an empty jar makes a lot of noise”.One of two opposing strategies for the design of RNA-based therapeutics present themselves; one obvious strategy is to avoid the utilization of long RNA duplexes in RNA complexes where stability and a long half-life is desired. Alternatively, purposefully introduced modified bases might increase half-life even for long RNA duplexes. A second strategy is to deliver an abundance of long RNA duplexes or activate conformational changes to induce duplex formation in target cells. In this case, cells might recognize a danger-associated molecular pattern and might trigger a response of innate immunity such as apoptosis. For example, the PKR protein can be activated by helices longer than ~30 base pairs, which can trigger downregulation of protein production  or even cell death. This mechanism was utilized for the conditional activation of PKR-mediated cell death via a duplex consisting of the Bcr/Abl fusion oncogene  and an exogenous designed cognate antisense strand . In lighDesigners of RNAi-based therapeutics should be aware of recent results related to the processivity of components of the RNAi pathway such as Dicer. The introduction of mismatches of a Dicer substrate RNA will likely reduce the effectiveness of its inhibition function. On the other hand, cleavage of a helical region by Dicer may not necessarily be prevented by a small number of mismatches or even bulges ,37. Take2. Cell pellets were collected for the following experiments.HEK293T, HeLa, U2OS, and MCF-7 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS)  and penicillin/streptomycin antibiotics  at 37 °C and 5% COFor the purposes of enzyme digestion optimization and spike-in, we synthesized four RNA oligos using partial green fluorescent protein (GFP) mRNA sequences (same or reverse complement) as templates. Sequence A (5′-rArGrCrArGrArArCrArCrCrCrCrCrArUrCrGrGrCrGrArCrGrGrCrCrCrCrGrUrGrCrUrG-3′) was annealed with B (5′-rCrGrGrGrGrCrCrGrUrCrGrCrCrGrArUrGrGrGrGrGrUrGrUrUrCrUrGrCrUrGrCrUrGrC-3′), C (5′-rCrGrGrGrGrCrCrGrUrCrGrCrCrGrArUrUrGrGrGrGrGrUrGrUrUrCrUrGrCrUrGrCrUrGrC-3′), or D (5′-rCrGrGrGrGrCrCrGrUrCrGrCrCrGrArUrArGrGrGrGrUrGrUrUrCrUrGrCrUrGrCrUrGrC-3′) to generate three kinds of different RNA duplexes . Annealed A/B had 30 nt of perfect duplex with a 5-nt overhang at both 3′ ends, Annealed A/C contained one bulge in the middle of an RNA duplex, and annealed A/D contained an internal loop in the middle of an RNA duplex.6 cells were lysed on ice with 50 μL of radioimmunoprecipitation assay (RIPA) buffer  . After adding different annealed RNA oligos as spike-in, cell lysates were digested in an additional 100 μL of digestion buffer with RNase A/T1 (1:200) at 37 °C for 30 min. RNA was separated on 15% acrylamide Tris/borate/EDTA (TBE)  native gels and stained with SYBR gold . Images were obtained with Chemidoc Imaging system .RNase A/T1 cocktail   was used for enriching RNA duplexes. For optimization, 1 μg of annealed oligos were digested in 150 μL of digestion buffer , pH 7.4) with different concentrations of RNase A/T1  at 37 °C for 30 min. For optimizing the RNase A/T1 condition in the context of cellular RNA, 1 × 106 cell pellets before lysing the cells or extracting total RNA.For enrichment of cellular RNA duplexes, three different methods were performed as described below. To monitor the sensitivity of the following experimental procedure, 50 copies per cell of perfect RNA oligo duplexes  were added into 1 × 106 cells were lysed on ice with 50 μL of RIPA buffer. Cell lysates were digested in an additional 100 μL of digestion buffer with RNase A/T1 (1:200) at 37 °C for 30 min.For digestion of native cell lysate, 1 × 10N,N′-bis(2-ethanesulfonic acid) (PIPES), 1mM EDTA, 400 mM NaCl, 80% formamide, pH 6.4) . The reannealed RNA was digested in 150 μL of digestion buffer at 37 °C for 30 min.For removal of proteins and RNA reannealing, total RNA was extracted from cells with Trizol  following the manufacturer’s instructions. After heating at 95 °C for 3 min, total RNAs were hybridized at 42 °C overnight in 10 μL of hybridization buffer  following the manufacturer’s instructions. After heating at 95 °C for 3 min, treated RNAs were hybridized at 42 °C overnight in 10 μL of hybridization buffer. The reannealed RNA was digested in 150 μL of digestion buffer at 37 °C for 30 min.Digested RNA from cell lysate or reannealed RNA was extracted with acid phenol/chloroform  following the manufacturer’s instructions. After treatment with a DNAse I kit , RNA was separated on 15% acrylamide TBE native gels and stained with SYBR gold (ThermoFisher Scientific). Images were obtained with Chemidoc Imaging system .The digested RNA products were separated on 15% acrylamide TBE native gels. RNA duplexes around 18–50 bp were sliced from gels and broken down by gel breaker tubes . RNAs were eluted overnight at 4 °C, and gel debris were filtered out by 5-μm filter tubes (IST Engineering). Eluted RNAs were precipitated by ethanol.Since RNase A/T1 leaves a 3′ phosphate, RNA duplexes were treated with Shrimp Alkaline Phosphatase (rSAP)  for 30 min. After phenol/chloroform extraction and ethanol precipitation, RNA duplexes were ligated with adaptors as previously reported  with minEluted ligation products were phosphorylated using polynucleotide kinase  and 1 mM ATP for 30 min. After phenol/ chloroform extraction and ethanol precipitation, the products were used for 5′ adaptor ligation with T4 DNA ligase at 16 °C overnight. The 5′ adaptor sequence was 5′-AmMC6/CTACACGACGCTCTrGrUrUrArArCrArG, which is partially complementary with the 3′ adaptor to aid in ligation, and generates a Y-shaped structure in the final product. Ligated products were purified using phenol/chloroform extraction and ethanol precipitation.After denaturing the ligated products for 3 min at 95 °C, reverse transcription was performed using ThermoScript™ Reverse Transcriptase  at 60 °C for 1 h with a specific primer (CCTTGGCACCCGAGAATTCCA). The complementary DNA (cDNA) products were then amplified by PCR using Phusion High-Fidelity DNA polymerase (NEB) to generate a deep sequencing library. The reaction was carried out at 25 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 15 s using the 5′ end primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and the 3′ end primer (CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA).® Reagent Kit v3 (150 cycles)  as per the manufacturer’s instruction on Illumina MiSeq.The PCR products were separated on 15% acrylamide TBE native gels. Sizes of DNA around 143 bp were sliced from gels and broken down by gel breaker tubes (IST Engineering). DNAs were eluted overnight at 4 °C, and gel debris were filtered out by 5-μm filter tubes (IST Engineering). Eluted DNAs were precipitated by ethanol. The concentration of DNA was measured with a Qubit fluorometer (ThermoFisher Scientific). The sizes of the DNAs were confirmed using an Agilent Technologies 2100 Bioanalyzer with High-Sensitivity DNA Kit . Sequencing was performed with Illumina MiSeqSequencing data (in FASTQ format) was obtained for 15 datasets listed in https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE117821). The data discussed in this publication were deposited in the National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus (GEO)  and are Unless a specific tool is mentioned in the description of the data analysis, the R programming language was utilized for data processing.Reference transcript sequences were compiled using four databases: (1) ENSEMBL , (2) LNReads were preprocessed in the form of a sliding window quality score filtering using the Sickle program with default options . Reads wThe RNAduplex program was utilized to identify read-pairs with long reverse-complementary regions . The proThe coverage of reads with respect to the three different treatments was computed by computing the coverage for each transcript . Next, the per-position read coverage was averaged over all positions of a transcript. The resulting per-transcript coverage values were averaged over all transcripts of a gene in order to compute per-gene average coverage values. Utilizing the gene type classification (based on the classification provided by the ENSEMBL database), one can, in this fashion, compute an average coverage value per gene class. For each gene class, the ratio of the average coverage for two treatments was used as enrichment .We searched for chimeric reads that could be matched to two different transcripts in a manner that suggests that endo-ligase-ligated RNA fragments were located in close proximity, effectively performing an RNA proximity ligation experiment . We requIn conclusion, we present a novel experimental protocol coupled with computational analysis applied to several cell line datasets with genomic information about RNA–RNA interactions. We found evidence that RNA–RNA interactions were far less common than one would expect by simply analyzing transcriptome sequences. Also, RNA–RNA interactions were more ubiquitous when removing proteins from cell extracts. The diversity of results from different cell lines suggests that RNA–RNA interactions are to a large extent stochastic events. These findings have important implications for the design of therapeutic RNAs resulting in two strategies of either avoiding or purposefully introducing long RNA helices.


Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was to design and prepare a specific mRNA, without 5’ cap and poly (A) tail transcribed in vitro transcription of the designed construct consisting of 5’ and 3’ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the Core gene of HCV. To design the modified mRNA, the 5’ cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay.Candidate mRNA was prepared by GFP and Core genes were achieved.Cell transfection results showed that despite the absence of 5’ cap and poly (A) tail, the structure of the mRNA was stable. Moreover, the successful expressions of Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.Our findings indicated the effectiveness of a designed IVT-mRNA harboring the  Hepatitis C virus (HCV) is a blood-borne pathogen and an enveloped, single-stranded, and positive-sense RNA virus belonging to Flaviviridae family,2. It isA wide variety of vaccines have been designed so far such as protein-based vaccines like recombinant glycoproteinE1/glycoprotein E2 adjuvanted with MF59 and recombinant HCV core antigen formulated with the T-cell adjuvant IMX, inactivated cell culture-derived HCV virions, virus-like particles presenting HCV envelope proteins, viral-vectored vaccines expressing conserved HCV NS3-NS5B non-structural proteins (Ad6-Nsmut/AdCh3-Nsmut and MVA-NSmut/AdCh3-NSmut vaccines), as well as DNA plasmid encoding the HCV proteins 3/4a,12.in vitro, production of high dose vaccines in a short time is achievable, which allows the rapid production of a vaccine for a new antigen during pandemics[in vitro transcription using a bacteriophage RNA polymerase and a DNA template encoding the antigen of interest [mRNA is a transient carrier of genetic information that is naturally metabolized and does not integrate to the genome. The synthetic mRNA can be easily designed to express any antigen with high efficiency, and it can also act as an adjuvant in vaccine formulations. Since mRNA, in contrast to DNA, is located and acts in the cytoplasm, it easily can transfect all cell types, especially hard-to-transfect cells. Another advantages of using mRNA in a treatment approach are its cost-effective manufacturing and its easy scalability. As mRNA transcription is possible pandemics-17. Typiinterest .Vaccine development using mRNA approaches have advantages over other vaccine methods. mRNA vain vitro transcribed- messenger RNA (IVT-mRNA), as a gene carrier, in subunit vaccines to supply therapeutic proteins. in vitro protein expression of brome mosaic virus and poliovirus RNA has been confirmed[Recently, studies have used confirmed,22, and confirmed. Studiesconfirmed,25. Althconfirmed, IVT-mRNconfirmed. In ordeCore gene and Core/signal peptide (SP) in human DCs.Our aim here was to design and prepare a synthetic mRNA that en codes the most conserved protein of HCV, core protein and also to determine its ability to encode the protein in DCs. In the present study, we have made a sequence consisted of 5’ and 3’ UTRs of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) fragments that enable eIF4G capture for the initiation of translation to express HCV PstI restriction enzyme. This plasmid was constructed under the control of T7 bacteriophage promoter with 5’ UTR, ORF, IRES, and 3’ UTR. The 5’ UTR and 3’ UTR were from human hsp70 along with IRES sequences of human DCs This synthetic construct was provided commercially of HCV-1a strain Tehran 12  was ligated into the pGE-30446-HCE vector using y, China .Core gene using overlap extension PCR method  from pEGFP-N1 vector was amplifiedIn order to secrete the Core protein from the DCs, the sequence of the SP of the CD86 marker (accession number: P42081) on the surface of the DCs was added to the 5’ region of the R method . Four paBstBI restriction enzyme. The accuracy of the constructs was verified by sequencing. and ligaHindIII and GsuI restriction enzymes . The IVT-mRNAs synthesis was performed using RiboMAX™ Large-Scale RNA Production Systems-SP6 and T7 Kit  according to the manufacturer’s instruction. DNA templates were purified using a PCR purification kit and used for in vitro transcription. Transcription of 5-10 µg of the DNA templates was carried out in a final 20–200 µl reaction mixture at 37 °C for 4 hours in order to generate uncapped IVT- mRNA. To remove the template DNA, 1 μl of RQ1 RNase-Free DNase  was added to the IVT reaction mixture and incubated at 37°C for 15 min. Purification of mRNA was performed after DNaseI digestion by Total RNA Purification Kit , according to the instructions provided by manufacturer. The purity and quality of the RNAs were determined using spectrophotometric analysis and run on the agarose gel (1.1%). The mRNAs were stored in the RNase-free water at -80 °C.IVT-mRNAs were synthesized from pGE-Core, pGE-SP-Core, and pGE-GFP vectors , and 1D http://rna.urmc.rochester.edu/RNAstructureWeb). The free energy of the mRNA was calculated using a dynamic planning algorithm to predict the most stable molecule. RNA structure software not only displays the thermodynamic properties of the sequence but also includes an interactive graphical representation of the predicted secondary structure[The secondary structure of designed mRNA was analyzed using the RNA structure software package  were ready for treatment.Human monocyte-derived DCs were generated as described elsewhere. Briefly2 incubator at 37 °C for 18-48 h prior to testing for transgene expression. The expressions of GFP and HCV core protein were measured 24-48 h after transfection by fluorescence microscopy  and Western blotting assay.moDCs were transfected with 1.5 μg of IVT-mRNA using Lipofectamine 2000 , an RNA transfection reagent, according to the manufacturer’s instructions. The modification of IVT-mRNA with lipofectamine was performed as per manufacturer’s protocol. The moDCs were then incubated in a COAlkaline phosphatase as the first and second antibodies, respectively. The protein was visualized by adding BCIP/NBT solution as a detection substrate.After the transfection of imoDCs with IVT Core and GFP mRNA, the transfected and control cells were sonicated in a protein lysis buffer containing 50 mM of Tris, 50% glycerol, 0.1% Triton X-100, 1 mM of anti-protease (phenylmethylsulfonylfluoride). The acetone-precipitated proteins were loaded on 10% SDS-polyacrylamide gel and subsequent Western blot analysis was carried out using positive anti-HCV antibody serum specimens, which had been diluted 1:100 in 1× TBS buffer. After washing, the membrane was incubated with secondary peroxidase-conjugated anti-human IgG antibody for 2 h, followed by washing with TBST buffer. The bands were visualized using the chromogenic substrate 3,3’-diaminobenzidine . The untransfected cells were included as negative controls. The expression of GFP protein in the transfected cells was analyzed in the same way using 1:1500 diluted anti-GFP- antibody  and 1:5000 diluted anti-rabbit antibody conjugated with The structure of synthesized fragment containing 5’ UTR, T7 promoter, multiple cloning site, IRES, and 3’ UTR (915 bp) with and without Core (1497 bp) and SP-Core (1565 bp) proteins coding sequences was predicted using RNA structure program. RNA structure utilizes the nearest neighbor parameters and thermodynamic parameters to predict the lowest free energy structure of RNA. In genePstI restriction site, PCR test was conducted using specific primers  electrophoresis  the lack of efficiency against highly variable and/or persistent pathogens, (B) the development of a cheaper and more flexible technology to use in the absence of resources, and (C) better adaptation for individual or rapid production. Considering the mentioned issues and the characteristics of synthetic mRNA, it seems that synthetic mRNA could be of particular importance in the future of vaccine development, especially for highly variable infectious agents,38.Synthetic mRNA allows creating the optimal immunogenic characteristics of a pathogen by eliminating unnecessary or harmful aspects of a pathogen. Due to its chemical and pharmacokinetic nature, IVT-mRNA may mimic the RNA of viruses; however, it causes an infection in a transient manner. To desiInitiating a translation independent of the 5’ cap has been well understood for viral mRNAs. For the first time in poliovirus, an alternative mechanism was found to start the translation, which occurs independent of the 5’ cap. Some viet al.[6A residue were used for in vitro translation assays. The results showed that the m6A in the 5’ UTR can bind to the eukaryotic initiation factor 3. The analysis of the transcriptome profiles showed that the translation of mRNAs from 5’ UTR m6A decreases with the deletion of m6A methyl transferase[in vivo[et al.[Meyer et al. have defansferase. Also, ie[in vivo. Anothervo[et al. have exavo[et al.. The effvo[et al..in vitro have shown that the absence of 5’ cap and poly (A) compensates with the presence of IRES sequences in the upstream and downstream regions of the gene in the structure of the mRNA, and despite this defect, the translation process is done properly. Considering that most RNA viruses have the same structure and that such a structure is naturally responsive to the expression of viral proteins, it can be modeled to design and synthesize mRNA expressing variant antigens.The analyses of studies followed the translation process without 5’ cap and poly (A) In this study, we considered the natural pattern of the RNA structure of the virus for the synthesis of the IVT-mRNA. Designed HCV RNA does not possess 5’ cap and poly (A) tail . Instead, the IRES element (340 nucleotides) is located close to the 5’ end of the viral genome. The resWe designed a modified HCV antigen-coding mRNA, and computational evaluation showed its stability to create a stable secondary structure. Core antigen-coding sequence of HCV was successfully cloned into GE-30446-HCE vector, and its expression in the imoDCs transfected by transcribed IVT-mRNA confirmed the accuracy of designed mRNA. Considering the stability of the designed mRNA and its efficient expression in the effector cells such as moDCs, the synthesized IVT-mRNA can be used for further attempts in the development of RNA vaccine against HCV.


Theoretical minimal RNA rings code by design over the shortest length once for each of the 20 amino acids, a start and a stop codon, and form stem-loop hairpins. This defines at most 25 RNA rings of 22 nucleotides. As a group, RNA rings mimick numerous prebiotic and early life biomolecular properties: tRNAs, deamination gradients and replication origins, emergence of codon preferences for the natural circular code, and contents of early protein coding genes. These properties result from the RNA ring’s in silico design, based mainly on coding nonredundancy among overlapping translation frames, as the genetic code’s codon-amino acid assignments determine. RNA rings resemble ancestral tRNAs, defining RNA ring anticodons and corresponding cognate amino acids. Surprisingly, all examined RNA ring properties coevolve with genetic code integration ranks of RNA ring cognates, as if RNA rings mimick prebiotic and early life evolution.Distances between RNA rings were calculated using different evolutionary models. Associations between these distances and genetic code evolutionary hypotheses detect evolutionary models best describing RNA ring diversification.Here pseudo-phylogenetic analyses of RNA rings produce clusters corresponding to the primordial code in tRNA acceptor stems, more so when substitution matrices from neutrally evolving pseudogenes are used rather than from functional protein coding genes reflecting selection for conserving amino acid properties.Results indicate RNA rings with recent cognates evolved from those with early cognates. Hence RNA rings, as designed by the genetic code’s structure, simulate tRNA stem evolution and prebiotic history along neutral chemistry-driven mutation regimes. Several early life hypotheses assume that the first genes combined functions of structural RNAs  and protein coding genes , groups codons into palindromic , and 5′- and 3′-extremity dominant . CDA > 0 associates with amino acids that are cognates of class II tRNA synthetases, CDA < 0 associates with cognates of class I tRNA synthetases . Class INatural genomes are frequently characterized by gradients in nucleotide biases, proportional to times spent single stranded during replication. This is typically due to hydrolytic deaminations, resulting in the observed nucleotide bias gradients across genomes due to replication –37 and/oSurprisingly, deamination gradients occur in theoretical minimal RNA rings. These overall start at the 5′ extremity of the RNA ring’s anticodon , as predThese evidences for RNA rings functioning as replication origins match their similarity with ancestral tRNAs , and thePrevious sections show that the simple design of RNA rings implies several properties expected for ancestral multifunctional RNAs, including coding for a peptide, functioning as replication origin, and as plausible proto-tRNAs . FurtherRNA secondary structures can be clustered into two main groups, presumed ancestral tRNA-like secondary structures, and presumed rRNA-like secondary structures. In rRNA-like RNAs, percentages of unpaired nucleotides within stems (forming ‘bulges’) among unpaired nucleotides, is greater than in tRNA-like RNAs. These are likely targets for enzymatic degradation and hence reflect greater regulation of RNAs forming rRNA-like secondary structures , 50.The concept that rRNA-like RNAs are derived from tRNA-like RNAs was tested on tRNAs from all three kingdoms of life and from giant viruses. Cloverleaves formed by tRNAs with each of the possible cognate amino acids were ranked on the tRNA-rRNA secondary structure axis. These estimates derived from secondary structure properties were compared with the genetic code integration orders of the cognate amino acids from various hypotheses on these orders . The worGenetic code inclusion orders of RNA ring cognate amino acids are determined according to the RNA ring’s anticodon, which is predicted from homology with ancestral tRNA loops . This inThese analyses show progressive emergence of the various properties from RNA rings with early cognates, to those with cognates having late genetic code integration ranks. This pattern is not trivial and we have no explanation for it: RNA rings result from rational in silico design, and presumably did not evolve one from the other, and/or from a common ancestor, as any evolutionary scenario would imply. Hence, we do not understand how RNA rings mimick evolutionary trends in so many properties, without any biological-historical context.This issue is addressed here, using pseudo-phylogenetic analyses of RNA rings. In other words, do clusters of RNA rings, based on sequence similarities, associate with evolutionary hypotheses on the order of genetic code integration of their predicted cognate amino acid? This would mean that RNA rings concentrate information on the evolution of the biomolecular translation machinery. This information comes from the genetic code’s codon-amino acid assignments, which are the major information used in the design of RNA rings. This could be either because the genetic code structure embeds information related to the historical processes that formed it, and/or that its structure determines the biomolecular prebiotic and/or early life evolution.The design of RNA rings is based on coding constraints. Their similarity with tRNAs is an unintended result from this design. This tRNA similarity defines the RNA ring’s anticodon and its cognate amino acid. The cognate amino acid genetic code integration order defines RNA ring evolutionary ranks. Hence, in order to understand how RNA ring comparisons reflect prebiotic and early life evolution, we focus on comparisons among RNA rings, aligning them according to their coding properties, with the stop codon at their 3′ extremity Table .Table 1The 25 RNA rings are compared using a simple distance between sequences, considering each combination of two RNA rings, aligned after splicing them at the 5′ extremity of their stop codon. Identical nucleotides at a given position have distance “0”. Non-identical ones have distance “1”. These position-specific distances are summed over the complete RNA ring length, producing pairwise distances among RNA rings. These theoretically range from “0” to “22”, because RNA rings are 22 nucleotides long. Table P = 0.00003, two tailed t-test). After Bonferroni correction considering the 1225 correlations calculated, this corresponds to P = 0.03763, which is still statistically significant at P < 0.05.The distance matrix in Table Note that the genetic code integration hypothesis that associates best with distances among RNA rings is one among the three tRNA-derived hypotheses. This is in line with the RNA ring proto-tRNA hypothesis.The previous section shows that RNA ring clusters match a primitive code in tRNA stems. This raises the question whether distances among RNA rings could be interpreted in an evolutionary sense, considering that RNA rings are rational constructs, and a priori did not evolve one from the other by point mutations. Nucleotide sequence evolution is characterized by a strong bias for transitions, meaning purine-to-purine and pyrimidine-to-pyrimidine mutations , as opposed to transversions, purine-to-pyrimidine and pyrimidine-to-purine point mutations . Transitions are more frequent, reflecting on average shorter evolutionary distances between sequences. Hence, when differences between two RNA rings could be interpreted as due to transitions, distances are set to “0.5”, while when these are due to transversions, distances remain as previously “1”. Table P = 0.00002, two tailed t-test). After Bonferroni correction considering the 1225 correlations calculated, this corresponds to P = 0.02050, which is still statistically significant at P < 0.05.The hypothesis that RNA rings evolved from each other predicts that associations with genetic code inclusion hypotheses for RNA ring cognate amino acids should be stronger for D2 than D1. This is not the case: only 50.37% of all comparisons between associations of genetic code inclusion hypotheses with D1 vs D2 indicate stronger associations with D2. For three genetic code inclusion hypotheses, significant majorities of associations were stronger for D2 than D1 (more than 17 cases among 25 comparisons for each hypothesis): Fox’s proteinoid hypothesis , yields For specific RNA rings, D1 yields significantly more stronger associations than D2 for 7 RNA rings , and vice versa for 9 RNA rings , according to two tailed sign tests. RNA rings with recent cognate amino acids have more genetic code integration order hypotheses for which D2 produces stronger associations than D1 than presumably more ancient RNA rings in 91.8% of the genetic code integration hypotheses examined. Effects are strongest for the RNY hypothesis , the JimP < 0.05 (two tailed tests) for 158 cases, among which 89.9% were positive correlations. The strongest association is between D3 from RNA ring 13, and the primitive acceptor stem code . RNA ring 13 is the barycenter of the sequence space formed by the 25 RNA rings, and its predicted anticodon matches glycine. Hence, it is a likely candidate for the most primitive RNA ring. The heatmap in Fig. r = − 0.5958, two tailed P = 0.00167).Distance D2 is a simplified approximation of known nucleotide substitution patterns. We used observed substitution rates in natural pseudogenes  Table 55 to estiD3 in Table Analyses in the previous section use estimates of sequence distances derived from observed nucleotide substitutions in pseudogenes, which seem to reflect mainly neutral evolution, apparently driven by nucleotide physicochemical properties. Table A further alternative hypothesis is that similarities in codon usages of RNA rings produced a clustering compatible with the tRNA acceptor stem primitive code. We calculated D5, a matrix of similarities between RNA rings according to codon usages. Associations between genetic code integration hypotheses and D5 are much weaker than with D3, excluding that results are due to confounding effects by codon usages.Comparisons between strengths of associations between genetic code inclusion order hypotheses and D1 vs D2 distances among RNA rings imply that ancient RNA rings arose spontaneously, perhaps by template-free polymerization , 64, andPreviously published analyses show how RNA ring properties mimick properties of modern genes (part above horizontal line in the scheme in Fig. This study, and most of our future endeavours, explore evolution upstream of RNA rings, corresponding to the area below the horizontal line in Fig. Distances among theoretical minimal RNA rings converge most with the genetic code integration hypothesis of amino acids derived from the primitive code in tRNA acceptor stems. This convergence is greatest when distances are calculated using the substitution model derived from nuclear pseudogenes. Other substitution models, such as models integrating effects of natural selection on amino acid replacements, produce weaker patterns. Hence, theoretical RNA rings evolved along physicochemical constraints affecting nucleotide substitutions, apparently devoid of effects on their coding properties on amino acid sequences, in line with a pre-translational origin of diversification of RNA rings that would at a later stage become the population of primordial coding and decoding RNAs. The RNA ring system appears as a useful synthetic simulator of prebiotic evolution . This anThe 25 theoretical minimal RNA rings are aligned considering their coding frame as presented in Table The model for D3 develops the principle that distances between non-identical nucleotides differ according to which nucleotide pair is considered. It is based on frequencies of observed nucleotide substitutions in pseudogenes, presumably neutrally evolving sequences. Model D4 uses also observed substitution frequencies for estimating distances between specific nucleotide pairs, but as these were observed for protein coding genes, meaning that substitution frequencies are affected by natural selection due to constraints on protein function. Empirical data on substitution frequencies for D3 and D4 are from Gojobori et al. . Note thr = 1. In order to obtain a distance, we used 1-r as distance D5. The corresponding D5 matrix is used in calculations, as are distance matrices D1-D4.The model for D5 compares codon usages of RNA rings and tests whether patterns of evolution could be confounded by similarities between RNA rings in codon usages, independently from the exact sequence alignment in Table For each distance matrix D1 to D5, further analyses consider separately each row of the matrix, which corresponds to distances from a given focal RNA ring to each of the remaining “target” RNA rings. Correlations between each of these 25 sets of distances and the genetic code integration order of the cognate amino acid of the target RNA ring are calculated. This is done for each of the genetic code integration hypotheses listed by Trifonov . Overall


Streptococcus pneumoniae. Intriguingly, its structural homologue, RumA, can only catalyze the methylation of U1939, while RlmC is the dedicated enzyme for m5U747 in Escherichia coli. In this study, we describe the structure of RlmCD in complex with its cofactor and the RNA substrate containing U747 at 2.00 Å or U1939 at 3.10 Å. We demonstrate that multiple structural features collaborate to establish the dual enzymatic activities of RlmCD. Of them, the side-chain rearrangement of F145 was observed to be an unusual mechanism through which RlmCD can discriminate between U747- and U1939-containing RNA substrate by switching the intermolecular aromatic stacking between protein and RNA on/off. An in-vitro methyltransferase assay and electrophoretic mobility shift assay were performed to validate these findings. Overall, our complex structures allow for a better understanding of the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of the RumA-type enzyme and the potential development of antibiotic drugs against S. pneumoniae.Methyltransferase RlmCD was previously shown to be responsible for the introduction of C5 methylation at both U747 and U1939 of the 23S ribosomal RNA in  Streptococcus pneumoniae.Methylation of nucleotides widely occurs on ribosomal RNAs (rRNAs) in all living organisms; some clusters at the functional centers of rRNA are important for protein translation. In pathogenic bacteria, it has been shown that modulation of the methylation state of some rRNA nucleotides serves as a clinically important mechanism to confer resistance to ribosome-targeting antibiotics. Because some methyltransferases can introduce methylation at different target sites, understanding the functional roles of each individual methylation site in rRNA has become meaningful but remains challenging. In this research, using protein-RNA crystal structures, we demonstrate that multiple structural determinants collaborate to establish the dual enzymatic activities of RlmCD, which has been previously shown to catalyze C5 methylation at both U747 and U1939 of 23S rRNA in some gram-positive bacteria. This structural biology study provides a new perspective for us to understand the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of RumA-type enzyme and the potential development of antibiotic drugs against  E. coli, respectively, with a variety of methylation types including 1-methylguanosine (m1G), 3-methyluridine (m3U), 5-methyluridine (m5U), 5-methylcytidine (m5C) and 2-methyladenosine (m2A) . In. In29]. To uncover the structural features determining the multi-selectivity of RlmCD for RNA substrates, further work has been conducted for this report to obtain the crystal structures of RlmCD in complex with re-designed U747 RNA substrates and the U1939 RNA substrate. Through careful structural comparison between RlmCD complexed with U747 and U1939 RNA substrates, we identified multiple crucial residues that behave differently in two specific recognitions. Further in-vitro MTase and EMSA assays were employed to confirm our observations from the structures. The structural insights we gained in this research provide a new perspective for us to understand the delicate mechanism that RlmCD employs to recognize different RNA substrates.740GGCACGUm5UGAAAAGUGCC757-3′) analogous to the major portion of the U747-methylated 23S rRNA hairpin 35 in S. pneumoniae  or 12-nt  U747-containing RNA analogue (743C-G754 and 744C-G753) was therefore crystalized with the construct of 1–454 of RlmCD containing a E443Q mutation, using S-adenosyl methionine (SAM) as the cofactor. The 743–754 base-pair in U747SL was modified to a G-C from an A-U to increase the stability of the RNA structure. A similar RNA design was also applied to co-crystalize the RlmCD with SAM and an 8-nt U1939-containing RNA stretches    to underSL complex structure crystalized in space group P1, each asymmetry unit contains two complex molecules that pack together through RNA-RNA intermolecular base stacking  as the search model , and thestacking . All strα are 0.997 Å for U747L complex and 0.995 Å for U1939L complex), without obvious relative motion between different domains upon the RNA binding  in RlmCD-SAH-U747SL show no obvious interaction with RlmCD, extending outward from the RNA catalytic groove. Given the higher resolution of RlmCD-SAH-U747L complex structure, further analysis of protein-RNA interactions for U747-containing RNA substrates was mainly based on the RlmCD-SAH-U747L ternary complex. Interestingly, in the RlmCD-SAH-U1939L complex structure, the U1939-containing RNA substrate also adopts a very similar conformation to that of U747L, even though their sequences are totally different  from the C-terminal catalytic domain by either a hydrogen bond or face-to-edge aromatic interaction. The first three residues and E443 are all highly conserved in RumA-like enzymes . BecauseL. The influence on the MTase activity and RNA binding of RlmCD by R127 and Q162 was therefore investigated by site-directed mutagenesis  binds at the catalytic groove formed by the central and catalytic domains of RumA, while the 3′-side hairpin (G1945-C1961) binds in the cleft formed between the N-terminal OB fold domain and the catalytic domain [L structure we determined in this research contains only the A1936-U1943 of 5′-end loop, we therefore focused on a comparison of the RNA binding within the catalytic grooves of RumA and RlmCD. The majority of nucleotides within A1936-U1943 maintain a very similar conformation in the two complex structures, except A1936 and A1937  that are responsible for its dual enzymatic activity towards C5 methylation of U747 and U1939, especially when these residues are all strictly conserved in YefA .L. Considering the opposite effects of Q162A mutation on MTase activities of RlmCD for U747 and U1939 RNAs, we concluded that although Q162 may not seem to be an evolutionary mutation beneficial for RlmCD MTase activity towards U1939, it does establish the binding specificity of RlmCD for U747-containing RNA.Of these residues, N249 behaves differently in interactions of RlmCD and two different RNA substrates but with a more significant impact on U747 methylation, confirming our previous conclusion that the linker containing N249 is a newly evolved structural determinant that helps RlmCD to obtain additional MTase activity for U747 . As anotL and U1939L show that F145 adopts multiple conformations for its side-chain under different circumstances , and RumA (F148). Unlike the RumA structures in which the conformations of F148 side-chain remain almost the same with and without RNA substrates, the structures of RlmCD in the absence and presence of U747mstances . Upon biL and U1939L unveil multiple structural features to explain how RlmCD can specifically recognize U747 RNA while it remains enzymatically functional for the methylation of U1939. The aromatic side-chain rearrangement of F145 was observed to be an unusual strategy used by RlmCD to discriminate between two different sites on 23S rRNA by switching on/off the intermolecular π-π stacking between protein and RNA; dit Konté and coworkers recently reported a similar mechanism employed by RRM3 (RNA recognition motif 3) of CUG-BP2 (GUG triplet repeats RNA binding protein) to discriminate its two U-rich RNA substrates [Overall, our crystal structures of RlmCD complexed with U747bstrates . Both th5U RNA MTases evolved from a common ancestor, which is most likely an RumA-like enzyme that originated amongst bacteria [E. coli m5U MTase, RlmC and TrmA [It has been suggested that all contemporary mbacteria . RumA hoand TrmA , are resand TrmA . In receand TrmA .5U sites on 23S rRNA unique to B. subtilis and S. pneumoniae, or is this feature prevalent in gram-positive bacteria? Based on our structural findings in this research, a further bioinformatics study should be employed to provide hints to answer this question.Although both RlmCD and RlmC can introduce C5 methylation at U747 of 23S rRNA, their RNA-recognition mechanisms may differ, as the residues contributing to the enzymatic activity of RlmCD for U747  are not conserved in RlmC, and RlmC does not possess a TRAM domain . Most liS. pneumoniae is the major cause of bacterial pneumonia and meningitis, as well as bloodstream, ear, and sinus infections. Due to the global antibiotic resistance crisis, it has been estimated that ~1.2 million illnesses and ~7,000 deaths per year are caused by S. pneumoniae infection. Its drug resistance has been listed as one of serious threats to human health by the CDC [5U747 locates in the loop region of hairpin 35, protruding into the large subunit tunnel for extension of the nascent peptide. Although U747 methylation has been recently found to contribute to TEL susceptibility through promoting efficient RlmAII-mediated G748 methylation in S. pneumoniae, its precise functional role must be further illustrated. The m5U747 is close to NPET and itself may become a target for the design of new antibiotics against diseases and syndromes induced by S. pneumoniae. the CDC . In 23S 5U747 in S. pneumoniae or other related pathogens [In this research, although multiple residues were shown to contribute simultaneously to the specific recognitions of RlmCD for two different sites on 23S rRNA, the single mutant F145A alone can abolish the MTase activity of RlmCD for U747 while its enzymatic activity for U1939 remains almost intact, exhibiting a relatively ‘clean’ effect in decoupling the dual MTase activities of RlmCD; however, this result must be further confirmed by in-vivo experiments. F145A therefore provides us with an unprecedented opportunity to illustrate the functional role of U747 methylation in cells without affecting the methylation level of U1939 in 23S rRNA under normal conditions and certain stress conditions, which may eventually assist in the development of new antimicrobial drugs targeting at mathogens .S. pneumoniae genomic DNA via PCR. The full-length RlmCD (residue 1–543) and C-terminal truncated RlmCD (residue 1–454) were subcloned into a modified pET28a (Novagen) vector with an 8×His-SUMO tag and a ULP1 cleavage site (pET-SUMO vector) at the N terminal site. All single residue mutants were generated using MutanBEST kit (TaRaKa) and verified by DNA sequencing. The plasmids were transformed into E. coli BL21-Gold (DE3) cells.The RlmCD (sp_1029) ORF was amplified from 600 reached 0.8, and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. After induction, cells were grown at 16°C for an additional 24 hours before harvesting. The cell pellets were suspended with binding buffer , and lysed by sonication. After centrifugation, the supernatant was purified using Ni-NTA affinity chromatography (Qiagen). The eluted SUMO-tag protein was loaded on Superdex 200(16/60)  equilibrated with binding buffer for further purification. The elution sample was mixed with ULP1 enzyme against storage buffer  to remove the SUMO tag. After overnight cleavage, the mixture was further purified by Superdex 200(16/60) , again with storage buffer.For wild-type and mutant protein expression, cells were grown in LB medium at 37°C until the ODL RNA (5′-745GUUGAAAA752-3′), U747SL RNA (5′-743CCGUUGAAAAGG754-3′), FAM-U747SL RNA (5′-FAM-743CCGUUGAAAAGG754-3′), U1939L RNA (5′-1935AAAUUCCU1943-3′), 30-nt FAM-U1939 RNA (5′-FAM-1933GCGAAAUUCCUUGUCGGGUAAGUUCCGACC1962-3′) were purchased from TaRaKa Bio Inc. and dissolved in diethyl pyrocarbonate (DEPC)-treated water to a final concentration of 2 mM. The 30-nt U1939 RNA (5′-1933GCGAAAUUCCUUGUCGGGUAAGUUCCGACC1962-3′) for the in-vitro MTase assay was obtained using in-vitro RNA transcription by T7 RNA polymerase, as previously described [Unmodified and 5′-6-carboxyfluorescein (FAM)-labelled RNA oligomers, including U747escribed . The nuc2 and 10% DEPC at 298 K for 1 hour.RlmCD (1–454) E443Q mutant was concentrated to ~6 mg/mL in storage buffer . RlmCD concentration was determined by its ultraviolet absorption at wavelength 280 nm. The extinction coefficient of full-length RlmCD was 0.78 and RlmCD (1–454) was 0.71. The enzyme-cofactor-RNA covalent complex was prepared by incubating 0.117 mM RlmCD with 0.234 mM SAM and the 0.129 mM RNA substrate at a molecular ratio of 1:2:1.1 in storage buffer supplemented with 1 mM MgClL for data collection were finally refined under 0.1 M HEPES, pH 7.5, 36%(w/v) PEG600; Crystals of RlmCD-SAH-747SL were finally refined under 0.2 M ammonium acetate, 0.1 M sodium acetate, pH5.2 20% (w/v) PEG3350; Crystals of RlmCD-SAH-1939L were finally refined under 0.1 M MES, pH 5.5, 0.15 M ammonium sulfate, 25% PEG4000. All the pH was adjusted at 298K.The initial crystallization trial was set up using the hanging drop vapor diffusion method at 293 K. Each sample was mixed with buffer in a 1:1 ratio to equilibrate against a 100-μL reservoir solution. The crystals started to appear after 1 week at 293 K. Crystals of RlmCD-SAH-747The size suitable crystals (fitted with 0.1–0.2 mm cryoloop) were transferred directly into cryo-protectant flash-frozen into liquid nitrogen. The cryo-protectant was prepared by mixing 7 μL crystallization buffer with 3 μL glycerol. X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) Beamline 19U1 or 17U. Intensity data were indexed and scaled by HKL2000. All complex structures were solved by molecular replacement with the program MOLREP in CCP4i using apo RlmCD (PDB ID: 5XJ1) as the search model . The RNA2. The final concentrations of FAM-labelled U747SL RNA or 30-nt U1939 RNA and SAH were 0.5 and 5μM. The final concentrations of RlmCD or its mutants are 0, 0.1, 0.2, 0.4, 0.8, 1.0, 2.5 and 5.0 μM, as indicated in 2O, 1.5 mL 40% Acrylamide: Bis-acrylamide (19:1), 0.6 mL 10X TBE buffer, 30 μL 10% APS, and 3 μL TEMED. Subsequently, gels were scanned using a Typhoo FLA 7000 biomolecular imager.EMSA was performed in a 10-μL reaction mixture containing 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, and 1 mM MgClThe MTase assay was performed as previously described .S1 FigSL (A) and RlmCD-SAH-U1939L (B). Electron density maps with 2Fo-Fc calculated at 1.0σ shown for SAH, RlmCD C408, and all RNA nucleotides. (C) The proposed catalytic mechanism of m5U MTase RlmCD.Close-up of active site in RlmCD-SAH-U747(TIF)Click here for additional data file.S2 FigSL complex, each asymmetry unit contains two complex molecules (lime and pink) that pack together through RNA-RNA intermolecular base-stacking. (B) Superimposition of complex structures of RlmCD-SAH-U747L (marine) and RlmCD-SAH-U747SL (pink) with an RMSD for Cα atoms of 0.4 Å. (Inset) Close-up of overall structures of U747L and U747SL. Orientation of structures is slightly adjusted for clarity.(A) In crystal of RlmCD-SAH-U747(TIF)Click here for additional data file.S3 FigL (A) and RlmCD-SAH-U1939L (B).Schematic view of protein-RNA interactions in RlmCD-SAH-U747(TIF)Click here for additional data file.S4 FigL (except U747) and surrounding RlmCD residues. (D) Aromatic stacking in RlmCD-SAH-U747L structure. RlmCD residues in different complex structures are shown in blue and yellow, respectively. (E-G) Interaction details between individual nucleotides of U1939L (except U1939) and surrounding RlmCD residues. (H) Aromatic stacking in RlmCD-SAH-U1939L structure.(A-C) Interaction details between individual nucleotides of U747(TIF)Click here for additional data file.S5 FigConserved residues are shown in white on a red background, and similar residues are shown in red in a blue rectangle. Conserved residues participating in U747 recognition between RlmCD and YefA  are labeled with a black asterisk.(TIF)Click here for additional data file.S6 FigFree RNA and shifted protein-RNA complex are labelled.(TIF)Click here for additional data file.S7 FigFree RNA and shifted protein-RNA complexes are labelled. Two shifted bands for protein-RNA complexes are observed in all EMSA experiments of U1939 RNA and we proposed that the upper band may be induced by the nonspecific binding of 30-nt RNA for an extra RlmCD molecule under high protein concentrations.(TIF)Click here for additional data file.S8 FigInset) Close-up of overall structures of U1939L RNAs in two models. (B) Stick model of U1939L RNA in 3.24 Å RlmCD-SAH-U1939L complex structure. 2Fo-Fc electron density map is calculated at 1.0σ.(A) Superimposition of 3.10 Å (yellow) and 3.24 Å (aquamarine) RlmCD-SAH-U1939L complex structures. ((TIF)Click here for additional data file.


West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV. West Nile virus (WNV), a positive-stranded RNA virus in the Japanese encephalitis serocomplex, is a mosquito-borne flavivirus of significant world-wide public health concern. Isolated in Uganda in 1937, the virus is endemic in Africa, parts of Eurasia, the Americas, and Australia ,3. FirstInfections are typically asymptomatic or subclinical, although about 20% of WNV cases develop mild, flu-like symptoms that last between two and seven days. In less than 1% of cases, WNV infection progresses to neuroinvasive disease, characterized by meningitis, encephalitis, and/or acute flaccid paralysis . In a suTo address this need, we optimized extraction protocols for WNV RNA from whole blood (WB) and urine (UR) for PCR detection in order to evaluate the persistence of WNV RNA in a variety of serially-collected body fluid specimens from a large cohort (n = 184) of WNV-infected study participants in Southeast Texas.Prior to testing of the archived body fluid specimens from the Houston West Nile Cohort (HWNC), our methods for RNA extraction from frozen WB and UR specimens were evaluated for efficacy in the recovery of WNV RNA. Controlled spiking of specimens from WNV-negative subjects validated that the original extraction protocol conferred a limited recovery of WNV RNA from WB specimens (19% average across the three spiking loads), while a complete restoration of WNV RNA recovery from WB was attained with a slightly adjusted procedure . As simiA total of 352 WB specimens from 184 HWNC participants with available WB samples were tested for the presence of WNV RNA, with a mean time of collection of 1612 days post onset (DPO) . A total of 59 of the 352 WB specimens (16.8%) tested positive for WNV RNA, collected from 19 different subjects within 126 DPO. Five subjects with only negative specimens had the first available specimen collected within 150 DPO (range 15–705 DPO).The viral load in WNV RNA-positive WB specimens was relatively low for all specimens: below the lower limit of quantification for six of the specimens and ranging between 1.0 and 21.2 cp/μl for viral loads that could be quantified. A trend of decreasing WNV RNA concentration was demonstrated in specimens consecutively-collected from the same patient over time. When estimating the time to loss of WNV RNA detection in WB, temporal data for the thirteen subjects with multiple WB specimens positive for WNV RNA were interval-censored, while those for the remaining six subjects with single positive specimens were right censored. The time data for the 165 subjects with only negative WB specimens was left-censored for this analysis. Weibull distribution modeling of the time to loss of WNV RNA detection in WB was calculated. The median time for loss was at 79 DPO with the 95th percentile placed at 119 DPO .Nine acutely-infected participants were prospectively enrolled in the cohort during the 2018 West Nile season, from which a total of 38 UR specimens were collected. The median time of UR specimen collection was 24 DPO , and the first collection available for each subject ranged from 4–21 DPO. A total of 10 UR specimens tested positive for WNV RNA, all collected within the first 18 DPO and representing samples collected from 7 participants. The two remaining participants did not have detectable virus in their urine at enrollment (18 and 21 DPO) . The virWe evaluated the effects of freezing on RNA detection for four of the aforementioned WNV RNA-positive UR specimens. Initial viral loads for these UR samples pre-freezing ranged from below the level of quantification (0.2 cp/μl) to 1.03 cp/μl. Aliquots of the fresh UR specimens were frozen at −80 °C for at least 130 days, then thawed, extracted, and run by PCR to assess reproducibility of viral RNA detection post-freezing. Following 161, 177, and 198 days of storage at −80 °C, three of the previously-positive UR specimens from two different participants tested negative by PCR using our optimized method of extraction. In two cases, these values were 0.6 and 0.19 cp/μl less than the viral loads calculated for the same UR specimen prior to freezing. One of the four UR specimens exhibited a higher viral load after 199 days of freezing (0.39 cp/μl), when compared to the viral load from the original collection date (BLQ). Thirty-four saliva specimens were collected from the 2018 cohort participants between 4 and 108 DPO and tested for WNV RNA. Of these specimens, two tested positive from a single participant, collected 4 DPO and 9 DPO. Viral loads for both WNV RNA-positive saliva specimens were too low to determine.Four semen specimens from three participants were screened for WNV RNA. Specimens were collected 9–34 DPO , and three were negative in accordance with the Ct unit cutoff value defined in our protocols. However, a single semen specimen collected on 22 DPO met the criteria for a positive sample, although it was very close to the defined cutoff (39.7 vs. a cutoff of 40.0).Our comparison of extraction methodologies using spiked specimens demonstrates that small adjustments to QIAamp MinElute Virus Spin Kit protocol facilitate the complete recovery of WNV RNA for PCR detection from “as is” WB specimens, despite undergoing a freeze-thaw cycle, similar to what is already published for ZIKV . AlthougIncluded in our analysis were 352 WB specimens collected in the range from disease onset to 8.8 years post WNV infection, of which 59 were positive for WNV RNA with a low level of target RNA quantity. Consistent with previous studies for WNV and other flaviviruses, such as dengue virus and ZIKV, all WNV-positive specimens detected were collected within 126 DPO ,30,31,32A possible, although unlikely, restriction of our study was the potential for WNV RNA degradation in WB specimens during storage. We anticipate this effect to be minimal because, unlike the harsh chemical composition of UR, the serum fraction of WB is a perfectly formulated buffer and widely used for the cryopreservation of viral specimens. In fact, eight of the WB specimens that we identified as WNV-positive were in frozen storage for almost six years and one had been archived for nearly 11 years.We recovered WNV RNA from 10 of the 38 UR specimens, from 7 participants up to 18 DPO, adding further to the burgeoning evidence for detection of WNV RNA in UR in the acute stage of the disease ,22,40 anIt was recently demonstrated that low levels of ZIKV in spiked urine specimens are susceptible to diminished detection by PCR after storage for 10 and 30 days at −80 °C, which was reversed with the addition of preservatives before storage . AlternaTwo of the 34 saliva specimens tested were positive for WNV RNA. No previous reports exist for the presence of WNV RNA in human saliva, though viral RNA has been recovered from the saliva of participants infected with ZIKV ,42 and dThe presence of viral RNA in semen and its potential to contribute to the sexual transmission of arboviruses has been well-established in the case of ZIKV  and descOptimizing detection of WNV in body fluids is crucial for research and diagnostics. Although the use of archived UR specimens in the investigation of chronic viral shedding may be limited by storage time, WB specimens prove to be valuable specimens for the prolonged detection of WNV RNA. The persistence of WNV in WB of affected individuals was confirmed by this study, which also facilitated calculation of the best estimate to date for the long-term presence of WNV in systemic circulation within infected participants. In future studies, it will be important to determine how WNV RNA persistence correlates to immunity and complications of disease. The HWNC was initiated in Texas in 2002. WNV-infected participants were identified through surveillance by the city of Houston and Harris County local health departments. WNV-positive cases were also located through blood donation screenings performed by the Gulf Coast Regional Blood Center. All detected WNV cases were invited to participate in the WNV longitudinal cohort study. No exclusion criteria were defined for age, gender, race, or ethnicity. Several control subjects, who did not have clinical or diagnostic evidence of WNV infection, were also enrolled in the study. Consents were obtained from cases and controls willing to participate via approved IRB protocols. All participants provided written consent to participate. Over 300 participants were enrolled in the HWNC between 2002 and 2018. The study protocol and consent form were approved by the University of Texas Health Science Center at Houston Committee for the Protection of Human Subjects  and Baylor College of Medicine Institutional Review Board .Upon enrollment into the HWNC cohort, an extensive interview was conducted to collect baseline characteristics ranging from demographic and health history data to physical functioning index and mental state score. An abbreviated questionnaire was used during follow-up interviews at six months intervals to keep track of relevant clinical symptoms. During initial and follow-up visits with the HWNC participants, serum, plasma, WB, and UR specimens were collected, aliquoted by 1 mL, and stored at −80 °C. For participants enrolled in 2018, saliva and semen were also collected and stored. All specimens collected in 2018 were tested for WNV RNA within 7 days of collection or as otherwise indicated. Fresh WB and UR specimens from WNV-negative donors were spiked with WNV virus, frozen at −80 °C for 24 h, and then processed for RNA extraction. The interval of freezing served to mimic the effect of storage temperature and freeze/thaw cycle of the archived specimens. The viral specimen used for spiking was a cell culture supernatant of Vero cells infected with WNV strain 2004Hou3, isolated from a squirrel in Houston, TX in 2004 [g, and elution incubation done for 5 min at 56 °C. In the current study, 200 µL aliquots of WB samples were processed as is, without obtaining packed cells fraction and without increasing lysis volume, as in Gorchakov et al. For UR specimens, 70 µL of UCB  were added to the still-frozen 1 mL aliquots and then thawed with constant mixing. UR specimens were centrifuged at 3,000 × g for 15 min, pellets were dissolved in the QIAGEN kit lysis mix , and processed by the manufacturer’s protocol starting with the Protease incubation step. RNA extraction from the HWNC WB and UR specimens was performed according to the optimal procedures described above.Frozen spiked specimens were thawed, and RNA was extracted with a QIAamp MinElute Virus Spin kit (QIAGEN) directly by the manufacturer’s protocol with elution in 30 µL of AVE buffer. Optimized RNA extraction procedures for WB (packed cells fraction) and UR specimens were previously determined for this extraction kit . AccordiTo screen the saliva and semen specimens of participants, RNA was extracted with a QIAamp MinElute Virus Spin kit (QIAGEN) directly by the optimized manufacturer’s protocol with elution in 30 µL of AVE buffer. This protocol was previously determined to be sufficient for extraction with other flaviviruses .In order to screen the extracted specimens for WNV RNA presence, 5 µL of extracted RNA from each specimen were used in a single 20 µL rRT–PCR reaction with TaqMan Fast Virus 1-Step Master Mix  on a ViiA 7 Real Time PCR System (Thermo Fisher Scientific) with a TaqMan WNV NS1 assay developed in our laboratory. The following controls were included in each rRT–PCR run: positive WNV RNA control, extraction negative control, and no template control. In addition, each extracted RNA specimen was tested in a separate rRT–PCR reaction with human RNase P TaqMan assay  as the iWNV RNA recovery from spiked negative specimens was considered acceptable if the specimen Ct values was within one Ct unit of the expected Ct value  × 100%The time of body fluid specimen collection was measured in DPO and corresponded to the difference in days between the date of specimen collection and either the date of West Nile disease symptoms onset or, for asymptomatic cases, the date of positive index blood donation. Subjects with at least one set of specimens collected in the same year as their WNV diagnosis, but without indication of acute infection at the time of collection , were excluded from these analyses. In the case of subjects with an unknown date of infection, the onset date was imputed as 15 September of the year of WNV diagnosis (no such imputation had to be done for the specimens with DPO <150). The time until loss of WNV RNA detection in a given body fluid was defined in DPO of the specimen with the negative WNV RNA test after the last positive WNV RNA test, so as to not exclude cases with possible intermittent shedding. All WNV cases were assumed to have positive tests for WNV RNA in both WB and UR at the time of disease onset. Data were right-censored for subjects with last available specimens that were still positive. Data were left-censored for subjects with only negative available specimens. Data were interval-censored if the time between the last positive and the first negative specimens was over one week for a given subject. STATA 12.1 software  was used to generate a Weibull distribution model in order to estimate the survival function for the time until loss of WNV RNA detection in WB as well as the median time and 95th percentile time for the outcome.