Bryozoan functional groups encompassed 20 taxa and Celleporaria atlantica  dominated the framework at all cores. While the prevalent mesotrophic conditions may have driven suspension-feeders’ dominance over photoautotrophs and mixotrophs, we propose that a combination of historical factors with the low storm-disturbance regime of the tropical South Atlantic also contributed to the region’s low diversity, and underlies the unique mushroom shape of the Abrolhos pinnacles.In major modern reef regions, either in the Indo-Pacific or the Caribbean, scleractinian corals are described as the main reef framework builders, often associated with crustose coralline algae. We used underwater cores to investigate Late Holocene reef growth and characterise the main framework builders in the Abrolhos Shelf, the largest and richest modern tropical reef complex in the South Western Atlantic, a scientifically underexplored reef province. Rather than a typical coralgal reef, our results show a complex framework building system dominated by bryozoans. Bryozoans were major components in all cores and age intervals , accounting for up to 44% of the reef framework, while crustose coralline algae and coral accounted for less than 28 and 23%, respectively. Reef accretion rates varied from 2.7 to 0.9 mm yr Such living coralgal reefs may reach thousands of square kilometers in shallow tropical and subtropical regions and encompass the world’s most biodiverse marine ecosystems2. Reef distribution, structure and morphology vary as a function of the geological, climatic and biogeographic histories of each ocean basin, coupled with modern climate-oceanographic conditions. For instance, benthic community structure, including the relative abundance of reef-builders and eroders, may vary sharply in small spatial scales of a few meters, being influenced by depth, light, nutrient, sedimentation, current and turbulence4. In addition, biodiversity patterns and community structure also respond to latitudinal and longitudinal gradients of thousands of kilometers, within and among ocean basins5.Shallow-water tropical and subtropical reefs are rigid carbonate structures built mainly by corals and crustose coralline algae (CCA) during the Cenozoic7. Reef structures develop as well-known morphological types that range from patch and fringing reefs to barrier reefs and atolls8. The tropical South-western Atlantic (SWA) represents one of the world’s smallest and poorest coral reef provinces9, with high endemism levels of up to 50%. In the SWA, the largest and richest coral reefs occur in the Abrolhos Shelf 11. The unusual mushroom-shaped morphology of the Abrolhos reefs and the low coral richness of the SWA was noted in the 19th Century13, but framework building within this entire region remains poorly understood.In the tropical and subtropical portions of the Indo-Pacific and the Caribbean it is well established that reef frameworks are built by species-rich assemblages of scleractinian corals and coralline algae14. Emergent and quasi-emergent reefs developed during the Holocene and occur in a near-shore and a mid-shelf arc, 12 and 60 km offshore respectively15, while mesophotic reefs with erosive topographies occur in the mid-outer shelf and near the shelf edge11 , in association with other corals and CCA16.The outer Abrolhos Shelf is largely covered by rhodoliths (CCA-structured nodules) and the inner shelf is covered by siliciclastic-carbonate mixed soft sediments16 and elsewhere, within and outside the tropical SWA. We also compiled meteoceanographic historical data in order to support our interpretation of reef morphology, biodiversity and benthic assemblage structure, within the context of the low-disturbance regime of SWA reefs.Here we examine the Late Holocene (~2000 yrs BP) framework of reef pinnacles with tops at 4, 15, and 25 m water depth in the Abrolhos Shelf, based on samples obtained from rotary drilling within the mid-shelf arc. We analyzed accretion rates and reef-builder assemblages, and compared our data with available information from Abrolhos near-shore reefsMussismilia braziliensis, M. harttii  and Millepora spp.  was the most conspicuous coral species. The marginal pinnacles with tops below 10 m depth lacked the mushroom-shaped morphology . These reefs occurred either in a shallow  and in a deeper margin  of the reef area. In general, pinnacle distribution showed a greater density in the shallower region, transitioning to more sparsely-distributed pinnacles in the deeper margin of the reef Fig. . These ipp. Fig. . Conversogy Figs  and 2B a14C AMS dating of coral, CCA and bryozoan components, reef sequences span 387–757 (4 m depth core), 463–1,199 (15 m depth core), and 224–1,897 cal years BP (25 m depth core)  to 2.7 mm.yr−1 in the shallowest pinnacle (top at 4 m) (Table −1 (25 m).Based on m) Table . AverageCelleporaria atlantica  dominated the framework at all cores and depths, followed by two undescribed species, Parasmittina sp. nov. 1 and Stylopoma sp. nov. 2. Five species occurred at all depths , Parasmittina sp. nov. 1, and Stylopoma sp. nov. 1 and 2), while three were restricted to the 25 m depth pinnacle , Gemelliporina glabra  and Metrarabdotos sp.). Multilayer encrusting was the most common growth form, with 10 species growing as colonies with more than two consecutive layers, six of which with more than 10 layers. Bryozoans were frequently intergrown with CCA. The few erect bryozoan species  and Crisia sp.) occurred mainly at the 25 m depth pinnacle , whereas the cores from walls and deeper reefs were dominated by sciaphyllic taxa such as Sporolithon  and encompassed 12 taxa. The cores from both the shallower and intermediate depth pinnacles’ tops comprised species typical of the shallow-water local assemblage–28% and hon Figs  and S3.Favia sp.), and massive slower-growing “stress-tolerant” species. These latter include both shallow water (e.g. Mussismilia) and wider depth-ranging species . The only branching forms were fast-growing shallow-water hydrocorals (Millepora spp.).Corals and hydrocorals reached 23% of the framework in the shallower pinnacle, decreasing to 9–12% in the deeper ones Figs  and S3. 18 described a reef framework “dominated by corals”, with bryozoans being “very common and sometimes abundant”. The first study of the drowned reefs off the Amazon river19 also described a mixed-framework composed by polychaetes, foraminiferans, bryozoans and mollusks.The reef framework of the Abrolhos mid-shelf pinnacles deviates from the archetypal coralgal reefs of the Indo-Pacific and the Caribbean, being dominated by suspension-feeding bryozoans interlayered with CCA, with minor contributions from corals. Instead of being a mere local outlier, this particular framework-building system may be more widespread than previously acknowledged in marginal coralline provinces such as the SWA. For instance, the single coring study from the Abrolhos nearshore reef arc20. Encrusting bryozoans are rare on the surface of the Abrolhos pinnacles tops, where CCA and corals dominate the benthic assemblage, being more frequent on the dim walls21. Experimental work with colonization plates22 revealed that bryozoans were the second most abundant group, following CCA. They were particularly fast-growing on the shaded surfaces of22, especially C. atlantica , are only recorded in Joulters Reefs, Bahamas23. These reefs thrive in shallow water (4 m deep), with a succession of cheilostome bryozoans (9 species) dominating the base (60–70% of the framework) and corals and CCA in the upper portions.Bryozoan dominance in Late Holocene-modern tropical reefs have rarely been reported. To our knowledge, small patchy reefs built by bryozoans, especially 24. Shallow-water reefs dominated by bryozoans were widespread in the Paleozoic25, with scleractinian reefs becoming increasingly important since the Late Triassic27. Concurrently, paleolatitudinal patterns of sediment-producing bryozoans shifted from a pan-tropical distribution in the Paleozoic to a more extratropical range during the Post-Paleozoic28. Giant Eocene shelf edge bryozoan mounds are described along the Great Australian Bight, the largest Cenozoic cold-water carbonate province30. Although temperate and deep assemblages are not a perfect analogue to Abrolhos, increased bryozoan diversity and biomass occurred during Pleistocene lowstands in southern Australia30, associated with increased subtropical convergence and upwelling. In Miocene reefs in the Kutai Basin (Indonesia), bryozoans are restricted to coral sheetstone facies and diverse bryozoan assemblage occur encrusting the undersides of thin platy corals embedded in a muddy matrix32. These species-rich coral communities formed low-relief buildups lacking rigid frameworks in nearshore turbid environments33. By contrast, bryozoans are absent in deposits with high siliciclastic input, and in rigid frameworks of tabulate and branching corals and coralline algae, which formed under lower terrestrial influx32.When bryozoans are considered in the context of longer geological time-scales, the present is not a reliable key to the past35. The Abrolhos reefs are within 15–30 m water depths and under 21.7–29.6 °C, i.e. within the ranges where coralgal reef growth predominates circutropically36. Bryozoan dominance in the reef framework seems to be primarily associated to the region high nutrient and turbidity. The eastern coast of tropical South America receives substantial riverine input and terrigenous sedimentation37. Dissolved inorganic nitrogen and phosphorous concentrations in Abrolhos are above commonly assumed eutrophication thresholds38, and phytoplankton productivity is significantly high39. Remote sensing-derived chlorophyll a annual values range from 0.38 to 0.79 mg.m−340, corresponding to a persistent mesotrophic condition41 that is enhanced by shelf-edge upwelling during the wet season (spring/summer). Turbidity is overall high and increases seasonally due to re-suspension of biogenic sediments42. Therefore, oceanographic conditions deviate sharply from those of the archetypal Indo-Pacific and Caribbean coralgal reefs, determining the unique reef-building assemblages. With the exception of oceanic islands44, Brazilian reefs develop under similar high turbidity and nutrient levels19, comprising foremost examples of turbid zone reefs with low diversity but significant coral cover46. Nevertheless, modern and ancient examples include turbid zone reefs that can present high coral diversity46, and even high bryozoan diversity32, but the latter largely occur at low biomasses and encrusting underneath coral surfaces31, which deviates from the high bryozoan abundance recorded in the Abrolhos mid-shelf reefs.Modern bryozoans occur at all latitudes, but tend to form significant accumulations only in eutrophic/turbid and deep/cold waters48, creating large habitat patches under different succession stages. Such extreme events change reef morphology and also preclude dominance by slow-growing “stress-tolerant” species. While historical factors  may underlie the low species richness and functional diversity of SWA coral assemblages, the marginal conditions (e.g. turbidity) and low disturbance regime may be major drivers of the region’s low coral diversity. In addition, the low disturbance regime does not impose significant constraints  to horizontal accretion of carbonate framework, preventing the breakage of the huge lateral expansions of the oddly shaped pinnacles. Previous mechanisms explaining the mushroom-shaped morphology of the Abrolhos reefs accounted only for the limitations in upward accretion due to the sea level drop during the last 4,000 years10. However, while sea level plays a major role in reef growth, it does not seem to be the single driver of the peculiar morphology of the Abrolhos reefs. Flat pinnacles tops occur in the subsurface, at depths of up to 10 m, and our results indicate ongoing vertical growth. For instance, a depth vs age analysis from the shallower pinnacle  does not support a sea-level control of its shape10.Another potential driver of reef growth and diversity along the eastern Brazilian margin is the region’s low disturbance regime, with very rare tropical storms and hurricanes Fig. . Akin toDespite being relatively small and species-poor, tropical SWA reefs provide relevant insights on global patterns of reef building and on the meso-scale drivers of reef biodiversity. Our results challenge the coralgal-framework paradigm of modern shallow water tropical reef building. The Abrolhos mid-shelf reefs deviate from coral reefs in which mixo- and autotrophic organisms  are the main builders. Instead, at least during the last 2,000 yr., these reefs have been largely built by bryozoans, which are heterotrophic suspension-feeders, with a secondary role of CCA and a species-poor coral assemblage. These findings suggest that the role played by bryozoans in reef building may be underestimated in other shallow water tropical reef systems under mesotrophic conditions.14, including the side scan images in Fig. 13. The mid-shelf chapeirões consist of 2–20 m height pinnacles, with diameters of 2–50 m and laterally expanded tops near the surface. Near-shore, pinnacles are shorter but similarly shaped, often coalescing to form extensive shallow banks and patch reefs15.Detailed descriptions of the Abrolhos Shelf reefs Fig.  are prov49. Core recoveries and depths were calculated by moving the recovered samples to the top of each core run, following standard International Ocean Discovery Program (IODP) protocols50. The high resolution core images were displayed in Corelyzer 2.0.250 and the percent surface area of each of the main lithologies and reef framework types calculated.Coring was carried out using a diver-operated, submarine hydraulic rotary drill. Cores were taken in three depths  and habitats  along the mid-shelf arc. For each depth and habitat, two along side cores were taken, with the exception of the 25 m deep pinnacle wall, where only one core was recovered. All cores were sliced in two halves and photographed in high resolution. The main reef framework types, sedimentary facies and sample context in the cores were logged using a combination of sample material, petrographic thin sections and digital images following procedures laid out inhttp://calib.qub.ac.uk/calib. The Marine13 calibration curve was applied assuming a global marine reservoir effect of 400 years and a Delta R of 85 ± 25  for regional correction. Only samples interpreted as in situ  were chosen for dating purposes. Vertical accretion rates were calculated using mean ages and the stacked core sample depths. The maximum potential depth error of each dated core sample was directly related to the actual core recovery of each core run  were radiocarbon dated at Beta Analytics-Florida (USA) via Accelerator Mass Spectrometry (AMS). Dates are reported as calendar years BP (“present” = 1950 CE) using the 2 sigma confidence interval. Calibration was carried out using Calib 7.1 available at All the processed data, including drilling sites coordinates, C14 results, core composition and taxa information are provided as tables in the Supplementary Information file.https://www.ncdc.noaa.gov/ibtracs/index.php?name=wmo-data, spanning from 1985 and 2015. Global level scleractinian coral richness data was compiled from3 and digitalized as shape files representing layers with <50, 51–100, 101–200, 201–300 and >400 species. Satellite image is an Ikonos - 2, with spatial resolution of 4 m. A supervised classification was carried out using Mahalanobis Distance algorithm51. All explicit spatially data were introduced and processed in a Geographic Information System (GIS).The storm dataset was compiled from NOAA National Climatic Data Center, World Data Centre for Meteorology - All data generated or analysed during this study are included in this published article and its Supplementary Information files.supplementary information


In contrast to the transcriptome-wide detection methods, the techniques used for monitoring purposes need to be cheap, fast and easy to perform. This review considers modern approaches for site-specific detection of methylated nucleotides in RNA. We also discuss the potential of third-generation sequencing methods for direct detection of RNA methylations.RNA methylations play a significant regulatory role in diverse biological processes. Although the transcriptome-wide discovery of unknown RNA methylation sites is essential to elucidate their function, the development of a bigger variety of detection approaches is desirable for multiple reasons. Many established detection methods for RNA modifications heavily rely on the specificity of the respective antibodies. Thus, the development of antibody-independent transcriptome-wide methods is beneficial. Even the antibody-independent high-throughput sequencing-based methods are liable to produce false-positive or false-negative results. The development of an independent method for each modification could help validate the detected modification sites. Apart from the transcriptome-wide methods for methylation detection  To date, 171 RNA modifications are known according to the MODOMICS database, of which 72 include methyl groups . HoweverN6-methyladenosine (m6A) is also removable and hence an RNA modification can be reversible sparked broad scientific attention in the past few years. An exciting hypothesis about the reversibility of m6A was proposed, stating that its levels would be mediated not only by its writers, but also by its erasers, removing this modification in a dynamic manner [6A, also 1-methyladenosine (m1A) was recently discovered to be reversible in tRNA and mRNA [RNA was shown to contain methylated bases in 1958 , and manand mRNA . AccessiIn the current review, we focus on recent advances in the field of methylation detection in RNA and discuss innovative approaches for site-specific detection of methylation, including the ones that still need further development. We will also discuss approaches for monitoring the presence of methylated nucleotides at a specific position in RNA. We will not address the well-established methods for transcriptome-wide mapping of RNA modifications coupled to NGS, because they have been extensively reviewed elsewhere ,9. Figur2.et al. [6A, 5-methylcytidine (m5C), 3-methyluridine (m3U) and 5-methyluridine (m5U) in short synthetic oligoribonucleotides was recently shown to be possible with a mass spectrometer equipped for low-energy collisionally activated dissociation experiments [Early methods for detection and quantification of modified nucleosides/nucleotides were based on their physico-chemical properties. The separation of RNA monomers was performed using TLC  or by het al. –13. The et al. . Direct eriments .6A sites in Rous sarcoma virus RNA were already mapped in 1985 [The early approaches for precise localization of RNA modifications relied on the RNase digestion and fingerprinting in combination with paper electrophoresis and TLC. For example, the m in 1985 .Another group of approaches for methylation detection in RNA is based on reverse transcription (RT). The detection of modified RNA nucleotides is possible if their presence influences the reverse transcriptase either by blocking or stalling RT, or by inducing misincorporation into cDNA opposite the modification . The imp6A detection at single-nucleotide resolution is based on the combination of RNase H site-specific cleavage, splinted ligation, ribonuclease digestion and TLC  [6A. However, this method is extremely laborious and time-consuming.A remarkable method for mSCARLET) . In prinIn the current review, we focus on recent advances in the field of site-specific detection of methylation in RNA. The approaches are broadly categorized based on their working principle.3.6A, m1A and m5C. Modification-specific antibodies in conjunction with NGS and  cross-linking were used to map m5C, m1A and m6A at the transcriptome-wide level [6A-specific antibody was shown not to distinguish between m6A and N6,2′-O-dimethyladenosine (m6Am) [Antibodies are commercially available for many methylated RNA residues, such as mde level –22. Howee (m6Am) . Moreovee (m6Am) .6A detection were developed, which, however, do not provide information about the m6A position in the sequence [The application of methylation-specific antibodies is not limited to transcriptome-wide detection in combination with NGS. They have also been used for immuno-northern blotting . Recentlsequence .6A in a bulge loop of an RNA–DNA duplex. The m6A is specifically recognized by an antibody in the one-bulge loop, but not in a fully matched RNA–DNA hybrid. The biotinylated DNA probe was designed to target an m6A-containing region, but lacked a nucleotide pairing with m6A. The developed approach was applied to detect m6A2030 in 23S rRNA of Escherichia coli total RNA. A significant difference between the one-bulge-inducing probe and the full-match probe is achieved in case of prior RNA fragmentation. However, the approach does not allow to achieve specificity in intact RNA samples. In addition, high background signals are observed in a control experiment for a non-methylated adenosine [Another recently reported immunochemical approach is based on the immunorecognition of mdenosine .4.1A, m3U, 3-methylcytidine (m3C) and 1-methylguanosine (m1G)), the base-pairing and therefore the RT signatures can be affected. One example of the established specific RT signatures for methylated RNA residues is m1A [Some of the methylated RNA nucleotides naturally block RT, facilitating the development of RT-based methods. In case the methyl group is present on the Watson–Crick edge . The evolved enzyme variant was applied to the NGS protocol for the analysis of a known m6A site in E. coli tRNAVal. An error rate of 14.3% was observed at the known m6A site. However, the misincorporation with error rates greater than 10% was also observed opposite 5-methyluridine and at the 5′-end of the RNA molecule. This suggests that the engineered enzyme is sensitive also to RNA modifications other than m6A, and the developed approach is ineffective for m6A detection at the 5′-end of the molecule, where the rates are inaccurate due to the low coverage.Detection of RT-silent modified RNA residues can be improved by engineering the reverse transcriptase enzymes to introduce signatures opposite the modification. Aschenbrenner  variant ,32. In c the m6A a. The evEscherichia coli MazF toxin was recently identified as the first m6A-sensitive RNA cleavage enzyme [6A)CA-3′. Based on this finding, a FRET-based assay was developed to determine the methylation status of RNA (b). However, it should be noted that MazF was also sensitive to m1A and is thus not suitable for distinguishing between methylation at those two positions. In addition, MazF does not cleave double-stranded sequences, thus hindering m6A detection in a structured RNA [Proteins other than DNA polymerases can also be sensitive to RNA methylation, including RNA-binding proteins (such as human single-stranded RNA-binding protein Pumilio 2 ), endorie enzyme . This ens of RNA b. Howeveured RNA .6A detection allowed discrimination between A and m6A, when T4 DNA ligase was used [6A. On the basis of this discovery, a PCR-based approach was developed for m6A detection [N6-methylation, the ligation is significantly hindered, which can be detected in the quantitative PCR (qPCR) of the ligation product. For better discrimination between A and m6A, one of the probes should be modified with two ribonucleotides, as shown in figure 2c. The method also enables the quantification of the fraction of RNA containing m6A at this position. This quantification was validated for one of the known m6A sites in MALAT1 lncRNA in the polyA+ RNA isolated from different cell types.The first developed ligation-based method for mwas used . Recentletection . Two DNA5.6A, which still forms the A–U base-pair including the hydrogen bond between the N6-position of adenosine and O4 in uridine. Hence, the presence of m6A reduces the thermodynamic stability of RNA duplexes likely due to the steric hindrance [6A presence at specific RNA positions was based on the melting properties of the RNA–DNA duplex in the presence of a modified nucleotide. The method developed by Golovina et al. [a). Analysis of fluorescence during melting of the produced duplex allowed detection of m6A at three specific positions of rRNA, one position of tRNA and one position of snRNA. The high specificity was also achieved in bulk cellular RNA. Although this method is simple to perform, it only has limited use due to the high concentration of the RNA sample required (0.4 µM). The possible use suggested by the authors could be screening of putative methyltransferase (MTase) knockout or knockdown cell lines in a search for unknown MTases [Even if methylation in RNA is present on the Watson–Crick edge, it does not necessarily impede base-pairing. A prime example is mindrance , but staindrance . One of a et al.  requiresn MTases .Figure in situ hybridization’ (MR-FISH)—allows to monitor RNA methylation at specific sites in single cells. To achieve this, Ranasinghe et al. developed molecular beacons as hybridization probes sensitive to methylation in their complementary rRNA sequences in fixed cells. Another molecular beacon complementary to a remote non-methylated sequence in the same RNA strand was used for internal calibration (b). The method was shown to be sensitive to two adjacent m62A bases, m1G and m3U. Interestingly, also the presence of m6A was shown to destabilize the duplex to some degree. Since this technique only requires simple equipment, it could be used in diagnostic tests for identification of antibiotic-resistant bacteria [Another approach for detecting of RNA methylation at specific positions used DNA hybridization probes that are sensitive to methylation of their complementary RNA sequences . This meibration b. The mebacteria .6A were used in another technique, in which the RNA template was reverse transcribed with deoxythymidine triphosphate analogue bearing a selenium atom at the 4-position (4SeT). The normal adenosine could base-pair with this analogue without any visible differences to A–T pairing, but the incorporation of 4SeT opposite m6A site was significantly stalled due to the perturbation of both hydrogen bonding and base stacking (c). The RT stalling took place for different reverse transcriptases used, but was affected by the reaction conditions, e.g. the incubation temperature or concentrations. To not only detect the presence of m6A at defined sites, but also to locate it for unknown samples, an m6A demethylase (FTO)-assisted strategy in combination with NGS was developed, resulting in m6A identification at single-nucleotide resolution. Up to date, the strategy was not applied for m6A identification in a real biological sample [The base-pairing properties of mstacking c. The RTl sample .6.in vivo or in cells. Thus, one of the approaches for identification of direct targets of RNA cytosine MTases is based on the disruption of the methyl transfer by the suicide inhibitor 5-azacytidine (5-aza-C). For this, HeLa cells expressing an epitope-tagged m5C-RNA MTase DNMT2 were grown in presence of 5-aza-C, which was incorporated into nascent RNA [5C tRNA MTase [Although most approaches for mapping RNA methylation sites in biological samples start from the RNA isolation from the cells/tissues, some approaches require a first step performed cent RNA . Later tNA MTase .S-adenosylmethionine (SAM) as a methyl donor, although some exceptions are known [l-selenohomocysteine, which gets converted by cellular methionine adenosyltransferases to a SAM analogue bearing a propargyl group instead of the methyl one  transfers an allyl group from the corresponding SAM analogue, albeit with low yield. In this case, the RT signature is induced not via the click-chemistry, but via iodination of the allyl group, which leads to formation of N1,N6-cyclized adenosine. Labelling of the isolated total RNA in presence of METTL3–METTL14 and allyl-SAM could potentially be used in future for transcriptome-wide discrimination of A from m6A. For this, a much higher allyl transfer activity should be achieved [Shu et al.  discusseachieved .7.Many specialized NGS-based protocols were developed for detection of different RNA modifications. They often rely on the antibody recognition or on specific chemical treatment of the analysed modified base . Owing tet al. [6A, m5C and m5U on the fidelity of M-MuLV, AMV and ProtoScript II enzymes was studied [The NGS protocols greatly depend on the fidelity of the reverse transcriptase, which has long been known to be affected by the presence of different modifications. Recent studies aimed to systematically investigate the effect of several RNA nucleobase modifications on the performance of some commercially available RT enzymes. Thus, Potapov et al.  develope studied .5C, m5U and m6A). Others  yielded sequencing profiles distinct from adenosine and guanosine, respectively. However, it has to be noted that the polymerase responses varied depending on the sequence contexts. In addition, it is possible that modification-dependent patterns observed in this study may overlap with those of untested RNA modifications. Nevertheless, this study is an important proof of concept for simultaneous detection of several RNA modifications. In future, further investigations of the modification-specific RT signatures or protein engineering might lead to the discovery of an RT enzyme with more pronounced discrimination.Another recent study hypothesized that different RNA modifications would cause distinguishable effects in deep sequencing data  and therefore elucidated the fingerprints of the modified RNA nucleobases . Synthet8.Third-generation sequencing, also known as single-molecule sequencing, is a class of diverse high-throughput sequencing technologies currently under development, whose working principles differ from NGS (often referred to as second-generation sequencing). In contrast to the latter, these emerging methods do not require breaking of the nucleotide strand to smaller segments nor amplification by PCR. Early experimental studies show the potential of third-generation technologies for detection of modified RNA nucleotides.E. coli 16S rRNA [6A and m5C was studied for the synthetic RNA strands. The average current level was found to be locally perturbed near the modified positions. This preliminary observation was only made for two base modifications present in synthetic and fully modified RNA and is therefore not yet suitable for detection of RNA methylation in real biological samples. A higher sensitivity might be achieved in the future after further technological advances such as additional pore engineering.Nanopore sequencing detects single DNA or RNA molecules that are captured in the nanopore and translocated through it. To facilitate the capture and the sequencing, the RNA is ligated to a modular oligonucleotide adapter bearing a proprietary motor protein which regulates the RNA migration through the pore. A constant electric field is applied to the system, and the observed electric current is characteristically disrupted when a nucleotide passes through the pore, which makes the sequencing possible. The first attempt to read modified RNA nucleosides using direct nanopore sequencing was carried out in 2017 using the 16S rRNA . One of 16S rRNA . The per6A affected binding of the phospholinked nucleotide both in the synthetic RNA template and at the known site in mRNA when the HIV RT enzyme was used [Another emerging NGS technology is single-molecule real-time detection of reverse transcription (SMRT). Originally developed for DNA, it was also adapted for RNA sequencing. The RT of the RNA molecule is visualized due to the fluorophores attached to the terminal phosphate groups on the dNTPs. The removal of the label during the nucleotide incorporation allows for real-time monitoring of cDNA synthesis. The presence of mwas used .6A, probably due to its outstanding biological role. The future of the field is likely to be linked to the development of second- and third-generation sequencing technologies, which are now becoming more accessible.In summary, the field of detection of methylation sites in RNA is rapidly growing. New methods are in development not only for transcriptome-wide detection of unknown modification sites, but also for fast and easy monitoring of the presence of the methylated nucleoside at a given position. Most of the recent methods focus on the detection of m


Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. The high operational cost of microfluidic capillary electrophoresis systems along with paucity of alternative methods for the quantitative assessment of rat RNA integrity constitute potential hurdles to the systematic implementation and reporting of RNA integrity assessment in rat studies. This manuscript describes the adaptation of an alternative RT-qPCR-based 3′:5′ assay as an additional option for the quantitative assessment of rat RNA integrity. Two PCR primer sets were designed on the 3′ and 5′ regions of a rat housekeeping gene to evaluate RNA integrity by measuring the relative expression (3′:5′ ratio) of these amplicons. The 3′:5′ ratios were then compared to Agilent Bioanalyzer’s RNA integrity number (RIN) for a wide range of RNA samples originating from different tissues, cultured cell lines and rat strains that were prepared freshly, stored for years at −80 °C, purchased commercially or intentionally degraded. The 3′:5′ ratios and RIN values presented similar assessment of RNA integrity status from intact to heavily degraded samples. Based on the LOWESS regression of this large comparison dataset, 3′:5′ ratio threshold criteria equivalent to RIN cut-off values can be proposed for the selection of RNA samples for RT-qPCR analyses. This qPCR-based assay is easy to implement, cost-effective, and provides a reliable quantification of RNA integrity to assist in the selection of rat RNA samples suitable for downstream RT-qPCR gene expression analyses. RNA samTraditionally, RNA integrity was evaluated qualitatively by inspecting the intensities of the 28S and 18S ribosomal RNA (rRNA) bands following agarose gel electrophoresis. More recently, manufacturers have developed automated microfluidics-based electrophoretic systems that calculate a quantitative RNA quality score based on the analysis of digitalized electropherograms by proprietary algorithms . RIN valAs most gene expression studies target protein coding genes, RT-qPCR-based methods such as the 3′:5′ assays were proposed to evaluate messenger RNA (mRNA) integrity status . This 3′in vivo and in vitro. A recent literature survey revealed that more than half of rat toxicological studies using RT-qPCR do not describe RNA quality control measures and that only about one in five reported RNA integrity assessment by electrophoretic-based methods . Although this lack of reporting does not necessarily imply the absence of appropriate RNA quality controls, such widespread omissions nevertheless impede the proper evaluation of the reliability of gene expression data in rat toxicological studies. While agarose gel electrophoresis requires large quantities of RNA and only allows a qualitative evaluation of RNA integrity, the more quantitative microfluidics-based platforms imply further operational costs and require additional equipment that may not be accessible to all laboratories. The development of a simple, affordable and easily implementable alternative method to quantitatively assess rat RNA integrity may facilitate adherence to RNA quality control measures and reporting in rat toxicological studies. The 3′:5′ assay originally developed for human and using probe-based Taqman dye possess many of these attributes and can be adapted to different species and fluorescent detection chemistries [Rat is a commonly used species for the assessment of chemical toxicity mistries ,11,13. I22.12 in cell culture media supplemented with 100 IU/ml penicillin + 100 μg/ml streptomycin. C6 glial cells from American Type Cell Culture , were cultured in F-12K medium containing 2.5% (v/v) foetal bovine serum (FBS) and 15% (v/v) horse serum. They were grown to confluence before RNA isolation. PC12 pheochromocytoma cells from ATCC were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% (v/v) FBS, 10% (v/v) horse serum and 2 mM L-glutamine. Upon exposure to nerve growth factor (NGF), dividing PC12 cells differentiate by developing axon-like projections [l-glutamine). The cell culture media was replaced by fresh media after one day in culture and total RNA was extracted on the fourth day of culture.All cells were grown at 37 °C in a humidified incubator containing 5% COjections . FreshlyDeveloping rat brains were harvested from PND14 and PND21 Sprague-Dawley pups following decapitation without anesthesia. The hippocampi were dissected immediately from the brains, flash-frozen in liquid nitrogen and stored at −80 °C until RNA isolation. Further details about rat perinatal exposures, tissue harvesting and RNA extraction can be found in the original developmental neurotoxicity study . Animals2.2C6, PC12 and CGC cultures were washed with 1× Phosphate-Buffered Saline (PBS) prior to RNA isolation. The cells were lysed directly on the culture dish using the lysis buffer provided in Qiagen’s RNeasy Mini Plus kit for total RNA isolation, and genomic DNA was removed using Qiagen’s gDNA Eliminator columns following the manufacturer’s protocols . Total RNA from juvenile rat hippocampus was extracted using TRIzol , and further purified using Qiagen’s RNeasy Mini Plus kit and gDNA Eliminator columns following the manufacturer’s protocols. Total RNA samples from various juvenile and adult tissues and different rat strains (n = 34) were purchased from Zyagen , see Table S2.2.3A Nanodrop 1000 spectrometer  was used to measure absorbance at 260 nm (A260) to evaluate RNA concentration. RNA purity was estimated using the A260/A280 ratio and only samples presenting a ratio greater than 1.8 were kept for further analyses. RNA integrity was assessed by an Agilent 2100 Bioanalyzer, using the Agilent RNA 6000 Nano Kit . The Solaris RNA Spike Control kit  was used to assess the presence of inhibitors in a subset of RNA samples . A PCR-based approach developed in-house  was used2.4Phosphoglycerate kinase 1  is well-suited for this 3′:5′ assay. The Pgk1 gene possesses few pseudogenes and produces a relatively long transcript that presents a well-characterized exon-intron structure. While the low number of pseudogenes and exon-spanning primers will limit the potential interference from inadvertent genomic DNA contamination [Pgk1 gene were designed (www.bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm). Primer–BLAST searches were conducted to check the specificity of these primer sets (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). In silico PCR analyses of these primer sets at rat UCSC genome browser (genome assembly RGSC 6.0/rn6 at https://genome.ucsc.edu/cgi-bin/hgPcr) were performed to assess cross-match to any non-target sequence such as pseudogenes. The expected amplicon sequences were used to query the rat ENSEMBL database (http://useast.ensembl.org/Rattus_norvegicus/Info/Index) to ensure the absence of Single Nucleotide Polymorphisms (SNPs) at primer binding sites that would impair quantitative PCR efficiency [http://mfold.rna.albany.edu/?q=mfold) [The ubiquitously expressed housekeeping gene mination , the lendesigned  using thficiency . The potq=mfold) . The pri2.5® SYBR® Green PCR kit, Qiagen) in a reaction volume of 20 μl. The RT-qPCR reaction mix was denatured at 95 °C for 10 min and then subjected to 45 amplification cycles  [The reverse transcription reactions were performed in a total volume of 20 μl using 0.5–2 μg of total RNA, 500 ng/μl Anchored Oligo-dT primers (Thermo Fisher Scientific), and 200 U Superscript III™ reverse transcriptase (Invitrogen) following manufacturer’s first-strand cDNA synthesis protocol. All cDNA samples were diluted in 9 volumes of sterile de-ionised water and stored at −20 °C until RT-qPCR analyses. Semi-quantitative RT-PCR reactions were carried out using PlatinumTaq (Invitrogen) in a C1000™ Thermal Cycler  for 35 cycles . Quantitative PCR was performed with an iCycler iQ™5 Real-Time Detection System (Bio-Rad Laboratories) using 8 μl diluted cDNA, 0.4 μM of the forward (1 μl) and reverse (1 μl) primer, and 10 μl 2× SYBR-Green I dye master mix  of each diluted RNA sample was taken into four separate tubes. While one tube served as the control (0 min heat), the other three tubes were exposed to 90 °C heat for 5, 10 or 20 min in a thermocycler (Tables S1 and S2). For each tube, 1.0 μl of RNA sample was analyzed by Agilent Bioanalyzer, and 10 μl of RNA sample (2 μg) was used for cDNA synthesis as described above.2.7Pgk1 forward (1 μl) and reverse (1 μl) primers , one for each primer set. RT-qPCR reactions were carried out in a total volume of 20 μl containing diluted cDNA (8 μl), 2 × SYBR-Green I dye (10 μl) and 0.4 μM of the appropriate  primers , as deschmittgen .2.8LOcally WEighted Scatterplot Smoothing (LOWESS) regression  was appl2.9Transforming acidic coiled-coil containing protein 2 (Tacc2) was measured in intact, moderately degraded or highly degraded RNA samples following a previously described RT-qPCR experimental approach [Gapdh and Pgk1) were used for the normalization of Tacc2 expression, according to a modified ΔΔCq method [Tacc2 [Gapdh [Pgk1 are presented in Table S3. Statistically significant differences in Tacc2 mRNA abundance between dividing and differentiating PC12 cells were determined using a two-tailed Student’s t-test (p < 0.05).High quality RNA samples from dividing n = 3) and differentiating PC12 cells (n = 3) were subjected to heat degradation as described in Section  and diffq method . The prid [Tacc2 , Gapdh [33.1Pgk1), which also presents a well characterized intron-exon structure  isolated in-house from C6 cells and PND14 hippocampus tissues were subjected to degradation by heat treatment at 90 °C for 0, 5, 10 and 20 min. These samples (n = 24) consisting of intact, moderately degraded and highly degraded RNAs , were assessed using the 3′:5′ assay. As the level of RNA degradation in C6 and PND14 hippocampus samples increased, there was a corresponding decrease in RIN values c, e, and3.2RNA samples isolated from 29 different tissues, three cell types and five rat strains were used to methodically compare 3′:5′ ratios to the commonly used RIN values. These RNA samples were freshly prepared, stored for years at −80 °C, experimentally- or accidentally-degraded, or purchased from a commercial supplier (Tables S1 and S2). High quality RNA (RIN > 9.8) prepared freshly from Cerebellar Granule Cells (CGCs) and PC12 cells, or C6 RNA samples stored at −80 °C for three years presented 3′:5′ ratios ranging from 1.4 to 2.5. Hippocampus RNA samples from PND21 rat pups stored at −80 °C for seven years showed RIN values between 7.7 and 8.7 and 3′:5′ ratios between 1.1 and 2.8. Of the 34 commercial RNA samples assessed, 21 tissues  showed RIN value >8.0, and their 3′:5′ ratio values ranged between 1.2–3.5. Another seven RNA samples  were moderately degraded (RIN 5.0–8.0) and presented 3′:5′ ratios ranging from 1.8 to 5.3. Accidental degradation of the remaining six RNA samples  during their transport resulted in low RIN values (2.5–4.8) and high 3′:5′ ratios (5.2–199.5).3.3RIN values and 3′:5′ ratios for all the RNA samples tested (listed in Tables S1 and S2) were plotted on a graph and a non-parametric LOWESS regression was performed to better visualise the relationship between these two metrics . As the 3.4Tacc2 is involved in the organization of centrosomal tubules in proliferating cells [Tacc2 relative expression was significantly lower in differentiating PC12 cells, when assessed using intact or moderately degraded RNA samples (3′:5′ ratio <8.0)  these cells exit the mitotic cycle and begin to differentiate by developing axon-like projections . Given tng cells , we hypoio <8.0) . Howeverld value . These e4in vivo or in vitro toxicology studies, the development of an alternative option for rat RNA integrity assessment may contribute to improved monitoring and reporting of RNA quality controls. The 3′:5′ assay is one such option, but this approach had not yet been adapted for the rat species.Our survey of RT-qPCR-based gene expression analyses in rat toxicological studies Fig. S1) clearly supports the claims that RNA quality controls generally receive insufficient attention  clearly . SystemaPgk1), which also presents a well characterized intron-exon structure. We then designed primer sets located far apart on the Pgk1 transcript that spanned exon-exon boundaries and generated amplicons of a similar size . These 3′:5′ ratios and RIN values plotted in Cq threshold of 3.0 [3 = 8). Discrepancy with the 3′:5′ ratio threshold of 5.0 proposed by Nolan et al. [For the rat 3′:5′ assay to be used in a concrete experimental setting, it was critically important to empirically determine practical 3′:5′ ratio thresholds to guide the selection of RNA samples suitable for downstream applications. Based on the LOWESS regression of the scatterplot presented in d of 3.0 , which cn et al. , may be n et al. ,11,13, tTacc2 relative expression in proliferating and differentiating PC12 cells using intact or degraded RNA samples .Finally, we compared  samples . AlthougTacc2 gene expression is yet another reminder of the importance of proper RNA quality controls for reliable and reproducible RT-qPCR analyses. However, for studies specifically attempting to measure gene expression in RNA samples presenting moderate to severe enzymatic degradation (from post-mortem samples for example), further characterization of the 3′:5′ assay may be needed. Contrary to heat treatment, enzymatic-mediated RNA degradation is a non-random process initiated by either 3′-end polyA tail/5′-cap removal, followed by exonucleolytic decay or endonucleolytic cleavage [This concrete example of the application of the 3′:5′ assay to the measurement of cleavage . Consequ5This study describes the adaptation of a 3′:5′ RNA integrity assay for the rat species. Comparison of 3′:5′ ratios and RIN values for a wide range of rat RNA samples revealed a good association between these two RNA integrity metrics. Based on RIN cut-off values for the selection of RNA samples for downstream RT-qPCR analyses, this extensive comparison allows the empirical determination of equivalent 3′:5′ ratio thresholds. For the vast majority of samples assessed, both methods produced concordant categorization of RNA sample suitability for RT-qPCR applications. Finally, we provided a concrete example of the application of the 3′:5′ assay in an experimental protocol, which constitutes yet another reminder of the importance of proper RNA quality controls. Based on these results, we conclude that the described 3′:5′ assay, which can be easily performed in any laboratory measuring rat gene expression by RT-qPCR, represents a valid alternative option for the quantitative evaluation of RNA integrity.The authors declare no competing financial interests.


Clostridium difficile infections.There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of  Currently, gut microbiome profiling largely relies on next-generation sequencing, which is slow and expensive. Here, the authors develop a low-cost, paper-based synthetic biology platform that allows species-specific quantification of bacterial mRNAs and clinically relevant host biomarkers. It is also an important modulator of gastrointestinal function, including inflammatory bowel disease (IBD)7, childhood malnutrition9, and cancer immunotherapy treatment11. Increasing evidence suggests that host–microbiome interactions also play a key role in these health conditions14. Despite the progress made in our understanding of the overall gut microbiome and the roles of individual species, large-scale longitudinal studies are needed to more directly investigate the causal relationship between microbial and host changes during disease states and responses to treatment. Current methods for profiling the gut microbiome typically involve deep sequencing coupled with high-throughput bioinformatics. These techniques are expensive, slow, and require significant technical expertise to design, run, and interpret. To reduce costs, researchers often batch samples for sequencing, which can lead to significant increases in turn-around time. These limitations have severely restricted the large-scale prospective monitoring of patient cohorts that is necessary to provide more granular data on microbial changes and human health15. Here we present a synthetic biology platform that addresses the need for affordable, on-demand, and simple analysis of microbiome samples that can aid in monitoring large-scale patient cohorts.The gut microbiome is an essential contributor to numerous processes in human health and disease, including proper development of the immune system17. The platform is comprised of two synthetic biology technologies. The first technology is a molecular sensor called an RNA toehold switch that can be designed to bind and detect virtually any RNA sequence18. The second is an in vitro cell-free transcription–translation system that is freeze-dried onto paper disks for stable, long-term storage at room temperature16; upon rehydration, the cell-free system can execute any genetic circuit. We combined these two technologies to form an abiotic platform for rapid and inexpensive development and deployment of biological sensors. Recently, we reduced the limit of detection of this platform to three femtomolar (fM) by adding an isothermal RNA amplification step called NASBA (nucleic acid sequence based amplification)17. We demonstrated the utility of our platform in detecting the presence or absence of clinically relevant RNAs, including those of Ebola16 and Zika17 viruses, but we were not able to quantify their concentrations.Our lab has developed a paper-based diagnostic platform for portable, low-cost detection of biologically relevant RNAs19, and another, oncostatin M (OSM), which may have an immediate impact on clinical decision-making in the treatment of IBD20. We validate our method against RT-qPCR using clinical samples from patients with IBD. Finally, we demonstrate an additional potential clinical application of our RNA detection platform using the example of Clostridium difficile infection (CDI), where differentiating active infection from passive colonization has been fraught with difficulty21. Our method shows markedly different toxin mRNA expression levels in two toxigenic C. difficile strains that would otherwise be indistinguishable by standard DNA-based qPCR diagnosis.Here, we address the need for affordable, on-demand, and simple analysis of microbiome samples by advancing our paper-based diagnostic platform for use as a research tool to quantify bacterial and host RNAs from stool samples Fig. . To democis by sequestration of the ribosome binding site (RBS) and start codon. Translation is activated upon the binding of a trans-acting trigger RNA to the toehold region of the switch, which relieves the sequestration of the RBS and allows translation of the downstream gene 24 to design toehold switch sensors that target the V3 hypervariable region of the 16S rRNA for each target species. The candidate sensors were constructed to regulate the expression of the GFPmut3b gene25, and tested using in vitro transcribed trigger RNAs (36 nucleotides) in paper-based, cell-free reactions  RNA. Thus, unprocessed rRNA from stool samples is beyond the limit of detection of toehold switch sensors alone, which is approximately 10–30 nMles Fig. 26 and caE. coli. We first tested the standard universal primer set routinely used to amplify the V3 region from 16S rDNA27 for sequencing applications. We used total RNA extracted from an E. coli monoculture to screen the primers. NASBA reactions were performed for 90 min on 1 ng of total RNA and then applied to paper-based reactions containing the E. coli 16S toehold switch sensor. Unexpectedly, these primers were not able to amplify the 16S V3 region from total RNA . In order to investigate why the universal primer set performed poorly, we mapped the primer locations to chemical structure probing data for E. coli 30S ribosomal subunits28 and found that the forward primer targeted nucleotides that were not structurally accessible  and measured the activation of each sensor when challenged with each of the 10 trigger RNAs .To address the specificity limitations of the 16S sensors, we devised a bioinformatic pipeline to identify mRNAs that are unique to any given bacterial species Fig. . Our pipWe followed the same steps described for 16S rRNA sensor development to develop sensors for the species-specific mRNAs. We tested candidate toehold switch sensors in paper-based reactions and selected the best performing sensor for each species  contained 1 ng of total RNA plus 9 ng of yeast tRNA and sample (2) included 1 ng of RNA from each of the 10 individual species. For each NASBA primer set, we observed equivalent activation of the toehold switch sensors by RNA amplified from samples (1) and (2). Additionally, the outputs from sample (3) were equivalent to the toehold switch sensor alone for each species indicating that there was no amplification of the test target in sample (3)  total RNA isolated from an individual species; (2) a mixed sample comprised of total RNA from each of the 10 species; and (3) a mixed sample containing total RNA from all species except for the one corresponding to the NASBA primer set being tested. To keep the total concentration of a given sample constant, we supplemented samples (1) and (3) with yeast tRNA (Ambion), which is commonly used to increase the complexity of mRNA standards in RT-qPCR, because reverse transcription efficiencies change with the total amount of RNA in a reaction(3) Fig. . These r33. In a previous application of the paper-based diagnostic platform, we demonstrated that the toehold switch sensors exhibit a linear response to trigger RNA inputs in the low nanomolar to micromolar range17. A mathematical model of NASBA reactions suggested that femtomolar to picomolar concentrations of RNA could be amplified to within the toehold detectable linear range, and 10-fold concentration differences would be distinguishable if NASBA reactions were stopped prior to completion . To mimic the complexity of a total RNA sample, we diluted our standards into yeast tRNA (50 ng/μl). NASBA reactions with varied amplification times (30 min–3 h) were carried out on mRNA standards to determine the duration that allowed us to distinguish concentrations that differed by 10-fold  from commercial human stool (Lee BioSolutions) and compared quantification of mRNA standards in this background to standards in a yeast tRNA background. We detected our species-specific mRNAs in stool RNA background, but the signal output for any given standard concentration was higher in total stool RNA than in the yeast tRNA background  and oncostatin M (OSM), a cytokine that has recently been found to predict the efficacy of anti-tumor necrosis factor (TNF)-alpha therapies in IBD patients20. To validate our sensors, we performed NASBA and toehold reactions on 50 ng of total RNA from human peripheral leukocytes (Takara Bio 636592) and demonstrated that we could detect each of the four transcripts  and C. difficile toxin  are slow, labor-intensive, and technically challenging37. The diagnostics currently in wide-spread use, such as enzyme-linked immunoassays (EIA) for C. difficile toxins and DNA-based qPCR assays for C. difficile toxin genes, offer greatly improved performance characteristics but have their own limitations21. The EIA tests have high clinical specificity, but reports of false-negatives and low sensitivity relative to toxigenic culture37 have led to the development of DNA-based qPCR assays for C. difficile toxin genes. This method is extremely sensitive for the presence of toxigenic C. difficile bacteria; however, it cannot distinguish between patients that are carriers with symptoms due to another cause and those with active CDI21. These cases are especially challenging for clinicians, and there is a debate on which testing methodology yields the highest combination of sensitivity and specificity for clinically meaningful CDI21. New ultrasensitive assays to detect C. difficile toxins are in development, but they require highly specialized and expensive laboratory equipment and in some cases have a 60-h turnaround time38. Our paper-based platform has the potential to address these limitations by providing a rapid, easy-to-use method for the diagnosis of active CDI based on the detection of C. difficile toxin mRNA , a high toxin producing strain40. We performed NASBA and toehold reactions on 25 ng of total RNA from each strain and demonstrated that we could detect toxin mRNA from both C. difficile strains  and paper-based (37 °C) reactions, existing laboratory incubators or thermocyclers may be used, or affordable incubators can be built for use in low-resource settings. Altogether, the low-cost and portable nature of our platform makes it uniquely suited for use in resource-limited environments.Our platform provides an easy to use, low-cost method for quantifying microbial and host RNAs from complex biological samples. Its flexibility allows for reactions to be freeze-dried for use outside of a laboratory setting. All reactions can also be run fresh, as they were done here, for researchers that do not have access to a lyophilizer. Specialized lab equipment is not required to develop our sensors or run the reactions. Since our toehold switch sensors can be used to regulate the production of any protein output, reactions may be monitored on a standard microtiter plate reader, if available, or an affordable, easy-to-build, portable electronic reader that quantifies change in absorbance from LacZ production46. Our limit of detection in total stool RNA ranges between 30 aM and 3 fM, depending on the specific toehold switch sensor. While this does not match the sensitivity of RT-qPCR (3 aM)47, we believe there are applications where our current limits of detection are sufficient. Future optimization of toehold switch sensor design and NASBA reaction conditions may continue to improve this sensitivity.The major advantages of our platform over RT-qPCR are cost and the ability to analyze multiple RNA transcripts at once. Using our platform, we can quantify mRNAs in 3–5 h at a cost of approximately $16 per transcript using commercially available kits as reagents (accounting for triplicate reactions and mRNA standard). This can be reduced to under $2 per transcript by using cell-free extracts prepared in-house, which are suitable for our platform , depending on machine and run type, and can take anywhere from 4 to 72 h49 yielded sensitive detection of nucleic acids with the ability to discriminate between single nucleotide differences52. While detection of single nucleotide polymorphisms (SNPs) is important, for example in tracking the epidemiology of viruses, the ability of the toehold switch sensors to tolerate SNPs enables the use of a single sensor to detect multiple strains. Although RT-RPA can be used to amplify RNA, as with RT-qPCR it cannot specifically amplify RNA without thorough DNase treatment to remove genomic DNA. Since NASBA uses reverse transcription to create DNA with a T7 promoter to then transcribe that template into RNA, it is highly resistant to DNA contamination53. Our method and the CRISPR enzyme-based diagnostics, SHERLOCK51 and DETECTR52, could be complementary tools, the selection of which will depend on the sample type (DNA or RNA), and whether the detection of single nucleotide differences is desired.Our paper-based platform is one of several new synthetic biology platforms that can be used for nucleic acid detection. Recent advances using the CRISPR associated enzymes Cas12a and Cas13 along with recombinase polymerase amplification (RPA)Our method for detecting and quantifying RNA sequences could be applied to a broad range of studies including samples from other human anatomical sites, and our approach is easily adaptable to a wide range of biological targets, including viruses, fungi, and eukaryotic nucleic acids from either stool or tissue samples. Furthermore, with continued optimization of sample processing, our method could be adapted for point-of-care use. Such a diagnostic platform could have many applications, including pre-screening enrollees in the field for prospective trials of therapeutic manipulations of the microbiome, at-home monitoring of research participants, and eventually for tracking changes in patient disease activity. Our easy-to-use synthetic biology platform has the potential to meet both research and clinical point-of-care needs.24 using the series B toehold switch design from Pardee et al.17 The script can be found in Supplementary Note Toehold switch sensors were designed with NUPACKDNA encoding trigger RNAs or mRNA standard sequences were ordered from Integrated DNA Technologies and amplified by PCR to create a linear template with a T7 promoter. RNA was transcribed from the DNA templates using a HiScribe T7 High Yield RNA Synthesis Kit, according to the manufacturer’s protocol . RNA was then purified using a Zymo RNA Clean and Concentrator kit (R1018), according to the manufacturer’s protocol. Following purification, DNA template was degraded by DNase digestion using the TURBO DNA-free DNase kit  for 1 h according to the manufacturer’s protocol.Cell-free reactions were performed using the PURExpress In Vitro Protein Synthesis Kit . The cell-free reactions consisted of NEB Solution A (40%), NEB Solution B (30%), RNase inhibitor , linear DNA constructs encoding toehold switch sensors (1.875 nM), and trigger RNA for a total of 5.5 µl. Paper disks  were punched out using a 2 mm biopsy  and placed in a 384-well plate (Corning 3544). 1.4 µl of the cell-free reaction mixture was applied to paper disks in triplicate. GFP expression  was monitored on a plate reader (Molecular Devices SpectraMax M5) every 5 min for 2 h at 37 °C.Sensor candidate designs from NUPACK were tested in paper-based reactions containing 1.875 nM of linear sensor DNA and 2 µM trigger RNA (36 nucleotides). GFP production rates were calculated  for reactions with sensor alone and sensor plus trigger. To select the best sensor, an activation ratio was calculated for each sensor candidate by dividing the sensor plus trigger production rate by the sensor alone production rate. Sensors were chosen based on the highest activation ratio and lowest sensor alone production rate. A minimum activation ratio of 5-fold is necessary to achieve desired sensitivity.Initial denaturation of total RNA consisted of a 2-min incubation at 95 °C followed by a 10-min incubation at 41 °C of 1.0 µl sample input, 1.675 µl reaction buffer , 0.825 µL nucleotide mix , 0.2 µl of 6.25 µM primers, 0.03 µl water, and 0.025 µl of RNase inhibitor (Roche) per 3.75 µl reaction. Afterwards, 1.25 µl of enzyme mix (Life Sciences NEC-1-24) was added to each reaction and the resulting 5.0 µl NASBA reactions were incubated for 30–180 min at 41 °C. Then 1.0 µl of NASBA product was added to the cell-free reaction mixture for a total of 5.5 µl.2 (VWR 97062-848), 75 mM KCl (VWR BDH7296-0), 10 mM DTT (Sigma GE17-1318-01), 40 mM Tris-HCl pH 8.5 (VWR RLMB-005), 15% DMSO, 2 mM each ATP, UTP, and CTP, 1.5 mM GTP, 0.5 mM ITP, 1 mM each dNTP , 0.25 µM each primer. Enzyme mix: 5 U/ml RNaseH (New England Biolabs M0297L), 1000 U/ml reverse transcriptase , 2500 U/ml T7 RNA polymerase , 43.75 mg/ml BSA. Initial denaturation of sample was performed as above, after which 1.25 µl enzyme mix was added to each reaction.Final concentrations of buffer components in each NASBA reaction: 13.2 mM MgClt = 50 min, resulting in values of RFU/min. The GFP production rates were then averaged over the replicates for each reaction condition.Paper-based reactions were analyzed by calculating GFP production rates for each reaction condition. GFP production rates were calculated by first subtracting the average background fluorescence measured from triplicate paper-based reactions that did not contain sensor DNA or trigger RNA. Then, the minimum value of each individual reaction was adjusted to zero by subtracting the average of its first three time points  from each time point. The zero-adjusted data were then fit to the equation: In quantification experiments, the GFP production rate for each sample was normalized to the GFP production rate for a single mRNA standard , Bifidobacterium breve (ATCC15700), Bifidobacterium longum subsp longum (ATCC 15707), Bacteroides fragilis (ATCC 25285), Bacteroides thetaiotaomicron (ATCC 29148), Clostridium difficile (ATCC BAA-1382), and Eubacterium rectale (ATCC 33656) were obtained from ATCC. Faecalibacterium prausnitzii A2–165 (DSM 17677) and Roseburia hominis (DSM 16839) were obtained from DSMZ. Freeze-dried samples were rehydrated with their respective growth mediums and grown for 24–48 h in liquid culture on a shaker at 200 rpm. For experiments testing RNA isolated from pure cultures, 12 ml of bacterial culture was diluted 1:2 into RNAProtect before removing from the anaerobic chamber for RNA extractions.The cultures were lysed at room temperature using 200 µl of 15 mg/ml of lysozyme in TE buffer and 20 µl of proteinase K (Qiagen). RNA was then extracted using the RNeasy Mini kit (Qiagen 74104), according to the manufacturer’s instructions. RNA samples were then DNase digested using TURBO DNA-free DNase kit  for one hour.E. coli (MG1655) was grown in Luria-Bertan (LB) medium (Difco). B. adolescentis was grown in Bifidobacterium medium (prepared according to DSMZ 58: Bifidobacterium medium). B. breve, B. fragilis, and B. longum subsp longum were grown in brain heart infusion-supplemented (BHIS) medium (prepared according to ATCC medium: 1293). B. thetaiotaomicron, E. rectale, and R. hominis were grown in cooked meat medium (CMM) purchased from Hardy Diagnostics. F. prausnitizii was grown in CMM with an additional 1% glucose. C. difficile was grown in BHIS for the species-specific RNA testing, and grown in either TY medium , or TY medium with 2% glucose and 10 mM cysteine for toxin RNA testing.54. Each frozen stool was homogenized using a mortar and pestle cooled with liquid nitrogen55, and 150 mg of each sample was loaded into each glass bead tube. Mechanical lysis was performed using a MoBio vortex adapter and a Vortex Genie 2 (Scientific Industries Inc) at maximum speed for 10 min. The manufacturer’s protocol was followed for RNA extraction with optional on-column DNase digestion included. Resulting RNA samples were then further DNase digested using TURBO DNA-free DNase kit  for one hour.Commercial stool specimens were purchased from Lee Biosolutions and provided as frozen specimens. Clinical stool samples were provided by Dr. Ashwin Ananthakrishnan as anonymized specimens from the Prospective Registry in IBD Study at Massachusetts General Hospital. Approval was provided by the Partners Healthcare Human Subjects Research Committee. Informed consent was obtained from all subjects. Both commercial and clinical stool samples were stored at −80 °C and processed using the RNeasy Powermicrobiome Kit , which was selected for its ability to isolate high quality RNA from stoolE. coli and B. fragilis were grown to mid-log phase and spiked into a commercial stool sample (Lee Biosolutions) before RNA extraction. Bacteria cultures ranging from 10 µl to 1.5 ml were spun down before being re-suspended in PM1 buffer and added to 150 mg of stool. C. difficile was grown to stationary phase in TY medium and TY medium supplemented with 2% glucose and 10 mM cysteine. Two ml of stationary C. difficile culture was spun down and re-suspended in PM1 buffer and added to 150 mg of stool. All samples were processed with the RNeasy PowerMicrobiome kit, according to the manufacturer’s instructions, with an extended 30-min lysis step for C. difficile spike-ins. RNA samples were then split into two samples, one that was DNase digested with the TURBO DNA-free DNase kit  for one hour and one that did not receive DNase treatment so that it could be used for DNA based qPCR.29. These programs use multiple bioinformatics approaches to reduce each bacterial species to a “bag of genes” and identify the set of genes or gene parts that is specifically associated with a target species or clade and not associated with any others. We extracted the Metaphlan2 markers for a given target species and used BLAST30 alignments against available genomes for our target species to ensure that the markers were present. We then assessed these preliminary markers for expression in the human fecal microbiome by using BLAST alignments against a human stool transcriptome database that we created from repositories of publically available adult human stool meta-transcriptome sequencing reads. Keeping only the markers that are expressed in human stool, we again tested for specificity by performing BLAST alignments against a pan-bacterial database that we created from all publically available reference and draft bacterial genomes. We selected the most specific markers with the highest expression in human stool and created toehold switch sensors to target these RNA sequences. In the case of C. difficile, expression was extremely low for all Metaphlan2 markers from the standard human stool transcriptome database. This was not unexpected since this species is reported to be very lowly abundant in normal healthy populations. To develop sensors for this species, we instead screened for expression using transcriptomic data from C. difficile cultures in various conditions available in public repositories.Our computational pipeline employs components from previously developed phylogenetic assignment tools, including Metaphlan and Metaphlan27 RNA copies per reverse transcription reaction. cDNA synthesis from stool samples was performed with 300 ng input RNA per reaction using Superscript III , according to the manufacturer’s protocol, using gene-specific primers  was used to generate standards for absolute quantitation based on calculations designed to incorporate 1 to 1056. E. coli BL21(DE3)ΔlacZ (gift of Takane Katayama) were grown in 400 ml of LB at 37 °C at 250 rpm. Cells were harvested in mid-exponential growth phase (OD600 ~ 0.6), and cell pellets were washed three times with ice cold Buffer A containing 10 mM Tris-Acetate pH 8.2, 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM DTT, and flash frozen and stored at −80 °C. Cell pellets were thawed and resuspended in 1 ml of Buffer A per 1 g of wet cells and sonicated in an ice-water bath. Total sonication energy to lyse cells was determined using the sonication energy equation for BL21 StarTM (DE3), [Energy] = [Volume (µL)] − 33.6] × 1.8−1. A Q125 Sonicator (Qsonica) with 3.174 mm diameter probe at a frequency of 20 kHz was used for sonication. A 50% amplitude in 10 s on/off intervals was applied until the required input energy was met. Lysate was then centrifuged at 12,000 rcf for 10 min at 4 °C, and the supernatant was incubated at 37 °C at 300 rpm for 1 h. The supernatant was centrifuged again at 12,000 rcf for 10 min at 4 °C, and flash frozen and stored at −80 °C until use. Using a previously published cell-free reaction protocol57, reaction mixtures were composed of 26.6 % (v/v) of in-house lysate, 1.5 mM each amino acid except leucine (1.25 mM), 1 mM DTT, 50 mM HEPES (pH 8.0), 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/mL tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine, 30 mM 3-PGA, 2% PEG-8000, 0.5 % (v/v) Protector RNase Inhibitor (Roche), 2 nM LacZ sensor plasmid DNA, 2 uM RNA trigger, and 0.6 mg/mL chlorophenol red-ß-D-galactopyranoside  for lacZ sensor. Optimal potassium glutamate (40–140 mM) and magnesium glutamate (2–8 mM) concentrations were determined for lacZ reporter product. Reactions were first assembled on ice without CPRG, incubated at 37 °C for 30 min, chilled on ice for 5 min, and then CPRG was added to reaction. 1.4 µl of reaction mixture was then applied to pre-blocked 5% BSA 2 mm paper disks, placed in a black, clear bottom 384-well plate  and incubated at 37 °C for 1.5 h for the detection of lacZ expression.Cell extract was prepared as described by Kwon and JewettAll code used in this work is available in Supplementary Note All toehold switch sensors from this work have been deposited at AddGene. AddGene #110696-110717, 111907-111909. All other data supporting the findings of this study are available within the article and its Supplementary Information files, or are available from the authors upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules,  Flaviviridae family, a group of single-stranded (ss) RNA enveloped viruses. Members of this family also include the human pathogens dengue virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, Japanese encephalitis virus and hepatitis C virus (HCV)1. ZIKV is an arthropod-borne virus and transmission is primarily caused by the bite of infected Aedes species mosquitoes2, but it can also be spread perinatally3, sexually4 or by blood transfusions5. ZIKV infection in humans is generally asymptomatic7; however, a significant proportion of infected individuals (~20%) develop neurological conditions, including Guillain-Barré syndrome (GBS), which is the most frequent cause of acute flaccid paralysis not associated with poliovirus in adults, and microcephaly, in newborns. An increase in the incidence of GBS and microcephaly has been associated with outbreaks of ZIKV in Micronesia (2007), French Polynesia (2013), and Brazil (2015)10. Accordingly, the World Health Organization Public Health Emergency Committee declared ZIKV a global public health emergency of international concern11.Zika virus (ZIKV) is an emerging human pathogen of the 14. The polyprotein undergoes successive proteolytic processing to generate three structural proteins: the capsid protein, the precursor of the membrane protein and the envelope protein, as well as seven nonstructural proteins . The different non-structural proteins are involved in the essential steps of the viral replication cycle inside the host cell. Among them, NS5 is the largest (903 amino acids) and most conserved viral protein15. NS5 includes an N-terminal domain containing methyltransferase activity (residues 1–262) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain (residues 275–903). A short linker interdomain formed by residues 263–274 covalently connects both enzymatic activities17. The crystal structures of the whole ZIKV NS5 protein and the RdRp domain alone have been recently solved18. ZIKV RdRp exhibits a typical encircled right-hand configuration with palm, fingers and thumb subdomains, and six conserved motifs (A–F) that are common to other viral RdRps. These motifs are critical for its polymerase activity, as they are involved in RNA and nucleotide binding, coordination of metal ions, and catalysis19. The catalytic aspartates are located in conserved motifs A (D536) and C (GDD tract at positions 665–667). These aspartates constitute the catalytic triad responsible for nucleotide transfer to nascent RNA. The process entails the coordination of two divalent cations by these residues that are essential to the catalytic process20.ZIKV has a positive-sense ssRNA genome of approximately 10.8 kb in length, which encodes a single polyprotein of 3400 amino acids flanked by untranslated RNA regionsversus conventional DNA-dependent DNA synthesis – viral RdRps are key targets for direct-acting antiviral agents21. The recent development of nucleoside and non-nucleoside analogs  targeting RdRps of diverse members of the Flaviviridae family have generated encouraging results29, including sofosbuvir, the first HCV RdRp NAI approved by the U.S. Food and Drug Administration for its potent antiviral efficacy and good tolerance in humans30. While NNIs typically require no intracellular modification to elicit their inhibitory activity, as they bind directly to allosteric sites on RdRps, NAIs generally require phosphorylation by the host cell machinery to be active. Phosphorylated nucleoside analogs are thus able to bind to the RdRp active site and compete with natural NTPs for their incorporation into the nascent RNA. Despite recent significant progress, no drugs are yet licensed for the treatment of disease caused by any mosquito-borne flavivirus, including ZIKV .Owing to substantial differences in the mechanisms of replication in RNA viruses and the host cell – RNA-templated RNA synthesis de novo, which can be then quantified in real-time using a fluorescent dye (SYTO 9), which detects the presence of double-stranded (ds) RNA generated after the copy of single-stranded homopolymeric RNA. We also demonstrate the potential value of this protocol to screen antiviral compounds by using known broad-spectrum antivirals, which reproduce their inhibitory activities in this assay. The versatility of this method was further established with RdRps from two distinct viruses: foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV). The implications of these results for high-throughput approaches are discussed.In this study, we outline the development of a reliable method for rapid detection of ZIKV polymerase activity in real-time. We show that recombinant ZIKV RdRp can synthesize RNA Eschericia coli BL21(DE3)-pRIL cells were produced according to standard protocols34. The following reagents were purchased from Applichem: LB medium (powder), ampicillin (used at 100 μg/mL) and chloramphenicol (used at 17 μg/mL), MnCl2, ammonium acetate, NaCl, MgCl2, ZnCl2, glycerol, imidazole, isopropyl-β-D-1-thiogalactopyranoside (IPTG), 1,4-dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF) and Tris base. DpnI restriction enzyme, BSA, Phusion® High-Fidelity (HF) DNA polymerase and Gibson Assembly® Master Mix were obtained from New England Biolabs. Stock solutions of ATP and GTP, SYTOTM 9 green fluorescent dye and HisPurTM Ni-NTA resin were purchased from ThermoFisher Scientific. pET28a and pET16b expression vectors were obtained from Novagen. Nucleotide analogs ribavirin 5′-triphosphate and cordycepin 5′-triphosphate were obtained from Jena Bioscience and Sigma-Aldrich, respectively. SYBR® Green II fluorescent dye, Tween 20, heparin, EDTA and oligonucleotides were also purchased from Sigma-Aldrich. ssRNA polyuridylic acid (poly-U), ssRNA polyadenylic acid (poly-A) and ssRNA polycytidylic acid (poly-C) were purchased from Amersham Biosciences. Radiolabeled nucleotides [α-32P]ATP and [α-32P]GTP  were obtained from Perkin Elmer. FMDV viral protein genome-linked 1 (VPg1) was synthetically prepared as previously described35.Electro-competent 16) from isolate PRVABC59 13 was cloned into the pET16b expression plasmid between NdeI and XhoI sites using the Gibson assembly method. To do this, the vector backbone was amplified using pET16b as template; the PCR reaction contained 1 × Phusion HF buffer, 200 μM dNTPs, 0.5 units of Phusion® HF DNA polymerase and 0.25 μM of a specific pET16_Fw and pET16_rv primer pair (Supplementary Table NS5 gene. PCR amplification was similar to that described above but using the specific primers NS5 short_pET16_Fw and NS5_pET16_rv (Supplementary Table E. coli BL21(DE3)-pRIL cells. After plasmid extraction from three independent bacterial colonies, nucleotide sequencing determined that two DNA samples contained the correct construct. The resulting plasmid pET16a-ZIKV-NS5RdRp encodes for ZIKV RdRp fused to an HHHHHHHHHHSSGHIEG amino acid tract in its N-terminus that is used for affinity purification using HisPurTM Ni-NTA resin The predicted molecular weight of this protein is 75 kDa.The ZIKV NS5 RdRp domain  FMDV 3D polymerases, were obtained in previous studies37, using primers NS5B_HCV_Fw and NS5B_Δ21_HCV_rv , 2, 10%, glycerol, 10 mM imidazole, and 0.1% (v/v) Tween 20] and sonicated on ice for 6 cycles of 20 s alternating with 5 cycles of 10 s. Cell debris was pelleted at 11,000 rpm for 30 min at 4 °C and the supernatant mixed with 800 μL of Ni-NTA resin previously equilibrated with 20 volumes of lysis buffer without ammonium acetate (BWE buffer). The lysate was incubated with the resin in batch method with gentle mixing during 1 h at 4 °C. The unbound fraction was then removed by decantation and the resin was then loaded onto a column and extensively washed with 20 × column volumes of BWE buffer and 20 × column volumes of BWE buffer containing 25 mM imidazole. The resin was further washed with increasing concentrations of imidazole . Finally, the His-tagged protein was eluted in 400 μL of BWE buffer containing 400 mM imidazole. The sample was dialyzed for 3 hours at 4 °C against 200 volumes of dialysis buffer . Samples obtained from different purification batches were pooled, quantified, aliquoted and stored at −80 °C until further use. Expression and purification of the recombinant ZIKV NS5RdRp-GNN (with D665N and D666N substitutions) was carried out following the same protocol. Likewise, the expression and purification of HCV NS5bΔ21 and NS5bΔ21-GNN polymerases was carried out following the same protocol described for ZIKV NS5. The protocol for the expression and purification of FMDV 3D polymerases has been described previously36.The bacterial pellets recovered from 200 mL cultures were resuspended in 20 mL of lysis buffer  was added to each well of a black 96-well flat-bottom plate. Reactions were initiated by the addition of 1 μL of FMDV 3D  and plates were incubated at 37 °C for 30 min. Fluorescence intensity was recorded as above.Polymerase activity assays with HCV NS5bΔ21 were performed as described above for ZIKV RdRp. For the detection of FMDV 3D polymerase activity, 49 μL of reaction mix ATP and 40 μg/mL poly-U; or 500 μM GTP, 16 nM [α-32P]GTP and 20 μg/mL poly-C. Reactions were performed at 30 °C and initiated by addition of of 1 μL of either ZIKV or HCV recombinant polymerases to reach a final concentration of 1 μM in the assay. The reaction was stopped at different time points by addition of 2 μL of formamide loading buffer . The reaction products were resolved by electrophoresis in 20% polyacrylamide gels containing 1 × TBE buffer and 8 M urea, at 30 W for 120 min. De novo synthesized polynucleotides using poly-U and [α-32P]ATP as substrates were detected either by autoradiography or by phosphorimaging .Similar procedures were used for both ZIKV NS5 RdRp domain and HCV NS5bΔ21 polymerases. Radioactivity assays were performed in 10 μL reactions containing 50 mM Tris-HCl, pH 7.5, 2.5 mM MnClm determinations were obtained by plotting the velocity of the reaction as a function of nucleotide or ssRNA template concentrations using nonlinear regression. IC50 values were obtained by fitting the velocity data to a four-parameter logistic equation. Kinetic parameters and IC50 values were calculated using Sigmaplot, version 11. Z’ factor was calculated according to Zhang et al.39: Z’ = 1 − [(3SDc+ + 3SDc−)/(meanc+ − meanc−)] where “c+” is the activity obtained in a standard assay and “c−” is the nonspecific activity obtained in a control performed in the absence of MnCl2.Fluorometric results were expressed as mean ± SD. Statistical significance was analyzed by two-way ANOVA using GraphPad Prism, version 7, as specified in the figure legends. KThe RdRp domain of ZIKV NS5 and a catalytic inactive mutant (GNN) were purified as described in Methods. Recombinant proteins were ≥95% pure as judged by PAGE analysis and Coomassie brillant blue R-250 staining Fig. .Figure 142. For the preliminary evaluation of ZIKV NS5 RdRp domain activity in vitro, we adapted a polymerization assay based on the detection of radioactive nucleotides incorporated by the polymerase. This method makes use of a homopolymeric ssRNA as a template in the absence of any primer, since it has been previously demonstrated that ZIKV NS5 can initiate RNA synthesis de novo42. The reactions were performed in the presence of radioactive-labeled nucleotides, and polymerization products were resolved by PAGE. RNA synthesis in the absence of primer was observed both in the presence of poly-U and [α-32P]ATP as template and nucleotide substrates , we performed the same assay but in the presence of radioactive nucleotides that were less competent for viral RNA synthesis: [α-32P]GTP to poly-U and [α-32P]ATP to poly-C. As shown in Fig. de novo RNA synthesis. Thus, these results show that both homopolymeric templates, poly-C and poly-U, can be used by ZIKV to initiate RNA replication, as has been previously documented42.Previous studies suggested that, under certain circumstances, 38. We anticipated that binding of this intercalating agent to dsRNA generated by ZIKV RdRp polymerization activity would lead to an increase in the emitted fluorescence.Based on the above results, we next sought to detect polymerization activity using a fluorescence-detection method. For this aim, we attemped to establish an assay to quantify RNA synthesis activity as the relative increase in fluorescence emitted by SYBR Green II dye after binding to dsRNA. This procedure was adapted from methods previously described to detect dsDNA synthesis by the human primase-polymerase PrimPol43 or human PrimPol38. Our resulted suggested that SYBR Green II acts as an inhibitor of ZIKV RdRp activity. Thus, we decided to test other fluorescent dyes for the real-time detection of newly synthesized dsRNA. It has been reported that SYTO 9 dye shows lower interference on polymerization assays when binding to dsDNA45. In contrast to the assays with SYBR Green II, we found that both end-point and real-time polymerization assays resulted in similar increases in fluorescence when using poly-U as template  also led to complete loss of polymerase activity  or barely detectable (using poly-U) increase in fluorescence. In contrast to real-time experiments, we found significant increases in polymerase activity in an end-point experiment where the dye was added after the reaction was completed Fig. . Previouate Fig. . Accordiate Fig. . ZIKV Rdity Fig. . LikewisTo further investigate the possible use of SYTO 9 dye to detect activity in the presence of other templates, we used poly-C. However, we detected increases in fluorescence only in end-point reactions, when SYTO 9 was added after the recation was complete, and not in a continuous reading assay when it was added before initiating the reaction Fig. . These r2 and enzyme) affected RdRp activity. The presence or absence of DTT and NaCl in the assay had little effect on RNA synthesis, which was only slightly impeded at high concentrations , whereas maximum activity was recorded with 2.5 mM MnCl2  and a Km for ATP of 561 ± 38 μM  and ribavirin 5′-triphosphate . Both compounds reduced the polymerase activities , followed by the NAIs, 3′dATP (54 ± 7 μM) and RTP (946 ± 46 μM). To confirm that the decrease in fluorescence was linked to the inhibition of RNA synthesis, we repeated these experiments using radioactive-labeled nucleotides. We found a reduced polymerization activity in the presence of inhibitors  that correlated with the aforementioned IC50 values . Each experiment was carried out in triplicate and on independent days. Relative activity values were determined as the velocity of polymerization recorded during the first 10 min of the reaction. The mean Z’ value obtained was 0.62, which according to published standards, qualifies our method as an excellent assay for high-throughput screening application39.The robustness and suitability of this assay as a prospective, high-throughput method to screen polymerase antiviral compounds was examined by calculating the Z′ value, a standard statistical measure to evaluate the quality for high-throughput platformsde novo using radiolabeling  but not an inactive mutant, and the activity was dependent on the presence of Mn2+  and HCV (recombinant NS5B) Fig. . We founing Fig.  and the ing Fig. , which in2+ Fig. . We alsoPg1 Fig. . Overall59. Although promising developments in this direction have been achieved (reviewed in32), there are as yet no antiviral agents licensed against ZIKV at the clinical level. Owing to significant differences in the mechanisms of replication between cellular DNA and viral RNA genomes, the latter involving the synthesis of RNA molecules templated by RNA, RdRps are attractive targets for the development of specific antiviral treatments. The use of antivirals against non-RdRp viral polymerases, such as human immunodeficiency virus and hepatitis B virus reverse transcriptases, and herpes virus DNA polymerase, supports the suitability of this group of enzymes as therapeutic targets60. Accordingly, the development of a fast and reproducible method for the screening of compounds with anti-ZIKV properties is a promising advance.There is an urgent need to develop new treatments for ZIKV infection and to control its rapid geographical spread. Different approaches for the discovery of potential small-molecule inhibitors include the screening of chemical libraries, molecular modeling and virtual screening66. However, there are several practical limitations to these approaches, such as the requirement for radioactive substances, entailing additional biosafety measures63, or an arduous experimental setup66 when compared with fluorescence-based methods64. Indeed, an advantage of fluorescence-based methods over traditional approaches is the absence of radioactive compounds, which facilitates their broad use in different laboratory settings without specific facilities or training requirements. An additional advantage of our strategy is that it allows the time-resolving determination of the polymerase activity, which we believe gives further insight into the mechanisms of replication and inhibition.Several methods for high-throughput drug screening against virus RdPs have been described and validated41, or costly fluorescent-labeled RNA substrates40. Our method has the advantages of being a more economically affordable alternative, as it uses inexpensive homopolymeric RNA as a template substrate instead of labeled synthetic heteropolymeric RNA. Our approach would also allow for assay scale-up to high-throughput formats , for the rapid testing of small-molecule compound libraries64.There have been previous attempts to develop high-throughput activity assays to identify drug inhibitors specifically against ZIKV RdRp. These methods include the use of either radioactive nucleotidesin vitro, which is of use in the identification of prospective antivirals. As part of our studies we have shown that RTP elicits a mild inhibition on ZIKV RdRp polymerization in vitro. We posit that the observed inhibition might be a consequence of reduced polymerase efficacy to elongate molecules where an RTP residue is incorporated67. RTP is not a chain terminator and its incorporation into the viral RNA has been linked to an increase in transition mutation rates in vitro and in cell culture for different RNA viruses, including ZIKV71.Fluorometric measurements of polymerase activity in the absence/presence of three representative inhibitors  revealed a positive correlation with the results using a traditional assay based on radiolabeled nucleotides, further validating the fluorescence-based approach for the screening of antiviral compounds. The possibility of visualizing dsRNA synthesis in real-time increases the sensitivity of the assay, allowing an accurate determination of replication kinetics in the presence or absence of the drug and the predicted affinity constants of the compound tested. This method also permits the characterization of inhibitory molecules 72, which in the presence of a fluorophore can further increase their inherently elevated stability44. Thus, these RNA-dye complexes might impede the effective elongation by the viral RdRp.Under different experimental conditions, including the use of different concentrations of SYBR Green II and SYTO 9, we found that poly-C RNA was not a suitable substrate for the detection of polymerase activity in real-time. Conversely, both poly-A and poly-U homopolymers were effective as template molecules in real-time assays. We hypothesize that the inhibition of polymerase activity is produced by the interaction of RdRp with the dye during dsRNA synthesis. It is possible that poly-G synthesis as a result of replicating poly-C can lead to non-canonical G-quadruplex structuresIn conclusion, we have demonstrated that the procedures developed here can be easily adapted to measure polymerization activity of several viral RdRps, strengthening our method as a universal procedure for the development of high-throughput tools to characterize viral polymerases  and to screen small-molecule libraries to identify antiviral drugs. In particular, we believe that this platform can be a useful tool for the development of therapeutics against ZIKV and other flaviviruses, which are currently unavailable.Supplementary Information