Clear seasonal patterns were documented for metabolic indices, zooxanthellae density and rETR, while cyclic diel patterns only occurred for yield and rETR, and RNA/DNA ratio. Significant daily variation was observed for RNA/DNA ratio, Chl a concentration, yield and rETR. Results suggest that zooxanthellae density and protein concentrations are good long-term indicators of coral health whose variance is largely seasonal, while RNA/DNA ratio and rETR can be used for both long-term  and short-term (diel) coral monitoring. Chl a can be used to describe changes between days and yield for both diel and daily variations. Correlations between health indices and light history showed that short-term changes in irradiance had the strongest impact on all health indices except zooxanthellae density for A. digitifera; for A. spicifera no correlation was observed at all. However, cumulative irradiance over the several days before sampling showed significant correlations with most health indices suggesting that a time-lag effect has to be taken into account when interpreting diel variations in coral condition.Coral health indices are important components of the management assessments of coral reefs, providing insight into local variation in reef condition, as well as tools for comparisons between reefs and across various time scales. Understanding how such health indices vary in space and time is critical to their successful implementation as management tools. Here we compare autotrophic and heterotrophic coral health indices, examining specifically the temporal variation driven by the local environmental variation, at three scales . We compared metabolic indices  and autotrophic indices (Chlorophyll  Photosynthesis in the symbionts of corals provide a carbon source for the coral host that is energy-rich but nitrogen-poor, while the zooxanthellae benefit from high nitrogen and phosphorus metabolic waste products of the corals Since metabolic rates in the coral host depend on photosynthetically derived and translocated products Seasonal cycles have been observed for a variety of indices describing coral metabolism such as protein concentration a concentration, zooxanthellae density, RNA/DNA ratio and protein concentration) for two dominant Acropora species as well as driving physico-chemical factors for those seasonal changes Acropora species for a better understanding for how useful metabolic and autotrophic indicators are for short-term as well as long-term monitoring projects. In this study we define coral health as relative physiological rates – thus our autotrophic and metabolic measurements are used as indicator for physiological changes inside the coral occurring under a range of both normal and extreme (La Niña) conditions. Measurements were taken during a normal winter season and during an extreme summer season (La Niña) and so include a wide range of normal as well as abnormal variations of physiological indices. Thus the aim of this study is to determine 1) how changes in metabolic indices and autotrophic indices occur on a diel, daily and seasonal basis for A. spicifera and A. digitifera, 2) how indices which express diel changes are correlated and 3) which physico-chemical factors are most likely to predict metabolic indices (RNA/DNA ratio and protein concentration) on a diel basis and 4) the time-lag between changes in light and the response of health indices.Our earlier work at Ningaloo Reef determined seasonal changes in health indices , is located at Ningaloo Reef, along the North-West Cape of Western Australia . Sandy Bay lagoon was chosen as hydrodynamic patterns have been described previously Acoropra digitifera and Acropora spiciferaA.digitifera a caespito-corymbose coral, and A. spicifera, a plate coral. Coral samples were collected four times daily  over four days (when weather conditions were convenient) during winter (August 2010) and summer (February 2011) at Station 4 (A. digitifera and A. spicifera) were tagged at six stations in the reef lagoon  concentration, RNA/DNA ratio, protein concentration during winter 2010, summer 2011 and additionally in autumn 2010 . Corals were sampled while snorkelling; with in situ measurement on the colony for yield and rETR and removal of 1–5 cm length coral pieces from the middle of the coral colony (tip of the branch) for each of the other health indices (see below).Two different coral species, tation 4 . Februar2) and Chl a concentration in the tissue (µg cm−2) were stored at −20°C prior to analysis. Methods for zooxanthellae density and Chl a in coral tissue were adapted from Siebeck et al. Coral samples (∼3–5 cm long) for the analysis of zooxanthellae density (per cma concentration was determined following the methods of Jeffrey and Humphrey g) for 15 min. The supernatant was discarded and the pellet re-suspended in 10 ml of acetone (100%) and Chl a extracted for 24 h in the dark (−20°C). After centrifugation at 3500×g for 10 min fluorescence was measured in a T700 fluorometer (Turner Designs) and standardized to coral surface area.Chl −1 for DNA and RNA, duplicates) and control homogenates were added to three 96 deep-well microplates. Plate one was treated with TE buffer (15 µl) while plate two with TE buffer (7.5 µl) and RNAse (7.5 µl) and plate three with RNAse (7.5 µl) and DNAse (7.5 µl). Plates were incubated (35 min for plate one and two and 60 min for plate three) and Ribogreen added to each well. Plates were read in a microplate reader (485 nm excitation and 528 nm emission). RNA was calculated by subtracting the fluorescence reading of plate two from plate one while DNA was calculated by subtracting the fluorescence reading of plate three from plate two. Calculating of RNA and DNA concentrations was based on the standard curves of each plate. Finally the ratio between DNA and RNA concentration was determined.Samples for RNA/DNA and protein analysis were stored at −80°C prior to analysis. RNA content varies with metabolic demand and is correlated with new protein synthesis, while DNA content is largely stable −1 to 1.5 mg ml−1 protein duplicates) in a 96 deep-well microplate. After adding 25 µl of reagent A and 200 µl of reagent B to each well, absorbance was read after 15 min at 750 nm. Protein content was calculated based on standard curve of each plate and all protein concentrations standardized afterwards to DNA values.For protein analysis, a standard DC Protein Kit was used −2 s−1) Variations in the effective quantum yield (yield) of photosystem II (PSII), a way to assess the photosynthetic performance of PSII, were measured using an underwater pulse-amplitude modulated (PAM) fluorometer  on 10 sections of the tagged coral colony. The fiber was placed at a fixed distance (1 cm) in front of the coral tissue. The yield (ΔF/Fm′) was measured by exposing 10 sections of the colony separately to a 0.8 s period of saturating light , phytoplankton and zooplankton.Light and temperature were measured using temperature loggers (Hobo Pendant Data Logger) that were deployed between 3 to 7 days before the sampling.Short-term current speed was estimated using drifters. Two crucifix design drifters a, and picoplankton .Water quality samples were taken for the analysis of dissolved nutrients, total nitrogen (TN), Chl x (NO3+ NO2) and NH4), water samples (40 ml) were filtered through 0.45 µm filters and stored at −20°C, before flow injection analysis (FIA) with detection by absorbance at specific wavelengths for nitrate (NO3)/nitrite (NO2) (QuikChem FIA+Lachat 8000 series). Briefly, Nitrate was reduced to nitrite through a copperized cadmium column that than reacted with sulphanilamide under acid conditions to form a diazonium ion. The diazonium ion is coupled with N- (1-naphthyl) ehthylenediamine dihydrogenchloride that has an absorbance maximum at 520 nm (Quikchem Method 31-107-04-1-A) For the analysis of dissolved inorganic nitrogen (DIN) , unfiltered water samples (50 ml) were stored at −20°C until analysis. TN was determined from autoclave digests with potassium persulphate  Organic nitrogen (ON) was calculated by subtracting DIN from TN.a was filtered onto 0.7 µm filters (Whatman GF/F) and the filters stored at −20°C in the dark until fluorometric analysis of duplicated 90% acetone extracts was carried out Seawater samples (1 L) for Chl Synechoccocus, Prochlorococcus and picoeukaryotes), 1.5 ml seawater was fixed with gluteraldehyde  for 10 minutes and then snap frozen in liquid nitrogen. Samples were analysed using flow cytometry following the technique of Patten et al. −1 for 100 s. Nitrogen (N) and carbon (C) content of picoplankton was calculated based on fixed factors for picoeukaryotes (39.2 fg N and 836 fg C ) Prochloroccocus (4 fg N and 46 fg C) and Synecoccocus (30 fg N and 213 fg C) Prochloroccocus, Synechoccocus and picoeukaryotes.To determine concentrations of autotrophic picoplankton groups (−1 for 50 m around the station) and preserved with formaldehyde (ca. 5%). Dry weight analyses were carried out for zooplankton between 100 and 1000 µm. Wet zooplankton samples were added to a pre-weighed container after cleaning samples of salt with DI water (30 s) and dried in an oven (60°C for 24 hours). Samples were then cooled down in a desiccator and weighted for subsequent calculation of dry weight (DW) Zooplankton was sampled with a plankton net  and analysed with Type I  sum of squares with permutation of residuals under a reduced model (9999 permutations). Health indices, which showed diel patterns were in addition tested for day/night differences (morning and noon combined and evening and midnight combined) with the same design as described above but night/day instead of times per day. Log-transformation was done based on results of a draftman plot as well as Grubbs’ test to reduce outliers and make data more continuous. Pair-wise tests were performed when significant differences occurred. To determine correlations between health indices, that displayed diel patterns the coefficient of determination R2 was tested with DistLM (Primer 6) based also on Euclidean dissimilarity matrix after log-transformation .A four-factor PERMANOVA was performed in PRIMER Version 6  with species, seasons and times per day as fixed terms and days nested in season (crossed factors) a concentration in the water, nitrogen and carbon content of picoplankton, zooplankton (dry weight) and DIN and ON were tested with DistLM (Primer 6) based on Bayesian information criterion (BIC)  To determine which physico-chemical factors best explain changes in protein concentration and RNA/DNA ratio on a diel basis, health indices and physico-chemical factors  between health indices and light history, were determined using DistLM (Primer 6) based on Euclidean dissimilarity matrix after log-transformation of health indices for integrated light during 0, 1, 3, 6, 12, 24, 36 and 48 hours prior to sampling . Diel as well as seasonal data was included to get a broader light spectrum.A. digitifera and A. spicifera between summer 2011 (February) and winter 2010 (August)  were tested for (August) . Diel pa1, 179 = 58.22, p<0.001; se: F1, 179 = 27.44, p<0.01; sp×se: F1, 179 = 2.41, p<0.01), RNA/DNA ratio  and zooxanthellae density . However, pair-wise tests for protein concentration revealed that only A. spicifera showed seasonal differences (p<0.01) with higher protein concentrations in summer than in winter, while for zooxanthellae density significant differences were only found for A. digitifera (p<0.001) based on higher values during winter  with highest values occurring during summer compared to winter for both species (Significant differences between species (sp) and seasons (se) were found for protein concentration  due to higher concentrations in summer for A. spicifera than for A. digitifera  while no differences occurred in winter between species for Chl a concentration per surface area (p>0.05) , with only rETR expressing seasonal differences with higher rates during summer  (Chl (p>0.05) . No seasp<0.001) .7, 178 = 10.53, p<0.001), Chl a , yield  and rETR  exhibited significant differences between days within each season. Significant changes throughout the day were observed for yield as well as rETR  even though this pattern was not consistent throughout all days (da (se) ×di: F13, 167 = 7.55, p<0.001; F13, 164 = 34.37, p<0.001)  even though day/light variations were only apparent for some and not all days. For rETR, daily variation lost importance when testing day/night differences with day/night differences occurring for summer and winter . For RNA/DNA, diel patterns were observed within a day but without a consistent pattern (i.e. changing between days)  . In genep<0.001)  and pairp<0.001) . Signifien days) . Howevera per cell was positively correlated with yield values  and yield was also positively correlated with rETR . R-values increased when only those values measured during day were taken into account and night values excluded . No correlation was found between RNA/DNA ratio with Chl a per cell, yield or rETR for either A. digitifera or A. spicifera within the whole day or only during daytime.Correlations between health indices which changed on short-term basis showed that Chl a concentration, zooplankton, N-content of picoplankton and C-content of picoplankton) were tested for best prediction of coral metabolic indices , separately for A. digitifera and A. spicifera during winter and summer. During winter changes in protein concentration during the day for A. digitifera were correlated with ON in the water, while RNA/DNA ratio showed strongest yet non-significant relationship with light. In summer, for A. digitifera, protein concentrations were not correlated with temperature or light, while RNA/DNA ratio were best explained by nitrogen provided by picoplankton and organic nitrogen  correlated with protein concentrations, while temperature was negatively, and carbon supplied by picoplankton positively correlated with variability in RNA/DNA ratio. In summer light showed strongest but non-significant correlation with protein concentration while Chl a concentration in the water column showed a negative correlation with RNA/DNA ratio of A. spicifera .Correlations between light and health indices showed that light measurements taken at the time of sampling showed highest correlations for protein concentration, RNA/DNA ratios, rETR and Chl ntration . Howevergitifera . Yield va concentration and zooplankton showed similar patterns with lowest concentrations occurring at noon and highest concentrations in the evening while in winter Chl a concentration declined throughout the day and zooplankton peaked at noon. Highest DIN concentration was found in the evening for both seasons while ON showed lowest values in the evening for summer and no real change throughout the day in winter  and short-term (diel and daily) changes in environmental factors. Previous work A. spicifera were generally higher than of A. digitiferaA. digitifera on a diel basis and for A. spicifera protein synthesis was correlated with light across seasons Our results suggest that protein concentration is relatively stable on a diel basis, suggesting that this index could be used primarily to determine changes in seasonal and long-term coral health. This is in agreement with previous work which showed that protein concentration only shows detectable changes after weeks or months of perturbation, generally associated with changes in particulate food availability Acropora species but also integrated light level from up to three previous days were correlated with the RNA/DNA ratio. However, the lack of correlation between RNA/DNA ratio and rETR, an indicator for photosynthetic activity RNA/DNA ratio showed significant differences between species, between seasons, between days and times of the day. This index is therefore an appropriate indicator for short as well as long-term changes in metabolic rates of corals. Because daily and diel variations are smaller than seasonal differences, the RNA/DNA ratio also provides an index of coral health that integrates appropriately across time scales. Seasonal changes in RNA/DNA ratio have been seen for corals 6 and 5×106 cells per cm2In general, zooxanthellae densities in our study fell within the range of previous studies, ranging between 0.5×10A. digitifera. Previous studies have shown that it takes 24 hours for zooxanthellae densities to be modified A. spicifera, changes in zooxanthellae density do not seem to be directly driven by light history within either hours or days, suggests that zooxanthellae density is driven primarily by other factors, such as nitrogen uptake A. digitifera zooxanthellae densities can be used to describe long-term  changes in coral health. This is in agreement with other studies which also observed seasonal changes in zooxanthellae densities The strongest correlation of incident light with zooxanthellae density occurred more than 24 hours prior to sampling in Daily changes in physico-chemical factors such as temperature, light and nutrient concentrations might have been not large enough to result in measurable changes in zooxanthellae density. Seasonal environmental variation is much greater, resulting in larger variation in symbiont densities. Temperature, nutrients and light were all observed to be important in driving measurable variation in coral health indices a per unit coral surface area, or for Chl a per zooxanthellae. Our measured daily variations suggest Chl a can be a good indicator for short-term (days) changes in physico-chemical impacts on coral health. Daily changes in light intensity have also been known to impact coral Chl a concentrations a concentrations, suggesting there are no real time-lag effects. In regard to seasonal changes no significant variation was detected in this study in contrast with previous studies that have shown significant variations in Chl a concentration throughout the year in the studied species a might be suitable to describe seasonal changes in physico-chemical factors in case these changes are big enough, however it should be kept in mind that daily changes might also influence these patterns.We found no seasonal or diel difference for Chl A. spicifera are in accordance with previous studies showing highest values at night, a decrease in the morning, low values around noon and an increase during afternoon towards evening A. digitifera, with lowest values in the morning might be due to different genetic types of Symbiodinium Acropora species’ photosystems react in similar ways to variations in light levels. However, yield values showed only a low correlation with short-term light history (R = 0.24 and 0.26) suggesting that other physico-chemical factors are more important for changes in yield. Despite previous studies which showed seasonal changes in yield values due to temperature °C between winter and summer. Overall for the studied Acropora species yield does not seem to be a good indicator for determining any seasonal differences, however yield appears more appropriate indicator for short-term changes since it reacts quickly to variations in light intensity. However since our study fell within a La Niña year and light values during summer were lower than normal Diel patterns in photosynthetic yield of Symbiodinium sp., colony morphology, animal behaviour (polyp extension and contraction) as well as the light history of a particular location from which fluorescence measurements are taken Seasonal, daily and diel changes in rETR were clearly observed in our study, suggesting this measure was a good short-term and long-term indicator of coral health. Higher values at noon than in the morning are in the same range as those seen in previous studies a concentration in coral tissue is a better indicator for daily changes but should be used carefully to describe seasonal changes in coral health, while photosynthetic yield is good indicator to describe diel and daily variations for the two studied Acropora species at Ningaloo Reef. The fact that Chl a per cell is positively correlated with yield suggests that photo-adaptation processes in the antennae and PSII are related A. digitifera relies more strongly on autotrophy, which is in accordance with our earlier work Overall, we conclude that zooxanthellae density and protein concentrations are good long-term indicators of changes in coral health since they express seasonal but not diel or daily changes, however species-specific variations must be taken into account. RNA/DNA ratios and rETR can be used as long-term as well as short-term indices changes in coral health. However diel changes in RNA/DNA ratios remain unresolved. Chl Table S1Four-factorial PERMANOVA for health indices of two Acropora species.(DOCX)Click here for additional data file.Table S2Pair-wise correlations  for diel variations of A) effective quantum yield and B) relative electron transport rate.(DOCX)Click here for additional data file.Table S3Pair-wise correlations  for diel variations for RNA/DNA ratios.(DOCX)Click here for additional data file.


Tobacco mosaic virus (TMV) has been studied for many years. The RNA of TMV moves cell-to-cell in a non-encapsidated form in a process depending on virus-encoded movement protein (MP). Here, we discuss the current state of the art in studies using TMV and its MP as a model for RNA transport. While the ability of plants to transport viral and cellular RNA molecules is consistent with RNA transport phenomena in other systems, further studies are needed to increase our ability to visualize viral RNA (vRNA) in vivo and to distinguish RNA-transport related processes from those involved in antiviral defense.Studies during the last 25 years have provided increasing evidence for the ability of plants to support the cell-to-cell and systemic transport of RNA molecules and that this process plays a role in plant development and in the systemic orchestration of cellular responses against pathogens and other environmental challenges. Since RNA viruses exploit the cellular RNA transport machineries for spreading their genomes between cells they represent convenient models to investigate the underlying mechanisms. In this regard, the intercellular spread of  Tobacco mosaic virus (TMV) is a rod-shaped virus with a positive-sensed RNA genome that encodes 126 kD and 183 kD subunits of replicase, a 30 kD movement protein (MP) and a 17.5 kD coat protein (CP). The virion consists of the viral RNA (vRNA) protected by 2130 subunits of assembled CP. The virus has been a paradigm for RNA virus movement since the requirement of its MP for virus movement was defined    interacts with microtubules (Wright et al., It will also be important to determine how far the observations described for TMV and summarized herein also apply to the movement of other RNA viruses. Mechanisms involved in the cell-to-cell movement of viruses have been reviewed in several recent overview articles Lucas,  and showHowever, a role of RNA-enriched mobile particles in the transport of RNA viruses and other RNA molecules is certainly consistent with similar observations in other systems. These include neuronal granules for transport of mRNAs along axons and dendrites for localized translation at synapses (Kiebler and Bassell, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA∶polymerase complexes, providing a mechanism for the genesis of viral genome∶polymerase complexes observed in vivo.The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its  in vivo.Infectious pancreatic necrosis virus (IPNV) is highly contagious and causes severe disease in fish. As a result of intensive rearing conditions it has become a serious problem for the salmon and trout farming industries. IPNV, like many other viruses, replicates its genome using a protein (a ‘polymerase’) that is itself encoded by the viral genome. Unusually, in infectious IPNV particles the polymerase is found chemically linked to the viral genome. We have determined the atomic structure of IPNV polymerase using X-ray crystallography, revealing some significant differences in the fold of the protein chain compared to other well-characterized viral polymerases. By mutating an amino acid residue at the beginning of the protein we show how the chemical linkage to the viral genome can be disrupted. This provides an elegant mechanism for the attachment of the viral genome to the polymerase observed  Flaviviridae), Foot-and-Mouth disease virus (family Picornaviridae) and bacteriophage Φ6 (family Cystoviridae)] share a similar structure and mechanism of catalysis and are therefore potential targets for generic antiviral drugs palm, fingers and thumb domains and contain a number of conserved sequence motifs (A–G), the conserved residues GDD within motif C being essential for catalytic activity Virus-encoded RNA-dependent RNA polymerases (RdRPs) are vital components in the life cycle of RNA viruses, being responsible for both genome replication and transcription. RdRPs from viruses that are considered unrelated -UTP  or [α32P]-GTP (replication). TNTase, TdNTase and self-guanylylation assays contained only labeled nucleotides of [α32P]-UTP (TNTase), [α32P]-dTTP (TdNTase) or [α32P]-GTP (self-guanylylation). Reverse transcription was performed with 1 mM dATP and dGTP and 0.2 mM dCTP supplemented with 0.3 µM (0.1 mCi/mL) labeled [α32P]-dTTP. After 2 h incubation, reactions were stopped by the addition of 2× or 10× loading buffer 32P]-GTP labeled self-guanylylation and replication assays were terminated with 7× SB loading buffer The RdRP from bacteriophage Φ6 was expressed and purified as described previously template  and founFigure S1Binding of N-terminal tail to the VP1 active site cleft. The N-terminal tail bound in the active site clefts of (A) Mg-bound ΔC55 VP1, and (B) ΔC55 VP1 (‘large unit cell’). Unbiased FO-FC electron density, calculated before the N-terminal tail was added to the models, is shown in green (3.0 σ). The final refined structures of the polymerase domain and N-terminal tail are shown as a cartoon (white) and sticks (carbon atoms orange), respectively. In (B), the polymerase domain from which the N-terminal tail arises is shown as a cartoon (orange).(TIFF)Click here for additional data file.Figure S2Reorganization of the C-terminal extension of full-length VP1. (A) Full-length VP1 with the C-terminal extension in the commonly observed conformation (chains A–C) is shown as a white (residues 3–687) and magenta (residues 688–799) cartoon. 2FO-FC electron density calculated from the final refined model is shown in blue (1.0 σ); for clarity density is shown only within a 2 Å radius of residues 688–799. (B) Full-length VP1 where the C-terminal extension is refolded (chain D) is shown as a white cartoon (residues 3–687) and a magenta Cα trace (residues 688 onwards). Electron density is as in (A), shown within a 3 Å radius of the Cα trace. Given the low resolution of the diffraction data we were unable to reliably build a main chain into the density and dock the sequence from residue 688 onwards in chain D. We have therefore excluded this region of the structure from the refinement, the deposited coordinates for this segment having zero occupancy to indicate this fact.(TIFF)Click here for additional data file.Figure S3Biochemical characterization of IPNV VP1. Screening the optimal reaction conditions for the IPNV VP1 in terms of (A) Mn2+ concentration, (B) Mg2+ concentration, (C) temperature, (D) ammonium acetate (NH4Ac) concentration, and (E) pH. Results are normalized against the highest attained polymerization activity for each RdRP in each experiment. Two ssRNA templates of different lengths  were used. Interestingly the optimal temperature for RNA polymerization and final RNA yield obtained depends on the length of the ssRNA template. VP1 efficiently processes the short template (sΔ+13) at 37°C, whereas when a long template (s+13) is applied the RdRP is most active at 45–50°C. Higher reaction temperatures lead to increased degradation of the ssRNA template and a subsequent drop in RNA synthesis. The product yield with short ssRNA templates is much higher than with long, although in all cases the yield is sufficiently high to be detected by ethidium bromide (EtBr) staining following agarose gel electrophoresis (not shown).(TIFF)Click here for additional data file.Figure S4Oligonucleotide displacement assay. (A) Schematic representation of the secondary structure at the 3′ terminus of sΔ+ (upper panel) and of partially double-stranded sΔ+ RNA hybrids with 10 nucleotide (nt) and 20 nt 3′ overhangs (lower panels). The blue and red lines (lower panel) indicate the position at which the complementary RNA oligonucleotide anneals. (B) The RNA synthesis activity of full-length VP1 using sΔ+ ssRNA hybridized to short RNA oligonucleotides in order to produce RNA hybrids with different 3′ overhangs (0–575 nt) as template. The results have been normalized against unhybridized ssRNA sΔ+13 template (Native). The pre-initiation behavior of the VP1 polymerase is similar to that described for the Φ6 RdRP +13 template.(TIFF)Click here for additional data file.Figure S5De novo reverse transcription activity of wild-type VP1. Left panel: Agarose gel electrophoresis and autoradiography of VP1 catalyzed primer independent reverse transcription. sΔ+13 ssRNA templated reverse transcription in the presence of [α32P]-dTTP leads to the formation of a labeled RNA/DNA hybrid (lane 1). Treatment with a mixture of DNases (lane 2) completely removes the signal. RNase treatment (lane 3) does not affect the sample intensity, indicating that the label becomes incorporated into the newly synthesized complementary DNA strand. A combination of RNases and DNases removes the signal completely (lane 4). Right panel: Agarose gel electrophoresis and autoradiography of PCR amplification of the labeled cDNA using Phusion HF DNA polymerase (Finnzymes Ltd.). Amplification of the RNA/DNA hybrid was not successful (lane 1) but removal of the template RNA by RNase digestion yielded a clearly visible DNA product (lane 3). The DNase (lane 2) and DNase/RNase (lane 4) digested samples did not yield any amplification products. In order to determine the fidelity of reverse transcription, ssDNA gained following RNAse treatment was PCR amplified and the resulting DNA was sequenced. The quantity and quality of the resulting DNA sequence was compared to the activity of a commercial primer-dependent Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega). The amount of dsDNA obtained after PCR amplification was comparable to the amount obtained by M-MLV reverse transcriptase. Throughout the sΔ+13 sequence, the VP1 RdRP and the M-MLV reverse transcriptase displayed two identical mismatches, both of which were attributed to the T7 RNA polymerase.(TIFF)Click here for additional data file.Figure S6Birnavirus VP1 sequence alignment. Selected representative birnavirus VP1 sequences are shown as follows (UniProt IDs in parentheses): IPNV, Infectious Pancreatic Necrosis virus strain Jasper (P22173); AY-98, marine birnavirus isolated from Plecoglossus altivelis (Q7T8V1); IBDV, Infectious Bursal Disease virus strain D78 (Q9Q6Q5); IBDV-L, Infectious Bursal Disease virus stain Lukert (Q49HH7); BSV, Blotched Snakehead virus (Q8AZL8); TV1, Tellina virus 1 (Q2PBR4); DSX, Drosophila X virus (Q91CD5). Numbering is for IPNV VP1 and moderately/highly conserved residues are shown in white type face with grey/black backgrounds, respectively. Note the conservation of residues that interact with the K+ ion , the only exceptions being the conservative substitution N514D in Drosophila X virus (PDF)Click here for additional data file.Figure S7The N-terminus of IPNV VP1 lies close to the (deoxy)nucleotide entrance tunnel. The structure of VP1 is shown as a molecular surface for the well-folded polymerase domain  and a cartoon for the N-terminal tail (orange). GTP molecules (spheres) modeled into the active site based on a structural superposition of the Φ6 RdRP initiation complex (PDB ID 1HI0) can be seen through the VP1 (deoxy)nucleotide entrance tunnel, revealing the proximity of the N-terminal tail to the exterior opening of this tunnel.(TIFF)Click here for additional data file.Table S1Data collection and refinement statistics. Numbers in parentheses refer to the appropriate outer shell.(PDF)Click here for additional data file.Table S2RNA templates used in this study.(PDF)Click here for additional data file.Text S1Supplementary Materials and Methods.(PDF)Click here for additional data file.


This labeling method thus constitutes a significant improvement compared to the above-mentioned single-atom labels, which require RNA concentrations in the low millimolar range; less material is needed and potential aggregation problems are minimized. Moreover, the 2′-SCF3 group represents an isolated spin system, therefore proton decoupling  is not necessary, thus making the label metrologically very straightforward. Similar to methyl groups, trifluoromethyl groups allow the prolongation of coherence lifetime based on transverse relaxation optimized spectroscopy (TROSY), accounting for an additional advantage in measurements of large RNA molecules or RNA–protein systems.Herein, we present a novel high-performance fluorine sensor for RNA, the ribose 2′-deoxy-2′-trifluoromethylthio unit thiocarbonyl]adenosine by treatment with pyridinium poly(hydrogen fluoride) (HF/pyridine) in the presence of 1,3-dibromo-5,5-dimethylhydantoin (DBH), however, yields were extremely low (22 %). In our own attempts, we were unable to increase the reported yields. Efforts to apply a new class of electrophilic trifluoromethylation reagents based on hypervalent iodine(III) derivatives (Togni reagents)3 uridine derivative. However, this pyrimidine nucleoside turned out to be unstable, its decomposition resulting in formation of 2,2′-anhydrouridine.Originally, we considered to develop a new 3 nucleosides, which appears rather unorthodox at first sight. Although such a label would most likely thermodynamically destabilize an RNA double helix ,19F NMR applications for probing structure and folding of RNA, binding of small molecules and RNA, or protein–RNA interactions, for which this label can be easily positioned in single-stranded regions, prompted us to pursue this goal.These observations prompted us to develop a novel concept for RNA labeling, namely with 2′-SCF1 . Subsequently, the 5′-OH group was protected as dimethoxytrityl (DMT) ether to give compound 3, and conversion into the corresponding phosphoramidite 4 was achieved in good yield by reaction with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Starting from compound 1, our route provides 4 in 60 % overall yield in three steps with three chromatographic purifications; in total, 1.9 g of 4 was obtained in the course of this study.We started our endeavors with 2′-deoxy-2′-mercaptouridine 3 uridine building block was tested, using the solid-phase synthesis methodology for 2′-O-TOM-protected RNA.3NH2 in ethanol/H2O, followed by treatment with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF). Salts were removed by size-exclusion chromatography on a Sephadex G25 column, and RNA sequences were purified by anion-exchange chromatography under strong denaturating conditions  required during RNA solid-phase synthesis for transformation of PIII to PV. Therefore, no special adaptions of the standard synthesis cycle Next, the preparation of RNA with the novel 2′-SCF3-modified RNA encouraged us to evaluate the new label in 19F NMR applications. In the following, we present three examples: 1) probing of the secondary structure of bistable RNA sequences; 2) verification of RNA–protein interactions; and 3) attesting rationally designed riboswitch modules.The efficient synthetic access to 2′-SCF5) that exists in slow conformational exchange of two distinct secondary structures (5′ and 5′′), as confirmed by comparative imino proton NMR spectroscopy5 a. When we labeled this RNA with 2′-SCF3 at uridine-26 (6), the label lies within a 4 nt loop of fold 6′ while it is located in a 7 nt internal bulge of fold 6′′. The assignment of secondary structures 6′ and 6′′ by 19F NMR spectroscopy is depicted in Figure 6′. The imino proton NMR spectra of modified (6) and unmodified (5) sequences are nearly identical, thus demonstrating that the equilibrium position has not been influenced by the label. This tendency was confirmed for a second bistable RNA  and hence underscores the applicability of the label for secondary-structure probing. Even more satisfying was the observation that in E. coli lysate at a very low RNA concentration of 10 μm, the two folds were readily detectable  that comprises a characteristic 14 nt recognition loop to form a high-affinity complex in the nanomolar range. This implies a ligand-induced rearrangement of the secondary structure, and thus provides the typical characteristics of a riboswitch.3 labeling and 19F NMR spectroscopy, we showed that the new label allows the straightforward assignment and quantification of the different RNA conformations  involved for this riboswitch module UCG, showed a ΔΔG°298K of 1.9 kcal mol−1 . The reason for the destabilization is most likely the preference for the C2′-endo conformation of the modified nucleoside. This theory was supported experimentally and by MD simulations for 2′-SCH3 moieties.3 functionalities, we synthesized a short RNA strand, 5′-UGU(2′-SCF3-U)GC, and determined 3J (H1′–H2′) coupling constants by 2D 1H,1H-DQF COSY NMR experiments . For the 2′-SCF3 uridine, a value of 9.9 Hz was determined, accounting for a population of 98 % of C2′-endo ribose conformation, which is indeed a strong indication that this modification would cause interference if forced into a C3′-endo conformation, as it demands an A-form RNA double helix.24One issue that remains to be addressed in more detail is positioning of the label within single-stranded RNA regions. This is advisable because the attachment of 2′-SCF19F NMR applications for 2′-SCF3-modified RNA molecules introduced here make this labeling concept a compelling new tool for probing of RNA structure and function, in particular when protein or small-molecule interaction partners are involved. Moreover, we stress that the chemical synthesis of this label is extremely robust and can be performed on large scale. Incorporation of the label into RNA is compatible with standard solid-phase synthesis and deprotection protocols, and thus highly convenient. Additionally, the potential expansion of the labeling concept to all four standard nucleosides holds promise for flexible and widespread applications in order to explore structure and dynamics of biologically relevant RNA sequences through in vitro and in vivo 19F NMR spectroscopic methods.Taken together, the


Shift transitions in dominance on coral reefs from hard coral cover to fleshy macroalgae are having negative effects on Caribbean coral reef communities. Data on spatiotemporal changes in biodiversity during these modifications are important for decision support for coral reef biodiversity protection. The main objective of this study is to detect the spatiotemporal patterns of coral reef fish diversity during this transition using additive diversity-partitioning analysis. We examined α, β and γ fish diversity from 2000 to 2010, during which time a shift transition occurred at Mahahual Reef, located in Quintana Roo, Mexico. Data on coral reef fish and benthic communities were obtained from 12 transects per geomorphological unit (GU) in two GUs (reef slope and terrace) over six years . Spatial analysis within and between the GUs indicated that the γ-diversity was primarily related to higher β-diversity. Throughout the six study years, there were losses of α, β and γ-diversity associated spatially with the shallow (reef slope) and deeper (reef terrace) GUs and temporally with the transition in cover from mound corals to fleshy macroalgae and boulder corals. Despite a drastic reduction in the number of species over time, β-diversity continues to be the highest component of γ-diversity. The shift transition had a negative effect on α, β and γ-diversity, primarily by impacting rare species, leading a group of small and less vulnerable fish species to become common and an important group of rare species to become locally extinct. The maintenance of fish heterogeneity (β-diversity) over time may imply the abetment of vulnerability in the face of local and global changes. One of the central topics in conservation is the analysis of species diversity patterns and composition for the purpose of differentiating, characterizing and preserving natural communities One way of assessing variation in diversity is through the additive partitioning of diversity This analysis has recently been used in Caribbean and Pacific coral reefs to perform additive partitioning for fish diversity in hierarchical scaled studies and to analyze the variables that modulate the β-diversity of coral reef benthic communities Montastraea complex In the last four decades, many coral reefs in the Caribbean Sea have been affected by a shift in benthic coral reef communities from a dominance of hard coral cover to one of fleshy macroalgae Coral reefs are spatially structured across a depth gradient from the coast to the open ocean. In the Caribbean, the reef lagoon, crest and front are the systems that receive direct impacts from coastal development and tourism. Deep geomorphological structures may also be considerably affected, but as these structures are farther from the coast (600–1000 m) and in deeper water (>20 m), they may be more resistant to climate change and coastal development. A pioneering study of shifts in the benthic structure of the reef fronts of eleven coral reefs in Quintana Roo, Mexico demonstrated that coral reefs associated with intensive coastal tourism development were largely dominated by fleshy macroalgae (82%) and that the coral cover (13%) was poor, whereas reefs with few coastal tourism activities exhibited a more balanced coverage of fleshy macroalgae and corals (38% and 22% respectively) In this paper, our primary objective was to explore patterns in coral reef fish diversity across two different spatial  and temporal (years) scales using the additive components of total diversity. In particular, we hypothesized that this shift would decrease fish γ-, β- and α-diversity, although it is expected that in a threatened or collapsed system, α-diversity would increase at the expense of a proportional amount of β-diversity. Our second objective was to quantify the additive diversity partitions within two geomorphological units (reef slope and terrace) in each study year and assess the main coral reef benthic drivers associated with α- and β-diversity change. Although the loss of fish diversity with loss of coral cover has been widely documented in coral reef ecology The coral reef system of Mahahual is located in the northern part of the Mesoamerican Barrier Reef System in the state of Quintana Roo, within the touristic area known as Costa Maya  . In 2000, Mahahual’s reef was listed as one of the better reefs, in terms of its condition, in the Mexican Caribbean Montastraea system was located: the reef slope (ca. 12 m depth) and terrace (ca. 18 m depth). Within each geomorphological unit, four sampling transects were selected at three different fixed sites that were separated from one another by 250–300 m  and GUs: reef slope+terrace) and a one-decade time scale (from 2000 to 2010) was used. Fish and benthos were censused using 50-m-long and 2-m-wide transects positioned at depths where the largest coral reef 50–300 m . At eachThe benthic community was surveyed by recording it using an underwater video camera at a distance of 40 cm above the substrate along each 50 m transect χ2) goodness-of-fit The number of species and optimum sampling effort were evaluated based on observed (Mao Tau function) and expected species (bootstrap) using transect-based rarefaction curves transects) within the reef slope, the reef terrace, and the reef as a whole (combining both reef slope and terrace). β-diversity was evaluated at two spatial scales ) within the reef slope and reef terrace and for the entire reef (reef slope+reef terrace).We measured α-diversity and β-diversity as components of γ-diversity among subdivisions of fish assemblages on the basis of transects, geomorphological units and years. The proportion of total diversity within subdivisions in a given dimension (i.e. space and time) provides a natural measure of similarity among the subdivisions transects+βtransects+βGU. We estimated α- and β-diversity using PARTITION v3 Diversity partitioning is an analytical procedure in which a measure of total species diversity is decomposed at spatial or temporal levels in a hierarchical sampling design The β-diversity within each geomorphological unit and within the reef was measured by pairwise comparisons among all transects, using presence-absence data. We used Lande’s β-diversity (β = S−α ¯), which was estimated with the equation β = (b+c)/2, where b is the total number of species that occur in the neighboring transect but not in the focal one and c is the total number of species that occur in the focal transect but not in the neighboring one 0.82) to meet statistical assumptions. We tested the statistical assumptions with a Shapiro-Wilk normality test and Levene equal variance test Due to the lack of independence among years , a two-way crossed ANOVA with a mixed-effects model (type model III) was used to contrast the -diversity (average species richness per transect) and β-diversity for presence-absence data within geomorphological units and within each year An additional method was applied to evaluate fish assemblage changes between the reef slope and terrace over the years. A two-way crossed permutational multivariate analysis of variance (PERMANOVA), based on a mixed-effects model following the two-way ANOVA design, was used to assess the spatiotemporal variation in species composition and abundance. This particular design included/permitted the following: i) the geomorphological unit×year interaction, which tested the generality of species turnover of geomorphological units over years; ii) the year factor, which tested the temporal variability among the six studied years; and iii) the geomorphological unit factor, which tested the consistency of species turnover between geomorphological units, above and along with the potential variability in its change among years. The PERMANOVA was performed using a Bray-Curtis similarity matrix built with previously fourth-root-transformed data. The statistical significance of the PERMANOVA was tested with 10,000 residual permutations under a reduced model and type II  sums of squares transects, βtransects and γ-diversity with coral reef benthic community for each geomorphological unit in each year. Redundancy analysis was performed using the CANOCO v4.5 Software Canonical redundancy analysis (RDA) was used to visualize and describe the relationships among the components of fish α ¯p<0.05).A total of 11,134 individuals were recorded from 33 families, 58 genera and 118 species. The families Serranidae, Pomacentridae, Haemulidae, Labridae, Lutjanidae, and Scaridae accounted for 54% of the total species richness. The observed and expected transect-based rarefaction curves indicated that the sampling effort for geomorphological units and for the overall reef was representative of species richness. The observed species curves obtained using the bootstrap exhibited no differences at the geomorphological unit or reef levels between 2000, 2005, 2006, 2007, 2008 and 2010 , where the geomorphological units were observed to separate according to year sampled , octocorals, sponges, boulder corals , tabular corals , encrusting corals (Agaricia spp.) and leaf corals (Agaricia tenuifolia).The RDA triplot presented a good fit and high statistical significance  in a negative linear relationship, and hence, they were not included in the analysis. Fleshy macroalgae presented an important negative correlation with α ¯transects, βtransects and γ-diversity, and they increased at the geomorphological unit level from the years 2005 to 2010 , which results in reduced structural complexity Montastraea annularis. RDA outcomes evidenced that mound corals had a strong negative correlation (high collinearity) with fleshy macroalgae, suggesting a high Montastraea complex decrease through the studied years. In contrast, in the years from 2005 to 2010, β-diversity had a strong negative relationship with boulder corals such as P. astreoides, which was mainly observed on the reef slope and terrace in 2010 , reef slope (B), and the whole reef (C) in the years 2000, 2005, 2006, 2007, 2008, and 2010.(PDF)Click here for additional data file.Figure S2Species rarity based on abundance and incidence by geomorphological units and whole reef. Numbers of singletons and doubletons (A). Numbers of unique and duplicate species (B).(TIF)Click here for additional data file.Table S1Shared and unshared fish species from reef terrace, reef slope, and reef between years. Singletons = S, doubletons = D, unique species = U, duplicate species = DU, and invasive specie = ***.(PDF)Click here for additional data file.Table S2The top 10 species that contribute more strongly to the dissimilarity (SIMPER). Average dissimilarity = AD.(PDF)Click here for additional data file.


Hence, aptamers can be thought of as a nucleic acid analog to antibodies. However, several viewpoints hold that the potential of aptamers arises from interesting characteristics that are distinct from, or in some cases, superior to those of antibodies. This review summarizes the recent achievements in aptamer programs developed in our laboratory against basic and therapeutic protein targets. Through these studies, we became aware of the remarkable conformational plasticity and selectivity of RNA, on which the published report has not shed much light even though this is evidently a crucial feature for the strong specificity and affinity of RNA aptamers.Aptamers are short, single-stranded nucleic acid sequences that are selected  The conequences . Importaequences  or as biequences . Moreoveequences .A characteristic of RNA aptamers is the high potential to create a vast set of tertiary structures, which depend on the different primary sequences. Therefore, it is even likely that some RNA aptamers can fold into structures that resemble protein structures of interest. This idea arose in our previous studies of the structure–function relationship of translation factors, in which we discovered that translation factors mimic the shape of tRNA. One of them, a polypeptide release factor that is required for protein termination, encodes a tripeptide that serves as an ‘anticodon’ to decipher stop codons in mRNA .We speculate that RNA has high potential to create many different tertiary structures, much more than ever thought. The ‘RNA world’ hypothesis  providesIn this review, we present an overview of the structure and function of representative RNA aptamers raised against a variety of human proteins and sensor molecules in our laboratory. This will contribute to our basic understanding of the potential of RNA and the global applications of aptamers.Although the 3D structures of RNA aptamers are commonly solved by X-ray crystallography or NMR spectroscopy , only thWe selected a 23-nucleotide, high-affinity RNA aptamer against the Fc region of human IgG1 hFc1; , the selThe solved structure showed several interesting features. First, the structure of the aptamer-bound hFc1 was superimposable upon the uncomplexed form of the hFc1 structure , indicatM-fold , but even so, it achieved remarkably strong affinity  is a pro-inflammatory cytokine produced primarily by a subset of CD4Kd) of Apt21-2 to hIL-17A and mIL-17A was estimated to be 48.5 and 701.3 pm, respectively  had an IC50 of 200–300 nm  peptide in complete Freund’s adjuvant, and PEG21-2idT was administered intraperitoneally  every other day. The appearance of EAE was significantly delayed in mice administered with 3 and 10 mg/kg PEG21-2idT, with incidence and symptoms reduced markedly in a dose-dependent manner (Apt21-2 is composed of 13 ribose 2′-fluoropyrimidines (ribonuclease-resistant) and 20 unmodified purines . The phat manner . Mice wen = 10) every other day after GPI immunization. PEG21-2idT resulted in significant improvement in the incidence of arthritis and the clinical scores of symptoms in a dose-dependent manner (n = 10) i.p. every day from day 8 after GPI immunization, a time point at which RA was already established. Importantly, PEG21-2idT injection at day 8 significantly suppressed the progression of arthritis even after it had developed , and 2 PEG21-2idT efficacy tests were conducted. Doses of PEG21-2idT  were administered i.p. to DBA/1 mice  inhibits inflammatory lesions and neurological symptoms in EAE and RA mouse models. Although PEG21-2idT was generated against hIL-17, it also exhibited weaker affinity to mIL-17. The intriguing finding in this study relates to the fact that PEG21-2idT was 1–2 orders of magnitude less effective against mIL-17A than it was against hIL-17A, but even so, could exert therapeutic impact in EAE and RA mice. This strongly suggests that the currently generated anti-IL-17A aptamer has potent therapeutic potential for human autoimmune diseases. Such approaches are in progress toward clinical trials.The IL-17 cytokine family is composed of six structurally related proteins . Of these, IL-17A and IL-17F are the most closely related to each other, sharing 55% amino acid sequence homology and four conserved cysteine residues at the C-terminal. These conserved cysteine residues participate in the formation of intermolecular disulfide bonds, leading to the formation of homodimeric  and heterodimeric (IL-17A/F) structures  assay  and blocin vitro . These fH1) cell–mediated autoimmune diseases, CD4+ CD25+ regulatory T (Treg) cells have recently received a great deal of attention as negative regulators of MS pathogenesis  is a heparin-binding growth factor and exerts pleiotropic effects, including cell proliferation, cell migration, angiogenesis and fibrinolysis in a variety of tissues . MK overdiseases . Thus, eKd of 0.9 nm  is 38 nts in length and has a uding MS .Aptamers have also been generated to dyes and fluorophores such as malachite green  and sulfThe choice of chromophore is crucially important for widespread practical application of aptamer probes to live cell imaging. Although malachite green has been successfully applied to the binary aptamer probe, it remains uncertain whether malachite green is the best choice for intracellular imaging. It is worth mentioning that malachite green very efficiently generates singlet oxygen upon irradiation and is used for targeted damage of mRNA constructs ; therefoOn the basis of these considerations, we isolated an RNA aptamer against the cyanine dye Cy3 (Cy3apt), a widely used, membrane-permeant and nontoxic fluorophore . The parThe shortened derivative of Cy3apt is composed of two separate hairpin modules . Althougin vitro and in vivo sensor system. As the selected aptamer is composed of unmodified  nucleotides, the in vivo sensor system is designable by expressing the binary Cy3 aptamer in test cells and directing its cellular localization, if necessary, using a variety of expression vectors. Moreover, our binary aptamer probe is composed of distinctly folded modules and can be applied to monitor a tertiary RNA–RNA interaction.Sequence-specific detection of nucleic acids is crucial to disease diagnosis, genome study and mRNA monitoring in living cells. Among the numerous nucleic acid analysis methods of particular interest are those that provide immediate visible or fluorescent response after hybridization to complementary nucleic acid analytes, thus offering easy and instant detection of specific DNA and RNA. The binary Cy3 aptamer probe generated in this study will facilitate this approach in view of the fact that it, for the first time, enabled us to deal with a pair of aptamers and Cy3, a commonly used, membrane-permeant, and nontoxic dye, for developing an In nature, many RNA molecules and motifs exhibit specific functions that require the formation of a specific 3D structure, rather than simply a linear carrier of genetic code information. The classical examples of such structural, protein-noncoding RNAs (ncRNAs) are tRNA and rRNA, which play key roles in the central dogma of molecular biology . In addiin vitro selection based on the ribozyme activity. A model selection targeting a GAAA loop successfully identified motifs previously known as GAAA loop receptors  by SELEX with or without negative selection against fully differentiated A-9 cells (derived from the connective tissues of an adult mouse) from an RNA pool randomized to over 60 nts (N60) with 2′-fluoropyrimidine modifications to resist ribonucleases . The bou32P]-labeled L2-2 was examined against mESCs and five differentiated mouse cell lines: connective tissue (A-9 cells), embryonic fibroblast (NIH 3T3 cells), muscle tissues (C2C12 cells), liver cancer (Hepa 1–6 cells) and neuroblastoma (NB2a cells). The data indicated that this aptamer bound efficiently to mESCs, but failed or only weakly bound, if at all, to the differentiated mouse cell lines (The binding specificity of [The mESC markers are known to be down-regulated during the course of differentiation . Surfaceetc. (Given that anti-mESC aptamers could bind to artificially created, induced pluripotent stem cells (iPSCs; etc. . CollectKd on the picomolar scale, 100-times stronger affinity than that of normal antibody–antigen interactions. Certain aptamers are more than 50 nts long for specific and high-affinity binding to their target proteins. Occasionally, these proteins lack RNA recognition motifs or an intrinsic, strong affinity to RNA, and the high affinity of the aptamer is achieved through capture of the protein’s global conformation. In contrast, the anti-hFc1 aptamer achieved strong and specific binding to hFc1 mainly by van der Waals contacts and hydrogen bonds rather than via electrostatic forces, unlike most known RNA–protein interactions. These findings demonstrate that RNA has great potential to form a vast set of tertiary structures and to achieve high affinity: We refer to this property as ‘RNA plasticity’. This conformational plasticity and selectivity can be achieved by multiple interactions, which are applicable to many protein targets with low or no affinity to nucleic acids. These results provide us with a solid and promising basis for steps to create novel RNA molecules with distinct structures and with therapeutic potential superior to that of antibodies.In this laboratory, RNA aptamers were selected against a variety of human proteins and a chemical reagent, and the key features were summarized in this review. Although many properties of the selected aptamers were similar to those of antibodies, the aptamers also exhibited superior features. Selected aptamers occasionally had a in vitro manipulation of activity and/or stability, less immunogenicity or toxicity, and scalable chemical production. In contrast to costly cell-based production of antibodies, the production costs of RNA aptamers will also be greatly reduced with the development of oligonucleotide-based therapies. Therefore, RNA aptamers offer a beneficial therapeutic approach for the treatment for diseases not only from a therapeutic standpoint, but also from the perspective of healthcare economics. Therapeutic antibodies are being rapidly developed worldwide. In 2010, the US FDA approved 31 therapeutic antibodies, and the antibody therapeutics market is expected to achieve an excess of US$ 40 billion in 2010. Moving forward, aptamer therapeutics is not at a disadvantage. Several characteristics grant aptamers potential superior to that of therapeutic antibodies, including increased binding affinity, To our great surprise, the completion of the Human Genome Project showed the existence of a large amount of ncRNAs. There are two classes of ncRNAs: one that includes antisense RNA and microRNA, and functions by sequence complementarity to target mRNA or DNA, whereas another functions independently of sequence complementarity by forming a functional 3D structure to target protein or macromolecules as an apparent equivalent to a protein. We believe that the former class of ncRNA is likely the tip of the ‘ncRNA iceberg’, regardless of it having received much attention. The latter class of RNAs, which may be referred to as ‘natural aptamers’, might play a crucial role in ncRNA function as well. Therefore, it is important to study both artificial and natural aptamer RNAs to consolidate the superior potential of RNA. The current study of RNA aptamers would be highly beneficial to a comprehensive understanding of genome-coded ncRNA function as well as to the development of RNA medicine.


Sediments are widely accepted as a threat to coral reefs but our understanding of their ecological impacts is limited. Evidence has suggested that benthic sediments bound within the epilithic algal matrix (EAM) suppress reef fish herbivory, a key ecological process maintaining reef resilience. An experimental combination of caging and sediment addition treatments were used to investigate the effects of sediment pulses on herbivory and EAMs and to determine whether sediment addition could trigger a positive-feedback loop, leading to deep, sediment-rich turfs. A 1-week pulsed sediment addition resulted in rapid increases in algal turf length with effects comparable to those seen in herbivore exclusion cages. Contrary to the hypothesised positive-feedback mechanism, benthic sediment loads returned to natural levels within 3 weeks, however, the EAM turfs remained almost 60% longer for at least 3 months. While reduced herbivore density is widely understood to be a major threat to reefs, we show that acute disturbances to reef sediments elicit similar ecological responses in the EAM. With reefs increasingly threatened by both reductions in herbivore biomass and altered sediment fluxes, the development of longer turfs may become more common on coral reefs. Coral reefs are among the most biodiverse ecosystems on the planet. Despite the ecological and physical complexity associated with this biodiversity, they are remarkably fragile. The increasing rate of disturbances associated with climate change and other anthropogenically induced stressors has repeatedly highlighted that acute stressors can have long lasting impacts ,2. For ein situ as the reef matrix is eroded into sediments by physical, biological and, chemical processes [The source of sediment on coral reefs affects its physical and chemical properties. Fine marine (primarily carbonate) and terrigenous (primarily silicate) sediments can be transported long distances in suspension and/or driven by hydrodynamic activity . While trocesses .While benthic sediments are highly dynamic on offshore coral reefs ,9. The cOn hard coral reef substrata some sediment exists in sand patches, but a large proportion of the benthic sediment is bound within algal turfs –16. The On ‘healthy’ coral reefs, EAMs are maintained as well-cropped productive turfs by herbivores, usually reef fishes . The intThe interactions between benthic sediment and herbivory suggest the possibility of a positive feedback loop. Any disturbance leading to increased EAM sediment load, such as exposure to a sediment plume from storms, flooding or dredging could deter herbivores. The reduced top down pressure on the EAM is likely to result in an increase in EAM length , allowinAll procedures were conducted according to the ethics guidelines of James Cook University, Townsville , and permitting requirements of the Great Barrier Reef Marine Parks Authority (permit number: G10/33755.1). Data are available in the supplementary material .2 plots were haphazardly delineated on the flat EAM. Full cages were constructed of a steel bar frame, 25 cm high, enclosing the 1 m2 plot, attached to the reef using epoxy putty and enclosed with galvanised 4 cm wire mesh. Partial cages were constructed of vertical panels identical to those used in the fully caged treatments and were deployed to control for any effect of the cage treatments on the EAM or sediment, while allowing herbivores access to graze the EAM.Experimental manipulations were conducted at two sites on the exposed reef crest southeast of Lizard Island in the northern section of the Great Barrier Reef, Australia. The experiment was conducted between December and March, in the southern hemisphere summer. The sites had similar topographies, were at a depth of 2-4 m, and separated by over 800 m. Sites were characterised by sparse coral colonies, separated by expanses of short well-cropped EAMs on a flat, low-complexity reef matrix. At each site, six fully caged, six open (uncaged) and six partially caged 1 m-2 was maintained for one week. This sediment load was equivalent to that on the adjacent reef flat (approximately 40 m leeward of the crest), which was determined by weighing dried sediment samples collected using an electric vacuum sampler from 20 replicate 5.8 × 10-3 m2 rings . The seT0), then at weekly intervals for 6 weeks post-manipulation (T1-6), and again after three months (T7).The sediment depth and EAM turf length in each plot were recorded using the depth probe of vernier calipers (n = 20 reps for each plot). Measurements were made as sediment was initially manipulated (T0) correcting for initial variation among plots. The data were then analysed using 2- and 3-way analyses of variances (ANOVAs) of the T7 data. Site was categorised as a random factor and was found to have no significant effect or interaction. It was therefore pooled to increase the power of the analysis [EAM turf length data were standardised to percent increase over initial turf length  the greatest increase in the length of algae in the EAM was seen in the caged plots and a significant difference in turf length was observed between the caged and open plots . While no other factors or interactions were significant  and in the time × caging interaction . No other factors showed significant interactions  increases in the length of turfs in EAMs on a coral reef . HoweverThe EAMs on the exposed crest used in this study are heavily grazed by coral reef fishes, which maintain short, productive algal turfs . PreviouThe EAMs in the present study grew in response to reduced herbivory (cages), increased sediments, or a combination of these factors. Growth in the cages was probably a result of the reduction in grazing ,35. WhilRemarkably, after the initial growth of the algal filaments the new, longer, EAMs remained for at least three months in both caged and open plots. The three treatments followed different trajectories: (1) by reducing herbivory, cages maintained a long EAM regardless of sediments . (2)We found that the growth of EAMs after a one-week sediment pulse was equivalent to, or greater than, that seen in simulated herbivore removals using cages. While increased sediment is often considered deleterious to reefs, most information pertaining to its effects are at a physiological scale, considering the impact on individual organisms ; little sensu [The manipulation used in this study is comparable to that of other short-term (acute) perturbations caused by natural or anthropogenic disturbances. Storms and cyclones can move sediment from areas with high natural loads, such as lagoons or reef flats, which, as with this study site, may be only a few tens of meters away . Storms sensu ) is likeIt is essential to note that our observations are of the effects of ‘natural’ carbonate dominated sediments, which characterise offshore coral reefs. The disturbance induced by this study, therefore, represents the best-case scenario for the effects of sediments on reefs. Terrigenous sediments, with higher silicate and nutrient loads alongside a high likelihood of associated pollutants , are likely to have even greater impacts ,63.Although our study did not find evidence to support the existence of a positive feedback leading to sediment-laden turfs, a clear and lasting effect was observed following an acute disturbance, with the development of a long-turfed EAM. Longer EAMs on reefs have the potential to reduce coral settlement , provideTable S1Raw data.‘Time’ column is measured in weeks. Plots 1-9 are site 1 and 10-18 site 2. ‘Turf/Sed’ column indicates what is being measured.(PDF)Click here for additional data file.Table S22-way ANOVA of T7 turf length data .(PDF)Click here for additional data file.Table S3RM MANOVA results of EAM depth over time, across caging and sediment treatments, pooled between sites . Assumptions of sphericity were violated by univariate tests, as such multivariate tests (Pillai’s Trace) were used.(PDF)Click here for additional data file.Table S4RM MANOVA results of sediment depth over time, across caging and sediment treatments, pooled between sites . Assumptions of sphericity were violated by univariate tests, as such multivariate tests (Pillai’s Trace) were used.(PDF)Click here for additional data file.


Arabidopsis α-importins and analyses of their gene expression patterns, we discuss functional redundancy and specialization within this transport receptor family. In addition, we consider how pathogen effector proteins that promote infection by manipulating host cell nuclear processes might compete with endogenous cargo proteins for nuclear uptake.Nuclear translocation of immune regulatory proteins and signal transducers is an essential process in animal and plant defense signaling against pathogenic microbes. Import of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors termed importins, typically dimers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complex. Here, we review recent reports of importin-α cargo specificity and mutant phenotypes in plant- and animal–microbe interactions. Using homology modeling of the NLS-binding cleft of nine predicted  Cargos may contain one (monopartite) or two (bipartite) NLS sequence motifs and directional binding to and release from the importin-α/β heterodimer is imposed by the nucleotide-binding state of Ran, a small guanosine-5c states . The Ranc states .Figures B). The flexible IBB domain not only connects importin-α to importin-β but also contains a cluster of basic amino acids that competes with NLS-cargos for binding to the ARM-repeat domain of importin-α. Thus, the IBB domain is involved in regulating both formation of the trimeric import complex in the cytoplasm and release of cargo in the nucleus after the IBB domain is freed from importin-β by RanGTP  domain followed by a series of ten armadillo (ARM) repeats that form the NLS-binding cleft . However, there is controversy from different profiling techniques regarding the levels and tissue-specificity of importin-α5, α7, and α8 expression  DUO1 suggests that importin-α8 is a DUO1 target gene that is specifically expressed in the male germ line  suggesting that it lacks both the capacity to bind importin-β and the auto-inhibitory mechanisms that are conserved in the other α-importins. Therefore, it remains to be tested if importin-α8 can function as a NTR and whether the loss of the IBB domain is a consequence of specialization in pollen development.Although the portin-α , severalbidopsis . Conceivpatterns , and nucpatterns . In Arabpression . For exaerm line . These dArabidopsis can only partially be rationalized by tissue-specific expression of single paralogs. Alternatively, multiple paralogs might have evolved to transport specific cargos. Indeed, the NLS from the rice COP1 protein binds in vitro the two rice importins α1a and α1b, but not importin-α2  forms the core of the major and minor NLS binding sites in Arabidopsis α-importins . Previous comparative analysis revealed that major determinants of specificity are (i) the amino acid positioned three residues upstream of the conserved Trp, and (ii) residues that constitute the loops connecting the H3 and H1 helices . As some plant NLSs specifically bind to the minor NLS binding site  to detect microbe-associated molecular patterns (MAMPs) and defend against pathogens . In addiChanges in nuclear translocation rates are often achieved via post-translational protein modifications leading to conformational changes that expose or conceal NLSs or nuclear export sequences (NESs). For example, gene expression changes in mammalian innate immunity are largely governed by the induced nuclear translocation of the NF-κB family of Rel-type TFs. Nuclear accumulation of NF-κB is controlled by its association with IκB proteins. Depending on the type of IκB, these proteins either sequester NF-κB in the cytoplasm by masking its NLS, or prevent its ability to bind to chromatin due to a strong NES in IκB that directs dominant nuclear export over nuclear import . Signal-Drosophila are also controlled at the level of nuclear transport. Upon activation of the Toll signaling cascade, NF-κB/Rel-type TFs translocate to the nucleus in a process that is dependent on nuclear transport factor-2 (NTF-2), an essential component of nuclear trafficking that acts as nuclear import receptor for RanGDP to replenish the nuclear Ran pool  proteins are a family of latent cytoplasmic TFs, consisting of seven members in mammals. Upon cytokine activation of the canonical STAT-signaling pathway, tyrosine phosphorylation induces STAT homo- or hetero-dimerization and subsequent importin-α-dependent nuclear import . Activatof STAT1 . This inFurther examples of vertebrate immune regulatory proteins that contain NLSs and can shuttle into the nucleus are the NLRs CIITA and NLRC5. Both these proteins function through association with DNA-binding proteins to regulate MHC class II and class I gene expression, respectively . CorrelaArabidopsis RPS4, snc1 and RRS1-R, tobacco N, barley MLAs, and potato Rx  phenotype (Agrobacterium requires active nuclear import of the transfer DNA/protein complex (T-complex). Two Agrobacterium effectors, VirD2 and VirE2 are essential for plant transformation and both proteins carry NLSs, providing a molecular link between the T-complex and the host’s nuclear import machinery (Arabidopsis α-importins, only a knock-out of importin-α4 impairs host transformation (rat phenotype is not only complemented by importin-α4 overexpression but also by overexpression of six other Arabidopsis α-importins. This suggests that although importin-α4 is the most relevant NTR for the T-complex other α-importins can complement loss of importin-α4 function when their protein levels are increased. These results are in agreement with findings in yeast which show that nuclear import of different NLSs, with varying affinities for importin-α, is largely governed by the rate of NLS/importin-α complex formation (A knock-out of henotype . Transfoachinery . Althougormation . Signifiormation . Thus, nin vitro studies reported dissociation constants in the low nanomolar range based on indirect affinity measurements (in vitro by isothermal titration calorimetry and found Kd values of ~3 and ~48 μM, respectively (d values in the low nanomolar range are difficult to reconcile with the finding that in vivo importin-α-mediated nuclear import cannot be saturated even by ~20-fold molar excess of NLS-cargo suggesting that the actual dissociation constants in the cytoplasm are significantly higher, possibly due to competitive binding of other cytoplasmic proteins to importin-α (d values in the low micromolar range in mammalian cells and substantiates the idea that formation of the NLS/importin-α complex in the cytoplasm is the rate-limiting event for nuclear import ( d values interfere with dissociation of the NLS/importin-α complex in the nucleus and prevent recycling of importin-α to the cytoplasm (Notably, the “optimal” binding affinity of a NLS for importin-α is still controversial. Several urements . Two othectively . Kd valuportin-α . Indeed,r import . Artificytoplasm . Consequytoplasm , 2010.A significant number of host-targeted pathogen effector proteins localize entirely to host cell nuclei, indicating active nuclear import or passive diffusion through the NPC and sequestration in the nucleus ,b. Generin vitro and in vivo using emerging quantitative cell biology approaches are required to understand the complex regulation of nuclear import. Finally, many nuclear proteins do not have canonical NLS motifs. Although other import routes such as direct binding to importin-β (The reports discussed in this review substantiate the idea that tissue-specific expression, importin-α protein levels and sequence variation in the NLS binding cleft determine which importin-α functions as NTR for a cargo protein. However, more thorough analyses of plant NLS/importin-α complexes both portin-β  or bindiportin-β  can accoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The synthesis involves the introduction of a nonbridging 18O atom into the phosphate group during the oxidation step of the synthetic cycle by using 18O water as the oxygen donor. The 18O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The 18O/16O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of 18O-labeled RNA, and this technique was used to determine the blood concentration of 18O-labeled RNA after administration to mice. 18O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of  Since the key discoveries in RNA interference (RNAi) by Fire et al. , the pac32P]ATP to the 5′-hydroxyl terminus of an RNA oligomer catalyzed by T4 polynucleotide kinase. The disadvantage of radioisotope labeling, however, is the risk associated with increased radiation exposure as well as the short half-life of some radioactive isotopes, in particular phosphorus.The conventional methods of labeling RNA are fluorescent and radioisotope labeling. While both of these methods have advantages, they also have disadvantages. Thus, fluorescent labeling is in principle sensitive enough to allow detection of a single molecule , but it 18O, which would avoid the disadvantages of fluorescent and radioisotope labeling outlined above. The properties of chemical equivalence and lack of radioactive decay make 18O well suited for labeling molecules for use as tracers in basic and clinical studies. Except for the labeling of proteins for quantitative proteomics  and isotope microscopy can be used for the quantification and imaging of 18O-labeled RNA in biological material.Here, we report the synthesis of 21mer RNA with tert-butyldimethylsilyl (TBDMS) amidites  as described by Bellon (N-methylimidazole in THF/pyridine (8:1), and the oxidizing agent was 0.01 M iodine in THF/pyridine/water (78:20:2). All reagents and solvents used were commercially available. 18O-labeled RNA oligomers were synthesized in the same way except that the solvent for the oxidizing agent was THF/pyridine/18O water (78:20:2). 18O-enriched water (99.2 atom %) was purchased from Taiyo Nippon Sanso . After synthesis, the protecting groups of the RNA oligomers were removed. The purity of the crude RNA product was confirmed by reverse-phase high-performance liquid chromatography (HPLC) and liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-ESI-Q-Tof MS). After ethanol precipitation, RNA oligomers were purified by preparative reverse-phase HPLC. Purified RNA oligomers to be used for the preparation of siRNA were dissolved in 30 mM HEPES-KOH buffer, pH 7.4, containing 100 mM potassium acetate and 2 mM magnesium acetate. siRNAs were prepared by annealing the purified short complementary strands by incubation at 90°C for 10 min and then at room temperature for 1 h. Duplex formation was confirmed by 15% polyacrylamide gel electrophoresis followed by staining with ethidium bromide.RNA oligomers were synthesized on commercially available controlled-pore glass solid supports with a pore size of 1000 Å  in columns installed in an Expedite model 8909 nucleic acid synthesizer . Synthesis was carried out on a 1-μmol scale from the y Bellon . The detHPLC was performed on a Shimadzu LC-10A system  equipped with an XBridge Oligonucleotide Separation Technology (OST) C18 column . The following solvent system was used for analytical and preparative HPLC: buffer A  and buffer B . Elution was carried out with a 5–10% linear gradient of buffer B in buffer A. Mass spectra were obtained with a Waters Acquity ultra performance liquid chromatography (UPLC) system coupled with a Waters Synapt G2 ESI-Q-Tof mass spectrometer. The UPLC system was used with a Waters Acquity UPLC OST C18 column . The following solvent system was used for liquid chromatography–mass spectrometry: solvent A  and solvent B (50% methanol). Elution was carried out with a 5–50% linear gradient of solvent B in solvent A. The mass spectrometer was operated in negative ion mode. External calibration was performed with sodium iodide, and the Transform software (Waters) was used for spectral deconvolution.18O-labeled uridine 20mer (20 μM) were prepared in 50 mM sodium phosphate buffer at pH 3, 4, 5.5, 7 and 8.5. Solutions were incubated at 37°C and samples were analyzed by LC-ESI-Q-Tof MS after 1, 3, 7, 14, 21 and 28 days.Solutions of Circular dichroism (CD) spectra of 10 μM siRNA solutions in 50 mM sodium phosphate buffer, pH 7.5, were recorded at 25°C with a JASCO J-720 W spectropolarimeter  in a 1-cm-path-length quartz cell at a rate of 50 nm/min and digitized at a resolution of 0.1 nm. The bandwidth was 1 nm and the response time was 8 s. For each determination, at least five spectra were accumulated and the averaged spectrum was subjected to noise reduction.Tm values were calculated with Tm Analysis Software (Shimadzu Scientific Instruments).Melting curves of siRNA in 50 mM sodium phosphate buffer, pH 7.5, were recorded with a UV-1800 UV-VIS spectrophotometer . The absorbance at 260 nm was monitored as the temperature was raised from 25 to 95°C at a rate of 0.5°C/min. 2 atmosphere.The cell lines A549  and HCT116  were obtained from the European Collection of Cell Cultures . The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and McCoy’s 5A medium , respectively, containing 10% heat-inactivated fetal bovine serum  and incubated at 37°C in a humidified 5% CO4 cells) suspended in 0.4 ml DMEM containing 10% fetal bovine serum were transfected with 98.5 μl siRNA targeting the human GAPDH gene in Opti-MEM reduced-serum medium  by using 1.5 μl Lipofectamine 2000 (Invitrogen) and then seeded on 24-well plates. Negative-control cells were treated with 100 μl Opti-MEM or 1.5 μl Lipofectamine 2000 plus 98.5 μl Opti-MEM (mock). Cells transfected with siRNA were examined 48 h after transfection and used for RNA analysis. Total RNA was extracted from the cultured cells with the RNeasy Mini Kit , and cDNA was synthesized by using SuperScript III (Invitrogen) according to the supplier’s instructions. To determine the mRNA knockdown level, quantitative real-time polymerase chain reaction (PCR) was carried out with the following primers : human GAPDH, forward 5′-GGAGAAGGCTGGGGCTCATTTGC-3′ and reverse 5′-TGGCCAGGGGTGCTAAGCAGTTG-3′; and the human β-actin gene, forward 5′-GCCACGGCTGCTTCCAGCTCCTC-3′ and reverse 5′-AGGTCTTTGCGGATGTCCACGTCAC-3′. Synthesized cDNAs were mixed with primers in LightCycler Fast Start DNA Master SYBR Green I reaction mix . Quantitative real-time PCR was performed with a LightCycler DX400 instrument (Roche Diagnostics), and the reaction conditions were as follows: 40 cycles of denaturation at 95°C for 5 s, annealing at 62°C for 15 s and polymerase extension at 72°C for 15 s (human GAPDH), or 40 cycles of denaturation at 95°C for 5 s, annealing at 65°C for 15 s and polymerase extension at 72°C for 15 s (the human β-actin gene). The data obtained from the assays were analyzed with LightCycler Software version 4.1 (Roche Diagnostics). The mRNA knockdown levels of each sample were normalized to the β-actin transcript level. The experiments were performed in triplicate.HCT116 cells  interfaced with a thermal conversion elemental analyzer (Thermo Fisher Scientific). Wild-type C57BL/6 male mice purchased from Nihon SLC  were maintained in Mie University’s animal house. The experimental protocol was approved by the Committee for Animal Investigation of Mie University. 18O-labeled control siRNA (100 or 500 μg) in water for injection was administered intravenously. Animals were sacrificed 5, 30 and 60 min after RNA administration, blood was collected in heparinized tubes and plasma was prepared by centrifugation at 900g. For the calibration curve, pooled plasma prepared from untreated mice was spiked with 18O-labeled control siRNA . For IRMS, plasma samples were lyophilized. Experiments were performed in triplicate. The relative difference in 18O/16O ratio between the sample and the internal standard, δ18O, was expressed as parts per thousand according to the equation δ18O (‰) = [(Rsample − Rstandard)/Rstandard] × 1000, where R denotes the 18O/16O ratio. The internal standard was Vienna Standard Mean Ocean Water.5 cells/dish) and incubated at 37°C in humidified 5% CO2. After 24 h, the culture medium was replaced by 2 ml of fresh medium and the cells were transfected with unlabeled or 18O-labeled control siRNA (490 μl) in Opti-MEM  by using Lipofectamine 2000 (10 μl). Twenty-four hours after transfection, the cells on the silicon wafer were washed with phosphate-buffered saline, immobilized with 2.5% glutaraldehyde in phosphate-buffered saline at 37°C for 1 h and dehydrated by soaking successively in 20, 40, 60, 70, 80, 90 and 100% ethanol followed by tert-butanol at room temperature for 10 min each. The transfected cells were frozen at 4°C for 24 h and dried under reduced pressure for isotope microscopy. Samples were coated with a 30-nm layer of gold to avoid the accumulation of positive charge due to the primary beam of the isotope microscope.A549 cells were seeded on a piece of a silicon wafer (7 × 7 mm) in a 35-mm dish  active pixel sensor (SCAPS) detector. The SCAPS detector features a wide dynamic range, no dead time and direct detection of ions. Small structures in biological materials, such as organelles or vesicles, are not always exposed at the sample surface. Direct imaging is useful to reach targets on the order of a few tens of nanometers in size embedded in a matrix on the order of a few microns in size because a high-intensity primary beam can be used without degrading the spatial resolution.There are two approaches to obtaining an isotope map by secondary ion mass spectrometry (SIMS): scanning and direct imaging. In scanning, isotopic data are collected sequentially from each point of the analyzed area with a focused primary ion beam and images are reconstructed by computer. In direct imaging, stigmatic ion optics are used with a high-efficiency 2D 2) was homogeneously irradiated with a 20-keV Cs+ primary beam with a beam current of ∼5 nA. Secondary ion images of 12C14N, 16O, 18O and 16O were obtained sequentially for the same field for each analytical sequence. The exposure times were 5 s for 12C14N, 5 s for 16O and 500 s for 18O isotopography. A 50-µm contrast aperture was used, and the secondary ion contributions, except for objective isotopes, were cut by the exit slit. The typical spatial resolution under the conditions of isotopography was ∼0.3 µm, and the width of a pixel of SCAPS corresponds to 0.2 µm on the sample surface. An image-processing method with a moving average of 3 × 3 pixels was applied to 18O/16O isotopography. The resulting spatial resolution of the isotope ratio image was 0.6 µm. Other analytical methods for isotopography were carried out as described elsewhere  was used to visualize the isotope distribution in the cells, a technique known as isotopography. The sample surface  and a control siRNA that had no gene-silencing target in the human genome, both 21 nucleotides in length , and their yield and purity estimated by their absorbance at 260 nm were nearly identical . This somewhat low value may be explained by a combination of an 18O/16O kinetic isotope effect and reaction with water from moisture in the air. The discrepancy may be decreased by using 18O water with a higher degree of isotopic purity and excluding moisture from the air. In any case, these results confirm that the 18O-labeled RNAs were correctly synthesized and that one 18O atom was incorporated into each phosphate group ∼85% of the time under our experimental conditions.We designed a siRNA targeting the human n length . The senycle see . It was dentical . Thus, tligomers  show tha34–35 Da . This is18O into the 3′-phosphate groups of RNA during endonuclease digestion. However, this method can only be used on a laboratory scale, and the 18O label is incorporated only into the 3′-phosphate group. In addition, Dai et al.   ,16 or 10 complex  in the o18O label in the RNA oligomer under physiological conditions is an important consideration. Any back exchange between 18O-labeled RNA and the surrounding 16O water would result in less accurate quantitative analysis and less sensitive imaging, thus limiting the value of the labeling technique. In the absence of ribonuclease, 18O/16O exchange is not observed in 3′-labeled RNA up to 8 h , 16O sense with 18O antisense (16O/18O), 18O sense with 16O antisense (18O/16O) and 18O sense with 18O antisense (18O/18O). On polyacrylamide gel electrophoresis, target and control siRNAs with all combinations of labeled and unlabeled strands showed a single band corresponding to a double-stranded oligomer ∼20 bp in length . This shows that the 18O-labeled RNA oligomers annealed normally to both 18O-labeled and unlabeled complementary strands to form a duplex.For labeled RNA to be used as a tracer, it should ideally have the same physicochemical and biological properties as the corresponding unlabeled RNA. To demonstrate the equivalence of Tm values (Supplementary Table S1). All of the target siRNA duplex combinations had the same Tm value to within 0.6°C and all of the control siRNA duplex combinations to within 0.5°C. Thus, 18O labeling of the backbone phosphate groups had little or no effect on the strength of base pairing.Melting curves were acquired for the four siRNA duplex combinations (data not shown) and used to calculate Supplementary Figure S4). This CD spectral pattern typically indicates the A-form RNA duplex  of 0.9999. We next estimated the blood concentration of control siRNA after intravenous administration to mice. Naked RNA oligomer in the blood is rapidly degraded by ribonucleases and eliminated by the kidney. After administration of naked RNA oligomer (100 or 500 μg), the blood concentration-time curves . The 12C14N images (16O and 18O images (18O ≈ 0.2%), also show the shapes of the cells. All of these images (12C14N, 16O and 18O, reflecting the natural abundance of these isotopes. In contrast, the 18O image (18O/16O ratio image, or isotopograph (18O enrichment in the cytoplasm around the nucleus, presumably in endosomes. The 18O enrichment in the foci was up to 5% above the natural abundance, whereas the 18O in the cytoplasm surrounding the foci and in the nucleus remained at its natural abundance (18O in color-coded form, with red indicating the highest abundance. Similar localization patterns of fluorescently labeled siRNA have been reported by Harborth et al. (18O-labeled RNA was taken up by the cells and accumulated in endosomes, and that the localization of the 18O-labeled RNA in the cells was correctly detected by isotope microscopy.We set out to use isotope microscopy to generate a 2D image of the distribution of N images A and E, 8O image G and theopograph H, show mbundance . The isobundance D and H sh et al. . These r18O signal was not only from intact RNA but also from degradation products. However, RNA introduced into cells with a transfection agent is reported to be detectable 48 h after transfection (18O signals detected with SIMS represent at least some intact RNA. As far as we know, this is the first application of isotope microscopy to the imaging of biological samples containing 18O-labeled compounds. Previous localization studies of RNA have usually been performed with fluorescently labeled RNA. However, fluorescence methods have disadvantages. One is the presence of covalently attached fluorophores, which not only make the labeled molecule chemically different from the unlabeled molecule, but in addition are usually bulky and hydrophobic and thus alter all the more the physicochemical properties of the labeled molecule. Another disadvantage is the possible loss of the fluorophore from the labeled molecule. In contrast, 18O-labeled RNA appears to be both chemically and biologically equivalent to the corresponding unlabeled RNA. In addition, any desired subset of phosphate groups in the oligomer, or all of them, can be labeled at will. These represent distinct advantages of 18O over fluorescent labels in cellular localization analysis. Imaging technology that combines 18O labeling with isotope microscopy is expected to be a useful biological and pharmaceutical research tool.As with the IRMS study, we assume that the observed sfection . Because18O atom in each phosphate group by using 18O water as the oxygen donor to phosphate during the oxidation step of the synthetic cycle. The 18O label was stable under physiological conditions for at least 28 days and the 18O-labeled RNA was physicochemically and biologically indistinguishable from the corresponding unlabeled RNA by electrophoresis, CD, Tm and RNAi activity. These properties of 18O-labeled RNA make it suitable for use as a tracer in basic and clinical studies of RNA drugs such as antisense oligomers, siRNA, miRNA and aptamers. 18O-labeled RNA lends itself to the quantification of RNA in blood by IRMS and to the imaging of the intracellular distribution of the RNA by isotope microscopy. These methods have the potential to avoid the present difficulties with the quantification and visualization of RNA in biological samples by radioisotope and fluorescent labeling, and we expect them to contribute the development of RNA therapeutics.We have prepared RNA labeled with one nonbridging Supplementary Data are available at NAR Online: Supplementary Table 1 and Supplementary Figures 1–4.