BARE retrotransposon, despite its organization into just one open reading frame, produces three distinct classes of transcripts. One is capped, polyadenylated, and translated, but cannot be copied into cDNA. The second is not capped or polyadenylated, but is destined for packaging and ultimate reverse transcription. The third class is capped, polyadenylated, and spliced to favor production of a subgenomic RNA encoding only Gag, the protein forming virus-like particles. Moreover, the BARE2 subfamily, which cannot synthesize Gag and is parasitic on BARE1, does not produce the spliced sub-genomic RNA for translation but does make the replication competent transcripts, which are packaged into BARE1 particles. To our knowledge, this is first demonstration of distinct RNA pools for translation and transcription for any retrotransposon.The replication of Long Terminal Repeat (LTR) retrotransposons, which can constitute over 80% of higher plant genomes, resembles that of retroviruses. A major question for retrotransposons and retroviruses is how the two conflicting roles of their transcripts, in translation and reverse transcription, are balanced. Here, we show that the  Long terminal repeat (LTR) retrotransposons or Class I transposable elements are ubiquitous in the eukaryotes and can comprise over 80% of the large genomes of plants ,2. They Pol, which encodes the enzymes reverse transcriptase (RT), RNAse H (RH), aspartic proteinase (PR), and integrase (IN). The stoichiometry between gag and pol products is critical for replication because the assembly of the VLP requires excess Gag relative to the enzymes [gag and pol [Copia superfamily of retrotransposons [copia of Drosophila deletes virtually all of pol, 2950 nt, generating an RNA dedicated to Gag translation [The structural Gag is often in a separate open reading frame (ORF) from  enzymes ,6. A com and pol . Howevernsposons  and alsonsposons . While anslation , this hanslation .Another major conundrum is that reverse transcription of retrotransposon and retrovirus RNA, which destroys the RNA template, conflicts with its further translation . AlternaBARE retrotransposon of superfamily Copia accounts for over 10% of the barley genome [BARE1 has no frameshift between gag and pol [BARE2 cannot express Gag [BARE1 and BARE2 produce multiple classes of RNA transcripts from two TATA boxes, of which only 15 to 25% are polyadenylated [BARE RNAs serve for translation, which ones are packaged, and does BARE use an alternative to frameshifting for Gag production. We were able to demonstrate not only different RNA pools for translation and reverse transcription but also a novel splicing pattern for Gag synthesis.Although retrotransposons comprise much of most plant genomes, the details of their lifecycle have been investigated for only a few. The y genome ,15. BARE and pol ,17. A varess Gag ,18. BAREenylated . MoreoveHordeum vulgare L.) plant materials and callus cultures used for RNA isolation, as well as the methods used to isolate the RNA, are described in the .Barley  using a kit  with small changes and a custom adapter. To examine 3’ polyadenylation of polyribosome-associated RNA, 3’ RLM-RACE was carried out. Details of both methods are presented in the The presence of a 5’ 7-methylguanosine cap on BARE1 and BARE2, RT-PCR was carried out using primer AP4  within the Arabidopsis genome and by SplicePredictor (http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi) against maize and human genomes, supporting the interpretation that the shorter BARE transcript is a splicing product.The presence of the short form of sequence . These atachyon  and for and rice ,23. The BARE2 genomic or RNA sequences  PCR, respectively by pyrophosphatase decapping and phosphatase 5’ dephosphorylation. Pyrophosphatase-mediated cap removal generates a 5’ phosphate in its place, which will allow ligation of an RNA adapter and PCR. Uncapped 5’ ends can be ligated directly without pyrophosphatase treatment.BARE elements containing internal domains and not from read-through transcripts of solo LTRs. Capped RNAs transcribed from BARE were found in all tissues examined  and 1682 (numbering from AJ279072). All start sites corresponded to those found in the earlier RACE-PCR data for TATA1 in BARE1 and BARE2 [In a complementary experiment, the uncapped transcripts, which originate only from TATA1, were cloned and sequenced from total RNA of embryo and callus . The stand BARE2 .BARE1 and BARE2 are capped; those from BARE1 are spliced about half the time and BARE2 transcripts are not spliced. Whereas translated cellular RNAs are generally both capped and polyadenylated, plant viral RNAs and most positive-strand RNA viruses are translated not only without caps but also without poly(A) tails [BARE, polyadenylated RNAs isolated from leaf and callus were subjected to RLM-PCR diagnostic for the presence of caps and then the PCR products sequenced. Caps were present in both BARE1 and BARE2 products from the polyadenylated RNA fraction; these transcripts start only after TATA2  tails . To clarer TATA2 . In contBARE1 and BARE2 RNA species, we investigated which pool is translated by examining RNA in polyribosomal translation complexes. As described above, the BARE2 transcripts neither express Gag nor are spliced, raising the question of whether they are nonetheless present among the polyribosomal RNAs. Polyribosomes were isolated; the 28S and 18S ribosomal RNAs were effectively concentrated into the pellet  for BARE1 and 25nt downstream of TATA2 for BARE2 (AJ279072) as before [Given the multiple e pellet . The supnd BARE2 . BARE1 as before . For coms before .BARE1 and BARE2 transcripts in the polyribosomes, specific primer pairs were used. The BARE2 polyribosomal transcripts are more abundant than those of BARE1  in VLPs. It is within the VLPs that transcripts are replicated into cDNA, which is then transported back to the nucleus for integration. TATA1 transcripts of the parasitic BARE2, which cannot produce its own Gag, are likewise packaged into VLPs. This is consistent with the conserved dimerization and packaging signals in BARE2 and with the predominance of BARE2 over BARE1 in the genomes of barley [In contrast to translation, for replicative competence retrotransposons must avoid the packaging, reverse transcription, and integration of spliced RNA. Several lines of evidence support the view that cription . Second,ave none ,31. The f barley .BARE, fairly little is known about the nature of the packaged RNA. The yeast Ty1 is thought to package uncapped RNA, although also capped RNA may be present [BARE TATA1 transcripts were not detected with caps, they could be initially capped and then very efficiently decapped. Intriguingly, Ty1 mRNA, Gag, and VLPs co-localize to P-bodies, components of which enhance retrotransposition [For LTR retrotransposons other than  present . Althougposition . The P-bposition ,47. At aBARE retrotransposon [BARE1 and BARE2 generated from a distinct promoter are reserved for replication into cDNA. For translation, BARE1 appears to increase production of Gag vs Pol by splicing about half of its capped, polyadenylated transcripts. However, BARE2 parasitizes BARE1 Gag; its replicative transcripts are packaged into BARE1 VLPs. To our knowledge, this is first demonstration of distinct RNA pools for translation and transcription for any retrotransposon.In summary, the deceptively simple structure of the single ORF in the ansposon , compareansposon , masks aBARE is regulated. Though it remains to be seen if the expression mechanism used by BARE is general, elements related to BARE are widespread and active [Wis-2 [Angela [OARE-1 [RIRE1 [SORE1 [BARE is part of a group of abundant and phylogenetically diverse retrotransposons of similar structure. Therefore, the replication mechanism of BARE may have wide relevance.At present, we are exploring the role of stress, developmental stage, and tissue on the function and relative abundance of these RNA species in order to understand how replication and propagation of d active . Togethee [Wis-2 , Angela  [Angela , OARE-1  [OARE-1 , RIRE1 [1 [RIRE1 , and SOR1 [SORE1  familiesMaterials S1An extended description of the plant materials, RNA isolation procedures, 5’ cap assays, 5’ RLM-RACE, 3’ RLM-RACE, as well as polyribosome RNA isolation and RT-PCR, are presented.(PDF)Click here for additional data file.Table S1BARE1 (Accession Z17327) or BARE2 (AJ279072), their ability to amplify products from BARE1 or BARE2, and their orientation.The primers used in this work are described by their name, sequence, matching region in (PDF)Click here for additional data file.


RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers.Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5’ pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors.To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65°C should reduce the constraints of RNA secondary structure in RNA ligation experiments.Mutational analysis of the heat stable RNA ligase from  TM, ThermoPhageTM) is used for ssDNA circularization -ATP (Perkin-Elmer), 10 mM Mg+2 for 30 min at 65°C in 10 mM bis-Tris propane-HCl buffer with corresponding pH@ 25°C indicated in the figures. For ATP binding assay, the same reactions were performed at pH 7.0 and 8.5 for the wild type MthRnl and the K246A mutant respectively with variable concentration (0–200 μM) of ATP. Reaction products were separated on 10–20% Tris-Glycine SDS polyacrylamide minigels (Invitrogen), visualized with Simply Blue Safe Stain (Invitrogen) and after gel drying exposed to a PhosphoStorage Screen (Bio-Rad) and scanned on a Typhoon 9400 Imager . Visualized radioactive bands were quantitated using Image Quant TL software . Alternatively, protein bands were isolated and radioactivity counted with a Liquid Scintillation Analyzer 2100TR (Packard).Self-adenylation of the enzymes were performed in 10 μl of a reaction mixture containing 10 pmol of MthRnl or MthRnl mutant (460 ng), 200 μM ATP and 1 μCi of α-[+2, 5 pmol 5’-phosphorylated 3’ blocked DNA, 10 pmol of MthRnl or MthRnl mutant (460 ng), 100 μM ATP for 60 min at 65°C in 200 μl PCR tubes using an S1000 Thermal Cycler (BioRad), followed by inactivation of the enzyme at 90°C for 3 min. After the addition of 5 μl formamide loading buffer, the reaction mixture was separated on 15% Urea–TBE denaturing polyacrylamide minigels (Invitrogen), stained with SYBR Gold (Invitrogen) and visualized using AlphaImager HP (Alpha Innotech). All parameters that are different from the standard reaction conditions are indicated in the figure legends.The ssDNA adenylation assay was performed in 10 μl of reaction mixture containing 10 mM bis-Tris propane-HCl buffer, pH 6.5, 10 mM MgThe deadenylation/hydrolysis reactions were performed under conditions described for the ssDNA adenylation reaction, except that pre-adenylated DNA was substituted for 5’ phosphorylated DNA, and ATP was omitted from the reaction.+2 for RNA ligation or 5 mM Mn+2 for ssDNA ligation, 5 mM DTT, 5 pmol of acceptor substrate, 8 pmol of pre-adenylated donor substrate, 20 pmol (monomer) MthRnl or MthRnl mutant. Assays were performed as described for ssDNA adenylation reaction. For quantitation, gel images were analyzed using Quantity One software (Bio-Rad). All parameters that are different from the standard reaction conditions are indicated in the figure legends.The ligation of pre-adenylated ssDNA or RNA donors with a ssDNA or RNA acceptor without ATP was performed in a reaction mixture  containing 10 mM bisTris-Propane buffer, pH 7.0 (@25°C), 10 mM Mg+2, 1 mM DTT and 200 U (~10 pmol) of truncated T4Rnl2 or 10 U (~50 pmol) of T4Rnl1. Reactions were incubated for 2 hours at 25°C. Standard reactions with a 5’-phosphorylated donor, various acceptors and ATP using T4Rnl1 were performed according to the manufacturer’s protocol (NEB). Reactions were stopped by adding 5 μl formamide loading buffer, heat inactivated at 90°C for 3 min and the products were separated, stained and visualized as described for the ssDNA adenylation above.The ligation of pre-adenylated ssDNA or RNA with an acceptor substrate using T4 RNA ligase 1 or truncated T4 RNA ligase 2 (NEB) without ATP were carried out in single-turnover conditions and performed in 10 μl containing 5 pmol of the RNA acceptor, 8 pmol of pre-adenylated donor substrate in 10 mM Tris-HCl pH 7.5 buffer, 10 mM MgLM and AZ are employees of New England Biolabs, a company that sells mutant Mth RNA ligase and other proteins for RNA and DNA research.AZ designed, carried out experiments and drafted the manuscript. LM discussed experimental data and conclusions and prepared the manuscript. All authors read and approved the final manuscript.


Populus spp,This poster will discuss recent validation of the platform for a subset of these applications in detail. The AdvanCE™ FS system successfully scored all SSR samples with resolution as low as 2 bp. Assessment of the quality/concentration of gDNA is also demonstrated with a new separation matrix, for separation of large DNA fragments. Separation and sizing of large cDNA was also demonstrated with good resolution and accuracy. A method for rapid and sensitive detection of total RNA concentration and quality will be discussed, as well as the sizing and quantification of NGS libraries.The AdvanCE™ FS platform offers rapid separation and ample resolution with excellent sensitivity and dynamic range, to benefit a variety of applications in genomic research.Central to genomic research is the analysis of nucleic acids, which requires versatile, sensitive and high throughput technologies that can deliver accurate results in a short time. Reported herein are recent developments for sizing and/or quantifying nucleic acids (dsDNA and RNA) on a parallel capillary electrophoresis platform with LED-based fluorescence detection (AdvanCE™ FS system). This flexible multi-capillary platform has been employed for applications such as SSR, gDNA analysis, RNA analysis, quantification of next generation sequencing (NGS) libraries, and the separation and sizing of large cDNA from


Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes. D. melanogaster and C. elegans have three paralogs and in mice, six different paralogs have been described The importin α  family comprises soluble transport factors that mediate the movement of proteins from cytoplasm to the nucleus in interphase cells Here, we studied the function of importin α7  by gene targeting in mice and show that it is essential for early embryonic development. We show that importin α7 is a maternal protein present in oocytes. Oocytes lacking this protein can be fertilized but display a reduced ability for the first cleavage and a complete arrest at the two-cell embryo stage. Expression analyses for several marker genes of zygotic genome activation (ZGA) showed that ZGA is severely disturbed in these embryos.ΔIBB ΔIBB) express an mRNA, which contains a cryptic translational start site in exon 3 and thus codes for an importin α7 protein lacking the importin β-binding domain (−/−) containing a gene trap cassette in intron 1 of the importin α7 gene. Western blot analyses of different tissues confirmed the absence of importin α7 protein in α7−/− animals (ΔIBB/ΔIBB and α7−/−) were analysed in parallel. They were viable, without obvious morphological abnormalities, and histological analyses of heart, kidney, liver, lung, and spleen showed no pathological phenotypes.To clarify the specific function of importin α7 in mice, we generated importin α7 knockout mice by deleting exon 2 of the gene . Unexpecg domain . This do animals . Both moΔIBB/ΔIBB and α7−/− female mice with wildtype males yielded no offspring, while heterozygous females of both lines were completely normal and fertile. To determine the cause of female infertility in importin α7-deficient mice, we examined ovarian histology of importin α7ΔIBB/ΔIBB females. Ovaries from these animals were indistinguishable from those of control females and all stages of follicle development and corpora lutea were evident , zygotes with two visible pronuclei could be recovered from oviducts of α7 females . FurtherΔIBB/ΔIBB females with successful pronuclear formation showed a markedly decreased capability to develop into two-cell embryos  of 5-bromo-2-deoxyuridine (BrdU), suggesting that DNA replication is not disturbed in importin α7-deficient embryos , importin α5 and murine endogenous retrovirus-like gene (MuERV-L) was analysed in oocytes and early embryos of importin α7-deficient and control females. While wildtype two-cell embryos displayed expression of all these genes, none of these transcripts could be detected in importin α7-deficient two-cell embryos, suggesting a defect in ZGA (MT1A) could be detected in two-cell stages of importin α7-deficient embryos, albeit at a markedly lower level than in wildtype.The developmental block of importin α7-deficient embryos coincides with the onset of ZGA. ZGA is an essential step for maternal-to-zygotic-transition and results in a novel gene expression profile which establishes the totipotent state of each blastomer in the cleavage-stage embryo t in ZGA . However−/− females after mating with importin α5−/− males lacZ cassette under control of the importin α5 promoter, leading to the formation of lacZ mRNA when the ZGA marker gene importin α5 is transcribed. Wildtype embryos displayed strong staining for β-galactosidase in the late two-cell stage, while no staining could be detected in importin α7-deficient embryos, confirming the RT-PCR data , a two-cell stage delay (CTCF), arrest at the two-cell stage  or arrest at later embryonic stages . A crucial step in the maternal-to-zygotic-transition is the activation of the embryonic genome Mater, Npm2, Zar1 and brg1 have been shown to display a severe disturbance of ZGA We show here that importin α7 is a maternally expressed protein. Presently, fourteen maternal-effect genes are known in vitro in M16 medium (Sigma) at 37 °C under 5% CO2.Local German authorities  approved the animal studies with standards corresponding to those prescribed by the American Physiological Society . Superovulation and embryo culture was performed as described elsewhere 2+ and Mg2+ -free M16 medium containing 2 mM Sr2+ at 37 °C under 5% CO2. To obtain diploid parthenogenetic embryos, the oocytes were cultured 7–8 hours in the presence of 5 µg/ml cytochalasin B (Sigma). Efficiency of pronuclear formation was analyzed 10–12 h after treatment. Oocytes were observed under an inverted microscope with Nomarski optics (Zeiss). Those oocytes that formed visible pronuclei were recorded as activated and cultivated further.Parthenogenetic activation of oocytes was performed as described elsewhere The generation of the anti-importin α7 antibody is described elsewhere 12 µg tissue extract were loaded on a 10% SDS gel. After transfer of proteins, the PVDF membrane was blocked by Tris-buffered saline/0.1% Tween (TBST) with 5% skim milk powder and subsequently incubated with primary anti-importin α7 antibody  or anti-importin α4 antibody  at 4 °C over night. On the next day, the membrane was incubated with secondary antibody against rabbit IgG conjugated with horseradish peroxidase  for 1 h at room temperature and detection was performed using the ECL Super Signal West Dura reagent (Pierce). For loading control, staining of the Western blot membranes was performed with Ponceau S solution (Sigma Aldrich) according to the manufacturer's instructions.2, and 0.2 U/µl Taq DNA polymerase (Invitrogen). For detailed information on primer sequences and PCR conditions see Total RNA was isolated from unfertilized oocytes and embryos using the RNeasy Mini Kit (QIAGEN) and incubated in a 20 µl reaction mixture containing 100 U of Super Script II Reverse Transcriptase (Invitrogen) and 200 ng of random primer (Roche) at 25 °C for 10 minutes, followed by 30 minutes incubation at 42 °C and inactivation for 15 minutes at 70 °C. The cDNA was diluted to 0.5 embryos equivalent/µl. For PCR, a 10 µl reaction mixture consisted of 1 µl of the cDNA solution, 10 ng of each primer, 2 µM dNTP, 20 mM MgClhttp://www.med.umich.edu/tamc/laczstain.html). For control, wildtype embryos were treated with α-amanitin  over night prior to fixation. After staining, embryos were placed in wash buffer und stored at 4 °C. For visualization, a Leica DMI6000B microscope (Leica) with a Leica DFC 420 camera (Leica) was used.Cultured two-cell embryos were rinsed in phosphate buffer, fixed for 5 minutes in fixation solution, washed 3 times for 5 minutes in wash buffer and incubated with X-gal stain overnight at 37 °C  were purchased form Sanger Gene Trap Resource and directly used for blastocyst injection.For the generation of α7−/− was cloned in a targeting vector The knockout construct for importin α4−/− mice, we performed RT-PCR with RNA isolated from tail biopsies. Genotyping of importin α7ΔIBB/ΔIBB and α4−/− mice was performed using PCR on genomic DNA. The primer sequences and PCR conditions are listed in From all injected ES cell clones we obtained germline chimeras, which were subsequently bred with C57Bl/6 mice. Both colonies of α7-mutant mice were maintained by breeding the resulting heterozygous mice. Importin α4-deficient mice were backcrossed to C57Bl/6 genetic background, bred to the homozygosity and, since they were fertile, maintained as −/− colony. For genotyping of importin α7Ovaries from 16 week old mice were isolated and paraffin embedded. Slides with sections were deparaffinized and rehydrated by incubation in descending ethanol series. Slides were incubated for 3 minutes in hematoxylin, subsequently rinsed with water for 20 minutes and placed in eosin for 6 minutes. After dehydration by ascending ethanol series, slides were mounted in Eukitt (Kindler). For visualization, a Leica DMI6000B microscope (Leica) with a Leica DFC 420 camera (Leica) was used.After isolation embryos were incubated in M16 medium until 24 hours post Ovogest injection to get late zygotes in which the first round of replication is completed. Zygotes were transferred to M16 medium supplemented with BrdU (Sigma) to a final concentration of 50 µM and incubated overnight. At the end of the incubation period, two-cell embryos were rinsed in PBS, fixed in 3.7% paraformaldehyde (PFA) in PBS at room temperature for 15 minutes and permeabilised in 0.1% Triton X-100 in PBS/1% normal donkey serum (NDS) for 30 minutes. After washing in PBS/0.05% Tween/0.1% NDS, embryos were placed in 2 M HCl/PBS for 10 minutes to denature the DNA. Embryos were then extensively washed in PBS/0.05% Tween/0.1% NDS and blocked with PBS/0.05% Tween/5% NDS for 1 h at room temperature. Detection of BrdU-DNA was performed using a monoclonal anti-BrdU antibody  and a secondary anti-mouse IgG antibody coupled to Cy3 . Embryos were incubated with 5 µg/ml Hoechst 33258 (Hoechst) for 15 minutes and mounted with Fluorescence Mounting Medium (DAKO). BrDU staining was quantified using the Image J program.For visualization of the meiotic spindle, oocytes isolated from oviducts of superovulated mice were collected, washed in PBS and fixed in 3.7% PFA in PBS at room temperature for 15 minutes. After permeabilisation in PBS/0.25% Tween for 5 minutes at room temperature, oocytes were transferred to blocking buffer /2% normal goat serum/0.1 M glycine/0.01% Triton X-100) and incubated at 4° overnight. Then, oocytes were incubated with a FITC conjugated anti-α-tubulin antibody  for 2 h at 37 °C. At the end of the incubation period, oocytes were washed in PBS/0.1% BSA, DNA was counterstained with 5 µg/ml Hoechst 33258 for 15 minutes and mounted with Fluorescence Mounting Medium.For visualisation of nuclear pores, zygotes isolated from oviducts of superovulated and mated mice were collected, washed in PBS and fixed in 3.7% PFA in PBS at room temperature for 15 minutes. After washing, zygotes were permeabilised for 20 minutes in PBS/0.5% Triton X-100/0.1% BSA, transferred to blocking buffer and incubated at 4 °C overnight. Incubation with anti-nucleoporin antibody  was performed for 2 h at 37 °C. At the end of the incubation period, oocytes were washed in PBS/0.1% BSA and incubated with Cy2-conjugated donkey anti-mouse IgG antibody . After washing DNA was counterstained with 5 µg/ml Hoechst 33258 for 15 minutes and mounted with Fluorescence Mounting Medium. Immunofluorescence was detected using the Leica DMI 6000B fluorescence microscope with a Leica DFC 350FX fluorescence camera (Leica).Statistical analysis was performed using Pearson's chi-square test.Figure S1−/− females does not interfere with the expression of the other α importins. The sequence of importin α2 (Kpna7) was identified by homology of sequence tags from GenBank database to other α-importins and subsequent sequencing of the respective cDNA clones. The sequence of one complete clone was deposited as entry AY950703.Expression analysis of importin α1, α2, and α3 in oocytes and early embryos. RT-PCR shows the maternal expression of importin α1 and α2 mRNAs in wildtype oocytes and the zygotic activation of importin α3. The absence of importin α7 mRNA in embryos from importin α7(JPG)Click here for additional data file.Table S1Primer sequences and conditions for PCR.(DOC)Click here for additional data file.


RNAexinv is an interactive java application that performs RNA sequence design, constrained to yield a specific RNA shape and physical attributes. It is an extended inverse RNA folding program with the rationale behind that the generated sequences should not only fold into a desired structure, but they should also exhibit favorable attributes such as thermodynamic stability and mutational robustness. RNAexinv considers not only the secondary structure in order to design sequences, but also the mutational robustness and the minimum free energy. The sequences that are generated may not fully conform with the given RNA secondary structure, but they will strictly conform with the RNA shape of the given secondary structure and thereby take into consideration the recommended values of thermodynamic stability and mutational robustness that are provided.The output consists of designed sequences that are generated by the proposed method. Selecting a sequence displays the secondary structure drawings of the target and the predicted fold of the sequence, including some basic information about the desired and achieved thermodynamic stability and mutational robustness. RNAexinv can be used successfully without prior experience, simply specifying an initial RNA secondary structure in dot-bracket notation and numerical values for the desired neutrality and minimum free energy. The package runs under LINUX operating system. Secondary structure predictions are performed using the Vienna RNA package.http://www.cs.bgu.ac.il/~RNAexinv.RNAexinv is a user friendly tool that can be used for RNA sequence design. It is especially useful in cases where a functional stem-loop structure of a natural sequence should be strictly kept in the designed sequences but a distant motif in the rest of the structure may contain one more or less nucleotide at the expense of another, as long as the global shape is preserved. This allows the insertion of physical observables as constraints. RNAexinv is available at  Before elaborating on the inverse problem, one should begin by mentioning the classical RNA folding problem that aims to predict the secondary structure of a given RNA sequence. Software packages are nowadays available that contain RNA thermodynamic parameters -3 and prabet κ =  for RNAssequence , varioussequence ,7 that dsequence  and INFOsequence , an extesequence , which asequence ,11) mentHere we describe the software implementation of a new method for the extended inverse RNA problem. Unlike the method in  that utiWe now elaborate in more detail on the various stages that the method consists of, starting from the first. For our requirements, using RNAinverse with a random start point is preferable over more "fixed" inverse RNA folding strategies such as INFO-RNA . In a diStarting with the sequence that was generated by RNAinverse from the Vienna RNA package with a random start point, we search for local optima using iterative mutating. Neighboring sequences are assessed according to an objective function that takes into account all the parameters: structure, thermodynamic stability, and mutational robustness. The combined objective function is given bywhere the neutrality is a number between 0 and 1, dG is the minimum free energy in kcal/mol, and distances are calculated using RNAdistance in the Vienna RNA package .The above objective function, as can be noticed in the third term, minimizes the distance between the desired shape and the mfe shape of the input. The distance is that as defined by tree edit distance over the Shapiro representation  . Increasing the number of steps will definitely increase CPU time but might increase the goodness of the results until a point when it will stride in the same local minimum.1) The number of minimization steps . Increasing it will increase CPU time but might help the local search overcome shallow local minima.2) The maximum distance of the look ahead  Trying to tie the ends of the structure together .Setting each parameter can influence the accuracy of RNAexinv result and the time to complete the run. Each user needs to balance the CPU time dedicated for each run and the number of repetitive runs. On the one hand, without enough CPU time for each run, the runs will not exploit their full potential. On the other hand, with too much CPU time for each run, the time will be wasted running in circles around the same minimum instead of starting from a different point to better examine the landscape.The output of RNAexinv is demonstrated in Figures We compared our suggested extension with the standard (non-extended) inverse RNA folding problem, in particular against RNAinverse , withouthttp://www.tbi.univie.ac.at/~ivo/RNA and is freely available for download in http://www.cs.bgu.ac.il/~RNAexinv.In examining its biological relevance, RNAexinv can be used for designing sequences that adhere to the extended inverse RNA folding problem suggested in , but in Project name: RNAexinvProject home page: http://www.cs.bgu.ac.il/~RNAexinvOperating system(s): web access: not applicable, stand-alone: LINUXProgramming language: C, JavaOther requirements: stand alone:Java 1.4.0 or higher, GNU C compilerLicense: NoneAny restrictions to use by non-academics: NoneThe authors declare that they have no competing interests.DB and AA conceived the study, coordinated and participated in software design and drafted the manuscript. AC worked on software design, carried out development and implementation, and participated in drafting the manuscript. All authors read and approved the final manuscript.


As a benchmark we have used reverse transcription-quantitative PCR. The results show that reads per kilobase per million (RPKM) and trimmed mean of M-values (TMM) normalization systematically leads to biased gene expression estimates. Biological scaling normalization (BSN), designed to handle differences in total expression, showed improved accuracy compared to the two other methods in estimating transcript level dynamics. The results have implications for past and future studies using RNA-sequencing on samples with different levels of total or polyA+ RNA.Methods for normalization of RNA-sequencing gene expression data commonly assume equal total expression between compared samples. In contrast, scenarios of global gene expression shifts are many and increasing. Here we compare the performance of three normalization methods when polyA RNA sequencing (RNA-seq) is frequently used for global gene expression analysis. RNA-seq generates short reads from fragmented RNA molecules and the number of reads is proportional to the abundance and length of the transcripts + RNA amounts between the 1-cell stage and the ∼1000-cell stage 3.5 h post-fertilization (hpf) + RNA levels several fold Although equal global gene expression levels are generally acknowledged as an important assumption in all of the aforementioned methods, it is rarely tested. We recently showed that in zebrafish embryos, approximately 70% of maternal transcripts undergo cytoplasmic polyadenylation prior to onset of zygotic transcription, leading to a 50–70% increase in the retrieved polyA+ RNA amounts as scales to normalize different developmental stages To account for this, we recently proposed an approach which uses experimentally measured polyAij] is the relative abundance of transcript i in sample j, Rij is the number of reads for gene i in sample j, and Rj is the total number of reads in sample j. We calculate the average library size:Where [EWhere n is the number of samples. A pseudo library size is obtained by multiplying the average library size with a stage specific scaling factor:ij] value to get the normalized dataset: The scaling factors can be obtained both mathematically and experimentally, as will be demonstrated later. The next step in the normalization procedure is to reassign the pseudo read libraries to each gene based on the previously estimated  = Rij/(Rj*ZjTMM)), this is done afterwards in BSN (see equation 3). RPKM, as an intermediate approach, normalize without adding any scaling factor ([Eij] = Rij/Rj). The rationale behind both RPKM and TMM normalization is similar, and distinct from BSN; they assume little difference in total expression between compared samples. If such a difference exists, it will be reduced after normalization. Rather, BSN seek to retain biological differences between samples, with the assumption that more RNA detected at a particular stage would correspond to more genes being up-regulated.Similarly to TMM normalization, our method contains a scale which aims at representing change in total expression, also termed the “global fold-change” + RNA levels during the first 6 h of zebrafish development. The first 2.5 h are characterized by substantial increase of polyA+ RNA To test our hypothesis we have validated BSN and compared it to RPKM and TMM normalization under conditions of global increase and decrease of polyAj throughout this paper. This measure represents the change in total RNA or polyA+ RNA, depending on which population is under study. Two methodologies were used to gain an estimate of the fluctuations of RNA levels in the embryo, one biological and one mathematical. First we isolated and measured the amount of total and polyA+ RNA from equal numbers of embryos at different developmental time points before zygotic genome activation (ZGA)  and after (3.5 hpf and 5.5 hpf). These time periods are referred to as pre- and post-ZGA samples from here on. Total RNA levels did not change significantly between stages, but we did observe a decreasing trend  see . We denourements . Moreove+ RNA and/or type of primers used to generate cDNA (random or oligo(dT) primers), we performed parallel experiments of the same samples using different combinations of template and primers. The results demonstrate that detection of increase in mRNA abundance pre-ZGA depends on an enrichment of transcripts in the polyA+ RNA fraction rather than in total RNA , the BSN values are closest to the qPCR benchmark in all cases ; however this is independent of normalization method. On average, the fold changes post-ZGA were 32% and 64% lower for BSN, compared to RPM and TMM. The BSN method also performed best when primer efficiency calculations were used for adjusting fold-changes, and discrepancies between qPCR and RNA-seq were reduced , and between 3.5 hpf and 5.3 hpf (post-ZGA). Spike-in RNA was added to the Trizol reagent before RNA isolation and polyAipts see . Transcripts see . Overallipts see . For allipts see . For sodll cases . For som reduced . Taken tComparisons between normalization methods and qPCR for transcripts varying post-ZGA using dataset 2 revealed the same trend as for dataset 1; however, there was less discrepancy between qPCR and RNA-seq results . BSN nor+ RNA levels during development is well documented + RNA levels (BSN), compared to methods aimed at making samples similar . Our results show that the normalized expression values were consistently best approximated by BSN when compared to a RT-qPCR benchmark. Only a few transcripts increasing prior to ZGA were best approximated by RPM normalized values; TMM-normalized values on the other hand were in all cases the least accurate. As expected, both the TMM and RPM normalization strategies leads to underestimation during the increase (pre-ZGA) and overestimation during the decrease (post-ZGA) of polyA+ RNA amount, RPM less so than TMM normalization Fluctuation in polyAiTMM, is a good estimator of global fold change, with high correlation to laboratory measurements. It could therefore be tempting to rely solely on this cost-effective and efficient mathematical approach. However, we strongly encourage laboratory validation of the estimated global fold-changes since the TMM method does not always detect global changes. In the aforementioned example of a global increase in expression after c-Myc stimulation Despite low accuracy of TMM normalization under the circumstances studied here, the TMM scaling factor, ZWe have shown improved accuracy of normalizing gene expression data using BSN compared to RPM and TMM during developmental stages that display global increases and decreases in mRNA content. Application areas of this approach are expected to be numerous, ranging from comparing gene expression during development, disease, and tissue- and cell-type specification. More generally, our results should be a strong argument for testing the assumption of equal RNA levels in all gene expression dynamics studies.All studies were performed in accordance with animal experiment legislation and guidelines. In Norway zebrafish embryos are not regulated prior to they start feeding at day five post-fertilization. Consequently, no approval was needed for experiments in this study of embryos up till 6 hours post fertilization.+ RNA was extracted from equal volumes of total RNA from each stage using the MicroPoly(A)Purist™ Kit . The amount of total and polyA+ RNA was measured with a NanoDrop 2000  and a Qubit® RNA Assay Kit , respectively. RNA integrity was measured using the Agilent RNA 6000 Nano chip on a Bioanalyzer 2100 .Wild-type zebrafish from AB background were maintained in the zebrafish facility of the Institute of Molecular and Cell Biology, Singapore. Embryos were grown in embryo medium at 28°C and staged according to standard morphological criteria + RNA extraction as well as in RT and qPCR reactions. For cDNA synthesis we used Superscript III First Strand Synthesis System . For a subset of transcripts, we performed RT on total and polyA+ RNA using either oligo(dT)20 or random hexamer primers, and generated four groups of cDNA: (1) total RNA + oligo(dT), (2) total RNA + random primers, (3) polyA+ RNA +oligo(dT), (3) polyA+ RNA + random primers /ErefΔct ref (ctl-sample)). The fold-change values were log2-transformed.mRNA levels for 20 transcripts at each developmental stage were measured by RT-qPCR. The transcripts were chosen systematically to represent different expression patterns. We used an equal number of embryos used synthetic kanamycin RNA to adjust for differences in RNA yield  primers . For the+ selected RNA. Dataset 1 using Poly(A) Purist kit  and dataset 2 using PolyA purist-MAG-kit (Ambion). The total RNA starting material was in both cases extracted with Trizol (Invitrogen). For comparison of TMM values we also downloaded a dataset based on total RNA http://www-huber.embl.de/users/anders/HTSeq/).We used RNA-seq data from two different previously published studies for comparison of normalization methods. The first dataset (“dataset 1”) was generated using SOLiD3 technology jTMM scaling factors using the R package “edgeR” and the “calcNormFactors” command jTMM, were obtained as for TMM-normalization  jBio, were obtained through PolyA+ RNA laboratory measurements and calculation of the percentage polyA+ RNA of the total RNA. Each stage specific percentage was then divided by the 1-cell stage percentage to generate the ZjBio scales. Replicates in dataset 2 were first normalized within each group through multiplying concentrations (eq. 1) with the average library size for each group. The average library size across all stages were then calculated (eq. 2). Scaling factors were obtained using “edgeR” and the command “calcNormFactors” on a set of replicate averaged values. These scales were used to obtain stage specific library sizes (eq. 3). Concentrations of the transcripts were then again calculated and multiplied with the stage-specific library size (eq. 4). The resulting normalized counts all had correlation coefficients >0.99. All raw and normalized values are available in dataset S1 and S2.The raw counts were normalized against the total number of mapped reads per million (RPM), to represent RPKM For comparison of normalization methods we selected transcripts with a dynamic expression pattern. Fold-changes for two different time points were calculated depending on whether transcripts expression levels changed or not: i) from the 1-cell to the 3.5 hpf stage (“pre-ZGA”) and/or ii) from 3.5 to 5.3 hpf (“post-ZGA”). Fold changes were calculated as the ratio of 3.5 hpf to 1-cell and as the ratio of 5.3 hpf to 3.5 hpf and subsequently log2-transformed.File S1Table S1 and Figures S1–S6. Table S1. List of primers. Forward and reverse primers used for qPCR. Figure S1. Changes in total and polyA+ RNA during development. a) Amount of total RNA per embryo at different developmental stages. b) Amount of polyA+ RNA per 100 embryos at different developmental stages. Vertical bars represent standard errors. Figure S2. The TMM scaling factor. a) The TMM scaling factor estimated using dataset 1 and 2. We observe very similar values. b) The TMM scaling factor obtained using the replicates in dataset 2. The TMM values are very reproducible. c) The TMM scale factor when RNA-seq data based on total RNA was used. Figure S3. Comparison of scales. We either square-root transformed or used that scales directly and compared the normalized fold-changes to RT-qPCR results. a) Transcripts with dynamic change pre-ZGA. b) Transcripts with decreased abundance post-ZGA. c) Transcripts with increased expression post-ZGA. Vertical bars represent standard deviations. Figure S4. Comparison of RT-qPCR results depending on RNA template  and primers (random or oligo(dT) primers) for setd3 (a), gtf2e2 (b) and yy1a (c). The increase pre-ZGA is dependent on template (setd3 and gtf2e2) and not primer type. Figure S5. Efficiency calibrated fold-changes for a subset of transcripts. Vertical bars represent standard deviations. Figure S6. Comparison normalization methods using dataset 2 for transcripts with decreased expression post-ZGA (a) and increased expression post-ZGA (b). Vertical bars represent standard deviations.(PDF)Click here for additional data file.


Picornaviruses contain stable RNA structures at the 5′ and 3′ ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5′ end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved. Picornaviridae, is a group of small, nonenveloped RNA viruses. The genus cardiovirus includes two species, encephalomyocarditis virus (EMCV) and Theilovirus, comprising Theiler's murine encephalitis virus (TMEV), Theiler's virus of rats, and Saffold virus (SafV), a human pathogen that is closely related to TMEV pol and some relatively stable processing intermediates performing activities in viral RNA replication distinct from their final cleavage products Cardiovirus, a genus of the family of pol. Minus-strand RNA serves as a template for new plus-strand RNA molecules. RNA synthesis takes place in the replication complex at the outer surface of virus-induced membrane vesicles The cardiovirus infectious cycle involves a number of distinct steps. Following cell entry and virion uncoating, the monocistronic viral RNA acts as an mRNA, directing the synthesis of the large polyprotein. Proteolytic processing of the polyprotein leads to the liberation and activation of the viral proteins involved in viral RNA replication. Replication of the viral plus-strand RNA starts with the synthesis of a complementary minus-strand RNA in the cytoplasm by the RdRp 3DReplication of the viral RNA depends on the assistance of other viral and host proteins. Initiation of a minus-strand synthesis demands positioning of the RdRp in close proximity to the 3′-end of the plus-strand, whereas the enzyme should be placed at the 3′-end of a minus-strand to initiate a progeny plus-strand. Since the 3′-ends of the complementary strands are dissimilar, the replication machinery recognizes two different types of origins of replication to initiate synthesis of plus and minus-strands, referred to as oriL and oriR, respectively In enteroviruses, OriL is well defined and is a multifunctional element involved in both viral genome replication and translation GCUA or GCUU tetraloop closed by a C-G, G-C, or U-A base pair.As a first approach to gain more insight into the secondary structure of the cardiovirus 5′UTR SLA, a multiple alignment was made of available RNA sequences in combination with in silico folding using MFold GCUA and GCUU tetraloops have been described previously UCGAUAUC sequence and might be folded into a stable GAUA tetraloop closed by UC/UC tandem pyrimidine base pairs. Tandem noncanonical pyrimidine base pairs have been found previously in the Y-stem of the poliovirus 3′UTR Tertiary structures of stable 2O/D2O. Iminoproton resonances were observed for G and U residues forming base pairs within the stem, including the U3-G20 non-Watson-Crick base pair. Iminoproton resonances could be assigned neither to U residues in the proposed CU/UC base pairs nor to the G residue of the potential GAUA tetraloop. So, these experiments demonstrate that the apical loop of EMCV SL-A consists of an unstructured octaloop.In order to determine the possible formation of GAUA tetraloop and CU/UC base pairs, NMR experiments were performed on a short RNA hairpin comprising the upper 22 nucleotides of the mengovirus SL-A . Figure Previously, in vivo SELEX experiments were performed on the stem-loop D (SLD) of the poliovirus 5′ cloverleaf and showed a clear correlation between structure of the apical SL-D stem-loop and binding of the viral RdRp Viruses were grown and further characterized by sequence analysis of the 5′UTR and the protein 3CD encoding region. In total, 218 individual plaques were analyzed whereby 18 different variants, including the wild-type sequence, were found . Except To further characterize sequence and structural requirements of SL-A for mengovirus replication, additional mutations were introduced into the mengovirus cDNA , Table 2Additional mutants were constructed in which the length of the stem was changed. The length of the stem was extended by either replacement of loop residues, thereby creating Watson-Crick base pairs or by inserting extra base pairs into the stem. Mutation U41G  changed Until now, the length of the stem was modulated by the replacement of loop residues. To examine the effect of stem length without affecting the loop sequence, two additional mutants were made. The length of the SL1 stem was shortened with one base pair in the mutant ΔC40/ΔG49 . This muThe OriL RNA element is an important structural domain in the picornavirus genome. A stable RNA structure can be found at the 5′ proximal end of the genomic RNA but apart from kobuviruses, aphthoviruses, and enteroviruses, there is hardly any experimental data on the role of the OriL in the replication cycle of other picornaviruses. In this study, we used mengovirus, a strain of EMCV to study sequence and structural requirements of the apical loop of the 5′ stem-loop A of cardiovirus. We describe results of mutational analysis of the mengovirus oriL by in vivo SELEX and site directed mutagenesis.In previous studies, we and others showed that the binding of enterovirus 3CD to the oriL requires an apical tetraloop in stem-loop D folded in YNMG geometry The results obtained with the mutants in which nucleotides in the apical loop and the upper part of the stem were deleted, inserted or modified, support the hypothesis that EMCV replication requires a unstructured or loosely-structured apical SL-A loop in combination with a stem of defined length. This was shown by the lack of replication of the ΔU41C42/U47C48 mutant which contains a regular RNA stem with a GAUA tetraloop that is further stabilized by the closing C-G base pair Previously, Nateri and colleagues 5′-ACAAUUUG-3′ octaloop closed by a U-A base pair. Although the loop was unstructured and flexible, helical stacking of ACA bases at the 5′-end of the loop was observed. The flexibility of the loop and the upper part of the apoB mRNA stem-loop appeared to be essential for protein binding. A more defined structure of an octaloop was described by Lebars and colleagues II). The hairpin contains a 5′-GUUGAAAA-3′ octaloop that is closed by a Watson-Crick C-G base-pair. The loop folds independently of the stem and is stabilized by a sheared G-A base-pair. NMR spectroscopy revealed that the loop structure is well defined and have a stretch of stacked nucleotides formed by the GAAA bases, preceded by two U bases stabilized by stacking between the two G bases. A certain degree of structure formed by non-Watson-Crick base pairs and base-stacking can be recognized in the octaloops described above. Until now, no evidence for interactions between bases in the mengovirus SL-A loop was found. Despite the unstructured and flexible nature of the RNA octaloops in the NRA and apoB mRNA, these elements are involved in specific RNA-protein interactions.RNA stem-loop structures can function in a number of ways. Terminal loops and internal bulges can form sites interacting with proteins or other nucleic acid partners. Previously, a number of stem-loop structures containing octaloops were described The in vivo SELEX approach was previously used in a study to the structure-function relationship of the stem-loop D apical loop of the poliovirus OriL element, the binding-site for the viral polymerase A similar influence of length of RNA helical parts on virus replication was found for the enterovirus OriR element It is likely that the constitution of the apical stem of SL-A is a determinant for the formation of tertiary RNA structures, essential for virus replication. Multiple alignment of EMCV SL-A sequences demonstrates a high degree of conservation of both stem and loop of this RNA element. This seems a contradiction with the observed tolerance of random apical loop sequences. However, minor differences in replication efficiency between various sequences supporting wild-type like replication may finally result in the selection of the most optimal constitution found in nature.Nucleotide and amino acid sequences were aligned using ClustalW 2O, 10% D2O at 15°C and 800 MHz using a jump return pulse for water suppression The 22-mer RNA oligonucleotide was purchased from ABI GmbH . The final concentration of the RNA sample was 0.5 mM. The pH was adjusted to 6.8 (meter reading). NMR spectra were acquired on Varian Inova 600 MHz and 800 MHz spectrometers. For the assignment of exchangeable imino protons, two-dimensional NOESY spectra were recorded in 90% HMutations were introduced into an infectious mengovirus cDNA by PCR using synthetic oligonucleotides . An oligonucleotide containing 10 random residues in 5′ SL-A apical loop and the closing base pair sequence was used as forward primer. The infectious mengovirus cDNA construct pM16.1 The 5′ SL-A mutants were generated by PCR amplification using the infectious mengovirus cDNA clone pM16.1 ATCC. The cells were grown in minimal essential medium, MEM Glutamax (Invitrogen), supplemented with 10% fetal bovine serum (FBS), 100 U penicillin per ml and 100 mg streptomycin per ml (Sigma).Virus propagation and viral RNA transfections were performed with mouse fibroblasts (L929 cells) and HeLa cells. L929 cells and HeLa cells were obtained from in vitro transcription by T7 RNA polymerase as previously described 2 for eight hours. Then, the cultures were subjected to three cycles of freezing/thawing. Cellular debris was removed by centrifugation for 15 min at 2000× g. Cellular lysates were used for plaque assay.pM16 and RzpM-Luz cDNAs containing 5′ SL-A mutations were linearized by digestion with BamHI. PCR products comprising the full length mengovirus cDNA including a 5′ T7 RNA polymerase promoter and the randomized 5′ SL-A apical loop or linearized cDNAs served as templates for 2 and 1.3% Na-bicarbonate in 0.6% agar (1∶1 mixture with 2×M199). At 48 h postinfection, cells were either fixed and stained with crystal violet Six-well dishes with >90% confluent monolayer of L929 cells were incubated with 10-fold virus dilutions in MEM for 1 h and rocked every 15 min. After absorption, the cells were overlaid with 2.5 ml of M199 (Sigma) containing 3% FBS, 1% glutamine, 0.5% gentamicine, 0.5% penicillin, 0.5% Funguzone, 1% MgClViral RNA extraction was performed using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma). RNA was reverse transcribed using Super RT (HT Biotechnology) and random hexamer primers. cDNA was PCR-amplified using Phusion DNA polymerase and specific oligonucleotides comprising the genomic region between position 1 and 350. SL-A mutations were confirmed by sequence analysis.in vitro transcribed RzpM-Luz RNA, translation of the viral RNA gives rise to luciferase activity in the cytoplasm. Replication competent wild-type and mutant viral RNA will produce progeny plus-strand RNA and consequently display an enhanced luciferase activity. L929 cells, grown in 24-well plates to a confluence of 80%, were transfected as described above. At 2, 4, 6, and 8 hours post-transfection cells were lysed in 100 µl Passive lysis buffer (Promega). Luciferase activity was assayed using the Luciferase Assay System (Promega) and quantified using a Promega GloMax luminometer.Upon transfection of cells with


Highly hydrogen bonded base interactions play a major part in stabilizing the tertiary structures of complex RNA molecules, such as transfer-RNAs, ribozymes and ribosomal RNAs.We describe the graph theoretical identification and searching of highly hydrogen bonded base triples, where each base is involved in at least two hydrogen bonds with the other bases. Our approach correlates theoretically predicted base triples with literature-based compilations and other actual occurrences in crystal structures. The use of ‘fuzzy’ search tolerances has enabled us to discover a number of triple interaction types that have not been previously recorded nor predicted theoretically.Comparative analyses of different ribosomal RNA structures reveal several conserved base triple motifs in 50S rRNA structures, indicating an important role in structural stabilization and ultimately RNA function. Tetrahymena ribozyme , E. coli [PDB: 2awb] and D. radiodurans [PDB: 1nkw] to the H. marismortui [PDB: 1ffk] structure were carried out using least squares superposition on the phosphate backbone atoms by the program LSQKAB , T. thermophilus [PDB: 2j01] and D. radiodurans [PDB: 1nkw] 23S rRNA structures. The superposition for the formations in all four structures shows good structural conservation. To our knowledge, there have been no previous discussions or hypotheses with regard to the possible functions of this novel stacked UAU Hoogsteen, Watson Crick triples motif. This motif is situated at a junction which joins the three subdomains of domain V , is also involved in an A-minor interaction with A76 of P-site tRNA being lower than our cut-off point. The inter-domain interactions and helix stabilization functions carried out by base triples may be important, albeit collective factors, in determining the correct folding of the RNA molecule to enable its catalytic functions.Triples in the vicinity of other ribozyme active sites have previously been observed; an example being the base triple sandwich in the structure of the  ribozyme, where tet al.[H. marismortui take part in interactions close to the tunnel, including the ACC triple adjacent to the hypothesized peptidyl transferase center discussed in the previous section. Multiply hydrogen bonded base interactions may contribute significantly towards the conformational integrity of the tunnel.The polypeptide exit tunnel is the passage through which nascent proteins pass as they are synthesized. This tunnel begins immediately below the peptidyl transferase center, and is approximately 100Å in length. We founet al. as approSeveral types of triples appear to occur more than once within one structure, but are not presently seen in any other class of RNA structure. For example, the new ACC1 triple Figure  occurs a2+ ions) which play important roles in the stabilization of interbase interactions[2+) in crystal structures unless they are at least 2.0Å in resolution; very few RNA structures match this criterion. This problem has been discussed, for example, by Banatao et al (2003) [At present the methodology is restricted to base-base interactions. Although this has revealed new types of interactions, it is clear that base to backbone interactions are also of great importance. While ieractions. Here thl (2003) , who obshttp://mfrlab.org/grafss/nassam/.The results of our survey reveal that multiply hydrogen bonded base triples have a high degree of conservation between comparable ribosomal structures, therefore suggesting significant collective contributions towards the overall folding and stabilization of the RNA molecule. By annotating triple patterns and correlating theoretically predicted base triples from NAIL with the literature-based compilations in the NCIR database, we were able to discover a number of multiply hydrogen bonded base triple formations that had not been previously recorded and/or had not been predicted theoretically. The same annotation approach has enabled motif discovery by putting into context known patterns of interactions such as the stacked UAU Hoogsteen, Watson Crick motif. The value of expertly curated databases such as NCIR  and SCORThe authors declare that they have no competing interests.MFR built the search database, designed the research methodology, carried out the base triple searches, analysed the data and drafted the manuscript. AMH designed the alternative triple pattern approach and carried out the initial NASSAM optimizations. PW participated in the design and coordination of the work. PJA conceived the study, wrote the algorithm, participated in the design, coordination and drafting of the manuscript. All authors read and approved the final manuscript.Supplementary Figure S1 and Supplementary Tables S1-S3 in PDF format.Click here for file


One of the key issues in the theoretical prediction of RNA folding is the prediction of loop structure from the sequence. RNA loop free energies are dependent on the loop sequence content. However, most current models account only for the loop length-dependence. The previously developed “Vfold” model (a coarse-grained RNA folding model) provides an effective method to generate the complete ensemble of coarse-grained RNA loop and junction conformations. However, due to the lack of sequence-dependent scoring parameters, the method is unable to identify the native and near-native structures from the sequence. In this study, using a previously developed iterative method for extracting the knowledge-based potential parameters from the known structures, we derive a set of dinucleotide-based statistical potentials for RNA loops and junctions. A unique advantage of the approach is its ability to go beyond the the (known) native structures by accounting for the full free energy landscape, including all the nonnative folds. The benchmark tests indicate that for given loop/junction sequences, the statistical potentials enable successful predictions for the coarse-grained 3D structures from the complete conformational ensemble generated by the Vfold model. The predicted coarse-grained structures can provide useful initial folds for further detailed structural refinement. For example, Dima and co-workers The ability to predict RNA 3D structure is critical for understanding RNA functions. Recent developments in A key issue in the predictions of RNA stability is how to compute the loop free energy. For hairpins and RNA secondary structures in general, the nearest neighbor model, which assumes that the total free energy is an additive sum of the free energy of each elements , and other models The prediction of sequence-dependent loop free energy requires a model that goes beyond simple fitting with the experimentally measured empirical parameters. The formation of the intraloop base pairs and stacks would cause significant restriction of the loop conformational space and the loop entropy. It is practically impossible to exhaustively measure the loop free energy for all the different possible intraloop contacts for the different sequences and loop lengths. Therefore, evaluation of loop free energy with an ensemble of possible intraloop base pairing/stacking interactions cannot be achieved by experiment alone. We also need a computational model. The main purpose of the present study is to develop a statistical potential model that enables predictions of loop and junction three-dimensional structures from the sequence.In general, there are two classes of physics-based models for RNA structure prediction: molecular dynamics . Specifically, we aim to derive a set of dinucleotide statistical potentials Our RNA folding model is based on a virtual bond-based RNA conformational model  Xeon(R) CPU 5150 @ 2.66 GHz on Dell EM64T cluster system, the 3000-cycle iteration process took about 30 minutes and a 300-step iteration took less than 6 minutes.The convergence speed of the iteration depends on the selection of convergence criteria. For instance, if the convergence threshold parameter in Comparison between the two sets of the derived potential parameters shows that the “true” potentials tentials , suggestWe test the statistical potentials for the accuracy in loop/junction structure prediction for a large number of sequences. For a given sequence, we generate the full ensemble of the virtual bond loop/junction conformations using our Vfold model. Each conformation is then scored by the total statistical potential, which is evaluated as the sum of the statistical potentials . All the loops and junctions  have lengths from 3 nt to 8 nt. Within these 2227 RNA molecules, 1609 have structures determined by X-ray crystallography, which contain 7459 RNA loops and junctions (TEST-II), and 934 of these 1609 RNAs have high-resolution structures (TEST-III).The January 2012 version of PDB database contains 2227 structures that contain at least one strand of RNA sequence. These structures range from hairpin-loop structures to RNA-protein complexes or RNA-DNA hybrids. We found 8452 loops and junctions in the 2227 PDB structures  v.s. 6222 (success rate = 83%) for TEST-II, and 1070 (success rate = 95%) v.s. 1001 (success rate = 89%) for TEST-III, respectively. Our test results show that the numbers of the correct predictions with the statistical potentials TEST-I, 7089 v.s. 6949 for TEST-II and 1102 v.s. 1065 for TEST-III  and TEST-II (the 7459 x-ray structures in TEST-I) databases, we randomly selected 3229 RNA loops/junctions from the database TEST-I and categorize them according to the types of RNA loops and junctions. The 3229 RNA loops and junctions contain 1406 hairpin loops, 255 internal/bulge loops, 184 pseudoknot loops, 1265 multibranched loops and 119 junctions. The correct (top-1) predictions with For the The March 17, 2012 version of the Leontis dataset contains 642 RNA sequences with structures of resolution better than 4.0 Å. The 3DNA software identified a total of 435 RNA loops and junctions with lengths ranging from 3 nt to 8 nt, excluding the 5′/3′-terminal dangling regions. Our calculations show that the difference between the diamond lattice-represented structures and the PDB structures varies for the different loop lengths and sequence contents with RMSD from 0.74 Å to 3.93 Å , and theTEST-I, TEST-II and TEST-III, constructed from the January 2012 version of PDB database. Our test results  for TEST-II, and 96% (1077 out of 1119) for TEST-III, respectively. results  show a sTEST-I, TEST-II and TEST-III, are shown in Moreover, the predictions of the top 20 lowest-potential conformations for the loops and junctions in all the three test datasets, datasets  show thaMotivated by the biological significance to predict sequence-dependent loop and junction structures, we have developed a knowledge-based scoring functions/potentials to predict the structures of RNA loops and junctions. We use a coarse-grained conformational model  to sample RNA loop/junction conformations. From the known RNA structures, we extract a set of sequence-dependent dinucleotide-based statistical potentials using two methods. In the first method, the statistical potentials  structure as the predicted native structure. Extensive tests indicate that the statistical potentials can successfully predict the native structure for a large class of loop and junction sequences and that  dataset .The present approach has several advantages. First, the extraction of the statistical potential We have performed extensive benchmark tests using the different databases. However, a direct comparison between our model other methods is not very straightforward. This is because our model is a coarse-grained model while other models mainly focus on the all-atom structures. Furthermore, our model aims to fold a low-resolution structure from the sequence without using any input information such as homologous templates, while other models mainly focus on the prediction of the native structures from near-native folds. Future development of our method, which may give all-atom structures from the low-resolution folds, would make direct comparison between our model and other models possible.Applications of the present statistical potentials to the Vfold structure prediction model We use the Vfold model to generate the full conformation ensemble of a given RNA loop or junction sequence. The Vfold model is a virtual bond-based RNA folding model ic state  and the d gauche  [55], , aThe quasi-chemical approximation-based statistical potentials are the “extracted” energy-like parameters derived from the observed density in the database of native structures To extract the “true” energy . The conformation ensemble is used in the iterative calculation.Calculate the observed dinucleotide frequencies Choose a set of initially guessed potentials. In this work, we start with the “extracted” quasi-chemical approximation-based potentials  We use the RNA 2009 database  RNA loops and junctions are constructed by all the unpaired nucleotides in RNA structure. We detect the loops and junctions by removing the nucleotides involved in the base pairs. Furthermore, we remove all the loop structures with modified bases or non-RNA atoms and molecules. Advances in the knowledge of the structures of both the canonical (Watson-Crick and wobbles) base pairs and non-canonical base pairs  In addition, the time consumption to enumerate the loop conformations on a diamond lattice increases exponentially with the length of the loop Larger loops often involve tertiary interactions with other subunits of RNA structures, which are not accounted for in this study. As a result, we found 152 loops/junctions with the length ranging from 3 nt to 8 nt in the RNA09 dataset. These loop and junction structures are used as the training dataset in our iterative approach.http://rna.bgsu.edu/nrlist/).To further test the influence of the use of the different training dataset on the statistical potentials and the predictive power of the statistical potentials, we also use another non-redundant high-resolution RNA structure dataset, collected by Leontis' lab  structure as the “coarse-grained correct structure”; see Step 1 in


Mechanistically, the phenotype of butenolide-treated Danio rerio (zebrafish) embryos was similar to the phenotype of the pro-caspase-3 over-expression mutant with pericardial edema, small eyes, small brains, and increased numbers of apoptotic cells in the bodies of zebrafish embryos. Butenolide also induced apoptosis in HeLa cells, with the activation of c-Jun N-terminal kinases (JNK), Bcl-2 family proteins, and caspases and proteasomes/lysosomes involved in this process. This is the first detailed toxicity and toxicology study on this antifouling compound.Butenolide [5-octylfuran-2(5H)-one] is a recently discovered and very promising anti-marine-fouling compound. In this study, the acute toxicity of butenolide was assessed in several non-target organisms, including micro algae, crustaceans, and fish. Results were compared with previously reported results on the effective concentrations used on fouling (target) organisms. According to OECD's guideline, the predicted no effect concentration (PNEC) was 0.168 µg l Its embryogenesis is also very well characterized and a database of developmental defects (www.zfin.org) in zebrafish (Danio rerio) is available. Developmental defects in zebrafish caused by small molecules can be linked to a specific genetic pathway known to cause the same defect Biofouling is one of the most serious problems in the maritime industry and aquaculture development. In the marine environment, submerged surfaces are often colonized by marine organisms that have come to be called biofoulers, which are marine organisms that attach to submerged surfaces. Biofoulers increase the weight, drag and surface corrosion of ships, and lead to huge costs to maintain mariculture systems and seawater pipelines The antifouling compound 5-octylfuran-2(5H)-one (here referred to as butenolide) was synthesized by Shanghai Medicilon Inc  The chemical structure for this compound is shown in −1) were added to each well. After incubation at 37°C for 4 h, 100 µl of dimethyl sulfoxide (DMSO) were added to each well and incubated for 20 min. The absorbance of each well was then measured at 570 nm by a Thermo scientific Multiskan® FC multiplate photometer . The inhibitory effect or cytotoxicity of butenolide was then calculated based on the following equation:−1) were added to each well and incubated for 4 h. They were centrifuged for 5 min at 1000 rpm and 100 µl of DMSO was added to the pellet and incubated for 20 min to solubilize the dark-blue formazan. Absorbance of the solution was measured at 570 nm and the cytotoxicity was calculated as described above.Skeletonema costatum, in the exponential growth stage were diluted 20 fold in an f/2 culture medium (Cell cultures of e medium . The cel50 and assessment factor (AF) The predicted no effect concentration (PNEC) was calculated according to the equation below using the lowest no effect concentration (NEC) or L(E)C−1 butenolide from the two-cell stage or the control embryos were fixed at 48 hours post fertilization (hpf) in 10% formalin in PBS overnight at 4°C. The samples were dehydrated in ethanol and infiltrated in paraffin resin  following the manufacturer's instructions. The specimens were then sectioned at 7 µm using a Leica Reichert-Jung 820-II Histocut Microtome . Histological hematoxylin-eosin (HE) staining of the sections was subsequently carried out using standard protocols Zebrafish embryos treated with 1.25 µg ml−1 butenolide from the two-cell stage or the control embryos were fixed at 24 hpf in 4% paraformaldehyde in PBS for 45 min at room temperature. Whole-mount TUNEL staining of zebrafish embryos was performed using a fluorescein in situ cell death detection kit  according to the manufacturer's protocol. Images were taken under a Nikon MULTIZOOM AZ100 fluorescent microscope with a GFP-B filter set  and a SPOT® FLEX color camera as described above.Zebrafish embryos treated with 1.25 µg ml−1) were separated on 10% SDS-PAGE and transferred onto a Hybond ECL nitrocellulose membrane . After blocking, the membranes were incubated for 3 h at room temperature or overnight at 4°C with antibodies at a dilution of 1∶1000. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of 1∶5000 for 1 h and developed using Immobilon™ Western detection reagents .Information on the antibodies used in the experiments is summarized in HeLa cells were cultured in 60 mm Petri-dishes. Cells at different time points after butenolide treatment were collected and lysed in NP-40 lysis buffer  in the presence of protease inhibitors. Whole cell lysates (60 µg lane−1 butenolide. The inhibitors tested were 10 µM ALLN (N-Acetyl-Leu-Leu-Nle-CHO), 10 µM MG132 , 50 µM Z-VAD-FMK , and 50 µM SP600125 . The cytotoxicity was measured at 4 h by the MTT assay as described above. To investigate the effects of over-expression of Bcl-2 family proteins on butenolide-induced apoptosis, HeLa cells seeded in 35 mm Petri dishes were transfected with 1 µg of empty vector, Bcl-2, Mcl-1 or Bcl-XL constructs by lipofectamine 2000  and incubated in an MEM medium containing 5% FBS at 37°C for 24 h before the application of 100 µg ml−1 of butenolide. The Bcl-2, Mcl-1 and Bcl-XL constructs tagged with green fluorescent protein were generously provided by Professor Donald C. Chang of HKUST. The cell toxicity was measured after 3 h of butenolide treatment by counting dead cells and total cell numbers under a fluorescent microscope.To test the inhibitors of the apoptosis regulators, HeLa cells were seeded into a 24-well culture plate. Inhibitors were added 1 h before the application of 100 µg ml50 was 0.0168 µg ml−1 (Hydroides elegans larvae) Skeletonema costatum; crustaceans: Balanus amphitrite larvae, Melita longidactyla, Tigriopus japonicus and Daphnia magna; and fish: Lutjanus erythropterus and Danio rerio embryos) as well as two additional marine taxonomic groups (Bugula neritina larvae and Hydroides elegans larvae) http://ecb.jrc.ec.europa.eu/documents/TECHNICAL_GUIDANCE_DOCUMENT/EDITION_2/tgdpart2_2ed.pdf), the AF was set at 1000 in the calculation, resulting in a PNEC of 0.0168 µg l-1. According to guidelines for the testing of chemicals from the Organization for Economic Co-operation and Development (OECD) (http://www.oecd.org/dataoecd/6/14/2483645.pdf), theassessment factor shall be 100 and thus, the PNEC would be 0.168 µg l-1. The positive controls for these toxicity tests are provided in The results of acute toxicity tests of butenolide in several marine and freshwater non-target organisms belonging to different taxonomic groups are summarized in Zebrafish embryos developed pericardial edema (edema around the heart), poor blood circulation, small brains and small eyes within 55 hours of treatment with butenolide (from the 2-cell stage). The severity of these symptoms increased as the concentration of butenolide increased . For exaThe concentration of butenolide that led to toxicity in HeLa, Ptk2, Sf9, HL-60 and K562 cells was similar, whereas the primary neuron cells were more sensitive to butenolide treatment than the other cell types . BecauseAs revealed by the Western blot analysis , the casIn the early stages (within 2 h) of treatment with butenolide, MAPKs family members JNK  and ERK  were activated (phosphorylated), whereas p38 was activated later (after PARP cleavage) . The JNKThe expression level of the Bcl-2 family protein Bax did not change with butenolide treatment . HoweverThe pan-caspase inhibitor z-VAD-fmk To assess the toxicity of a new antifouling compound, we calculated the predicted no effect concentration (PNEC) according to two standards with different AFs. When calculated and compared using the OECD's standard, the PNEC of butenolide is among the highest in representative alternative new biocides H. elegans, B. neritina, and B. amphitrite were found to be among the most sensitive species to butenolide. Species-selectivity ratios/indices were used to assess the species-selectivity of herbicides Regarding the selection of test species, we investigated more organisms than the minimum requirements, which requires three species from three trophic levels, and preferably two additional marine taxonomic groups for PNEC calculations biocides , suggest50/EC50) were based on single species. When used on fouling organisms, the pharmaceutical ratio assessed the specificity of the compound's effect, with a higher value suggesting higher specificity toward the fouling process and lower off-target side effects. The pharmaceutical ratio of butenolide was quite high in target organisms  D. rerio (3.67). The exact mechanism for this huge difference is unclear, but one possibility is that butenolide has a very specific effect on the fouling process in fouling organisms but much less specific effects on non-target organisms. The variation in the L(E)C50 of butenolide in different non-target species is within an order of magnitude, which is quite low among new antifouling biocides 50 varies by at least an order of magnitude between target organisms; and LC50 of non-target crustaceans was much lower than that of the crustacean B. Amphitrite. The pharmacology of butenolide in different species should be investigated to clarify the reasons for these inter-species differences.The species-selectivity ratio of butenolide is much lower than its pharmaceutical ratios. The pharmaceutical ratios .During zebrafish organogenesis, the heart is the first organ formed in the developing embryo, and the development of other organs depends on blood circulation in the embryo. Pericardial edema was the first symptom observed after treatment with butenolide, and blood circulation in the embryos was in turn affected. It is possible that the developmental defects/retardation in the brains and eyes were caused by insufficient blood circulation. We therefore focused only on pericardial edema and looked for similar phenotypes. Using the key words “pericardial edema” to search the zebrafish embryo developmental defect database, we found that the symptoms caused by butenolide were similar to the phenotype found in pro-caspase-3 over-expression mutants However, there were some differences between the pro-caspase-3 over-expression mutants and the butenolide-treated embryos. In the pro-caspase-3 mutants, some retina cells were lost in the eyes, especially in the photoreceptor cell layer, and the number of apoptotic cells in the retina increased 50s in the tested cell lines were higher than in the non-target organisms. Although the reason for this discrepancy is unclear, it is important to understand that the direct effect on cells differs from the most prominent effects on the whole organism, and that the host species, cell type and culture method could all influence the sensitivity of the cell to butenolide. Among several cell types, HeLa cells were chosen for molecular toxicology study, because they are very consistent, robust, and easy to manipulate, and butenolide's effective concentration in this cell line was similar to that in most of the other tested cell lines, including the insect cell line Sf9. The concentration of butenolide for the molecular mechanism study was 100 µg ml−1. This concentration was just high enough to cause sufficient cytotoxicity , which was verified by FACS analysis (data not shown); 10 µM purvalanol A (a CDK1 inhibitor) did not inhibit butenolide-induced apoptosis (data not shown), suggesting that butenolide may have less effect on the cell cycle.Besides apoptosis, we also tested the effect of butenolide on several other cell signaling pathways. Butenolide treatment did not change the proportion of interphase cells -2H-furan-5-one], which is structurally similar to the butenolide studied here, exhibited novel anti-parasitic activities specifically against Trypanosoma brucei bruceiIn the cyprid larvae of the marine fouling organism −1 according to the European Chemicals Bureau, and 0.16 µg l−1 according to the Organization for Economic Co-operation and Development (OECD). As of now, the fouling species Hydroides elegans was found to be the most sensitive species to butenolide treatment and it could be chosen for chronic toxicity tests. Mechanistically, butenolide induced apoptosis in both zebrafish embryos and HeLa cells. JNK activation, Bcl-2 family members, caspases and proteasome/lysosome activation were involved in butenolide-induced apoptosis in HeLa cell lines. The results reported here increased our understanding on butenolide's toxicity and toxicology. For risk assessment, the degradation, bioaccumulation, and the predicted environment concentration (PEC) of butenolide should be further measured and calculated In summary, the antifouling compound butenolide's PNEC was 0.016 µg lTable S1Positive controls for toxicity tests. All concentration units are µg ml−1.(DOC)Click here for additional data file.Table S2The effect of butenolide on zebrafish hatching.(DOC)Click here for additional data file.Table S3The toxicity of butenolide in several cell lines. All concentration units are µg ml−1.(DOC)Click here for additional data file.


The transfection efficiency of exogenous RNA mediated by SDS-CTAB increases if vesicles assemble in the presence of the reporter RNA; finally, freezing abrogates the transfection ability. The results of our experimental strategy suggest that catanionic vesicles may be adopted in gene therapy and control of antiproliferative diseases.Catanionic vesicles are supramolecular aggregates spontaneously forming in water by electrostatic attraction between two surfactants mixed in nonstoichiometric ratios. The outer surface charges allow adsorption to the biomembrane by electrostatic interactions. The lipoplex thus obtained penetrates the cell by endocytosis or membrane fusion. We examined the possible cytotoxic effects and evaluated the transfection efficiency of one vesicle type as compared to known commercial carriers. We show that the individual components of two different vesicles types, CTAB (cetyltrimethylammonium bromide) and DDAB (didodecyldimethylammonium bromide) are detrimental for cell survival. We also assayed the cytotoxicity of SDS-DDAB vesicles and showed dose and time dependency, with the DDAB component being  P, originally introduced by Israelachvili and coworkers [V) and the area of the polar head (a) multiplied by the length of the chain (L) being part of the same surfactant (P = V/aL). The modulus of P is suggestive of the type of structure/shape that surfactants tend to assume upon aggregation. The formation of vesicles is, therefore, possible when the packing parameter reaches an optimal value thus leading to the formation of a double layer [Catanionic vesicles are supramolecular aggregates formed by mixing in non-stoichiometric ratios cationic and anionic surfactant species . Surfactoworkers . This pale layer . Previous work from our laboratory, where the phase diagram was presented, indicated that to form quasispherical vesicles, it is necessary to mix the surfactants at nonequimolar concentrations since at stoichiometric ratio the two components precipitate . Concerning the potential use of these complexes in biotechnological and biomedical applications, it should be pointed out that the interaction of vesicles with other molecules, such as DNA, RNA, or other biopolymers, results in the formation of the above mentioned lipoplex. This represents a potential tool to deliver genetic material across the cell membrane via plasma-membrane fusion and/or endocytosis. As a matter of fact, previous work from our laboratory showed that it is possible to form complexes between DNA and vesicles with an excess positive charge at the surface, which could be potentially delivered within the cell , 6. HoweIn the present work, we report on the cytotoxic action of both the individual surfactants and vesicles formed by the same ones, on HEK-293 cultured cells. In addition, we quantitatively evaluated the transfection, mediated by SDS-CTAB vesicles, of an exogenous RNA and measured the level of translation of the reporter protein. Strictly speaking, in fact, one should talk in terms of messenger RNA translation rather than gene expression. The data discussed here clearly indicate that after transfection, the nucleic acid is translated into protein with the correct structure and at abundant level. The novelty of the data discussed here consists of the fact that naked RNA, a very vulnerable biomacromolecule, is protected by the interaction with vesicles. Finally, these data add further evidence that vesicles may find a use in biotechnology and gene therapy.2O. Purification was made by dissolving the individual species in hot ethyl alcohol, under stirring with subsequent precipitation and filtering the surfactant solution by cold acetone. The salts were recovered and vacuum-dried at 70°C. Their purity was determined by conductivity methods in water at 25.0°C. Mixtures of the single species were prepared in conductivity water  and heated above the respective Krafft points of each surfactant [Vesicles were prepared and characterized as previously published and extensively discussed , 6, 9. Brfactant . Thereafin vivo were done in a Human Embryonal Kidney cell line (HEK-293). In general, 1 × 105 cells per well were seeded on 12 well polylysine-treated plates without antibiotics. Cells were allowed to reach at least 80% confluence prior to transfection. We used Plasmid pOT.CAT-A98 to generate CAT-A98 mRNAs. The RNAs used for transfection were transcribed in vitro using a commercial kit (RiboMax) and were phenol-chloroform-extracted (5 : 1 v/v at pH 4.7), ethanol-precipitated, and further purified on Sephadex G-50 or G-25 columns ; CAT-A98 mRNAs were 5′-capped and polyadenylated. As control, CAT-A98 mRNAs were transfected using Lipofectamine 2000 (Invitrogen) in Opti-MEM culture medium (Invitrogen). Briefly, transfections were performed with RNA and Lipofectamine at a ratio of 1 : 2.5 as previously published [Cells were maintained according to standard cell culture protocols. Transfection experiments ublished . CAT wasublished .Cell viability was evaluated by the colorimetric Mosmann assay  on both P < 0.05.All experiments and measurements were independently replicated 3 times. Statistical analysis was done by one-way ANOVA test  followedThe vesicular mixtures were prepared as previously reported , 5, 6, 9per se exhibited a dramatically higher toxicity than CTAB (not shown). In this work, we compared the cytotoxic action of the individual surfactants SDS, CTAB, and DDAB on HEK-293 cells. Treatment time was four hours at increasing vesicle concentrations. The four-hour treatment time was chosen since this is required in the subsequent transfection experiments. In any case, it is worth noting that the CTAB component is more toxic than SDS . MoreoveIn a second series of experiments, we have compared the toxic effects of two different vesicles, SDS-CTAB and SDS-DDAB, after four hours of exposure and in the same range of concentrations used in the previous experiments. As shown in Chloramphenicol-Acetyltransferase (CAT) is a bacterial enzyme whose messenger RNA (mRNA) can be translated into active protein in eukaryotic cells. We transfected the CAT mRNA into HEK-293 cells using SDS-CTAB vesicles. The rationale of these experiments is that CAT is not normally present in higher cells; therefore, the detection of this enzyme is the diagnostic sign that the CAT mRNA has been successfully transferred across the plasma membrane by the vesicles and, subsequently, translated into protein within the cytoplasm matrix. We quantified the intracellular concentration of enzyme by the immunoenzymatic assay ELISA; finally, the level of CAT was normalized to the total protein content of the cells. μM vesicles) with respect to Lipofectamine used as control . This strongly suggests that actually the RNA molecule is internalized and protected within the vesicle. Subsequently to transfection, the CAT mRNA is released in the cytoplasm where it can be translated into protein. In the last set of experiments discussed here, we probed the role of the storage temperature; as a matter of fact, previous evidence from our laboratory [We compared the efficiency of RNA intracellular delivery of our vesicles with Lipofectamine, a commercially available liposome transfection system. Transfection of naked CAT mRNA represented the negative control and, evidently, is almost totally hydrolyzed by the RNases normally present in the cytoplasm. As matter of fact, messenger RNA exists in a quasilinear molecular configuration which is easily hydrolyzed by the resident RNases. In the first experiments, we added the CAT mRNA to preformed vesicle. One can expect that in this case, the RNA is anchored via electrostatic interactions to the surface of the vesicles. The results of boratory , 8 suggeboratory . This isIn conclusion, the data discussed in this work evidence that it is possible to define an experimental situation where the cell mortality caused by the exposure to the vesicles is minimal. This is a crucial aspect to maximize the transfection efficiency of catanionic vesicles. The formation of vesicles in the presence of the molecule to be delivered inside the cell is of crucial importance for its internalization within the inner aqueous medium. Furthermore, the vesicles are stable at room temperature and have a relatively long shelf life. They must be stored at room temperature and should not be frozen since this would disrupt their supramolecular structure thus abolishing their capacity of molecular delivery. Catanionic vesicles efficiently deliver genetic material across the cell membrane. Transfection efficiency increases if vesicles are formed in the presence of RNA. Treatment with RNase suggests the internal localization of RNA the nucleic acid.