S)-3′-((2-amino-3-(4-methoxyphenyl)-1-oxopropyl)amino)-3′-desoxy-N,N-dimethyladenosine), which possesses an amide instead of an ester junction, represents such a stable conjugate.5RNA–peptide conjugates that mimic acylated tRNA termini are valuable compounds for structural and functional studies of the ribosomal elongation cycle, particularly if they contain a hydrolysis-resistant linkage between the RNA and the peptide moiety.The total syntheses of 3′-aminoacyl- and 3′-peptidyl-RNA represent substantive challenges for organic chemists;2O3, a metal-free version for cysteine thiol reduction was reported by Danishefsky and co-workers.13] both promote valine—a rather abundant amino acid (6.6 % as opposed to 1.7 % frequency for cysteine)—as a practicable ligation site.Within this framework, we considered here an important extension of the NCL-based strategy towards 3′-peptidyl-tRNA mimics, specifically their desulfurization . As NCL Involving RNA–peptide conjugates in metal- or radical-based desulfurization appears difficult at first sight, as RNA is rather fragile under the required conditions. Many hurdles are to be expected, such as irreversible adsorption of the nucleic acid on the metal surface or unspecific phosphodiester cleavage catalyzed by the metal ions involved.17N-(2-aminoethyl)carbamoyl)benzylthioesters; the latter are referred to as ABT thioesters (m cysteinyl-RNA and 8 mm peptide thioester in Tris buffer (1 m) at pH 8.0, in the presence of urea (7 m) and tris(carboxyethyl)phosphine  for S-tBu removal in situ,v/v)) for formation of more reactive thioesters.To begin our endeavors, we synthesized a series of 3′-cysteinylamino-3′-deoxy-RNAs and short peptide 4-phosphine (TCEP) in combination with a water-soluble radical initiator dihydrochloride, V-50), comparable to the protocol for cysteine reduction in peptides by Wan and Danishefsky.1 to 7). The major byproduct was isolated, characterized by mass spectrometry, and assigned as desulfurized starting material, namely, 3′-alanylamino-3′-deoxy-RNA. This side product is most likely generated from unreacted 3′-cysteinyl-RNA; however, it might also form earlier during side chain deprotection of Cys(S-tBu)S-tBu deprotection  were at much higher cost with respect to overall yields of the isolated target conjugates.1 to 7) with Xaa-Ala (Xaa=any amino acid) ligation sites, synthesized by the approach presented here. To define the limits, we also examined the principal accessibility of Xaa-Val sites in 3′-peptidyl-tRNA mimics. We decided in favor of β,β-dimethylcysteine 3UGAU3CGAUCAC3AC2A-3′-NH-VGFFM-NH28 in low but acceptable yield, leaving room for further optimization; unreacted and desulfurized starting material were the major byproducts. Another issue we wanted to test was the compatibility of the elaborated desulfurization conditions with DNA, which might be more sensitive than RNA towards free radicals and could therefore be harmed.9 and were pleased to observe good yields. We confirmed this extension to DNA for a second 3′-peptidyl-DNA conjugate (10) with an internal alanine ligation site.Many of the conjugates synthesized here represent tRNA acceptor stem–loop structures carrying an arginine-containing short peptide. Such conjugates have resisted direct access by solid-phase synthesis in which both peptide and RNA are assembled on the same functionalized solid support, followed by cleavage and deprotection of the whole conjugate.We note that we focused on free-radical-mediated methods for desulfurization from the beginning of our investigation as an early report by Hecht and co-workers on the desulfurization of thio-modified nucleosides in tRNA indicated severe limitations for metal-based approaches.In summary, we have demonstrated a convenient synthetic strategy towards hydrolysis-resistant 3′-peptidyl-tRNA mimics. By relying on NCL and subsequent desulfurization, sequences that are inaccessible by other methods  become possible for this important class of RNA conjugates.Desulfurization procedure: After native chemical ligation of a 3′-peptidyl-RNA conjugate, which was carried out as described previously,m ammonium citrate solution was added to the upper reservoir, followed by centrifugation for another 20 min. After a final washing step with H2O (500 μL), the upper solution containing the crude NCL product was lyophilized to dryness and subsequently dissolved in freshly prepared, degassed desulfurization reagent stock solutions. Final concentrations were: 0.6 mm conjugate, 240 mm sodium phosphate (pH 7.5), 200 mm TCEP, 16 mm V-50, and 4 mm glutathione. The reaction mixture was sonicated for 10 s and incubated under argon atmosphere for 6 to 12 h at 37 °C. Analysis of the reaction was performed by direct injection of an aliquot onto an anion-exchange chromatographic column (Dionex DNA-Pac PA100). Purification was also performed via anion-exchange chromatography, followed by desalting on a C18 SepPakPlus cartridge (Waters) and lyophilization. All conjugates were analyzed by LC–ESI mass spectrometry to confirm their expected molecular weights (see Supporting the Information for details).


Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5PolDV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5PolDV contains a specific priming site for adenosine 5′-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn2+ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5PolDV ensures the conservation of the 5′-adenosine by strongly discriminating against viral templates containing an erroneous 3′-end nucleotide in the presence of Mg2+. In the presence of Mn2+, NS5PolDV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.The dengue virus (DV) is an important human pathogen from the  Flavivirus genus of positive-strand RNA viruses contains important human pathogens such as yellow fever virus, West Nile virus, Japanese encephalitis virus and dengue virus (DV). The Flavivirus genome ends are strictly conserved as 5′-AG…CU-3′. We demonstrate here the primary role of the DV polymerase in the conservation of the first and last genomic residue. We show that DV polymerase contains an ATP-specific priming site, which imposes a strong preference for the de novo synthesis of a dinucleotide primer starting with an ATP. Furthermore, the polymerase is able to indirectly correct erroneous sequences by producing the correct primer in the absence of template and on templates containing incorrect nucleotides at the 3′-end. The correct primer is productively elongated on either correct or incorrect templates. Our findings provide a direct demonstration of the implication of a viral RNA polymerase in the conservation and repair of genome ends. Other polymerases from other RNA virus families are likely to employ similar mechanisms.The 5′- and 3′-ends of RNA virus genomes have evolved towards efficient replication, translation, and escape from defense mechanisms of the host cell. Little is known about how RNA viruses conserve or restore the correct ends of their genomes. The  The 5′ genome end may carry a cap structure to ensure both genome stability and efficient translation Flavivirus genus within the +RNA virus family of Flaviviridae together with viruses of the genera Hepacivirus and PestivirusFlavivirus genus comprises around 50 virus species Flavivirus NS5 RdRp domains have been determined for DV E.coli DdDp I), RNA-dependent DNA polymerase (prototype HIV reverse transcriptase) and DNA-dependent RNA polymerases , their apo-structure is usually likened to a right hand comprising fingers, palm and thumb subdomains. Viral RdRps contain an encircled active site having connecting elements between the fingers and thumb subdomains. Active sites of viral RdRps performing de novo RNA synthesis are additionally closed in their initiation conformation due to the existence of structural elements allowing the stable positioning of the first NTP into a priming site Flaviviridae RdRps studied so far initiate RNA synthesis de novo. Accordingly, Flavivirus RdRp domain structures contain a “priming loop” in the thumb subdomain closing the catalytic site Flavivirus RdRps. They might play the role of an initiation platform to which the base of the priming NTP stacks as it was shown for bacteriophage φ6 de novo initiation complexes of DV and WNV RdRps favor Trp795 in the role of the initiation platform DV belongs to the Flaviviridae lack a poly(A) tail at the 3′-end. A remarkable trait of Flavivirus genomes is the strict conservation of the 5′- and 3′-end dinucleotides as 5′ AG…CU 3′. The molecular basis for this strict conservation of the 5′- and 3′-end dinucleotides and/or the use of the same starting nucleotide for +RNA and -RNA strand synthesis by the viral polymerases is not known. Its Hepacivirus and Pestivirus counterparts have to display higher nucleotide tolerance. They are able to initiate with (A/G)C and G(G/U), respectively, since the 5′- and 3′-ends of Hepacivirus genomes of different genotypes correspond to 5′ (A/G)C…GU 3′ and the genomes of pestiviruses to 5′ GU…CC 3′. Interestingly, genomes and antigenomes of non-segmented -RNA (ns-RNA) paramyxoviruses, whose RdRps perform de novo RNA synthesis, start with a conserved 5′-AC Genomes of Flavivirus genome ends is entirely polymerase-encoded. We demonstrate ATP-specific de novo initiation using the RdRp domain of DV protein NS5 (NS5PolDV) and specific 10-mer oligonucleotidic RNA templates corresponding to the 3′-end of genomic +RNA and -RNA. We document the existence of a built-in ATP-specific priming site of NS5PolDV. This specific site is one of the means by which NS5PolDV ensures that the DV genome and antigenome start with an A, the others being several correction mechanisms including the generation of non-templated pppAG primers as well as the preferential formation and elongation of pppAG even on templates with non-cognate 3′-ends. Finally, we show that the ATP-specific priming site is part of the putative priming loop coming from the thumb subdomain. There, residue H798, and not W795, is essential for de novo initiation and may act as a priming platform stabilizing the ATP priming nucleotide. DV RdRp is actively involved in the conservation of the correct ends of the genome proving thus a direct example of how RNA viruses maintain the integrity of their genomes. The mechanisms described here may more broadly apply to other RNA viruses having viral RdRps able to initiate RNA synthesis de novo.Here we show that the strict sequence conservation of DV) using small specific templates corresponding to the 3′-ends of the genome (+RNA) and the antigenome (-RNA). Templates are comprised of 10 nucleotides and are predicted to be devoid of stable secondary structure  and on DV103′- corresponding to that of the antigenome 5′-ACUAACAACU-3′ (right). We used either [α-32P]-GTP  or [γ-32P]-ATP  as the radioactive nucleotide. For the catalytic ion, either Mg2+ (panel A) or Mg2+ supplemented with Mn2+ (panel B) were used at their optimum concentrations 5 mM for Mg2+ and 2 mM for Mn2+de novo RNA synthesis. When DV103′+ is used as a template, pppAG is formed as well as pppAGA and pppAGAA. When all NTPs are used, pppAG accumulates with time as does pppAGA in the case of DV103′+ and pppAGU in the case of DV103′-. After the synthesis of trinucleotides NSPolDV adopts a processive RNA synthesis elongation mode to continue synthesis up to full-length products  as radiolabeled nucleotide in order to visualize exclusively de novo RNA synthesis products starting with ATP. The pattern observed with Mg2+ is reproduced when Mn2+ is present  is at the origin of the conservation of the first nucleotide, we tested a set of DV103′- variants with different 3′-ends. In addition to the correct DV103′- CU, we used DV103′- CC, DV103′- CA and DV103′- CG in the presence of the corresponding priming NTP and GTP. The expected primer products are pppAG, pppGG, pppUG and pppCG, respectively. 2+ as the catalytic ion. Remarkably, the CU template only is proficient for product synthesis (pppAG). RNA primer synthesis on other templates is almost undetectable. We conclude that in the presence of Mg2+ as a catalytic ion the DV RdRp priming-site accommodates exclusively ATP.The first nucleotide of 2+ was used instead of Mg2+, the pppAG primer was generated even in the absence of the template, albeit to a lower extent , the latter by initiation internal to the template. No product is formed in the absence of the template. Finally, DV103′-CG allows formation of pppCG which is not formed in the absence of the template. In conclusion, NS5PolDV keeps the strict preference for an ATP as the priming nucleotide in the presence of Mn2+ when no template is present. Nevertheless, the use of templates with an altered 3′-nucleotide can force NS5PolDV to start the de novo RNA synthesis with the corresponding base-paired priming nucleotide, and also allows internal initiation.To our surprise, when Mnr extent . This isDV has a marked specificity for ATP. This preference is strict in the presence of Mg2+. It is equally strict for dinucleotide synthesis in the presence of Mn2+ and in the absence of template. The specificity for ATP as the starting nucleotide is lost when Mn2+ is used in the presence of templates with incorrect 3′-ends; only then NS5PolDV is able to form pppNG products as efficiently as pppAG.Collectively, these observations confirm that the priming site of NS5Pol2+ and/or Mn2+ the built-in ATP-specific priming site drives NS5PolDV-mediated RNA synthesis starting with pppA. The dinucleotide pppAG is accumulated during RNA synthesis on templates with the correct 3′-end . Note that all templates are present at the same concentration, which should not correspond to the situation in vivo. We conclude that in the presence of incorrect templates and Mg2+, NS5PolDV discriminates against these templates and forms pppAG only on the correct template  by replacing residues T794-A799 between T793 and K800 by two glycines (see close-up in m) for both proteins (wild type (wt) NS5PolDV Tm 49.0°C ± 0.5°C, NS5PolDV TGGK Tm 48.4°C ± 0.05°C).NS5PolNS5PolDV  allowed de novo initiation over the 3′-end of a single-stranded RNA template but may favor the accommodation of double-stranded RNA. Its RNA synthesis initiation and elongation activity was first tested using a “minigenomic” RNA template consisting of 224 nucleotides of the 5′-end of the DV genome fused to 492 nucleotides of the 3′-end de novo RNA synthesis product is generated corresponding to the size of the template. Secondly, an elongation product is generated by back-primed RNA synthesis. There, the 3′-end (…AACAGGUUCU-3′) forms a short hairpin annealing the last di-nucleotide to nucleotides -6 and -7 (underlined in the sequence) and is then elongated 2+ or Mn2+ as catalytic ions. The left and right panels of DV TGGK produces a range of elongated products of different lengths. This might be due to the accommodation of long hairpins, which then create longer products than the template but shorter than the elongation product of wt NS5PolDV.The TGGK mutant is expected to have an open active site, which impedes correct ATP-specific De novo RNA synthesis initiation by wt NS5PolDV and the TGGK mutant were then tested on DV103′-, in the absence of a template and on DV103′+ using Mn2+ as the catalytic ion, ATP and GTP containing αGTP. DV, NS5PolDV TGGK is not able to catalyze de novo initiation on DV103′-. Secondly, NS5PolDV TGGK does not catalyze pppAG formation without template (panel 2). In contrast, it is able to catalyze de novo initiation on DV103′+ presenting ca. 32% of wt activity (panel 3). In order to understand this apparent contradiction, we used γATP instead of αGTP as radioactive NTP. It became clear that NS5PolDV TGGK was unable to generate the pppAG primer product (panel 4). We conclude that the product observed with αGTP corresponds to pppGA formed by internal de novo initiation being only possible on DV103′+. When using Mg2+ as catalytic ion again we did not observe formation of the de novo RNA synthesis initiation product pppAG on either template .Two aromatic residues, W795 and H798, within the priming loop were proposed to play a particular role in providing an initiation platform to which the base of the priming ATP could establish a stacking interaction DV and the W795A and H798A mutants were tested using the minigenomic RNA template and either Mg2+ or Mn2+ as catalytic ions  corresponds to pppGA generated by internal RNA synthesis initiation . We thus conclude that residue H798 is essential for the formation of the correct ATP-specific priming site and may act as a priming platform.We then compared the capacities of wt and all mutant NS5Polsee also ; and theDV) alone is responsible for maintenance of A and U as first and last nucleotides of the DV genome, respectively. NS5PolDV was used instead of full-length NS5 in the frame of this study in order to avoid any interference of the RNA-binding, NTP-binding, or enzymatic activities of the N-terminal domain of NS5. We report that NS5PolDV is endowed with several structural and mechanistic features converging to the specific de novo synthesis and elongation of the correct ATP-initiated primer even on templates that lack the correct corresponding U at the 3′-end. The first and last nucleotides of the genome are strictly conserved in the genus Flavivirus thus the results presented here may apply to the entire genus.In this study, we present evidence that the dengue virus NS5 polymerase domain . Even in the absence of any template and in the presence of Mn2+ , pppAG is still a major product  nucleotide(s) by the terminal transferase activity of NS5. This activity has also been listed as another way of repairing terminal damage of viral RNA genomes DV we have observed this activity before 2+  contains a specific ATP-binding site 2+, non-templated 5′-initiation was not observed 2+ to correctly initiate RNA synthesis on erroneous templates as observed for NS5PolDV here. It is generally believed that Mg2+ is the activating cofactor of polymerases in vivo because viral RdRp properties observed with Mg2+in vitro are more consistent with properties observed biologically. A second reason for giving the preference to Mg2+ is its cellular abundance in comparison to Mn2+  and DV103′- (5′-ACUAACAACU-3′) were synthesized at the RIBOXX GmbH Dresden and by Dharmacon. The templates are devoid of stable secondary structure when submitted to the Mfold server 103′+ and no folding for DV103′-).DVO-dimethoxytrityl-2′-O-acetyl-2N-dimethylformamide guanosine. The dinucleotide AG was assembled at a 8-µmol scale in Twist oligonucleotide synthesis columns  (Glen research) using 5′-O-DMTr-2′-O-pivaloyloxymethyl-6N-phenoxyacetyladenosine]-3′-O- and following a previously described procedure Chemical synthesis of the diribonucleotide AG was performed on an ABI 394 synthesizer (Applied Biosystems) from commercially available (Link Technologies) long chain alkylamine controlled-pore glass (LCAA-CPG) solid support with a pore size of 1000 Å derivatized through the succinyl linker with 5′-N,O-bis-trimethylsilylacetamide , CH3CN (1.875 ml), CCl4 (1.875 ml) and triethylamine (0.25 ml) was added under argon and left to react for 5 h at 30°C. After washing and drying the support, the TBAPP solution  was applied to the column and left to react for 18 h at 30°C. The solution was removed and the support was washed with dry CH3CN (4×8 ml). Finally, the column was dried by 1-min argon flush.A solution (8 ml) of 1 M diphenyl phosphite (1.6 ml) in dry pyridine (6.4 ml) was manually passed with a glass syringe through the columns containing AG still attached to the solid support and left to stand for 30 minutes at room temperature. After several washings, the oxidation solution containing imidazole  in 3CN (6.8 ml) was applied to each column for 3 min. Subsequently, a 30% aqueous ammonia solution was applied to each column in three batches  for 30 min each. The three ammonia fractions were collected in screw-capped glass vials and were left to react at 30°C for 1.5 h. The fully deprotected pppAG was transferred to 50 ml round-bottomed flasks and isopropylamine  was added to the solutions. Then the mixtures were evaporated under reduced pressure at 30°C until the volumes were reduced to 0.5 ml. The residues were redissolved in water (1.5 ml), transferred to 2 ml Eppendorf-vials and then lyophilized.A 0.1 M solution of 1,8-diazadicyclo-undec-7-ene (DBU) (1.2 ml) in anhydrous CH18, 100 Å, 3 µm, 4.6×70 mm for analysis and Nucleodur C18, 100 Å, 7 µm, 8×125 mm for purification, Macherey Nagel). The following solvent system was used: 5% CH3CN in 50 mM TEAB buffer, pH 8 (buffer A) and 80% CH3CN in 50 mM TEAB buffer, pH 8 (buffer B). Flow rates were 1 ml.min−1 and 2 ml.min−1 for analysis and semi-preparative purposes, respectively. Elution was performed with a linear gradient of 0% to 10% buffer B in buffer A in 20 min. The fractions containing the pure pppAG were pooled in a 100 ml round-bottomed flask and were concentrated to a volume of 0.5 ml under reduced pressure at 30°C. The residue was coevaporated ten times with 2 ml of water. The residue was redissolved in 1.5 ml water, transferred to 2 ml Eppendorf-vials and lyophilized. MALDI-TOF mass spectra were recorded on a Voyager-DE spectrometer  using a 10∶1 (m/m) mixture of 2,4,6-trihydroxyacetophenone/ammonium citrate as a saturated solution in acetonitrile/water  for the matrix. Analytical samples were mixed with the matrix in a 1∶5 (v/v) ratio, crystallized on a 100-well stainless steel plate and analyzed. UV quantitation of pppAG was performed on a Varian Cary 300 Bio UV/Visible spectrometer by measuring absorbance at 260 nm. Two µmol of pure pppAG were obtained corresponding to 25% total yield. Lyophilized aliquots of 100 nmol have been stored at −20°C for several months without any sign of degradation.Analytical and semi-preparative HPLC was performed on a Dionex DX 600 HPLC system equipped with reverse-phase columns  as defined in E.coli (Tuner (Novagen) or NEB Express (New England Biolabs)) cells carrying helper plasmid pRare2LacI (Novagen). Expression was carried out in Luria broth overnight at 17°C after induction with 50 µM IPTG, addition of 2% EtOH and a cold shock (2 h at 4°C). Sonication was done in 50 mM sodium phosphate lysis buffer, pH 7.5, 500 mM NaCl, 20% glycerol, 0.8% Igepal (10 ml of this lysis buffer for around 2 g cell pellet from 1l culture) in the presence of DNase I (22 µg/ml), 0.2 mM benzamidine, protease inhibitor cocktail (SIGMA), 5 mM β-mercaptoethanol and 1 mg/ml lysozyme after 30 min incubation at 4°C. After centrifugation the soluble fraction was incubated in batch with 2 ml TALON metal-affinity resin slurry (Clontech) for 40 min at 4°C. Protein bound to the beads was washed once with 10 volumes of sonication buffer containing 1 M NaCl and 10 mM imidazole and once with the former buffer without Igepal. Protein fractions were then eluted with sonication buffer containing 250 mM imidazole, no Igepal and 250 mM glycine. After dialysis into 10 mM Tris buffer, pH 7.5 containing 300 mM NaCl, 20% glycerol, 250 mM glycine and 1 mM DTT the protein was diluted with the same volume of this buffer without NaCl and loaded onto a HiTrap heparin column . Pure NS5PolDV was then eluted in a single peak applying a gradient from 150 mM to 1 M NaCl. Alternatively, gel filtration was used as a second purification step using a Superdex 75 HR 16/60 column  and the dialysis buffer. NS5PolDV was stored at −20°C at a concentration of 40 to 60 µM after a final extensive dialysis into 10 mM Tris buffer, pH 7.5 containing 300 mM NaCl, 40% glycerol and 1 mM DTT. Purity was higher than 98% as judged by SDS-PAGE.The gene coding for N-terminal HisDV expression plasmids were generated using the kit QuikChange (Stratagene). Protein expression and purification was done as for the wt protein. Analysis by gel filtration showed a single peak eluting at the same volume as wt NS5PolDV.Mutant TGGK, W795A and H798A NS5Polm) values of wt and mutant NS5PolDV were determined using a thermofluor-based assay 2O) was added to 21.5 µl protein solutions at a concentration of 0.5 or 1 mg/ml (6.7 or 13.4 µM) in storage buffer. Thermal denaturation of the proteins was followed by measuring fluorescence emission at 575 nm (excitation 490 nm). Tm values were calculated using GraphPad Prism software and the Boltzmann equation as in Melting temperature . Reactions were started by addition of a mixture of HEPES buffer, KCl, catalytic ions and UTP and CTP when used . After given time points samples were taken and reactions stopped by adding an equal volume of formamide/EDTA gel-loading buffer. Reaction products were separated using sequencing gels of 20% acrylamide-bisacrylamide (19∶1), 7 M Urea with TTE buffer , 0.5 mM EDTA). RNA product bands were visualized using photo-stimulated plates and the Fluorescent Image Analyzer FLA3000 (Fuji) and quantified using Image Gauge (Fuji). The oligoG marker was produced as explained in Reactions were done in 50 mM HEPES buffer, pH 8.0 containing 10 mM KCl, 10 mM DTT and template, NS5Polin vitro transcription and tests carried out as described in The minigenomic template was produced by DV, 500 µM NTP except for UTP (4 µM), [3H]-UTP at 0.2 µCi/µl and either 5 mM MgCl2 or 2 mM MnCl2. Reactions were started by the addition of a mixture of HEPES, KCl, catalytic ions, CTP, and UTP. After 30, 60, 90, and 120 min 10-µl samples were taken and diluted into 50 µl of 100 mM EDTA, pH 8.0 to quench the reaction. Samples were then transferred onto a DEAE filter mat. Non-incorporated [3H]-UTP was removed by washing with 300 mM ammonium formate and the radioactively labeled product quantified in counts per minute (cpm) using liquid scintillation counting. Product formation was then plotted against time and initial velocities calculated in cpm/min.Reactions analyzed by filter-binding and liquid scintillation counting contained 50 mM HEPES buffer, pH 8.0, 10 mM KCl, 10 mM DTT, 100 nM RNA template, 200 nM NS5PolDV, 500 µM NTP except for UTP (4 µM), [α-32P]-UTP at 0.4 µCi/µl, and 5 mM MgCl2. Reactions were started by a mixture of HEPES, KCl, MgCl2, CTP and UTP and stopped after 60 and 120 min by adding an equal volume of sample buffer . Samples were denatured for 10 min at 70°C and 1/10 of loading buffer  added. Samples were then analyzed on a 1.2% agarose-formaldehyde gel in 20 mM MOPS buffer pH 7.0, 5 mM sodium acetate, 1 mM EDTA. Gels were dried and RNA product bands visualized using photo-stimulated plates and the Fluorescent Image Analyzer FLA3000 (Fuji) and quantified using Image Gauge (Fuji).Reactions analyzed on formaldehyde-agarose gels contained 50 mM HEPES buffer, pH 8.0, 10 mM KCl, 10 mM DTT, 100 nM RNA template, 200 nM NS5PolDV serotype 2 strain New Guinea C was generated using the Swiss-model server DV serotype 3 (PDB code 2J7W DV and the RdRp of bacteriophage φ6 in complex with a template RNA strand and initiating NTPs (PDB code 1HI0) were then superimposed using the three catalytic aspartate residues of both proteins. The structural model of the initiation complex of NS5PolDV serotype 2 was then generated by changing the RNA template to UUCU (3′-end of the DV genome) and the initiating NTP to ATP, and by manually adapting the conformation of the priming loop using the UCSF Chimera software A homology model of NS5Pol


Neurotransmitter transporters of the SLC1 and SCL6 family are found on presynaptic neurons and on glia cells. The function of these transporters is the termination of neurotransmission by the rapid removal of the neurotransmitter molecules from the synaptic cleft. These transporters couple substrate transport to ion gradients of sodium and chloride. Almost all of the eucaryotic transporters have been described to function as oligomers. However, the forces stabilizing the oligomeric state are not well understood. No crystal structures of eukaryotic transporters are available, but recently crystal structures of bacterial homologs thereof have been solved: GltPh (SLC 1 family) was found as a trimer, LeuT (SLC6 family) was crystallized as a dimer. These homologous crystal structures allow rationalizing on the driving forces that stabilize the eukaryotic counterparts.The crystal structures of LeuT and GltPh were obtained from the Protein Data Bank (PDB). We identified the interfaces between the protomers and analyzed hydrogen bonding, hydrophobic and hydrophilic interactions as well as size and width of the interface area.We investigated the protein-protein interfaces between the transporter protomers and identified the dominant forces that stabilize the oligomer. These consist of hydrophobic interactions between the aliphatic side chains within the interface and of polar interactions by hydrogen bonds between hydroxyl groups.The contributions of different forces to the stability of oligomer assemblies vary between proteins. While the hydrophobic mismatch is a prominent contributor to the stability of the GltPh transporter, it plays a minor role for LeuT, where helix packing and aromatic interactions seem to dominate.


Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using  Non-coding RNAs (ncRNAs) have become powerful tools for synthetic biology. There are now examples of engineered ncRNA systems that control nearly all aspects of gene regulation . In addiStaphylococcus aureus plasmid pT181 were recently configured in simple architectures that evaluated genetic not-or (NOR) logics, and in transcriptional cascades that propagated signals directly as antisense RNA molecules with no intermediate protein species , with the introduced sequences split between overhangs on the forward and reverse primers. Sequences for all chimeras in this study can be derived from Supplementary Tables S1–S3. As indicated in Supplementary Figure S1, all sense plasmids had the p15A origin and chloramphenicol resistance, and all antisense plasmids had the ColE1 origin and ampicillin resistance. The J23119 E. coli consensus promoter (http://partsregistry.org/Part:BBa_J23119), modified to include a SpeI site right before the start of transcription, was used for all sense and antisense in vivo transcription.A table of all the plasmids used in this study can be found in s et al. . ChimeriE. coli strain TG1. Plasmid combinations were transformed into chemically competent E. coli TG1 cells, plated on Difco LB + Agar plates containing 100 µg/ml carbenicillin and 34 µg/ml chloramphenicol, and incubated overnight (∼17 h) at 37°C. Plates were taken out of the incubator and left at room temperature for ∼7 h. At least three colonies were used to inoculate 300 µl of LB containing carbenicillin and chloramphenicol at the concentrations above in a 2-ml 96-well block (Costar 3960), and grown ∼17 h overnight at 37°C at 1000 rpm in a Labnet Vortemp 56 bench top shaker. Four microliters of this overnight culture were then added to 196 µl (1:50 dilution) of M9 minimal media  containing the selective antibiotics and grown for 3 h at the same conditions as the overnight culture. One hundred microliters of this culture was then transferred to a 96-well plate (Costar 3631) containing 100 µl of phosphate buffered saline (PBS). Fluorescence  and optical density  were then measured using a Biotek SynergyHT plate reader.All experiments were performed in E. coli TG1 cells that do not produce GFP . The block contained three replicates of each control. OD and fluorescence values for each colony were first corrected by subtracting the corresponding values of the media blank. The observed OD range across all experiments was 0.03–0.16. The ratio of fluorescence to OD (RFU/OD) was then calculated for each well (colony), and the average RFU/OD of TG1 cells without GFP was subtracted from each colony RFU/OD value. In all experiments except the 14 × 14 matrix, three colonies of each sense/antisense plasmid combination were picked per experiment. Experiments were repeated from fresh colony transformations on two separate days. Owing to the size of the matrix experiment, two Vortemp incubators had to be used. Four colonies were picked for each plasmid combination—two placed in each incubator. Data points were thrown out if the OD for a single colony was lower by a factor of two compared with the OD of the other colonies picked that day. We suspected that after the overnight incubation these colonies did not come out of the stationary phase, resulting in a low OD. Averages of RFU/OD were calculated over the 2 days, and error bars represent the standard deviations of at least five colonies. For each antisense/attenuator pair, attenuation  was calculated as the percent decrease in RFU/OD of cells containing both the attenuator and antisense plasmids, versus the RFU/OD of cells containing the attenuator plasmid and a no-antisense control plasmid. The ON level of engineered chimeric attenuators—RFU/OD of cells with the no-antisense control plasmid and an attenuator plasmid—was compared with the pT181 ON level and was reported as a ratio to the pT181 ON level (Fusion ON/pT181 ON).On each 96-well block, there were two sets of controls—a media blank (M9) and All minimum free energy structures reported were predicted by RNAStructure . SequencPrevious work on antisense RNA translational and transcriptional regulators showed that antisense/target specificity could be changed through mutations to the sequences that initiate RNA–RNA interaction ,10,19,28A compilation of naturally occurring antisense RNA translation regulators provided a starting source of RNA–RNA interaction sequences . These rMany natural loop–loop antisense-mediated translational regulators require the interaction of multiple hairpin loops for regulation . For simTo create chimeric transcription attenuators, we first needed to determine the RNA sequence to use from TransSysM, as well as the sequence from the pT181 hairpin to replace. Previous work on the pT181 system revealed that the sequence before position C21 A in the Candidate RNA sequences from the TransSysM hairpin were chosen based on its predicted structural features that mimic those of the pT181 attenuator hairpin. The first and simplest choice was to minimize the fused sequence by using just the predicted loop of the translational regulator hairpin  . The functionality of chimeric attenuators was assessed by measuring two quantities: ON and OFF level. The ON level  was compared with the pT181 ON level, and is reported as a ratio to the pT181 ON level. The OFF level (attenuation) is reported as the percent decrease in relative fluorescence in the presence of antisense RNA . Fusion 1 had an ON level comparable with that of pT181 (1.00), but did not shut OFF in the presence of antisense RNA (24%). Fusion 2 had significantly lower ON and OFF levels of 0.72 and 43%, respectively, indicating fundamental problems in its function. However, Fusion 3 proved to be a functional attenuator, with an ON level within error to that of pT181 (1.06), and an OFF level of 79%.Following previous work on the pT181 system ,33, we tLlac0-1 promoter. An induction curve measurement of Fusion 3 under different concentrations of IPTG showed that its dynamic range could also be tuned in the same manner . In addition, the pT181 attenuator was previously shown to be functionally modular in its ability to regulate different reporter proteins of unrelated sequence , and an OFF level of 81%. However, Fusion 5 had a poor OFF level with only 27% attenuation. To address this, we tried a second pT181 fusion position within the previously noted region found to be important for changing antisense/attenuator interaction specificity . TransSysC was not included in these tests because of the sequence homology of the top of the hairpin to the top of the TranSysM hairpin  and three different fusion positions from the TransSysM and TransSysR hairpins . We hypothesized that this unexpected result could be due to specific differences between loop–loop and loop–linear RNA-binding mechanisms and took steps to optimize both of these levels beginning with the OFF level.The results from our loop–loop chimeras above led us to engineer an attenuator including the first interior loop of the TransSysI hairpin indicated by the dashed line at I1 A. In vivtenuator C, which Supplementary Figure S1). This effectively adds an additional 368 nucleotides (nt) to the antisense RNA, which is predicted to fold into a very complex and large RNA structure . We hypothesized that the chimeric antisense RNA sequence could be interacting with part of this terminator sequence, thus preventing its binding to the attenuator target. We therefore sought to remove this structural interference by using a smaller terminator sequence on these plasmids. The 30-nt t500 terminator forms a compact hairpin composed of a 7 G-C pair stem . Switching to the t500 terminator on the linear antisense plasmid resulted in a 30% improvement in OFF level for Fusion 13 .To optimize the ON level, we tried including more of the RNA sequence from the TransSysI hairpin by adding the interior loops designated by the dashed lines at I2 and I3 A. The reUsing the knowledge gained from all of our positive results above, we then engineered a chimeric attenuator using a second loop–linear antisense-RNA regulator from the hok/sok post-segregational killing system of plasmid R1  TransSyF.Once we confirmed that the chimeric strategy was a route to engineering functional transcriptional attenuators, we sought to expand the library of attenuators even further by applying previously reported mutational strategies ,10 to thSupplementary Figure S7), four of the mutants resulted in additional functional attenuators   B.Supplementary Figure S8). These mutations resulted in the working attenuator, Fusion 15m5  (Previous work on the IS10 system (TransSysI) demonstrated the ability to engineer a family of orthogonal translational regulators through rationally designed mutations . The repFF: 73%) B. MutatiFF: 73%) B.Supplementary Figure S9). Several attenuators showed a slight increase in average fluorescence in the presence of antisense RNA, which is indicated as a negative attenuation. However, in all but one case, these were within error of the no antisense condition for that attenuator . Remarkably, this matrix showed that many of our engineered chimeric attenuators function independently of each other. Using 20% cross-reactivity as our cutoff for orthogonality, we were able to isolate two 7 × 7 submatrices of mutually orthogonal chimeric attenuators. Six of the attenuators are the same in the two families , with a choice for the seventh attenuator between Fusion 6 and Fusion 3m2. Flow cytometry profiles were obtained for each of the final eight attenuators in the final matrix to show that the cell populations were unimodal . In addition, attenuation levels calculated with the flow cytometry data were identical to the bulk fluorescence/OD measurements, indicating that the observed range of OD had no effect on measured attenuation .Using our strategy, we created a total of 11 new functional chimeric transcriptional attenuators. Our next task was to test their feasibility as components of synthetic genetic circuitry by checking for orthogonality between each other. Orthogonal regulators show minimal cross-talk between non-cognate antisense/attenuator pairs . CombiniIn this work, we have demonstrated a modular strategy for creating new orthogonal RNA transcriptional regulators. In doing so, we have overcome one of the central hurdles for expanding the utility of these regulators as building blocks of RNA-based genetic circuitry . Our strSupplementary Figure S2). Although the limited dynamic range of these attenuators (2.5–6 fold) could be a concern for use in complex circuitry, it was previously shown that for the pT181 attenuator, dynamic range could be improved by composing two attenuators in series  suggested that the antisense would form a 7-base pair stem with bases from TrrnB, whereas it would only pair to one base from the t500 sequence. It appears that for loop–linear systems to work, the antisense RNA must be somewhat unstructured so that it is free to bind to the structured attenuator hairpin loop. This suggests that the utilization of minimal terminators could be a design principle going forward with engineering unstructured RNAs.For loop–linear chimeras, we found that minimizing excess RNA sequences on antisense transcripts was essential for proper function. In particular, for chimeras from the IS10 (TransSysI) system, we found that switching from the 368-nt TrrnB terminator to the 30-nt t500 terminator on the antisense RNA plasmid allowed proper ON/OFF switching. Predicted MFE structures of the antisense RNA for Fusion 15 with TrrnB and t500 . However, we were able to engineer two new functional attenuators from each fusion that was mutated, with one being orthogonal to its parent chimeric attenuator. This suggests that while a more complete understanding of these mutations is required, mutations to the loop and upper hairpin regions may be general orthogonal design principles of loop–loop RNA–RNA interaction mechanisms.To expand the available library of attenuators, we applied previously reported mutational strategies to our newly engineered chimeric attenuators. This also gave us the opportunity to test the validity of the design principles underlying these mutational approaches. In the loop–loop attenuator case, we applied a strategy from previous work on the pT181 attenuator, which showed that mutations in both the loop and upper hairpin regions are required for orthogonality . As obseSupplementary Figure S8), despite the fact that these same mutations led to orthogonality in the parent translational system IS10 (TransSysI)  . This pransSysI) . Our resOverall, the mutational studies did result in at least one new functional attenuator from each chimeric system. Combined with their parent chimeras, we created 11 new attenuators, bringing the total transcriptional attenuator library to 14. Of these, eight showed promise for use in genetic networks as independently acting components. The orthogonal set included at least one chimeric attenuator from four of the five translational systems we tested, demonstrating the robustness of our approach, and the promise for expanding the number of orthogonal components in the future.One interesting result was the observed orthogonality between the loop–loop chimeras, as they all had the same 6-base loop sequence (5′-TTGGCG-3′). To further examine the sequence determinants of orthogonality amongst our attenuators, we created a sequence alignment of the chimeric region for the 14 attenuators included in our orthogonality matrix test . The seqWe anticipate that further work to refine and elucidate these design principles, coupled with bioinformatic analysis to automatically identify other RNA interaction sequences found in nature, would create a systematic and rapid approach to creating new orthogonal RNA transcription regulators.This work has led to a strategy to create orthogonal RNA transcriptional regulators that decouple RNA sensing from regulatory actuation. Underlying this strategy is the hypothesis that RNA transcriptional attenuators are structurally modular—RNA interaction ‘domains’ can be exchanged between different systems while keeping the transcriptional actuation ‘domain’ functional. The fact that our strategy is clearly robust provides support of this modularity hypothesis. As the number of natural non-coding RNA regulators continues to be discovered at a rapid pace, our modular, robust strategy will accelerate the creation of even larger families of orthogonal RNA regulators that could facilitate breakthroughs in our ability to construct RNA genetic circuitry.Supplementary Data are available at NAR Online: Supplementary Tables 1–3, Supplementary Figures 1–10, Supplementary Methods and Supplementary References .National Science Foundation Graduate Research Fellowship Program (NSF GRFP) (to M.K.T.); Defense Advanced Research Projects Agency Young Faculty Award (DARPA YFA) [N66001-12-1-4254 to J.B.L.]. J.B.L. is an Alfred P. Sloan Research Fellow. Funding for open access charge: Cornell University discretionary funds to J.B.L.Conflict of interest statement. None declared.


We investigated the fine-scale spatial heterogeneity of high-frequency temporal variability in temperature, dissolved oxygen concentration, and pH experienced by benthic organisms in a shallow coastal coral reef. We used a spatio-temporal sampling design, consisting of 21 short-term time-series located along a reef flat-to-reef slope transect, coupled to a long-term station monitoring water column changes. Spectral analyses revealed sharp gradients in variance decomposed by frequency, as well as differences between physically-driven and biologically-reactive parameters. These results highlight the importance of environmental variance at organismal scales and present a new sampling scheme for exploring this variability in situ.Spatial and temporal environmental variability are important drivers of ecological processes at all scales. As new tools allow the  The spatial and temporal distribution of aquatic organisms is tightly coupled to underlying physical, chemical, and biological changes that are distributed unequally over a wide range of scales e.g. . Most ofin situ, hypotheses about how organisms will acclimate and adapt The development of faster, more accurate and affordable autonomous sensors has triggered studies describing patterns of natural variability in the framework of global change, particularly for pH Tropical coral reefs are particularly appropriate for an investigation of high-frequency environmental fluctuations because dramatic changes in temporal variability can occur over very short distances for several reasons. First, coastal systems are boundaries between ocean, land, and atmosphere and are subjected to a wide array of fluctuations from all three sources. Second, reef shorelines can be very abrupt, and steep physical and chemical gradients are established across changes in depth. Concurrent with these physical gradients are gradients in primary productivity, which can drive fluctuations in oxygen and pH over relatively small temporal and spatial scales. Third, tropical systems are characterized by a relatively low seasonality and strong diurnal forcing; therefore, high-frequency variability may be comparatively more important than in temperate environments. In addition, the need to understand patterns of natural variability is particularly acute on coral reefs, because corals show narrow tolerance levels and are sensitive to global change Moku o Lo‘e (Coconut Island), home to the Hawai‘i Institute of Marine Biology, is a small island of about 12 ha located in Kāne‘ohe Bay, a tropical semi-enclosed embayment at the eastern shore of the island of O‘ahu in the Hawaiian archipelago. It is situated on the southern basin of the bay lagoon, where water residence time is relatively long Seasonality is defined by dry (May to October) and rainy (October to April) seasons. NE trade winds are prevailing throughout the year, but occasionally intense south “Kona” winds occur in winter. A weak thermocline is usually established in summer at a depth of about 8.5 m Diurnal and semidiurnal components are the only significant tidal constituents In order to explore distribution in variance across spatial and temporal scales, we established 21 random-stratified sampling locations along a ∼32 m reef flat-to-reef slope transect across a fringing reef. At each site along the transect, two-week long times series of environmental parameters were obtained non-simultaneously throughout a period of 11 months. At an adjacent long-term station, we obtained a similar time series for the entire experimental period. This is an approximation to an idealized  setup with a grid of sensors deployed simultaneously.2]) and pH sensors . Only temperature, pH and [O2] data were used in this study. Salinity could not be used in the statistical analyses because its distribution was not normal, and the time series were spiky. Therefore, the spectral analyses resulted in flat, anomalous spectral densities.The transect was oriented eastward off the southeastern shore of Coconut Island . Starting 0.5 meters from shore, the transect ran along the reef flat for ∼24 m and then down the reef slope for another ∼7 m to a depth of 4.5 m . Sites w2]. As the aim was characterizing water column background conditions, rather than bottom conditions, the instrument was placed in midwater, attached facing down to a pole at a depth of ∼1.7 m over a ∼3 m deep bottom.The long-term station was located a few meters south of the transect, just off the easternmost point of Moku o Lo‘e, on a site with a shorter reef flat than the transect. It consisted of a multi-parametric instrument  with sensors for temperature, conductivity, pH and , and pH, respectively. In relative terms, the contribution of the different frequencies to total variance was also remarkably constant throughout the year, with one exception: lowest frequency contribution on temperature variance changed considerably , while the relative importance of weekly frequencies was higher for temperature.Overall, daily frequencies contained the most variance , account−1) also increased offshore, but only in the case of the biologically driven parameters (pH and [O2]). Finally, distance to shore appeared to be a better linear predictor of changes in all variance components than depth  vs. their intensity expressed in terms of variance. However, a more natural way to evaluate the contribution of each frequency to overall variability may be achieved by looking at the amplitude of fluctuations. Assuming that values are normally distributed , amplituef slope . SpatialFinally, spectral slopes also increased linearly with distance to shore . Close tWe used two complementary spectral analyses to assess the distribution of temporal variability along a cross-reef transect: variance decomposition at different frequency ranges, and fitting of spectral slopes at the high-frequency end of the spectra. The first assesses the contribution of the most important frequencies to variability; the second is a common way to characterize patchiness in the distribution of a scalar e.g.  and is b2], individuals on the reef flat experience a highly variable environment, and those on the reef slope a relatively constant one. Here, we first discuss the suitability of the spatial and temporal sampling design and its potential application in other systems; second, we address what physical and biological processes may be driving the observed patterns; and finally, we discuss the ecological relevance of these results in the light of the ongoing discussions about the responses of organisms and ecosystems to natural variability and climate change.Results show dramatic changes in the regime of temporal fluctuations over spatial scales of meters , 6 and 7Our observational design, combining mobile short-term deployments of sensors and a long-term stationary suite of sensors, made use of limited resources to provide information on the spectral distribution of environmental variance across a spatial transect. Auto-spectral analyses were adequate tools for characterizing variance distribution in this design because variability along the transect was much higher than the long-term temporal dynamics. However, auto-spectra would not be appropriate in systems where differences between sampling periods are pronounced . In such cases, an alternative approach would be to normalize each of the two-week time series on the transect to its corresponding period in the long-term station, using cross-spectral techniques. This normalization allows direct comparison of the spectra of fluctuations across transect locations. In our dataset, the spatial patterns obtained using the cross-spectral approach were very similar to the results from the auto-spectral approach we present  over the reef flat than through the deeper water column (2–5 m) of the reef slope. This leads to rapid and intense physical, chemical, and biological responses to high-frequency atmospheric forcing on the reef flat. Most physical and biological processes dominant in coastal systems, such as light, tides, waves, or turbulence, are strongly influenced by water column height. Not surprisingly, the relative importance of daily variance decreases offshore whereas that of weekly variance increases .Distance to shore reflects horizontal mixing between the reef flat and the bay waters. Limited horizontal mixing may result in long water residence times in the reef flat, depending mostly on tidal oscillations and wind In any case, the combined effects of depth and distance to shore result in clear differences in fluctuation regime between reef flat and reef slope: 1) processes generating variability are most intense and/or amplified in the reef flat, and 2) there is limited mixing between reef flat and slope. The pattern and scale of temporal variability reflect the underlying processes.At the weekly scale, the longest temporal scale in our sampling, variability is most likely related to weather e.g. . This is2] and pH  was fast enough to resolve any part of the turbulent inertial subrange Finally, at the highest frequencies of our sampling, turbulence is likely to be the most important factor contributing to variability because it is the main mechanism transferring variance from large to small scales and will cause mixing of scalars between reef flat and slope. A scalar under isotropic turbulence is expected to show a spectral slope of −5/3 e.g. . Thus, tSpectral slopes offshore lie between −5/3 and −1. These values are indicative of turbulent diffusivity driving changes at these scales. However, slopes gradually decreased onshore, to reach values of −3 at the sites nearest to shore. This indicates that physical or biological processes other than water motion are driving variability We have shown robust spatial patterns in the regime of environmental fluctuations across a reef flat at relatively high frequencies. But, to what extent do changes occurring at these frequencies impact the life of reef organisms, and what role do they have in defining their spatial distribution? Such questions are obviously out of the scope of the present study, which solely deals with environmental variability. However, our approach may be useful in defining the limits of natural variability that individuals are experiencing, thus allowing for hypothesis-driven field studies, and providing a basis for better predictions of biological responses to change.The amplitudes of oscillations in temperature, pH and oxygen observed at short timescales on this small transect are comparable to long-term trends . At the For example, corals can acclimatize to changes in fluctuation frequency Spatial patterns in high-frequency environmental variability could also translate into different distributions of biological organisms and processes. Temporal and spatial variances are correlated across scales e.g. . FrequenThese are important issues to address as global climate change and ocean acidification alter the pattern of environmental fluctuations. Experimental works have mostly addressed how changes in baselines may affect individuals and populations, but there is an increased awareness of the importance of variance at organism scales e.g. . Under cThe sampling scheme that we used allowed us to explore the spatial distribution of temporal variability making use of limited resources. Spectral analyses of time series reveal patterns in the regime of environmental fluctuations at all frequencies resolved in the study that occur over very short distances making it a good model to explore the acclimatization and adaptation of organisms to high-frequency variability.Furthermore, the spectral analyses allow a quantification of the amplitude of fluctuations occurring at specific scales. The results show that the fluctuations at relatively high frequencies and also at weekly scales are comparable in magnitude to diurnal, seasonal and longer trends . This pain situ with new molecular tools (e.g. in situ responses to high frequency fluctuations, and provide clues about what scales are worth exploring.In general, spectral analyses cannot by themselves distinguish the underlying mechanisms that generate environmental variance. They can, however, reveal and quantify patterns in variance that organisms in different places will experience. Whether and how organisms may sense and respond to these different regimes of fluctuations, and whether this can translate into long-term spatial changes in community structure, can now be addressed ols e.g. . The ana


To prepare Ds-containing DNA templates with long chains, the Ds-Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds-containing DNA templates and a biotin-linked Pa substrate (Biotin-PaTP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining the Ds-Px and Ds-Pa pairs with modified Pa substrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5- The site-specific incorporation of functional nucleotide analogs into RNA molecules is performed by chemical synthesis, posttranscriptional modification, and enzymatic incorporation of nucleotide analogs as substrates. Among them, chemical synthesis is a commonly employed method. However, it is only capable of synthesizing small RNA molecules. Other site-specific modifications of RNA, by posttranscriptional modification pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) /[216-bp DNA fragments]). Therefore, the Ds misincorporation opposite a natural base per replication was calculated as approximately 0.004% (≈(0.096% − 0.054%)/10). Consequently, the Ds misincorporation into the nontemplate strand would occur, and thus the misincorporation rates of Diol1-dPxTP and dDsTP opposite the natural bases in templates during PCR amplification would be 0.008%, which is as high as that of the noncognate natural base pairings.We also assessed the misincorporation of Biotin-per base . In addiral NTPs . This miDs-Px pair was retained in the amplified 282-bp DNA fragments, and the misincorporation rate of the unnatural bases opposite the natural bases was 0.008% per base per replication. We employed the natural base substrates at relatively high concentrations (600 μM each), as compared to the unnatural base substrates  in the fusion PCR, to reduce the misincorporation rate. By using this method, the biotin-linked unnatural base was site-specifically incorporated at predetermined positions of RNA transcripts with selectivities ranging from 72% to 92%, depending on the sequence contexts around the unnatural base. In addition, the misincorporation rate of the biotin-unnatural base opposite the natural bases was around 0.05% per natural base. These selectivity and misincorporation rates were obtained using 2 mM unnatural and natural base substrates in T7 transcription. The incorporation selectivity (90−96%) and the misincorporation rate of Biotin-Pa depend on the ratio of Biotin-PaTP to the natural base NTPs in transcription [Pa base, this method could be applied to a wide range of site-specific labeling and functionalization of large RNA molecules.In this study, we demonstrated the two-unnatural-base-pair system for the site-specific labeling of large RNA molecules by fusion PCR and T7 transcription. In fusion PCR, more than 97% of the cription . Thus, tcription , 17. Sin


SNORD116 small nucleolar RNAs and the spliced exons of the host gene, 116HG. However, both the molecular function and the disease relevance of the spliced lncRNA 116HG are unknown. Here, we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription and increases in size in post-natal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to the dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1 and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates the diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders.Prader–Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of non-coding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple  Prader–Willi syndrome (PWS), one of the leading genetic causes of obesity in children , is charHBII-85/SNORD116, making PWS the first human disease found to be caused by loss of non-coding RNA  (SNORD116) and a spliced long non-coding RNA (lncRNA) host gene (116HG) that is stably retained in the nucleus (sno-lncRNA) was recently described that binds the splicing factor Fox2 (sno-lncRNA has not been confirmed and the relevance to the phenotype of PWS is currently unknown. Mice with an engineered deletion of the Snord116 repeat cluster (Snord116del) recapitulate the PWS phenotype of altered metabolism and growth deficiency, although not obesity  mice, corresponding to the metabolic phenotype of the reduced respiratory exchange ratio (RER) and the increased energy expenditure in this PWS mouse model.In this study, we use a combination of 116HG lncRNA in mammalian brain development, RNA fluorescence in situ hybridization (FISH) using probes targeting the splice junction of 116HG or 115HG . The 116HG and 115HG nuclear clouds observed were similar to small ‘grape’ structures seen previously in primary neuronal cultures . Both 116HG and 115HG lncRNA clouds significantly increased in diameter during the first week of post-natal life . The Snord116 DNA overlapped with 116HG, but not 115HG, and the two DNA loci (Snord116 and Snord115) co-localized in adult kidney, but not brain, nuclei  consistent with the neuronal tissue-specific chromatin decondensation of this locus  (116HG. Reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) on RNA isolated by ChIRP showed 25–30% of input 116HG RNA was retrieved by 116HG-specific oligos but not by non-specific control (NSC) oligos  was specifically retrieved by 116HG ChIRP in an RNA-dependent manner , biotinylated 116HG also retrieved RBBP5 in a conformation-dependent manner, requiring slow-cooling following denaturation . An unbiased proteomic screen of 116HG ChIRP identified the Xist associating protein SAF-A, unlike RBBP5.Other lncRNAs that form subnuclear RNA clouds, such as  regions ,16. In o (ChIRP) –22 usingntiation . An in vein SAF-A, althoug116HG lncRNA nuclear subdomain suggested that 116HG may interact in trans with other gene loci, DNA isolated by ChIRP was analyzed by next-generation sequencing (ChIRP-seq). Sequencing libraries were made from ChIRP DNA samples from adult WT or Snord116del+/− mouse brain using 116HG-specific or NSC oligos. As expected based on qPCR analysis , 116HG ChIRP-seq peaks from the WT brain were enriched at the Snord116 and the Snord115 region  of the  μm of thion Fig. .116HG-specific ChIRP peaks associated with 2403 genes . Combined RNA/DNA FISH experiments confirmed the enriched co-localization of these ChIRP target genes with the 116HG lncRNA cloud, compared with two negative control regions . 116HG-specific ChIRP genes included the mammalian target of rapamycin (Mtor), transcriptional regulator Creb-binding protein (Crebbp) and imprinted insulin growth factor receptor (Igf2r).In addition, analysis revealed 116HG on transcript levels genome wide by comparing WT versus Snord116del+/− mice by RNA-seq analysis of the adult cortex. 6467 genes were significantly altered  in the Snord116del compared with the WT cortex, with 94.2% up-regulated in the Snord116del cortex . Comparison of Snord116del dysregulated genes to ChIRP-seq peaks revealed that 50% (1201) of genes identified by ChIRP had altered expression levels in the Snord116del cortex . GO analysis of genes identified by ChIRP and RNA-seq analysis was enriched for protein and cellular metabolic processes, chromatin modification, cellular biosynthetic processes, neurogenesis and central nervous system development . Most ChIRP peaks were located within genes or distal to transcription start sites (TSSs)  +6 h and Zt +16 for RNA-seq analysis of cortical transcripts. At Zt +6, 6467 genes were altered in the Snord116del cortex . Genes altered in the Snord116del cortex at Zt +6 significantly overlapped with genes previously described to be diurnally regulated . Dendrogram analysis of gene expression revealed that WT and Snord116del from Zt +16 were more closely related than WT and Snord116del from Zt +6 . However, Snord116del mice exhibited a significant decrease in the RER compared with WT littermates during light hours . The RER reflects the relative source of energy for the animal with values closer to 1 representing increased carbohydrate oxidation and values closer to 0.7 representing increased fat oxidation. An increase in the RER occurs during dark compared to light hours in both WT and Snord116del mice . In contrast to WT mice, Snord116del mice have decreased RER during light hours, which explain the decreased fat mass and body weight in Snord116del mice . Snord116del mice also showed increased energy expenditure compared with WT mice during both light and dark hours , suggesting that their central circadian clock is not disrupted and that the metabolic phenotype is due to the central dysregulation of energy expenditure, not changes in feeding or activity. Our results suggest that a key feature of the Snord116del phenotype is altered the regulation of diurnal energy source due to loss of 116HG modulation of diurnally regulated circadian and metabolic genes during light hours.We sought to relate altered diurnal transcript levels to the metabolic phenotype of adult del mice , which wurs Fig. . Snord11Snord116 locus was first hypothesized 10 years ago to be responsible for the pathogenesis of PWS  by SNORD115  mice were obtained from Jackson Labs  and housed in a 24-h light/dark cycle , temperature controlled room and fed a standard diet of Picolab mouse chow 20 . Animal genotyping was performed using PCR from tail DNA as described previously (Snord116del+/− and WT littermates.B6(Cg)-Snord116eviously . HeterozHuman postmortem cerebellum of brain 1209 was received frozen and was fixed in 10% formalin, embedded and sectioned (5 µm). Tissue was obtained through the NICHD Brain and Tissue Bank for Developmental Disorders located at the University of Maryland School of Medicine.in vitro biotinylated RNA pull-down of proteins, one hemisphere of a WT adult C57BL/6 brain was fixed in 3.7% formaldehyde/1× phosphate buffered saline (PBS) and homogenized. Nuclei were resuspended in RNA immunoprecipitation/formamide buffer . For ChIRP, one hemisphere of a WT adult C57BL/6J or Snord116del brain was fixed in 3.7% formaldehyde/1× PBS and homogenized. Nuclei were resuspended in ChIRP lysis buffer . For RNA-sequencing analysis, the brains from adult Snord116del and WT littermates were collected and cortices were dissected and placed in RNALater (Ambion) before RNA isolation.For RNA, DNA and RNA/DNA FISH analyses, tissue was prepared as described previously . For the115HG and then scoring for complete, partial or no signal from 116HG within the area defined by 115HG. DNA FISH measurements were taken using a 100 × oil objective with a 2 × zoom and pixel counts converted to micron distance. All measurements for a given experiment were taken with the same exposure times and microscope settings appropriate for the fluorescence intensity. RNA FISH on human postmortem cerebellum samples was performed using custom directly labeled, single-stranded oligo library synthesis probes (Agilent). Significant changes tested by two-tailed Student's t-test.FISH was performed as described previously . OverlapSlides were prepared as for RNA FISH and hybridized with locked nucleic acid probes (Exiqon) overnight at 46°C and washed 3× in 50% formamide/2× saline-sodium citrate (SSC) and 2× in 2× SSC at 46°C, fixed with 3.7% formaldehyde/1× PBS at RT for 5 min, washed in 1× PBS and dehydrated through a series of ethanol washes . DNA FISH probes were then applied and slides were denatured for 3 min at 85°C. Washing and detection was performed as for RNA or DNA FISH alone.Immunofluorescence and laser scanning cytometer (LSC) analysis was performed as described previously .t-test.Slides were analyzed on an Axioplan 2 fluorescence microscope  equipped with a Qimaging Retiga EXi high-speed uncooled digital camera, appropriate fluorescent filter sets and automated xyz stage controls. Images were captured with appropriate filter sets at 0.1 µm sections and multiple Z-slices were combined for final images. For phospho-S6 quantification, slides were scanned on a laser scanning cytometer (CompuCyte) with a 20× objective. Nuclei were contoured using 4′,6-diamidino-2-phenylindole fluorescence, the contours extended by 15 pixels, and cells were gated for fluorescence above background. All slides were scanned on identical voltage, photomultiplier tube and threshold settings. Brain regions were partitioned based on the composite image from all scan fields. Significant changes tested by Student's 200 pmol of biotinylated DNA oligos (Invitrogen) were added to 1 mg of material and incubated at 4°C for 4 h. C-1 streptavidin beads (Invitrogen) were used to retrieve biotinylated DNA oligos with hybridized RNA:protein complexes. Proteins were analyzed by immunoblotting. For RNAse sensitivity, Superasin was omitted and 10 μg of RNAse A (Ambion) was added to sonicated material and incubated at 37°C for 30 min before biotinylated probe was added. For mass-spec identification, proteins were run on an sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and stained with Imperial protein stain. A 1 cm square piece of gel from 80–140 kDa was excised and submitted to the UC Davis Proteomics core for mass-spec identification.Template DNA was linearized and 250 ng of DNA was IVT using Biotin RNA labeling Mix (Roche), DNAse I (NEB) treated and nucleotides removed using Micro-Biospin columns (Biorad). IVT RNA was denatured at 65°C for 5 min and then slow-cooled to 4°C. RNA was quantified using a Nanodrop spectrophotometer, 100 pmol RNA was added to 1 mg of lysate and incubated for 2 h at RT. C-1 streptavidin beads were used to retrieve biotinylated RNA and bound proteins were analyzed by immunoblotting.Primary antibodies: anti-GAPDH clone 6C5 , anti-RBBP5 (Bethyl), anti-MeCP2 , anti-FOX2, anti-histone H3-total (Abcam), anti-SAF-A/HNRNP-U  and anti-Ezh2 (Active Motif). Secondary antibodies: GARIG-Alexa-488 (Life Technologies), anti-DIG-Alexa-488, anti-biotin-Rhodamine (Jackson), GARIG-HRP and GAM-HRP (Biorad).g for 5 min at 4°C. 100 μl was saved as input for DNA purification and 100 μl for RNA purification. ChIRP was performed as described previously (Brain cell lysate was sonicated on high in a Bioruptor sonicator (Diagenode) at 4°C until solution was no longer turbid and size of fragments was 200–800 bp by gel analysis. Insoluble material was pelleted by centrifugation at 16 000Snord116del males from Zt +6 and two Snord116del males from Zt +16 were analyzed.RNA was purified from dissected cortices using RNEAsy columns (Qiagen) following the manufacturer's instructions and analyzed on a Bioanalyzer. Samples with an RNA integrity number > 7 were used to prepare libraries using the NuGEN Encore Complete RNA-seq library kit following the manufacturer's instructions for multiplexing, with cDNA fragmentation performed on a Bioruptor for 1 h at 4°C. Three WT males from Zt +6, two WT males from Zt +6, two Sequencing libraries for ChIRP-sequencing and RNA-sequencing were analyzed for quality on a Bioanalyzer (Agilent) and were sequenced on a Hi-seq-2000 at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley.Mus musculus mm9 genome, and peaks were called using model-based analysis for ChIP-Seq (MACS)  and fold-enrichment over input (>20). Since no sequence motifs were identified using all the called 116HG ChIRP peaks from two biological replicates, we used a 10 kb window to identify peaks that appeared in the same genomic region in both WT-116HG ChIRP samples. 8105 MACS identified peaks replicated between the two WT-116HG ChIRP samples. In contrast, of 8300 MACS-called peaks combined from the three negative control ChIRP samples , there was <0.05% overlap between samples and only 985 peaks were within 10 kb of 116HG-ChIRP identified peaks. The BED file with corresponding confident peaks was then used to generate an associated gene list by mapping to closest gene within 2 kb of the 8105 replicated peaks. Genes associated with 116HG peaks were submitted for DAVID GO analysis  ,39. The Mus musculus mm9 genome using Tophat2 with a reference gene transfer format guide. Reads per sample were: WT-Zt6-1 56,801,996, WT-Zt6-2 64,288,201, WT-Zt6-3 63,022,540, Snord116del-Zt6-1 53,278,255, Snord116del-Zt6-2 40,243,885, WT-Zt16-1 63,985,980, WT-Zt16-2 56,483,862, Snord116del-Zt16-1 54,310,542 and Snord116del-Zt16-2 61,584,399. Cuffdiff was used to identify significant changes of transcript levels between paternal inherited +/−Snord116del and WT mice , the diurnally regulated gene set (P = 2.2 × 10−16) and the WT Zt6/Zt16 gene set (P = 2.2 × 10−16).Reads were aligned against the +/−Snord116del mice were analyzed by CLAMS. Locomotion, food and water intake, O2 consumption and CO2 emission were automatically monitored. To allow the acclimation of mice to CLAMS chambers only the final four full light/dark cycles were used in analysis of energy expenditure. Significant changes tested by two-tailed Student's t-test.Mice were placed in Oxymax/CLAMS (Columbus Instruments) at the UC Davis Mouse Metabolic Phenotyping Center and monitored for 7 days with six full 24-h light/dark cycles. Only male After being removed from CLAMS chambers, mice were weighed, anesthetized with isofluorane and scanned using dual energy X-ray absorptiometry .Supplementary Material, Table S7). Significant changes tested by the Mann–Whitney U-test.qPCR analyses were performed using Sybr Green (Bioline) on an ABi ViiiA 7 in 384-well formats with custom primers  and the Prader-Willi Foundation. Funding to pay the Open Access publication charges for this article was provided by Agilent, Inc.Work funded by


Renal cell carcinoma (RCC) has high metastatic potential, which requires early diagnosis to optimize the chance of cure. Metastasis of RCC to the head and neck region is less common and metastasis to the buccal mucosa is extremely rare. This phenomenon occurs mostly in patients with generalized dissemination, especially with lung metastases. In this article we report a case of buccal mucosa metastasis from RCC in a 65-year-old man who presented 19 years after undergoing a left radical nephrectomy for clear cell RCC. Surgical excision of the buccal lesion was performed without evidence of recurrence or new metastatic lesions after 6 years of followup. To our knowledge, this is the first case of metastasis to the buccal mucosa from a RCC reported in the literature. Metastasis of RCC to the buccal mucosa represents an extremely rare finding and is regarded to have very poor prognosis. The metastasis of RCC to the head and neck region is relatively uncommon and can be found in 8–16% of all cases , usuallyIn this paper, we report a rare case of metastasis to the buccal mucosa from an RCC that occurred 19 years after left radical nephrectomy. A65-year-old otherwise healthy male patient presented to clinic with a sensation of discomfort in his left cheek. His past history is significant for left radical nephrectomyperformed 19 years earlier for clear cell renal cellcarcinomawithrenalveinthrombosis (pT3aN0M0), and there were no other sites of disease evident upon current presentation. Computed Tomography scan detected a 10 mm × 8 mm mass suggestive of malignant lesion in theleftbuccalmucosa  which waRCC represents 3% of all adult malignancies . Male toRCC is the most common malignancy of the kidney , 8 and rRCC is the third most frequent neoplasm reported to metastasise to the head and neck region, after breast and lung carcinoma –5. ApproThe thyroid gland is the most common site in the head and neck region for RCC metastasis , 5. OnlyAlmost 25% of patients with RCC have distant metastases at the time of diagnosis. This trend has remained stable over the last two decades despite advances in methods of detection, especially abdominal imaging techniques .The tumour can metastasise after long periods of latency, with reports of up to 20  or even RCC invades the local vascular network of the kidney, spreading through the systemic circulation . If therCT scan is the radiologic investigation of choice in assessing the extent of the metastatic lesion , 10. AngManagement of these patients should be individualised based on the presence or absence of metastasis to different organs and the patient's general health , 4. SurgThe goal of surgical treatment of buccal metastasis is usually palliative providing patients with comfort and pain relief and at the same time preventing bleeding and infections . The rolIn conclusion, the presenceof metastasis inoral mucosadue to RCC is rareand does not necessarily implypoorprognosisas has been describedfor metastases in thehead and neck. However, reviewingthe literature, wesuggest that the treatmentof metastaticRCC shouldbe individualised.Asurgical approach tailored tosolitary lesions, as in our case,may be beneficialfor controlling metastatic disease.


In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA–protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA–His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient  Recombinant approaches for the production of stable and structured RNA in vivo have progressively emerged to produce stable structured RNA in vivo  were unsuccessful. It was impossible to obtain simultaneous expression of both RNA and protein. The protein alone was recovered after 3 h of induction followed by cell lysis, whereas after one night of induction, only the overexpressed RNA was detected.With the purpose of co-expressing a protein together with a tRNA–RNA fusion in 1 origin ,34. We sE. coli . High levels of expressions of both RNA and protein were observed. Therefore, these three examples validate the feasibility of co-expressing RNA and protein in E. coli using two plasmids.To synchronize the RNA and the protein production, we substituted the second T7 promoter in the pACYCDuet-1 vector by the bacterial tac promoter . The resE. coli . A smallin vitro . The phiin vitro  was clonE. coli. Two new plasmids, pProRNA and p44K, were thus designed . Plasmid p44K carries the ampicillin resistance gene. Although it does not contain the lacI gene, the co-expression of the protein and the RNA before IPTG addition is locked in E. coli LaqIq strains like JM101 or DH5 α turbo. A. aeolicus AtmRNA/MS2 coat protein couple. The tmRNA is similarly produced in both plasmids.Thereafter, we aimed to design a single plasmid that could simultaneously express an RNA and a protein. Such a plasmid would simplify cloning procedures and would reduce the pressure of selection by the use of only one antibiotic. Moreover, it would allow concomitant use of compatible commercial plasmids such as those coding for rare codons (pRARE) commonly used for the production of heterologous proteins in designed . PlasmidE. coli . In our hand, there is no difference in the yield of RNA or protein production according to the plasmids used to achieve the co-production . The success of the co-production in vivo with TrmI was evidenced by the comparison of the NMR spectra of tRNALys3 with those of the tRNA overproduced alone in E. coli. Indeed, the NMR spectrum of tRNALys3 co-produced with TrmI contains the signal of the m1A58 methyl and exhibits the chemical shift variation of the imino proton H3 of Ψ55 as expected for a tRNALys3 bearing the m1A58 modification. Therefore, the co-production system is suitable for in vivo RNA processing by heterologous enzymes.In our group, we have been studying for many years the annealing process between tRNAiral RNA ,38,39. Wcription . Howevercription , are lacn TrmI, ,41] toge togeLys3E. coli bacteriophage MS2 is resistant to RNase digestion. Bacteriophage MS2, which infects E. coli, adopts a simple ribonucleoprotein structure composed of 180 molecules of the MS2 coat protein, one copy of a maturase and one copy of genomic RNA. The MS2 coat protein makes up the bulk of the bacteriophage and assembles into an icosahedral structure of about 26 nm in diameter. Interestingly, pseudo-viral empty particles can be synthesized in vivo and in vitro from the coat protein alone . This indicates that the presence of the MS2 operator hairpin does not impair the overall folding of the recombinant RNA.Next step was to check whether the AtRNA technology could produce functional RNAs. The malachite green aptamer was previously used to probe the functionality of recombinant RNAs produced with tRNA or 5 S rRNA scaffolds ,21. To uE. coli JM101 strains were transformed by both plasmids (pACYCT2-MS2coat and pBSTNAV-AtRNA). The expression of AtRNA is constitutive. We tested the induction of the expression of the protein by addition of IPTG, either in exponential or stationary phase of culture growth. The protein was not produced when induced in the stationary phase presumably because the intracellular concentration of AtRNA was maximal during this period. On the contrary, induction in the exponential phase (absorbance at 660 nm around 0.6) proved to be suitable to overexpress both AtRNA and MS2 coat protein. Four hours after induction, the concentration of AtRNA and MS2 coat protein reached 25 and 27 mg/l of culture, respectively. We analysed the co-production of AtRNA and MS2 coat protein obtained in these optimal conditions. After ultrasonicating bacteria, we examined by size exclusion chromatography the supernatant recovered by centrifugation . fugation A. As a rfugation B. These fugation A. This oo 100 nm C. Altogetrans-translation, we were trying to produce the A. aeolicus tmRNA using the pBSTNAV vector in E. coli. Trans-translation is a highly sophisticated process in bacteria that recycles ribosomes stalled on defective mRNAs and adds a short tag-peptide to the C-terminus of the incomplete polypeptide as degradation signal [for review, see  is a multidomain RNA of 347 nt. It comprises two functional domains, the tRNA domain partially mimicking a tRNA and the mRNA-like region (MLR), which encodes the tag peptide, surrounded by four pseudo-knots. This molecule is, therefore, a case study for our ‘tRNA scaffold’ approach, as it naturally contains a tRNA domain. Although the overproduction is good, the A. aeolicus tmRNA undergoes systematic degradation by nucleases during its expression in E. coli  or co-transformed by the pBSTNAV-AtmRNA and the pACYCT2-coat plasmids (lane 2) are displayed in A. aeolicus AtmRNA is overexpressed alone . Diffracting crystals were rapidly obtained, which we are currently optimizing.The possibility to co-purify the AtRNA/His6-MS2 coat protein leads us to investigate if this RNA–protein complex were amenable to X-ray crystallography study  or a single plasmid. We demonstrated that these expression systems can be successfully used by producing several binary RNA–protein complexes and via the efficient purification of the complex from crude extracts by affinity chromatography by means of a fusion tag. Our methods of RNA–protein co-expression can offer attractive methods for expressing proteins that are poorly expressed or incorrectly folded because of the absence of their RNA partner. Our method offers a unique preparative system for overexpressing RNA–protein complexes in E. coli, with yields of several milligrams of purified material, which is a prerequisite to structural biology studies. There are a multitude of potential applications of this technology, including RNA production and purification, RNA modifications and the production of RNA–protein complexes. Finally, if needed, the pACYCT2 plasmid can be easily modified to co-express multiple proteins in combination with a recombinant RNA target embedded in a tRNA scaffold.In this article, we have developed plasmids for co-expressing RNA–protein couples in in vivo. The in vivo expression strategy that we have designed is efficient for expressing structured RNA, at least up to 250-nt long. In addition, our method abolishes the requirement of T7 transcripts with the 5′-end starting with G or GG. However, our previously designed plasmid to produce RNA in E. coli has suffered from two limitations: (i) poorly structured or unstructured RNA gets partially or totally degraded and is poorly expressed; and (ii) purification of the RNA requires phenol-extraction steps, which cannot be easily parallelized or transposed to high throughput approaches. Moreover, it is frequently important to work under ‘native’ conditions, especially for large RNA. Phenol extraction often requires ethanol precipitation, which can cause large and highly helical RNAs to form irreversible aggregates.The first application deals with the production of RNA in large quantities E. coli cytoplasm. After cell lysis and VLPs purification by size exclusion chromatography, RNA can be recovered by heat treatment, whereas chemical extraction may decrease the quantity of RNA as described for armored RNA  between a fluorescent RNA and a protein fused with Green Fluorescent Protein (GFP) for instance.The use of tRNA scaffold for producing RNA targets has already been successfully applied to eriments . Thus, oSupplementary Data are available at NAR Online: Supplementary Figures 1–5.European Union (FSG-V-RNA); the ‘Agence Nationale de la Recherche’ (TriggeRNA and RNAUREUS); the French national research center (CNRS); the University Paris Descartes. Funding for open access charge: Institutional funds or Department fund.Supported by the 6th framework program of the Conflict of interest statement. None declared.