Unprecedentedly high degree of chiral amplification using dynamic propeller conformation of triphenylamine. vice versa depending on its partners. These achievements could be realized using the dynamic propeller conformation of TPA and provide new insights into supramolecular assemblies and the supramolecular chiral amplification of helices.An unprecedentedly high degree of chiral amplification of supramolecular helices in a sergeants and soldiers system was realized using a propeller-shaped molecule, triphenylamine (TPA), as the monomer. One sergeant controlled the handedness of 500 soldiers in supramolecular helices. We further demonstrated that a TPA derivative could switch its role from sergeant to soldier and  The helix is intrinsically chiral and is always either left- or right-handed. To fully exploit the potential of helices, it is important to selectively synthesize or isolate helices with a single handedness. However, left- and right-handed helices are enantiomers, and their thermodynamic stabilities are identical. Hence, to selectively form one-handed helices, it is necessary to differentiate their stabilities kinetically or thermodynamically. For this purpose, a well-established method is the employment of chiral auxiliaries for racemic helices at the molecular and supramolecular levels. Owing to the introduction of other chiral sources, left- and right-handed helices become diastereomers of each other, and consequently, one of the helical structures becomes thermodynamically preferred. However, the presence of chiral auxiliaries inevitably alters the conformation of the helices and their physical properties. Therefore, it is desirable to control the handedness of helices using a minimal amount of chiral auxiliaries via chiral amplification.The helix has attracted scientists owing to its structural beauty, its vital roles in biological systems, and Yashima, one-handed helices consisting of only achiral monomers have been successfully isolated, even in the absence of a chiral source. The helical polymers memorize the induced handedness by the action of chiral sources, even after the removal of the chiral sources. However, most reported helical polymers and supramolecular helices are dynamic and undergo racemization after removal of the chiral sources. In 1989, Green and his colleagues demonstrated the first chiral amplification system for helices in which a one-handed helical conformation of dynamic helices could be obtained by copolymerization of the achiral monomers with a small fraction of the chiral monomers. Because the chiral monomers dictate the handedness of the helices, which mainly consist of achiral monomers, this system is referred to as the sergeants  and soldiers  principle. This concept was later proven to be valid even for supramolecular helices. Thanks to the systematic study by Meijer and coworkers using benzene-1,3,5-tricarboxamides (BTA) derivatives, now we have deep insights on the mechanism and important parameters for chiral amplifications in supramolecular helices. However, the degree of chiral amplification in the sergeants and soldiers system is still at most approximately 100 (one sergeant per 100 soldiers) for both dynamic helical polymers and supramolecular helices. Because the concept of sergeants and soldiers is widely used for various helices, it is desirable to further extend this limit for both fundamental science and practical applications.As represented by the pioneering works of OkamotoHere, we report an unprecedentedly high degree of amplification of supramolecular chirality in a sergeants and soldiers system using a propeller-shaped aromatic core in which only one sergeant controls the helicity of 500 soldiers in a supramolecular helix, on average . Further In particular, they have suggested that TPA derivatives having amide groups in its side chains self-assemble into supramolecular helices and TPA derivatives selectively adopt (P)- or (M)-propeller chirality according to the handedness of the supramolecular helices in which they are assembled. Inspired by these pioneering works, we wondered how such propeller chirality of TPA would affect the degree of chiral amplification. We then first performed a density functional theory (DFT) computational study to predict the optimal conformation of trimeric OMeA C in cyclohexane exhibited characteristic exciton splitting at 312 nm and 328 nm (3(R)C displayed the mirror-image CD spectrum under the same conditions, indicating the formation of one-handed supramolecular helices (A and 3(S)C in their assembled states  derivatives.ic AOMe , a simplhirality . We thenhirality  and inveFig. S19b, a charaFig. S19c. Tapping Fig. S20, possibld states , we conc3(R)C and 3(S)C. As shown in 3(R)C and 3(S)C exhibited clear mirror-image spectra, with the exception of a few wavenumber ranges highlighted in gray in which the solvent absorption interference was not negligible. The VCD peaks between 1639 and 1584 cm–1 correspond to the stretching vibration of the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O bond and the bending vibration of the N–H bond, respectively C and A/3(S)C at a mole ratio of 9 to 1. We obtained VCD spectra nearly identical to those of 3(R)C or 3(S)C alone, indicating that TPA adopts a single propeller chirality according to the helicity of the supramolecular helices  measurements of methylcyclohexane solutions of  Fig. S22,14b,15 helices .A and 3(S)C and found that one-handed helices of A could be obtained by mixing an unusually small fraction of 3(S)C with A. In other words, the chiral information of the side chains of 3(S)C was efficiently amplified into the handedness of the supramolecular helices of A. To accurately estimate the degree of chiral amplification of the mixed system of A/3(S)C, we performed the sergeants and soldiers experiments using various fractions of 3(S)C C without noticeable changes in the CD spectral shapes. By plotting the g-values of the peak tops at 234, 252, 312, and 373 nm, we detected minor discontinuous changes of the g-values at an approximately 40 mol% fraction of 3(S)C (A and 3(S)C were nearly identical, the peak tops of 3(S)C were slightly red-shifted by 1–2 nm on average C are slightly different. Hence, we assumed that co-assemblies of A and 3(S)C adopt a helical conformation similar to that of either the homo-assembly of A or that of 3(S)C and that the transition of this helical conformation occurs at an approximately 40 mol% fraction of 3(S)C, resulting in the observed discontinuous changes in the g-values. Therefore, in the following studies, we used the g-value at a 20 mol% fraction of 3(S)C as the value for the one-handed supramolecular helices of A. To clarify the magnitude of the changes in the g-values, we used net helicity as a probe for the degree of chiral amplification. As shown in the inset of 3(S)C manipulated the handedness of the supramolecular helices of 333 molecules of A on average. At this high degree of chiral amplification, the average length of the supramolecular helices may affect the result. However, the degree of chiral amplification of this system exhibited no change, even at a 30-fold diluted concentration  . The samof C3(S) . AlthougFig. S21a, indicat Fig. S24, suggest have revealed that two important parameters, mismatch penalty (MMP) and helical reversal penalty (HRP), govern the degree of chiral amplification in sergeant and soldier systems. The MMP is an energetic penalty incurred by a system when chiral sergeants are incorporated into non-preferred helices, whereas the HRP is the energetic barrier that maintains the handedness of supramolecular helices. In general, as these two parameters increase, one-handed helices can be obtained with a smaller amount of sergeants. As expected from the above results, a mixed system of A and 3(S)C exhibited the highest MMP and HRP values ever reported C (A and 3(S)C. We investigated the degree of chiral amplification in the mixed systems with A and its chiral analogues 1m(S)C, 1p(S)C, and 2m,m(S)C, respectively, whose names indicate the number and positions of the stereogenic centers in their side chains C resulted in the lowest chiral amplification, and 1m(S)C and 2m,m(S)C resulted in nearly identical degrees of chiral amplification to that of 3(S)C, suggesting that the role of side-chain chirality is completely different at the para- and meta-positions. Because side chains at the meta-positions sterically hinder the rotation of the phenyl groups to which the side chains are attached, side chains at these positions must affect the propeller conformation of TPA more than those at the para-position. Consequently, the MMP of 1m(S)C originating from the mismatch between the chiral side chains and the propeller chirality might be larger than that of 1p(S)C. An identical trend was observed for soldiers -mA and -pA, whose long alkyl chains at the meta- and para-positions are replaced by methyl groups C, -pA resulted in a nearly identical degree of chiral amplification to that of A, whereas -mA exhibited a much lower degree of chiral amplification C had a higher degree of chiral amplification than that of A and 3(S)C (inset in -pA was fully biased by the addition of only 0.2 mol% 3(S)C. Because -pA starts to self-assemble at a higher temperature during the cooling process than A and -mA  . All anand C3(S)  and S21†fication . From thinset in ; the han Hence, to obtain a high degree of chiral amplification in these dynamic systems, sergeants and soldiers must co-aggregate efficiently. The MMP of the supramolecular helices increases after introducing larger numbers of chiral side chains per sergeant. However, this approach is incompatible with efficient co-aggregation because the chiral side chains decrease the association constant of the sergeants for soldiers because of steric hindrance. However, in the TPA system, because the sergeants and soldiers are both propeller-shaped, the presence of propeller chirality does not lead to the segregation of the sergeants and soldiers, which may be no less important than the MMP and HRP values of TPA systems for chiral amplification. Furthermore, the degree of chiral amplification can be compared over the various TPA derivatives because their helical conformations are always nearly identical, in contrast to supramolecular helices based on planar π-conjugated molecules, such as perylene bisimide. In addition, it is rare that the degree of chiral amplification drastically changes just by altering the position of the stereogenic centers. By utilizing these unique features, we demonstrated that 1p(S)C can switch its role from sergeant to soldier if the partner was changed from A to 3(R)C. As shown in g-value of the mixed system of 1p(S)C and 3(R)C was saturated with the addition of a 4 mol% fraction of 3(R)C C/1p(S)C (1p(R)C or 1p(S)C, if 5 mol% 3(R)C was present. Because 1p(S)C behaves as a sergeant in the mixed system with 1p(S)C and A  states. Fig. S32. By cont)/C1p(S)  and S33†S) and A  and S26†S) and A . Note thvia the dynamic propeller conformation of TPA. Therefore, this study provides valuable insights into the construction of helical structures and a novel design strategy for supramolecular chiral amplification.In conclusion, we have demonstrated unprecedented chiral amplification of supramolecular helices consisting of TPA derivatives in which the handedness of the helices was fully controlled using a 0.2 mol% fraction of sergeants. Such an unprecedentedly high degree of chiral amplification was achieved


RP and SP-configurated RNA diastereomers using a combination of anion-exchange and reversed-phase HPLC, and comment on troubleshooting if their separation appears difficult. Furthermore, we demonstrate the stereochemical assignment of short RP and SP mP-modified RNA diastereomers based on 2D ROESY NMR spectroscopy and we report on the impact of the mP modification on thermal and thermodynamic stabilities of RNA-DNA hybrid and RNA-RNA duplexes.Methylphosphonate(mP)-modified RNA serves as valuable probe to evaluate biomolecular interactions between the nucleic acid backbone and binding partners, such as proteins or small molecules. Here, we describe an efficient workflow for the synthesis of RNA with a single mP modification in diastereomerically pure form. While the automated assembly of mP-modified RNA is straightforward, its deprotection under basic conditions is challenging; a carefully optimized step-by-step procedure is provided. In addition, we demonstrate purification and separation strategies for the  All oligonucleotides are synthesized on a nucleic acid synthesizer (ABI 392) on a 1.5 μmole scale. The solutions of amidites and the solution of the activator 5-(Benzylthio)-1H-tetrazole (Carbosynth) in acetonitrile are dried over activated molecular sieves  overnight. Although 2′-deoxy-5′-O--N2-isobutyrylguanosine 3′--phosphonamidite has to be dissolved in tetrahydrofuran, the corresponding 2′-OCH3 guanosine phosphonamidite is applied in acetonitrile. Compared to unmodified phosphoramidites, a longer coupling time for methylphosphonamidites (6 min) is advisable.In modern solid-phase RNA synthesis, protected nucleoside phosphoramidite building blocks are sequentially coupled to a solid support-bound nucleoside. Each cycle starts with a detritylation step  . Nucleobamidites  and poly2.2O-silyl groups follows standard conditions, with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF). In our hands, both procedures gave unsatisfying yields and qualities of crude RNA products indicating severe degradation. The detailed protocol below is the result of a comprehensive and careful optimization of deprotection conditions to give high quality crude products.Deprotection and cleavage from the solid support is the most critical step in the preparation of mP-modified RNA . For cle2.2.13 in H2O), ethanol, acetonitrile, ethylenediamine.Aqueous ammonium hydroxide solution (28% NH2 is bubbled through until pH 8.0 is obtained. The volume is adjusted to 1000 mL with nanopure water. The buffer solution is stored at 4 °C and diluted to 0.1 M just before use.1 M Triethylammonium bicarbonate (TEAB) stock buffer: Triethylamine  is added to 800 mL nanopure water and CO1 M Triethylammonium acetate (TEAA) buffer: Glacial acetic acid  is dissolved in 750 mL of nanopure water. Then, 101 g triethylamine  is slowly added and the two layers are mixed carefully in an ice bath. The pH is adjusted to 7.0 with triethylamine. Finally, the volume is adjusted to 1000 mL with nanopure water. The buffer can be stored at 4 °C for several months.4NF·3H2O) in tetrahydrofuran (THF).1 M Tetrabutylammonium fluoride trihydrate (BuNanopure water provided by a Millipore-Q System (18 Ω/cm).GE Healthcare), size exclusion column , HPLC system, Dionex DNAPac® PA-100 column (4 mm × 250 mm).Parafilm, 2 mL micro tubes, 50 mL and 100 mL round-bottomed flask, rotary evaporator, high vacuum pump, ÄKTAprime plus system ((1)Transfer the solid support from the synthesis column into a 2 mL screw cap micro tube.(2)3 in H2O)/ethanol/acetonitrile  and agitate the mixture for 30 min at room temperature.Add 0.5 mL of a mixture of concentrated aqueous ammonium hydroxide solution (28% NH(3)Add 0.5 mL of ethylenediamine and agitate the mixture for 5 h at room temperature.(4)Filter the supernatant into a 100 mL round-bottomed flask and wash the remaining solid support twice with ethanol/water . The supernatant and the washings are combined in the 100 mL round-bottomed flask.(5)Add 1 mL of 1 M triethylammonium bicarbonate (TEAB) buffer (pH 8.0).(6)Note: The 100 mL flask is recommended due to possible strong foaming of the solution. Rotavapor bath temperature should be set to or below 40 °C. To avoid degradation of the RNA, try to keep evaporation time as brief as possible.The solution is evaporated and a clear oil is obtained. (7)Dry under high vacuum for 20 min.(8)4NF·3H2O) in THF.Add 1 mL of 1 M tetrabutylammonium fluoride trihydrate (Bu(9)Note: Without agitation of the two phases, the deprotection will not work properly.The suspension is agitated at 37 °C under light exclusion for 16 h. (10)Add 1 mL of 1 M of triethylammonium acetate (TEAA) (pH 7.0).(11)Note: Do not evaporate to dryness.Reduce the volume of the solution by evaporating THF. (12)GE Healthcare, HiPrep™ 26/10 Desalting; 2.6 × 10 cm; Sephadex G25) connected to an ÄKTA Prime Plus system  to get rid of salts. Elute with H2O at a flow rate of 1 mL/min and collect the RNA in a 50 mL round-bottomed flask.Apply the remaining solution to a size exclusion column ((13)The collected fraction containing the RNA is evaporated to dryness using a rotary evaporator. Rotavapor bath temperature should be set to 40 °C or below.(14)Note: At this point the sample might be stored at −20 °C for several weeks.Resuspend the crude RNA in 1 mL of nanopure water (stock solution). Stepwise procedure for the deprotection of mP-modified RNA and cleavage form the solid support after synthesis: 2.3RP and SP diastereomers are separated by reversed-phase chromatography (Dionex ASI-110), pump (Dionex P580), column thermostat (Jetstream 2 Plus), and UV detector (Dionex UVD-170U).The crude mP-RNA products are analyzed and purified by anion-exchange chromatography. tography . For bot2.3.1Dionex DNAPac PA-100 column (4 × 250 mm) at 80 °C. RNA stock solution (2 μL) is diluted to 100 μL and injected into the HPLC system. Flow rate: 1 mL/min; eluent A: 25 mM Tris·HCl (pH 8.0), 6 M urea; eluent B: 25 mM Tris·HCl (pH 8.0), 0.5 M NaClO4, 6 M urea; gradient: 0–60% B in A within 45 min or 0–40% B in 30 min for short sequences up to 15 nucleotides; UV detection at 260 nm.The crude RNA products are analyzed by anion-exchange chromatography on a 2.3.1.1Eluent A (25 mM Tris·HCl (pH 8.0), 6 M urea): Urea  and 100 ml 250 mM Tris·HCl (pH 8.0) stock solution are dissolved in nanopure water and adjusted to 1000 mL.Eluent B (25 mM Tris·HCl (pH 8.0), 6 M urea, 0.5 M NaClO4): Urea , 100 ml of 250 mM Tris·HCl (pH 8.0) stock solution and sodium perchlorate monohydrate  are dissolved in nanopure water and adjusted to 1000 mL.2.3.2Dionex DNAPac PA-100 column (9 × 250 mm) at 80 °C. Flow rate: 2 mL/min; eluent A: 25 mM Tris·HCl (pH 8.0), 6 M urea; eluent B: 25 mM Tris·HCl (pH 8.0), 0.5 M NaClO4, 6 M urea; UV detection: 260 nm. To avoid column overload, small aliquots of the crude 1 mL-RNA stock solution are purified, starting with 20 μL to optimize the gradient (gradient steepness Δ = approximately 8–10% eluent A in B within 20–30 min (for RNA of up to 30 nt)). Fractions containing the pure product are desalted using a C18 Sep-Pak® cartridge. The cartridge is equilibrated with 2 × 10 mL CH3CN (HPLC grade), 3 × 10 mL CH3CN/H2O , 3 × 10 mL H2O and 3 × 10 mL 0.1 M TEAB  into a 50 mL round bottom flask and evaporated to dryness using a rotary evaporator. The purified RNA is resuspended in 1 mL of nanopure water and the yield is determined by UV photometrical analysis. At this point, the sample can be stored at −20 °C for several months. The integrity of the purified RP/SP RNA is analyzed by liquid chromatography–electro spray ionization-mass spectrometry (LC–ESI-MS).The crude deprotected mP-RNA is purified on a semipreparative 2.3.3PR and PS mP-RNA diastereomers is performed by reversed-phase chromatography on a Waters XBridge C18 column  at 40 °C. An aliquot of PR/PS RNA  from the 1 mL-stock solution is diluted into 100 μL nanopure H2O and injected. Flow rate: 1 mL/min; eluent A: 0.1 M ammonium acetate, pH 7.0; eluent B: acetonitrile (HPLC grade); gradient: steepness Δ 3–5% B in A within 25 min; UV detection at 260 nm.Analysis of 2.3.3.1Eluent A : A 1 M stock solution is prepared. Ammonium acetate  is dissolved in 800 mL nanopure water and the pH is adjusted to 7.0 with an ammonium hydroxide solution (28% NH3 in H2O). The volume is adjusted to 1000 mL with nanopure water and the stock solution was stored at 4 °C. Finally, 0.1 M ammonium acetate buffer is prepared by diluting 100 mL of the stock solution with 900 mL of nanopure water. The buffer can be stored at 4 °C for several months.Eluent B: Acetonitrile .GE Healthcare Resource RPC  column because of higher loading capacity, at 40 °C. Flow rate of 2 mL/min; eluent A: 0.1 M ammonium acetate, pH 7.0; eluent B: acetonitrile (HPLC grade); UV detection: 260 nm. To optimize the gradient for separation, only about 20 μL of the stock solution is used. In general, gradients in the range of 0–4% B in A within 25 min for sequences of up to 30 nucleotides are well suited. About 100 μL-aliquots of the stock solution can be injected without overloading of the column. Fractions containing the diastereomerically pure RNA are collected in falcon tubes, diluted with an equal volume of 0.1 M TEAB buffer (pH 8.0) and loaded onto a C18 Sep-Pak® cartridge , 3 × 10 mL H2O and 3 × 10 mL 0.1 M TEAB buffer). After rinsing the loaded column three times with 10 mL H2O, the diastereomerically pure RNA was eluted with 30 mL CH3CN/H2O  into a 50 mL round bottom flask and evaporated to dryness.The mP-RNA diastereomers are separated on a reversed-phase RP and SP mP-RNAs, respectively, are each dissolved in 1 mL of nanopure H2O; these stock solutions can be stored at −20 °C for months. Yields are determined by UV photometrical analysis. To check the diastereomerical purity, 200 pmol of each separated diastereomer is re-analyzed on the XBridge™ C18 column  at 40 °C. Flow rate: 1 mL/min; eluent A: 0.1 M ammonium acetate, pH 7.0; eluent B: acetonitrile (HPLC grade); gradient: steepness 3–5% B in A within 25 min; UV detection at 260 nm.The 2.4®MS, C18 2.5 μm; 1.0 × 50 mm) at 21 °C; flow rate: 30 μL/min; eluent A: 8.6 mM Et3N, 100 mM 1,1,1,3,3,3-hexafluoroisopropanol in H2O (pH 8.0); eluent B: methanol; gradient: 0–100% B in A within 30 min; UV detection at 254 nm.The molecular weights of the purified oligonucleotides are analyzed by mass spectrometry , using a2.52O containing 10 mM KH2PO4 and 50 mM KCl, pH 6.4. The final RNA concentration is about 0.2 mM. The 1H 1D-NMR spectra are acquired using a double pulsed field gradient spin–echo pulse sequence. The ROESY 1H,1H NMR spectra are acquired on a 600 MHz spectrometer (Bruker Avance II+) equipped with a proton-optimized triple resonance NMR ‘inverse’ (TCI) CryoProbe Prodigy (5 mm). The ROESY spectra are acquired at 278 K. The size of the data matrices are 2048 × 300 complex data points, the number of scans is 144, the interscan delay is 1.5 s, and the mixing time is set to 150 ms, resulting in a total measuring time of 20 h for each spectrum.The RNA sample is lyophilized and dissolved in D2.62HPO4, pH 7.0, 150 mM NaCl) is added to give the desired concentration and the solution is transferred into a quartz cuvette (1 cm [800 μL] or 0.1 cm [300 μL] path (Hellma)). The samples are degassed for 2 min in an ultrasound bath and then covered with a layer of silicon oil (dimethylpolysiloxane) to avoid evaporation during the measurements at elevated temperatures. Absorbance versus temperature profiles are recorded at 250 and 260 nm on a UV–VIS spectrophotometer (Varian Cary 100) equipped with a multiple cell holder and a peltier temperature control device. Data are collected after a complete cooling and heating cycle at a rate of 0.7 °C/min. In general, melting curves are measured at five different concentrations ranging from about 1 to 40 μM for bimolecular melting transitions and at two different concentrations for monomolecular melting transitions. KaleidaGraph (Synergy software) was used for the analysis of UV melting profiles. The accuracy of the melting point determination by this method is ±0.3–0.5 °C.An aliquot of the RNA sample is taken from the stock solution and lyophilized to dryness. Melting buffer , or alternatively, methylamine and ammonia in water (AMA) [N4 amino group to avoid transamination at C4 by methylamine [O-TOM groups are deprotected by tetrabutylammonium fluoride in tetrahydrofuran [O-TOM groups of mP-modified RNA and did not require further optimization [The preparation of diastereomerically pure RNA with a single mP backbone modification involves several steps. While the automated solid-phase synthesis of these RNA derivatives follows standard synthesis cycles that do not require adaptations and utilize commercially available 2′-er (AMA) . Under thylamine ,32. Secodrofuran . Howeverdrofuran . Also, odrofuran  gave crudrofuran . This prmization .3G–mP–A instead dG–mP–A linkage (data not shown). We also note that triethylammonium bicarbonate buffer can be replaced by triethylammonium acetate buffer leading to the same results.To further demonstrate the effectiveness of the optimized procedures, they were tested and confirmed for a 27 nt RNA of the same sequence but containing a 2′-OCHRP and SP diastereomers of mP-modified RNA are migrating with almost identical retention times, in particular, when the RNAs are longer than 5–10 nucleotides  under denaturating conditions  at elevated temperatures (60–80 °C). In our experience, these conditions are especially powerful to separate slightly shorter  strands from the target RNA, in particular for RNAs larger than 15–20 nucleotides. The same conditions are also successfully applied to analyze  and purileotides .RP/SP mixture of mP-modified RNA. Two buffer systems, namely triethylammonium acetate (TEAA)/acetonitrile (ACN) buffer (pH 7.0) and ammonium acetate (NH4OAc)/ACN buffer (pH 7.0) were evaluated applying different gradients and temperatures. Interestingly, we found that the NH4OAc/ACN buffer system had better separation power in particular for RNA with more than 15–20 nucleotides (We therefore recommend to utilize reversed-phase chromatography (RP-HPLC) for the separation of the AE-purified leotides .4OAc/ACN buffer system (0–4% B in A within 25 min) also allowed for the direct separation of the crude deprotected RNA products by RP-HPLC , the use of a rather flat gradient for the NH RP-HPLC  with no 3 mP-modified RNA HPLC-conditions comparable to the 2′-deoxy mP-modified counterparts can be applied. However, in general, we observed less difference in retention time between RP and SP 2′-OCH3 mP-RNA diastereomers and therefore their separation required slightly shallower gradients to achieve baseline-separation.Furthermore, we note that for the separation of 2′-OCH4OAc/ACN buffer system on a reversed-phase GE Healthcare Resource RPC  column at 40 °C with a flow rate of 2 mL/min using different gradients has proven very powerful for separation of mP-modified oligoribonucleotides of a length of up to 30 nucleotides. However, as for phosphorothioates [RP mP-RNA diastereomer generally elutes before the SP diastereomer on reversed-phase C18 columns [To summarize, the NHthioates ,35, the  columns .RP and SP mP-modified RNA diastereomers is reliably performed by NMR spectroscopy as originally described by Löschner et al. for mP-modified 2′-deoxydinucleosides [1H,1H NMR measurements (SP diastereomer shows crosspeaks from the methylphosphonate methyl group to aromatic protons of the neighboring nucleobases and to protons at the 2′-position of deoxyguanosine (dG H2′), the RP diastereomer does not show any of these correlations GGGA was investigated and a very similar ROESY correlation pattern obtained that allowed for unambiguous stereochemical assignment . We alsoGAGGA)/5′-d(TCCTCTCG) (GAGGA)/5′-r(UCCUCUCG) (Tm) and thermodynamic data  in aqueous buffer system  were derived from the slope and intercept of the linear regression of the data points in a 1/TM versus  plot; these data are summarized in Tm value of 50.6 °C which is decreased by 4.8 °C when the central G-p-A sequence is replaced by dG-p-A (Tm = 45.8 °C). When the charged phosphodiester is replaced by the neutral methylphosphonate group, stability is further diminished. The reduction depends on the mP configuration. The RP diastereomer forms a more stable duplex (Tm = 42.0 °C) compared to the SP diastereomer (Tm = 40.7 °C), with a difference in Tm of 1.3 °C. Noteworthy, when we measured the duplex stability using the 1:1 mixture of diastereomers, the Tm value was found to be approximately the arithmetic mean of the Tm values measured for the two separated isomers (Tm = 41.4 °C) (data not shown).Methylphosphonate backbone modifications impact on the base pairing stability of nucleic acids. Studies on the thermodynamic stabilities of 2′-deoxy- or 2′-methoxy mP-containing oligonucleotides are rare in literature –20,23,39CCTCTCG)  and of tCCUCUCG) . The theRP and SP diastereomer melted slightly higher at 59 °C and 60 °C, respectively. However, no thermodynamic data was extracted because of the non-two-state melting of this system.At this point we mention that we previously investigated the thermal stabilities of the sarcin-ricin (SRL) stem loop RNA with a mP modification in the loop region . While tRP-configurated methylphosphonate retains higher stability compared to the SP-configurated methylphosphonate.Taken together, our data confirms that the mP modification is destabilizing when positioned in a double helical base-paired region while it has a minor effect when positioned in a single stranded loop region. Moreover, the 4RP and SP-configurated diastereomers by anion exchange and reversed-phase HPLC and provide troubleshooting comments if their separation appears difficult. Furthermore, we have outlined the stereochemical assignment for short RP and SP mP-modified RNA diastereomers by 2D ROESY NMR and have reported on the influence that the mP modification exerts on thermal and thermodynamic stabilities of DNA-RNA hybrid and RNA-RNA duplexes.In this article, we have described an efficient workflow for the synthesis of RNA with a single mP backbone modification in diastereomerically pure form. Synthesis of mP-modified RNA is straightforward. We have elaborated powerful conditions for the most critical step that is basic deprotection of the synthesized RNA under concomitant cleavage from the solid-support; a detailed step-by-step procedure is provided. In addition, we have demonstrated a path to purify and separate the http://dx.doi.org/10.1016/j.ymeth.2016.03.024.Supplementary data associated with this article can be found, in the online version, at Supporting Fig. S1Supporting Fig. S2Supporting Fig. S3


Following cell based counter screening and binding validation, we characterized a hit compound’s anti-viability, activation of PEDF signaling-related genes, anti-wound healing, and anti-cancer signaling properties. This hit compound has potential for future development as a lead compound for treating tumor growth and inhibiting angiogenesis.Laminin receptor (67 LR) is a 67 kDa protein derived from a 37 kDa precursor (37 LR). 37/67 LR is a strong clinical correlate for progression, aggression, and chemotherapeutic relapse of several cancers including breast, prostate, and colon. The ability of 37/67 LR to promote cancer cell aggressiveness is further increased by its ability to transduce physiochemical and mechanosensing signals in endothelial cells and modulate angiogenesis. Recently, it was demonstrated that 37/67 LR modulates the anti-angiogenic potential of the secreted glycoprotein pigment epithelium-derived factor (PEDF). Restoration of PEDF balance is a desirable therapeutic outcome, and we sought to identify a small molecule that could recapitulate known signaling properties of PEDF but without the additional complications of peptide formulation or gene delivery safety validation. We used an  RPSA 50 was calculated as described.PC-3 cells were seeded overnight in a white reflective 96 well plate (Costar) at 1,500 cells per well. Media was aspirated and cells were treated with 100 nM, 500 nM, 1 µM, 10 µM, 20 µM, 40 µM, 60 µM, 80 µM, or 100 µM C3 diluted in the previously described media with the addition of 0.5% DMSO. Compounds were incubated for 24 hours at 37°C with 5% CO2 and assayed for BrdU incorporation using the manufacturer’s kit and protocol . Relative light units were quantified from Brd-U HRP using a GloMax MultiMode Reader. C3 signal was normalized to PC-3 cells exposed only to media with 0.5% DMSO. An ICPC-3 cells were seeded overnight in white reflective 96 well plates at 15,000 cells per well. Media was aspirated and cells were treated for 6, 12, 24, or 48 hours with 1 µM, 10 µM, or 50 µM C3 diluted in the previously described media with the addition of 0.5% DMSO. Media was aspirated and cells were assayed for caspase 3/7 activity using a CaspaseGlo kit (Promega). Relative luciferase units were quantified using a GloMax MultiMode Reader.5 cells per well and incubated with 50 µM C3 or DMSO equivalent in 1× RPMI-1640, 1X anti-anti, and 2% FBS (ThermoFisher) for 48 hours. PC-3 cells were lifted using 20 mM EDTA in 1X DPBS and pelleted before isolating total RNA using the RNeasy Mini Kit (Qiagen). 1 µg of RNA per sample was reverse transcribed using the amfiRivert cDNA synthesis master mix (GenDEPOT). Real-time PCR wells contained 1 µL cDNA template, 2× SYBR Green Master Mix (Applied Biosystems), and 20 µM forward and reverse primers. Primer sequences are provided in Table PC-3 cells were seeded in 6 well plates at 3 × 105 cells per well in complete media until the cells reached confluency. Three horizontal and three vertical scratches were made in the confluent monolayers using a standard 200 µL pipette tip and cellular debris were removed by washing with 1× DPBS three times. 1× RPMI-1640 supplemented with C3 or DMSO was added and cells were incubated for 48 hours as described previously. 0 hour and 48 hour images were acquired using a bright field microscope (VWR) and repositioning at 48 hours was achieved using x,y stage coordinates after washing the monolayer three times with 1× DPBS to remove debris. Images were uploaded and analyzed for scratch geometry using the tscratch program [PC-3 cells were seeded in 6 well plates at 3 × 10 program . First, 5 cells per well in six well plates in complete media and treated with 10 µM C3 or 0.1% DMSO for 72 hours with a media change  at the 48 hour mark. After 72 hours, cells were lifted using 20 mM EDTA in 1X DPBS, pelleted, snap frozen in liquid nitrogen and stored at –80°C until digested for mass spectrometry. A moderate 10 µM dose was chosen from the BrdU and t-scratch functional assays.PC-3 cells were seeded at 2.5 × 10Prior to digestion, cells were lysed using the Barocycler NEP2320 . 50 µl of 100 mM ammonium bicarbonate was added to the cells, and they were lysed at 4°C under 35,000 psi for 45 minutes. 100 µg of protein was isolated for digestion using an acetone precipitation. After removing acetone, samples were reduced and alkylated, and sequence grade Lys-C/Trypsin (Promega) was used to enzymatically digest the extracted protein. All digestions were carried out in the Barocycler NEP2320 at 50°C under 20,000 psi for 1 hour. Digested samples were cleaned over C18 spin columns (Nest Group) and dried. Resulting pellets were resuspended in 97% purified water/3% acetonitrile (ACN)/0.1% formic acid (FA).The samples were analyzed using the Dionex UltiMate 3000 RSLC Nano System coupled to the Q Exactive™ HF Hybrid Quadrupole-Orbitrap MS (Thermo Scientific). Peptides were loaded onto a trap column (20 µm × 350 mm) and washed using a flow rate of 5 µl/minute with 98% purified water/2% ACN/0.01% FA. The trap column was then switched in-line with the analytical column after 5 minutes. Peptides were separated using a reverse phase Acclaim PepMap RSLC C18 (75 µm × 15 cm) analytical column using a 120 minute method at a flow rate of 300 nl/minute. Mobile phase A consisted of 0.01% FA in water and a mobile phase B consisted of 0.01 % FA in 80% ACN. The linear gradient started at 5% B and reached 30% B in 80 minutes, 45% B in 91 minutes, and 100% B in 93 minutes. The column was held at 100% B for the next 5 minutes before being brought back to 5% B and held for 20 minutes. Sample was injected into the QE HF through the Nanospray Flex™ Ion Source fitted with an emission tip from Thermo Scientific. Data acquisition was performed monitoring the top 20 precursors at 120,000 resolution with an injection time of 100 milliseconds.Raw files obtained from the QExactive were uploaded using MaxQuant version 1.5.5.1 . The folt-test with a false discovery rate of 0.05 and an S0 of 0.1. A 1.5 log2 fold cutoff change was applied for Ingenuity Pathway Analysis .The identified protein groups generated by the MaxQuant program were uploaded to the Perseus program version 1.5.3.0 . Site on4 cells were seeded per well into a clear 96 well plate with 50 µL of preformed matrigel media and treated with 1, 10, or 50 µM C3 or 0.1% or 0.5% DMSO in 1× DMEM, 10% FBS (ThermoFisher), and 1× anti-anti. Calcein was added following the manufacturer’s protocols (Cellbiolab) and tube formation images were captured using an inverted fluorescence microscope .EA.hy926 cells were cultured as described earlier. 3 × 1015,000 PC-3 cells were seeded per well in a white reflective 96 well plate and transfected next day using 200 ng of a c-MYC binding site luciferase (pBV-Luc wt MBS 1–4) reporter plasmid and 4 ng of cytomegalovirus (CMV)-LacZ  plasmid for six hours using DMRIE-C transfection reagent according to manufacturer’s instructions. pBV-Luc wt MBS1-4 was a gift from Bert Vogelstein (Addgene plasmid # 16564). Transfection reagent was removed and replaced complete media for an additional 18 hours. Cells were then treated with 1, 10, or 50 µM of C3 for 24 hours. Media was aspirated, 1× passive lysis buffer was added, and cells were freeze-thawed after being stored at –80°C. Luciferase activity detection was measured using the Luciferase Assay System (Promega) and a Glomax Discover luminometer (Promega).t-test was used for pairwise comparisons and one-way ANOVA was used for group comparisons with significance set at p < 0.05. In the case of multiple comparisons, the Holm-Sidak test was used or a false discovery rate was applied.Statistical analysis was performed using GraphPad Prism 6. All assays were run in triplicate with values shown as the mean ± SEM unless otherwise indicated. Student’s


Cytochrome P450 2A5 (Cyp2a5), a mouse enzyme orthologous of human CYP2A6, catalyzes a number of toxicologically important reactions, including the metabolism of nicotine, aflatoxin B1, and several other xeno- and endobiotics. Cyp2a5 expression is complex and not yet fully understood. We investigated inter-strain differences in the activity and mRNA expression of hepatic Cyp2a5. Cyp1a1/2 and Cyp2b9/10 activities were evaluated for comparative purposes. Data on the interstrain differences in the expression and activity of Cyp2a5 are important to select a suitable mouse model for studying CYP2A6-mediated metabolism.O-deethylase) and Cyp2b9/10 (bezyloxyresorufin-O-debenzylase) activities while only Cyp2a5 was enhanced by pyrazole . Inductions of Cyp2a5 activity by PYR and PB were accompanied by increases of Cyp2a4/5 mRNA. PYR and PB did not upregulate heme oxygenase-1 (hmox-1) mRNA expression in any strain, a finding that is apparently at odds with the notion that Cyp2a5 and hmox-1 inductions are coordinated events.Activity of Cyp2a5 (coumarin 7-hydroxylase) was highest in DBA-2 and DBA-1, intermediate in B6D2F1 (hybrid) and low in the remaining strains . Contrasting with the activity, background levels of Cyp2a4/5 mRNA did not differ between high- and low-activity murine strains. Phenobarbital  increased Cyp2a5, Cyp1a1/2  contains supplementary material, which is available to authorized users. CYP2A) encompasses 23 genes and pseudogenes, including four genes identified in mice , three in rats  and three in humans  [Cyp2a5 gene is expressed in the liver and in extra-hepatic tissues , and its activity and expression in the liver is female-predominant [The cytochrome P450 2A gene subfamily  and it can be either up- or down-regulated by pathophysiological conditions such as infections, liver cancer and inflammatory stimuli [Cyp2a5/CYP2A6. The mode by which liver pathological conditions regulate Cyp2a5/CYP2A6 expression and activity, however, remains to be elucidated.Regulation of Cyp2a5/CYP2A6 expression is complex and not yet fully understood.  stimuli –9. HypotCyp2a5 gene locus on chromosome 7, one for high activity and the other for low activity [Strain differences in the constitutive activity of coumarin 7-hydroxylase  and coumarin metabolism have been reported and the activity recorded in the DBA-2 mice is generally higher than that found in other strains –12. Mousactivity , 13, 14.Cyp2a4 or Cyp2a5 genes in the liver. Additionally, we investigated whether the up-regulation of Cyp2a5 activity and expression by known inducers  was necessarily associated with clinical manifestations of liver damage and enhanced expression of heme oxygenase-1 (hmox-1). Data from this study are expected to be of help for selection of a suitable mouse model in toxicological studies dealing with xenobiotic compounds metabolized by Cyp2a5/CYP2A6.This study was designed to investigate interstrain differences in the constitutive activities of Cyp2a5, Cyp1a1/2 and Cyp2b9/10, and Cyp2a5 mRNA expression in the mouse liver. To the authors’ knowledge, no previous study has investigated differences between murine strains in the expression of ®, Curitiba, PR, Brazil) and filtered tap water. The research project was approved by the “Ethics Committee on the Use of Animals of the Oswaldo Cruz Foundation” (CEUA-FIOCRUZ). All procedures were conducted in accordance with Brazilian animal protection and welfare legislation and international guidelines [Female mice, 8–10 weeks old, from the Oswaldo Cruz Foundation (FIOCRUZ) breeding stock  were used. Six mice of the same strain were housed per cage (standard plastic cage with stainless steel cover lids) and white wood shavings were used as bedding. The animals were maintained under controlled environmental conditions  with free access to a commercial rodent diet  was from Cristália Produtos Químicos Farmacêuticos LTDA . All other chemicals used in the experiments were of high analytical grade.Benzyloxy-, ethoxyresorufin, coumarin, EDTA, pyrazole, Bradford reagent, BSA, β-NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, resorufin, umbelliferone and glycine were from Sigma Chemical Co . Phenobarbital  injections of pyrazole , phenobarbital  or phosphate buffered saline solution only  for 3 consecutive days, and were euthanized by cervical dislocation 24 h after the last injection. Blood was taken from the retro orbital sinus immediately before the cervical dislocation. Animals were always treated and killed between noon and 2:00 pm. Mice (N = 18 of each strain) were allocated at random (N = 6 per group) to one of the treatment groups .®, Molecular Devices, USA).After euthanasia, livers were quickly removed, freed from fat and extra tissue, weighed and frozen in liquid nitrogen. Liver microsomal fraction (LMF) was prepared essentially as described by De-Oliveira et al. , except Coumarin 7-hydroxylase activity  was assayed essentially as reported by van Iersel et al.  with a fO-dealkylases  were assayed in 96-well microplates as described by Kennedy and Jones [®, Molecular Devices, USA) with excitation and emission wavelengths set at 530 nm and 590 nm, respectively.Benzyloxy- (Cyp2b9/10) and ethoxy- (Cyp1a1/2) resorufin-nd Jones  with som®, Belo Horizonte, MG, Brazil) adapted to a multi-well plate spectrophotometer reader , and absorbance was registered at 505 nm.Serum alanine (ALT) and aspartate aminotransferase (AST) activities were determined by a colorimetric method using a commercially available kit (Bioclin® and quantified using a Nanodrop® spectrophotometer while cDNA was synthesized using the High Capacity RNA-to-cDNA kit® (Applied Biosystems®) and a T100™ thermocycler (BioRad®). TaqMan® gene expression assays were purchased from Applied Biosystems® . Real-time reactions were performed in a Step One Plus real-time thermocycler (Applied Biosystems®). The relative quantification of the target genes was made using the Q-Gene software application (Equation 3 of the manuscript) [mRNA was extracted from the liver tissue with TRI Reagentuscript) .U test with Bonferroni correction. Statistical evaluation was performed using GraphPad Prism version 5.01 for Windows , and differences were considered statistically significant at a value of P < 0.05.Data following a normal distribution (e.g. enzyme activities) were analyzed by one-way analysis of variance (ANOVA) and Dunnett’s post hoc test. For data that are known not to be normally distributed , the Kruskal–Wallis test was used, followed by the Mann–Whitney Results from this study showed that constitutive (non-induced) activity of COH in the liver microsomal fraction markedly varied among mouse strains . The DBA-2 (D2) strain—the oldest of all inbred strains of mice—ranked first for COH activity (mean ± SEM: 228.4 ± 24.8 pmol/mg ptn/min), while DBA-1 (D1) (142.5 ± 28.3 pmol/mg ptn/min) exhibited the second highest activity. Of the remaining murine strains, CBA, BALB/cAn (BALB) and C57BL/6 (B6) presented COH activities ranging from 32.9 ± 5.0 to 44.5 ± 6.0 pmol/mg ptn/min, while the lowest activities were found in C57BL/10 (B10) (24.4 ± 3.8 pmol/mg ptn/min) and Swiss Webster (SW) mice (24.1 ± 5.3 pmol/mg ptn/min). Owing to its additive mode of inheritance, Cyp2a5 (COH) activity in the liver of the hybrid B6D2F1 (F1) (113.3 ± 11.6 pmol/mg ptn/min) was in between those of its parental high- (D2) and low- (B6) activity strains  enhanced COH activity in all strains  and Cyp2b9/10 (BROD) constitutive activities were far less pronounced than those observed with Cyp2a5 (COH) activity, and ratios of the highest to the lowest strain activity were 2.6 and 2.5-fold for Cyp1a1/2  and Cyp2b9/10 , respectively , a finding that is consistent with the notion that this heterocyclic diazole compound is a selective inducer of Cyp2a, 2j and Cyp2e1 activities in the liver tissue .PB, on the other side, markedly enhanced activities mediated by Cyp1a1/2 and Cyp2b9/10 in all mouse strains, thereby confirming that it causes a pleiotropic induction of hepatic monooxygenase activities. Treatment with PB provoked a nearly fourfold increase in Cyp1a1/2 activity (EROD) in all mouse strains, except for BALB, the IF of which was 7.5  and both are expressed in the mouse liver. Despite their high degree of similarity, Cyp2a4 and Cyp2a5 genes code for enzymes that have distinct substrate specificities: while the former mediates 15-α-hydroxylation of steroid hormones , the latter catalyzes the 7-hydroxylation of coumarin. A single amino acid substitution (Phe209Leu) seems to be sufficient to convert the specificity of Cyp2a5 reaction from coumarin 7-hydroxylase to testosterone 15-α-hydroxylase [Cyp2a4 and Cyp2a5, so that levels of both mRNAs were quantified jointly. As shown in Fig. roxylase . PrimersUsing a PCR-enhanced diagnostic analysis based on Squires and Negishi’s method , Hahnema4, cocaine, griseofulvin, thioacetamide, metals such as cadmium, indium and cobalt and also by some infectious diseases such as hepatitis, malaria and fascioliasis [Cyp2a5 expression, however, is not completely elucidated.A number of studies demonstrated that liver Cyp2a5 can be induced by a myriad of structurally unrelated organic and inorganic chemicals such as PYR, PB, CClioliasis , 27, 28.ioliasis . The exaCyp2a5 gene transcript levels were predominantly due to mRNA stabilization [Results presented here showed that PYR increased levels of total Cyp2a4/5 mRNA in all strains Fig. , a findilization . Subsequlization , 31, 32.The induction of Cyp2a5 by PB, on the other side, seems to involve enhanced translational efficiency and/or protein stabilization in addition to increased transcription rates. For instance, while increasing Cyp2a protein and Cyp2a5 activity (COH), PB did not change levels of Cyp2a4/5 mRNA in the liver of male D2 mice . NonetheThe most striking finding of the interstrain comparison was that, while presenting much higher constitutive activities of Cyp2a5 Table , D2 and It is known that some xenobiotic metabolizing enzyme mRNAs do not correlate well with protein expression and consequently with enzyme activities. Chang et al. , for insmax determined in liver microsomes from D2 was higher than that measured in microsomes from AKR and C57BL/6, the reaction Km did not differ either between D2 and AKR [m was essentially the same, strain distinct COH constitutive activities should result from different amounts of Cyp2a5 protein in liver microsomes. As far as the authors are aware, no previous study has compared the constitutive levels of Cyp2a4/5 (or Cyp2a5) mRNAs between mouse strains using a quantitative real-time PCR method (qPCR). The lack of quantitative data on the Cyp2a5 protein expression levels in the evaluated mouse strains, however, is an important limitation of this study.In principle, increased enzyme activity when mRNAs levels remain unchanged could be explained either because of protein stabilization (which increases protein levels) or due to post-translational/allosteric mechanisms . Since we did not quantify protein levels in the present study, it was not possible to exclude one of these two explanations for the inter-strain differences in the constitutive activity of Cyp2a5. The interpretation that inter-strain differences in Cyp2a5 activity might result from differences in degree of protein stabilization (and protein levels), however, is consistent with enzyme kinetic data provided by other studies. Although COH V and AKR , or betw and AKR . Since KCyp2a5 overexpression [Since Cyp2a5 expression and activity are up-regulated by hepatotoxic agents and some liver pathological conditions , 9, it wpression , 37. PYRpression , 150 mg/pression  or one spression . In the hmox-1 [Some authors postulated that all pathophysiological conditions and hepatotoxic chemicals that up-regulate Cyp2a5 share the common feature of altering cellular redox state in the liver tissue , 39, 40.hmox-1  and in thmox-1 , a concuhmox-1 expression in the livers of BALB, CBA, B6, B10, D2, F1 and SW mice  in the liver of B6 mice 24-h after treatment with a high dose of PYR . Unlike the PYR dose regimen used in our study (100 mg/kg bw/d ays × 3 days), the PYR dose employed by Nichols and Kirby [Data from this study, however, showed that PYR, at a dose regimen that induced Cyp2a5 activity (and Cyp2a4/5 mRNA expression) in the absence of signs of hepatotoxicity, failed to enhance nd Kirby  induced 2 and MeHg were observed in mouse primary hepatocytes [hmox-1 and Cyp2a5 expression, the up-regulation of hmox-1 gene was more rapid and transient as compared to that of Cyp2a5. Moreover, contrasting with a lack of induction of Cyp2a5 expression, an enhanced expression of hmox-1 was still present in hepatocytes from Nfr2 null mice [hmox-1 gene. In other words, while Nfr2 would be critical for regulating Cyp2a5 in any case, inactivation of Bach 1 would induce hmox-1 in the Nfr2 null mouse.Differences in the response of hmox-1 and Cyp2a5 to treatment with PbClatocytes . Althougull mice . AccordiCyp2a4/5 gene transcripts of high-activity strains  did not differ from those of low-activity mice , distinct constitutive activities did not result from different transcription rates and/or mRNA half-lives. The absence of data on Cyp2a5 protein levels was an important limitation of this study. Owing to the lack of quantitative data on protein expression levels it was not possible to elucidate whether constitutive activities of Cyp2a5 correlated with protein levels. Differences in protein stabilization and/or in post-translational/allosteric mechanisms are possible explanations for strain differences in Cyp2a5 constitutive activity.In conclusion, results from this study showed that constitutive activity of Cyp2a5 (COH) in the liver of D2 and D1 were clearly higher than that of the outbred SW or the inbred strains BALB, CBA, B6 and B10. As expected from an additive mode of inheritance, Cyp2a5 activity of the hybrid B6D2F1 was intermediate between those of its high- and low-activity parents . Since background levels of Cyp2a4/5 expression, but did not affect Cyp1a1/2 and Cyp2b9/10 activities in the liver of mice from any strain. As expected from a pleiotropic inductor, PB increased activities of Cyp2a5, Cyp1a1/2 and Cyp2b9/10 as well. In CBA, B10 and SW PB-caused induction of Cyp2a5 activity was unaccompanied by a clear enhancement of Cyp2a4/5 mRNA levels, a finding that is consistent with the prevailing notion that PB-mediated CYP2A induction involves mainly actions at translational and/or post-translational levels (Fig. It was also shown that PYR up-regulated Cyp2a5 activity and els Fig. . Finally


Xenopus laevis that developmental activation of the checkpoint kinase Chk1 at the MBT results in the SCFβ-TRCP-dependent degradation of a limiting replication initiation factor Drf1. Inhibition of Drf1 is the primary mechanism by which Chk1 blocks cell-cycle progression in the early embryo and is an essential function of Chk1 at the blastula-to-gastrula stage of development. This study defines the downregulation of Drf1 as an important mechanism to coordinate the lengthening of the cell cycle and subsequent developmental processes.The early cell divisions of many metazoan embryos are rapid and occur in the near absence of transcription. At the mid-blastula transition (MBT), the cell cycle elongates and several processes become established including the onset of bulk transcription and cell-cycle checkpoints. How these events are timed and coordinated is poorly understood. Here we show in   •Xenopus MBT results in the degradation of Drf1Activation of Chk1 at the •β-TRCP dependentDrf1 degradation is SCF•Chk1 blocks the cell cycle in the early embryo through inhibition of Drf1•Inhibition of Drf1 is an essential function of Chk1 during gastrulation Xenopus laevis that the checkpoint kinase Chk1 causes SCFβ-TRCP-dependent degradation of the replication factor Drf1 at the MBT. Inhibition of Drf1 is an essential function of Chk1, guaranteeing timely cell-cycle elongation.Embryonic divisions lengthen at the mid-blastula transition (MBT) in many metazoa. Collart et al. show in  The early embryonic development of many animals, particularly those that develop externally, involves a rapid expansion in cell numbers. These fast early cell divisions exhibit very little zygotic transcription and rely on maternally supplied products . After aXenopus (One mechanism responsible for timing the events of the MBT involves the nuclear to cytoplasmic (N/C) ratio . In the Xenopus .Xenopus laevis embryos that the increasing N/C ratio titrates out four replication initiation factors Drf1, Treslin, Recq4, and Cut5 , with Drf1 being predominant during cleavage divisions  complex, which is required for replication initiation . In pre-chk1 together with either dbf4 or drf1. Over-expression of wild-type chk1 resulted in cell-cycle arrest as expected  plays any role in Chk1-mediated control of cell-cycle progression in Xenopus. To explore this, we used a mutant of Cdk1 (cdk1-AF) that cannot be inhibited by Wee1/Myt1 phosphorylation because the inhibitory phosphorylation sites threonine 14 and tyrosine 15 are mutated to alanine and phenylalanine, respectively  delays it becoming limiting for S-phase progression at the MBT . To test the MBT C–4E, and the MBT D and 4E, the MBT C. Conver the MBT . Togethein vitro. Since Chk1 and SCFβ-TRCP regulate Drf1 levels at the MBT in vivo, we wondered whether it is this pathway or the out-titration of Drf1 that causes this protein to become limiting at the MBT in vivo. By directly comparing SCFβ-TRCP inhibition with Drf1 over-expression, we observed that stabilization of Drf1 levels, together with the over-expression of Cut5, Treslin, and Recq4, while sufficient for an extra division at the MBT, was not sufficient to reduce cell-cycle asynchrony during the MBT cycles 12 or 13 , immunoprecipitated β-Trcp from MBT-stage embryos  remained stable, an N-terminal fragment (13–467) was degraded at the MBT like the full-length protein , where Φ represents a hydrophobic residue, X represents any amino acid, and pS represents phosphoserine . We did  protein D. Import protein D. To tes of Drf1 E. We conXenopus, also lacks canonical β-Trcp interaction motifs  did not interact with β-Trcp  has little effect on the cell cycle during divisions 12–15 . While over-expression of all four factors allows synchronous cleavages across cycles 12–15  in part caused by Chk1 (grapes) activation, resulting in even greater extension of S phase and the introduction of G2 phase in cycle 14 .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Philip Zegerman  and implemented by the Home Office in the UK, were followed.Regulations for the use of Xenopus research facility designed by Aqua Schwarz. Tanks contained dechlorinated water at 19°C, pH 7.5 and conductivity between 900-1100 μS/cm. Ammonia and nitrite levels were at 0 ppm. The light/dark cycle in the room was 12h/12h. Tanks were populated with 3 females or 6 males. Frogs were fed a diet of 3 x 1 g of 5LP3 frog diet advanced protocol (from labdiet), per frog and per week.Frogs were obtained from NASCO and kept in a Females were induced by injection of 400 U Chorulon (Human Chorionic Gonadotrophin), followed by injection of 50 U PMSG (Pregnant Mare’s Serum Gonadotrophin) from Intervet within 10 to 12 days. Males were killed by injection of an overdose (160 mg) of the anesthetic MS222 dissolved in water.Adult males and females were only used to obtain sperm and eggs respectively.All experiments were performed on embryos.Xenopus laevis were obtained by artificial fertilisation. They were maintained in 10% normal amphibian medium (NAM) 2.4H2O, 1 mM MgSO4.7H2O, 0.1mMEDTA, 0.02 mM NaH2PO4.2H2O, 0.08 mM Na2HPO4.2H2O) (Xenopus embryos were injected at the one or two cell stage (as indicated in the figure legends) with antisense morpholino oligonucleotides (MOs) (dissolved in water) obtained from GeneTools, LLC or with sense RNA obtained by in vitro transcription (as indicated in the figure legends).Embryos of O4.2H2O)  at 20 °CO4.2H2O) . XenopusXenopus embryos using the TriPure reagent (Invitrogen), followed by a LiCl precipitation. cDNA was prepared by reverse transcription, using the Transcriptor First Strand cDNA kit (Roche).Total RNA was isolated from Xenopus laevis chk1 was amplified by PCR and cloned between the EcoRI and XhoI sites of pCS2. cDNA encoding a dominant negative Asp148 to Ala mutant  at the following positions  was cloned between the EcoRI and XhoI sites of Myc-pCS2. The coding sequence of Xenopus laevis cdk1 (Cdk1) and Cdk1AF  was amplified by PCR and cloned between the EcoRI and XhoI sites of Myc-pCS2.The coding sequence of To obtain sense RNA from these constructs, plasmids were digested with Not1 followed by in vitro transcription using SP6 RNA polymerase.treslin, cut5, drf1 and recq4 containing plasmids  were diAnti-Drf1 and anti-Dbf4 were used at concentrations of 1/10000 and 1/3000 respectively in PBS with 0.1% Tween and incubated overnight. at 4°C. Anti-HA, anti-Myc and anti-βTrcp were used at a concentration of 1/1000 in PBS+0.1%Tween and incubated 1 hr at room temperature. Anti-Chk1 was used at a concentration of 1/100 in PBS+0.1%Tween and incubated 1 hr at RT. Goat anti-rabbit (Thermo Scientific 31466) and horse anti-mouse (Vector Laboratories PI 2000) HRP coupled antibodies were used as secondary antibodies and used at concentrations of 1/10000 in PBS+0.1%Tween and incubated for 1 hour at room temperature.Xenopus embryos were frozen in liquid nitrogen and solubilised in lysis buffer containing 1% NP40, 150 mM NaCl, 20 mM Tris pH 7.5, 2 mM EDTA, 50 mM NaF, 1 mM sodium pyrophosphate, supplemented with protease inhibitors (Roche). Yolk and lipids were extracted with Freon and lysates were cleared by centrifugation. Precipitations were performed by overnight incubations with mouse monoclonal anti-Myc coated magnetic beads at 4°C. Unbound proteins were removed by washing four times with lysis buffer and once with phosphate-buffered saline at 4°C. Bound proteins were harvested by boiling in sample buffer, and they were resolved by SDS-polyacrylamide gel electrophoresis.Xenopus embryos were frozen in liquid nitrogen and solubilised in lysis buffer containing 1% NP40, 150 mM NaCl, 20 mM Tris pH 7.5 and 2 mM EDTA, supplemented with protease inhibitors (Roche). Yolk and lipids were extracted with Freon and lysates were cleared by centrifugation. Proteins were precipitated with trichloroacetic acid . After washing the pellet in ice-cold acetone, proteins were dissolved in 1x restriction enzyme buffer 3 from NEB. MnCl2 was added to a final concentration of 0.066 mM. Proteins were then resolved by SDS polyacrylamide gel electrophoresis with resolving gel – 4% acrylamide, acrylamide/bisacrylamide = 29/1, 0.5% agarose, 0.035 mM MnCl2, 375 mM Tris pH 8.8, 0.1% SDS, 0.001 % Temed, 0.05% APS, 0.0125 mM Phos-tag acrylamide (Alpha Laboratories Ltd) and stacking gel – 3% acrylamide, acrylamide/bisacrylamide = 29/1, 125 mM Tris pH 6.8, 0.001% Temed, 0.05% APS. The gel was incubated 3 X 15 min in 10 mM EDTA before Western blotting.Xenopus embryos were dissolved in lysis buffer  and treated with RNAse A for 2 hours at 37°C  and then overnight with proteinase K at 55°C . After phenol/chloroform extraction and ethanol precipitation of the sample, DNA pellets were dissolved in H2O and loaded onto a 1% agarose gel.Movies were made with a Leica MZ FL III microscope at 20°C and analysed with ImageJ (Fiji) software. Numbers of cells and embryos analysed are indicated in the figure legends.Numbers of embryos injected for phenotypical analysis are indicated in the figures and figure legends.The quantification of bulk genomic DNA was performed on three biological replicates and the intensity of the bands was scanned and analysed with ImageJ software.C.C., P.Z., and J.C.S. conceived and designed the experiments, which were all performed by C.C. P.Z. wrote the paper.