Arabidopsis thaliana seedlings.Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs. However, many of the details during plant CCV formation are not completely elucidated. The analysis of isolated CCVs is therefore important to better understand the formation of plant CCVs, their cargos and the regulation of clathrin-mediated transport processes. In this article, we describe an optimized method to isolate CCVs from  Plasma membrane associated receptors, transporters and lipids play an important role in coordinating extracellular signals with intracellular responses. The regulation of plasma membrane proteins is therefore critical for optimal plant development and growth. Besides transcriptional control, endocytosis and subsequent endosomal trafficking of plasma membrane proteins is also essential for correct environmental responses . The endArabidopsis is the eight core-component complex TPLATE (TPC) that is important for clathrin recruitment at the plasma membrane and for the initiation of CME , link the clathrin coat to the plasma membrane bilayer and recognize specific cargo proteins for endocytosis. Another important adaptor complex in n of CME . Once thn of CME . The endn of CME . Clathrin of CME .Arabidopsis mutants. CCVs were originally isolated and purified from pig brains in which they are highly abundant . ARA7pro:mRFP-ARA7 line has been described previously  are supplemented with the following protease inhibitors: 500 μM PMSF and 7.3 μM pepstatin A, 2.1 μM leupeptin-hemisulfate and 1 mM DTT.All the solutions were prepared using ddHlication  with som•500 mM PMSF and 7.3 mM pepstatin A solved in DMSO. Store at -80°C in 200 μL aliquots.•2O. Store at-80°C in 200 μL aliquots.2.1 mM leupeptin-hemisulfate in ddH•2O. Store at -80°C in 200 μL aliquots.1 M dithiothreitol (DTT) in ddH•2 (magnesium chloride), 3 mM EDTA (ethylenediaminetetraacetic acid), 1 mM EGTA (ethylene glycol tetraacetic acid). pH adjusted with 5 N KOH; filter (pore size ≤ 0.4 μm) and store at 4°C until use. Prepare 150 mL and use to prepare CIB∗ and the sucrose solutions∗.Clathrin Isolation Buffer (CIB), pH 6.4: 100 mM MES monohydrate [2-(N-morpholino) ethanesulfonic acid monohydrate], 0.5 mM MgCl•∗: Prepare 100 mL.CIB•∗: Use analytical grade sucrose to prepare 2.5 mL, 11 mL, and 7 mL of 50%, 25%, and 12% w/v sucrose solutions in CIB, respectively. Can be prepared a day before as sucrose takes time to dissolve at higher concentrations. Filter (pore size ≤ 0.4 μm) and store at 4°C until use. Supplement the sucrose solutions with the protease inhibitors before use (see above).Sucrose solutions•2O/30% Ficoll solution∗: Prepare 15 mL of 90% w/v D2O, 30% w/v Ficoll, 100 mM MES monohydrate, 0.5 mM MgCl2, 3 mM EDTA pH 8.0, 1 mM EGTA pH 8.0. Incubate 1 h at 60°C while mixing. Adjust pH to 6.4 with 10 N NaOH and bring to the final volume with ddH2O. Can be prepared a day before. Store at 4°C until use. Supplement the solution with the protease inhibitors before use (see above).90% D•2O/5% Ficoll solution∗: Prepare 15 mL of 18% w/v D2O, 5% w/v Ficoll, 100 mM MES monohydrate, 0.5 mM MgCl2, 3 mM EDTA pH 8.0, 1 mM EGTA pH 8.0. Incubate 1 h at 60°C while mixing. Adjust pH to 6.4 with 10 N NaOH and bring to the final volume with ddH2O. Can be prepared a day before. Store at 4°C until use. Supplement the solution with the protease inhibitors before use (see above).18% D•5x SDS sample Buffer : 310 mM •4 (Osmium tetraoxide). Make 10 μL aliquots and store at -30°C.4% w/v OsO•Methylamine tungstate  ready-to-use 2% solution in water at pH 6.8. Store at 4°C. The solution is stable for up to 1 year after first use.•TM Centrifuge 5424R or equivalent) and accompanying rotor .Laboratory standard refrigerated centrifuge  and accompanying rotor .Refrigerated high-speed centrifuge  with a rotor for 50 mL disposable tubes .Refrigerated high-speed centrifuge  compatible with different rotors and tubes for differential and density gradient ultracentrifugation .High-speed ultracentrifuge (Beckman Coulter Optima•Ultra-Clear centrifuge tubes (14 × 95 mm) for density gradient centrifugations (Beckman Coulter).•Polycarbonate tubes with Cap Assembly for use in Ultracentrifuge Rotors (Beckman Coulter).•Clean mortar and pestles.•Disposable sterile needles 1.6 × 25 mm – 16 G × 1″.•Refractometer.•TEM square mesh support grids, 200 mesh, nickel (Plano GmbH).•Non-magnetic steel forceps.•Scanning electron microscope .2O/Ficoll linear gradient. Medium density fractions are subsequently subjected to ultracentrifugation to obtain a pellet that contains the isolated intact CCVs.For the isolation of CCVs, membranes are first enriched from plant total extracts by ultracentrifugation. The membrane fraction is then separated on a sucrose step gradient and further separated on a D∗) immediately before use. The overview of the whole procedure is schematically presented in Figure Keep all the solutions, mortars and pestles at 4°C overnight and pre-chill centrifuges prior to use. Prepare solutions indicated with an asterisk ((1)2O/30% Ficoll solution∗ and 15 mL 18% D2O/5% Ficoll solution∗.Prepare 15 mL 90% D(2)2O/30% Ficoll solution∗ to each of the two Ultra-Clear centrifuge tubes (14 × 95 mm).Gently add 7 mL of 90% D(3)2O/5% Ficoll solution∗ without disturbing the bottom layer.Slowly fill up the two Ultra-Clear centrifuge tubes (14 × 95 mm) with 7 mL of 18% D2O/5% Ficoll solution∗.Note: The ultracentrifuge tubes must be completely filled up. If the amount of solution is not enough, add more 18% D(4)Close the centrifuge tubes with a plastic film and lay them down very carefully on a small tray. Keep the tubes horizontally in a stable position at 4°C for at least 6 h.Note: Alternatively, the gradient can be generated using a gradient maker.(1)∗. Collect the lysate and keep in a 50 mL tube on ice. Repeat until all seedlings are processed. Distribute the lysate equally into two 50 mL conical tubes. Save 500 μL in a 1.5 mL tube and label as “Lysate” for later analysis.Prepare 30 g of 7-days-old seedlings. In a cold room, grind 5 g of the seedlings in a clean mortar with 5 mL ice cold CIB(2)g at 4°C. Carefully collect the supernatant with a 25 mL disposable sterile pipette into a fresh 50 mL conical tube and keep on ice.Centrifuge the lysate for 10 min at 1,000 ×(3)∗ and collect the lysate in a fresh 50 mL tube. Centrifuge the lysate for 10 min at 1,000 × g at 4°C. Carefully collect the supernatant and add it to the previously obtained supernatant from step 2. Save 500 μL of sample in a 1.5 mL tube and label as “S1” for later analysis.To increase the yield of CCVs, transfer the pellets from the two tubes to a mortar and grind the pellets with 6 mL CIB(4)g at 4°C. Save 500 μL of sample in a 1.5 mL tube and label as “S30” for later analysis.In a cooled high-speed centrifuge, centrifuge the supernatant for 25 min at 30,000 ×(5)g at 4°C.Transfer the supernatant to two 26.3 mL polycarbonate ultracentrifuge tubes and centrifuge for 1 h at 120,000 ×(6)∗, 11 mL 25% (w/v) sucrose solution∗ and 7 mL 12% (w/v) sucrose solution∗.While waiting for the centrifugation, prepare the tubes for the sucrose step gradient. Prepare 2.5 mL 50% (w/v) sucrose solution(7)∗. Slowly add 5 mL of 25% sucrose solution∗ and subsequently add 3 mL of 12% sucrose solution∗ into each tube without disturbing the other layers. Keep the tubes in a tube rack at 4°C until use.Prepare two Ultra-Clear centrifuge tubes (14 × 95 mm), and first layer gently 1 mL of 50% (w/v) sucrose solution(8)Discard the supernatant.(9)∗ each. Transfer the homogenate into two 1.5 mL tubes and centrifuge for 10 min at 16,000 ×g at 4°C.Resuspend the two pellets in 1 mL CIB(10)∗ each. Centrifuge 10 min at 16,000 ×g at 4°C. Pool the supernatant together. This should yield a final volume of approximately 5 mL. Save 200 μL in a 1.5 mL tube and label as “SGL” for “sucrose gradient load” for later analysis.Transfer the supernatant to a 15 mL conical tube. Resuspend the pellets remaining in the 1.5 mL tubes with 1.5 mL of CIB(11)g at 4°C.Carefully layer 2.4 mL of the supernatant on each of the sucrose step gradient prepared in step 7. Centrifuge 50 min at 116,000 ×Note: During this step, carefully handle the ultracentrifuge tubes to avoid disturbing the gradient. Select slow acceleration and slow deceleration.(12)After the centrifugation, collect the 12% (w/v) layer and the 12%/25% (w/v) interface. Transfer each of the collected layers into a fresh 26.3 mL polycarbonate ultracentrifuge tube.Note: Avoid taking the 25% (w/v) fraction to prevent contamination with denser membrane structures.(13)∗ each. Sucrose concentration should be less than 5% (w/v). Centrifuge 50 min at 200,000 ×g at 4°C.Dilute the content of both 26.3 mL polycarbonate ultracentrifuge tube with 13 mL CIB∗ to dilute the sucrose concentration to below 5% (w/v), if necessary.Note: Measure the refractive index of the diluted content using a refractometer. Add more CIB(14)∗. Transfer the homogenate into two 1.5 mL tubes and centrifuge for 5 min at 16,000 ×g at 4°C.Discard the supernatant and resuspend each of the two pellets in 300 μL CIB(15)∗ each. Centrifuge 5 min at 16,000 ×g at 4°C. Add the supernatant to the 1.5 mL tube, which should result in a final volume of approximately 700 μL. Save 100 μL in a 1.5 mL tube and label as “DFGL” for “D2O/Ficoll gradient load” for later analysis.Transfer the supernatant to a fresh 1.5 mL tube. Resuspend the pellets left after step 14 in 50 μL CIB(16)2O/Ficoll gradient prepared in 2.4.1.Carefully remove 500 μL of the DNote: Skip this step if linear gradient was prepared with a gradient mixer.(17)2O/Ficoll gradient without disturbing the gradient. Centrifuge 14 h at 80,000 × g at 4°C.Layer 300 μL of the supernatant obtained in step 15 on each DNote: During this step, carefully handle the ultracentrifuge tubes to avoid disturbing the gradients. Select slow acceleration and slow deceleration.(18)After the centrifugation, keep one of the ultracentrifuge tubes on ice and make a hole in the bottom of the other tube with a G16 needle. Fractionate the entire gradient in 0.2 mL aliquotes by collecting the drops.(19)Repeat step 18 with the remaining tube.(20)Measure the sucrose percentage (w/w) equivalent of each fraction from one gradient with a refractometer. CCVs are present in the fractions between 12% and 15% (w/w) sucrose equivalent (refractive index [at 20°C] = 1.35093 – 1.35568). Collect the corresponding fractions and transfer them to a fresh 26.3 mL polycarbonate ultracentrifuge tube.(21)Repeat step 20 with the fractions from the second gradient.(22)∗ each. Sucrose percentage (w/w) equivalent of the solution should be less than 5 % (w/v). Centrifuge 1 h at 174,000 ×g at 4°C.Dilute the content of the 26.3 mL polycarbonate ultracentrifuge tubes with 8 mL CIB∗ to dilute the sucrose (w/w) equivalent concentration to below 5% (w/v), if necessary.Note: measure the refractive index of the diluted content using a refractometer. Add more CIB(23)∗. Transfer the homogenate to the other 26.3 mL polycarbonate ultracentrifuge tube and resuspend the pellet. Transfer the resulting homogenate in 1.5 mL tubes and centrifuge 5 min at 16,000 ×g at 4°C.Discard the supernatant and resuspend the pellet in one 26.3 mL polycarbonate ultracentrifuge tubes with 70 μL CIBNote: Remove the supernatant completely with a pipette without disturbing the pellet.(24)∗. Centrifuge 5 min at 16,000 ×g at 4°C.Transfer the supernatant to a fresh 1.5 mL tube. Resuspend the pellet left after step 23 in 10 μL CIB(25)Transfer the supernatant to the tube from step 24. The total volume should be 80 μL. This contains the enriched CCVs. Save 70 μL in the tube and label it as “CCV” for later analysis and the remaining 10 μL for electron microscope analysis.(1)Glow discharge carbon-coated 200-mesh nickel grids.(2)Immobilize carbon-coated 200-mesh nickel grids by clasping with non-magnetic negative steel forceps.(3)4 solution . Mix thoroughly by pipetting.In an appropriate fume hood, on a bit of plastic film mix one part of the CCV fraction and three part of 4% (w/v) OsO(4)Carefully deposit 1 μL of the CCV mixture  on the face of each of the immobilized grids. Let dry under the fume hood.(5)Place a droplet of Nano-W ready-to-use 2% solution onto a plastic film. Transfer 10 μL of Nano-W ready-to-use 2% solution on each of the dried mixture-coated grids. Incubate for 1 min. Using a wedge of Whatman paper, wick away excess fluid until the grids are nearly dry. Let dry under the fume hood.Note: Avoid touching the surface of the grids with paper. Accordingly, touch the edges of the Nano-W ready-to-use 2% solution droplet and remove as much solution as possible.(6)Image the grids using a scanning electron microscope in scanning transmission electron microscope (STEM) mode with 30 kV. A 25,000 × magnification  is sufficient to distinguish CCVs from uncoated vesicles.Samples collected during the purification were heated for 5 min at 65°C in 1 × Laemmli buffer and analyzed by immunoblot using antibodies against specific organelles/cellular compartments. SDS/PAGE and immunoblotting were performed according to standard protocols. Antibodies used for immunoblotting were anti-CHC (Agrisera), anti-BIP2 (Agrisera), anti-Sec21p (Agrisera), anti-TOC75 (Agrisera), anti-V-ATPase (Agrisera), anti-RFP .n = 1714 vesicles).To determine the presence of intact CCVs, we examined isolated CCVs from 7-day-old wild-type seedlings under a scanning electron microscope. Negative staining and scanning electron microscopy analysis of the sample showed the presence of intact CCVs Figure . Since uTo further evaluate the purity of CCVs we analyzed the obtained samples by immunoblotting using an anti-CHC antibody and antibodies specific to intracellular organelles Figure . CHC wasWe conducted the same experiment using 7-d-old seedlings expressing the late endosome marker mRFP-ARABIDOPSIS RAB GTPase 7 (ARA7) under the control of its native promoters . When anArabidopsis seedlings grown under standard conditions. This enables the quantitative and morphological analyses on CCVs and CCV-associated proteins from different Arabidopsis mutant backgrounds or upon treatment with compounds of interest. We verified the successful enrichment and isolation of intact CCVs applying this method by immunoblotting and scanning electron microscopy.The method presented here is suitable for the isolation of CCVs from NM, TB, M-KN, and EI optimized the method. NM, TB, and ML conducted negative staining and analysis by SEM. NM and EI wrote the manuscript with the help of ML.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Systematic crystallographic studies of the Cax1−EuxCd2Sb2 series indicated expansion of the unit cell upon an increase in the Eu content, in accordance with a larger ionic radius of Eu2+ vs. Ca2+. The Cax2−EuxCdSb2 composition with x ≈ 0.6 adopts the non-centrosymmetric space group, Cmc21, although the parent ternary phase, Ca2CdSb2, crystallizes in the centrosymmetric space group, Pnma. Two non-equivalent Ca sites in the layered crystal structure of Cax2−EuxCdSb2 get unevenly occupied by Eu, with a preference for the interlayer position, which offers a larger available volume. Similar size-driven preferred occupation is observed in the Eux11−CaxCd6Sb12 solid solution with x ≈ 1.Investigation of the quaternary system, Ca–Eu–Cd–Sb, led to a discovery of the new solid solutions, Ca Zintl phases constitute a unique family of intermetallic compounds with polar chemical bonding. Due to a significant electron transfer, formal charges can be assigned to parts of crystal structures in such compounds, considering that all elements in a given composition achieve a stable closed-shell (octet) electron configuration . In Zint14MnSb11 and related compounds [The energetic separation of the bonding and antibonding states in Zintl phases typically result in semiconducting properties ,8. Sinceompounds ,10,11.Chemical modifications, such as doping, proved to be a convenient tool to optimize electronic properties of Zintl phases. In addition, introduction of extra components can sometimes yield unexpected multinary compounds with new structural features ,13,14. Ix1−EuxCd2Sb2 (x ≈ 0.3–0.9) were prepared by the flux technique, using molten lead as a flux. Elemental Ca, Eu, Cd, Sb, and Pb  were loaded in alumina crucibles with the molar ratio (Ca + Eu): Cd: Sb: Pb = 1:2:2:10. The crucibles were sealed in evacuated fused silica tubes. After that, the sealed tubes were moved to programmable muffle furnaces, where the reactions were carried out at 1223 K for 24 h (ramp rate 200 K/h). The Pb flux was removed at 773 K (cooling rate of 5 K/h) by centrifugation, after which the tubes were brought back into the glovebox and crack-opened. In all cases, the main products were well-formed single crystals of Cax1−EuxCd2Sb2. However, the refined Ca: Eu ratios were always different in comparison with the nominal compositions. For example, the sample with the starting ratio, Ca:Eu = 1:1, produced Cax1−EuxCd2Sb2 crystals with x ≈ 0.7 (Ca:Eu ≈ 1:2.3), whereas the reaction with nominal Ca:Eu = 1:3 yielded crystals with x ≈ 0.9 (Ca:Eu ≈ 1:9). In the latter sample, small single crystals of the Cax2−EuxCdSb2 composition with x ≈ 0.6 were also found as a side product. Attempts to produce this solid solution in larger quantities starting from the composition, Ca:Eu:Cd:Sb:Pb = 1:1:1:2:10, and using the same temperature profile as before resulted in Eux11−CaxCd6Sb12 with x ≈ 1 as the main phase.All weighing and loading manipulations were performed inside an Ar-filled glovebox with oxygen and moisture levels of less than 1 ppm. Caα radiation, λ = 1.5418 Å). Data were gathered in a Θ–Θ mode between 10° and 75° with a step size of 0.05° and 2 s/step counting time. The diffractometer was operated inside a nitrogen-filled glovebox to prevent possible deterioration of the samples upon exposure to air.PXRD patterns were collected on a Rigaku Miniflex diffractometer . Data were gathered at 200 K, maintained by a stream of cold nitrogen gas. The raw data were integrated using the program, SAINT [F2 using SHELXL [http://www.ccdc.cam.ac.uk/conts/retrieving.html .Suitable single crystals were picked under dry Paratone-N oil and mounted on low-background plastic loops. Data collection was performed on a Bruker SMART APEX CCD diffractometer equipped with monochromated Mo Km, SAINT . Semiempm, SAINT . Crystalg SHELXL . Atomic g SHELXL . Full de2,  using the SIESTA code [x2−EuxCdSb2 structure with x ≈ 0.6. The AE1 and AE2 sites were treated as fully occupied by Eu and Ca, respectively . The Perdew-Burke-Ernzerhof parametrization of the generalized gradient approximation (GGA) functional was employed [x2−EuxCdSb2 were relaxed down to residual forces of less than 10−2 eV/Å. Double zeta polarized orbitals were employed as a basis set. The Brillouin zone was sampled by a 18 × 12 × 10 k-point grid and the mesh cut-off was set to 800 Ry after checking for convergence. Strong correlations of the Eu 4f orbitals considered by applying the GGA + U approach with Ueff = 7 eV . For coimonides ,24, we aanalysis  as implex1−EuxCd2Sb2 solid solution crystallizes in the space group, Pm1, and adopts the CaAl2Si2 structure type (Pearson code hP5), which can be also viewed as a ternary derivative of the La2O3 type (x1−EuxCd2Sb2 with x = 0.29(1) are presented in The Ca2O3 type a. Atomic4 tetrahedra alternating with hexagonal layers of AE atoms, where AE = Ca + Eu. The AE atoms are six-fold coordinated by the adjacent Sb atoms, forming almost perfectly regular octahedra. Assignment of the formal charges to the atoms in the structure yields an electron-balanced composition according to the notation (AE2+)(Cd2+)2(Sb3−)2. The end members of these series with x = 0 (CaCd2Sb2) and x = 1 (EuCd2Sb2) are also known and crystallize in the same structure [AETM2Pn2 composition  adopt this type [The crystal structure accommodates two-dimensional slabs of corner- and edge-sharing CdSbtructure . In addihis type ,28,29. Mhis type ,34,35,36x1−EuxCd2Sb2 series, the lattice parameters and the unit cell volume gradually increase on going from the Eu-poorer to the Eu-richer compositions  = 1.00 Å, r(Eu2+) = 1.17 Å [2Si2 structure.In the Caositions b, in acc= 1.17 Å ). The VeAE2+ cation in AECd2Sb2 (AE = Ca + Eu), the AE–Sb contacts become progressively longer upon increasing the level of Eu substitution , was discovered as a by-product in the sample containing Cax1−EuxCd2Sb2 as the main phase. The crystal structure of Cax2−EuxCdSb2 belongs to the Yb2CdSb2 type , in contrast to the parent ternary phase, Ca2CdSb2, crystallizing in its own type  [AE2+)2(Cd2+)(Sb3−)2, where AE stands for Yb or Ca. The main difference between the two types stems from non-identical stacking of the layers built up of corner-sharing CdSb4 tetrahedra. Whereas in the non-centrosymmetric Yb2CdSb2, the adjacent Cd-Sb layers are related only by translation, in Ca2CdSb2, similar layers alternate along the stacking direction with their crystallographically inverted counterparts. A crystal of the new solid solution with the composition, Caup Pnma) . The two2CdSb2 and Ca2CdSb2 can be understood as two different ways of occupying tetrahedral voids in otherwise isostructural Yb–Sb or Ca–Sb substructures [2CdSb2 structure [2CdSb2 remains the only representative of its type. In this regard, it is important to mention that the Zn-variants with the same composition are totally different (defect ZrBeSi-type structure) [2CdSb2”, with either a Ca2CdSb2 or Yb2CdSb2 structure type, is not known to exist (to date). Apparently, the Ca2CdSb2 structure becomes destabilized upon isovalent substitution of Ca with Eu, yielding a solid solution with the non-centrosymmetric Yb2CdSb2 atomic arrangement  , and theructure) ,43. Withangement a. Furthex2−EuxCdSb2 (x ≈ 0.6), two nonequivalent AE sites (AE = Ca + Eu) are located between the Cd-Sb layers (AE1) and within the layers (AE2), respectively. The former site adopts a distorted octahedral coordination by the Sb atoms, while the latter position displays a square-pyramidal coordination , Bax2−xACdSb2  [x2−EuxCdSb2 [In the crystal structure of Cadination b. Due to Eu, Yb) , and Yb2EuxCdSb2 ,39.U scheme for the idealized CaEuCdSb2 composition after optimizing the lattice parameters and atomic coordinates. The electronic density of states (DOS) is shown in 2 displays an electronic bandgap of about 0.5 eV. This value is larger than the 0.1 eV bandgap calculated for the isostructural YbEuCdSb2 [EF) mainly show the Sb(5p) character, indicating a significant electron transfer onto the Sb atoms. A strong hybridization of these states with Cd(5s) and Cd(5p) occurs in the energy window, −5.5 eV < E–EF < 0. The s, p, and d states of Ca and Eu also demonstrate a sizeable contribution in this energy region. A high peak of the Eu(4f) character is localized around E–EF = −3.7 eV. The converged magnetic moment on the Eu atom is 7.0 µB, in accordance with the 4f7 ground term of the Eu2+ ion. Similar to the case of YbEuCdSb2, the Eu f orbitals cannot be considered completely inert in this case, since an apparent hybridization with the Sb(5p) states takes place around the peak position. In addition, a “tail” of the 4f peak extends up to the top of the valence band, hybridizing further with the Sb states. Well below the Fermi level, the DOS is dominated by the Sb(5s) states, corresponding to the Sb lone pairs, and by the occupied Cd(4d) states.Electronic structure calculations were performed within the GGA+bEuCdSb2 . This isEF gives rise to a broad domain of antibonding character. This finding suggests that the typically core-level 4f orbitals may play a role in the crystal structure formation/selection. The involvement of the 4f states in chemical bonding can be important, alongside atomic size factors, for understanding the stability of compounds that are known to form only with certain rare-earth elements, such as the binary RE3Bi7 phases with RE = Nd and Sm [EF, with a small domain of antibonding states in the near vicinity of the Fermi level  revealed optimized bonding interactions for the Ca–Sb and Eu–Sb pairs c,d. Mostd and Sm . The Cd–mi level d. A simitructure .x11−CaxCd6Sb12 crystallizes isotypically to the parent Eu11Cd6Sb12 compound , x ≈ 1 in the studied crystal must be close to the upper limit of the Ca substitution for Eu, since the starting mixture contained a large surplus of Ca . The crystal structure likely becomes unstable at higher Ca contents due to geometric/size factors. Accordingly, the pure ternary Ca phase has not been reported. This result is in line with the study on the isostructural solid solution, Eux11−YbxCd6Sb12, where the Sr11Cd6Sb12 type is retained only up to x ≈ 2 [2+ and Yb2+: r(Ca2+) = 1.00 Å, r(Yb2+) = 1.02 Å for coordination number 6 [Euto x ≈ 2 . The simnumber 6 .11Cd6Sb12 and isostructural compositions has been described in detail previously [4 tetrahedra propagating along the monoclinic b axis , except AE5, are six-fold coordinated by Sb. AE5 resides in a five-fold coordination environment with an average AE–Sb bond length of 3.35 Å. A six-fold coordination of AE5 can be completed by including an Sb atom located at a much longer distance of 3.98 Å. Similarly, the six-fold coordination of the AE1 site (<d(AE–Sb)> = 3.43 Å) can be regarded as a highly distorted square antiprism (coordination number 8) with two additional Sb atoms at 4.02 Å and 4.03 Å from AE1. Although such long distances can hardly be considered bonding interactions, when considered, they provide an estimate of the available volume offered by the Sb coordination environment around each AE position. In fact, the occupancy of the AE sites by Eu correlates well with the volume of such “extended” polyhedra  states may be relevant for chemical bonding in this phase. Finally, the Eux11−CaxCd6Sb12 solid solution adopts the same structure as the parent Eu11Cd6Sb12 compound (Sr11Cd6Sb12 type). The distribution of Eu over the non-equivalent AE sites (AE = Eu + Ca) correlates well with the available volume offered by these positions. Although the pure calcium counterpart does not exist, the Eux11−CaxCd6Sb12 appears to extend up to x ≈ 1. In the realm of Zintl chemistry, formation of mixed crystals has been widely employed as a tool for electronic property optimization. Results of the present study demonstrate that, in addition to this capability, solid solutions may provide a way of stabilizing crystal structures not observed for the end members of the corresponding series.In summary, using a Pb flux synthesis, we have discovered three new series of solid solutions in the Ca–Eu–Cd–Sb system. The Ca


Circular Dichroism (CD) spectra show the disruption of daunomycin dimers, suggesting the end-stacking and groove-binding of the daunomycin monomer. Proton and phosphorus-31 Nuclear Magnetic Resonance (NMR) spectroscopy show a sequence-specific interaction and a clear proof of absence of intercalation of the daunomycin chromophore between base quartets or stacking between G-quadruplexes. Restrained molecular dynamics simulations using observed short interproton distance contacts depict interaction at the molecular level. The interactions involving ring A and daunosamine protons, the stacking of an aromatic ring of daunomycin with a terminal G6 quartet by displacing the T7 base, and external groove-binding close to the T1–T2 bases lead to the thermal stabilization of 15 °C, which is likely to inhibit the association of telomerase with telomeres. The findings have implications in the structure-based designing of anthracycline drugs as potent telomerase inhibitors.The anticancer drug daunomycin exerts its influence by multiple strategies of action to interfere with gene functioning. Besides inhibiting DNA/RNA synthesis and topoisomerase-II, it affects the functional pathway of telomere maintenance by the telomerase enzyme. We present evidence of the binding of daunomycin to parallel-stranded tetramolecular [d-(TTGGGGT)] The steady-state response increases with concentration in the range between 30–960 μM  analysis using fluorescence shows a slope change between approximately linear regions at a mole fraction of daunomycin of ~0.50 and 0.66, yielding stoichiometry ratios of ~1:1 and 2:1 for the daunomycin:[d-(TTGGGGT)]4 complex ]4 at D/N = 1.0–4.0  in free daunomycin and T7H6/G6H8 protons in free DNA (~1.5 × 10−10 m2/s) are greater than that for bound DNA (~1.0 × 10−10 m2/s) at D/N = 2.0 at 25 °C. This shows that ligand-bound DNA undergoes diffusion at a slower rate as a stable complex upon binding 4. Intranucleotide and sequential internucleotide NOE correlations, between base H8/H6 and deoxyribose H1′/H2′/H2′′ protons ]4  was made4  by rigor spectra ,40,41. T protons . A singl protons , confirm4 was titrated with daunomycin at D/N = 0.0–4.0 and monitored by 1H and 31P NMR at several D/N ratios at three different temperatures: 25 °C, 30 °C, and 40 °C. Two-dimensional 1H-1H NOESY, 1H-13C HSQC, and 1H-31P HMBC spectra were recorded at D/N = 1.0, 2.0, 3.0, and 4.0 at 25 °C. 1H-1H NOESY and 1H-13C HSQC spectra were also recorded at 35 °C at D/N = 1.0, 2.0, and 3.0. The progressive addition of daunomycin to 1.16 mM of [d-(TTGGGGT)]4 resulted (3 protons shows that there is no loss of fourfold symmetry of [d-(TTGGGGT)]4 upon complexation. The unambiguous assignment of all non-exchangeable and exchangeable protons of [d-(TTGGGGT)]4, as well as daunomycin protons in complex at D/N = 1.0–4.0, was accomplished (4 (4 complex (1H-1H NOESY (1H-13C HSQC (1H-13C HSQC spectra (3 resonance (3.67 ppm). The carbons attached to them resonate at ~119 ppm  and deoxyribose H1′ (82–92 ppm)  of deoxyribose H5′/5′′ (3.7–4.4 ppm) separate out from those of 4OCH3, 7H, 5′H, 4′H, and 3′H protons (3 resonances (1.7–2.8 ppm) that overlap with deoxyribose H2′/H2′′ (2.2–3.0 ppm) have 13C signals close to that for free drugs in the range 22–35 ppm . The existence of a single set of resonances for both ligand and DNA show that a well-defined stable complex is formed, which refers to the major conformer present in the ligand–DNA complex.The quadruplex [d-(TTGGGGT)]resulted a,b in thmplished  by an anished  . The 13C protons . Similar2–35 ppm , which a4 resulted in the severe broadening of G6NH, T7H6, and T7CH3   showed that daunosamine sugar may interact with the DNA backbone in the bound complex. Although DNA peaks sharpened at D/N = 2.0, the daunomycin peaks remained broader than the DNA base and imino signals. The existence of only one set of daunomycin signals requires the ligand to be in fast exchange among all of the possible bound conformations; that is, due to either of the two sites or both DNA sites being occupied by the ligand. The hydroxyl protons at the 6, 11, 9 and 4′ positions were not observable in any spectra of complex, even at low temperatures, that is, 5 °C.The initial addition of 0.2 and 0.5 mole equivalents of daunomycin to [d-(TTGGGGT)]nd T7CH3 a,b, whic complex ,39,40,41drifting . On the .15 ppm) , as well.15 ppm) a,b at D/ractions ,43. Appaprotons, , so thatprotons,  due to t/N = 4.0 . The cha1H-31P HMBC spectra using 3J and 4J coupling with (H3′)n/(H5′/H5′′)n+1 and (H4′)n/(H4′)n+1, respectively in free  to a more flexible BII conformation  with 1 Kcal/mole greater energy induces a downfield shift ~1.5–2.0 ppm, which is required to open the base pairs from 3.4 Å to 6.8 Å to permit intercalation between two base pairs. Besides, the widening and narrowing of the ester O-P-O bond angle due to distortions caused in the DNA backbone lead to small upfield and downfield shifts, respectively. Purely electrostatic interactions between the drug and the DNA in drug–DNA complexes produce small upfield shifts. The dispersion in 31P chemical shifts may also result from different populations of BI and BII conformational states [3 moiety to phosphates of DNA or the widening of the ester bond. The absence of a large downfield shift completely rules out the opening of base pairs to allow classical intercalation at any step along the DNA. These results clearly prove that daunomycin binds to d-(TTGGGGT)]4 externally, and certainly not by the classical intercalation mode of binding.The phosphorus-31 NMR signals were assigned using standard strategies by  in free  and comp in free  at 25 °C upfield . The drigeometry . Theoretgeometry ,46 that l states ,46. The 4 complexes closely resemble that of free [d-(TTGGGGT)]4 having right-handed helix and anti glycosidic bond rotation in all guanines. The presence of all sequential NOEs of H8/H6 with H1′/H2′/H2′′ , these NOEs were also seen at a short mixing time (200 ms), so the chances of a combination of transferred NOEs or exchange effect of two orientations of a single molecule can be excluded. There were no detectable NOEs from daunomycin to any part of the interior G4 plane, and this plane therefore appears to have no contact with the ligand. Also, the chemical shift of G3/G4/G5 protons were not significantly influenced by ligand binding, although the G3H8 showed line broadening. We observed some extra resonance peaks of low intensity, particularly in 1.2–1.8 ppm range, which was apparent in NOESY spectra at D/N > 2.0. Some of these signals give weakly intense intermolecular NOEs with ring D protons of daunomycin. Such NOEs, which do not match with the sharp major assigned TCH3 peaks, were not considered for analysis. These presumably arise due to a presence of a minor conformation of complex.The sequential NOE cross-peaks among adjacent guanine imino protons and imino to base protons  confirm H2′/H2′′  indicateobserved . In addiobserved , indicat protons . Three N4. The restrained Molecular Dynamics (rMD) simulations approach that was used to arrive at a conformation that fits into observed NOE data included 138 intra-DNA and 11 intra daunomycin ] complex , in whic/2′H/3′H ,38. On t 3′H/5′H . The NOE complex . Besides complex  would bemplex 4 apparently follows a three-state process  in which an intermediate conformation of G-quadruplex DNA exists that has G3NH, G6NH in unfolded form, and G4NH and G5NH as still folded, so that Tm1 ~65 °C (±5 °C). Meanwhile, Tm2 may be comparatively quite high, but this could not be ascertained, as data was not recorded beyond 80 °C 4 e.g., T1/T2/T7 nucleotide residues. This may be evidenced from the imino protons of thymine not being observed, since they were in fast exchange with solvent orimeter . The the± 0.1 °C . This in± 0.1 °C . In the  solvent , as mentrmograms  with NH rmograms , it may /N ratio d, which 4 leads to the thermal stabilization of the DNA quadruplex. The observed ΔTm ~15 °C is significantly higher than the values at 0 °C and 5 °C that have been reported in the literature for 21-mer human telomeric DNA in Na+ A in Na+  and K+ 2, etc. 2, etc. ,45,47. G etc. 4 complex using fluorescence spectroscopy. Two sets of samples, that is, free daunomycin and bound daunomycin in a daunomycin–DNA complex, were prepared separately. In a set of daunomycin–DNA complexes, the total concentration of DNA and daunomycin was kept constant at 7 μM, while the relative mole fraction of daunomycin and DNA was varied. In the other set of free daunomycin, the concentration of daunomycin in a sample was kept identical to that of the corresponding sample of the daunomycin–DNA complex. The fluorescence emission was measured at λem = 592 nm using λex = 480 nm at a constant temperature of 25 °C. The difference in the fluorescence intensity of free (F0) and bound (F) daunomycin  corresponding to each sample in two sets was plotted as a function of the mole fraction of daunomycin. The inflection point indicates the change in slope, which gives the stoichiometry of the daunomycin bound to the G-quadruplex according to the following equation:n is the stoichiometry of the complex.A continuous variation analysis procedure i.e., Job Plot, was used to establish the binding stoichiometry of the daunomycin [d-(TTGGGGT)]4 was kept constant as 15.0 μM, with daunomycin added progressively to obtain various D/N ratios in one set of experiments. In another set of experiments, daunomycin concentration was kept constant at 400 μM, with DNA added stepwise to it to obtain various D/N ratios at 25 °C. The final spectra were averaged over two scans, then baseline was subtracted and smoothed by using the Savitzky–Golay algorithm provided by Chirascan software 4.5.1848.0 .Circular dichroism experiments were performed using a model Chirascan  spectropolarimeter equipped with a temperature-controlled cell holder using a quartz cuvette of 10-mm path length. Before igniting the xenon lamp and throughout the course of the experiments, the instrument was continuously purged with pure nitrogen gas using a nitrogen generator . The CD spectra were recorded at 25 °C using a 10-mm quartz cuvette in the range of 200–600 nm, bandwidth = 1 nm, step size = 0.5 nm, and time per point = 0.25 s. During titrations, the concentration of [d-(TTGGGGT)]4 was prepared by heating DNA sequence d-(TTGGGGT) in 10 mM of KBPES buffer (in 90% H2O and 10% D2O solvent) containing 10 mM of K2HPO4, 1 mM of EDTA, and 100 mM of KCl (pH 7.0) at 90 °C for 5 min, and kept overnight at room temperature. Drug stock was prepared in water directly, and different drug (D) and nucleic acid (N) ratios, D/N, were obtained from the titration of the drug accordingly. 1H and 31P resonances were calibrated using 0.1 µL of 0.1 M of trimethylsilyl propionic acid (TSP) as the standard internal reference, and 85% phosphoric acid as the external reference, respectively. All of the NMR spectra were recorded in a 500-MHz Bruker Avance NMR spectrometer equipped with a TXI (triple inverse) probe, BBO (Broad Band Observed) probe, and BVT (Variable Temperature Unit) at Central NMR Facility, IIT Roorkee, India. One-dimensional (1D) 1H spectra were recorded with the zgpr pulse program from Bruker library parameters for achieving water suppression with data points (TD) = 64 K, number of scans (NS) = 64–128, pulse width (P1) = 11.49–14.9 µs, spectral width (SW) = 20 ppm, and relaxation delay (D1) = 2.0 s at different temperatures. 1D 31P spectra were recorded at different temperatures by pulse program zgpg30 with TD = 64 K, NS = 64–128, P1 = 13.60–14.9 µs, SW = 10 ppm, and D1 = 2.0 s. Thermal melting studies were performed by acquiring the 1D 1H NMR spectra of free DNA and daunomycin [d-(TTGGGGT)]4 complexes at D/N ratios = 1.0–4.0 in the range of 25 °C to 80 °C. All of the samples were incubated for 10 min at respective temperatures before performing any experiment. Two-dimensional (2D) 1H-1H NOESY experiments were done using the pulse program noesyphpr with States-TPPI mode in the TXI probe at 25 °C along with mixing times (τm) = 400 ms, 250 ms, and 200 ms, NS = 64, data size = 256 (t1) × 2048 (t2), P1 = 11.49–14.9 µs, SW = 20 ppm on both F1 and F2 dimensions, and D1= 1.9 s at D/N = 1.0, 2.0, 3.0, and 4.0. 2D 13C-1H Heteronuclear Single Quantum Coherence (HSQC) experiments were carried out with a TXI probe to find out the proton and carbon correlation of free daunomycin, free DNA, and daunomycin [d-(TTGGGGT)]4 complexes  at 25 °C using pulse program hsqcetgpsi with NS = 64, data size = 256 (t1) × 2048 (t2), SW = 200 ppm in the F1 dimension, and 20 ppm in the F2 dimension and D1 = 1.9 s. 2D 1H-31P Heteronuclear Multiple Bond Correlation (HMBC) experiments were recorded with a BBO probe using pulse program hmbcgpndqf (gradient) at 25 °C with NS = 64, data size = 256 (t1) × 2048 (t2), SW = 10 ppm along the F1 dimension and 20 ppm along the F2 dimension, and D1 = 1.9 s at D/N = 1.0, 2.0, 3.0, and 4.0. Interproton distances were calculated using the isolated spin pair approximation (ISPA) method from the 2D 1H-1H NOESY spectra of complex recorded at 25 °C using τm = 250 ms, as it was within the linear approximation range. The integration of well-separated cross-peak volumes was done using SPARKY software 3.114, and subsequently, distances were calculated using thymine H6-CH3 as a reference distance (2.95 Å), assuming the NOE cross-peak intensity to be inversely proportional to the sixth power of distance. A range of ±0.5 Å was used as the distance restraint to avoid errors during integration. Diffusion Ordered SpectroscopY (DOSY) spectra was acquired for free daunomycin, free quadruplex DNA [d(TTGGGGT)]4, and daunomycin-[d(TTGGGGT)]4 complex using the following acquisition parameters: pulse program = ledbpgppr2s, data points = 16 K, spectral width = 20.0 ppm in the F2 direction and 16 free induction decays in the F1 direction with number of scans (NS) = 8. During the experiment, the diffusion time (Δ or D20) = 50 ms and diffusion gradient length (δ or P30) = 2.2 ms were kept constant, while the gradient range varied from 5 G/cm to 95 G/cm in 16 steps with a linear ramp at 25 °C. The gradient shape SMSQ 10 and 100 were used with eddy current D21 = 5 ms. The diffusion coefficients were measured from the DOSY experiment using the relation between translational self-diffusion and the measurable NMR parameters, as given by the following equation:0 is the maximum peak intensity, Dt is the translational diffusion constant (m2/s), γH is the gyromagnetic ratio of a proton (2.67 × 104 G−1 s−1), δ is the duration of the gradient, Δ is the time between gradients, and Gz is the strength of the gradient (in G/cm). Data was plotted as −ln (I/I0) versus γH2δ2Gz2 (Δ − δ/3), and Dt was calculated from the slope of the plot. The diffusion coefficient (Dt) was obtained from the slope of the linear graph of −ln (I/I0) versus γH2δ2Gz2 (Δ − δ/3) using diffusion decay curve fitting carried out with the SimFit algorithm in Topspin 3.5.First, 1.16 mM of G-quadruplex DNA [d-(TTGGGGT)]fδ and bδ are the chemical shift (ppm) values of the free [d(TTGGGGT)]4 and daunomycin [d(TTGGGGT)]4 complexes, respectively, k0 and k1 are the proportionality constants, tC and CNt are the total concentrations of drug and nucleotide respectively, and bK is the binding affinity.To estimate the binding affinity of the drug, the NMR titration data of the drug–DNA complex was used. We tried to fit the data in non-linear curve fitting regression analysis by giving the user-defined equation in OriginPro 2018 .(3) δb−4 was obtained from PDB ID:139D, and was used further by replacing aromatic bases in the BIOPOLYMER module  for maintaining the potential setup. The initial structure of daunomycin was built using restraints obtained from the NOE cross-peaks builder module of INSIGHT II (version 2005). Distances were categorized as strong (s) = 2.0–3.0 Å, medium (m) = 3.0–4.0 Å, and weak (w) = 4.0–5.0 Å. The upper and lower bounds of distances were set as 0.5 Å for intermolecular restraints. A force constant of 10.0 kcal mol−1 Å−2 was used for each NOE contact. Daunomycin was placed manually at the position and distance obtained from the NOE cross-peaks and minimized using 1000 steps of steepest descent and conjugate gradient using a constant valence force field (CVFF) in DISCOVER software. Simulated annealing restrained molecular dynamics protocol was used as a tool to search for conformations that fit into the experimental NOE data and enable the visualization of 3D structural models. The molecule was heated to 800 K in steps of 100 K, so that the chances of a molecule being trapped in local minima was lost, and it reached global minima instead. The simulations were carried out for 1000 iterations, each with a time step of 1 fs at 800 K. The molecule was annealed from 800 K to 300 K with 100 K steps using MD runs of 1000 iterations of 1 fs each; thus, there was a total of 6 ps. In the end, all of the structures were minimized via 1000 steps of Steepest Descent until a predefined convergence limit of root mean square derivative of <0.001 Kcal mol−1 Å−1 was reached. The total of the 10 lowest structures was chosen for RMSD analysis out of 100 structures saved at regular intervals of 10 fs during the final step of molecular dynamics (1 ps).The restrained molecular dynamics (rMD) simulations were done on a Silicon Graphics Fuel workstation enabled with INSIGHT II  and the DISCOVER  software program. The initial structure of the G-quadruplex DNA [d-(TTGGGGT)]4 and its complexes with daunomycin at D/N ratios = 0.5, 1.0, 2.0, 3.0, 3.5, and 4.0 using a MicroCal VP-DSC instrument . All of the samples were prepared in 10 mM of KBPES buffer containing 10 mM of K2HPO4, 1 mM of EDTA, and 100 mM of KCl (pH 7.0). All of the samples were properly degassed before the experiment. Each sample was scanned from 25 °C to 120 °C with a scan rate of 60 °C/h at approximately constant pressure ~33 psi. The buffer was scanned repeatedly under similar conditions as that of the sample until a proper baseline was obtained. Then, 50 µM of free [d-(TTGGGGT)]4 (corresponding to a single-strand concentration of d-(TTGGGGT) as 200 µM) was scanned to obtain the melting profile of free DNA. Each set of complexes with increasing daunomycin concentrations and a fixed [d-(TTGGGGT)]4 concentration of 50 µM at D/N ratios = 0.5, 1.0, 2.0, 3.0 and 4.0 were scanned to obtain the melting profiles of the bound forms. Each scan was subtracted from the buffer baseline. The thermograms were then deconvulated and fitted in three-state or four-state models of unbound quadruplex DNA and its complexes using the Origin 7.1 software  that was provided with the instrument.Excess heat capacity as a function of temperature was measured to obtain thermal transitions of free [d-(TTGGGGT)]4 quadruplex DNA comprising telomeric DNA sequence from Tetrahymena thermophilia. Direct real-time binding experiments show specific interaction between two molecules. The daunomycin-[d-(TTGGGGT)]4 complex undergoes diffusion as a stable complex; complexes having stoichiometry of 1:1 and 2:1 coexist. The overall conformation of [d-(TTGGGGT)]4 is not significantly altered on interaction, and the daunomycin dimers present in free daunomycin are disrupted on binding to DNA. The NMR experiments confirm that a specific interaction between two molecules occurs. The presence of sequential NOEs along the sequence of DNA in 1H-1H NOESY spectra and the absence of large downfield shifts in 31P NMR studies give a clear proof of absence of classical intercalation to permit the stacking of the aromatic chromophore of daunomycin between G-quartets of DNA. Two binding sites, G6-T7 and T1-T2, are evident from an analysis of chemical shifts, line broadening, and 22 short interproton NOE contacts. Ring A protons—1H, 2H, 3H, 4OCH3—and daunosamine sugar protons—5′H, 5′CH3—are involved in interaction with DNA. End stacking with terminal G-tetrad G6 and groove-binding close to T1-T2 bases are found to exist. The binding leads to substantial thermal stabilization of [d-(TTGGGGT)]4 by ΔmT ~15 °C, which may result from an enhanced stacking of the G6 quartet with daunomycin chromophore at the G6pT7 site, as well as the presence of hydrogen bond and specific van der Waals contacts at grooves. The findings have implications in the structure-based designing of anthracycline drugs as potent telomerase inhibitors.The present study focuses on the binding of daunomycin to parallel-stranded tetramolecular [d-(TTGGGGT)]


In the present study, the cloning and nutritional regulation of elovl6 were determined in large yellow croaker. Sequence and phylogenetic analysis revealed that the full-length cDNA of elovl6 was 1360 bp, including an open reading frame of 810 bp encoding a putative protein of 269 amino acid that possesses the characteristic features of Elovl proteins. The transcript level of elovl6 was significantly increased in the liver of croaker fed the diets with soybean oil  or linseed oil  than that in croaker fed the diet with fish oil (enriched with 20: 5n-3 and 22: 6n-3). Correspondingly, the elovl6 expression in croaker’s hepatocytes treated with ALA or LA was remarkably increased compared to the controls. Furthermore, the transcription factors including hepatocyte nuclear factor 1α (HNF1α), CCAAT-enhancer-binding protein β (CEBPβ), retinoid X receptor α (RXRα), and cAMP response element-binding protein 1 (CREB1) greatly enhanced promoter activity of elovl6 in large yellow croaker, and the expression of transcription factors is consistent with the changes of elovl6 expression in response to fatty acids in vivo and in vitro. In conclusion, this study revealed that elovl6 expression in large yellow croaker could be upregulated by dietary ALA or LA via the increased transcriptional expression of transcription factors including hnf1α, cebpβ, rxrα, and creb1.Elongation of very long chain fatty acids protein 6 (Elovl6) is a key enzyme in fatty acid synthesis, which participates in converting palmitate (C16:0) to stearate (C18:0). Although studies of Elovl6 have been carried out in mammals, the nutritional regulation of  Long chain fatty acids (LCFAs) are predominantly synthesized in the liver by desaturations and elongations, which contributes a special function for normal physiological metabolism . Elongatelovl6 increased synthesis of 18:0 and 18:1n-7 [elovl6 was altered by dietary lipid supplementation [elovl6 in fish has been carried out, especially in marine fish.Elovl6 is highly expressed in the liver, adipose tissues, and brain ,6,7, whi 18:1n-7 . The recentation . Furtherentation ,15. AlthLarimichthys crocea), a particularly notable marine fish with high commercial value in southeast China, belongs to Sciaeni-dae family. It has been a useful model for studying fatty acid (FA) metabolism and lipid deposition of fish [elovl6 in response to dietary FA. These results may contribute to a further understanding of nutritional regulation and provide the basis for studying the fatty acid synthesis in fish.Large yellow croaker  , Oreochromis niloticus (97%), Salmo salar (95%), Oncorhynchus mykiss (95%), Cyprinus carpio (93%), and Danio rerio (93%).The full length of large yellow croaker cid (AA) . BLAST aSequence analysis of the large yellow croaker Elovl6 proteins showed that possessed the characteristic features of Elovl family members including histidine box (HXXHH) and six membrane-spanning domains . Phylogeelovl6 was determined in different tissues of large yellow croaker fed the diet with fish oil (FO), including the intestine, spleen, kidney, gill, liver, eye, heart, muscle, brain, and adipose tissues. The highest expression of elovl6 in large yellow croaker was observed in the liver, moderate level of expression was observed in the eye and brain, and the lowest expression was detected in the heart . The expression of the elovl6 gene in the liver of large yellow croaker fed the diets with soybean oil (SO), linseed oil (LO), or palm oil (PO) were significantly higher than that in croaker fed the diet with fish oil (FO) (p < 0.05), and the highest expression of elovl6 was observed in PO group (The relative mRNA expression of PO group .elovl6 expression. Compared with the control group (no supplementation), the relative expression of elovl6 in hepatocytes from large yellow croaker treated with 100 or 200 μM α-linolenic acid (ALA) for 12 h was significantly increased compared to the control group (p < 0.05) (elovl6 in hepatocytes from large yellow croaker treated with 200 μM linoleic acid (LA) and palmitic acid (PA) for 12 h was significantly higher than that of controls (p < 0.05) (Incubation of hepatocytes from large yellow croaker with fatty acids caused distinct changes in  < 0.05) A. The ex < 0.05) B,C.elovl6, an upstream sequence of 2021 bp adjacent to the start of elovl6 was cloned to characterize the transcriptional regulatory mechanism of elovl6. Using bioinformatics software including JASPAR, TRANSFAC, and TF binding, several transcriptional factors binding sites including hepatocyte nuclear factor 1α (HNF1α), CCAAT-enhancer-binding protein β (CEBPβ), peroxisome proliferator-activated receptor γ (PPARγ), transcriptional factor SP1 (SP1), retinoid X receptor α (RXRα), ChREB, SREBP1, SREBP2, LXRα, and cAMP response element-binding protein (CREB1) were predicted within the elovl6 promoter region of large yellow croaker. The results of dual-luciferase reporter assays showed that the transcription factor HNF1α, CEBPβ, RXRα, ChREB, SREBP2, and CREB1 upregulated the promoter activity of elovl6 by 1.78-fold, 2.35-fold, 2.32-fold, 1.80-fold, 1.51-fold, and 2.68-fold, respectively. Interestingly, PPARγ, SP1, SREBP1, and LXRα, as important transcription factors in regulation of elovl6 in mammals, showed no significantly regulatory effect on elovl6 promoter in large yellow croaker  (elovl6 in hepatocytes incubated with 4 and 8 μM RXRα antagonist for 1 h was significantly downregulated compared with the control group (p < 0.05) (Primary hepatocytes of large yellow croaker were treated with RXRα agonist (Bexarotene) or antagonist (UVI 3003) to verify the regulation of RXRα on  < 0.05) A. Furthe < 0.05) B.hnf1α gene in the liver of large yellow croaker fed the diets with SO and LO were significantly higher than that in croaker fed the diet with FO (p < 0.05), but there was no significant difference between the SO and LO groups (p > 0.05) (cebpβ gene in the liver of large yellow croaker fed the diets with LO was significantly higher than that in croaker fed the diet with FO and SO (p < 0.05)  (The expression of the  > 0.05) A. The ex < 0.05) B. Moreov > 0.05) C,D.hnf1α in hepatocytes of large yellow croaker treated with 100 or 200 μM ALA for 6 h or 12 h was significantly increased (p < 0.05)  (cebpβ in hepatocytes treated with 200 μM ALA for 6 h or 12 h was significantly increased (p < 0.05), and the expression in the group of 200 μM LA for 6 h or 12 h was also significantly increased (p < 0.05) (rxrα in hepatocytes treated with 100 or 200 μM ALA for 6 h or 12 h was significantly higher than that of the controls (p < 0.05), and the expression in the group of 200 μM LA for 6 h or 12 h was also significantly higher than that of the controls (p < 0.05) (creb1 in hepatocytes treated with 100 or 200 μM ALA for 6 h or 12 h was significantly increased (p < 0.05), and the expression of creb1 in hepatocytes with 100 or 200 μM LA for 12 h was also significantly increased (p < 0.05)  A. The ex < 0.05) B. Compar < 0.05) C,D. The  < 0.05) E,F. Comp < 0.05) G,H.elovl6 gene was cloned from large yellow croaker, and the cDNA exhibited characteristic features of Elovl protein family members, including a single histidine box (HXXHH) and six transmembrane regions [The Elovl6 protein has been confirmed to catalyze the key step in the elongation of C16: 0 to C18: 0 . Previou regions . Phylogeelovl6 is highly expressed in lipogenic organs and is sensitive to the diets [elovl6 in large yellow croaker was observed in the liver, and the relative mRNA expression of elovl6 was remarkably affected by fatty acids in vitro and in vivo. The qPCR data for the elovl6 expression in the liver of croaker fed the diets with PO and hepatocytes treated with PA was significantly higher than that of the controls. PA is the substrate for synthesis of C18:0 and C18:1n-9 as substrate [elovl6 to drive synthesis of C18:0 and C18:1n-9 using C16:0 as a substrate [elovl6 expression in the liver of croaker fed the diets with SO and LO was significantly higher than that in croaker fed the diet with FO. Correspondingly, the elovl6 expression in hepatocytes treated with LA and ALA was remarkably increased compared to the controls. LA or ALA is the substrate for LC-PUFA synthesis, which is catalyzed by fatty acid desaturases (Fads) and elongation of very long-chain fatty acids (Elovl) [elovl6 may be increased with the treatment of LA and ALA, which upregulated the transcriptional expression of elovl6.In mammals, he diets ,21. In tubstrate . Hence, ubstrate . Further (Elovl) ,24,25,26 (Elovl) . Thus, ielovl6 promoter luciferase reporter plasmid and the transcription factors expression plasmids were cotransfected into HEK293T cells in the dual-luciferase reporter assay. The studies in mouse suggested that the elovl6 promoter could be activated by SREBP1c and synergistically activated by both ChREB/Mlx and SREBP1c [elovl6 could be regulated by LXRα directly or indirectly [elovl6 could be regulated by the transcription factors including PPARα and PPARγ [elovl6 promoter activity [elovl6 promoter in large yellow croaker, and only the SREBP2 and ChREB showed activation of elovl6 promoter activity. The results indicated that the regulation mechanisms of elovl6 in fish might be different from mammals. It may be correlated with difference in the requirements of essential fatty acids and evolutionary spectacle in two species. Furthermore, transcription factors including HNF1α, CEBPβ, RXRα, and CREB1 could upregulate the activity of elovl6 promoter in large yellow croaker. Meanwhile, the RXRα agonist (Bexarotene) could greatly increase the transcriptional expression of elovl6, and the RXRα antagonist (UVI 3003) suppressed the expression of elovl6 in large yellow croaker. Thus, the transcription factor RXRα could be a potential target in future studies aiming to regulate the transcriptional expression of elovl6. The studies on regulatory effect of these transcription factors  in fish or mammals mainly focused on other genes related to lipid metabolism, with almost no study on elovl6, and the finding may contribute to a further understanding of the regulation mechanism of elovl6. In order to verify the speculation above,  SREBP1c ,28. Furtdirectly ,29. In mactivity . Howeverelovl6 could be regulated by dietary long-chain fatty acids (LCFAs) via transcription factors [hnf1α and cebpβ in the liver of large yellow croaker fed the diets with replacement of FO with SO or LO was increased. Correspondingly, the relative expression of hnf1α, cebpβ, rxrα, and creb1 in hepatocytes treated with LA and ALA was remarkably increased. This is consistent with the changes of elovl6 expression in response to fatty acids in vivo and in vitro, and the results suggested that the increase in expression of the transcription factors may be attributed to the increase of elovl6 expression in croaker treating with ALA or LA. Previous studies have suggested that that  factors ,6,28, whelovl6 was cloned from large yellow croaker and elovl6 contained the typical features of Elovl proteins. Elovl6 promoter activity was positively regulated by the transcription factors, HNF1α, CEBPβ, RXRα, and CREB1. Furthermore, large yellow croaker treated with LA or ALA resulted in greatly increased expression of elovl6, and this increase was likely regulated by transcription factors including HNF1α, CEBPβ, RXRα, and CREB1.In conclusion, the cDNA of Four isoproteic (41% crude protein) and isolipidic (12% crude lipid) diets were formulated with the fish oil (FO), soybean oil (SO), linseed oil (LO), and palm oil (PO) as the main lipid sources. The oils purchased from Great Seven Biotechnology Co., Ltd, Qingdao, China and fatty acid profiles of oils were detected . Large yIn the present study, all experimental procedures performed on fish and animal care were in strict accordance with the Management Rule of Laboratory Animals .Total RNA was isolated from the tissues of large yellow croaker using TransZol  followed by quality measurement on agarose gel electrophoresis and Nanodrop® 2000 . The RNA was reversely transcribed to cDNA by PrimeScriptTM RT reagent kit  according to the manufacturer’s instructions.elovl6 cloning were designed based on sequence (XM_010729493.3). One microliter of liver cDNA, 1 μL of each primer, 12.5 μL of PrimeSTAR® Max DNA Polymerase  and 9.5 μL RNA-free water was added in a 25 μL mixture. The PCR program was performed with the following parameters: 35 cycles of 98 °C for 10 s, 55 °C for 15 s and 72 °C for 1 min, followed by 72 °C for 10 min. RACE (rapid amplification of cDNA ends) primers were designed from the sequence of the first elovl6 fragment. SMARTerTM RACE cDNA Amplification Kit  was used to clone the 3′- and 5′-end sequence of elovl6 according to the manufacturer’s instructions. The PCR program was performed as described above. Then, the PCR products was cloned into pEASY-T1 simple cloning plasmid  for sequencing.PCR primers of the first fragment for elovl6 cDNA was aligned to orthologues from other vertebrate species. Phylogenetic analysis of the deduced AA sequences of the Elovl6 proteins from large yellow croaker was performed using Mega 4.0. and phylogenetic trees were formed by the neighbor joining method [The amino acid (AA) sequence of cloned large yellow croaker g method .elovl6, hnf1α, cebpβ, rxrα, and creb1 was performed in a quantitative thermal cycler  by SYBR Green real-time PCR kit  according to a previously described procedure [-ΔΔCT method was used to calculate the relative expression of genes [qPCR of the target genes including rocedure . The priof genes .elovl6 promoter cloning were designed according to the large yellow croaker genomic DNA . The program for elovl6 promoter cloning was performed by PrimeSTAR® Max DNA Polymerase  with the following parameters: 35 cycles of 98 °C for 10 s, 55 °C for 15 s and 72 °C for 1 min, followed by 72 °C for 10 min. Then, the product of PCR was inserted into pGL3-Basic plasmid  as a reporter plasmid.The SQ Tissue DNA Kit  was used to extracted Genomic DNA from the liver in large yellow croaker according to manufacturer’s instructions. The gene-specific primers for http://jaspar.genereg.net/), TRANSFAC (http://genexplain.com/transfac/) and TF binding (http://tfbind.hgc.jp/) were used to predict potential transcription factor binding sites in the promoter regions of elovl6 in large yellow croaker.JASPAR  using ClonExpress II One Step Cloning Kit  and primers , CEBPβ (GenBank: XM_010741628.3), PPARγ (GenBank: XM_010731330.3), SP1 (GenBank: XM_010732100.3), RXRα (GenBank: XM_019271558.2), ChREB (GenBank: XM_019254372.2), SREBP1(GenBank: KP342262), SREBP2(GenBank: XM_010754025.3), LXRα(GenBank: XM_019273432), and CREB1 (GenBank: XM_027291522.1) were cloned into pCS2 primers .HEK293T cells were cultured according to a previously described procedure . For traThe isolation and culture of primary hepatocyte from large yellow croaker was performed according to the method described previously . The fatt-test using SPSS 20.0 with p < 0.05 set to be statistically significant.The results were presented as means ± S.E.M. The data were analyzed by one-way ANOVA and Student’s


Chiral bisoxazoline ligands containing naphthyridine, pyridazine, pyrazole, and phenol bridging units were prepared and shown to form bimetallic complexes with various metal salts. X-ray crystal structures of bis-nickel naphthyridine-bridged, bis-zinc pyridazine-bridged, and bis-nickel as well as bis-palladium pyrazole-bridged complexes were obtained. Metal-centered asymmetric catalysis most commonly relies on monometallic complexes of various chiral ligands, among which chiral bisoxazolines have been highly successful in facilitating various Lewis acid-catalyzed asymmetric transformations –3. In ad1, in which the two different metals play distinct roles [2 which show cooperative reactivity between the two metal centers in the asymmetric ring opening of meso-epoxides [3 and their application to enantioselective Aldol reactions [A range of bi- and multi-metallic complexes have been utilized in asymmetric catalysis  6]. For. For6]. ct roles –15. Jacoepoxides . Trost eeactions –18 and aeactions –20, Marueactions –22, Wueseactions ,23–25, aeactions –46. In teactions –48.6–13 that can simultaneously bind to two metals PdCl]2 2 . Little 9 and 11 ,52. The 4.291 Å) . Tetraox9.432 Å) .A number of bisoxazoline ligands with different bridging units were designed. We rationalized that the presence of three binding sites per metal center would be ideal in order to achieve the desired 1:2 ligand to metal ratio, and to prevent the potential formation of 2:2 or other higher order complexes. Variation of the bridging moiety should allow for modulation of the distance between the metal centers. An important criterion for selecting bridging units was their known ability to engage in metal-binding, along with being readily available. This led to the selection of naphthyridine, pyridazine, pyrazole, and phenol building blocks. We opted to connect these linkers to oxazolines via amide bonds. The reasoning for this was twofold. Firstly, this should provide ligands with significantly improved stabilities over for instance imine linkers. In addition, each amide moiety, upon deprotonation (a requirement for complex formation), would provide a formal negative charge on the ligand, thus resulting in increased complex stability while reducing the number of spectator anions associated with the two metals. Different combinations of five- and six-membered chelate rings were considered, as those allow for further modulation of metal–metal distances. Convenient synthetic sequences were developed for six different ligands.Naphthyridine-bridged bisoxazoline ligands. The synthesis of naphthyridine bridged bisoxazoline ligand 16-H2 is outlined in 14, obtained via a known procedure from the corresponding diacid [15 [16-H2 in 65% yield. Upon deprotonation, 16-H2 provides a dianionic ligand with three nitrogen donor atoms per metal center. Ligand 16-H2 was found to undergo complex formation with various copper, zinc, palladium and nickel salts.g diacid , was allacid [15  to provi16·Ni2(OAc)2, obtained from ligand 16-H2 and two equivalents of nickel(ΙΙ) acetate. The asymmetric unit of the crystal 16·Ni2(OAc)2 contains two nickel(ΙΙ) centers held in close proximity by three donor nitrogen atoms per metal center and two differently bridged acetate ions inside the coordination sphere. The nitrogen atoms on the naphthyridine and amide moieties bind to the nickel(ΙΙ) center to form a five-membered metallacycle, subtending N(2)–Ni(1)–N(3) and N(5)–Ni(2)–N(6) angles of 81.53° and 80.34°, respectively. Additionally, the nitrogen atoms on the oxazoline and amide moieties form six-membered rings with the nickel(ΙΙ) center with N(1)–Ni(1)–N(2) and N(4)–Ni(2)–N(5) angles of 93.03° and 90.24°, respectively. All of the Ni–N distances are between 2.003 and 2.129 Å, typical for complexes of this type [10. The sixth coordination of Ni(2) is fulfilled by the amide oxygen of a second molecule of the complex (not shown in the figure for clarity). Apparently, the coordination environment involving three O-atoms from acetate groups (the Ni(1) situation) for one of the two metals is preferred in 16·Ni2(OAc)2, presumably for steric reasons, as crystal packing motivations would likely favor two situations as found for Ni(2), and possibly favoring formation of the 1D polymeric chain found here extending along the crystal b-axis. When this interaction is taken into account, both the nickel(ΙΙ) centers are in distorted octahedral environment. Overall, the ligand backbone of complex 16·Ni2(OAc)2 shows a helical arrangement. This helicity is facilitated by the innate stereogenic centers of the oxazoline moieties which is further extended by the flexibility afforded by the amide connections.his type –57. Inte22-H2 was designed as an analogue of ligand 16-H2 that possesses a shortened amide bridge to allow for the formation of bimetallic complexes with all five-membered chelate rings. Ligand 22-H2 was obtained starting from glycinol 17 and N-carboxybenzyl glycine (20) via the sequence of steps outlined in 22-H2 undergoes complex formation with various nickel and palladium salts [Naphthyridine ligand um salts .Pyrazole-bridged bisoxazoline ligands. The synthesis of an analog of bisoxazoline ligand 16-H2 in which the naphthyridine bridge is replaced with a pyrazole linker is shown in 25-H3 is a potentially trianionic as opposed to a dianionic ligand. A reaction of pyrazole-3,5-diacyl chloride 23, obtained by treatment of pyrazol-3,5-dicarboxylic acid with thionyl chloride, with aminophenyloxazoline 24 [25-H3 in 85% yield.oline 24  provided25-H3 with nickel(II) acetate provided the binuclear complex 25·Ni2(OAc), the X-ray structure of which is shown in Treatment of 30-H3 was prepared starting from the N-carboxybenzyl amino acid 26. Coupling of 26 with phenylglycinol (17) resulted in the formation of the amide 27. Subsequent treatment with p-toluenesulfonyl chloride and triethylamine in the presence of a catalytic amount of DMAP provided oxazoline 28. Following deprotection, the resulting amine 29 was allowed to react with half an equivalent of the pyrazole diacyl chloride 23 to yield the pyrazole-bridged bisoxazoline ligand 30-H3.The synthesis of a pyrazole containing ligand potentially capable of forming bimetallic complexes with all five-membered chelate rings is shown in 30-H3 readily forms complexes with various nickel, copper and palladium salts. 30·Pd2Br, obtained from ligand 30-H3 and two equivalents of palladium(ΙΙ) bromide. Each of the two palladium(ΙΙ) centers of the complex 30·Pd2Br is confined in a slightly distorted square planar geometry by three donor nitrogen atoms from the ligand and a bridging bromide ion. Unlike the situation in 16·Ni2(OAc)2, where the Ni–O bond from the carbonyl of an adjacent molecule yields both the five-coordinate Ni and 1D polymeric chain, there are no Pd–O bonds (not even dative) in 30·Pd2Br and the Pd atoms remain nearly square planar. The donor nitrogen atoms from pyrazole, amide and oxazoline moieties coordinate to the palladium centers, forming five-membered chelate rings, with the subtended N–Pd–N angles ranging from 79.43° to 82.16°. The two palladium centers are bridged by the bromide ion and the pyrazole segment. All of the Pd–N bond lengths fall within a relatively narrow range of 1.925 to 2.025 Å, and are typical for Pd–N bonds with this coordination geometry. As expected, the Pd–Br bond lengths are slightly longer (2.519 and 2.529 Å) than the Pd–N bonds. The bromide ion bridges the two palladium centers with a Pd–Br–Pd angle of 98.48°. The two nitrogen donor atoms from the pyrazole moiety forms the second bridging link between the two metal centers, thereby restricting the N(1)–Pd(1)–Br(1) and N(4)–Pd(2)–Br(1) angles to 89.89° and 89.73°, respectively. The resulting Pd(1)∙∙∙Pd(2) distance was observed to be 3.824 Å.Initial experiments have shown that the trianionic ligand Pyridazine-bridged bisoxazoline ligands. Ligand 32-H2 was synthesized from pyridazine diester 31 [15  centers, with each bound by three donor nitrogen atoms from the ligand skeleton and bridged by a chloride ion. The nitrogen atoms on the pyridazine and amide moieties form five-membered chelate rings upon coordination to the zinc centers, subtending N(2)–Zn(1)–N(1) and N(5)–Zn(2)–N(4) angles of 74.74° and 77.04°, respectively. The coordination of the nitrogen atoms on oxazoline and amide moieties with the two zinc centers forms six-membered rings with N(3)–Zn(1)–N(2) and N(6)–Zn(2)–N(5) angles of 87.05° and 88.55°, respectively. All of the Zn–N distances are within 2.023–2.177 Å, as expected for such complexes. Interestingly, the two chloride ions bind in discrete manners. While one of them acts as a bridge between the two zinc centers with a Zn(1)–Cl(1)–Zn(2) angle of 105.97°, the other chloride ion binds to Zn(2) only with a Zn(2)–Cl(2) distance of 2.241 Å. Consistent with having a bridging chloride atom, the Zn(1)∙∙∙Zn(2) distance of 3.857 Å is somewhat long, compared to the M∙∙∙M distances in two of the crystal structures mentioned here, namely, the Ni∙∙∙Ni in 16·Ni2(OAc)2 at 3.338 Å, shorter because of the smaller radius of the bridging μ2-O atom, and the 30·Pd2Br at 3.824 Å, shorter because of both the Pd coordination geometry and the bridging μ2-Br atom. The Ni∙∙∙Ni in 25·Ni2(OAc) at 4.176 Å is longer due to its longer three-atom bridge from OAc. In the Cambridge Structural Database (CSD), there are ten instances of Ni–N–N–Ni bond connectivity with sp2 N-atoms that are also bonded to a C-atom. However, all of those have trans related Ni atoms unlike the cis related here such that the Ni∙∙∙Ni distances range from 4.54 to 4.72 Å for those ten compounds. Similarly, for Pd–N–N–Pd and Zn–N–N–Zn, there are few and only all trans situations in the CSD with ranges of 4.83 to 4.84 Å and 4.53 to 4.78 Å, respectively, for the two Pd and three Zn compounds. For M–N–N–M with sp3 bridging N-atoms, only four instances are found – all with M = Zn and N–N from substituted aminohydrazido ligands and the distances are longer (4.93 to 5.04 Å) and the trans relationship maintains. Thus, the cis M–N–N–M geometry observed here is quite rare for M = Ni, Zn, Pd and is uncommon for M = any transition metal (about 90 instances in CSD). Different coordination environments are found for Zn(1) and Zn(2) in 32·Zn2Cl2. Zn(1) is found to exist in a square planar environment that experiences a significant tetrahedral distortion. In contrast, a distorted square pyramidal binding mode is observed for Zn(2).Upon deprotonation, Phenol-bridged bisoxazoline ligands. Ligands incorporating naphthyridine, pyridazine and pyrazole linkers discussed thus far bridge two metal atoms by attachment to two different nitrogen donor atoms. As a result, metal∙∙∙metal distances tend to be relatively long. Ligand 34-H3 was designed to explore the effect of a single atom linker, namely a phenoxy bridge. The tert-butyl group in the para-position was incorporated as it would likely increase the overall solubility of the ligand as well as its associated complexes. DCC-mediated coupling of 33 [15 led to the formation of phenol-bridged bisoxazoline ligand 34-H3 in a single step and moderate yield .


RNA aptamers selected to bind fluorophores and activate their fluorescence offer a simple and modular way to visualize native RNAs in cells. Split aptamers which are inactive until the halves are brought within close proximity can become useful for visualizing the dynamic actions of RNA assemblies and their interactions in real time with low background noise and eliminated necessity for covalently attached dyes. Here, we design and test several sets of F30 Broccoli aptamer splits, that we call fluorets, to compare their relative fluorescence and physicochemical stabilities. We show that the splits can be simply assembled either through one-pot thermal annealing or co-transcriptionally, thus allowing for direct tracking of transcription reactions via the fluorescent response. We suggest a set of rules that enable for the construction of responsive biomaterials that readily change their fluorescent behavior when various stimuli such as the presence of divalent ions, exposure to various nucleases, or changes in temperature are applied. We also show that the strand displacement approach can be used to program the controllable fluorescent responses in isothermal conditions. Overall, this work lays a foundation for the future development of dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates. DNA and RNA, which are best known for their roles in processing genetic information, are natural biopolymers that are also increasingly utilized in the design and construction of nucleic acid nanoparticles (NANPs) ,2,3,4. PThe responsive behaviors of NANPs can be directly visualized in living cells using complementary strands labeled with pairs of dyes that can undergo Förster resonance energy transfer (FRET). Tracing changes in FRET signals that result upon re-hybridization of labeled strands entering the compositions of cognate NANPs confirms the dynamicity of their behavior in real time ,21,22. TFor visualizing intracellular RNAs, the MS2-green fluorescent protein (GFP) system has been utilized to tag and image endogenous RNAs in cells . HoweverZ)-4--1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI), and to exhibit green fluorescence when bound [m and higher binding affinity for ligand DFHBI-1T, an optimized dye [The development of RNA aptamers which activate fluorophores upon binding has been a major improvement over these imaging techniques, offering a high signal-to-noise ratio, modularity for simple sequence incorporation and tagging, and real-time protein-free imaging in cells . Using sen bound ,38. The en bound ,40. A neized dye ,40,41,42Fluorescent RNA aptamers such as Spinach, Broccoli, and Mango have been used to monitor a variety of metabolites and proteins in mammalian and bacterial cells ,44,45,46Rationally designed (as described in Methods) split F30-Broccoli  aptamers were assembled in the presence of DFHBI-1T to assess their responses to an array of stimuli a which wWe first experimentally confirmed that fluorets can be assembled co-transcriptionally while activating DFHBI-1T fluorescence. We followed the protocols of previously established generalized in vitro methodology for the one-pot T7 RNA polymerase-driven co-transcriptional assembly of different RNA NANPs , includiIt is beneficial to trace the activity and involvement of various enzymes in real time. As a proof of concept, we tested if the presence of different nucleases can conditionally drive either assembly or disassembly of fluorescent fluorets. Hybrid DNA/RNA duplexes for each fluoret monomer with an excess of DNA were incubated with DNase, resulting in the assembly of complete RNA fluorets which were then further deactivated by RNase b. The inms  that removes the Mg2+ ions necessary for forming the tertiary structure of the aptamers required for binding and activating DFHBI-1T fluorescence. Importantly, the functional structure can be restored upon the subsequent addition of Mg2+ and the oscillation can continue based on repeated additions . After 3.5 h at 37 °C, the reaction was incubated with RQ1 RNase-free DNase  prior to purification using denaturing 8 M urea polyacrylamide gel electrophoresis . RNA bands were visualized under UV (short wavelength), cut, and eluted in crush and soak buffer , 2 mM EDTA) overnight. Precipitation of the RNA was done in 2.5 volumes of 100% ethanol for 3 h at −20 °C. Samples were rinsed with 90% ethanol, vacuum dried, and dissolved in double-deionized water (17.8 MΩ·cm).DNA strands were purchased from Integrated DNA Technologies   was added, followed by 30 min of incubation at 37 °C.F30-Broccoli RNA or Broc and Coli RNA strands composed of unmodified nucleotides were mixed in an equimolar ratio in double-deionized water. The samples were heated to 95 °C for 2 min, snap-cooled to 4 °C for 2 min, and assembly buffer (89 mM tris-borate (pH 8.2), 2 mM MgCl2, 5 mM each rNTP) and 10 µM DFHBI-1T in buffer. Samples in triplicate were placed at 37 °C on a Bio-Rad CFX96 Touch Real-Time PCR Detection System  for 3.5 h. Fluorescence was measured every minute, using 10 µM DFHBI-1T in buffer and transcription reactions at the above concentrations but without pDNA as negative controls.PCR-amplified DNA templates for complementary splits were added 1:1 as 20% final volume with T7 RNA polymerase (80 mM HEPES-KOH (pH 7.5), 2.5 mM spermidine, 50 mM DTT, 25 mM MgCl2. Native-PAGE gels were run for 20 min  at 4 °C and 300 V. Prior to staining, gels were washed 3 × 5 min in water and stained for 15 min in 10 µM DFHBI-1T in buffer (50 mM HEPES (pH 7.4), 100 mM KCl, 1 mM MgCl2) as previously reported [For analysis, 8% non-denaturing native-PAGE (37.5:1) was used in the presence of 89 mM tris-borate (pH 8.2) and 2 mM MgClreported . Gels we2+ and incubating for 2 min at room temperature and again using 10 mM EDTA.Fluorets were assembled as normal at a concentration of 4 µM with 10 µM DFHBI-1T in buffer and measured using a NanoDrop 3300 . An amount of 1 µL of 10 mM EDTA was added to the sample, which was incubated at room temperature for 2 min before measuring again with a NanoDrop 3300. This was repeated using 1 µL of 10 mM Mg2, 50 mM KCl) was added, followed by 20 min of incubation at room temperature. Each hybrid split was then added with its opposite hybrid split  in an equimolar ratio at a final concentration of 1 µM with 2/15 volume of RQ1 RNase-free DNase (New England BioLabs). This was incubated at 37 °C for 30 min, then 2/5 of the volume were added to RNase ONE™  at a volume equal to the RQ1 RNase-free DNase added previously. This was incubated at 37 °C for 30 min. Samples were visualized on an 8% non-denaturing native-PAGE (37.5:1) in the presence of 89 mM tris-borate (pH 8.2) and 2 mM MgCl2 run for 20 min at 4 °C and 300 V.Split Broc RNA strands were assembled with Coli DNA strands (and vice-versa) by mixing in an equimolar ratio in double-deionized water. The samples were heated to 95 °C for 2 min, snap-cooled to 4 °C for 2 min, and assembly buffer (89 mM tris-borate (pH 8.2), 2 mM MgClFluorets were assembled as previously described and then added in an equimolar ratio with either DNA for Broc or DNA for Coli prior to incubation at 37 °C for 30 min. RNA fluoret monomers were also assembled with their complementary DNAs as controls by mixing in an equimolar ratio in double-deionized water, heating to 95 °C for 2 min, snap-cooling to 4 °C for 2 min, and adding assembly buffer (89 mM tris-borate (pH 8.2), 2 mM MgCl2, 50 mM KCl) followed by 20 min of incubation at room temperature.2) and incubated for 30 min at 37 °C. Assemblies at a final concentration of 1.25 µM underwent a 4 °C to 80 °C thermal gradient with a step size of 0.5 °C per 5 s using a Bio-Rad CFX96 Touch Real-Time PCR Detection System. For the reactivation of fluorets, assemblies were programmed to undergo cooling from 80 °C to 4 °C with a step size of 2 °C per 45 s. Fluorescence of thermal deactivation was measured in triplicate for all fluorets using 10 µM DFHBI-1T in buffer and F30-Broccoli without DFHBI-1T (1.25 µM) as negative controls. Analysis of Tm ± SEM was done in GraphPad Prism Software  with a Boltzmann sigmoidal curve fit.Aptamers were assembled as previously described. For in vitro staining with DFHBI-1T, assemblies were mixed in a 1:1 volumetric ratio with 20 µM DFHBI-1T in buffer (100 mM HEPES (pH 7.4), 200 mM KCl, 2 mM MgCl1/2 was done in GraphPad Prism Software with a linear fit.Aptamers were assembled as previously described, mixed in a 1:1 volumetric ratio with 20 µM DFHBI-1T in buffer, and incubated for 30 min at 37 °C. An amount of 9 µL of assembled aptamer at a final concentration of 1.25 µM was added with 1 µL of 20% HBS and placed at 37 °C on a Bio-Rad CFX96 Touch Real-Time PCR Detection System. Fluorescence was measured every min, using 2% HBS with 10 µM DFHBI-1T in buffer and 1.25 µM of F30-Broccoli with 2% HBS without DFHBI-1T as negative controls. Analysis of Tp-value of less than 0.05 was considered to be statistically significant.Statistical analysis was done by one-way analysis of variance (ANOVA) using GraphPad Prism Software. All column means were compared by Tukey’s multiple comparison test. A In conclusion, the experimental design reported in this work is anticipated to lead the development of several robust strategies allowing for real-time fluorescence-assisted tracking of various processes at the nanoscale level. The reported system is made of biocompatible materials that can be used for a broad range of biological and nanotechnological applications both in vitro and potentially in vivo. The developed experimental schemes are expected to help address some fundamental questions such as co-transcriptional folding of RNAs, formation of multi-stranded RNA NANPs, and their responses to various stimuli. Such complex behaviors will definitely promote fields such as synthetic biology or applications such as the stimulation of differentiating cells during basic research and tissue engineering. Although only eight different variations in the split aptamer were tested, optimized fluorescence of the split was demonstrated and shown to be responsive to various stimuli.