Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a “DNA Aptaomics” approach to systematically design functional aptamers as well as to obtain high affinity aptamers.     

A Novel Catalytic Function of Synthetic IgG‐Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine

The synthetic IgG‐binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine‐utilizing luciferase. The Z domain derivatives (ZZ‐gCys, Z‐gCys and Z‐domain) were purified and the luminescence properties were characterized by comparing with coelenterazine‐utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ‐gCys (100%) > Z‐gCys (36.8%) > Z‐domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ‐gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z‐gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG‐binding region of the Z domain.     

Ice chromatography: current progress and future developments

Ice chromatography, in which water-ice particles are employed as a chromatographic stationary phase, has proven an efficient technique for probing the solution/ice interface. The preparation of fine ice particles has allowed us to not only obtain higher-resolution separation but also investigate the molecular processes occurring on the ice surface in more detail. Chromatographic investigations have revealed that two or more hydrogen bonds are simultaneously formed between a solute and the dangling bonds on the ice surface when the solute gives measurable retention. Several compounds, including estrogens, amino acids, and acyclic polyethers, have been successfully separated by ice chromatography with a hexane-based mobile phase. In addition, this method effectively probes the surface melting of the ice stationary phase and the liquid phase that coexists with water ice at thermodynamic equilibrium. The thickness of the surface liquid layer and the size of the liquid phase that grows inside an ice particle have been evaluated. The perspectives of this method are also discussed.      

Transmetalation in a Ce(III)‐phosphoester Crystalline Coordination Polymer with an Exceptionally High Selectivity for Yb(III) and Lu(III)

A novel crystalline coordination polymer containing Ce³⁺ and bis(4‐nitrophenyl) phosphate (L), CeL₃, was synthesized and its unique transmetalation selectivities toward Yb³⁺ and Lu³⁺ in the lanthanide series were evaluated. The relatively large difference in transmetalation selectivity between the neighboring Tm³⁺ and Yb³⁺ species is noteworthy because the reactivities of heavy lanthanides are generally considerably similar. The structural strain of the polymeric framework is likely responsible for this unusual trend. Powder X‐ray diffraction analysis indicated that, in the cases of only Yb³⁺ and Lu³⁺, large differences in their ionic sizes compared to that of Ce³⁺ in the parent framework may induce a structural strain after solid solution formation, while cleavage of the relatively weak Ce?O bond allows the formation of new Yb?O and Lu?O bonds with the incoming Yb³⁺ and Lu³⁺, respectively. Structural phase transitions likely caused by the heterogeneous nucleation of the Yb‐ (or Lu‐) type phase were also observed.     

Novel preparation of fluorinated isoindoles and their conversion to fluorinated benzoporphyrins

4,5,6,7-Tetrafluoroisoindole and their related compounds were prepared directly from the corresponding phthalonitriles by reduction of a hydride reagent such as DIBAL or catalytic hydrogenation in the presence of an acid. 4,5,6,7-Tetrafluoroisoindole was converted to fluorinated benzoporphyrins.     

Ice chromatography modification of solute retention on water-ice stationary phase

Ice chromatography, in which water-ice particles are used as the stationary phase, has high potential as a separation tool, and is an efficient approach to probe the molecular interaction occurring on the water-ice surface. In the present paper, to modify solute retention, several methods are proposed, that is, changing the mobile phase composition, the surface modification of water-ice, and the implantation of functional molecules into water-ice. The retention mechanism of ice chromatography is discussed through these retention modifications.     

Up to 4 orders of magnitude enhancement of crown ether complexation in an aqueous phase coexistent with ice

Ice chromatography measurements have revealed anomalous enhancements of crown ether complexation in a liquid phase coexistent with ice. The 4 orders of magnitude enhancement was confirmed for the complexation of dibenzo-24-crown-8 in sub-μm-sized liquid inclusions formed in ice doped with <1 mM NaCl or KCl. This enhancement became less pronounced with increasing dopant concentration.