Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.     

The dynamics of the calcium‐induced chain–chain association in the polyuronate systems

The calcium‐induced formation of strong, hydrophilic gels is the important feature of polyuronates, connected with most of their practical applications. The insight into the molecular details of gelling process dynamics is hardly feasible for both experimental and theoretical methods. Here, the application of the transition path sampling method for studying this problem is reported; the focus was on the poly(α‐L ‐guluronate) systems, treated as the representative for all polyuronate‐containing systems. The results allowed for identifying several distinct local minima of the free energy lying on the transition paths and visited by the system during the process of chain–chain association. These minima usually correspond to the intermediate structures in which the water molecules bridge calcium ion and carboxyl groups. This work emphasizes the importance of water and provides more complete understanding of the calcium binding by the polyuronate chains. © 2012 Wiley Periodicals, Inc.     

TReNDS—Software for reaction monitoring with time-resolved non-uniform sampling

NMR spectroscopy, used routinely for structure elucidation, has also become a widely applied tool for process and reaction monitoring. However, the most informative of NMR methods—correlation experiments—are often useless in this kind of applications. The traditional sampling of a multidimensional FID is usually time-consuming, and thus, the reaction-monitoring toolbox was practically limited to 1D experiments (with rare exceptions, e.g., single-scan or fast-sampling experiments). Recently, the technique of time-resolved non-uniform sampling (TR-NUS) has been proposed, which allows to use standard multidimensional pulse sequences preserving the temporal resolution close to that achievable in 1D experiments. However, the method existed only as a prototype and did not allow on-the-fly processing during the reaction. In this paper, we introduce TReNDS: free, user-friendly software kit for acquisition and processing of TR-NUS data. The program works on Bruker, Agilent, and Magritek spectrometers, allowing to carry out up to four experiments with interleaved TR-NUS. The performance of the program is demonstrated on the example of enzymatic hydrolysis of sucrose.     

Uranyl–Peroxide Capsule Self-Assembly in Slow Motion

Uranyl-peroxide capsules are the newest family of polyoxometalates. Although discovered 13 years previously with over 70 topologies reported, there is a lack in the fundamental understanding of assembly mechanisms, particularly the role of the alkali counterions. Herein, the reaction pathway and assembly of uranyl peroxide capsules is reported by tracking the conversion from K⁺ uranyl triperoxide monomer to the K⁺ uranyl-peroxide U₂₈ capsule by means of small-angle X-ray scattering and Raman spectroscopy. For the first time, the K⁺ uranyl-peroxide pentamer face is isolated and structurally characterized, giving credence to the long-held belief that these geometric faces serve as building blocks to the fully formed capsules. Once isolated and re-dissolved, the pentamer face undergoes rapid conversion to capsule forms, underlining its high reactivity that challenges its isolation. Calorimetric measurements of the studied species confirms the pentamer lies on the energy landscape between the monomer and capsule.     

The reactivity of SbVO<Subscript>5</Subscript> with T-Nb<Subscript>2</Subscript>O<Subscript>5</Subscript> in solid state in air

The reactivity of SbVO₅, a compound known since a short time, with T-Nb₂O₅ in solid state in air has been investigated over the whole component concentration range of a system built by these two reacting substances. The investigation results have shown that an equimolar mixture of SbVO₅ and T-Nb₂O₅ reacts with a subsequent formation of a hitherto unknown compound of the formula Nb₂VSbO₁₀. This compound has been characterized by the methods XRD, DTA/TG, and SEM. Its orthorhombic unit cell parameters have been calculated, and its stability in air up to 880 ± 10 °C has been proved. At this temperature, the compound melts incongruently with an accompanying deposition of solid Nb₉VO₂₅, i.e., of a compound that crystallizes in the binary oxide system V₂O₅–Nb₂O₅.     

Geometrical and electrical properties of indium tin oxide clusters in ink dispersions

The analysis of the TEM images of indium tin oxide (ITO) clusters in ink solutions deposited from ink dispersions reveals that their geometry arises from a diffusion limited cluster aggregation (DLCA) process. We model films of ITO clusters as built through deposition of DLCA clusters made of primary spherical nanoparticles of 13 nm in diameter. The deposition is then followed by a further compactification process that imitates sintering. We determine the conductivity of the sintered films by mapping the problem to that of the resistor network in which the contact regions between the touching spheres provide the dominant electric resistance. For a given volume fraction, conductivity of the sintered films is shown to be larger than that for the randomly packed spheres. However, the larger a typical radius of gyration of the clusters the smaller the enhancement. We also provide numerical tests for the routines used in the interpretation of the TEM images.