Syntheses of RNAs with up to 100 nucleotides containing site-specific 2'-methylseleno labels for use in X-ray crystallography

The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.     

Determination of the branching ratiosΓ(K L→3π0)/Γ(K L→π+ - 0) andΓ(K L→3π0)/Γ(K L→πev)

Improved branching ratios were measured for theK _L3⁰ decay in a neutral beam at the CERN SPS with the NA31 detector:

Photoelectric spectroscopy of oxide states with MOS structures atT=79 K

AtT=79 K illumination effects with visible and UV light on the drain current were studied forn-channel enhancement-type MOS transistors. The results show that the response of photoelectric measurements is due to electron excitation from oxide states into the silicon surface layer (positive changes of drain current). The oxide states lying near the bottom of the silicon dioxide conduction band are distributed in energy. Oxide states having captured a hole can be discharged by electrons excited from the silicon conduction or valence band (negative changes of drain current) in combination with a tunneling process.     

Approximate model for laser trepanning with microsecond Nd:YAG laser radiation

Trepanning of 200 m cooling holes in 2–4 mm thick CMSX-4 turbine material is done by a lamp-pumped Nd:YAG slab laser with pulse durations of 100–500 s and pulse energies up to 1 J determining the material removal mainly by melt expulsion. The high removal velocities achieved enable us to describe the motion of the melt front – the phase boundary of solid/liquid material – approximately. Compared to the complete solution including heat conduction and phase transition (Stefan problem) the approximate model is asymptotically accurate and its errors are controllable. The simulation is compared with experimental results of cross sections of trepanning kerfs, assuming that the melt is removed instantaneously by being expelled by a coaxial gas stream during trepanning. The assumption of temperature-independent material parameters is discussed. First results with an assumed Gaussian beam profile (M²=2) are compared with a refined simulation considering the measured spatial beam profile. PACS 42.60.Jf; 42.62.Cf; 44.05.+e; 46.15.Cc     

Gestaltsbestimmung des Hämoglobinmoleküls mittels der Röntgen-Kleinwinkelstreuung

Ohne Zusammenfassung     

Oxidation properties of InSb(110) Surfaces

The structural and electronic properties of InSb(110) surfaces, which were cleaned by argon bombardment annealing technique, have been investigated by LEED and surface conductivity measurements during oxygen adsorption. The diffraction patterns before and during exposure exhibit only diffraction spots which are compatible with the bulk periodicity. The exposures resulted in a gradual decrease of all beams. The surface conductivity increases during exposures. The magnitude of the adsorption induced changes is determined by the coverage and by the density of surface defects. In view of these results the oxidation process on the InSb(110) sur face is discussed.     

Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry

A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.1–50 and 0.5–100 nM, respectively. Fully ¹³C isotope-labeled and deuterated internal standards were used to achieve accurate quantification. The applicability of the method to analyze serum, urine, and cell culture supernatants was demonstrated by recovery experiments and the evaluation of matrix effects. Precision for the analysis of serum, urine, and cell culture supernatants ranged between 1.3% and 16.0%, 1.5% and 13.5%, and 1.0% and 17.4%, respectively. The method was applied to analyze changes in tryptophan metabolism in cell culture supernatants from IFN-γ-treated monocytes and immature or mature dendritic cells.     

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19F NMR

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of ¹⁹F NMR methods to characterize complex exchange processes with multiple excited states.