Investigation of asymmetry reduction for surface structuring and destructuring by laser remelting.

Lasers are widely used for structuring metallic surfaces by ablating material. An alternative approach for laser structuring is surface structuring by laser remelting (WaveShape), which is based onthe continuous remelting of a thin surface layer using laser radiation while simultaneously modulating the laser power. The structures are generated by redistribution of the molten material. The structure height and the structure wavelength of periodic structures created using WaveShape can be precisely adjusted by the adaption of various process parameters. However, the structures produced are mostly asymmetrical. An asymmetric structure refers to a structure that is not symmetrical and is inclined in or against the scanning direction. In the context of this work, the asymmetry of the structures was significantly reduced through two different process adaptations. As a first adaption, a compensation term is added to the laser power modulation, which is calculated from the difference profile between a target profile and a structured profile. With this adaption, the shape deviation of an asymmetrical structure could be decreased by 66 %. Asymmetry can be reduced efficiently, although the difference profile required must be determined from a preliminary process step. As a second adaption, a modulation of the scanning speed is investigated with which shape deviation can be decreased by 40 %. Asymmetry is not as effectively prevented as when using the first adaption, but the adaption can be performed without the difference profile. Another aim was to investigate the destructuring, i.e. the removal and therefore smoothing, of asymmetric structures. Using the inverse laser power modulation for destructuring, the structure height of a symmetrical structure can be reduced by 91 % while the structure height of an asymmetric structure can be reduced by 68 %. To increase the efficiency of destructuring of an asymmetrical structure, iterative destructuring was investigated. With two iterations of destructuring, the structure height was reduced by 90 %. As a second approach for more efficient destructuring of asymmetric structures an adaption of the laser power modulation via a compensation term was investigated. The structure height could be reduced by 86 %. In summary, results show that asymmetry can be prevented when structuring with WaveShape and that asymmetric structures can be destructured efficiently.

Synthesis of point-modified mRNA.

Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed 'point modification' in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatable and internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA.

Engineering of Thermostable ß-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

The ß-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-ßHAD), which catalyzes the NADP+ -dependent oxidation of ß-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-ßHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50 °C.