Susanne Dietrich successfully defended her dissertation in a truly outstanding defense-Congratulations!

Abstract

Sequence-specific detection and analysis of nucleic acids are central to molecular diagnostics, especially when rapid, amplification-free, and robust readout is required. Hybridization-based capture on solid supports is combined with controlled enzymatic digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), complemented by fluorescence-based readout and chip-based implementation.
First, an assay was established in which biotinylated capture strands are immobilized on magnetic beads to enrich complementary target oligonucleotides. Hybridization specificity was investigated using defined point mutations: two central mismatches already caused a signal loss, three or more mutations fully suppressed hybridization, whereas terminal mutations were tolerated but remained distinguishable by their mass. Single-strand (ss)-specific nucleases were employed to digest extending regions of longer targets directly on the beads. By combining S1 and mung bean (MB) nuclease digestion, overhangs were removed.
In a complementary study, the influence of matrix composition and deposition on MALDITOF MS performance for oligonucleotides was comprehensively evaluated. The ionic matrix 6-aza-2-thiothymine with 1-methylimidazole consistently yielded comparably low mass standard deviations and high mass accuracy, whereas the signal-to-noise ratio and precision of the commonly used 3-hydroxypicolinic acid depended on solvent composition and additives.
The hybridization concept was extended from a mass-spectrometric to an optical readout by employing the intercalating fluorophore GelRed, which preferentially binds double-stranded (ds) nucleic acids. This enabled discrimination of ss and ds nucleic acids at the capture surface and thus direct assessment of target presence without MALDI-TOF MS. Ss-specific capture was further transferred to Sepharose particles and evaluated by polyacrylamide gel electrophoresis of extracted 16S rRNA. The digestion process with MB nuclease was systematically tuned by multi-objective optimization, yielding four parameter sets that produced the desired fragment length (26 base pairs). Finally, the bead-based assay was transferred onto indium tin oxide (ITO) slides, enabling MALDI-TOF MS readout in a chip format. On these slides, negative and positive control samples were significantly distinguished.