Biology subjects

Göringer, H. U.

Publications and source records attributed to Göringer, H. U..

5 recordsLinked to original sources

bioRxiv2022

Core-shell DNA-cholesterol nanoparticles exert lysosomolytic activity in African trypanosomes

Trypanosoma brucei is the causal infectious agent of African trypanosomiasis in humans and Nagana in livestock. Both diseases are currently treated with a small number of chemotherapeutics, which are hampered by a variety of limitations reaching from efficacy and toxicity complications to drug-resistance problems. Here, we explore the forward design of a new class of synthetic trypanocides based on nanostructured, core-shell DNA-lipid particles. In aqueous solution, the particles self-assemble into micelle-type structures consisting of a solvent-exposed, hydrophilic DNA shell and a hydrophobic lipid core. DNA-lipid nanoparticles have membrane-adhesive qualities and can permeabilize lipid membranes. We report the synthesis of DNA-cholesterol nanoparticles, which specifically subvert the membrane integrity of the T. brucei lysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA shell with a spliced leader (SL)-RNA-specific DNAzyme, we target a second trypanosome-specific pathway (dual-target approach). The DNAzyme provides a backup to counteract the recovery of compromised parasites, which reduces the risk of developing drug resistance.

molecular biology↗

bioRxiv2021

Fuzzy RNA-recognition by the Trypanosoma brucei editosome

The recognition of RNA-molecules by proteins and protein complexes is a critical step on all levels of gene expression. Typically, the generated ribonucleoprotein complexes rely on the binary interaction of defined RNA-sequences or precisely folded RNA-motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA-molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome we identified multiple similar but nonidentical RNA-motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.523 ({lambda}2). We establish that a synthetic 4V(3E)-RNA is sufficient to compete for the editosomal pre-mRNA binding site and that it is able to inhibit RNA-editing in vitro. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA-folding space by recognizing a fuzzy continuum of RNA-folds that fit a consensus graph-descriptor. This provides a mechanism on how the protein complex is able to bind the structurally pleomorphic pool of pre- and partially edited mRNAs. We speculate that other fuzzy RNA-recognition motifs exist especially for proteins that interact with multiple RNA-species.

molecular biology↗

bioRxiv2020

Analyzing editosome function in high-throughput

Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.

molecular biology↗

bioRxiv2019

Cell-free prototyping of AND-logic gates based on heterogeneous RNA activators

RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages like short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multi-level regulated circuits. In this work, we combined a variety of small transcriptional activator RNAs (STARs) and toehold switches to build highly effective AND-gates. To characterise the components and their dynamic range, we used an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets. We analysed a prototype gate in vitro as well as in silico, employing parameterised ordinary differential equations (ODEs), where parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. Based on this analysis, we created nine additional AND-gates and tested them in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, offering a dynamic range comparable to the level of protein circuits. This study shows the potential of a rapid prototyping approach for RNA circuit design, using cell-free systems in combination with a model prediction.\n\nAbbreviationsTX-TL (transcription-translation), ODEs (ordinary differential equations), STARs (small transcriptional activator RNAs), MCMC (Markov chain Monte Carlo).

synthetic biology↗

bioRxiv2018

Bioinspired Design of Lysolytic Triterpenoid-Peptide Conjugates that Kill African Trypanosomes

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). Active TLF-ingredient is the primate-specific apolipoprotein L1 (ApoL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies only two are able to counteract the activity of ApoL1, which illustrates its evolutionary optimized design and trypanocidal potency. Here we ask the question whether a synthetic (syn)TLF can be synthesized using the design principles of the natural TLF-complexes but relying on different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell binding, uptake and lysosomal transport-moduls and the synthetic peptide GALA as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis demonstrating proof-of-principle for the bioinspired, forward-design of a synTLF.

synthetic biology↗