H. Ulrich Göringer - Academia.edu (original) (raw)

Papers by H. Ulrich Göringer

Research paper thumbnail of A putative RNA helicase of the DEAD ☐ family from Trypanosoma brucei

Molecular and Biochemical Parasitology, 1995

Research paper thumbnail of Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

The EMBO Journal, Feb 5, 2009

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific R... more Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of B20S and B35-40S and present the three-dimensional structures of both complexes by electron microscopy. The B35-40S complexes consist of a platform density packed against a semispherical element. The B20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The B20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Research paper thumbnail of Kinetoplastid RNA editing involves a 3′ nucleotidyl phosphatase activity

Nucleic Acids Research, Feb 3, 2009

Research paper thumbnail of Charge Reduction and Thermodynamic Stabilization of Substrate RNAs Inhibit RNA Editing

PLOS ONE, Mar 5, 2015

African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcri... more African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.

Research paper thumbnail of The RNA chaperone activity of the Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs

Scientific Reports, Jan 19, 2016

Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction that... more Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. The reaction is catalyzed by editosomes and requires guide (g)RNAs as templates. Recent data demonstrate that the binding of pre-edited mRNAs to editosomes is followed by a chaperone-type RNA remodeling reaction. Here we map the changes in RNA folding using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that pre-mRNAs in their free state adopt intricately folded, highly stable 2D-structures. Editosome binding renders the pre-mRNAs to adopt 2D-conformations of reduced stabilities. On average about 30% of the nucleotides in every pre-mRNA are affected with a prevalence for U-nucleotides. The data demonstrate that the chaperone activity acts by increasing the flexibility of U-residues to lower their base-pairing probability. This results in a simplified RNA folding landscape with a reduced energy barrier to facilitate the binding of gRNAs. The data provide a first rational for the enigmatic U-specificity of the editing reaction. Results Pre-edited mRNAs adopt thermodynamically highly stable 2D-structures. To map changes in the structural landscape of pre-edited mRNAs upon binding to editosomes we used the SHAPE (selective 2′-hydroxyl

Research paper thumbnail of Nanopore-Based Direct RNA Sequencing of the Trypanosoma brucei Transcriptome Identifies Novel lncRNAs

Genes

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene... more Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay, to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized to date. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known, because of the inherent cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here, we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome Trypanosoma brucei, with a special emphasis on the inven...

Research paper thumbnail of Nanopore-based direct RNA sequencing of theTrypanosoma bruceitranscriptome identifies novel lncRNAs

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene... more Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression posttranscriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example, by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized today. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known because of the inherent cDNA recoding and DNA amplification limitations of short-read RNA sequencing. Here we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosomeT. bruceiwith a special emphasis on the inventory of lncRNAs. ...

Research paper thumbnail of Core-shell DNA-cholesterol nanoparticles exert lysosomolytic activity in African trypanosomes

Trypanosoma bruceiis the causal infectious agent of African trypanosomiasis in humans and Nagana ... more Trypanosoma bruceiis 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 theT. bruceilysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA she...

Research paper thumbnail of 6 Authors, Including

doi:10.1093/nar/gkp049 Kinetoplastid RNA editing involves a 3 ’ nucleotidyl phosphatase activity

Research paper thumbnail of Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing

Methods in molecular biology, 2020

Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D... more Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8 MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and as a consequence generates a simplified RNA folding landscape that is critical for the progression of the editing reaction cycle. Here we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (double-...

Research paper thumbnail of in Trypanosoma brucei Complexes gBP21 with Active RNA Editing Association of Guide RNA Binding Protein

Research paper thumbnail of On-Demand Production of Femtoliter Drops in Microchannels and Their Use as Biological Reaction Compartments

Analytical Chemistry, 2019

We present a method allowing to produce monodisperse droplets with volumes in the femtoliter rang... more We present a method allowing to produce monodisperse droplets with volumes in the femtoliter range in a microchannel on demand. The method utilizes pulsed electric fields deforming the interface between an aqueous and an oil phase and pinching off droplets. Water and xanthan gum solutions are considered as disperse-phase liquids, and it is shown that the method can be applied even to solutions with a zero-shear rate viscosity more than 10 4 times higher than that of water. The droplet formation regimes are explored by systematically varying the pulse amplitude and duration as well as the salt concentration. The dependence of the process on the pulse amplitude can be utilized to tune the droplet size. To demonstrate the applicability of the electric-fielddriven droplet generator, it is shown that the droplets can be used as versatile biological reaction compartments. It is proven that droplets containing a cell-free transcription-translation system execute gene transcription and protein biosynthesis in a timely and programmable fashion. Moreover, it is verified that biomolecules inside the aqueous droplets such as small RNAs can be diffusionally activated from the outside to induce a ligand-driven biochemical switch.

Research paper thumbnail of Parasite-specific aptamers as biosynthetic reagents and potential pharmaceuticals

Trends in parasitology, 2012

Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in v... more Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in vitro evolution method known as 'systematic evolution of ligands by exponential enrichment' (SELEX). SELEX represents an experimental platform to identify rare ligands with predetermined functionality from combinatorial nucleic acid libraries. Since its discovery about 20 years ago the method has been instrumental in identifying a large number of aptamers that recognize targets of very different chemistry and molecular complexity. Although aptamers have been converted into sophisticated biomolecular tools for a diverse set of technologies, only a limited number of aptamers have been selected as binding reagents for parasites or parasite-derived molecules. Here the published examples of aptamers that target Leishmania-, Trypanosoma- and Plasmodia-specific molecules are reviewed.

Research paper thumbnail of RNA Editing Accessory Factors — the Example of mHel61p

Nucleic Acids and Molecular Biology, 2008

The majority of mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of a U ... more The majority of mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of a U nucleotide-specific, insertion/deletion-type RNA editing reaction. The process converts nonfunctional pre-mRNAs into translatable molecules, and can generate protein diversity by alternative editing. A high molecular mass enzyme complex, the editosome, catalyzes the reaction. Editosomes provide a molecular platform for the individual catalytic steps of the reaction cycle.

Research paper thumbnail of Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

RNA, 2007

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affi... more Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable o...

Research paper thumbnail of Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination

Proceedings of the National Academy of Sciences, 1991

Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Esc... more Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.

Research paper thumbnail of The formation of mitochondrial ribonucleoproteincomplexes involving guide RNA molecules in Trypanosoma brucei

Proceedings of the National Academy of Sciences, 1994

Transcripts from mitochondrial (maxicircle) genes of kinetoplastid organisms undergo RNA editing ... more Transcripts from mitochondrial (maxicircle) genes of kinetoplastid organisms undergo RNA editing characterized by a series of reactions that insert and delete uridine nucleotides within the sequence of the pre-mRNAs. Guide RNAs, which complement fully edited mRNAs, provide the information for the edited sequence by an unknown mechanism. We report here that guide RNA molecules associate with other mitochondrial components to form four specific, stable ribonucleoprotein complexes. The complexes form very rapidly at a low monovalent cation concentration, and their formation is blocked by heparin or pretreatment of the mitochondrial lysate with SDS. ATP hydrolysis is not required but slightly stimulates complex association up to concentrations of 5 mM. The results are suggestive of a sequential assembly of the ribonucleoprotein complexes, and their possible involvement during the kinetoplastid RNA editing is discussed.

Research paper thumbnail of Mutant 16S ribosomal RNA: a codon-specific translational suppressor

Proceedings of the National Academy of Sciences, 1988

We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The supp... more We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211). The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4. It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations. The suppressor also does not allow mistranslation of the UGA-related trpA missense mutations UGG at positions 211 and 234, AGA at 211 and 234, CGA at 211, or UGU and UGC at 234. The suppressor mutation was mapped by genetic procedures to position 89 on the E. coli genetic map. Localization of the suppressor mutation to rrnB was achieved by cloning it in the low-copy-number plasmid pEJM007 by in vivo recombination from the chromosome. Recloning in bacte...

Research paper thumbnail of The secondary structure of guide RNA molecules fromTrypanosoma brucei

Nucleic Acids Research, 1995

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is chara... more RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with TmS between 33 and 390C and transition enthalpies of-32 to-38 kcaVmol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.

Research paper thumbnail of The guide RNA database (3.0)

Nucleic Acids Research, 1999

The RNA editing process within the mitochondria of kinetoplastid organisms is controlled by small... more The RNA editing process within the mitochondria of kinetoplastid organisms is controlled by small, transacting RNA molecules referred to as guide RNAs. The guide RNA database is a compilation of published guide RNA sequences, currently containing 254 entries from 11 different organisms. Additional information includes RNA secondary and tertiary structure models, information on the gene localisation, literature citations and other relevant facts. The database can be accessed through the World Wide Web

Research paper thumbnail of A putative RNA helicase of the DEAD ☐ family from Trypanosoma brucei

Molecular and Biochemical Parasitology, 1995

Research paper thumbnail of Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

The EMBO Journal, Feb 5, 2009

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific R... more Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of B20S and B35-40S and present the three-dimensional structures of both complexes by electron microscopy. The B35-40S complexes consist of a platform density packed against a semispherical element. The B20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The B20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Research paper thumbnail of Kinetoplastid RNA editing involves a 3′ nucleotidyl phosphatase activity

Nucleic Acids Research, Feb 3, 2009

Research paper thumbnail of Charge Reduction and Thermodynamic Stabilization of Substrate RNAs Inhibit RNA Editing

PLOS ONE, Mar 5, 2015

African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcri... more African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.

Research paper thumbnail of The RNA chaperone activity of the Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs

Scientific Reports, Jan 19, 2016

Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction that... more Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. The reaction is catalyzed by editosomes and requires guide (g)RNAs as templates. Recent data demonstrate that the binding of pre-edited mRNAs to editosomes is followed by a chaperone-type RNA remodeling reaction. Here we map the changes in RNA folding using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that pre-mRNAs in their free state adopt intricately folded, highly stable 2D-structures. Editosome binding renders the pre-mRNAs to adopt 2D-conformations of reduced stabilities. On average about 30% of the nucleotides in every pre-mRNA are affected with a prevalence for U-nucleotides. The data demonstrate that the chaperone activity acts by increasing the flexibility of U-residues to lower their base-pairing probability. This results in a simplified RNA folding landscape with a reduced energy barrier to facilitate the binding of gRNAs. The data provide a first rational for the enigmatic U-specificity of the editing reaction. Results Pre-edited mRNAs adopt thermodynamically highly stable 2D-structures. To map changes in the structural landscape of pre-edited mRNAs upon binding to editosomes we used the SHAPE (selective 2′-hydroxyl

Research paper thumbnail of Nanopore-Based Direct RNA Sequencing of the Trypanosoma brucei Transcriptome Identifies Novel lncRNAs

Genes

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene... more Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay, to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized to date. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known, because of the inherent cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here, we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome Trypanosoma brucei, with a special emphasis on the inven...

Research paper thumbnail of Nanopore-based direct RNA sequencing of theTrypanosoma bruceitranscriptome identifies novel lncRNAs

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene... more Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression posttranscriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example, by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized today. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known because of the inherent cDNA recoding and DNA amplification limitations of short-read RNA sequencing. Here we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosomeT. bruceiwith a special emphasis on the inventory of lncRNAs. ...

Research paper thumbnail of Core-shell DNA-cholesterol nanoparticles exert lysosomolytic activity in African trypanosomes

Trypanosoma bruceiis the causal infectious agent of African trypanosomiasis in humans and Nagana ... more Trypanosoma bruceiis 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 theT. bruceilysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA she...

Research paper thumbnail of 6 Authors, Including

doi:10.1093/nar/gkp049 Kinetoplastid RNA editing involves a 3 ’ nucleotidyl phosphatase activity

Research paper thumbnail of Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing

Methods in molecular biology, 2020

Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D... more Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8 MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and as a consequence generates a simplified RNA folding landscape that is critical for the progression of the editing reaction cycle. Here we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (double-...

Research paper thumbnail of in Trypanosoma brucei Complexes gBP21 with Active RNA Editing Association of Guide RNA Binding Protein

Research paper thumbnail of On-Demand Production of Femtoliter Drops in Microchannels and Their Use as Biological Reaction Compartments

Analytical Chemistry, 2019

We present a method allowing to produce monodisperse droplets with volumes in the femtoliter rang... more We present a method allowing to produce monodisperse droplets with volumes in the femtoliter range in a microchannel on demand. The method utilizes pulsed electric fields deforming the interface between an aqueous and an oil phase and pinching off droplets. Water and xanthan gum solutions are considered as disperse-phase liquids, and it is shown that the method can be applied even to solutions with a zero-shear rate viscosity more than 10 4 times higher than that of water. The droplet formation regimes are explored by systematically varying the pulse amplitude and duration as well as the salt concentration. The dependence of the process on the pulse amplitude can be utilized to tune the droplet size. To demonstrate the applicability of the electric-fielddriven droplet generator, it is shown that the droplets can be used as versatile biological reaction compartments. It is proven that droplets containing a cell-free transcription-translation system execute gene transcription and protein biosynthesis in a timely and programmable fashion. Moreover, it is verified that biomolecules inside the aqueous droplets such as small RNAs can be diffusionally activated from the outside to induce a ligand-driven biochemical switch.

Research paper thumbnail of Parasite-specific aptamers as biosynthetic reagents and potential pharmaceuticals

Trends in parasitology, 2012

Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in v... more Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in vitro evolution method known as 'systematic evolution of ligands by exponential enrichment' (SELEX). SELEX represents an experimental platform to identify rare ligands with predetermined functionality from combinatorial nucleic acid libraries. Since its discovery about 20 years ago the method has been instrumental in identifying a large number of aptamers that recognize targets of very different chemistry and molecular complexity. Although aptamers have been converted into sophisticated biomolecular tools for a diverse set of technologies, only a limited number of aptamers have been selected as binding reagents for parasites or parasite-derived molecules. Here the published examples of aptamers that target Leishmania-, Trypanosoma- and Plasmodia-specific molecules are reviewed.

Research paper thumbnail of RNA Editing Accessory Factors — the Example of mHel61p

Nucleic Acids and Molecular Biology, 2008

The majority of mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of a U ... more The majority of mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of a U nucleotide-specific, insertion/deletion-type RNA editing reaction. The process converts nonfunctional pre-mRNAs into translatable molecules, and can generate protein diversity by alternative editing. A high molecular mass enzyme complex, the editosome, catalyzes the reaction. Editosomes provide a molecular platform for the individual catalytic steps of the reaction cycle.

Research paper thumbnail of Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

RNA, 2007

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affi... more Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable o...

Research paper thumbnail of Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination

Proceedings of the National Academy of Sciences, 1991

Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Esc... more Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.

Research paper thumbnail of The formation of mitochondrial ribonucleoproteincomplexes involving guide RNA molecules in Trypanosoma brucei

Proceedings of the National Academy of Sciences, 1994

Transcripts from mitochondrial (maxicircle) genes of kinetoplastid organisms undergo RNA editing ... more Transcripts from mitochondrial (maxicircle) genes of kinetoplastid organisms undergo RNA editing characterized by a series of reactions that insert and delete uridine nucleotides within the sequence of the pre-mRNAs. Guide RNAs, which complement fully edited mRNAs, provide the information for the edited sequence by an unknown mechanism. We report here that guide RNA molecules associate with other mitochondrial components to form four specific, stable ribonucleoprotein complexes. The complexes form very rapidly at a low monovalent cation concentration, and their formation is blocked by heparin or pretreatment of the mitochondrial lysate with SDS. ATP hydrolysis is not required but slightly stimulates complex association up to concentrations of 5 mM. The results are suggestive of a sequential assembly of the ribonucleoprotein complexes, and their possible involvement during the kinetoplastid RNA editing is discussed.

Research paper thumbnail of Mutant 16S ribosomal RNA: a codon-specific translational suppressor

Proceedings of the National Academy of Sciences, 1988

We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The supp... more We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211). The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4. It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations. The suppressor also does not allow mistranslation of the UGA-related trpA missense mutations UGG at positions 211 and 234, AGA at 211 and 234, CGA at 211, or UGU and UGC at 234. The suppressor mutation was mapped by genetic procedures to position 89 on the E. coli genetic map. Localization of the suppressor mutation to rrnB was achieved by cloning it in the low-copy-number plasmid pEJM007 by in vivo recombination from the chromosome. Recloning in bacte...

Research paper thumbnail of The secondary structure of guide RNA molecules fromTrypanosoma brucei

Nucleic Acids Research, 1995

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is chara... more RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with TmS between 33 and 390C and transition enthalpies of-32 to-38 kcaVmol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.

Research paper thumbnail of The guide RNA database (3.0)

Nucleic Acids Research, 1999

The RNA editing process within the mitochondria of kinetoplastid organisms is controlled by small... more The RNA editing process within the mitochondria of kinetoplastid organisms is controlled by small, transacting RNA molecules referred to as guide RNAs. The guide RNA database is a compilation of published guide RNA sequences, currently containing 254 entries from 11 different organisms. Additional information includes RNA secondary and tertiary structure models, information on the gene localisation, literature citations and other relevant facts. The database can be accessed through the World Wide Web