Martin Hengesbach

Bio: Martin Hengesbach is an academic researcher from Goethe University Frankfurt. The author has contributed to research in topics: RNA & Riboswitch. The author has an hindex of 12, co-authored 49 publications receiving 562 citations. Previous affiliations of Martin Hengesbach include Heidelberg University & University of California, Santa Cruz.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

Abstract: The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

A Methyl Group Controls Conformational Equilibrium in Human Mitochondrial tRNALys

Abstract: The unmodified maturation intermediate in the biogenesis of human mitochondrial tRNALys adopts a nonfunctional conformation, resembling an extended hairpin. Single molecule fluorescence resonance energy transfer (FRET) studies reveal the additional presence of a small population in a functional cloverleaf conformation. Both conformations are in a dynamic equilibrium, interconverting on the 100 ms time scale. The post-transcriptional methylation of adenosine 9 to 1-methyladenosine is a crucial step in the biogenesis of the mature tRNA. This single methyl group, by disfavoring the nonfunctional conformation, shifts the observed equilibrium toward the functional cloverleaf and makes it available for further maturation. Here, single molecule FRET analysis makes dynamics and thermodynamics of these small RNAs conveniently accessible and elucidates the mode of action of a simple post-transcriptional modification.

Single‐Molecule FRET Reveals the Folding Dynamics of the Human Telomerase RNA Pseudoknot Domain

Abstract: Telomerase catalyzed synthesis of telomere DNA provides the foundation for DNA-protein structures called telomeres. Telomeres are required to evade DNA processing which may result in nucleolytic degradation and fusion of linear chromosomes. Human telomerase contains a protein reverse transcriptase (hTERT), telomerase RNA (hTR), and several additional proteins.[1] hTR provides the template sequence for hTERT during a novel reverse transcription reaction that produces short telomere DNA repeat sequences.[2] Additionally, hTR provides a flexible scaffold for the assembly and function of telomerase associated proteins[3] and has recently been suggested to contribute to enzyme catalysis.[4] Many pathogenic mutations have been mapped to hTR;[5] however the precise mechanisms of these mutations remain unclear.

Use of DNAzymes for site-specific analysis of ribonucleotide modifications

Abstract: Post-transcriptional ribonucleotide modifications are widespread and abundant processes that have not been analyzed adequately due to the lack of appropriate detection methods. Here, two methods for the analysis of modified nucleotides in RNA are presented that are based on the quantitative and site-specific DNAzyme-mediated cleavage of the target RNA at or near the site of modification. Quantitative RNA cleavage is achieved by cycling the DNAzyme and its RNA substrate through repeated periods of heating and cooling. In a first approach, DNAzyme-directed cleavage directly 5′ of the residue in question allows radioactive labeling of the newly freed 5′-OH. After complete enzymatic hydrolysis, the modification status can be assessed by two-dimensional thin layer chromatography. In a second approach, oligoribonucleotide fragments comprising the modification site are excised from the full-length RNA in an endonucleolytic fashion, using a tandem DNAzyme. The excised fragment is isolated by electrophoresis and submitted to further conventional analysis. These results establish DNAzymes as valuable tools for the site-specific and highly sensitive detection of ribonucleotide modifications.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

Abstract: SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

Principles Of Fluorescence Spectroscopy

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RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage

Abstract: The covalent modification of nucleic acids plays an important role in regulating the functions of DNA and RNA. DNA modifications have been analyzed in considerable detail, and the characterization of (cytosine-5) DNA methylation has been crucial for understanding the molecular basis of epigenetic gene regulation (Klose and Bird 2006). (Cytosine-5) methylation has also been documented in various RNA species, including tRNA, but the function of RNA methylation has not been firmly established yet (Motorin et al. 2010). Dnmt2 proteins were originally assigned to the DNA methyltransferase family, because of their strong sequence conservation of catalytic DNA methyltransferase motifs (Okano et al. 1998; Yoder and Bestor 1998). A recent study has suggested that Dnmt2-mediated DNA methylation is important for transposon silencing in Drosophila (Phalke et al. 2009). However, only a weak and distributive DNA methylation activity has been reported in various systems (Jeltsch et al. 2006). The ambiguities associated with the DNA methyltransferase activity of Dnmt2 have also prompted the search for alternative enzyme substrates, and resulted in the discovery of a tRNA methyltransferase activity of Dnmt2 (Goll et al. 2006). Purified recombinant human Dnmt2 methylated RNA preparations from Dnmt2 mutant mice, flies, and plants. Further experiments identified C38 in the anti-codon loop of tRNAAsp as the methylation target site of Dnmt2 (Goll et al. 2006). However, the functional relevance of the tRNA methyltransferase activity of Dnmt2 remains to be established. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile (Goll et al. 2006) under standard laboratory conditions. A distinct Dnmt2 mutant phenotype, caused by morpholino knockdown experiments, has so far been reported only in zebrafish, leading to lethal differentiation defects in the retina, liver, and brain (Rai et al. 2007). In addition, two studies have indicated increased stress tolerance in Dnmt2-overexpressing flies and amoebas (Lin et al. 2005; Fisher et al. 2006). However, the underlying molecular mechanisms have not been investigated yet.

Detecting RNA modifications in the epitranscriptome: predict and validate

Abstract: RNA modifications are emerging players in the field of post-transcriptional regulation of gene expression, and are attracting a comparable degree of research interest to DNA and histone modifications in the field of epigenetics. We now know of more than 150 RNA modifications and the true potential of a few of these is currently emerging as the consequence of a leap in detection technology, principally associated with high-throughput sequencing. This Review outlines the major developments in this field through a structured discussion of detection principles, lays out advantages and drawbacks of new high-throughput methods and presents conventional biophysical identification of modifications as meaningful ways for validation.

tRNA stabilization by modified nucleotides.

Abstract: Post-transcriptional ribonucleotide modification is a phenomenon best studied in tRNA, where it occurs most frequently and in great chemical diversity. This paper reviews the intrinsic network of modifications in the structural core of the tRNA, which governs structural flexibility and rigidity to fine-tune the molecule to peak performance and to regulate its steady-state level. Structural effects of RNA modifications range from nanometer-scale rearrangements to subtle restrictions of conformational space on the angstrom scale. Structural stabilization resulting from nucleotide modification results in increased thermal stability and translates into protection against unspecific degradation by bases and nucleases. Several mechanisms of specific degradation of hypomodified tRNA, which were only recently discovered, provide a link between structural and metabolic stability.

The RNA modification landscape in human disease.

Abstract: RNA modifications have been historically considered as fine-tuning chemo-structural features of infrastructural RNAs, such as rRNAs, tRNAs, and snoRNAs. This view has changed dramatically in recent years, to a large extent as a result of systematic efforts to map and quantify various RNA modifications in a transcriptome-wide manner, revealing that RNA modifications are reversible, dynamically regulated, far more widespread than originally thought, and involved in major biological processes, including cell differentiation, sex determination, and stress responses. Here we summarize the state of knowledge and provide a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. With the advent of direct RNA-sequencing technologies, we expect that this catalog will help prioritize those RNA modifications for transcriptome-wide maps.