A5057247374- RNA and protein synthesis mechanisms
- RNA modifications and cancer
- RNA Research and Splicing
- SARS-CoV-2 and COVID-19 Research
- Advanced biosensing and bioanalysis techniques
- Cancer-related gene regulation
- DNA and Nucleic Acid Chemistry
- Viral gastroenteritis research and epidemiology
- Viral Infections and Immunology Research
- Computational Drug Discovery Methods
- European history and politics
- Force Microscopy Techniques and Applications
- Molecular Biology Techniques and Applications
- vaccines and immunoinformatics approaches
- German Literature and Culture Studies
- Bacterial Genetics and Biotechnology
- Advanced Fluorescence Microscopy Techniques
- Genomics and Chromatin Dynamics
- COVID-19 Clinical Research Studies
- Metabolomics and Mass Spectrometry Studies
- RNA Interference and Gene Delivery
- Advanced NMR Techniques and Applications
- Epigenetics and DNA Methylation
- Advanced Proteomics Techniques and Applications
- Molecular Junctions and Nanostructures
Goethe University Frankfurt
2014-2024
Johannes Gutenberg University Mainz
2024
Robert Bosch (Germany)
2023
Goethe Institute
2015
University of California, Santa Cruz
2011-2014
Heidelberg University
2007-2011
KU Leuven
2007
University of Illinois Urbana-Champaign
2007
Universität Ulm
2007
Abstract The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights need for coordinated research to combat COVID-19. A particularly important aspect is development of medication. In addition viral proteins, structured RNA elements represent a potent alternative as drug targets. search drugs that target requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, worldwide consortium NMR researchers aims...
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these fold independently, in line with data from vivo ex-vivo structural probing experiments. These contain non-base-paired regions potentially harbor ligand-binding pockets. Here, performed an NMR-based screening poised...
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 additional presence small population functional cloverleaf conformation. Both conformations are dynamic equilibrium, interconverting on 100 ms time scale. post-transcriptional methylation adenosine 9 to 1-methyladenosine is crucial step mature tRNA. This single...
Do knot FRET: Single-molecule FRET was used to analyze the folding of human telomerase RNA (see scheme) and allowed discrimination between correctly folded (left) misfolded (right) structures. Analyzing effects various mutations showed that base pairing, triplex formation, Mg2+ ion binding act synergistically in pseudoknot structure a functional hTR construct. Detailed facts importance specialist readers are published as ”Supporting Information”. Such documents peer-reviewed, but not...
Gene repression induced by the formation of transcriptional terminators represents a prime example for coupling RNA synthesis, folding, and regulation. In this context, mapping changes in available conformational space transcription intermediates during synthesis is important to understand riboswitch function. A majority riboswitches, an class small metabolite-sensing regulatory RNAs, act as regulators, but dependence ligand binding subsequent allosteric switch on mRNA transcript length has...
Abstract Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover translation. Furthermore, the regulatory function N6-methyladenine (m6A) during heat shock has been uncovered, including enhancement phase separation potential RNAs. In response to acute stress, e.g. shock, orderly sequestration mRNAs in stress granules (SGs) is considered important protect transcripts from irreversible aggregation. Until recently, role N1-methyladenine (m1A) on remains...
More than 170 post-transcriptional modifications of RNAs have currently been identified. Detailed biophysical investigations these limited since large containing are difficult to produce. Further, adequate readout spectroscopic fingerprints important, necessitating additional labeling procedures beyond the naturally occurring RNA modifications. Here, we report chemo-enzymatic synthesis and several structurally similar fluorine-modified analogs further optimizing a recently developed...
The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature conserved three-way junctional purine aptamer, it adopts second (apoB) with fundamentally different secondary structure. Here, we characterized ligand-dependent dynamics of Asw by NMR single-molecule FRET (smFRET) spectroscopy. Both methods revealed an...
N6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of human N6-methyltransferase complex (MTC) formed by a heterodimer consisting METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins site-specific methylation assay that allows determination quantitative yields, our results show this methylates its target RNAs not only but also secondary structure dependent. Furthermore, we demonstrate role...
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 analysis modified nucleotides in RNA presented based on quantitative site-specific DNAzyme-mediated cleavage target at or near site modification. Quantitative is achieved by cycling DNAzyme its substrate through repeated periods heating cooling. In a first approach, DNAzyme-directed directly 5′...
The current outbreak of the highly infectious COVID-19 respiratory disease is caused by novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight pandemic, search for promising viral drug targets has become a cross-border common goal international biomedical research community. Within Covid19-NMR consortium, scientists support development against providing publicly available NMR data on proteins and RNAs. nucleocapsid protein (N protein) an RNA-binding involved...
SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The increased vaccines, thus necessitating for development new intervention strategies including anti-virals. Within international Covid19-NMR consortium, we have identified binders targeting RNA genome SCoV2. We established protocols production NMR characterization more than 80 % all SCoV2 proteins. Here, performed an screening using a fragment library binding 25 proteins hits also against...
Translational riboswitches located in the 5' UTR of messenger RNA (mRNA) regulate translation through variation accessibility ribosome binding site (RBS). These are result conformational changes riboswitch governed by ligand binding. Here, we use a combination single-molecule colocalization techniques (Single-Molecule Kinetic Analysis Transient Structure (SiM-KARTS) and Single-Molecule Ribosome Binding (SiM-KARB)) microscale thermophoresis (MST) to investigate adenine-sensing
Abstract Cellular RNA function is closely linked to structure. It therefore imperative develop methods that report on structural stability of and how it modulated by binding ions, other osmolytes, RNA‐binding ligands. Here, we present a novel method analyze the virtually any structured in highly parallel fashion. This can easily determine influence various additives stability, even characterize ligand‐induced stabilization riboswitch RNA. Current approaches assess include thermal melting...
With or without Me: The effect of a single methyl group on the free energies functional and nonfunctional conformations human mitochondrial lysine transfer RNA (tRNALys) has been studied. stabilization three distinct by Mg2+ ion binding energy changes resulting from methylation were characterized revealed preference for functionally relevant cloverleaf-shaped tRNA form (see picture). Supporting information this article is available WWW under...
The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute spectroscopic analysis of viral proteins and RNA. Here, report NMR chemical shift assignments protein Nsp3b, a domain Nsp3. 217-kDa large Nsp3 contains multiple structurally independent, yet functionally related domains including papain-like protease macrodomain (MD). In general, MDs SARS-CoV MERS-CoV were suggested to play key role in replication by modulating immune...
Temperature-dependent alternative splicing was recently demonstrated for intron 2 of the gene coding heat shock factor HsfA2 tomato plant Solanum lycopersicum, but molecular mechanism regulating abundance such temperature-dependent splice variants is still unknown. We report here on regulatory pre-mRNA structures that could function as regulators by controlling use sites in a manner. investigate at from S. lycopersicum using NMR- and CD-spectroscopy well in-line probing. The undergoes...
Investigation of single RNA molecules using fluorescence resonance energy transfer (FRET) is a powerful approach to investigate dynamic and thermodynamic aspects the folding process given RNA. Its application requires interdisciplinary work from fields chemistry, biochemistry, physics. The present gives detailed instructions on synthesis labeled with two fluorescent dyes interacting by FRET, as well their investigation single-molecule spectroscopy.
Abstract NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application restricted in RNA size most often requires isotope‐labeled or even selectively labeled Additionally, new pulse sequences, such as heteronuclear‐detected experiments, are introduced. We herein provide detailed protocols preparation via vitro transcription. This protocol covers all steps, from DNA template to transcription milligram quantities. Moreover, we present...
Abstract The SARS‐CoV‐2 genome has been shown to be m 6 A methylated at several positions in vivo . Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides core of transcriptional regulatory leader sequence (TRS−L) position A74, which is highly conserved and essential viral discontinuous transcription. Methylation A74 correlates with pathogenicity. Discontinuous transcription produces set subgenomic mRNAs that function as templates translation all structural...