Institutes
Refine
Document Type
- Article (32)
- Contribution to a Periodical (2)
- Preprint (1)
Has Fulltext
- yes (35)
Is part of the Bibliography
- no (35)
Keywords
- SARS-CoV-2 (9)
- NMR spectroscopy (8)
- RNA (5)
- COVID19-NMR (4)
- Non-structural protein (3)
- Solution NMR spectroscopy (3)
- Solution NMR-spectroscopy (3)
- 5′-UTR (2)
- Covid19-NMR (2)
- G-quadruplexes (2)
We investigated the folding kinetics of G-quadruplex (G4) structures by comparing the K+-induced folding of an RNA G4 derived from the human telomeric repeat-containing RNA (TERRA25) with a sequence homologous DNA G4 (wtTel25) using CD spectroscopy and real-time NMR spectroscopy. While DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favoured off-pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs. DNA G4s, in particular with regard to the conformation around the glycosidic torsion angle χ that uniformly adopts anti conformations for RNA G4s and both, syn and anti conformation for DNA G4s. Modified DNA G4s with 19F bound to C2′ in arabino configuration adopt exclusively anti conformations for χ. These fluoro-modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting the correlation between folding kinetics and pathways with differences in χ angle preferences in DNA and RNA, respectively.
Riboswitches are gene regulatory elements located in untranslated mRNA regions. They bind inducer molecules with high affinity and specificity. Cyclic-di-nucleotide-sensing riboswitches are major regulators of genes for the environment, membranes and motility (GEMM) of bacteria. Up to now, structural probing assays or crystal structures have provided insight into the interaction between cyclic-di-nucleotides and their corresponding riboswitches. ITC analysis, NMR analysis and computational modeling allowed us to gain a detailed understanding of the gene regulation mechanisms for the Cd1 (Clostridium difficile) and for the pilM (Geobacter metallireducens) riboswitches and their respective di-nucleotides c-di-GMP and c-GAMP. Binding capability showed a 25 nucleotide (nt) long window for pilM and a 61 nt window for Cd1. Within this window, binding affinities ranged from 35 μM to 0.25 μM spanning two orders of magnitude for Cd1 and pilM showing a strong dependence on competing riboswitch folds. Experimental results were incorporated into a Markov simulation to further our understanding of the transcriptional folding pathways of riboswitches. Our model showed the ability to predict riboswitch gene regulation and its dependence on transcription speed, pausing and ligand concentration.
Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry.
Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.
Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.
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.
The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio-frequency power to lossy aqueous samples, refolding of barstar from its cold-denatured state can be followed by real-time NMR spectroscopy. Since temperature-induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real-time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low-temperature-unfolded state, which can potentially induce multiple folding pathways. The high time resolution real-time 2D-NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state-correlated spectroscopy, an alternative folding pathway circumventing the rate-limiting cis-trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.
We propose a generalized modeling framework for the kinetic mechanisms of transcriptional riboswitches. The formalism accommodates time-dependent transcription rates and changes of metabolite concentration and permits incorporation of variations in transcription rate depending on transcript length. We derive explicit analytical expressions for the fraction of transcripts that determine repression or activation of gene expression, pause site location and its slowing down of transcription for the case of the (2’dG)-sensing riboswitch from Mesoplasma florum. Our modeling challenges the current view on the exclusive importance of metabolite binding to transcripts containing only the aptamer domain. Numerical simulations of transcription proceeding in a continuous manner under time-dependent changes of metabolite concentration further suggest that rapid modulations in concentration result in a reduced dynamic range for riboswitch function regardless of transcription rate, while a combination of slow modulations and small transcription rates ensures a wide range of finely tuneable regulatory outcomes.
Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.