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- NMR spectroscopy (14)
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G-quadruplexes (G4), found in numerous places within the human genome, are involved in essential processes of cell regulation. Chromosomal DNA G4s are involved for example, in replication and transcription as first steps of gene expression. Hence, they influence a plethora of downstream processes. G4s possess an intricate structure that differs from canonical B-form DNA. Identical DNA G4 sequences can adopt multiple long-lived conformations, a phenomenon known as G4 polymorphism. A detailed understanding of the molecular mechanisms that drive G4 folding is essential to understand their ambivalent regulatory roles. Disentangling the inherent dynamic and polymorphic nature of G4 structures thus is key to unravel their biological functions and make them amenable as molecular targets in novel therapeutic approaches. We here review recent experimental approaches to monitor G4 folding and discuss structural aspects for possible folding pathways. Substantial progress in the understanding of G4 folding within the recent years now allows drawing comprehensive models of the complex folding energy landscape of G4s that we herein evaluate based on computational and experimental evidence.
Riboswitches are highly structured elements in the 50-untranslated regions (50-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/ hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by longrange base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using highresolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.
Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding.
Oral presentations Background: We selected peptide ligands for the HIV-1 packaging signal PSI by screening phage displayed peptide libraries. Peptide ligands were optimized by screening spot synthesis peptide membranes. The aim of this study is the functional characterization of these peptide ligands with respect to inhibition of HIV-1 replication. Methods: Phage displayed peptide libraries were screened with PSI-RNA structures. The Trp-rich peptide motifs were optimized for specific binding on spot synthesis peptide membranes. The best binding peptide was expressed intracellularly in fusion with RFP or linked to a protein transduction domain (PTD) for intracellular delivery. The effects on virion production were analyzed using pseudotyped lentiviral particles. Results: After positive and negative selection rounds, phages binding specifically to PSI-RNA were identified by ELISA. Peptide inserts contained conserved motifs of aromatic amino acids known to be implicated in binding of PSI-RNA by the natural Gag ligand. The filter assay identified HKWPWW as the best binding ligand for PSI-RNA, which is delivered into several cell lines by addition of a PTD. Compared to a control peptide, the HKWPWW peptide inhibited HIV-1 replication as deduced from reduced titers of culture supernatants. As HKWPWW also binds to the TAR-RNA like the natural nucleocapsid PSI-RNA ligand, the effect on Tat-TAR inhibition will also be analyzed. Currently T-cell lines are established which stably express HKWPWW as well as a control peptide, which will be infected with HIV-1 to monitor the ability of HKWPWW to inhibit wild type HIV-1 replication. Conclusion: The selection of a peptide ligand for PSI-RNA able to inhibit HIV-1 replication proves the suitability of the phage display technology for the selection of peptides binding to RNA-structures. This enables the indentification of peptides serving as leads to interfere with additional targets in the HIV-1 replication cycle.
Simple Summary: Targeted therapies are of growing interest to physicians in cancer treatment. These drugs target specific genes and proteins involved in the growth and survival of cancer cells. Brain tumor therapy is complicated by the fact that not all drugs can penetrate the blood brain barrier and reach their target. We explored the non-invasive method, Magnetic Resonance Spectroscopy, for monitoring drug penetration and its effects in live animals bearing brain tumors. We were able to show the presence of the investigated drug in mouse brains and its on-target activity.
Abstract: Background: BAY1436032 is a fluorine-containing inhibitor of the R132X-mutant isocitrate dehydrogenase (mIDH1). It inhibits the mIDH1-mediated production of 2-hydroxyglutarate (2-HG) in glioma cells. We investigated brain penetration of BAY1436032 and its effects using 1H/19F-Magnetic Resonance Spectroscopy (MRS). Methods: 19F-Nuclear Magnetic Resonance (NMR) Spectroscopy was conducted on serum samples from patients treated with BAY1436032 (NCT02746081 trial) in order to analyze 19F spectroscopic signal patterns and concentration-time dynamics of protein-bound inhibitor to facilitate their identification in vivo MRS experiments. Hereafter, 30 mice were implanted with three glioma cell lines (LNT-229, LNT-229 IDH1-R132H, GL261). Mice bearing the IDH-mutated glioma cells received 5 days of treatment with BAY1436032 between baseline and follow-up 1H/19F-MRS scan. All other animals underwent a single scan after BAY1436032 administration. Mouse brains were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Results: Evaluation of 1H-MRS data showed a decrease in 2-HG/total creatinine (tCr) ratios from the baseline to post-treatment scans in the mIDH1 murine model. Whole brain concentration of BAY1436032, as determined by 19F-MRS, was similar to total brain tissue concentration determined by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS), with a signal loss due to protein binding. Intratumoral drug concentration, as determined by LC-MS/MS, was not statistically different in models with or without R132X-mutant IDH1 expression. Conclusions: Non-invasive monitoring of mIDH1 inhibition by BAY1436032 in mIDH1 gliomas is feasible.
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10
(2020)
The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.
Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via a RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and find that this FendrrBox is partially required for Fendrr function in vivo. We find that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs, associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in fibroblasts. We confirm the formation of RNA:dsDNA formation with target promoters. We find that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.
Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.