The architecture of the 10-23 DNAzyme and its implications for DNA-mediated catalysis

Funding Information: The authors acknowledge access to the Jülich‐Düsseldorf Biomolecular NMR Center. HG is grateful for computational support and infrastructure provided by the ‘Zentrum für Informations‐ und Medientechnologie’ (ZIM) at the Heinrich Heine University Düsseldorf and the John von Neumann Institute for Computing (NIC) (user ID: HKF7, VSK33). We thank Hannah Rosenbach for providing activity data. This work was supported by the German Research Foundation (DFG) (103/4‐1, ET 103/4‐3, and the Heisenberg grant ET 103/5‐1) to ME, the Volkswagen Foundation to ME and HG (project no. 9B798) and the European Union‘s Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie grant agreement no. 660258 to AV. Open Access funding enabled and organized by Projekt DEAL. Publisher Copyright: © 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.Understanding the molecular features of catalytically active DNA sequences, so-called DNAzymes, is essential not only for our understanding of the fundamental properties of catalytic nucleic acids in general, but may well be the key to unravelling their full potential via tailored modifications. Our recent findings contributed to the endeavour to assemble a mechanistic picture of DNA-mediated catalysis by providing high-resolution structural insights into the 10-23 DNAzyme (Dz) and exposing a complex interplay between the Dz's unique molecular architecture, conformational plasticity, and dynamic modulation by metal ions as central elements of the DNA catalyst. Here, we discuss key features of our findings and compare them to other studies on similar systems.publishersversionpublishe

Discovery of inhibitory fragments that selectively target Spire2−FMN2 interaction

Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2–FMN2 but not the Spire1–FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1H–15N HSQC nuclear magnetic resonance. Extensive structure–activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2–FMN2 interaction

Expanding crystallization tools for nucleic acid complexes using U1A protein variants

The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes

Molecular Architecture of a Network of Potential Intracellular EGFR Modulators Involving the Juxtamembrane Segment, ARNO, Phospholipids and CaM

Signaling of the Epidermal growth factor receptors (EGFRs) is a central cellular element and its dysregulation is related to a number of severe diseases. While ligand binding to the extracellular domain is the receptor’s most obvious regulatory element, also intracellular factors can act as modulators of EGFR activity. The juxtamembrane (JM) segment of the EGFR seems to be a key interaction interface of these cytoplasmic factors. However, very few JM-interacting molecules have been identified so far and even fewer is known about the molecular mechanism underlying JM-targeted receptor modulation. Here we report ARNO as a new EGFR-JM binding protein and provide high-resolution insights into its mode of interaction. We obtain comparable insights also for the known interaction partners Calmodulin and phospholipid bilayers containing different lipid compositions. Our results show clear similarities and distinct differences in each binding mode. Furthermore, we show that each interaction can be modulated by a set of additional orthogonal factors generating a distinctly regulated competitive network of possible EGFR modulators acting on the intracellular domain of the receptor. This newly identified interaction network and the obtained insights into the underlaying molecular mechanism may foster future EGFR-targeted therapeutic strategies

Influence of monovalent metal ions on metal binding and catalytic activity of the 10–23 DNAzyme

Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10–23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10–23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10–23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell‐Free Expression Systems

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell‐Free Expression Systems

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39