Collauto A , von Bülow S , Gophane DB , Saha S , Stelzl LS , Hummer G , Sigurdsson ST , Prisner TF .

CRC 902 Molecular Principles of RNA-based Regulation, RGP0026/2017 Human Frontier Science Program, Max Planck Society, 141062051 Icelandic Research Fund, Biological Magnetic Resonance Center Frankfurt (BMRZ)

	Figure 1. A) Structure of the EImUm spin label. B) Sequence of the 20‐mer duplex RNA I; the spin‐labeled nucleotides are displayed in red. C) Model of the duplex RNA I containing the two EImUm labels; the vertical line connects the nitroxide groups of the two spin labels.
	Figure 2. Q‐band 4‐pulse PELDOR background‐subtracted data normalized by the modulation depth (A) and the corresponding distance probability distributions obtained by model‐free analysis (B) for the duplex RNA I in a buffered solution (10 mM phosphate pH 7.0, 100 mM NaCl, 0.1 mM EDTA; black traces), in Xenopus lævis oocytes (red traces), in cytoplasmic extract (blue traces), and in a 200 mg mL−1 lysozyme solution (green traces). Multiple traces, where present, show results obtained from different samples. The original traces are reported in the Supporting Information (Figure S4) together with a validation of the distance probability distributions (Figure S5), confirming the statistical relevance of the reduction of the inter‐spin distance upon in‐cell internalization.
	Figure 3. RNA conformational dynamics in crowded LYZ solution as revealed by atomistic MD simulations with fully flexible RNA and proteins in explicit solvent. A) Simulation snapshot showing the RNA A‐helix (orange) in crowded LYZ solution (blue shades) at 200 mg mL−1 concentration. Na+ and Cl− ions are shown as pink and green spheres. Water is indicated by the transparent surface. B) Zoom‐in on the MD simulation snapshot showing basic residues interacting with the phosphate backbone of the RNA. Arginine labels are colored to distinguish the proteins they belong to. C) Compaction of the RNA. Reference idealized A‐helix structure (gray) and mean structures from simulations of the RNA in dilute (orange; absence of LYZ) and dense solution (green; 200 mg mL−1 LYZ) are compared for bases separated by about one helical turn. D) Average base‐base distances in the MD simulations of the dsRNA in dilute solution. E) Difference in the average base‐base distances in presence and absence of LYZ.

Azarkh, Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in-cell spin-label EPR. 2011, Pubmed, Xenbase

Azarkh, Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in-cell spin-label EPR. 2011, Pubmed , Xenbase
Azarkh, Gd(III)-Gd(III) Relaxation-Induced Dipolar Modulation Enhancement for In-Cell Electron Paramagnetic Resonance Distance Determination. 2019, Pubmed , Xenbase
Banáš, Performance of Molecular Mechanics Force Fields for RNA Simulations: Stability of UUCG and GNRA Hairpins. 2010, Pubmed
Bao, Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy. 2017, Pubmed , Xenbase
Barthelmes, Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy. 2015, Pubmed
Bass, Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs. 1994, Pubmed , Xenbase
Bleicken, gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility. 2019, Pubmed
Braun, Isoindoline-Based Nitroxides as Bioresistant Spin Labels for Protein Labeling through Cysteines and Alkyne-Bearing Noncanonical Amino Acids. 2020, Pubmed
Chiang, The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. 2005, Pubmed
Dalaloyan, Tracking Conformational Changes in Calmodulin in vitro, in Cell Extract, and in Cells by Electron Paramagnetic Resonance Distance Measurements. 2019, Pubmed
Dhimitruka, Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging. 2007, Pubmed
Dzatko, Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells. 2018, Pubmed
Eckmann, Xlrbpa, a double-stranded RNA-binding protein associated with ribosomes and heterogeneous nuclear RNPs. 1997, Pubmed , Xenbase
Erlenbach, Flexibilities of isoindoline-derived spin labels for nucleic acids by orientation selective PELDOR. 2016, Pubmed
Fessl, Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET. 2012, Pubmed
Fleck, SLIM: A Short-Linked, Highly Redox-Stable Trityl Label for High-Sensitivity In-Cell EPR Distance Measurements. 2020, Pubmed
Gao, RNA Hairpin Folding in the Crowded Cell. 2016, Pubmed
Giassa, Advances in the cellular structural biology of nucleic acids. 2018, Pubmed
Gophane, Hydrogen-bonding controlled rigidity of an isoindoline-derived nitroxide spin label for nucleic acids. 2013, Pubmed
Gophane, A semi-rigid isoindoline-derived nitroxide spin label for RNA. 2018, Pubmed
Gophane, Conformationally restricted isoindoline-derived spin labels in duplex DNA: distances and rotational flexibility by pulsed electron-electron double resonance spectroscopy. 2014, Pubmed
Grotz, Dispersion Correction Alleviates Dye Stacking of Single-Stranded DNA and RNA in Simulations of Single-Molecule Fluorescence Experiments. 2018, Pubmed
Hänsel, Evaluation of parameters critical for observing nucleic acids inside living Xenopus laevis oocytes by in-cell NMR spectroscopy. 2009, Pubmed , Xenbase
Igarashi, Distance determination in proteins inside Xenopus laevis oocytes by double electron-electron resonance experiments. 2010, Pubmed , Xenbase
Jagtap, Sterically shielded spin labels for in-cell EPR spectroscopy: analysis of stability in reducing environment. 2015, Pubmed
Jassoy, Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells. 2017, Pubmed , Xenbase
Jeschke, Direct conversion of EPR dipolar time evolution data to distance distributions. 2002, Pubmed
Karthikeyan, A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies. 2018, Pubmed , Xenbase
Kinoshita, In vivo evaluation of novel nitroxyl radicals with reduction stability. 2010, Pubmed
Kirilyuk, Synthesis of the tetraethyl substituted pH-sensitive nitroxides of imidazole series with enhanced stability towards reduction. 2004, Pubmed
Krafcikova, Monitoring DNA-Ligand Interactions in Living Human Cells Using NMR Spectroscopy. 2019, Pubmed
Krovat, Comparative mutational analysis of the double-stranded RNA binding domains of Xenopus laevis RNA-binding protein A. 1996, Pubmed , Xenbase
Krstić, Long-range distance measurements on nucleic acids in cells by pulsed EPR spectroscopy. 2011, Pubmed , Xenbase
Mahawaththa, Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments. 2018, Pubmed
Marko, Conformational flexibility of DNA. 2011, Pubmed
Martin, Determination of End-to-End Distances in a Series of TEMPO Diradicals of up to 2.8 nm Length with a New Four-Pulse Double Electron Electron Resonance Experiment. 1998, Pubmed
Martorana, Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling. 2014, Pubmed
Mascali, Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements. 2016, Pubmed
Matsumoto, CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes. 2000, Pubmed , Xenbase
Mustoe, Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing. 2018, Pubmed
Paletta, Synthesis and reduction kinetics of sterically shielded pyrrolidine nitroxides. 2012, Pubmed
Pannier, Dead-time free measurement of dipole-dipole interactions between electron spins. 2000, Pubmed
Piana, Water dispersion interactions strongly influence simulated structural properties of disordered protein states. 2015, Pubmed
Prisner, Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels. 2015, Pubmed
Pérez, Refinement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers. 2007, Pubmed
Qi, Gd(III)-PyMTA label is suitable for in-cell EPR. 2014, Pubmed , Xenbase
Rogozhnikova, Generation of Trityl Radicals by Nucleophilic Quenching of Tris(2,3,5,6-tetrathiaaryl)methyl Cations and Practical and Convenient Large-Scale Synthesis of Persistent Tris(4-carboxy-2,3,5,6-tetrathiaaryl)methyl Radical. 2013, Pubmed
Rouskin, Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. 2014, Pubmed
Ryter, Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. 1998, Pubmed , Xenbase
Saha, Site-directed spin labeling of 2'-amino groups in RNA with isoindoline nitroxides that are resistant to reduction. 2015, Pubmed
Schlagnitweit, Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy. 2019, Pubmed
Spitale, RNA SHAPE analysis in living cells. 2013, Pubmed
Stelzl, Resolving the Conformational Dynamics of DNA with Ångstrom Resolution by Pulsed Electron-Electron Double Resonance and Molecular Dynamics. 2017, Pubmed
Su, Site-Specific Tagging of Proteins with Paramagnetic Ions for Determination of Protein Structures in Solution and in Cells. 2019, Pubmed , Xenbase
Sun, RNA structure maps across mammalian cellular compartments. 2019, Pubmed
Theillet, Structural disorder of monomeric α-synuclein persists in mammalian cells. 2016, Pubmed
Tormyshev, Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins. 2020, Pubmed
Tyrrell, The cellular environment stabilizes adenine riboswitch RNA structure. 2013, Pubmed
Wojciechowski, Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd(3+)-DOTA. 2015, Pubmed , Xenbase
Wuebben, Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability. 2019, Pubmed
Yamaoki, The first successful observation of in-cell NMR signals of DNA and RNA in living human cells. 2018, Pubmed , Xenbase
Yang, In-Cell EPR Distance Measurements on Ubiquitin Labeled with a Rigid PyMTA-Gd(III) Tag. 2019, Pubmed
Yang, A Reactive, Rigid GdIII Labeling Tag for In-Cell EPR Distance Measurements in Proteins. 2017, Pubmed
Yang, In-Cell Trityl-Trityl Distance Measurements on Proteins. 2020, Pubmed
Yang, High Sensitivity In-Cell EPR Distance Measurements on Proteins using an Optimized Gd(III) Spin Label. 2018, Pubmed
Yesselman, Sequence-dependent RNA helix conformational preferences predictably impact tertiary structure formation. 2019, Pubmed
Zgarbová, Refinement of the Cornell et al. Nucleic Acids Force Field Based on Reference Quantum Chemical Calculations of Glycosidic Torsion Profiles. 2011, Pubmed
von Bülow, Dynamic cluster formation determines viscosity and diffusion in dense protein solutions. 2019, Pubmed