Assessing Roles of Cations in G‑Quadruplex-Based Nanowires by NMR

G-quadruplexes are uncanonical DNA structures exhibiting promising characteristics for designing DNA-based nanomaterial and nanodevices. The present NMR study addresses the role of cations, the requiring factor for G-quadruplex formation, in long G-quadruplexes. We investigated d­(TG₈T)₄ and its interactions with ¹⁵NH₄⁺ ions, which provided insights into cation localization within a model of G-quadruplex-based nanowire exhibiting eight stacked G-quartets. All interquartet cavities along the central channel were completely occupied by cations. Determined cation exchange rates demonstrated that ¹⁵NH₄⁺ ions move faster from the binding site between two G-quartets, both composed of all guanine residues in an anti-glycosidic conformation with respect to the binding site between all-syn/all-anti G-quartets. These results not only extend previous findings from calculated models of long G-quadruplexes and experimental studies on shorter G-quadruplexes but also establish correlation of kinetics of cation movement with the mode of G-quartet stacking, a potentially valuable relation for understanding G-quadruplex-type specific electronic properties

High-Resolution Structure of RNA G‑Quadruplex Containing Unique Structural Motifs Originating from the 5′-UTR of Human Tyrosine Kinase 2 (TYK2)

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor-associated tyrosine kinases together with highly homologous JAK1, JAK2, and JAK3 paralogues. Overexpression of TYK2 is associated with several inflammatory diseases, including severe complications during the COVID-19 infection. Since the downregulation of JAK paralogues could lead to serious health consequences or even death, it is critical to avoid it when designing drugs to suppress TYK2. To achieve the required specificity only for TYK2, researchers have recently selectively targeted TYK2 mRNA by developing antisense oligonucleotides. In this work, we expand the target space of TYK2 mRNA by showing that the mRNA adopts tetra-helical noncanonical structures called G-quadruplexes. We identified a TYKwt RNA oligonucleotide from the 5′-UTR of TYK2 mRNA, which adopts multiple different parallel G-quadruplexes that exist at equilibrium. Using NMR spectroscopy, we showed that some of the G-quadruplexes adopt unique structural motifs, mainly due to the formation of a stable GA bulge. Using guanine to uridine substitutions, we prepared the oligonucleotide TYK3_U6, which serves as an excellent model for the bulged G-quadruplexes formed by the TYKwt oligonucleotide. NMR structural analysis, including data on the residual coupling constants (RDC) of the loop regions, unveiled that the studied three-quartet parallel G-quadruplex contains many unusual structural features such as a G(U)A bulge, a guanine residue in the syn conformation, A and U residues stacked on the top G-quartet, and a well-defined adenine from a three-residue long propeller loop oriented in the groove, all of which could be valuable targets for future drug design

H NMR signal intensity ln(′) as a function of gradient field strength (g) at 25°C

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The filled squares represent A6 H2 signal intensity of 3′-MOE-2′,5′-RNA hairpin, while the open triangles represent A6 H2 of the duplex form. The open circles represent the average signal intensity of aromatic resonances of 2′-MOE-3′,5′ RNA duplex. The sample conditions were 3.0 mM strand concentration, 350 mM NaCl and pH* 7.4 in 100% HO

UV melting profiles of 3′-MOE-2′,5′-RNA at various oligomer and salt concentrations and pH

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The calculated fits of the first derivatives of a melting curves are presented as a function of temperature at the following solution conditions: () 1 M NaCl and pH of 6.0 over a concentration range of 8 μM (magenta), 17 μM (cyan) and 123 μM (green). The of high temperature transition is 48°C and low temperature transition is melted in the range of 15–23°C. () The 1 M NaCl and pH 7.5 over a concentration range of 9 μM (magenta), 39 μM (blue), 101 μM (green) and 266 μM (orange). The of high temperature transition is 45°C, whereas the of low temperature transition is in the range of 15–26°C. () The 50 mM NaCl and pH of 6.0. Concentrations are 9 μM (magenta), 23 μM (cyan), 39 μM (blue) and 169 μM (green). The of high temperature transition form is 45°C whereas the of low temperature transition appears under the broad transition profile of high temperature form. () The 50 mM NaCl and pH of 7.4. Concentrations are 17 μM (cyan) and 270 μM (orange). The of high temperature transition is near 42°C and low temperature transition melts in 16–20°C range

H NMR spectra of () imino and aromatic proton region and () methyl proton region of 3′-MOE-2′,5′-RNA as a function of pH and temperature

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The vertical scale of imino and aromatic region is doubled. The sample was 0.9 mM strand concentration, 50 mM NaCl in 90% HO/10% HO. Stars indicate C7(H7) resonances of duplex form whose intensities increase upon lowering pH and temperature

() Methyl–methyl and () aromatic–aromatic region of ROESY spectrum (100 ms mixing time) recorded at 25°C in 100% HO

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The assignments of the resolved exchange signals for hairpin (H) and duplex (D) forms are indicated. Sample conditions were 3.0 mM strand concentration, 350 mM NaCl and pH* 7.4

() Imino proton spectra of 3′-MOE-2′,5-RNA at 5°C as a function of pH

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The sample was 9 mM strand concentration, 50 mM NaCl in 90% HO/10% HO. Imino protons involved in Watson–Crick base pairs of duplex (D) are shown. () Schematic representation of the duplex–hairpin equilibrium of 3′-MOE-2′,5-RNA. Closed lines represent Watson–Crick base pairs

Non-terminal building blocks of 2′,5′- and 3′,5′-linked RNAs and imino proton spectra of 3′-MOE-2′,5′ RNA

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Shematic representation of 3′-MOE-2′,5′-RNA monomer (left) and 2′-MOE-3′,5′-RNA monomer (right). () Imino proton spectrum recorded in 90% HO/10% HO at 9.0 mM strand concentration, 50 mM NaCl, pH 6.9 and 20°C. Duplex (D) and hairpin (H) resonances are denoted

Comparison of () aromatic and () methyl region of H NMR spectra of 3′-MOE-2′,5′-RNA at various strand concentrations (0

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>2, 0.4, 2.6 and 3.0 mM from bottom to top). Arrows indicate signals of the duplex that are separated from signals of the dominant hairpin form. Spectra were recorded in 100% HO at 30°C, 50 mM NaCl and pH* 7.4

Aromatic () and imino () regions of H NMR spectrum of form of dG(TG) G-quadruplex at 298 K

<p><b>Copyright information:</b></p><p>Taken from "NMR evaluation of ammonium ion movement within a unimolecular G-quadruplex in solution"</p><p>Nucleic Acids Research 2007;35(8):2554-2563.</p><p>Published online 4 Apr 2007</p><p>PMCID:PMC1895886.</p><p>© 2007 The Author(s)</p> Plot of 2D N–H HSQC spectrum (). The cross-peak corresponding to ions in bulk is labeled as B, while those residing at the binding sites within the G-quadruplex are labeled as O, O and I