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Nucleic Acids Res
2025 Sep 05;5317:. doi: 10.1093/nar/gkaf894.
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Structural basis of bis-quinolinium ligands binding to quadruplex-duplex hybrids from PIM1 oncogene.
Ghosh A
,
Harnos J
,
Stadlbauer P
,
Sponer J
,
Lenarcic Zivkovic M
,
Trantirek L
.
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Our study investigates the interaction of two bis-quinolinium ligands, Phen-DC3 and 360A, with the quadruplex-duplex hybrid (QDH) derived from the promoter region of the PIM1 oncogene. While the QDH is polymorphic in vitro, with a hybrid and antiparallel conformation, we demonstrate that it predominantly adopts the antiparallel conformation within the intracellular environment of Xenopus laevis oocytes (eukaryotic model system). Notably, both ligands selectively bind to the hybrid QDH conformation in vitro and in a cellular context. High-resolution nuclear magnetic resonance (NMR) structures of the complexes between the hybrid QDH and the ligands reveal distinct binding modes at the quadruplex-duplex (Q-D) junction. Specifically, Phen-DC3 binds rigidly, while 360A dynamically reorients between two positions. Our findings provide a crucial paradigm highlighting the differences in structural equilibria involving QDH in vitro compared to its behavior in the intracellular space. They also underscore the potential to modulate these equilibria under native-like conditions through ligand interactions. The observed differences in the binding of Phen-DC3 and 360A lay the groundwork for designing next-generation bis-quinolinium compounds with enhanced selectivity for the Q-D junction. Methodologically, our study illustrates the potential of 19F-detected in-cell NMR methodology for screening interactions between DNA targets and drug-like molecules under physiological conditions.
EU Horizon 2020, 101068280 Marie Skłodowska-Curie, MUNI/JS/1849/2024 Masaryk University, MUNI/J/0004/2021) Masaryk University, P1-0242 Slovenian Research and Innovation Agency, 101094131 European Union's Horizon 2020, GF24-10605K Czech Science Foundation
Figure 1.(A) Chemical structures of bis-quinolinium-based G4 ligands, Phen-DC3 and 360A, with the atom numbering on the left and with highlighted atoms used for the definition of collective coordinate (i.e. angular phase) in the well-tempered metadynamics simulation on the right; two points, marked by the red edge, used for the definition of the angular phase are related to the ligand atoms as follows: the blue circle represents the geometric center of all the highlighted atoms, while the yellow circle is the geometric center of three nearby atoms of the six-membered ring. (B) Hybrid-1 and antiparallel QDH topologies formed by sequences SO7 and SO2, derived from the PIM1 gene promoter, respectively. Oligonucleotide SO8 is polymorphic and simultaneously forms hybrid (SO7-like) and antiparallel (SO2-like) QDHs in a ∼75%:20% ratio [36]. Sequences of SO7, SO2, and SO8 are shown in Table 1. G4 and duplex stem-loop units are colored gray and magenta, respectively. Guanines in syn glycosidic conformation are marked in bold. (C) Imino regions of the 1H NMR spectra of free SO7, SO2, and SO8 (left), and in the presence of 1:1 molar equivalents of Phen-DC3 (middle) and 360A (right), respectively. Signals belonging to free hybrid and antiparallel QDHs are marked with red asterisks and gray rectangles, while DNA–Phen-DC3 and DNA–360A complexes are denoted by green and blue dots, respectively. Signals belonging to ligand atoms are marked with L.
Figure 2.The imino regions of 1D 1H NMR spectra of (A) SO7–Phen-DC3 and (B) SO7–360A 1:1 complexes with corresponding imino-aromatic and imino-imino regions of 2D NOESY spectra (τm = 300 ms). Cross peaks labeled with red, orange, and dark blue rectangles correspond to G3•G25•G22•G7, G4•G8•G21•G26, and G5•G9•G20•G27 quartets, respectively. Cross-peaks labeled with magenta correspond to NOE connections between G-C base pair-forming residues. Ligand imino signals are marked according to the labels in Fig. 1A.
Figure 3.Intermolecular NOE contacts positioning Phen-DC3 between G5•G9•G20•G27 quartet, and G19–C10 base pair at the Q-D junction. (A) The region of the NOESY spectrum (τm= 300 ms) shows intermolecular NOE cross-peaks between the imino protons of SO7 and Phen-DC3. NOESY spectral region showing intermolecular NOE interactions between other protons of SO7 and Phen-DC3 is presented in Supplementary Fig. S19A. (B) Schematic depiction of key intermolecular interactions within the SO7–Phen-DC3 1:1 complex. NOESY spectrum was obtained in 20 mM potassium phosphate buffer, pH 7.1, 20 mM KCl at 298.2 K with 0.5 mM SO7 and Phen-DC3 concentrations.
Figure 4.Intermolecular NOE contacts positioning 360A between G5•G9•G20•G27 quartet and G19–C10 base pair at the Q-D junction. (A) The region of the NOESY spectrum (τm= 300 ms) shows intermolecular NOE cross-peaks between the imino protons of SO7 and 360A. NOESY spectral region showing intermolecular NOE interactions between other protons of SO7 and 360A is presented in Supplementary Fig. S19B. (B) Schematic presentation of key intermolecular interactions within the SO7–360A 1:1 complex. NOESY spectrum was obtained in 20 mM potassium phosphate buffer, pH 7.1, 20 mM KCl at 298.2 K with 0.5 mM SO7 and 360A concentrations.
Figure 5.Lowest-energy NMR solution structures of (A) SO7–Phen-DC3 (PDB: 9GVI) and (B) SO7–360A (PDB: 9GVV) 1:1 complexes. Bird’s- and side-view into the binding pocket of (C) SO7–Phen-DC3 and (D) SO7–360A complexes at the Q-D junctions. G-quartet-forming guanines and residues forming G–C base pairs in the stem-loop are marked gray and magenta, respectively. Phen-DC3 is colored green, while 360A is blue. All the other residues are depicted in light brown.
Figure 6.(A) Comparison of in vitro (gray) and in-cell (black) 19F NMR spectra of SO8-F and 1:1 SO8-F-Phen-DC3 and SO8-F-360A complexes, respectively. (B) Images of the X. laevis oocytes before and after progesterone treatment. Progesterone was dissolved in DMSO and added to the samples at a concentration of 1 μM. Negative control represents oocytes without DNA/DNA–ligand microinjection but with the addition of pure DMSO. The buffer sample contains Ori-Ca2+ buffer with the addition of DNA dissolving buffer. In the case of other samples, prefolded DNA and 1:1 DNA–ligand constructs were microinjected into ∼300 X. laevis oocytes. The evidence of maturation (white spot) is marked with red rectangles.
