Table 1.
Characterization of selected G-rich sequences from the IAV genome.
Fig 1.
Scheme of the experimental design and workflow of the study.
Fig 2.
The results of the inhibition of cDNA synthesis catalyzed by the reverse transcriptase in the presence of TMPyP4; the resultant gels (upper) and the plots of band intensity vs. ligand concentration (bottom) for 1q/1qm, 7q/7qm, and 11q/11qm variants (panel A, panel B, and panel C, respectively).
The most representative gel results for each sequence are presented here. The IC50 values were obtained as the average of three replicates.
Fig 3.
The results of the inhibition of cDNA synthesis catalyzed by the reverse transcriptase in the presence of BRACO-19; the resultant gels (upper) and the plots of band intensity vs. ligand concentration (bottom) for 1q/1qm, 7q/7qm, and 11q/11qm variants (panel A, panel B, and panel C, respectively).
The most representative gel results for each sequence are presented here. The IC50 values were obtained as the average of three replicates.
Table 2.
The binding parameters from the calorimetric titration of the wild-type and mutated variants of 1Q, 7Q, and 11Q with TMPyP4 and BRACO-19 ligands.
Fig 4.
Representative raw data (upper panels) and integration of the peaks (bottom panels) with the best fit of the ‘One Set of Sites’ or ‘Two Sets of Sites’ model (only in the case of wild-type sequences and TMPyP4 ligand).
The data were obtained after titrations of 1Q, 7Q, and 11Q G-quadruplexes and 1Qm, 7Qm, and 11Qm mutants with TMPyP4 (A) or BRACO-19 (B) ligands.
Fig 5.
Representative UV melting curves of RNA oligomers in the presence or absence of ligands with Tm values obtained by monitoring the melting profile at 295 nm.
The Tm values were obtained as the average of three replicates.
Fig 6.
Circular dichroism spectra for pre-folded RNA G4s oligomers: 1Q (A), 7Q (B), and 11Q (C) with TMPyP4 increasing concentrations.
Fig 7.
Circular dichroism spectra for pre-folded RNA G4s oligomers: 1Q (A), 7Q (B), and 11Q (C) with BRACO-19 increasing concentrations.
Fig 8.
The differences in nucleotide composition of G-rich sequences from the Cal2009 and PR8 strains used in our study.
Red color indicates the point mutations; the G-tracts are underlined.
Fig 9.
Circular dichroism spectra of 1qPR8, 7qPR8, and 11qPR8 oligomers.
Fig 10.
The fluorescence titration spectra of the TMPyP4 ligand in the absence and presence of pre-folded RNA G4s oligomers: 1Q (A), 7Q (B), and 11Q (C). The spectrum of TMPyP4 alone in the buffer is indicated as a light green dashed line.
Fig 11.
The fluorescence titration spectra of the BRACO-19 ligand in the absence and presence of pre-folded RNA G4s oligomers: 1Q (A), 7Q (B), and 11Q (C). The spectrum of BRACO-19 alone in the buffer is indicated as a purple dashed line.
Fig 12.
Three main stages of building the 1Q model. A) Generating RNA strands; B) Intramolecular folding; C) Intermolecular folding. The RNA molecules are shown using the new ribbon representation and highlighted with a white, semi-transparent surface. The two strands forming the G-quadruplex are coloured blue and red. The four layers of tetrads are shown in liquorice representation and coloured red, yellow, green, and blue.
Fig 13.
Simulation box with 1Q bimolecular G-quadruplex in the new ribbon representation, explicit water solution with ions (represented as transparent bubbles) during molecular dynamics simulation.
The pink spheres are potassium ions in the channel of the core between the stacked G-tetrads (left panel). Representative structures of the 1Q G-quadruplex obtained during 300 ns of molecular dynamics simulations, superimposed using the phosphate groups of the core (consisting of 16 guanines) as the fitting centres. The two strands forming the G-quadruplex are shown in a tube representation of the RNA backbone and are colored blue and red. The core of the molecule remains stable, while the loops do not reach equilibrium during the entire simulation (right panel).
Fig 14.
Scheme of the IAV minireplicon system used in this study.
Fig 15.
The inhibition of Cal2009 and PR8 minireplicon activity in HEK 293T cells after TMPyP4 or TMPyP2 treatment.
All data are presented as the means from two biological replicates, each subjected to three technical replicates. Error bars reflect the SD.