Thermodynamic parameters for the binding of RAL to LTR32.

<p>Titration of LTR32, at three different concentrations: 9 nM (black), 20 nM (red), and 30 nM (blue). Curve treatment provided a 1∶1 stoichiometry for the complex formation and an average Kd of ≈6 nM for the binding affinity. Samples were in phosphate buffer pH 6, I = 0.05, at 5°C, MgCl₂ 5 mM final concentration.</p

MD simulations of the RAL-LTR34 and RAL-LTR32 complex systems (PFV oligonucleotides), using GROMACS with the AMBER force field.

<p>(A) Time evolution of RMSD (root mean square deviation) values based on all the heavy atoms for the two LTR34 trajectories (black: LTR34-1 and blue: LTR34-2). RMSD calculations for a single trajectory were also performed using the sugar C4′ atoms (green: LTR34-1) and repeated for LTR34 devoid of 3′-AT (purple). (B) Time evolution of RMSD values of LTR32 for two trajectories (black: LTR32-1 and blue: LTR32-2). (C) RMSF (root mean square fluctuation) variations of sugar C4′ atoms for LTR34 and (D) RMSF variations of sugar C4′ atoms for LTR32.</p

UV-absorption analysis of oligonucleotides.

<p>Spectra of RAL 20 µM (black) together with 20 µM LTR32 (green), 20 µM LTR34 (blue), 20 µM LTR30 (red), in phosphate buffer pH 6, I = 0.05, and MgCl₂ 5 mM final concentration.</p

Calculated binding parameters for the complexes of RAL with LTR32 and LTR34.

<p>The free energy ΔG_MMPBSA from two trajectories for each system (LTR34-1, 2 and LTR32-1, 2) and averaged over 500 frames from each trajectory. Energies and standard deviations are given in kcal/mol. E_ele: Coulombic energy; E_vdw: van der Waals energy; E_MM: total molecular mechanics energy (E_ele+E_vdw); G_PB: polar solvation free energy based on Poisson-Boltzmann; G_SASA: Non-polar solvations free energy based on SASA; TΔS: the entropy contribution to the binding calculated by the QH; ΔG: the total free energy.</p

Circular dichroism analysis of oligonucleotides-drug complexes.

<p>Spectra of LTR34 (A) and LTR32 (B) at 10 µM (black) and difference spectra [LTR32/34 (10 µM)+RAL (10 µM, red; 20 µM, green; 40 µM, blue; 60 µM, orange; and 80 µM, purple)−LTR32/34 (10 µM)], in phosphate buffer pH 6, I = 0.05, and MgCl₂ 5 mM final concentration.</p

Effects of RAL on the distance and the fraying of unprocessed LTR ends.

<p>Time evolution of the distance (nm) between the mass of RAL (D) and that of the terminal bases (T and A) of LTR34-1 (black:D-T and red: D-A) and LTR34-2 (purple: D-T and blue: D-A). Interaction of RAL with the terminal bases decreases the distance between the drug and the bases and also reduces moderately the end fraying.</p

Snapshots from the two 100 ns trajectories of RAL in complex with unprocessed LTR (LTR34-1 and 2, top) and processed LTR (LTR32-1 and 2, bottom).

<p>RAL is colored in slime green and bases in sandy brown, except for atoms at interacting distances which are colored using the usual code (hydrogen in white; nitrogen in blue; and oxygen in red) except for carbons, while the Mg²⁺ ion is represented by magenta ball. Selected snapshots are 0 ns (the initial structure), 50 ns and 100 ns.</p

Quantitative analysis of RAL binding to oligonucleotides.

<p>Fluorescence anisotropy titration of the four oligonucleotides LTR32 (black), LTR34 (red), LTR32-I (blue) and LTR30 (green) at 20 nM by increasing concentrations of RAL (from 10⁻¹² M to 10⁻⁴ M). Kds obtained from titrations of LTR32 and LTR34 at 20 nM are indicated near the corresponding curves.</p