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The ‘hidden side’ of spin labeled oligonucleotides: Molecular Dynamics study focusing on the EPR-silent components of base pairing

Nitroxide labels are combined with nucleic acid structures and studied using electron paramagnetic resonance experiments (EPR). Detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (Ç) labels (in both 2’-O methylated and not-methylated forms) on RNA duplexes. Further, we also investigate sequence-dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. Further, to understand the effect of labeled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabeled structures. Based on the structural analysis, we can conclude that TEMPO-based and Ç labels do not significantly perturb the base pair arrangements of the native oligonucleotide. TOAC Figure Molecular Dynamics (MD) analysis can complement and validate distance measurements determined by pulsed EPR spectroscopy and can provide information beyond the inter-spin distances. The diamagnetic components of DNA or RNA sequences (the ‘hidden side’ for DEER/PELDOR experiments, i.e. those components of base pairs not measurable by EPR experiments) may generate structural perturbations upon the insertion of nitroxide spin-probes. Those perturbations may drive the choice of the spin label suitable for the macromolecular structure under investigation. The most suitable spin probe should not perturb the native DNA/RNA structure generating any local structural changes effecting the entire structure. The abstract figure points out the ten regions that can be examined by MD analysis, while only four regions of the oligonucleotide are accessible in EPR analysis. Considering the schematic view of the duplexes and corresponding techniques suitable for detection of structural modification, in the labeled molecules, only 40% of the system under investigation is visible in EPR data, while 100% of the system can be studied by MD analysis.