PELDOR (pulsed electron–electron double resonance) is an established method to study intramolecular distances and can give evidence for conformational changes and flexibilities. However, it can also be used to study intermolecular interactions as for example oligerimization. Here, we used PELDOR to study the “end-to-end” stacking of small double-stranded (ds) RNAs. For this study, the dsRNA molecules were only singly labeled with the spin label TPA to avoid multispin effects and to measure only the intermolecular stacking interactions. It can be shown that small dsRNAs tend to assemble to rod-like structures due to π–π interactions between the base pairs at the end of the strands. On the one hand, these interactions can influence or complicate measurements aimed at the determining of the structure and dynamics of the dsRNA molecule itself. On the other hand, it can be interesting to study such intermolecular stacking interactions in more detail, as for example their dependence on ion concentration. We quantitatively determined the stacking probability as a function of the monovalent NaCl salt and the dsRNA concentration. From these data, the dissociation constant was deduced and found to depend on the ratio between the NaCl salt and dsRNA concentrations. Additionally, the distances and distance distributions obtained predict a model for the stacking geometry of dsRNAs. Introducing a nucleotide overhangs at one end of the dsRNA molecule restricts the stacking to the other end, leading only to dimer formations. Introducing such an overhang at both ends of the dsRNA molecule fully suppresses stacking, as we demonstrate by PELDOR experiments quantitatively.
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