Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy. 
Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology.  The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached.  We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR.  In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created.  In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously.  This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction.  We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center.  The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif.  The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.