Conformation and dynamics of nucleotides in bulges and symmetric internal loops in duplex DNA studied by EPR and fluorescence spectroscopies

► Bulges and loops were studied by both EPR and fluorescence spectroscopies using the probe Ç/Çf. ► One-base bulge was in a temperature-dependent equilibrium between looped-out and stacked states. ► Bases in two- and three-base bulges were stacked at all temperatures, resulting in DNA bending. ► Bases were stacked in symmetrical two- to five-base internal loops, according to EPR data. ► Unexpectedly high fluorescence for the smaller loops indicated local structural perturbations. The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe Ç, which becomes fluorescent (Çf) upon reduction of the nitroxide functional group, along with EPR and fluorescence spectroscopies. A one-base bulge was in a conformational equilibrium between looped-out and stacked states, the former favored at higher temperature and the latter at lower temperature. Stacking of bulge bases was favored in two- and three-base bulges, independent of temperature, resulting in DNA bending as evidenced by increased fluorescence of Çf. EPR spectra of Ç-labeled three-, four- and five-base symmetrical interior DNA bulges at 20°C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37°C increased as the loops became larger. A considerable variation in fluorescence between different loops was observed, as well as a temperature-dependence within constructs. Fluorescence unexpectedly increased as the size of the loop decreased at 2°C. Fluorescence of the smallest loops, where a single T·T mismatch was located between the stem region and the probe, was even larger than for the single strand, indicating a considerable local structural deformation of these loops from regular B-DNA. These results show the value of combining EPR and fluorescence spectroscopy to study non-helical regions of nucleic acids.