The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe C, which becomes fluorescent (C-f) 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 C-f. EPR spectra of C-labeled three-, four- and five-base symmetrical interior DNA bulges at 20 degrees C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37 degrees 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 degrees C. Fluorescence of the smallest loops, where a single TT 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. (C) 2012 Elsevier Inc. All rights reserved.