Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe

Previously, we developed the deoxycytosine analog (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 resolution crystal structure of within a decamer duplex A-form DNA confirmed that forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base displayed a ∼20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR-or fluorescence-based structural and dynamics studies.