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

Electron Paramagnetic Resonance (EPR) spectroscopy and fluorescence spectroscopy are complementary biophysical techniques used to examine the structure and dynamics of macromolecules. We have previously described the bi-functional spectroscopic probe Ç for the study of nucleic acid structure and dynamics using EPR and fluorescence spectroscopy. As with any newly designed spectroscopic probe, the utility, functionality, and the structural effects of the probe on the nucleic acid must be examined in detail. Initial EPR, fluorescence, and thermal denaturation studies indicated that the phenoxazine-derived spin-labeled deoxycytosine analog Ç forms a structurally non-perturbing base-pair with deoxyguanosine in DNA. Here we extend the analysis of the spectroscopic probe by presenting a detailed crystallographic study of this label based on small molecule crystal structures of the nucleoside base ç and its phenoxazine analog as well as a 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA. The DNA crystal structure confirms that the spin-labeled deoxycytosine analog forms a non-perturbing base-pair with deoxyguanosine. Interestingly, this structure and also the one of the phenoxazine base show the label in a planar conformation, whereas the structure of the free spin label base ç has a bend at the oxazine linkage. Density function theory (DFT) calculations reveal that both conformations are very close in energy and possess both the same frequency for bending at the oxazine linkage. These results are interpreted as a small degree of bending flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity in this 16-pi electron ring system. Within DNA, the amplitude of the bending motion is likely to be restricted due to steric hindrance. This detailed structural analysis shows that the spin label base ç can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies of oligonucleotides.