Structural studies by high-field NMR spectroscopy of a binary-addressed complementary oligonucleotide system juxtaposing pyrene and perfluoro-azide units

Recently, a new approach has been proposed to improve the site- specificity and efficiency of the modification of nucleic acid target sequences, the binary system of complementary-addressing nucleic acid sequences. The binary system comprises two oligonucleotides, one modified with a photosensitizing group and the other with a photoreactive group. The sites of chemical modification are arranged to bring the two chemical functions close enough together in space to allow efficient energy transfer from the photo-excited photosensitizer to an arylazide moiety which expels N2 to form a nitrene which subsequently covalently labels the target nucleic acid. Structural analysis performed by high-resolution 2D NMR spectroscopy (400 MHz and 600 MHz) are reported for the model binary system 1:2:3, where 1 is the target 12-mer pdGTATCAGTTTCT, 2 is a photoactivatable fluoroazide derivative dAGAAACp-L-Az and 3 is the photosensitizer derivative Pyr-pdTGATAC (here: Az is the p-azidotetrafluorobenzyl group, Pyr the pyrenyl-1-methylamino group, L a linker group). The assignment of oligonucleotide and modifying group protons was performed using 1H COSY, TOCSY and NOESY experiments. Comprehensive analysis of 1H NOESY spectra of 1:2:3 showed that terminal fragments of the complex [5'p-1T-2G-3A-4T-], [-21A-22T-23A-24C], [-8T-9T-10T-11C-12T] and [13A-14G- 15A-15A-17A-18C-] gave a continuous set of intra- and inter- nucleotide interactions, typical of regular double-stranded B-DNA. In contrast, the central region of the complex composed of 5C, 6A, 7G, 19T and 20G nucleotide residues, nearest the Pyr and Az groups, was found to be distorted. Thus some signals from aromatic and/or sugar-ring protons of the above nucleotide residues were extremely broadened or almost absent. Moreover, some intra- and/or inter-nucleotide interactions, typical of the regular DNA duplex, were not detected for the [-5C-6A-7G-] and [-19T- 20G-] regions of the tandem system. Instead of that, some cross-peaks of low-intensity between the H2 proton of the Pyr group and 7G(H1'), 7G(H2'/H2''), 7G(H3'), 4T(H2''), 4T(H4') and 4T(H5'/H5'') were observed. Additional 1H -1H NOE-interactions between methylene protons of the linker group L and some sugar ring protons of 18C nucleotide residue were detected. A preliminary structural model, constructed using proton- proton distances between Pyr and the DNA and Az-L and DNA obtained from a 1H NOESY experiment at 300 ms mixing time as constraints for the refinement of the structure, displayed significant distortion from B-DNA of the double- strand-ed helix in the middle of the complex, (-5C-6A-7G, -18C-19T- 20G-). The Pyr group was located in what remains of the minor groove near 4T, 5C, 6A and 7G and the centroid of the azide ring less than 9A°from the centroid of the ring system of Pyr group.