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 N-2 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 IH COSY, TOCSY and NOESY experiments. Comprehensive analysis of IH NOESY spectra of 1:2:3 showed that terminal fragments of the complex [5'p-T-1-(2)G-(3)A-T-4-], [-(21)A-T-22-(23)A-C-24], [-T-8-T-9-T-10-C-11-T-12] and [(13)A-(14)G-(15)A-(15)A-(17)A-C-18-] 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 C-5, (6)A, (7)G, T-19 and (20)G 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 [-C-5-(6)A-(7)G-] and [-T-19-(20)G-] regions of the tandem system. Instead of that, some cross-peaks of low-intensity between the H2 proton of the Pyr group and (7)G(H1'), (7)G(H2'/H2 ''), (7)G(H3'), T-4(H2 ''), T-4(H4') and T-4(H5'/H5 '') were observed. Additional H-1-H-1 NOE-interactions between methylene protons of the linker group L and some sugar ring protons of C-18 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 H-1 NOESY experiment at 300 ms mixing time as constraints for the refinement of the structure, displayed significant distortion from B-DNA of the double-stranded helix in the middle of the complex, (-C-5-(6)A-(7)G, -C-18-T-19-(20)G-). The Pyr group was located in what remains of the minor groove near T-4, C-5, (6)A and (7)G and the centroid of the azide ring less than 9A degrees from the centroid of the ring system of Pyr group.