Refined high-field NMR solution structure of a binary-addressed pyrene/perfluoro azide complementary DNA oligonucleotide system shows extensive distortion in the central nick region

The structural analysis of the photoactivated binary system of complementar y-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectros copy and restrained molecular dynamics is reported. The binary system compr ised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanu cleotides, (dAGAAACp-L-Az (2) and Pyr-pdTGATAC (3)), complementary to neigh bouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p -azidotetrafludrobenzyl group (Az) via a linker group (L), and the other ol igonucleotide (3) is equipped with the photosensitizing pyrenyl-1-methylami no group (Pyr). We now extend the structural analysis of 1:2:3, which was p reviously based on qualitative 2D H-1-NMR data and thermodynamic analysis o f complex formation from UV-visible thermal denaturation experiments. In th e current work structural refinement was performed by separate molecular dy namics runs for six different starting structures based on 318 proton-proto n distance-range constraints, evaluated from the H-1-NOESY spectrum (tau(mi x)=200ms, 600MHz) using complete relaxation matrix analysis (NMIR/TRIAD/MAR DIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable proton s, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62 .5 degrees) with the bending point in the middle of the duplex in the regio n of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes disp laying a hinge-like: flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues T-4, C-5 and T-20. In contrast, the loca tion of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feat ure common to all six final azido group orientations was the outside locati on on the side of the major groove. The distance between the Pyr and Az gro ups varied from 11 Angstrom to 24 Angstrom for the six final structures (17 Angstrom, final average structure). The dynamics of duplex denaturation fo r 1:2:3 was probed by monitoring the temperature-induced NMR line broadenin g of the imino protons in a 1D variable temperature NMR experiment. The mel ting of 1:2:3 starts both from the ends and from the middle part of the dup lex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.