NMR-STUDIES OF DNA DUPLEXES SINGLY CROSS-LINKED BY DIFFERENT SYNTHETIC LINKERS

Molecular modelling studies resulted in the design of a variety of non -nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand a nd the 5'-end of the (-)-strand in DNA duplexes. Three of these linker s were synthesized and used to prepare singly cross-linked duplexes d( GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene -, a phosphate- and a second propylene-group and is thought to mimic t he backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of t he cross-linked duplexes are 65 degrees C for I and 73 degrees C for I I and III, as compared with 36 degrees C for the corresponding non-lin ked nonadeoxynucleotide duplex. The three cross-linked duplexes were s tructurally characterized by nuclear magnetic resonance spectroscopy. The H-1 and P-31 resonance assignments in the DNA stem were obtained u sing standard methods. For the resonance assignment of the linker prot ons, two-dimensional H-1-P-31 heteronuclear COSY and two-quantum-exper iments were used. Distance geometry calculations with NOE-derived dist ance constraints were performed and the resulting structures were ener gy-minimized. In duplex I, the nucleotides flanking the propylene-phos phate-propylene-linker do not form a Watson-Crick base pair, whereas i n duplexes II and III the entire DNA stem is in a B-type double helix conformation.