NMR INVESTIGATIONS OF DUPLEX STABILITY OF PHOSPHOROTHIOATE AND PHOSPHORODITHIOATE DNA ANALOGS MODIFIED IN BOTH STRANDS

Duplex formation from the self-complementary 12mer d(CGCGAATTCGCG) (Di ckerson dodecamer) in which all phosphodiester linkages were replaced by phosphorothioate or phosphorodithioate linkages was studied using v ariable-temperature H-1 and P-31 NMR spectroscopy, Melting temperature s of the dodecamer, measured spectrophotometrically, showed significan t decrease upon sulfur substitution (T-m 49 degrees C for the phosphor othioate and 21 degrees C for the phosphorodithioate, compared with 68 degrees C for the unmodified oligomer, in 1 M salt), Hyperchromicity observed upon melting of the dithioate was surprisingly low. NOESY spe ctra of the monothioate showed a cross-peak pattern characteristic for a right-handed duplex, Imino proton resonances of the duplex, shown b y the mono- and the dithioate, were similar to those of the parent com pound, In spite of monophasic melting curves, temperature dependence o f the imino proton resonances and phosphorus resonances of the phospho rodithioate indicated heterogeneity with respect to base-pairing, comp atible with the presence of a hairpin loop. Relaxation times (T-1) of the imino protons in the phosphorothioate, determined by the saturatio n recovery method, were considerably shorter than in the unmodified ol igomer, Base-pair lifetimes in the unmodified Dickerson dodecamer, det ermined by catalyst-dependent changes in relaxation rates of imino pro tons, were in the range of 2-30 ms at 20 degrees C, Strongly reduced b ase-pair lifetimes were found in the phosphorothioate analogue.