NMR Investigations of Duplex Stability of Phosphorothioate and Phosphorodithioate DNA Analogues Modified in Both Strands
Open Access
- 1 January 1996
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 24 (5) , 829-834
- https://doi.org/10.1093/nar/24.5.829
Abstract
Duplex formation from the self-complementary 12mer d(CGCGAATTCGCG) (Dickerson dodecamer) in which all phosphodiester linkages were replaced by phosphorothioate or phosphorodithioate linkages was studied using variable-temperature 1H and 31P NMR spectroscopy. Melting temperatures of the dodecamer, measured spectrophotometrically, showed significant decrease upon sulfur substitution (Tm 49°C for the phosphorothioate and 21°C for the phosphorodithioate, compared with 68°C for the unmodified oligomer, in 1 M salt). Hyperchromicity observed upon melting of the dithioate was surprisingly low. NOESY spectra of the monothioate showed a cross-peak pattern characteristic for a right-handed duplex. Imino proton resonances of the duplex, shown by the mono-and the dithioate, were similar to those of the parent compound. In spite of monophasic melting curves, temperature dependence of the imino proton resonances and phosphorus resonances of the phospho-rodithioate indicated heterogeneity with respect to base-pairing, compatible with the presence of a hairpin loop. Relaxation times (T1) of the imino protons in the phosphorothioate, determined by the saturation recovery method, were considerably shorter than in the unmodified oligomer. Base-pair lifetimes in the unmodified Dickerson dodecamer, determined by catalyst-dependent changes in relaxation rates of imino protons, were in the range of 2–30 ms at 20°C. Strongly reduced base-pair lifetimes were found in the phos-phorothioate analogue.Keywords
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