Substituting Inosine for Guanosine in DNA: Structural and Dynamic Consequences

Abstract

Inosine differs from the guanosine nucleoside only by the absence of the N2 amino group. Both nucleosides also have similar electrostatic potentials. Therefore, substituting I for G has been used to probe various properties of nucleic acids and to facilitate the interpretation of binding studies. In particular, the absence of the amino group permits the assessment of its importance in the binding of ligands to the minor groove of duplex DNA. It has been known for some time that an I-C base pair is of lower stability than a regular G-C base pair, which needs to be considered when making DNA constructs containing inosine. However, it is generally assumed that both base pairs are structurally highly similar. To test this assumption in an identical sequence environment, we have determined the fine structure of two hairpin DNA substrates that differ only in the substitution of an I-C base pair for a G-C base pair. The structures have been solved using nuclear magnetic resonance (NMR) restraints in conjunction with Mardigras and molecular dynamics. The structural data are complemented with thermodynamic and dynamic data to get a comprehensive evaluation of the consequences of G-C vs I-C base pair substitutions. Our data show a strong similarity in the structures of the hairpins, but a significant difference in the melting temperatures, T m. This difference is also reflected in the drastically decreased base pair lifetime of 7.4 milliseconds compared to the G-C base pair lifetime of 155 milliseconds. The substitution of I-C for G-C is to probe for specific effect due to the amino group is satisfactory, as long as the lowered thermal stability and the drastically increased local dynamics are considered.