E. coli RNase HI and the phosphonate-DNA/RNA hybrid: molecular dynamics simulations.

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3-O-CH2-P-O-5' or 3-O-P-CH2-O-5) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5ns). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg++. 4H2O chelate complex (bound in the active site) were analyzed in detaiL Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn 16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).

-CH2- lengthening of the internucleotide linkage in the ApA dimer can improve its conformational compatibility with its natural polynucleotide counterpart.

The complete family of ApA phosphonate analogues with the internucleotide linkage elongated by insertion of a -CH2- group was prepared and the hybridisation and structural properties of its members in interaction with polyuridylic acid were investigated using an original 2D Raman approach. Except for the conformationally restricted A(CH)pA(2'3'endo-5') modification, all of the isopolar, non-isosteric analogues form triplex-like complexes with poly(rU) at room temperature, in which two polymer strands are bound by Watson-Crick and Hoogsteen bonds to a central pseudostrand consisting of a 'chain' of A-dimers. For all of these dimers, the overall conformation of the triplexes was found to be similar according to their extracted Raman spectra. A simple semi-empirical model was introduced to explain the observed dependency of the efficiency of triplex formation on the adenine concentration. Apparently, for most of the modifications studied, the creation of a stable complex at room temperature requires the formation of a central pseudostrand, consisting of several adenine dimers. Molecular dynamics calculations were finally performed to interpret the differences in 'cooperative' behaviour between the different dimers studied. The results indicate that the exceptional properties of the Ap(CH2)A(3'-5') dimer could be caused by the 3D conformational compatibility of this modified linkage with the second (Hoogsteen) poly(rU) strand.