Our 200ns MD simulations show that even fully modified oligonucleotides bearing the 3'-O-P-CH2-O-5' (but not 3'-O-CH2-P-O-5') phosphonate linkages can be successfully attached to the surface of Human RNase H. It enables to explain that oligonucleotides consisting of the alternating 3'-O-P-CH2-O-5' phosphonate and phosphodiester linkages are capable to elicit the RNase H activity (while the 3'-O-CH2-P-O-5' phosphonates are completely inactive). Stability of the binuclear active site of Human RNase H was achieved using the one-atom model for Mg(2+) in conjunction with a polarized phosphate group of the scissile bond, which is wedged between both magnesium ions. The reference MD simulation (lasting for 1000ns), which was produced using a well-established seven-point (with dummy atoms) model for Mg(2+) led to essentially the same results. The MD run (lasting for 500ns) produced for the Thermus thermophilus Argonaute enzyme shows the transferability of our approach for the stabilization of a binuclear active site. Glu512 was bound in the T. thermophilus Argonaute active site to the 2'-OH of the nucleotide adjacent to the scissile phosphate and one of the two active-site divalent metal ions in exactly the same way as Glu186 in Human RNase H. Glu512 thus completes the catalytic tetrad of Argonaute.

Charles University Faculty of Mathematics and Physics Institute of Physics Ke Karlovu 5 Prague 2 121 16 Czech Republic

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5'-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes