Influence of conformationally restricted pyrimidines on the activity of 10-23 DNAzymes

Abstract

The catalytic core of a 10-23 DNAzyme was modified introducing conformationally restricted nucleosides such as (2′R)-, (2′S)-2′-deoxy-2′-C-methyluridine, (2′R)-, (2′S)-2′-deoxy-2′-C-methylcytidine, 2,2′-anhydrouridine and LNA-C, in one, two or three positions. Catalytic activities under pseudo first order conditions were compared at different Mg 2+ concentrations using a short RNA substrate. At low Mg 2+ concentrations, triple modified DNAzymes with similar kinetic performance to that displayed by the non-modified control were identified. In the search for a partial explanation of the obtained results, in silico studies were carried out in order to explore the conformational behavior of 2′-deoxy-2′-C-methylpyrimidines in the context of a loop structure, suggesting that at least partial flexibility is needed for the maintenance of activity. Finally, the modified 2′-C-methyl DNAzyme activity was tested assessing the inhibition of Stat3 expression and the decrease in cell proliferation using the human breast cancer cell line T47D.