Influence of LNA modifications on the activity of the 10–23 DNAzyme†

Abstract

The 10–23 DNAzyme is a catalytic DNA molecule that efficiently cleaves RNA in the presence of divalent cations such as Mg²⁺ or Ca²⁺. Following their discovery, the 10–23 DNAzymes demonstrated numerous advantages that quickly led them to be considered powerful molecular tools for the development of gene-silencing tools. In this study, we evaluate the efficiency of the 10–23 DNAzyme and an LNA-modified analog in cleaving human MALAT1, an RNA overexpressed in cancer cells. First, we perform in vitro assays using a 20 nt RNA fragment from the MALAT1 sequence, with 2 mM and 10 mM Mg²⁺ and Ca²⁺ as cofactors, to evaluate how LNA modifications influence catalytic activity. We found that the activity is increased in the LNA-modified DNAzyme compared to the unmodified version with both cofactors, in a concentration-dependent manner. Finally, the RNA-cleaving activity of the LNA-modified, catalytically active 10–23 DNAzyme was tested in MCF7 human breast cancer cells. We found that the DNAzyme persists for up to 72 h in cells and effectively silences MALAT1 RNA in a concentration-dependent manner as early as 12 h post-transfection.