Molecular crowding: impacts on the activity of the 10-23 DNAzyme

Abstract

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.