Mapping the Structure and Conformational Landscape of the 10–23 DNAzyme

Deoxyribozymes (DNAzymes) are programmable DNA catalysts with therapeutic and diagnostic potential. The RNA-cleaving 10–23 DNAzyme was the first DNAzyme shown to function using common bioavailable metal ion cofactors, establishing the potential for DNA-based RNA knockdown in vivo. Despite extensive biochemical characterization, structural knowledge on the 10–23 DNAzyme is limited, hindering efforts to rationally improve its activity for physiological applications. To address this need, we developed a T7 RNA polymerase-based protein scaffold that enables cryo-EM visualization of the 10–23 DNAzyme. Using this approach, we obtained a 4.5 Å reconstruction of the DNAzyme-substrate complex and used dimethyl sulfate (DMS) labeling to further examine DNAzyme dynamics. Our structural work supports a model in which the palindromic core folds into a pseudoknot stabilized by guanine stacking, creating a rigid element that organizes subsequent folding of the catalytic core and active site. DMS probing further indicates that magnesium binding collapses a flexible A9–A15 loop onto the pseudoknot, compacting the catalytic core. Together, these findings provide insight into 10–23 DNAzyme dynamics through a proposed metal-dependent hinged activation mechanism. The protein scaffolding approach may also serve as a broadly applicable framework for further structural investigations of DNAzymes.