Characterizing functional states of the 8-17 DNAzyme by pulsed EPR Spectroscopy
Year: 2026
Type: data-paper
Abstract: DNAzymes are single-stranded DNA molecules with enzymatic activity. Due to their promising therapeutic potential in the area of gene regulation, they have attracted significant attention over the past decades. The 8-17 DNAzyme is one of the best known RNA-cleaving DNAzymes, which can be designed to cleave a multitude of RNA substrates. Here, we describe the application of Pulsed Electron Double Resonance spectroscopy to investigate the structure and dynamics of three functional states of the 8-17 DNAzyme. Changes in the three-dimensional structure upon substrate binding and cleavage were investigated by measuring the distance and angles between two rigid spin labels that were incorporated into the two substrate binding arms. The interhelical distance increases from ~3.5 nm in the pre-catalytic state to ~5.2 nm after cleavage, while the substrate-free DNAzyme is pre-structured with a mean distance of ~6.1 nm. Formation of the catalytic core was also studied by PELDOR measurements between the spin labels and paramagnetic Mn2+ bound to the catalytic site. Our results show that the DNAzyme is pre-structured in the absence of the substrate, which forms a more compact pre-catalytic state upon substrate binding. Notably, divalent metal ions are not required for the pre-structuring or the substrate binding. However, divalent metal ions are essential for substrate cleavage, leading to the post-catalytic state. Our data also show that the catalytic core is present in all three states.
Institutions: Goethe University Frankfurt, University of Iceland
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7224852136