The 8-17 DNAzyme can operate in a single active structure regardless of metal ion cofactor

Abstract

DNAzymes - synthetic enzymes made of DNA - have long attracted attention as RNA-targeting therapeutic agents. Yet, as of now, no DNAzyme-based drug has been approved, partially due to our lacking understanding of their molecular mode of action. In this work we report the solution structure of 8-17 DNAzyme bound to a Zn²⁺ ion solved through NMR spectroscopy. Surprisingly, it turned out to be very similar to the previously solved Pb²⁺-bound form (catalytic domain RMSD = 1.28 Å), despite a long-standing literature consensus that Pb²⁺ recruits a different DNAzyme fold than other metal ion cofactors. Our follow-up NMR investigations in the presence of other ions - Mg²⁺, Na⁺, and Pb²⁺ - suggest that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. Based on these findings, we propose a model for 8-17 DNAzyme interactions with metal ions postulating the existence of only a single catalytically-active structure, yet populated to a different extent depending on the metal ion cofactor. Our results provide structural information on the 8-17 DNAzyme in presence of non-Pb²⁺ cofactors, including the biologically relevant Mg²⁺ ion.

Fig. 1. The 8-17 DNAzyme.

A consensus structure, (B) construct used in the crystallographic study, (C) construct used in this NMR investigation, (D) current paradigm for 8–17 DNAzyme interplay with divalent metal ion cofactors, (E) the tertiary fold observed in Pb²⁺-bound crystallographic structure, (F) the proposed catalytic mechanism.

Fig. 2. Zn²⁺-induced folding of 8–17_short.

A monitored by CD spectroscopy and (B) monitored by 1D ¹H-NMR. NMR resonances belonging to an initially present second spectral form are marked by red stars. The titration was performed in 10 mM sodium cacodylate buffer which originally contained no additional salts. Source data are provided as a Source Data file.

Fig. 3. The solution structure of 8-17 DNAzyme in the presence of Zn²⁺.

A the overall fold, (B) geometric details of Helix 4.

Fig. 4. Comparison between Pb²⁺ and Zn²⁺ stabilized structures.

Main panel uses a single conformer from the NMR bundle for clarity, while the insert shows the entire bundle.

Fig. 5. Na⁺- and Mg²⁺-induced folding of 8-17_short.

A titrations with Na⁺ (B) titrations with Mg²⁺. For each ion the folding was monitored by CD spectroscopy (top) and 1D ¹H-NMR (bottom). NMR resonances belonging to an initially present second spectral form are marked by red stars. The titration was performed in 10 mM sodium cacodylate buffer which originally contained no additional salts. Source data are provided as a Source Data file.

Fig. 6. Pb²⁺-induced folding of 8-17_short.

(A) monitored by CD spectroscopy and (B) monitored by 1D ¹H-NMR at millimolar DNA concentrations, (C) monitored by 1D ¹H-NMR at submilimolar DNA concentrations. NMR resonances belonging to an initially present second spectral form are marked by red stars while those to the form appearing at higher concentrations of Pb²⁺ with green ones. The titration was performed in 10 mM sodium cacodylate buffer which originally contained no additional salts. Source data are provided as a Source Data file.

Fig. 7. Ion induced folding of the 8-17 DNAzyme construct used in the previous crystallographic investigation (sequence in Fig. 1B).

CD-monitored titrations with four different metal ions: (A) Zn²⁺, (B) Na⁺, (C) Mg²⁺ and (D) Pb²⁺. Source data are provided as a Source Data file.

Fig. 8. The proposed model for the interplay between divalent metal ions and 8–17 DNAzyme structure and activity.

The top part of the scheme represents the conformational ensemble of the apo- form of the enzyme, while the effects of Pb²⁺ and Zn²⁺ ions are depicted below in the left and right panels, respectively. The occupation state of structural and catalytic metal binding sites is also shown in each case for the well-folded DNAzyme molecules. When the catalytic site is filled the DNAzyme is activated, as marked by the black, highlighted scissors.

Fig. 9. The study of Zn²⁺ binding to Na⁺-structured 8-17_short through chemical shift perturbations (CSPs).

A spectral changes in the imino region of 1D ¹H-NMR spectrum upon Zn²⁺ titration, (B) spectral changes in the aromatic region of ¹H-¹³C HSQC, (C) measured CSPs visualized on the 3D structure, color coded from 0.00 [ppm] (white) to ≥0.25 [ppm] (red). Source data are provided as Supplementary Data files.