Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions

Abstract

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu²⁺) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

Keywords: DNAzymes; hyperfine spectroscopy; metal soup; multiple binding; radical path.

Scheme 1

Secondary structures of the DNAzymes (deoxyribozymes) cleaving DNA: a) the first DNA‐cleaving‐DNA formed by 69 nucleotides has been followed by the b) 46mer as reduced structure containing the catalytic core. c) for the mono‐molecular 46‐mer a triplex version can be obtained. d) The bimolecular structure (c4s4) formed by substrate and enzyme oligomers, both containing four additional nucleotides (GCCG / CGGC base paring; c4s4 nomenclature derives from those four bases both on the catalyst and on the substrate replacing the GGA bulge fragment, to stabilize the secondary structure).

Figure 1

a) Modelled structure of the 46mer for MD simulations without Cu²⁺; strand regions and stem regions are highlighted. b) Time series distance profile between the phosphate atoms of A14 and A41 from the MD trajectory of 46mer with Cu²⁺ (1‐MD‐46mer Cu2⁺). c) Structures from MD ensemble with Cu²⁺, selected based on distance between phosphate atoms of A14 (blue) and A41 (orange) ranging from 0.5 to 5 nm.

Figure 2

a) Interaction between Cu2⁺ and the 46mer with a distance cut‐off of 0.4 nm was defined as residence time and the contact frequency as percentage of simulation time is shown for 1‐MD‐46mer‐ Cu²⁺ (resident time analysis, RTA). b) Structure from 1‐MD‐46mer‐ Cu2⁺ with a distance of 0.5 nm between A14 and A41, showing a plausible pre‐reactive complex where Cu2⁺ is interacting with residues from substrate and catalytic strands simultaneously.

Figure 3

a) CW‐EPR spectra for Cu‐c4s4 sample at X‐Band, measured at 120 K. The red dotted lines are the fitting of the experimental spectra (black line). On the right the Echo‐Detected Field‐Swept (EDFS) spectrum; the green arrow indicates the selected observed field for the 2D‐ESEEM experiment. b) Structure of the c4s4 complex with the isotopic labeled by ¹⁵N at Guanosine residue‐14 of the catalyst (c4). c,d) HYSCORE (HYperfine Sublevel COrRElation spectroscopy) 2D EPR experiments recorded at 9.72 GHz for the native c4s4 architecture containing Cu²⁺ and for the isotopically labeled structure depicted in b. By comparing the (+/+) quadrant of the HYSCORE experiments for the unlabeled c4s4 with the samples containing guanosine G14 (c4) sequence additional cross peaks with Δv = 2.7 MHz have been observed. These additional cross peaks are generated by the hyperfine coupling Cu^2+/15N (having ¹⁵N nuclear spin I = 1/2, the manifold of the coupling generating a doublet without the presence of double quanta transition and/or frequencies combination). ¹⁵N Larmor frequency (1.51 MHz) is indeed observed only on the HYSCORE experiments recorded on the sequences containing isotopologues (circled cross peaks. In addition, the comparison between the (‐/+) quadrant of the unlabeled/labeled samples shows the reduced cross peaks (double‐quanta transition for ¹⁴N). e,f) 3D plot for the HYSCORE spectra recorded at Q‐band (34 GHz) for (top) c4s4 unlabeled sample and for the c4s4 isotopically labeled (¹⁵N) (bottom) at Guanosine residue‐14 of the catalyst (c4).