Influence of monovalent metal ions on metal binding and catalytic activity of the 10–23 DNAzyme

Abstract

Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10–23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10–23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10–23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex.

A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing

“…2a ), and an electrophoretic mobility shift assay (EMSA) after hydrolysis and phosphatase treatment ( Supplementary Fig. 2b ), which were consistent with the presence of a terminal 2’,3’ cyclic phosphate, suggesting a transesterification pathway similar to other RNA-cleaving catalysts 61 including the classic 10-23 and 8-17 DNAzymes 62 , as well as the previously described “FR17_6” FANA XNAzyme 56 . Consistent with a transesterification involving deprotonation of the 2’hydroxyl group of the ribose at the cut site for in-line attack, methylation of this group by substitution with a 2’-O-Methyl-RNA residue in a modified substrate rendered it uncleavable ( Supplementary Fig.…”

The architecture of the 10‐23 DNAzyme and its implications for DNA‐mediated catalysis

“…Interestingly, very similar effects are found when changing the NaCl concentration from 100 to 500 m m (I, blue, in the absence of Mg 2+ ). Panels B,C,G,H,I combine and reproduce data reported in Refs [27,49].…”

“…Structural representations in (D, E) were prepared with pymol [51]. (G) Single turnover cleavage assay of Dz in the presence of 1 m m Mg 2+ and indicated concentrations of Na + (data taken from [49]). (H, I) NMR fingerprint spectra of precatalytic Dz:RNA complexes under indicated conditions.…”

“…Inhibiting effects induced by (demonstrated in [27]) that mimics hexahydrated Mg 2+ , as well as differential effects observed for Mn 2+ and Mg 2+ (demonstrated in [49]), further suggest that inner‐sphere contacts of the functionally relevant metal ions may also play a role in catalysis.…”

The inorganic chemicals that surround us: role of tellurium, selenium and zinc on behavioural functions in mammals

“…There is evidence that the positively charged ions of trace elements interact directly with molecular regions of high density charged micro environment residues, particularly with phosphate bonds in the DNA strands[15]. This interaction can change the three dimensional conformation of DNA which is the basic molecular requirement to modulate epigenetically the activity of gene regions to express determined translation products[16][17][18][19]. Thus, trace elements in theory have the physical chemical requirements to act as inducers, fulfilling a biological interaction in the cell metabolism.…”