A Stability Concept for Metal Ion Coordination to Single-Stranded Nucleic Acids and Affinities of Individual Sites

Abstract

The three-dimensional architecture and function of nucleic acids strongly depend on the presence of metal ions, among other factors. Given the negative charge of the phosphate-sugar backbone, positively charged species, mostly metal ions, are necessary for compensation. However, these ions also allow and induce folding of complicated RNA structures. Furthermore, metal ions bind to specific sites, stabilizing local motifs and positioning themselves correctly to aid (or even enable) a catalytic mechanism, as, for example, in ribozymes. Many nucleic acids thereby exhibit large differences in folding and activity depending not only on the concentration but also on the kind of metal ion involved. As a consequence, understanding the role of metal ions in nucleic adds requires knowing not only the exact positioning and coordination sphere of each specifically bound metal ion but also its intrinsic site affinity. However, the quantification of metal ion affinities toward certain sites in a single-stranded (though folded) nucleic acid is a demanding task, and few experimental data exist. In this Account, we present a new tool for estimating the binding affinity of a given metal ion, based on its ligating sites within the nucleic acid. To this end, we have summarized the available affinity constants of Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, Cd2+, and Pb2+ for binding to nucleobase residues, as well as to mono-and dinucleotides. We have also estimated for these ions the stability constants for coordinating the phosphodiester bridge. In this way, stability increments for each ligand site are obtained, and a dear selectivity of the ligating atoms, as well as their discrimination by different metal ions, can thus be recognized. On the basis of these data, we propose a concept that allows one to estimate the intrinsic stabilities of nucleic acid-binding pockets for these metal ions. For example, the presence of a phosphate group has a much larger influence on the overall affinity of Mg2+, Ca2+, or Mn2+ compared with, for example, that of Cd2+ or Zn2+. In the case of Cd2+ and Zn2+, the guanine N7 position is the strongest intrinsic binding site. By adding up the individual increments like building blocks, one derives an estimate not only for the overall stability of a given coordination sphere but also for the most stable complex if an excess of ligating atoms is available in a binding pocket saturating the coordination sphere of the metal ion. Hence, this empirical concept of adding up known intrinsic stabilities, like building blocks, to an estimated overall stability will help in understanding the accelerating or inhibiting effects of different metal ions in ribozymes and DNAzymes. CONSPECTUSThe three-dimensional architecture and function of nucleic acids strongly depends, among other factors, on the presence of metal ions. Most importantly, metal ions, needed to compensate the negative charge of the phosphate-sugar backbone, allow and induce folding of complicated RNA structures. On the other hand, metal ions bind to spec...

Biochemical Characterization of a Lanthanide-Dependent DNAzyme with Normal and Phosphorothioate-Modified Substrates

“…This assay agrees with the soft-hard acid-base theory, where Ce 3+ has a stronger affinity with oxygen, Cd 2+ and Cu 2+ bind sulfur ligands better, and Pb 2+ can bind both. 30 Therefore, using PS-modified substrate is a good way to obtain DNAzymes active with thiophilic metals, 31 which is consistent with the ribozyme literature. 32,33 Next, Ce 3+ and Cd 2+ were used to study the PO and PS systems, respectively.…”

“…Compared to most divalent metal ions, which bind to nucleotide acids with mM affinity (e.g. using pUpU as a model), 30 lanthanide binding yields µM affinity. 45 This is probably a primary reason that lanthanides are generally more active for Ce13.…”

Metal‐Induced Specific and Nonspecific Oligonucleotide Folding Studied by FRET and Related Biophysical and Bioanalytical Implications

“…Without a rigid scaffold, these oligonucleotides can be easily condensed by metal ions such as Na I and Mg II , [21][22][23][24] which also increases the FRET efficiency and may lead to false positive results ( Figure 1B). In other words, the information content from FRET is insufficient to distinguish specific (target induced) and non-specific (metal ion electrostatics related) folding.…”

An Ultrasensitive Light-up Cu2+ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate

“…The selection rounds are marked with a plus sign and a blue bar 8 (indicating positive selection using Cu 2+ ; 50 µM Cu 2+ for the first 8 rounds, 20 µM Cu 2+ for round 9 and 10 rounds, and 10 µM Cu 2+ for the last 2 rounds); or with a minus sign and a red bar (indicating negative selection using a mixture of Cd 2+ , Pb 2+ and Zn 2+ (20 µM each) for round 8-10, and Cd 2+ alone (10 µM) for round [11][12]. In some rounds, two negative selections were performed in tandem to improve metal specificity for Cu 2+ .…”

“…[8][9][10][11] DNAzymes are DNA-based catalysts that often require divalent metal ions for activity. [12][13][14] In vitro selection has been intentionally carried out in the presence of specific metal ions to isolate sensor DNAzymes. 15,16 A Cu 2+ biosensor was reported using a DNAzyme that performs oxidative DNA cleavage.…”