The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb2+ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni2+ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, Rp and Sp, indicated that Mg2+, Mn2+, Fe2+, and Co2+ had the highest Sp:Rp activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb2+, suggesting inner-sphere coordination. The pH-rate profiles showed that Pb2+ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent pKa and the pKa of metal-bound water. Combining previous reports and our current results, we propose that Pb2+ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.

Abstract Nickel is a highly important metal, and the detection of Ni2+ using biosensors is a long-stand analytical challenge. DNA has been widely used for metal detection, although no DNA-based sensors were reported for Ni2+. DNAzymes are DNA-based catalysts, and they recruit metal ions for catalysis. In this work, in vitro selection of RNA-cleaving DNAzymes was carried out using a library containing a region of 50 random nucleotides in the presence of Ni2+. To increase Ni2+ binding, a glycyl–histidine-functionalized tertiary amine moiety was inserted at the cleavage junction. A representative DNAzyme named Ni03 showed a high cleavage yield with Ni2+ and it was further studied. After truncation, the optimal sequence of Ni03l could bind one Ni2+ or two Co2+ for catalysis, while other metal ions were inactive. Its cleavage rates for 100 μM Ni2+ reached 0.63 h−1 at pH 8.0. A catalytic beacon biosensor was designed by labeling a fluorophore and a quencher on the Ni03l DNAzyme. Fluorescence enhancement was observed in the presence of Ni2+ with a detection limit of 12.9 μM. The sensor was also tested in spiked Lake Ontario water achieving a similar sensitivity. This is another example of using single-site modified DNAzyme for sensing transition metal ions.

Since 1994, deoxyribozymes or DNAzymes have been in vitro selected to catalyze various types of reactions. Metal ions play a critical role in DNAzyme catalysis, and Zn2+ is a very important one among them. Zn2+ has good biocompatibility and can be used for intracellular applications. Chemically, Zn2+ is a Lewis acid and it can bind to both the phosphate backbone and the nucleobases of DNA. Zn2+ undergoes hydrolysis even at neutral pH, and the partially hydrolyzed polynuclear complexes can affect the interactions with DNA. These features have made Zn2+ a unique cofactor for DNAzyme reactions. This review summarizes Zn2+ -dependent DNAzymes with an emphasis on RNA-/DNA-cleaving reactions. A key feature is the sharp Zn2+ concentration and pH-dependent activity for many of the DNAzymes. The applications of these DNAzymes as biosensors for Zn2+ , as therapeutic agents to cleave intracellular RNA, and as chemical biology tools to manipulate DNA are discussed. Future studies can focus on the selection of new DNAzymes with improved performance and detailed biochemical characterizations to understand the role of Zn2+ , which can facilitate practical applications of Zn2+ -dependent DNAzymes.

Detection of heavy metal contamination in the environment is an on-going analytical challenge. In effort of developing portable biosensors, deoxyribonucleic acid (DNA)-based designs have gained much attention for their high affinity and specificity to metals, stability, cost-efficiency, ease of modification, and batch-to-batch reproducibility. Specific sequences of DNA aptamers and DNAzymes provide grounds for rational designs of fluorescent, colorimetric, and electrochemical detection methods. Aptamers exert only a binding function, while DNAzymes can use heavy metals to catalyze specific chemical and biological transformations. This article starts with a brief introduction of heavy metals and their interactions with DNA. Then DNA aptamers and DNAzymes are respectively reviewed from their in vitro selection, representative DNA sequences, and design of biosensors. For signal transduction, various fluorescent, colorimetric, and electrochemical examples are described. Finally, future perspectives are discussed.

It has been proposed that Mg2+ and Fe2+ are very similar in interacting with ribozymes and some protein-based enzymes, but their activities with DNAzymes have yet to be studied. Here, the activity of Fe2+ as cofactor for a few RNA-cleaving DNAzymes is investigated. 17E is a well-studied DNAzyme that is active in the presence of many different divalent metal ions; it is highly active with Fe2+ with an apparent Kd of 29.7±2.3 μm and a kobs of 1.12±0.11 min-1 in the presence of 1 mm Fe2+ at pH 7.5. Fe2+ has 21-fold higher activity than Mg2+ . Six different DNAzymes are then tested, and only the DNAzymes active with Mg2+ (17E, 8-17, and E5) are active with Fe2+ . Fe2+ has 25 and one- to twofold higher activity than Mg2+ for the 8-17 and E5 DNAzymes, respectively. In pH>7 buffer and in presence of air, 1 mm Fe2+ results in a nonspecific degradation of the DNA strand due to reactive oxygen species (ROS). Cleavage reactions in anoxic environment and antioxidant ascorbate can be used to overcome the effect of oxidation. The findings provide insights for potential DNAzyme catalysis in the early Earth, and they further support the similarity between Mg2+ and Fe2+ in enzyme catalysis.

Metal ions play a critical role in the RNA-cleavage reaction by interacting with the scissile phosphate and stabilizing the highly negatively charged transition state. Many metal-dependent DNAzymes have been selected for RNA cleavage. Herein, we report that the Ce13d DNAzyme can use nonmetallic iodine (I2) to cleave a phosphorothioate (PS)-modified substrate. The cleavage yield exceeded 60% for both the Rp and Sp stereoisomers in 10 s, while the yield without the enzyme strand was only ∼10%. The Ce13d cleavage with I2 also required Na+, consistent with the property of Ce13d and confirming the similar role of I2 as a metal ion. Ce13d had the highest yield among eight tested DNAzymes, with the second highest DNAzyme showing only 20% cleavage. The incomplete cleavage was due to competition from desulfurization and isomerization reactions. This DNAzyme was engineered for fluorescence-based I2 detection. With EDTA for masking metal ions, I2 was selectively detected down to 4.7 nM. Oxidation of I- with Fe3+ produced I2 in situ, allowing detection of Fe3+ down to 78 nM. By harnessing nonelectrostatic interactions, such as the I2/sulfur interaction observed here, more nonmetal species might be discovered to assist DNAzyme-based RNA cleavage.