Waterloo Institute for Nanotechnology
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Browsing Waterloo Institute for Nanotechnology by Subject "Acid"
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Item DNA Adsorption by ZnO Nanoparticles near Its Solubility Limit: Implications for DNA Fluorescence Quenching and DNAzyme Activity Assays(American Chemical Society, 2016-06-07) Ma, Lingzi; Liu, Biwu; Huang, Po-Jung Jimmy; Zhang, Xu; Liu, JuewenZinc oxide (ZnO) is a highly important material, and Zn2+ is a key metal ion in biology. ZnO and Zn2+ interconvert via dissolution and hydrolysis/condensation. In this work, we explore their interactions with DNA, which is important for biointerface, analytical, and bioinorganic chemistry. Fluorescently labeled DNA oligonucleotides were adsorbed by a low concentration (around 5 mu g/mL) of ZnO nanoparticles, near the solubility limit. Right after mixing, fluorescence quenching occurred, indicating DNA adsorption. Then, fluorescence recovered, attributable to ZnO dissolution. The dissolution rate followed A(5) > T-5 > C-5. Dissolution was slower with longer DNA. The adsorption affinity was also measured by a displacement assay to be G(5) > C-5 > T-5 > A(5), suggesting that tightly adsorbed DNA can retard ZnO dissolution. Electrostatic interactions are important for DNA adsorption because ZnO is positively charged at neutral pH, and a high salt concentration inhibits DNA adsorption. Next, in situ formation of ZnO from Zn2+ was studied. First, titrating Zn2+ into a fluorescently labeled oligonucleotide at pH 7.5 resulted in an abrupt fluorescence quenching beyond 0.2 mM Zn2+. At pH 6, quenching occurred linearly with the Zn2+ concentration, suggesting the effect of Zn2+ precipitation at pH 7.5. Second, a Zn2+-dependent DNA-cleaving DNAzyme was studied. This DNAzyme was inhibited at higher than 2 mM Zn2+, attributable to Zn2+ precipitation and adsorption of the DNAzyme. This paper has established the interplay between DNA, Zn2+, and ZnO. This understanding can avoid misinterpretation of DNA assay results and adds knowledge to DNA immobilization.Item A DNAzyme requiring two different metal ions at two distinct sites(Oxford University Press, 2015-12-10) Zhou, Wenhu; Zhang, Yupei; Huang, Po-Jung Jimmy; Ding, Jinsong; Liu, JuewenMost previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na+ and a trivalent lanthanide (e.g. Ce3+), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na+-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb3+ luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na+-binding. The Na+ dissociation constants of Ce13d measured from the cleavage activity assay, Tb3+ luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce3+ might be replaced by G(23) in NaA43. Ce3+ functions by stabilizing the transition state phosphorane, thus promoting cleavage. G(23) competes favorably with low concentration Ce3+ (below 1 mu M). The G(23)-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes.Item A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence(Wiley, 2016-08-17) Zhou, Wenhu; Ding, Jinsong; Liu, JuewenA previous study of two RNA-cleaving DNAzymes, NaA43 and Ce13d, revealed the possibility of a common Na+ aptamer motif. Because Na+ binding to DNA is a fundamental biochemical problem, the interaction between Ce13d and Na+ was studied in detail by using sensitized Tb3+ luminescence spectroscopy. Na+ displaces Tb3+ from the DNAzyme, and thus quenches the emission from Tb3+. The overall requirement for Na+ binding includes the hairpin and the highly conserved 16-nucleotide loop in the enzyme strand, along with a few unpaired nucleotides in the substrate. Mutation studies indicate good correlation between Na+ binding and cleavage activity, thus suggesting a critical role of Na+ binding for the enzyme activity. Ce13d displayed a K-d of approximate to 20mm with Na+ (other monovalent cations: 40-60mm). The K-d values for other metal ions are mainly due to non-specific competition. With a single nucleotide mutation, the specific Na+ binding was lost. Another mutant improved K-d to 8mm with Na+. This study has demonstrated a Na+ aptamer with important biological implications and analytical applications. It has also defined the structural requirements for Na+ binding and produced an improved mutant.Item A Silver DNAzyme(American Chemical Society, 2016-04-05) Saran, Runjhun; Liu, JuewenSilver is a very common heavy metal, and its detection is of significant analytical importance. DNAzymes are DNA-based catalysts; they typically recruit divalent and trivalent metal ions for catalysis. Herein, we report a silver specific RNA-cleaving DNAzyme named Ag10c obtained after six rounds of in vitro selection. Ag10c displays a catalytic rate of 0.41 min(-1) with 10 mu M Ag+ at pH 7.5 with 200 mM NaNO3, while its activity is completely inhibited with the same concentration of NaCl. Ag10c is highly specific for Ag+ among all the tested metals. A catalytic beacon biosensor is designed by labeling a fluorophore and a quencher on the DNAzyme. Fluorescence enhancement is observed in the presence of Ag+ with a detection limit of 24.9 nM Ag+. The sensor shows a similar analytical performance in Lake Huron water. This is the first monovalent transition metal dependent RNA-cleaving DNAzyme. Apart from its biosensor application, this study strengthens the idea of exploring beyond the traditional understanding of multivalent ion dependent DNAzyme catalysis.