Wenhu Zhou

Metal Sensing by DNA

Metal ions are essential to many chemical, biological, and environmental processes. In the past two decades, many DNA-based metal sensors have emerged. While the main biological role of DNA is to store genetic information, its chemical structure is ideal for metal binding via both the phosphate backbone and nucleobases. DNA is highly stable, cost-effective, easy to modify, and amenable to combinatorial selection. Two main classes of functional DNA were developed for metal sensing: aptamers and DNAzymes. While a few metal binding aptamers are known, it is generally quite difficult to isolate such aptamers. On the other hand, DNAzymes are powerful tools for metal sensing since they are selected based on catalytic activity, thus bypassing the need for metal immobilization. In the last five years, a new surge of development has been made on isolating new metal-sensing DNA sequences. To date, many important metals can be selectively detected by DNA often down to the low parts-per-billion level. Herein, each metal ion and the known DNA sequences for its sensing are reviewed. We focus on the fundamental aspect of metal binding, emphasizing the distinct chemical property of each metal. Instead of reviewing each published sensor, a high-level summary of signaling methods is made as a separate section. In principle, each signaling strategy can be applied to many DNA sequences for designing sensors. Finally, a few specific applications are highlighted, and future research opportunities are discussed.

A DNAzyme requiring two different metal ions at two distinct sites.

Most 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 G23 in NaA43. Ce3+ functions by stabilizing the transition state phosphorane, thus promoting cleavage. G23 competes favorably with low concentration Ce3+ (below 1 μM). The G23-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.

A New Na(+)-Dependent RNA-Cleaving DNAzyme with over 1000-fold Rate Acceleration by Ethanol.

Enzymes working in organic solvents are important for analytical chemistry, catalysis, and mechanistic studies. Although a few protein enzymes are highly active in organic solvents, little is known regarding nucleic acid‐based enzymes. Herein, we report the first RNA‐cleaving DNAzyme, named EtNa, that works optimally in concentrated organic solvents containing only monovalent Na+. The EtNa DNAzyme has a rate of 2.0 h−1 in 54 % ethanol (with 120 mm NaCl and no divalent metal ions), and a Kd of 21 mm Na+. It retains activity even in 72 % ethanol as well as in DMSO. With 4 mm Na+, the rate in 54 % ethanol is >1000‐fold higher than that in water. We also demonstrated the use of EtNa to measuring the ethanol content in alcoholic drinks. In total, this DNAzyme has three unique features: divalent metal independent activity, Na+ selectivity among monovalent metals, and acceleration by organic solvents.

Aptamer-nanoparticle bioconjugates enhance intracellular delivery of vinorelbine to breast cancer cells

Abstract Targeted uptake of therapeutic nanoparticles in cell- or tissue-specific manner is an attractive technology since they can offer greater efficacy and reduce cytotoxicity on peripheral healthy tissues. In this study, AS1411 (AP), a DNA aptamer specifically binding to nucleolin that is overexpressed on the plasma membrane of breast cancer (BC) cells, was exploited as the targeting ligand of a nanoparticle-based drug delivery system. Vinorelbine (VRL) loaded PLGA-PEG nanoparticles (NP) were formulated by an emulsion/solvent evaporation method, and AP was conjugated to the particle surface using the EDC/NHS technique. The drug-loading efficiency and in vitro drug release studies were measured using HPLC. The resulting AP−NP/VRL formed spherical nanoparticles (<200 nm) with drug loading of about 7% and a stable in vitro drug release profile. Fluorescence microscopy was used to confirm the cellular uptake of the particles and targeted drug delivery. Moreover, cytotoxicity studies were carried out in two different cell lines, MDA-MB-231 BC cells and MCF-10A normal epithelial cells. AP-nucleolin interaction significantly enhanced in vitro cytotoxicity to nucleolin overexpressed cells, as compared with non-targeted nanoparticles, while there was no significant difference in cytotoxicity of the two types of nanoparticles on the nucleolin negative cells. The results further support that AS1411-functionalized nanoparticles are potential carrier candidates for targeted drug delivery towards BC.

2-Aminopurine-modified DNA homopolymers for robust and sensitive detection of mercury and silver

Heavy metal detection is a key topic in analytical chemistry. DNA-based metal recognition has advanced significantly producing many specific metal ligands, such as thymine for Hg2+ and cytosine for Ag+. For practical applications, however, robust sensors that can work in a diverse range of salt concentrations need to be developed, while most current sensing strategies cannot meet this requirement. In this work, 2-aminopurine (2AP) is used as a fluorescence label embedded in the middle of four 10-mer DNA homopolymers. 2AP can be quenched up to 98% in these DNA without an external quencher. The interaction between 2AP and all common metal ions is studied systematically for both free 2AP base and 2AP embedded DNA homopolymers. With such low background, Hg2+ induces up to 14-fold signal enhancement for the poly-T DNA, and Ag+ enhances up to 10-fold for the poly-C DNA. A detection limit of 3nM is achieved for both metals. With these four probes, silver and mercury can be readily discriminated from the rest. A comparison with other signaling methods was made using fluorescence resonance energy transfer, graphene oxide, and SYBR Green I staining, respectively, confirming the robustness of the 2AP label. Detection of Hg2+ in Lake Huron water was also achieved with a similar sensitivity. This work has provided a comprehensive fundamental understanding of using 2AP as a label for metal detection, and has achieved the highest fluorescence enhancement for non-protein targets.

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

RNA-cleaving DNAzymes provide a unique platform for developing biosensors. However, a majority of the work has been performed in clean buffer solutions, while the activity of some important DNAzymes in biological sample matrices is still under debate. Two RNA-cleaving DNAzymes (17E and 10-23) are the most widely used. In this work, we carefully studied a few key aspects of the 17E DNAzyme in human blood serum, including hybridization, cleavage activity, and degradation kinetics. Since direct fluorescence monitoring is difficult due to the opacity of serum, denaturing and nondenaturing gel electrophoresis were combined for studying the interaction between serum proteins and DNAzymes. The 17E DNAzyme retains its activity in 90% human blood serum with a cleavage rate of 0.04 min(-1), which is similar to that in the PBS buffer (0.06 min(-1)) with a similar ionic strength. The activity in serum can be accelerated to 0.3 min(-1) with an additional 10 mM Ca(2+). As compared to 17E, the 10-23 DNAzyme produces negligible cleavage in serum. Degradation of both the substrate and the DNAzyme strand is very slow in serum, especially at room temperature. Degradation occurs mainly at the fluorophore label (linked to DNA via an amide bond) instead of the DNA phosphodiester bonds. Serum proteins can bind more tightly to the 17E DNAzyme complex than to the single-stranded substrate or enzyme. The 17E DNAzyme hybridizes extremely fast in serum. With this understanding, the detection of DNA using the 17E DNAzyme is demonstrated in serum.

Tandem Phosphorothioate Modifications for DNA Adsorption Strength and Polarity Control on Gold Nanoparticles

Unmodified DNA was recently used to functionalize gold nanoparticles via DNA base adsorption. Compared to thiolated DNA, however, the application of unmodified DNA is limited by the lack of sequence generality, adsorption polarity control and poor adsorption stability. We report that these problems can be solved using phosphorothioate (PS) DNA. PS DNA binds to gold mainly via the sulfur atom and is thus less sequence dependent. The adsorption affinity is ranked to be thiol > PS > adenine > thymine. Tandem PS improves adsorption strength, allows tunable DNA density, and the resulting conjugates are functional at a low cost.

An Exceptionally Selective DNA Cooperatively Binding Two Ca2+ Ions

Ca2+ is a highly important metal ion in biology and in the environment, and thus there is extensive work in developing sensors for Ca2+ detection. Although many Ca2+‐binding proteins are known, few nucleic acids can selectively bind Ca2+. DNA‐based biosensors are attractive for their high stability and excellent programmability. We report a RNA‐cleaving DNAzyme, EtNa, cooperatively binding two Ca2+ ions but to only one Mg2+. Four DNAzymes with known Ca2+‐dependent activity were compared, and the EtNa had the best selectivity for Ca2+. The EtNa is 90 times more active in Ca2+ than in Mg2+. Phosphorothioate (PS) modification showed that both non‐bridging oxygen atoms at the scissile phosphate contribute equally to Ca2+ binding. The pH–rate profile suggests two concurrent deprotonation reactions. EtNa was further engineered for Ca2+ sensing, and found to have a detection limit of 17 μm Ca2+ and excellent selectivity. The detection of Ca2+ in tap water was performed, and the result was comparable with that by ICP‐MS. This study offers new fundamental insights into Ca2+ binding by nucleic acids and improved metal selectivity by having multiple cooperative metal binding sites.

A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence

A 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 Kd of ∼20 mm with Na+ (other monovalent cations: 40–60 mm). The Kd 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 Kd to 8 mm 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.

A Silver-Specific DNAzyme with a New Silver Aptamer and Salt-Promoted Activity

Most RNA-cleaving DNAzymes require a metal ion to interact with the scissile phosphate for activity. Therefore, few unmodified DNAzymes work with thiophilic metals because of their low affinity for phosphate. Recently, an Ag+-specific Ag10c DNAzyme was reported via in vitro selection. Herein, Ag10c is characterized to rationalize the role of the strongly thiophilic Ag+. Systematic mutation studies indicate that Ag10c is a highly conserved DNAzyme and its Ag+ binding is unrelated to C-Ag+-C interaction. Its activity is enhanced by increasing Na+ concentrations in buffer. At the same metal concentration, activity decreases in the following order: Li+ > Na+ > K+. Ag10c binds one Na+ ion and two Ag+ ions for catalysis. The pH-rate profile has a slope of ∼1, indicating a single deprotonation step. Phosphorothioate substitution at the scissile phosphate suggests that Na+ interacts with the pro-Rp oxygen of the phosphate, and dimethyl sulfate footprinting indicates that the DNAzyme loop is a silver aptamer binding two Ag+ ions. Therefore, Ag+ exerts its function allosterically, while the scissile phosphate interacts with Na+, Li+, Na+, or Mg2+. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag+ aptamer.