Waterloo Institute for Nanotechnology

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2-Aminopurine-modified DNA homopolymers for robust and sensitive detection of mercury and silver
(Elsevier, 2017-01-15) Zhou, Wenhu; Ding, Jinsong; Liu, Juewen
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 3 nM 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. (C) 2016 Elsevier B.V. All rights reserved.
3D bioprinting of liver-mimetic construct with alginate/cellulose nanocrystal hybrid bioink
(Elsevier, 2018-03-01) Wu, Yun; Lin, Zhi Yuan (William); Wenger, Andrew; Tam, Kam C.; Tang, Xiaowu (Shirley)
3D bioprinting is a novel platform for engineering complex, three-dimensional (3D) tissues that mimic real ones. The development of hybrid bioinks is a viable strategy that integrates the desirable properties of the constituents. In this work, we present a hybrid bioink composed of alginate and cellulose nanocrystals (CNCs) and explore its suitability for extrusion-based bioprinting. This bioink possesses excellent shear-thinning property, can be easily extruded through the nozzle, and provides good initial shape fidelity. It has been demonstrated that the viscosities during extrusion were at least two orders of magnitude lower than those at small shear rates, enabling the bioinks to be extruded through the nozzle (100µm inner diameter) readily without clogging. This bioink was then used to print a liver-mimetic honeycomb 3D structure containing fibroblast and hepatoma cells. The structures were crosslinked with CaCl2 and incubated and cultured for 3 days. It was found that the bioprinting process resulted in minimal cell damage making the alginate/CNC hybrid bioink an attractive bioprinting material.
3D N-doped hybrid architectures assembled from 0D T-Nb2O5 embedded in carbon microtubes toward high-rate Li-ion capacitors
(Elsevier, 2019-02) Tolami Hemmati, Sahar; Li, Ge; Wang, Xiaolei; Ding, Yuanli; Pei, Yu; Yu, Aiping; Chen, Zhongwei
Herein, a unique nitrogen-doped T-Nb2O5/tubular carbon hybrid structure in which T-Nb2O5 nanoparticles are homogeneously embedded in an in-situ formed nitrogen-doped microtubular carbon is synthesized, utilizing a facile and innovative synthesis strategy. This structure addresses the poor electron conductivity and rate capability that hinder T-Nb2O5's promise as an anode for Li-ion devices. Such a distinctive structure possesses a robust framework that has ultrasmall active nanocomponents encapsulated in highly conductive carbon scaffold with hollow interior and abundant voids, enabling fast electron/ion transport and electrolyte penetration. Moreover, nitrogen-doping not only ameliorates the electronic conductivity of the heterostructure, but also induces pseudocapacitance mechanism. When evaluated in a half-cell, the as-prepared material delivers a specific capacitance of 370 F g−1 at 0.1 A g−1 within 1–3 V vs. Li/Li+ and excellent cyclability over 1100 cycles. A high energy density of 86.6 W h kg−1 and high power density of 6.09 kW kg−1 are realized. Additionally, a capacitance retention as high as 81% after 3500 cycles is achieved in an Li-ion Capacitor (LIC) with activated carbon as the cathode and nitrogen-doped T-Nb2O5/tubular carbon as the anode.
Accelerating peroxidase mimicking nanozymes using DNA
(Royal Society of Chemistry, 2015-09-07) Liu, Biwu; Liu, Juewen
DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide.
Adsorption and Desorption of DNA on Graphene Oxide Studied by Fluorescently Labeled Oligonucleotides
(American Chemical Society, 2011-03-15) Wu, Marissa; Kempaiah, Ravindra; Huang, Po-Jung Jimmy; Maheshwari, Vivek; Liu, Juewen
Being the newest member of the carbon materials family, graphene possesses many unique physical properties resulting is a wide range of applications. Recently, it was discovered that graphene oxide can effectively adsorb DNA, and at the same time, it can completely quench adsorbed fluorophores. These properties make it possible to prepare DNA-based optical sensors using graphene oxide. While practical analytical applications are being demonstrated, the fundamental understanding of binding between graphene oxide and DNA in solution received relatively less attention. In this work, we report that the adsorption of 12-, 18-, 24-, and 36-mer single-stranded DNA on graphene oxide is affected by several factors. For example, shorter DNAs are adsorbed more rapidly and bind more tightly to the surface of graphene. The adsorption is favored by a lower pH and a higher ionic strength. The presence of organic solvents such as ethanol can either increase or decrease adsorption depending on the ionic strength of the solution. By adding the cDNA, close to 100% desorption of the absorbed DNA on graphene can be achieved. On the other hand, if temperature is increased, only a small percentage of DNA is desorbed. Further, the adsorbed DNA can also be exchanged by free DNA in solution. These findings are important for further understanding of the interactions between DNA and graphene and for the optimization of DNA and graphene-based devices and sensors.
Adsorption of DNA Oligonucleotides by Titanium Dioxide Nanoparticles
(American Chemical Society, 2014-01-28) Zhang, Xu; Wang, Feng; Liu, Biwu; Kelly, Erin Y.; Servos, Mark R.; Liu, Juewen
Titanium dioxide (TiO2) or titania shows great promise in detoxification and drug delivery. To reach its full potential, it is important to interface TiO2 with biomolecules to harness their molecular recognition function. To this end, DNA attachment is an important topic. Previous work has mainly focused on long double-stranded DNA or single nucleotides. For biosensor development and targeted drug delivery, it is more important to use single-stranded oligonucleotides. Herein, the interaction between fluorescently labeled oligonucleotides and TiO2 nanoparticles is reported. The point of zero charge (PZC) of TiO2 is around 6 in water or acetate buffer; therefore, the particles are positively charged at lower pH. However, if in phosphate or citrate buffer, the particles are negatively charged, even at pH ∼2, suggesting strong adsorption of buffer anions. DNA adsorption takes place mainly via the phosphate backbone, although the bases might also have moderate contributions. Peptide nucleic acids (PNAs) with an amide backbone cannot be adsorbed. DNA adsorption is strongly affected by inorganic anions, where phosphate and citrate can strongly inhibit DNA adsorption. DNA adsorption is promoted by adding salt or lowering pH. DNA adsorption is accompanied with fluorescence quenching, and double-stranded DNA showed reduced quenching, allowing for the detection of DNA using TiO2 nanoparticles.
Adsorption of DNA onto gold nanoparticles and graphene oxide: surface science and applications
(Royal Society of Chemistry, 2012-06-28) Liu, Juewen
The interaction between DNA and inorganic surfaces has attracted intense research interest, as a detailed understanding of adsorption and desorption is required for DNA microarray optimization, biosensor development, and nanoparticle functionalization. One of the most commonly studied surfaces is gold due to its unique optical and electric properties. Through various surface science tools, it was found that thiolated DNA can interact with gold not only via the thiol group but also through the DNA bases. Most of the previous work has been performed with planar gold surfaces. However, knowledge gained from planar gold may not be directly applicable to gold nanoparticles (AuNPs) for several reasons. First, DNA adsorption affinity is a function of AuNP size. Second, DNA may interact with AuNPs differently due to the high curvature. Finally, the colloidal stability of AuNPs confines salt concentration, whereas there is no such limit for planar gold. In addition to gold, graphene oxide (GO) has emerged as a new material for interfacing with DNA. GO and AuNPs share many similar properties for DNA adsorption; both have negatively charged surfaces but can still strongly adsorb DNA, and both are excellent fluorescence quenchers. Similar analytical and biomedical applications have been demonstrated with these two surfaces. The nature of the attractive force however, is different for each of these. DNA adsorption on AuNPs occurs via specific chemical interactions but adsorption on GO occurs via aromatic stacking and hydrophobic interactions. Herein, we summarize the recent developments in studying non-thiolated DNA adsorption and desorption as a function of salt, pH, temperature and DNA secondary structures. Potential future directions and applications are also discussed.
Adsorption of Nanoceria by Phosphocholine Liposomes
(American Chemical Society, 2016-12-13) Liu, Yibo; Liu, Juewen
Nanoceria (CeO2 nanoparticle) possesses a number of enzyme-like activities. In particular, it scavenges reactive oxygen species based on in-vitro and in vivo antioxidation studies. An important aspect of fundamental physical understanding is its interaction with lipid membranes that are the main components of the cell membrane. In this work, adsorption of nanoceria onto phosphocholine (PC) liposomes was performed. PC lipids are the main constituents of the cell outer membrane. Using a fluorescence quenching assay, a nanoceria adsorption isotherm was determined at various pH values and ionic strengths. A non-Langmuir isotherm occurred at pH 4 because of lateral electrostatic repulsion among the adsorbed cationic nanoceria. The phosphate group in the PC lipid is mainly responsible for the interaction, and the adsorbed nanoceria can be displaced by free inorganic phosphate. The tendency of the system to form large aggregates is a function of pH and the concentration of nanoceria, attributable to nanoceria being positively charged at pH 4 and neutral at physiological pH. Calcein leakage tests indicate that nanoceria induces liposome leakage because of transient lipid phase transition, and cryo-transmission electron microscopy indicates that the overall shape of the liposome is retained although deformation is still observed. This study provides fundamental biointerfacial information at a molecular level regarding the interaction of nanoceria and model cell membranes.
Amplifying the Macromolecular Crowding Effect Using Nanoparticles
(American Chemical Society, 2012-01-11) Zaki, Ahmed; Liu, Juewen; Dave, Neeshma
The melting temperature (Tm) of DNA is affected not only by salt but also by the presence of high molecular weight (MW) solutes, such as polyethylene glycol (PEG), acting as a crowding agent. For short DNAs in a solution of low MW PEGs, however, the change of excluded volume upon melting is very small, leading to no increase in Tm. We demonstrate herein that by attaching 12-mer DNAs to gold nanoparticles, the excluded volume change was significantly increased upon melting, leading to increased Tm even with PEG 200. Larger AuNPs, higher MW PEGs, and higher PEG concentrations show even larger effects in stabilizing the DNA. This study reveals a unique and fundamental feature at nanoscale due to geometric effects. It also suggests that weak interactions can be stabilized by a combination of polyvalent binding and the enhanced macromolecular crowding effect using nanoparticles.
Application of a constant hole volume Sanchez–Lacombe equation of state to mixtures relevant to polymeric foaming
(Royal Society of Chemistry, 2018) Park, Chul B.; von Konigslow, Kier; Thompson, Russell B.
A variant of the Sanchez–Lacombe equation of state is applied to several polymers, blowing agents, and saturated mixtures of interest to the polymer foaming industry. These are low-density polyethylene– carbon dioxide and polylactide–carbon dioxide saturated mixtures as well as polystyrene–carbon dioxide–dimethyl ether and polystyrene–carbon dioxide–nitrogen ternary saturated mixtures. Good agreement is achieved between theoretically predicted and experimentally determined solubilities, both for binary and ternary mixtures. Acceptable agreement with swelling ratios is found with no free parameters. Up-to-date pure component Sanchez–Lacombe characteristic parameters are provided for carbon dioxide, dimethyl ether, low-density polyethylene, nitrogen, polylactide, linear and branched polypropylene, and polystyrene. Pure fluid low-density polyethylene and nitrogen parameters exhibit more moderate success while still providing acceptable quantitative estimations. Mixture estimations are found to have more moderate success where pure components are not as well represented. The Sanchez–Lacombe equation of state is found to correctly predict the anomalous reversal of solubility temperature dependence for low critical point fluids through the observation of this behaviour in polystyrene nitrogen mixtures.
Application of Pyrene Fluorescence to the Characterization of Hydrophobically Modified Starch Nanoparticles
(American Chemical Society, 2018-06-25) Kim, Damin; Amos, Ryan; Gauthier, Mario; Duhamel, Jean
Several aspects of pyrene fluorescence were applied to gain an insight into the nature of the microdomains in hydrophobically modified starch nanoparticles (HM-SNPs), prepared by reacting SNPs with propionic and hexanoic anhydride to yield C3- and C6-SNPs, respectively. The fluorescence experiments took advantage of the inherent hydrophobicity of pyrene to bind onto the hydrophobic domains generated by the HM-SNPs, and its specific response to the polarity of its environment, to probe its accessibility to quenchers such as oxygen or nitromethane dissolved in water. The equilibrium constant KB for the binding of pyrene onto HM-SNPs, the ratio (I1/I3)o describing the relative hydrophobicity of the microenvironment experienced by pyrene, its lifetime (τSNP), and the rate constant of quenching of pyrene bound to the HM-SNPs by water-soluble nitromethane (kqSNP) were determined as a function of the degree of substitution and weight fraction (wt %) of the hydrophobic modifier. The C3- and C6-SNPs yielded similar parameters at low levels of hydrophobic modification, indicating higher hydrophobicity of the modified SNPs with increasing modification level. However, SNPs modified with more than 5 wt % of hexanoyl pendants all displayed enhanced hydrophobicity for the C6-SNPs relative to the C3-SNPs. This substantial enhancement is attributed to the formation of larger hydrophobic microdomains by the hexanoyl pendants of the C6-SNPs above the 5 wt % C6-modification threshold, which did not occur with the C3-SNPs. Finally, the size of the SNPs did not appear to influence their relative hydrophobicity. These experiments demonstrate how the fluorescence of pyrene can be harnessed to provide information about the relative hydrophobicity of HM-SNPs.
Aptamer-based biosensors for biomedical diagnostics
(Royal Society of Chemistry, 2014-06-07) Zhou, Wenhu; Huang, Po-Jung Jimmy; Ding, Jinsong; Liu, Juewen
Aptamers are single-stranded nucleic acids that selectively bind to target molecules. Most aptamers are obtained through a combinatorial biology technique called SELEX. Since aptamers can be isolated to bind to almost any molecule of choice, can be readily modified at arbitrary positions and they possess predictable secondary structures, this platform technology shows great promise in biosensor development. Over the past two decades, more than one thousand papers have been published on aptamer-based biosensors. Given this progress, the application of aptamer technology in biomedical diagnosis is still in a quite preliminary stage. Most previous work involves only a few model aptamers to demonstrate the sensing concept with limited biomedical impact. This Critical Review aims to summarize progress that might enable practical applications of aptamers for biological samples. First, general sensing strategies based on the unique properties of aptamers are summarized. Each strategy can be coupled to various signaling methods. Among these, a few detection methods including fluorescence lifetime, flow cytometry, upconverting nanoparticles, nanoflare technology, magnetic resonance imaging, electronic aptamer-based sensors, and lateral flow devices have been discussed in more detail since they are more likely to work in a complex sample matrix. The current limitations of this field include the lack of high quality aptamers for clinically important targets. In addition, the aptamer technology has to be extensively tested in a clinical sample matrix to establish reliability and accuracy. Future directions are also speculated to overcome these challenges.
Aptamer-Functionalized Hydrogel Microparticles for Fast Visual Detection of Mercury(II) and Adenosine
(American Chemical Society, 2012-04-25) Helwa, Youssef; Dave, Neeshma; Froidevaux, Romain; Samadi, Azadeh; Liu, Juewen
With a low optical background, high loading capacity, and good biocompatibility, hydrogels are ideal materials for immobilization of biopolymers to develop optical biosensors. We recently immobilized mercury and lead binding DNAs within a monolithic gel and demonstrated ultrasensitive visual detection of these heavy metals. The high sensitivity was attributed to the enrichment of the analytes into the gels. The signaling kinetics was slow, however, taking about 1 h to obtain a stable optical signal because of a long diffusion distance. In this work, we aim to understand the analyte enrichment process and improve the signaling kinetics by preparing hydrogel microparticles. DNA-functionalized gel beads were synthesized using an emulsion polymerization technique and most of the beads were between 10 and 50 μm. Acrydite-modified DNA was incorporated by copolymerization. Visual detection of 10 nM Hg2+ was still achieved and a stable signal was obtained in just 2 min. The gel beads could be spotted to form a microarray and dried for storage. A new visual sensor for adenosine was designed and immobilized within the gel beads. The adenosine aptamer binds its target about 1000-fold less tightly compared to the mercury binding DNA, allowing a comparison to be made on analyte enrichment by aptamer-functionalized hydrogels.
Aqueous intercalation-type electrode materials for grid-level energy storage: Beyond the limits of lithium and sodium
(Elsevier, 2018-08-01) Xing, Zhenyu; Wang, Shun; Yu, Aiping; Chen, Zhongwei
Intermittent, fluctuational, and unpredictable features of renewable energy require grid-level energy storage (GES). Among various types of GES, aqueous electrochemical storage is undoubtedly the most promising method due to its high round-trip efficiency, long cycle life, low cost and high safety. As the most encouraging candidate for aqueous electrochemical storage, aqueous rocking-chair batteries have been heavily investigated. Recently, intercalation-type aqueous batteries beyond the limits of Li+ and Na+ have caught researchers’ attention due to potentially higher capacity and better cyclability, and the number of publications in this nascent field since 2015 has dramatically increased. Therefore, it is highly demanded to summarize what have been learned in this field. In this first comprehensive review paper, we summarize these novel intercalation-type electrode materials and provide perspectives of opportunities and challenges for future research.
Arborescent Poly(L-glutamic acid)s as Standards to Study the Dense Interior of Polypeptide Mesoglobules by Pyrene Excimer Fluorescence
(American Chemical Society, 2018-10-01) Hall, Timothy; Whitton, Greg; Casier, Remi; Gauthier, Mario; Duhamel, Jean
A series of arborescent poly(l-glutamic acid)s of generations 0 to 3 (PGA(GY) with Y = 0–3) were randomly labeled with 1-pyrenemethylamine to yield several Py-PGA(GY) constructs with pyrene contents ranging between 2.5 and 22 mol %. The density (ρ) of the interior of the PGA(GY) samples was estimated in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) by conducting gel permeation chromatography and dynamic light scattering experiments to determine their molar mass and hydrodynamic diameter, respectively. It was determined that ρ increased with the generation number from PGA(G1) to PGA(G2), which promoted more contacts between the pyrene labels. The increase in the number of pyrene–pyrene contacts was quantified with the parameter Nblob obtained by analysis of the fluorescence decays for the Py-PGA(GY) samples in DMF and DMSO. In the analysis, Nblob represented the number of structural units, i.e., glutamic acid residues, comprised inside the volume probed by an exited pyrene, termed a blob. Inside a blob, pyrene excimer formation (PEF) could occur upon diffusive encounters between an excited- and a ground-state pyrene label. It was found that Nblob increased with the generation number, and larger Nblob values were retrieved in DMSO as compared to DMF because the oligo(l-glutamic acid) (OGA) side chains in the PGA(GY) samples underwent partial loss of helicity in DMSO, which increased their hydrodynamic volume and forced the side chains closer to each other, thus resulting in more pyrene–pyrene contacts and larger Nblob values. The trends observed for Nblob in DMF as a function of the generation number could be correlated theoretically with the degree of polymerization of the OGA side chains used to prepare the PGA(GY) samples and their internal density ρ. The good agreement found between the theoretical and experimental Nblob values confirms that pyrene is an excellent probe to study the complex interior of partially structured polypeptides.
Assembly of DNA-Functionalized Gold Nanoparticles with Gaps and Overhangs in Linker DNA
(American Chemical Society, 2011-04-28) Smith, Brendan D.; Dave, Neeshma; Huang, Po-Jung Jimmy; Liu, Juewen
DNA-directed assembly of gold nanoparticles (AuNPs) has been extensively studied because of its important applications in analytical chemistry, materials science, and nanomedicine. In a typical system, two DNA-functionalized AuNPs are assembled via a linker DNA to form large aggregates. In the majority of the previous reports, the linker DNA is fully base paired with no gaps or overhangs present. Introducing such nonbase-paired regions in the linker DNA has been recently shown to be important for making stimuli-responsive materials and in crystallization of such AuNPs. In this work, we systematically studied the effect of introducing gaps and overhangs in the linker DNA to understand the kinetics of assembly and the melting transition of these aggregates. We found that the assembly kinetics decreased with increasing linker DNA length. The melting temperature decreased with the loss of base stacking by introducing gaps as well as the steric effect of overhangs. Additional insights were obtained by measuring the melting curves of the free DNAs in the absence of AuNPs. For example, it appeared that DNA base stacking at the nick site was favored in assembled nanoparticles compared to that in free DNA. Our results indicate that, while it is possible to form AuNP assemblies with linker DNAs containing various types of unpaired regions, these kinetic and thermodynamic factors need to be considered when designing related sensors and materials.
Assembly of DNA-Functionalized Nanoparticles in Alcoholic Solvents Reveals Opposite Thermodynamic and Kinetic Trends for DNA Hybridization
(American Chemical Society, 2010-05-12) Smith, Brendan D.; Liu, Juewen
DNA has been a key molecule in biotechnology and nanotechnology. To date, the majority of the experiments involving DNA have been performed in aqueous solutions, which may be related to the perception that DNA hybridization is slower and less stable in organic solvents. All studies on the effect of organic solvents have focused on thermodynamic properties such as DNA melting temperature and the B-to-A form transition for very long DNAs, but not on the hybridization kinetics of short synthetic DNAs. We employed DNA-functionalized gold nanoparticles (AuNPs) as a model system and found that if the alcohol content is less than ∼30%, more alcohol leads to a faster DNA hybridization, although with a decreased melting temperature. The generality of this observation was independently verified with two molecular beacon systems (in the absence of AuNPs) using fluorophore and quencher-labeled DNAs. With 25% ethanol, the hybridization rates are three to four times faster than in the case with water. This discovery will extend the application of DNA bio- and nanotechnology to organic solvents with improved performance.
Attaching DNA to Nanoceria: Regulating Oxidase Activity and Fluorescence Quenching
(American Chemical Society, 2013-08-14) Pautler, Rachel; Kelly, Erin Y.; Huang, Po-Jung Jimmy; Cao, Jing; Liu, Biwu; Liu, Juewen
Cerium oxide nanoparticles (nanoceria) have recently emerged as a nanozyme with oxidase activity. In this work, we present a few important interfacial properties of nanoceria. First, the surface charge of nanoceria can be controlled not only by adjusting pH but also by adsorption of simple inorganic anions. Adsorption of phosphate and citrate gives negatively charged surface over a broad pH range. Second, nanoceria adsorbs DNA via the DNA phosphate backbone in a sequence-independent manner; DNA adsorption inhibits its oxidase activity. Other anionic polymers display much weaker inhibition effects. Adsorption of simple inorganic phosphate does not have the inhibition effect. Third, nanoceria is a quencher for many fluorophores. These discoveries provide an important understanding for further use of nanoceria in biosensor development, materials science, and nanotechnology.
Biochemical Characterization of a Lanthanide-Dependent DNAzyme with Normal and Phosphorothioate-Modified Substrates
(American Chemical Society, 2015-10-06) Vazin, Mahsa; Huang, Po-Jung Jimmy; Matuszek, Zaneta; Liu, Juewen
A trivalent lanthanide (Ln3+)-dependent RNA-cleaving DNAzyme, Ce13d, was recently isolated via in vitro selection. Ce13d is active in the presence of all Ln3+ ions. Via introduction of a single phosphorothioate (PS) modification at the cleavage site, its activity with Ln3+ decreases while all thiophilic metals can activate this DNAzyme. This property is unique to Ce13d and is not found in many other tested DNAzymes. This suggests the presence of a well-defined but general metal binding site. Herein, a systematic study of Ce13d with the PO substrate (using Ce3+) and the PS substrate (using Cd2+) is performed. In both the PO and PS systems, the highest activity was with ∼10 μM metal ions. Higher concentrations of Ce3+ completely inhibit the activity, while Cd2+ only slows the activity. A comparison of different metal ions suggests that the role of metal is to neutralize the phosphate negative charge. Both systems follow a similar pH–rate profile with a single deprotonation step, indicating similar reaction mechanisms. The activity difference between the Rp and Sp form of the PS substrate is <10-fold, which is much smaller than most known RNA-cleaving enzymes. Mutation studies identified eight highly conserved purines, among which the two adenines play mainly structural roles, while the guanines are likely to be involved in metal binding. Ce13d can serve as a model system for further understanding of DNAzyme biochemistry and bioinorganic chemistry.
Biomimetic sensing based on chemically induced assembly of a signaling DNA aptamer on a fluid bilayer membrane
(Royal Society of Chemistry, 2012-04-18) Dave, Neeshma; Liu, Juewen
The adenosine aptamer was split into two halves and linked to a fluid liposome surface; addition of adenosine resulted in aptamer assembly, which did not occur if the split aptamer was attached to silica nanoparticles, demonstrating the feasibility of using aptamer probes to study diffusion within lipid membranes.

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