Two Completely Different Mechanisms for Highly Specific Na+ Recognition by DNAzymes

Our view of the interaction between Na⁺ and nucleic acids was changed by a few recently discovered Na⁺‐specific RNA‐cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na⁺ binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140‐fold, and it cannot be rescued by thiophilic Cd²⁺ or Mn²⁺, whereas the activity of PS‐modified Ce13d can be rescued. Na⁺‐dependent activity assays indicate that two Na⁺ ions bind cooperatively in EtNa, and each Na⁺ likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na⁺ ion in a well‐defined Na⁺ aptamer, and this Na⁺ ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH–rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na⁺ fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na⁺ binding is detected by 2‐aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na⁺ mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well‐defined aptamer for Na⁺ binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na⁺ binding.     

Stimuli‐sensitive nanostructured poly(sodium 4‐styrene sulfonate): Synthesis,characterization, and study of metal ion retention properties

Stimuli‐sensitive polymers are a type of smart polymers having the capability to change their configuration or properties under adequate stimuli as heat, pH, magnetic field, mechanical strength, among other. The aim of this work was to synthesize nanostructured polymers with antibacterial properties capable to change their retention properties of divalent metal ions by external stimuli (pH and ionic strength). For that, a polymerizable nanostructured crosslinker (PNC) based on silver nanoparticles (AgNPs) and acrylic acid was synthesized. Later, NPSS was synthesized by free‐radical polymerization, characterized by different analytical techniques and its retention properties of divalent ions (Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺) were studied at different pHs and ionic strengths (5.0, 7.0, and 9.0; and 0.0, 0.5, and 1.5% NaCl, respectively). It was evidenced that AgNPs can be synthesized using acrylic acid as stabilizing agent, and later, be used for synthesis of NPSS by free‐radical polymerization. For NPSS, metal ion retention decreases as pH is increased; in addition, results suggest that the electrostatic interaction is not the only determining factor in the retention of ions. Other possible factors which would be affecting the retention are: water flow by swelling capacity and water flow by osmotic stress resulting of high salt concentration. NPSS showed antimicrobial activity against Escherichia coli and Staphylococcus aureus which was enhanced by incorporation of PNC based on AgNPs. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46001.     

An in Vitro–Selected DNAzyme Mutant Highly Specific for Na+ under Slightly Acidic Conditions

Sodium is one of the most common metal ions in biology; however, DNA-based sodium probes have only been reported recently. A Na⁺-specific RNA-cleaving DNAzyme named NaA43 is active with Na⁺ alone. In this work, we were using Co(NH₃)₆³⁺ as the intended metal cofactor for in vitro selection, but obtained a mutant of the NaA43 DNAzyme. The mutant was named NaH1, and differs from NaA43 by only two nucleotides. NaA43 has an optimal pH of 7.0, whereas the optimal pH for NaH1 is 6.0. This difference might be due to our selection having been performed at pH 6.0. NaH1 also displays an excellent selectivity for sodium relative to other competing monovalent ions, as well as a fast catalytic rate of (0.11±0.01) min⁻¹ with 50 mm Na⁺. At low Na⁺ concentrations, the selected DNAzyme exhibited a higher cleavage rate than NaA43 and thus a tighter apparent K_d of (12.0±1.6) mm Na⁺. Furthermore, the NaH1 DNAzyme was engineered into a fluorescent Na⁺ biosensor by attaching a fluorophore/quencher pair to the DNAzyme with a detection limit of 223 μm Na⁺. Preliminary work on detection of Na⁺ in serum was demonstrated as well. This study provides a useful mutant that works in a slightly acidic environment, which might be useful for sensing Na⁺ in acidic in vivo environments.     

Enzymatic recognition of 2'-modified ribonucleoside 5'-triphosphates: towards the evolution of versatile aptamers

The quest for effective, selective and nontoxic nucleic-acid-based drugs has led to designing modifications of naturally occurring nucleosides. A number of modified nucleic acids have been made in the past decades in the hope that they would prove useful in target-validation studies and therapeutic applications involving antisense, RNAi, aptamer, and ribozyme-based technologies. Since their invention in the early 1990s, aptamers have emerged as a very promising class of therapeutics, with one drug entering the market for the treatment of age-related macular degeneration. To combat the limitations of aptamers containing naturally occurring nucleotides, chemically modified nucleotides have to be used. In order to apply modified nucleotides in aptamer drug development, their enzyme-recognition capabilities must be understood. For this purpose, several modified nucleoside 5'-triphosphates were synthesized and investigated as substrates for various enzymes. Herein, we review studies on the enzyme-recognition of various 2'-sugar-modified NTPs that were carried out with a view to their effective utilization in SELEX processes to generate versatile aptamers.     

Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences. Modifications, including LNA monomers with locked sugar residues, were incorporated into the substrates to stabilize their structures. In functional substrates, the A2058 methylation target (on the 13- to 19-nucleotide top strand) was displayed in an unpaired sequence immediately following a conserved irregular helix, and these are the specific structural features recognized by Erm. Erm methylation was enhanced by stabilizing the top-strand conformation with an LNA residue at G2056. The bottom strand (nine to 19 nucleotides in length) was required for methylation and was still functional after extensive modification, including substitution with a DNA sequence. Although it remains possible that Erm makes some unspecific contact with the bottom strand, the main role played by the bottom strand appears to be in maintaining the conformation of the top strand. The addition of multiple LNA residues to the top strand impeded methylation; this indicates that the RNA substrate requires a certain amount of flexibility for accommodation into the active site of Erm. The combinatorial approach for identifying small but functional RNA substrates is a step towards making RNA-Erm complexes suitable for cocrystal determination, and for designing molecules that might block the substrate-recognition site of the enzyme.     

Characterization of Structure and Dynamics of the Guanidine-II Riboswitch from Escherichia coli by NMR Spectroscopy and Small-Angle X-ray Scattering (SAXS)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg²⁺ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg²⁺ and ligand binding.     

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 K_d of ～20 mm with Na⁺ (other monovalent cations: 40–60 mm ). 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 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.     

Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.     

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.     

An evaluation of multiple binary interaction parameters in cubic equations of state

Three cubic equations of state are utilized in regressing a large vapour-liquid equilibrium data bank using single and multiple binary interaction parameters. A second binary interaction parameter is shown to be both physically significant and useful in improving the match of experimental data, particularly in polar systems. The use of a third binary interaction parameter in the Trebble-Bishnoi equation of state is found to be of value in systems with large molecular size differences.     

Electronic Structure and Properties of the O2+2, SO2+ and S2+2 Diatomic Dications

Multiply charged ions show novel energetic features which can potentially have useful photophysical properties. In order to investigate these interesting type systems, energy interaction curves for the ground and excited electronic states of O²⁺₂, SO²⁺, and S²⁺₂ have been generated by the ab initio complete active space multiconfiguration self-consistent field (CAS-MCSCF) method. The calculations were carried out in a triple-zeta sp plus double-zeta d Gaussian function basis set using compact effective potentials to replace the core electrons. In order to gauge the accuracy of the results, analogous calculations were carried out on the valence isoelectronic N₂ and NO⁺ systems for which experimental information is available for comparison of geometric and spectroscopic properties. Diverse high energy spectroscopy experiments on O²⁺₂ are interpreted and reconciled using the calculated ground and excited state energy interaction curves. Special attention is paid to the ₃Σ₊^u state, whose location and character have been particularly puzzling. Almost all the dication molecular states studied show the characteristic avoided crossing of diabatic states which gives a trapped equilibrium structure and a barrier to dissociation. These features make the dication kinetically stable, but thermodynamically unstable with an exothermic dissociation energy. The spectroscopy experiments on O²⁺₂ show that these states are experimentally attainable.     

Probing Local Folding Allows Robust Metal Sensing Based on a Na+-Specific DNAzyme

Fluorescent metal sensors based on DNA often rely on changes in end-to-end distance or local environmental near fluorophore labels. Because metal ions can also nonspecifically interact with DNA through various mechanisms, such as charge screening, base binding, and increase or decrease in duplex stability, robust and specific sensing of metal ions has been quite challenging. In this work, a side-by-side comparison of two signaling strategies on a Na⁺-specific DNAzyme that contained a Na⁺-binding aptamer was performed. The duplex regions of the DNAzyme was systematically shortened and its effect was studied by using a 2-aminopurine (2AP)-labeled substrate strand. Na⁺ binding affected the local environmental of the 2AP label and increased its fluorescence. A synergistic process of Na⁺ binding and forming the duplex on the 5′-end of the enzyme strand was observed, and this end was close to the aptamer loop. Effective Na⁺ binding was achieved with a five base-pair stem. The effect on the 3′-end is more continuous, and the stem needs to form first before Na⁺ can bind. With an optimized substrate binding arm, a FRET-based sensor has been designed by labeling the two ends of a cis form of the DNAzyme with two fluorophores. In this case, Na⁺ failed to show a distinct difference from that of Li⁺ or K⁺; thus indicating that probing changes to the local environment allows more robust sensing of metal ions.     

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.     

Use of electrodialysis to remove silver ions frommodel solutions and wastewater

The feasibility of the removal of Ag⁺ ions in a model solution and a sample of rinse water provided from an industrial plating plantwas investigated in a batch electrodialysis system. The experiments were carried out using two different types of ion-exchange membranes. The effects of applied potential, pH value and initial silver concentration on the duration of electrodialysis and energy consumption were examined. Full removal of Ag+ ions was achieved from model solutions and the sample of rinse water. The most convenient applied voltage and energy consumption values to remove silver ions are reported. These results will be useful for designing and operating different capacities of electrodialysis plants for recovering Ag⁺ ions.     

Recovery of Zinc,Cadmium, and Lanthanum by Biopolymer Gel Particles of Alginic Acid

Abstract

Biopolymer gel particles of alginic acid were found to be a useful material for recovering zinc, cadmium, and lanthanum from aqueous solutions. The metals sorbed by the gel particles could be completely eluted by using dilute HCl solution of 0.1 kmol/m³. The distribution ratios of the individual metals between the gel and liquid phases were measured by using a batch method. The equilibrium data were consistent with predictions made assuming that sorption takes place with the ion-exchange reaction between metal ions and alginic acid. The maximum sorption capacity of the gel particles and the distribution equilibrium constants for the metals were determined by comparing the experimental data with the theoretical predictions. The observed effect of temperature on the distribution equilibrium was insignificant in the range from 15 to 35°C.     

Zn2+-Dependent DNAzymes: From Solution Chemistry to Analytical,Materials and Therapeutic Applications

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.     

An Efficient Lanthanide‐Dependent DNAzyme Cleaving 2′–5′‐Linked RNA

RNA can form two types of linkage. In addition to the predominant 3′–5′ linkage, 2′–5′‐linked RNA is also important in biology, medicine, and prebiotic studies. Here, in vitro selection was used to isolate a DNAzyme that specifically cleaves 2′–5′ RNA by using Ce³⁺ as the metal cofactor, but leaves the 3′–5′ counterpart intact. This Ce5 DNAzyme requires trivalent light lanthanide ions and shows a rate of 0.16 min^?1 in the presence of 10 μm Ce³⁺; the activity decreases with heavier lanthanide ions. This is the fastest DNAzyme reported for this reaction, and it might enable applications in chemical biology. As a proof‐of‐concept, using this DNAzyme, the reactions between phosphorothioate‐modified RNA and strongly thiophilic metals (Hg²⁺ and Tl³⁺) were studied as a function of pH.     

Key role of hydrates in determining ion rejection by polyamide membrane

Understanding the mechanism of ion rejection in the separation process remains a major hurdle in designing new membrane materials for seawater desalination. Here, we investigate the effects of water and aromatic polyamide (PA) membrane on the rejection of salt ions, such as Fe²⁺, Na⁺, and Cl^?, by molecular dynamic simulations. Results demonstrate that water plays a key role in salt ion rejection because salt ions preferentially interact with water molecules to form hydrates with multishell structures, which aids the rejection of various salt ions. For FeCl₂ aqueous solution, the innermost shell water molecules of a Fe²⁺ hydrate can hardly be replaced by other substances in their molecular dynamics simulations, so ions would be highly rejected if the effective diameter of a water channel (EDC) in the membrane is smaller than that of the innermost shell of a hydrate. However, the water molecules in the second water shell can be replaced by the atoms of the membrane when the EDC is smaller than the size of the second shell (about 9 Å). Besides, although the sizes of these ions approximate to those of water molecules, the rejection level of PA membrane to salt ions is higher than that to water, which is accounted for by the formation of hydrate. POLYM. ENG. SCI., 55:466–473, 2015. © 2014 Society of Plastics Engineers     

Poly(vinyl alcohol)/polyethyleneimine (PVA/PEI) blended monolithic cryogel columns for the depletion of haemoglobin from human blood

We have synthesized PVA/PEI monolithic cryogel columns chelated with Cu²⁺ ions as a model adsorbent, which is capable of binding haemoglobin (Hb) from human blood. The goal of this study is to perform the depletion of Hb via a single and easy process to be useful in proteomic studies. PVA/PEI-Cu²⁺ cryogel columns were subjected to adsorption studies of Hb from both aqueous solution and human plasma to evaluate the extent of interaction between cryogel columns and Hb. The effects of experimental parameters, such as pH, Hb equilibrium concentration, adsorption time, temperature, and ionic strength, on Hb adsorption capacity were investigated.     

Salt-Toggled Capture Selection of Uric Acid Binding Aptamers

Uric acid is the end-product of purine metabolism in humans and an important biomarker for many diseases. To achieve the detection of uric acid without using enzymes, we previously selected a DNA aptamer for uric acid with a K_d of 1 μM but the aptamer required multiple Na⁺ ions for binding. Saturated binding was achieved with around 700 mM Na⁺ and the binding at the physiological condition was much weaker. In this work, a new selection was performed by alternating Mg²⁺-containing buffers with Na⁺ and Li⁺. After 13 rounds of selection, a new aptamer sequence named UA-Mg-1 was obtained. Isothermal titration calorimetry confirmed aptamer binding in both selection buffers, and the K_d was around 8 μM. The binding of UA-Mg-1 to UA required only Mg²⁺. This is an indicator of successful switching of metal dependency via the salt-toggled selection method. The UA-Mg-1 aptamer was engineered into a fluorescent biosensor based on the strand-displacement assay with a limit of detection of 0.5 μM uric acid in the selection buffer. Finally, comparison with the previously reported Na⁺-dependent aptamer and a xanthine/uric acid riboswitch was also made.