Tuning Riboswitch‐Mediated Gene Regulation by Rational Control of Aptamer Ligand Binding Properties

Riboswitch‐mediated control of gene expression depends on ligand binding properties (kinetics and affinity) of its aptamer domain. A detailed analysis of interior regions of the aptamer, which affect the ligand binding properties, is important for both understanding natural riboswitch functions and for enabling rational design of tuneable artificial riboswitches. Kinetic analyses of binding reaction between flavin mononucleotide (FMN) and several natural and mutant aptamer domains of FMN‐specific riboswitches were performed. The strong dependence of the dissociation rate (52.6‐fold) and affinity (100‐fold) on the identities of base pairs in the aptamer stem suggested that the stem region, which is conserved in length but variable in base‐pair composition and context, is the tuning region of the FMN‐specific aptamer. Synthetic riboswitches were constructed based on the same aptamer domain by rationally modifying the tuning regions. The observed 9.31‐fold difference in the half‐maximal effective concentration (EC₅₀) corresponded to a 11.6‐fold difference in the dissociation constant (K_D) of the aptamer domains and suggested that the gene expression can be controlled by rationally adjusting the tuning regions.     

Optimization of Experimental Parameters to Explore Small‐Ligand/Aptamer Interactions through Use of 1H NMR Spectroscopy and Molecular Modeling

Aptamers constitute an emerging class of molecules designed and selected to recognize any given target that ranges from small compounds to large biomolecules, and even cells. However, the underlying physicochemical principles that govern the ligand‐binding process still have to be clarified. A major issue when dealing with short oligonucleotides is their intrinsic flexibility that renders their active conformation highly sensitive to experimental conditions. To overcome this problem and determine the best experimental parameters, an approach based on the design‐of‐experiments methodology has been developed. Here, the focus is on DNA aptamers that possess high specificity and affinity for small molecules, L ‐tyrosinamide, and adenosine monophosphate. Factors such as buffer, pH value, ionic strength, Mg²⁺‐ion concentration, and ligand/aptamer ratio have been considered to find the optimal experimental conditions. It was then possible to gain new insight into the conformational features of the two ligands by using ligand‐observed NMR spectroscopic techniques and molecular mechanics.     

SAM‐II Riboswitch Samples at least Two Conformations in Solution in the Absence of Ligand: Implications for Recognition

Conformational equilibria are increasingly recognized as pivotal for biological function. Traditional structural analyses provide a static image of conformers in solution that sometimes present conflicting views. From ¹³C and ¹H chemical exchange saturation transfer experiments, in concert with ligation and selective labeling strategies, we show that in the absence of metabolite, a Mg²⁺ (0–0.5 mm )‐bound apo SAM‐II riboswitch RNA exists in a minor (≈10 %) partially closed state that rapidly exchanges with a predominantly (≈90 %) open form with a lifetime of ≈32 ms. The base and sugar (H6,C6, H1′,C1′) chemical shifts of C43 for the dominant conformer are similar to those of a free CMP, but those of the minor apo species are comparable to shifts of CMPs in helical RNA regions. Our results suggest that these transient, low populated states stabilized by Mg²⁺ will likely enhance rapid ligand recognition and, we anticipate, will play potentially ubiquitous roles in RNA signaling.     

Optofluidics-based DNA structure-competitive aptasensor for rapid on-site detection of lead(II) in an aquatic environment

Lead ions (Pb²⁺), ubiquitous and one of the most toxic metallic pollutants, have attracted increasing attentions because of their various neurotoxic effects. Pb²⁺ has been proven to induce a conformational change in G-quadruplex (G4) aptamers to form a stabilizing G4/Pb²⁺ complex. Based on this principle, an innovative optofluidics-based DNA structure-competitive aptasensor was developed for Pb²⁺ detection in an actual aquatic environment. The proposed sensing system has good characteristics, such as high sensitivity and selectivity, reusability, easy operation, rapidity, robustness, portability, use of a small sample volume, and cost effectiveness. A fluorescence-labeled G4 aptamer was utilized as a molecular probe. A DNA probe, a complementary strand of G4 aptamer, was immobilized onto the sensor surface. When the mixture of Pb²⁺ solution and G4 aptamer was introduced into the optofluidic cell, Pb²⁺ and the DNA probe bound competitively with the G4 aptamer. A high Pb²⁺ concentration reduced the binding of the aptamer and the DNA probe; thus, a low-fluorescence signal was detected. A sensitive sensing response to Pb²⁺ in the range of 1.0–300.0 nM with a low detection limit of 0.22 nM was exhibited under optimal conditions. The potential interference of the environmental sample matrix was assessed with spiked samples, and the recovery of Pb²⁺ ranged from 80 to 105% with a relative standard deviation value of <8.5%. These observations clearly illustrate that with the use of different DNA or aptamer probes, the sensing strategy presented can be easily extended to the rapid on-site monitoring of other trace analytes.     

Comparison of solution and crystal structures of preQ1 riboswitch reveals calcium-induced changes in conformation and dynamics

Riboswitches regulate gene expression via specific recognition of cognate metabolites by their aptamer domains, which fold into stable conformations upon ligand binding. However, the recently reported solution and crystal structures of the Bacillus subtilis preQ(1) riboswitch aptamer show small but significant differences, suggesting that there may be conformational heterogeneity in the ligand-bound state. We present a structural and dynamic characterization of this aptamer by solution NMR spectroscopy. The aptamer-preQ(1) complex is intrinsically flexible in solution, with two regions that undergo motions on different time scales. Three residues move in concert on the micro-to-millisecond time scale and may serve as the lid of the preQ(1)-binding pocket. Several Ca(2+) ions are present in the crystal structure, one of which binds with an affinity of 47 ± 2 μM in solution to a site that is formed only upon ligand binding. Addition of Ca(2+) to the aptamer-preQ(1) complex in solution results in conformational changes that account for the differences between the solution and crystal structures. Remarkably, the Ca(2+) ions present in the crystal structure, which were proposed to be important for folding and ligand recognition, are not required for either in solution.     

Interaction study of a lysozyme‐binding aptamer with mono‐ and divalent cations by ACE

Binding between an aptamer and its target is highly dependent on the conformation of the aptamer molecule, this latter seeming to be affected by a variety of cations. As only a few studies have reported on the interactions of monovalent or divalent cations with aptamers, we describe herein the use of ACE in its mobility shift format for investigating interactions between various monovalent (Na⁺, K⁺, Cs⁺) or divalent (Mg²⁺, Ca²⁺, Ba²⁺) cations and a 30‐mer lysozyme‐binding aptamer. This study was performed in BGEs of different natures (phosphate and MOPS buffers) and ionic strengths. First, the effective charges of the aptamer in 30 mM ionic strength phosphate and MOPS (pH 7.0) were estimated to be 7.4 and 3.6, respectively. Then, corrections for ionic strength and counterion condensation effects were performed for all studies. The effective mobility shift was attributed not only to these effects, but also to a possible interaction with the buffer components (binary or ternary complexes) as well as possible conformational changes of the aptamer. Finally, apparent binding constants were calculated for divalent cations with mathematical linearization methods, and the influence of the nature of the BGE was evidenced.     

Conformational Changes in Calcium‐Sensor Proteins under Molecular Crowding Conditions

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca²⁺ sensors, which undergo conformational changes in response to oscillating intracellular Ca²⁺ concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca²⁺‐binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase‐activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca²⁺‐sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca²⁺ conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size‐exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein–water interface. Our study provides insight into how rather small signaling proteins that have very similar three‐dimensional folding patterns differ in their Ca²⁺‐occupied functional state under crowding conditions.     

Magnesium-Free Immobilization of DNA Origami Nanostructures at Mica Surfaces for Atomic Force Microscopy

DNA origami nanostructures (DONs) are promising substrates for the single-molecule investigation of biomolecular reactions and dynamics by in situ atomic force microscopy (AFM). For this, they are typically immobilized on mica substrates by adding millimolar concentrations of Mg²⁺ ions to the sample solution, which enable the adsorption of the negatively charged DONs at the like-charged mica surface. These non-physiological Mg²⁺ concentrations, however, present a serious limitation in such experiments as they may interfere with the reactions and processes under investigation. Therefore, we here evaluate three approaches to efficiently immobilize DONs at mica surfaces under essentially Mg²⁺-free conditions. These approaches rely on the pre-adsorption of different multivalent cations, i.e., Ni²⁺, poly-l-lysine (PLL), and spermidine (Spdn). DON adsorption is studied in phosphate-buffered saline (PBS) and pure water. In general, Ni²⁺ shows the worst performance with heavily deformed DONs. For 2D DON triangles, adsorption at PLL- and in particular Spdn-modified mica may outperform even Mg²⁺-mediated adsorption in terms of surface coverage, depending on the employed solution. For 3D six-helix bundles, less pronounced differences between the individual strategies are observed. Our results provide some general guidance for the immobilization of DONs at mica surfaces under Mg²⁺-free conditions and may aid future in situ AFM studies.     

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

Sensitive and selective detection of Pb²⁺ is of great importance to both human health and environmental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb²⁺ in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb²⁺. It was found that the aptamer probe had a high FA in the absence of Pb²⁺. This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb²⁺ to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb²⁺. Indeed, we observed a decrease in FA of aptamer probe upon Pb²⁺ binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb²⁺. Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb²⁺ in homogeneous solution. The change in FA showed a linear response to Pb²⁺ from 10 nM to 2.0 μM, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb²⁺ over other metal ions. The method is simple, quick and inherits the advantages of aptamer and FA.     

Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum

A rapid and cost-effective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in serum has been optimized and validated. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassays. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3′-end on aptamer and at neutravidin plate. Various factors such as incubation temperature, divalent ion – Mg²⁺ ion and treatment of serum solution were evaluated for the performance of ELAA. The aptamer was incubated for 1 h at 4 °C in the assay buffer containing 5 mM Mg²⁺ ion, and serum was diluted (1:9, serum:assay buffer) and filtrated through a 3 kDa dialysis membrane to extract the proteins present in the serum. Assay was performed with 0.01 μg mL⁻¹ of aptamer and 1.205 × 10⁻⁷ M DA-HRP conjugate using the optimized method. A dose–response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 1.0 × 10⁻¹² M and four orders (1.0 × 10⁻⁷ M to 5.0 × 10⁻¹¹ M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown a good agreement with the correlation coefficient (R² = 0.9872): Abs. (in serum) = 0.9612 × Abs. (in buffer) − 0.0556. The cross-reactivity evaluation demonstrated that norepinephrine showed some cross-reactivity (3.68%) whereas 3-methoxytyramine, epinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid showed almost no cross-reactivity (<1%). Percent recoveries of DA in serum were quite satisfactory (∼95%). This paper describes usefulness of the aptamer assay in monitoring DA in human serum.     

Electrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity

Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

Effect of Mg2+ Ions on the Stability of PolyA/2PolyU Three-Stranded Helices in Aqueous Solutions

The influence of Mg²⁺, Na⁺ and temperature on the conformational state of three-stranded helical polyA/2polyU (A2U) has been studied by the thermal denaturation method. At Na⁺ concentrations of 0.01–0.1 M , on heating the transition A2U→AU+U (the 3→2 transition) and then AU→A+U transition (the 2→1 transition) are observed. (AU is double helix polyA/polyU; A and U are single-stranded polyA and polyU, respectively.) With 0.01 M and 0.03 M Na⁺ these transitions occur at Mg²⁺ concentrations within (0 ÷ 0.003) M . At these ionic concentrations, there is a narrow temperature region (3 ÷ 5°C) at which double-helical AU formed by the 3→2 transition is resistant to heating. In 0.1 M Na⁺, a rise in the Mg²⁺ concentration leads to a continuous decrease in the temperature range of this region, and above a critical concentration of Mg²⁺ (ca. 3.6×10^–5 M )_cr there is only one transition (the 3→1 transition) instead of the successive transitions 3→2→1. The constants of Mg²⁺ ion association with polyU, polyA and A2U were calculated using equilibrium binding theory. The data obtained helped explain the reasons for the different phase diagrams for A2U + Mg²⁺ complexes in solution at high and low Na⁺ concentrations.     

Cation Complexation by a Lower Rim Calix(4)arene Derivative: Structural,Electrochemical and Thermodynamic Studies

The synthesis and characterization (¹H and ¹³C NMR) of a partially substituted lower rim p-tert-butylcalix(4)arene, namely, 5,11,17,23-tetra-4-tert-butyl-25,27-bis(diethylphosphate amino)ethoxy-26,28-dihydroxycalix[4]arene (1), are reported. The solution thermodynamics of the ligand in a variety of solvents at 298.15?K was investigated through solubility (hence standard Gibbs energy of solution) measurements while the calorimetric technique was used to derive the standard solution enthalpy. These data were used to calculate the standard entropy of solution. An enthalpy–entropy compensation effect is shown and, as a result, slight variations are observed in the transfer Gibbs energies of this ligand from the reference to other solvents. ¹H NMR, conductance and calorimetric measurements were carried out to establish the degree of interaction of the ligand with univalent (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺ and Ag⁺) and bivalent (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Pb²⁺, Cd²⁺, Hg²⁺, Cu²⁺, Zn²⁺) cations in acetonitrile, methanol, N,N-dimethylformamide and propylene carbonate. No complexation was found between this ligand and univalent cations in these solvents. As far as the bivalent cations are concerned, interaction between 1 and these cations was found only in acetonitrile. The versatile behaviour of this ligand with bivalent cations in this solvent is reflected by the formation of complexes of different stoichiometry. Thus the interaction of 1 with alkaline-earth (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) and Pb²⁺ metal cations leads to the formation of 1:2 (cation:ligand) complexes. However, for other bivalent metal cations (Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺) the complex stoichiometry was found to be 1:1. The results are discussed in terms of the key role played by acetonitrile in processes involving calix[4]arene derivatives.     

Metal‐Ion‐Mediated Tuning of Duplex DNA Binding by Bis(2‐(2‐pyridyl)‐1H‐benzimidazole)

Studies of double‐stranded‐DNA binding have been performed with three isomeric bis(2‐(n‐pyridyl)‐1H‐benzimidazole)s (n=2, 3, 4). Like the well‐known Hoechst 33258, which is a bisbenzimidazole compound, these three isomers bind to the minor groove of duplex DNA. DNA binding by the three isomers was investigated in the presence of the divalent metal ions Mg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺. Ligand–DNA interactions were probed with fluorescence and circular dichroism spectroscopy. These studies revealed that the binding of the 2‐pyridyl derivative to DNA is dramatically reduced in the presence of Co²⁺, Ni²⁺, and Cu²⁺ ions and is abolished completely at a ligand/metal‐cation ratio of 1:1. Control experiments done with the isomeric 3‐ and 4‐pyridyl derivatives showed that their binding to DNA is unaffected by the aforementioned transition‐metal ions. The ability of 2‐(2‐pyridyl)benzimidazole to chelate metal ions and the conformational changes of the ligand associated with ion chelation probably led to such unusual binding results for the ortho isomer. The addition of ethylenediaminetetraacetic acid (EDTA) reversed the effects completely.     

Mg-induced vesicles of tetradecyldimethylamine oxide and magnesium dodecyl sulfate

A Mg²⁺-induced vesicle phase was prepared from a mixture of tetradecyldimethylamine oxide (C₁₄DMAO) and magnesium dodecyl sulfate [Mg(DS)₂] in aqueous solution. Study of the phase behavior shows that at the appropriate mixing ratios, Mg²⁺–ligand coordination between C₁₄DMAO and Mg(DS)₂ results in the formation of molecular bilayers, in which Mg²⁺ can firmly bind to the head groups of the two surfactants. The area of the head group can be reduced because of the complexation. In this case, no counterions exist in aqueous solution because of the fixation of Mg²⁺ ions to the bilayer membranes. Therefore, the charges of the bilayer membranes are not shielded by salts. The birefringent solutions of Mg(DS)₂ and C₁₄DMAO mixtures consist of vesicles which were determined by transmission electron microscopy (TEM) images and rheological measurements. Magnesium oxide (MgO) nanoplates were obtained via the decomposition of Mg(OH)₂ which were synthesized in Mg²⁺-induced vesicle phase which was used as the microreactor under the existence of ammonia hydroxide. The morphologies and structures of the obtained MgO nanoplates have been characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The results indicate that the crystal growth is along the (1 1 1) direction which can be affected by the presence of a vesicle phase having a fixation of Mg²⁺ ions to the bilayer membranes.     

A novel resonance Rayleigh scattering aptasensor for dopamine detection based on an Exonuclease III assisted signal amplification by G - quadruplex nanowires formation

A new method was constructed for detecting dopamine based on aptamer-specific recognition and resonance Rayleigh scattering (RRS) of G - quadruplex nanowires (G - wires). The dopamine aptamer was used to recognize the target of dopamine, and the Exonuclease III was applied to cleave the hairpin DNA, and the G - wire formation was induced in the presence of K⁺ and Mg²⁺. This phenomenon was confirmed by polyacrylamide gel electrophoresis. Thus, a quantitative relationship between the RRS intensity of the G - wires and the dopamine concentration was established. The experimental conditions were optimized, such as the concentration of Mg²⁺, reaction temperature, reaction time and the concentration of Exonuclease III in the reaction system, and the interference substances were investigated, such as uric acid, ascorbic acid and serotonin. Under the optimal conditions, there was a good linear relationship between the RRS and the logarithm of dopamine concentration in the range from 5.0 × 10^-11 M to 1.0 × 10^-8 M (r = 0.995), with a detection limit of 1.2 × 10^-11 M. The novel method for dopamine detection showed excellent selectivity and high sensitivity, and could be used to detect dopamine in mice brain tissues.     

Calculation of the equilibrium constants and binding cooperativity parameters of ammonia with Group II metal cations in solution

The matrix model was used to analyze the distribution diagrams and formation functions of ammonia complexes [M(NH₃) _n ]²⁺ (n = 0−4) of Group II metal ions (Mg²⁺, Ca²⁺, Zn²⁺, Cd²⁺, and Hg²⁺) in solution. Intrinsic binding constants of the ligand (K _in) and mutual influence corrections (ω) for complex formation with aqua ions in solution were calculated. The equilibrium constants were calculated by the matrix method. The coordination sphere of Mg²⁺, Ca²⁺, and Zn²⁺ by ammonia in a cooperative manner; with Cd²⁺ and Hg²⁺, both cooperative and anticooperative binding occur concurrently. Possibilities for differentiation between tetrahedral and square planar coordination polyhedra on the basis of the characteristic features of ligand binding, determined by the matrix model, are discussed.