Utilizing the DNA Aptamer to Determine Lethal α-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA)

Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 μg/mL, while, in mushroom it was 0.372 μg/mL and in urine 0.337 μg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.     

Screening and preliminary application of a DNA aptamer for rapid detection of Salmonella O8

We report on a rapid method for the detection of Salmonella O8. It does not require an enrichment step but rather uses an aptamer as a probe that was selected by system evolution of ligands by exponential enrichment (SELEX) assay. Firstly, aptamer against Salmonella O8 was selected from a 78 bp random DNA library that was prepared in-vitro. The binding ability of the aptamers to target bacterium was examined by aptamer-linked immobilized sorbent assay. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. Next, this high affinity aptamer B10 was used to recognize Salmonella O8 via fluorescence microscopy. The selected aptamer has a high specificity and high affinity against its target. We believe that the resulting fluorescence in-situ labeling assay is a potentially useful alternative in rapid screening and detection of foodborne pathogens.

Fliplike motion in the thalidomide dimer: Conformational analysis of (R)-thalidomide using vibrational circular dichroism spectroscopy

The dynamic fliplike motion in the (R)-thalidomide dimer has been reported for the first time. The vibrational circular dichroism (VCD) spectrum of (R)-thalidomide in DMSO-d6 indicates the characteristic nu(CO) bands with opposite signs and reflects the structural property of the equatorial configuration of the phthalimide ring. On the other hand, the VCD spectrum of (R)-thalidomide in CDCl3 exhibits a different pattern of nu(CO) bands and suggests the fliplike motion in dimer forms. This novel insight for the dimer forms would be helpful for the understanding of the structure-activity relationship for thalidomide.     

STRUCTURE-SPECIFIC BINDING and PHOTOSENSITIZED CLEAVAGE OF BRANCHED DNA THREE-WAY JUNCTION COMPLEXES BY CATIONIC PORPHYRINS

Abstract— The interactions of cationic porphyrins with DNA oligonucleotides that form branched, three-way junction complexes (TWJ) were investigated using native gel electrophoresis, absorption spectroscopy and photochemical probing using DNA sequencing techniques. Meso-tetra(pa ra-N-trimethylaniliniumyrjporphine (TMAP), meso-tetra (4-JV-methylpyridiniumyl)porphine (T4MPyP) and meso-tetra(4- N -methylpyridiniumytyporphine (T3MPyP) were found to bind more tightly to DNA TWJ than to DNA duplexes. The binding to the junction DNA persists at high ionic strength, conditions that greatly decrease porphyrin binding affinity to duplex DNA. The TWJ DNA binding sites of TMAP and T4MPyP were localized to the junction region based on the observation of site- and structure-specific, porphyrin-sensitized photodamage to guanosine residues flanking the junction region.     

Microcalorimetrics studies of the thermodynamics and binding mechanism between L-tyrosinamide and aptamer

In recent years, several high-resolution structures of aptamer complexes have shed light on the binding mode and recognition principles of aptamer complex interactions. In some cases, however, the aptamer complex binding behavior and mechanism are not clearly understood, especially with the absence of structural information. In this study, it was demonstrated that isothermal titration calorimetry (ITC) and circular dichroism (CD) were useful tools for studying the fundamental binding mechanism between a DNA aptamer and L-tyrosinamide (L-TyrNH2). To gain further insight into this behavior, thermodynamic and conformational measurements under different parameters such as salt concentration, temperature, pH value, analogue of L-TyrNH2, and metal ion were carried out. The thermodynamic signature along with the coupled CD spectral change suggest that this binding behavior is an enthalpy-driven process, and the aptamer has a conformational change from B-form to A-form. The results showed that the interaction is an induced fit binding, and the driving forces in this binding behavior may include electrostatic interactions, hydrophobic effects, hydrogen bonding, and the binding-linked protonation process. The amide group and phenolic hydroxyl group of the L-TyrNH2 play a vital role in this binding mechanism. In addition, it should be noted that Mg(2+) not only improves binding affinity but also helps change the structure of the DNA aptamer.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements. The SPR studies indicated that two of these three aptamers distinguished the dicarboxylic acid moiety of the D-isomer from that of the L-isomer; however, the third aptamer did not show enantioselectivity.     

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K_d values for the in-capillary reaction of an aptamer and a target and that the obtained K_d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.     

Cocaine detection by structure-switch aptamer-based capillary zone electrophoresis

Aptamers, which are nucleic acid oligonucleotides that can bind targets with high affinity and specificity, have been widely applied as affinity probes in capillary electrophoresis (CE). Due to relative weak interaction between aptamers and small molecules, the application of aptamer-based CE is still limited in certain compounds. A new strategy that is based on the aptamer structure-switch concept was designed for small molecule detection by a novel CE method. A carboxyfluorescein (fluorescein amidite, FAM) label DNA aptamer was first incubated with partial complementary strand (CS), and then the free aptamer and the aptamer-CS duplex were well separated and determined by metal cation mediated CE/laser-induced fluorescence. When the target was introduced into the incubated sample, the hybridized form was destabilized, resulting in the changes of the fluorescence intensities of the free aptamer and the aptamer-CS duplex. The length of CS was investigated and 12 mer CS showed the best sensitivity for the detection of cocaine. The presented CE-LIF method, which combines the separation power of CE with the specificity of interactions occurring between target, aptamer, and CS, could be a universal detection strategy for other aptamer-specified small molecules.     

In vitro selection and characterization of deoxyribonucleic acid aptamers for digoxin

The low therapeutic index of digoxin necessitates careful monitoring of its serum levels. Most of digoxin immunoassays suffer from interferences with digoxin-like immunoreactive substances. Since aptamers have been shown to be highly specific for their targets, the aim of this study was to develop DNA aptamers for this widely used cardiac glycoside. Digoxin was coated onto the surface of streptavidin magnetic beads. DNA aptamers against digoxin were designed using Systematic Evolution of Ligands by Exponential enrichment method (SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR amplification. The PCR product was cloned and sequenced. Binding affinity was determined using digoxin–BSA conjugate, coated onto ELISA plate. Inhibitory effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium. Three aptamers (D1, D2 and D3) were identified. Binding studies of fluorescein-labeled truncated (without primer binding region) D1 and D2 and full length D1 anti-digoxin aptamers were performed and their corresponding dissociation constants values were 8.2 × 10⁻⁹, 44.0 × 10⁻⁹ and 17.8 × 10⁻⁹ M, respectively. This is comparable to what other workers have obtained for interaction of monoclonal antibodies raised against digoxin. There was little difference in binding affinity between full length and truncated anti-digoxin D1 aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between digoxin and ouabain in both tissue study and binding experiments. Our finding indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further studies might show its suitability for use in digoxin assays and as a therapeutic agent in life-threatening digoxin toxicity.     

A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis

Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve.     

Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs

[structure: see text] The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258 units with a polyether linker for cooperative binding with two remote A3T3 sites of DNA. The linker is introduced to the benzimidazole ring so that it is located at the convex side of the Hoechst unit. DNA binding affinity of the ligands was evaluated by measuring surface plasmon resonance (SPR), circular dichroism, and fluorescence spectra. Interestingly, the bidentate ligands (1) did not show affinity to DNA1 with a single A3T3 motif but showed selective affinity to DNA2 with two A3T3 motifs. The Long Bis-H (1L) having a long polyether linker showed specific binding to DNA2(6) with two A3T3 motifs separated by six nonbinding base pairs. The Long Bis-H (1L) has also shown specific binding to the three-way junction DNA4 with two A3T3 motifs. This study has demonstrated that DNA with double binding motifs can be selectively recognized by the newly designed bidentate ligands.     

In vivo Fluorescence Imaging of Tumors using Molecular Aptamers Generated by Cell‐SELEX

Poor sensitivity and low specificity of current molecular imaging probes limit their application in clinical settings. To address these challenges, we used a process known as cell‐SELEX to develop unique molecular probes termed aptamers with the high binding affinity, sensitivity, and specificity needed for in vivo molecular imaging inside living animals. Importantly, aptamers can be selected by cell‐SELEX to recognize target cells, or even surface membrane proteins, without requiring prior molecular signature information. As a result, we are able to present the first report of aptamers molecularly engineered with signaling molecules and optimized for the fluorescence imaging of specific tumor cells inside a mouse. Using a Cy5‐labeled aptamer TD05 (Cy5‐TD05) as the probe, the in vivo efficacy of aptamer‐based molecular imaging in Ramos (B‐cell lymphoma) xenograft nude mice was tested. After intravenous injection of Cy5‐TD05 into mice bearing grafted tumors, noninvasive, whole‐body fluorescence imaging then allowed the spatial and temporal distribution to be directly monitored. Our results demonstrate that the aptamers could effectively recognize tumors with high sensitivity and specificity, thus establishing the efficacy of these fluorescent aptamers for diagnostic applications and in vivo studies requiring real‐time molecular imaging.     

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

Background: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP)' with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution.Results: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G·A mismatches are flanked by sheared G·A and reversed Hoogsteen G·G mismatch pairs.Conclusions: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G·A mismatch formation. The recognition G·A mismatch stacks with a reversed Hoogsteen G·G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10¹⁴ molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to antithrombin DNA aptamers

We have applied surface plasmon resonance (SPR) spectroscopy, in combination with one-step direct binding, competition, and sandwiched assay schemes, to study thrombin binding to its DNA aptamers, with the aim to further the understanding of their interfacial binding characteristics. Using a 15-mer aptamer that binds thrombin primarily at the fibrinogen-recognition exosite as a model, we have demonstrated that introducing a DNA spacer in the aptamer enhances thrombin-binding capacity and stability, as similarly reported for hydrocarbon linkers. The bindings are aptamer surface coverage and salt concentration dependent. When free aptamers or DNA sequences complementary to the immobilized aptamer are applied after the formation of thrombin/aptamer complexes, bound thrombin is displaced to a certain extent, depending on the stability of the complexes formed under different conditions. When the 29-mer aptamer (specific to thrombin's heparin-binding exosite) is immobilized on the surface, its affinity to thrombin appears to be lower than the immobilized 15-mer aptamer, although the 29-mer aptamer is known to have a higher affinity in the solution phase. These findings underline the importance of aptamers' ability to fold into intermolecular structures and their accessibility for target capture. Using a sandwiched assay scheme followed by an additional signaling step involving biotin-streptavidin chemistry, we have observed the simultaneous binding of the 15- and 29-mer aptamers to thrombin protein at different exosites and have found that one aptamer depletes thrombin's affinity to the other when they bind together. We believe that these findings are invaluable for developing DNA aptamer-based biochips and biosensors.     

Probing high-affinity 11-mer DNA aptamer against Lup an 1 (β-conglutin)

Aptamers are synthetic nucleic acids with great potential as analytical tools. However, the length of selected aptamers (typically 60–100 bases) can affect affinity, due to the presence of bases not required for interaction with the target, and therefore, the truncation of these selected sequences and identification of binding domains is a critical step to produce potent aptamers with higher affinities and specificities and lowered production costs. In this paper we report the truncation of an aptamer that specifically binds to β-conglutin (Lup an 1), an anaphylactic allergen. Through comparing the predicted secondary structures of the aptamers, a hairpin structure with a G-rich loop was determined to be the binding motif. The highest affinity was observed with a truncation resulting in an 11-mer sequence that had an apparent equilibrium dissociation constant (K _D) of 1.7?×?10^?9 M. This 11-mer sequence was demonstrated to have high specificity for β-conglutin and showed no cross-reactivity to other lupin conglutins (α-, δ-, γ-conglutins) and closely related proteins such as gliadin. Finally, the structure of the truncated 11-mer aptamer was preliminarily elucidated, and the GQRS Mapper strongly predicted the presence of a G-quadruplex, which was subsequently corroborated using one-dimensional NMR, thus highlighting the stability of the truncated structure.     

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor

A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.     

Recognition of DNA three-way junctions by metallosupramolecular cylinders: gel electrophoresis studies

The interaction of metallosupramolecular cylinders with DNA three-way junctions has been studied by gel electrophoresis. A recent X-ray crystal structure of a palindromic oligonucleotide forming part of a complex with such a cylinder revealed binding at the heart of a three-way junction structure. The studies reported herein confirm that this is not solely an artefact of crystallisation and reveal that this is a potentially very powerful new mode of DNA recognition with wide scope. The cylinders are much more effective at stabilizing three-way junctions than simple magnesium di-cations or organic or metallo-organic tetra-cations, with the M cylinder enantiomer being more effective than P. The recognition is not restricted to three-way junctions formed from palindromic DNA with a central AT step at the junction; non-palindromic three-way junctions and those with GC steps are also stabilised. The cylinder is also revealed to stabilise other Y-shaped junctions, such as that formed at a fraying point in duplex DNA (for example, a replication fork), and other DNA three-way junction structures, such as those containing unpaired nucleotides, perhaps by opening up this structure to access the central cavity.