Multi-sample acoustic biosensing microsystem for protein interaction analysis

The current work describes a novel setup for multi-sample biomolecular analysis. It is based on the assembly of a dual acoustic device chip with a four-channel microfluidic module, forming an array of eight available domains for experiments. Initially, multiple detection was demonstrated via the specific interaction of neutravidin with four different biotinylated proteins, namely protein G, protein A, bovine serum albumin, and immunoglobulin G; results revealed a reproducibility between the microchannel domains better than 90%. Real-time analysis of the binding interactions was used to calculate the affinity and kinetic constants of the four biotinylated molecules binding to surface-immobilized neutravidin; this was the first time that this information was derived using a biosensing device and four biotinylated molecules. Interestingly, all calculated kinetic and affinity constants resemble those typical of antibody-antigen interactions, although the investigated specific binding was of avidin-biotin nature. Finally, under device pre-functionalization conditions, it was possible to probe eight interactions all together, exploiting the full capacity of the microsystem and reducing significantly the analysis time, contrary to the use of the standard acoustic device configuration. The outcome of this full-scale validation opens the way for the integrated acoustic platform to be implemented in even higher throughput detection for future diagnostic/biomedical applications, as well as in fundamental research studies regarding biomolecular interaction investigation and characterization.     

Molecular signaling aptamers for real-time fluorescence analysis of protein

Aptamers are a new class of nucleic acids that are selected in vitro for binding target molecules with high affinity and selectivity. They are promising protein-binding molecular probes that rival conventional antibodies for protein analysis. There have been recent advances in the development of molecular signaling aptamers that can transduce target protein binding to sensitive fluorescence signal changes. This facilitates the real time protein monitoring in homogenous solution as well as potentially in vivo. Different signaling strategies of using dual labeled aptamers based on fluorescence resonance energy transfer (FRET), one fluorophore labeled aptamers based on fluorescence anisotropy assay, or other label-free aptamers are reviewed.     

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.     

Single-strand DNA aptamers as probes for protein localization in cells.

The accurate localization of proteins in fixed cells is important for many studies in cell biology, but good fixation is often antagonistic to good immunolabeling, given the density of well-preserved cells and the size of most labeled antibody probes. We therefore explored the use of single-stranded oligonucleotides (aptamers), which can bind to proteins with very high affinity and specificity but which are only approximately 10 kD. To evaluate these probes for general protein localization, we sought an aptamer that binds to a widely used protein tag, the green fluorescent protein (GFP). Although this quest was not successful, we were able to solve several practical problems that will confront any such labeling effort, e.g., the rates at which oligonucleotides enter fixed cells of different kinds and the extent of nonspecific oligonucleotide binding to both mammalian and yeast cell structures. Because such localization methods would be of particular value for electron microscopy of optimally fixed material, we also explored the solubility of aptamers under conditions suitable for freeze-substitution fixation. We found that aptamers are sufficiently soluble in cold organic solvents to encourage the view that this approach may be useful for the localization of specific proteins in context of cellular fine structure.     

Proximity extension of circular DNA aptamers with real-time protein detection

Multivalent circular aptamers or ‘captamers’ have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the ‘proximity extension’ assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer–thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.     

Surface modification for DNA and protein microarrays

Microarrays of biomolecules are emerging as powerful tools for genomics, proteomics, and clinical assays, since they make it possible to screen biologically important binding events in a parallel and high throughput fashion. Because the microarrays are fabricated on a solid support, coating of the surface and immobilization strategy of the biomolecules are major issues for successful microarray fabrication. This review deals with both DNA microarrays and protein microarrays, and focuses on the various modification approaches for the two-dimensional surface materials and three-dimensional ones. In addition, the immobilization strategies including adsorption, covalent attachment, physical entrapment, and affinity attachment of the biomolecules are summarized, and advantage and limitation of representative efforts are discussed.     

Development of drug-loaded protein nanoparticles displaying enzymatically-conjugated DNA aptamers for cancer cell targeting

Molecular Biology Reports - Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we...     

High-throughput quantitative binding analysis of DNA aptamers using exonucleases

Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer–ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer–ligand pairs.     

Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization

Commercial porous polypropylene membranes were chemically modified with polyaniline (PANI) using ammonium persulfate as the oxidizer. The influence of polymerization conditions on the membrane properties was studied by adsorption analysis and membrane permeability. The PANI-coated polypropylene (PANI/PP) membranes possessed high affinity toward the proteins, which can be immobilized onto the membrane surface through physical adsorption or covalent immobilization. The quantity of immobilized horseradish peroxidase (HRP) and its activity depended on the quantity and quality (oxidation level) of PANI. The storage conditions for PANI/PP membranes containing immobilized HRP were studied. HRP immobilized on the PANI/PP membrane was shown to retain 70% of its activity after 3-month storage at +5 degrees C, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes.     

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K_d values between 20 and 240 nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.     

G-quadruplex DNA aptamers generated for systemin

Ligands specific to bioactive molecules play important roles in biomedical researches and applications, such as biological assay, diagnosis and therapy. Systemin is a peptide hormone firstly identified in plant. In this paper we report the selection of a group of DNA aptamers that can specifically bind to systemin. Through comparing the predicted secondary structures of all the aptamers, a hairpin structure with G-rich loop was determined to be the binding motif of these aptamers. The G-rich loop region of this binding motif was further characterized to fold into an antiparallel G-quadruplex by truncation-mutation assay and CD spectrum. The apparent equilibrium dissociation constant (K(d)) of one strong binding sequence (S-5-1) was measured to be 0.5 μM. The specificity assay shows that S-5-1 strongly bind to whole systemin, weakly bind to truncated or mutated systemin and does not bind to the scrambled peptide with the same amino acid composition as systemin. The high affinity and specificity make S-5-1 hold potentials to serve as a molecular ligand applied in detection, separation and functional investigation of systemin in plants.     

Selection of DNA aptamers for breast cancer

A method of selection of DNA aptamers to breast tumor tissue based on the use of postoperative material has been developed. Breast cancer tissues were used as the positive target; the negative targets included benign tumor tissue, adjacent healthy tissues, breast tissues from mastopathy patients, and also tissues of other types of malignant tumors. During selection a pool of DNA aptamers demonstrating selective binding to breast cancer cells and tissues and insignificant binding to breast benign tissues has been obtained. These DNA aptamers can be used for identification of protein markers, breast cancer diagnostics, and targeted delivery of anticancer drugs.     

Isolation and characterization of enantioselective DNA aptamers for ibuprofen

Single stranded DNA aptamers that can bind to ibuprofen, a widely used anti-inflammation drug, were selected from random DNA library of 10¹⁵ nucleotides by FluMag-SELEX process. Five different sequences were selected and their enantioselectivity and affinity were characterized. Three out of five aptamer candidates did not show any affinity to (S)-ibuprofen, but only to racemic form of ibuprofen, suggesting that they are (R)-ibuprofen specific aptamers. Another two aptamer candidates showed affinity to both racemic form and (S)-ibuprofen, which were considered as (S)-ibuprofen specific aptamers. The affinity of five ssDNA aptamers isolated was in a range of 1.5–5.2 μM. In addition, all of these five aptamers did not show any affinity to analogues of ibuprofen in its profen’s group (fenoprofen, flubiprofen, and naproxen) and the antibiotics of oxytetracycline, another control.     

Single-stranded DNA aptamers specific for antibiotics tetracyclines

Tetracyclines (TCs) are a group of antibiotics comprising of a common tetracycline (TET) nucleus with variable X(1) and X(2) positions on 5 and 6 carbon atoms, such as oxytetracycline (OTC) and doxycycline (DOX). In this study, the tetracycline group specific (TGS) ssDNA aptamers were identified by modified SELEX method by employing tosylactivated magnetic beads (TMB) coated with OTC, TET, and DOX, respectively, as targets and counter targets. Twenty TGS-aptamers were selected, of which seven aptamers, designated as T7, T15, T19, T20, T22, T23, and T24, showed high affinity to the basic TET backbone (K(d)=63-483 nM). The specificity of these TGS-aptamers to structural analogues followed the order in which the TCs was employed during SELEX process (OTC>TET>DOX) except aptamer T22, which was highly specific to TET than OTC or DOX. Aptamers that were specific to one target molecule but fail to bind the other structurally related TCs were eliminated during counter selection steps. Three aptamers, T7, T19, and T23 contained palindromic consensus sequence motif GGTGTGG. The remaining TGS-aptamers showed many consensus sequences that are truncated forms of this palindrome forming mirror image or inverted sequences. For example, GTGG or its inverted form, GGTG motif was found in all TGS-aptamers. A consensus sequence motif TGTGCT or its truncated terminal T-residue was found in most TGS-aptamers, which is predicted to be essential for high affinity and group specificity. These TGS-aptamers have potential applications such as target drug delivery, and detection of TCs in pharmaceutical preparations and contaminated food products.     

Electrochemical studies of kanamycin immobilization on self-assembled monolayer and interaction with DNA

The electrochemical characteristics of kanamycin onto self-assembled monolayer (SAM) modified gold electrode (SAM/Au) is investigated by cyclic voltammetry. In the potential range 0-0.6 V, Cu(II) yields a pair of stable redox waves at the bare gold electrode. E(pa) is located at 0.189 V and E(pc) at 0.254 V. In contrast, Cu(II) is reduced at a more positive potential and a decreasing current at the kanamycin SAM/Au electrode. Cu(II) and kanamycin can form a stoichiometry complex with chemical ratio of 2:1. The interaction of Cu(II)-kanamycin complex with calf thymus DNA is also studied in the solution. And the interactive mode between Cu(II)-kanamycin complex and DNA is verified by the fluorescence method. Binding constants (K) of the Cu(II)-kanamycin complex to DNA and binding site size (s) are calculated from voltammetric data and equal to 1.5 x 10(7) l/mol and 4 bp, respectively.     

Comparative thermodynamic analysis of thrombin interaction with anti-thrombin aptamers and their heterodimeric construct

Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs by means of a poly-(dT)-linker of 35 nucleotides (nt) in length. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using an optical biosensor Biacore-3000. The K _D values obtained for the hetero- and homodimeric constructs were correspondingly 25–30- and 2–3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of the K _D values within the temperature interval of 10–40°C has shown that affinity increases with the temperature decrease. The values of the enthalpy change ΔH upon formation of complexes of thrombin with the aptamers and the heterodimeric construct were basically the same. The value of the entropy change ΔS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5–2-fold higher than the ΔS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 to 37°C. However, at both temperatures the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that for the complexes with the aptamers.     

Computer aided analysis for biosensing and screening

Biosensors with animal and microbial cells immobilized close to the tip of a membrane electrode have been developed for chemical and drug testing. Our experimental results show that biosensors can be used for drug screening and to provide useful information about various cell-chemical interactions. A computer aided analysis (CAA) software package is being developed here using the biosensor for various screening purposes. This software package enables us to use a computer to analyze the biosensor dynamic responses. Computer simulation and parameter estimation techniques are used to select the best model and to describe the biochemical and pharmacologic effects of various chemicals and drugs on different cell lines.