An Aptamer-Based Biosensor for Direct, Label-Free Detection of Melamine in Raw Milk.

Melamine, a nitrogen-rich compound, has been used as a food and milk additive to falsely increase the protein content. However, melamine is toxic, and high melamine levels in food or in milk can cause kidney and urinary problems, or even death. Hence, the detection of melamine in food and milk is desirable, for which numerous detection methods have been developed. Several methods have successfully detected melamine in raw milk; however, they require a sample preparation before the analyses. This study aimed to develop an aptamer-DNAzyme conjugated biosensor for label-free detection of melamine, in raw milk, without any sample preparation. An aptamer-DNAzyme conjugated biosensor was developed via screening using microarray analysis to identify the candidate aptamers followed by an optimization, to reduce the background noise and improve the aptamer properties, thereby, enhancing the signal-to-noise (S/N) ratio of the screened biosensor. The developed biosensor was evaluated via colorimetric detection and tested with raw milk without any sample preparation, using N-methylmesoporphyrin IX for fluorescence detection. The biosensor displayed significantly higher signal intensity at 2 mM melamine (S/N ratio, 20.2), which was sufficient to detect melamine at high concentrations, in raw milk.

Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Sub-nanomolar Affinity for a Salivary Biomarker.

We have attained a chemically modified DNA aptamer against salivary α-amylase (sAA), which attracts researchers' attention as a useful biomarker for assessing human psychobiological and social behavioural processes, although high affinity aptamers have not been isolated from a random natural DNA library to date. For the selection, we used the base-appended base (BAB) modification, that is, a modified-base DNA library containing (E)-5-(2-(N-(2-(N6-adeninyl)ethyl))carbamylvinyl)-uracil in place of thymine. After eight rounds of selection, a 75 mer aptamer, AMYm1, which binds to sAA with extremely high affinity (Kd < 1 nM), was isolated. Furthermore, we have successfully determined the 36-mer minimum fragment, AMYm1-3, which retains target binding activity comparable to the full-length AMYm1, by surface plasmon resonance assays. Nuclear magnetic resonance spectral analysis indicated that the minimum fragment forms a specific stable conformation, whereas the predicted secondary structures were suggested to be disordered forms. Thus, DNA libraries with BAB-modifications can achieve more diverse conformations for fitness to various targets compared with natural DNA libraries, which is an important advantage for aptamer development. Furthermore, using AMYm1, a capillary gel electrophoresis assay and lateral flow assay with human saliva were conducted, and its feasibility was demonstrated.

Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity.

Many cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities.

High-throughput quantitative screening of peroxidase-mimicking DNAzymes on a microarray by using electrochemical detection.

Some guanine-rich DNA sequences, which are called DNAzymes, can adopt G-quadruplex structures and exhibit peroxidase activity by binding with hemin. Although known DNAzymes show less activity than horseradish peroxidase, they have the potential to be widely used for the detection of target molecules in enzyme-linked immunosorbent assays if sequences that exhibit higher activity can be identified. However, techniques for achieving this have not yet been described. Therefore, we compared the DNAzyme activities of more than 1000 novelistically designed sequences with that of the original DNAzyme by using an electrochemical detection system on a 12K DNA microarray platform. To the best of our knowledge, this is the first description of an array-based assessment of peroxidase activity of G-quadruplex-hemin complexes. By using this novel assay system, more than 200 different mutants were found that had significantly higher activities than the original DNAzyme sequence. This microarray-based DNAzyme evaluation system is useful for identifying highly active new DNAzymes that might have potential as tools for developing DNA-based biosensors with aptamers.

Hishot display--a new combinatorial display for obtaining target-recognizing peptides.

Display technologies are procedures used for isolating target-recognizing peptides without using immunized animals. In this study, we describe a new display method, named Hishot display, that uses Escherichia coli and an expression plasmid to isolate target-recognizing peptides. This display method is based on the formation, in bacteria, of complexes between a polyhistidine (His)-tagged peptide including random sequences and the peptide-encoding mRNA including an RNA aptamer against the His-tag. When this system was tested using a sequence encoding His-tagged green fluorescent protein that included an RNA aptamer against the His-tag, the collection of mRNA encoding the protein was dependent on the RNA aptamer. Using this display method and a synthetic library of surrogate single-chain variable fragments consisting of VpreB and Ig heavy-chain variable domains, it was possible to isolate clones that could specifically recognize a particular target (intelectin-1 or tumor necrosis factor-α). These clones were obtained as soluble proteins produced by E. coli, and the purified peptide clones recognizing intelectin-1 could be used as detectors for sandwich enzyme-linked immunosorbent assays. The Hishot display will be a useful method to add to the repertoire of display technologies.

ValFold: Program for the aptamer truncation process.

UNLABELLED: DNA or RNA aptamers have gained attention as the next generation antibody-like molecules for medical or diagnostic use. Conventional secondary structure prediction tools for nucleic acids play an important role to truncate or minimize sequence, or introduce limited chemical modifications without compromising or changing its binding affinity to targets in the design of improved aptamers selected by Systematic Evolution of Ligands by EXponential enrichment (SELEX). We describe a novel software package, ValFold, capable of predicting secondary structures with improved accuracy based on unique aptamer characteristics. ValFold predicts not only the canonical Watson-Crick pairs but also G-G pairs derived from G-quadruplex (known structure for many aptamers) using the stem candidate selection algorithm. AVAILABILITY: The database is available for free at http://code.google.com/p/valfold/

Effective isolation of RNA aptamer through suppression of PCR bias.

An aptamer is a short RNA or DNA molecule that binds to a specific target. The main strategy for obtaining aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Although various SELEX techniques have been devised and refined on the basis of the selection technique used, in most cases, the isolation of an aptamer still requires several trials or the use of special equipment. In the present study, we attempted SELEX in which PCR bias was suppressed by using RNA transcription to amplify nucleic acids. This procedure, which can be accomplished easily and inexpensively without special equipment, effectively simplifies the SELEX process. Using this SELEX, we obtained large numbers of RNA aptamers against the target that could not be isolated by standard SELEX. The results of our study suggest that exclusion of PCR bias may be far more important than previously assumed for isolating RNA aptamers via SELEX.

RNA aptamer binding to polyhistidine-tag.

Polyhistidine-tag (His-tag) is a powerful tool for purification of recombinant protein. His-tagged protein can be affinity-purified by using resins immobilizing Ni2+ or anti-His-tag antibodies. However, Ni2+-affinity-purification is prevented by the presence of divalent cations. The purification with antibodies has contamination of antibody peptides, which interferes with following analysis. In the present study, we isolated RNA aptamers binding to His-tag. The best clone, named shot47, bound to the target with low picomolar dissociation constant. In the presence of divalent cations, shot47 was substitutable for antibodies against His-tag on ELISA, immunoprecipitation, and Western blotting. Shot47 can be synthesized easily by in vitro transcription. Thus, shot47 would be applicable as a useful and cost-effective tool for biochemical analyses.

RNA aptamers specifically interact with the Fc region of mouse immunoglobulin G.

We have designed the in vitro selection method to obtain some aptamers such as a general antibody-probing agent, which might bind to the constant regions of mouse immunoglobulin G (IgG) subclasses. As a consequence, one of the selected aptamers found to recognize mouse IgG1, 2a, and 3 subclasses. According to the binding assay, it is suggested that this aptamer recognizes the constant regions of mouse IgG subclass. In addition, this aptamer could recognize the only native form of mouse IgGs but the denatured IgGs. These features show the advantage of the aptamer as an antibody-probing agent rather than the usual secondary antibodies.

The entire organization of transcription units on the Bacillus subtilis genome.

BACKGROUND: In the post-genomic era, comprehension of cellular processes and systems requires global and non-targeted approaches to handle vast amounts of biological information. RESULTS: The present study predicts transcription units (TUs) in Bacillus subtilis, based on an integrated approach involving DNA sequence and transcriptome analyses. First, co-expressed gene clusters are predicted by calculating the Pearson correlation coefficients of adjacent genes for all the genes in a series that are transcribed in the same direction with no intervening gene transcribed in the opposite direction. Transcription factor (TF) binding sites are then predicted by detecting statistically significant TF binding sequences on the genome using a position weight matrix. This matrix is a convenient way to identify sites that are more highly conserved than others in the entire genome because any sequence that differs from a consensus sequence has a lower score. We identify genes regulated by each of the TFs by comparing gene expression between wild-type and TF mutants using a one-sided test. By applying the integrated approach to 11 sigma factors and 17 TFs of B. subtilis, we are able to identify fewer candidates for genes regulated by the TFs than were identified using any single approach, and also detect the known TUs efficiently. CONCLUSION: This integrated approach is, therefore, an efficient tool for narrowing searches for candidate genes regulated by TFs, identifying TUs, and estimating roles of the sigma factors and TFs in cellular processes and functions of genes composing the TUs.

An RNA aptamer that discriminates bovine factor IX from human factor IX.

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.