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

AbstractMany 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

The entire organization of transcription units on the Bacillus subtilis genome

<p>Abstract</p> <p>Background</p> <p>In the post-genomic era, comprehension of cellular processes and systems requires global and non-targeted approaches to handle vast amounts of biological information.</p> <p>Results</p> <p>The present study predicts transcription units (TUs) in <it>Bacillus subtilis</it>, 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 σ factors and 17 TFs of <it>B. subtilis</it>, 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.</p> <p>Conclusion</p> <p>This integrated approach is, therefore, an efficient tool for narrowing searches for candidate genes regulated by TFs, identifying TUs, and estimating roles of the σ factors and TFs in cellular processes and functions of genes composing the TUs.</p

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

Genome Informatics 14: 396--397 (2003) Regulator Identification by Expression Profiles of

Introduction Computational prediction of the regulation relation among genes is the first step to reveal the expression regulation network. There are some problems including lack of knowledge for consensus sequences and ambiguous patterns of sequences recognized by transcription factors. The integration of information concerning to regulation relations such upstream DNA sequences and expression profiles of genes may be important for identifying regulation relations of genes reliably. So, we must predict transcription networks on the basis of various di#erent data source concerning the regulation relations. In the present study, we predicted transcription regulation relations of genes by several sfactors in Bacillus subtilis genome using genome sequence and microarray information. We addressed non-lethal mutants of sigma factors (sigD, sigE, sigF, sigG, sigH, sigK, sigW, sigX). In the present study, we proposed methodology for constructing regulation relation in transcription level ba

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

<div><p>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 <i>Escherichia coli</i> 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 <i>E. coli</i>, 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.</p></div

Target-binding activity of surrogate scFv isolated by Hishot display.

<p>Binding of surrogate scFv was measured by ELISA using horseradish peroxidase-conjugated anti-His-tag mAb. The ELISA plate was coated with proteins (5 µg/mL) and then blocked with BSA. Values represent the mean ± S.D. of triplicate determinations. (A) Specific binding activity of surrogate scFv against intelectin-1. The amino acid sequence of the random region is shown under the clone name (IA04 and IA10). Fixed Tyr is indicated as shaded characters. (B) Dose-dependency of the binding of purified surrogate scFv against intelectin-1. Values represent the mean of duplicate determinations. (C) Sandwich ELISA against intelectin-1, using surrogate scFv against intelectin-1 as the detector. Anti-intelectin-1 (capture mAb, 10:2D2) was coated at a concentration of 2 µg/mL, and intelectin-1 (0.1 µg/mL) was captured. The surrogate scFv against intelectin-1 was used at the concentration of 8 µg/mL. (D) Specific binding activity of surrogate scFv against TNF-α. The amino acid sequence of the random region is shown under the clone name (T4E). Fixed Tyr is indicated as shaded characters. BSA, bovine serum albumin; OVA, ovalbumin; Trf, transferrin; IgG, rabbit IgG; ITLN1, human intelectin-1; TNF-α, human tumor necrosis factor-α.</p

Concentration of target-binding clones by Hishot display using a random peptide.

<p>Rabbit IgG was used as a target. Hishot complexes in a library were collected with rabbit IgG on the plate, amplified by RT-PCR, and then re-ligated into pHishot1. Clones in the next round were shown as selected clones. The amount of binding peptide was measured by ELISA using rabbit IgG (target) and horseradish peroxidase-conjugated anti-His-tag mAb (detector). Values represent the mean ± S.D. of triplicate determinations.</p

Determining the best position for shot47 for collecting the Hishot complex.

<p>(A) Schema of the possible positions for shot47. Shot47 sequences were inserted into a gene of the His-tagged V_H fragment at 3 different positions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083108#pone.0083108.s001" target="_blank">Figure S1</a>). (B) Expression of His-tagged V_H from expression vector containing shot47. The expression level was measured by sandwich ELISA using anti-Ig antibody (capture) and horseradish peroxidase-conjugated anti-His-tag mAb (detector). (C) Amount of mRNA in the Hishot complex on a plate coated with BSA or anti-Ig (Ab). The collected mRNA was amplified by RT-PCR (15 or 20 cycles of amplification), resolved by agarose gel electrophoresis, and stained with ethidium bromide.</p