Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A.

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM.

Expression and purification of active protein kinases from wheat germ extracts.

In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and -enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins remains a challenging task. Current wheat-germ-based cell-free in vitro translation systems present a plausible alternative for protein synthesis since majority of eukaryotic proteins could be obtained in their native folded form with general protocols. The use of special in vitro translation vectors with ligation-independent cloning sites and cleavable affinity tags eliminates further bottlenecks of the protein producing procedure and makes this system a reasonable method for simultaneous generation of active kinases.

Selection and versatile application of virus-specific aptamers.

Although the significance of molecular diagnostics in routine plant virus detection is rapidly growing, the preferred methods are still antibody-based enzyme immunoassays. In the past decade, aptamers have been demonstrated to be viable alternatives of antibodies in many applications. We set out to select apple stem pitting virus (ASPV)-specific aptamers and to apply them as antibody substitutes in various immunoassay methods. The applied systematic evolution of ligands by exponential enrichment (SELEX) procedure resulted in highly discriminative aptamers selectively binding to the target virus coat protein even in complex protein matrixes. We developed protocols for exploitation of aptamers in diverse plant virus diagnosis methods, such as dot and Western blot analyses and enzyme-linked oligonucleotide assay (ELONA). Our selected aptamers proved to be superior to the available antibody in all aspects. In contrast to the antibody, the aptamers decorate both native and denaturated proteins, and ELONA produces higher signal intensity than traditional enzyme-linked immunosorbent assay (ELISA) with virus-infected plant extract. Summarily, our results present the selection and practical utilization of first plant virus-specific aptamers. Most important, the first application of ELONA for virus detection is demonstrated, which proposes a novel, more flexible, and cost-effective means of virus diagnostics.

Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging.

Specific detection of virus strains by affinity-based bioassays is often limited by the availability of ligands able to differentiate among close homologues of virus coat proteins. As viruses are prone to mutation, the ligand generation should, in addition, be fast enough to allow rapid identification of new varieties. These two criteria are difficult to be fulfilled by antibodies; however, they open up opportunities for aptamer-based detection. Here we report on the feasibility of selectively detecting the apple stem pitting virus (ASPV) coat proteins (PSA-H, MT32) using original DNA aptamers. Surface plasmon resonance (SPR) imaging was used together with aptamer-modified sensor chips to optimize the aptamer immobilization for highest sensitivity and to characterize the aptamer-virus coat protein binding. Different parameters affecting this binding, such as the aptamer flanking, surface coverage, and type of spacer molecules, were identified and their influence was determined. A direct label-free method is proposed for assessing the ASPV based on the detection of the respective virus coat proteins in plant extracts.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

BACKGROUND: The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. RESULTS: We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. CONCLUSION: Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin.

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence.

Identification of a DNA methylation-dependent activator sequence in the pseudoxanthoma elasticum gene, ABCC6.

ABCC6 encodes MRP6, a member of the ABC protein family with an unknown physiological role. The human ABCC6 and its two pseudogenes share 99% identical DNA sequence. Loss-of-function mutations of ABCC6 are associated with the development of pseudoxanthoma elasticum (PXE), a recessive hereditary disorder affecting the elastic tissues. Various disease-causing mutations were found in the coding region; however, the mutation detection rate in the ABCC6 coding region of bona fide PXE patients is only approximately 80%. This suggests that polymorphisms or mutations in the regulatory regions may contribute to the development of the disease. Here, we report the first characterization of the ABCC6 gene promoter. Phylogenetic in silico analysis of the 5' regulatory regions revealed the presence of two evolutionarily conserved sequence elements embedded in CpG islands. The study of DNA methylation of ABCC6 and the pseudogenes identified a correlation between the methylation of the CpG island in the proximal promoter and the ABCC6 expression level in cell lines. Both activator and repressor sequences were uncovered in the proximal promoter by reporter gene assays. The most potent activator sequence was one of the conserved elements protected by DNA methylation on the endogenous gene in non-expressing cells. Finally, in vitro methylation of this sequence inhibits the transcriptional activity of the luciferase promoter constructs. Altogether these results identify a DNA methylation-dependent activator sequence in the ABCC6 promoter.