The effect of RNA base lesions on mRNA translation.

The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell. Here we report on the effect of the commonly known RNA base-lesions 8-oxo-rG, 8-oxo-rA, ε-rC, ε-rA, 5-HO-rC, 5-HO-rU and the RNA abasic site (rAS) on ribosomal translation. To this end we have developed an in vitro translation assay based on the mRNA display methodology. A short synthetic mRNA construct containing the base lesion in a predefined position of the open reading frame was (32)P-labeled at the 5'-end and equipped with a puromycin unit at the 3'-end. Upon in vitro translation in rabbit reticulocyte lysates, the encoded peptide chain is transferred to the puromycin unit and the products analyzed by gel electrophoresis. Alternatively, the unlabeled mRNA construct was used and incubated with (35)S-methionine to prove peptide elongation of the message. We find that all base-lesions interfere substantially with ribosomal translation. We identified two classes, the first containing modifications at the base coding edge (ε-rC, ε-rA and rAS) which completely abolish peptide synthesis at the site of modification, and the second consisting of 8-oxo-rG, 8-oxo-rA, 5-HO-rC and 5-HO-rU that significantly retard full-length peptide synthesis, leading to some abortive peptides at the site of modification.

Base pairing and miscoding properties of 1,N(6)-ethenoadenine- and 3,N(4)-ethenocytosine-containing RNA oligonucleotides.

Two RNA phosphoramidites containing the bases 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) were synthesized. These building blocks were incorporated into two 12-mer oligoribonucleotides for evaluation of the base pairing properties of these base lesions by UV melting curve (Tm) and circular dichroism measurements. The Tm data of the resulting duplexes with the etheno modifications opposing all natural bases showed a substantial destabilization compared to the corresponding natural duplexes, confirming their inability to form base pairs. The coding properties of these lesions were further investigated by introducing them into 31-mer oligonucleotides and assessing their ability to serve as templates in primer extension reactions with HIV, AMV, and MMLV reverse transcriptases (RT). Primer extension reactions showed complete arrest of the incorporation process using MMLV RT and AMV RT, while HIV RT preferentially incorporates dAMP opposite εA and dAMP as well as dTMP opposite εC. The properties of these RNA lesions are discussed in the context of its putative biological role.

Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices.

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar-attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms.

Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays.

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk.

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.

A PNA microarray for tomato genotyping.

The design and development of a PNA microarray designed for the simultaneous identification of several SNPs characteristic of seven different tomato varieties is described. Highly selective arginine-based monomer containing PNAs (Arg-PNAs) have been used in order to obtain very selective probes. Seven modified PNA probes were synthesised and their binding properties in solution were studied. PNA-microarrays based on these probes were prepared and applied to SNP discrimination in model experiments using oligonucleotide mixtures simulating the different sequences of the seven tomato varieties. The strength and the limitations of such a system for SNP recognition are thoroughly discussed.

Patterning of peptide nucleic acids using reactive microcontact printing.

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

Affinity and selectivity of C2- and C5-substituted "chiral-box" PNA in solution and on microarrays.

Two peptide nucleic acids (PNAs) containing three adjacent modified chiral monomers (chiral box) were synthesized. The chiral monomers contained either a C2- or a C5-modified backbone, synthesized starting from D- and L-arginine, respectively (2D- and 5L-PNA). The C2-modified chiral PNA was synthesized using a submonomeric strategy to avoid epimerization during solid-phase synthesis, whereas for the C5-derivative, the monomers were first obtained and then used in solid-phase synthesis. The melting temperature of these PNA duplexes formed with the full-match or with single-mismatch DNA were measured both by UV and by CD spectroscopy and compared with the unmodified PNA. The 5L-chiral-box-PNA showed the highest T(m) with full-match DNA, whereas the 2D-chiral-box-PNA showed the highest sequence selectivity. The PNA were spotted on microarray slides and then hybridized with Cy5-labeled full match and mismatched oligonucleotides. The results obtained showed a signal intensity in the order achiral >2D-chiral box >5L-chiral box, whereas the full-match/mismatch selectivity was higher for the 2D chiral box PNA.

Arginine-based PNA microarrays for APOE genotyping.

Four modified PNAs containing one chiral monomer bearing two arginine-derived side chains, with the correct configuration for specific and stable DNA binding, were synthesized, complementary to two DNA tracts in the APOE gene containing SNPs related to the insurgence of Alzheimer's disease. PNA binding performances were first tested in solution against complementary and mismatched oligonucleotides by measuring melting temperatures, and showed high specificity in SNP recognition. In order to set up a new diagnostic platform for APOE genotyping, PNA microarrays were then developed with the synthesized modified PNAs. PNA probe deposition protocols on microarrays were optimized in order to minimize cross-contamination due to carry over. The microarrays obtained by arginine-based PNA deposition were incubated with complementary and mismatched oligonucleotides, showing excellent mismatch recognition on the microarray platform. The specificity of the microarrays was finally tested with oligonucleotide mixtures simulating the real genotype profiles. Six different hybridisation patterns related to six different genotypes in the APOE gene were found to be clearly distinct in microarray experiments, demonstrating the potential of this approach for highly specific genetic analysis.