Polycyclic aromatic hydrocarbon skin permeation efficiency in vitro is lower through human than pigskin and decreases with lipophilicity.

Polycyclic aromatic hydrocarbons (PAH) are persistent environmental pollutants, which occasionally appear as contaminants in consumer products. Upon dermal contact, transfer of PAH into the stratum corneum (s.c.) and migration through the skin may occur, resulting in this class of highly toxic compounds to become bioavailable. In this study, dermal penetration through human and porcine skin of 24 PAH, comprising broad molar mass (M: 152-302 g/mol) and octanol-water partition coefficient (logP: 3.9-7.3) ranges, was evaluated via Franz diffusion cell in vitro assays. More lipophilic and potentially more toxic PAH had decreased permeation rates through the rather lipophilic s.c. into the more hydrophilic viable (epi-)dermis. Furthermore, human skin was less permeable than pigskin, a commonly used surrogate in skin penetration studies. In particular, the s.c. of human skin retains a greater share of PAH, an effect that is more pronounced for smaller PAH. Additionally, we compared the skin permeation kinetics of different PAH in pigskin. While small PAH (M < 230 g/mol, logP < 6) permeate the skin quickly and are detected in the receptor fluid after 2 h, large PAH (M > 252 g/mol, logP ≥ 6) do not fully permeate the skin up to 48 h. This indicates that highly lipophilic PAH do not become bioavailable as readily as their smaller congeners when transferred to the skin surface. Our data suggest that pigskin could be used as a surrogate for worst case scenario estimates of dermal PAH permeation through human skin.

Less efficient skin penetration of the metal allergen Pd2+ compared to Ni2+ and Co2+ from patch test preparations.

BACKGROUND: Contrary to Ni2+- and Co2+-induced allergic contact dermatitis (ACD), reactions against Pd2+ are rare. However, Pd2+ activates a larger T cell fraction in vitro, suggesting an inefficient skin penetration. OBJECTIVES: This study compares Ni2+, Co2+ and Pd2+ skin penetration from commonly used diagnostic patch test preparations (PTPs) and aqueous metal salt solutions. METHODS: Using Franz diffusion cell assays, we applied the metals in PTPs (5% NiSO4, 1% CoCl2, 2% PdCl2 and 3% Na2PdCl4) and in solution to pigskin for 48 h, thereby mirroring the time frame of a patch test. The different compartments were analysed individually by inductively coupled plasma mass spectrometry. RESULTS: Metal ions were mainly retained in the upper stratum corneum layers. After application of PTPs, concentrations in the viable skin were lower for Pd2+ (1 and 7 µM) compared to Ni2+ and Co2+ (54 and 17 µM). CONCLUSIONS: Ni2+ and Co2+ penetrated the skin more efficiently than Pd2+ and thus may sensitize and elicit ACD more easily. This was observed for ions applied in petrolatum and aqueous solutions. We hypothesize that the differently charged metal complexes are responsible for the varying skin penetration behaviours.

A multi-technique approach for the quantification of 60 plasticizers and selected additives using GC- and LC-MS/MS and its application for beverages in the BfR MEAL study.

The development of multi-analyte methods is always challenging, especially when the target compounds derive from many different substance classes. We present an approach to analyze up to 60 additives - mainly plasticizer - including 28 phthalates and 32 further compounds such as sebacates, adipates, citrates, fatty acid amides, among others. Our multi-analyte multi-technique approach combines a single sample preparation step with one GC-MS/MS and two LC-MS/MS quantification methods. We demonstrate the applicability for beverages by a full validation in tomato juice matrix and determining the recoveries in apple juice, mulled wine, and spirits. The approach features good reproducibilities and high precisions with limits of quantification in the low µg·kg-1 food range, enabling the method to be applied for enforcement and especially for exposure investigations. In course of the BfR MEAL study, 16 pooled beverage samples were examined and - if at all - analytes were found only in very low concentrations.

4-Aminobenzoic acid, 2-phenoxyethanol and iodine used as tracers in a short-term in vivo-kinetics study for tattoo ink ingredients: Mass spectrometry method development and validation.

Tattoos have been gaining popularity in recent years, leading to a growing interest in researching tattoo inks and the tattooing process itself. Since the exposure to soluble tattoo ink ingredients has not yet been investigated, we here present the method validation for a short-term biokinetics study on soluble tattoo ink ingredients. The three tracers 4-aminobenzoic acid (PABA), 2-phenoxyethanol (PEtOH) and iodine will be added to commercially available tattoo inks, which will subsequently be used on healthy study participants. Following the tattooing process, blood and urine will be sampled at specific time points and analysed for these tracers. For this purpose, a method using liquid chromatography separation coupled to a quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) in positive and negative ESI mode for the quantification of PABA, PEtOH and selected metabolites and an inductively-coupled plasma (ICP)-MS method for the determination of iodine were developed and validated. For LC-QTOF-MS analysis, the most applicable additives for LC eluents (0.01 % formic acid for positive and 0.005 % acetic acid for negative mode) were identified. Protein precipitation with acetonitrile was chosen for sample preparation. The methods were validated for selectivity, specificity, carryover, linearity, limit of detection (LOD) and quantification (LOQ), matrix effects, accuracy and precision, stability under different conditions and dilution integrity according to national and international guidelines with an allowed maximum variation of ±15 %. The LC-QTOF-MS method met the imposed guideline criteria for most parameters, however, some metabolites showed strong matrix effects. Validation of the ICP-MS method revealed that the KED-H2 collision mode is superior to the standard analysis mode due to enhanced method accuracy. The methods were validated for the relevant matrices plasma, urine, tattoo ink and tattoo consumables and proved to be applicable for the main target substances in the short-term biokinetics study. A proof-of-concept study showed successful quantification of iodine and PABA metabolites. The PEtOH metabolite was also quantified, but showed strong matrix effects in urine. Therefore standard addition was selected as an alternative quantification method.

Migration of polycyclic aromatic hydrocarbons from a polymer surrogate through the stratum corneum layer of the skin.

In this study, we determined partition (Ksc/m) and diffusion (Dsc) coefficients of five different polycyclic aromatic hydrocarbons (PAH) migrating from squalane into and through the stratum corneum (s.c.) layer of the skin. Carcinogenic PAH have previously been detected in numerous polymer-based consumer products, especially those dyed with carbon black. Upon dermal contact with these products, PAH may penetrate into and through the viable layers of the skin by passing the s.c. and thus may become bioavailable. Squalane, a frequent ingredient in cosmetics, has also been used as a polymer surrogate matrix in previous studies. Ksc/m and Dsc are relevant parameters for risk assessment because they allow estimating the potential of a substance to become bioavailable upon dermal exposure. We developed an analytical method involving incubation of pigskin with naphthalene, anthracene, pyrene, benzo[a]pyrene and dibenzo[a,h]pyrene in Franz diffusion cell assays under quasi-infinite dose conditions. PAH were subsequently quantified within individual s.c. layers by gas chromatography coupled to tandem mass spectrometry. The resulting PAH depth profiles in the s.c. were fitted to a solution of Fick's second law of diffusion, yielding Ksc/m and Dsc. The decadic logarithm logKsc/m ranged from -0.43 to +0.69 and showed a trend to higher values for PAH with higher molecular masses. Dsc, on the other hand, was similar for the four higher molecular mass PAH but about 4.6-fold lower than for naphthalene. Moreover, our data suggests that the s.c./viable epidermis boundary layer represents the most relevant barrier for the skin penetration of higher molecular mass PAH. Finally, we empirically derived a mathematical description of the concentration depth profiles that better fits our data. We correlated the resulting parameters to substance specific constants such as the logarithmic octanol-water partition coefficient logP, Ksc/m and the removal rate at the s.c./viable epidermis boundary layer.

Continuous removal of single cell layers by tape stripping the stratum corneum - a histological study.

Studies on the penetration of toxicologically or pharmaceutically relevant substances through the skin and, more specifically, through the stratum corneum (s.c.) often rely on the well-established method of tape stripping. Tape stripping involves the removal of skin layers by means of adhesive tape, which is usually followed by quantification of dermally applied substances in these layers. However, the amount of s.c. removed by each individual tape strip is still a matter of scientific debate. While some studies imply that the amount of s.c. adhering to each tape strip decreases with increasing depth into the s.c., others observed a constant removal rate. All these studies rely on the quantification of the amount of s.c. captured on individual or pooled tape strips. Here, we present an approach whereby we measured the amount of s.c. remaining on excised porcine skin in the process of tape stripping. Staining and bloating of the s.c. allowed to measure its thickness and to count individual s.c. layers, respectively. Histologically, we show that the s.c. remaining on the skin decreased linearly as a function of strips taken. We found that each tape strip removes about 0.4 µm of s.c., which corresponds to approximately one cellular layer. With a high coefficient of determination (r2 > 0.95), we were able to linearly correlate the thickness of the remaining s.c., the number of remaining cell layers and the number of tape strips applied. Furthermore, we elaborate on possible reasons for the discrepancies reported in the scientific literature regarding the amount of s.c. removed by each tape strip.

Peat and disinfectant powder used in swine husbandry systems - quantification of oral intake using toxic metals as potential markers.

The use of enrichment and bedding materials in pig husbandry intends to comply with the animals' behavioural needs to perform natural exploratory behaviour, which is strongly connected to foraging behaviour. It can thus be assumed that pigs will ingest a certain material quantity possibly posing a risk to animal health and food safety as previous studies identified contaminants in enrichment and bedding materials. However, risk assessment requires knowledge about the effective amount of ingested material. Voluntary material intake of pigs with free access to peat and disinfectant powder was estimated by measuring the tissue levels of toxic metals originating from the respective materials in 28 pigs (seven groups, n = 4) via inductively coupled plasma mass spectrometry and comparing the results to tissue levels of pigs fed with known amounts of metals. Additionally, as markers of consumption, n-alkanes and acid insoluble ash naturally occurring in the materials and titanium dioxide, added as an external marker to disinfectant powder, were analysed in pigs' faeces. Tissue levels of toxic metals as well as marker analyses in pigs' faeces could prove material consumption. Results revealed mean voluntary intake levels of peat and disinfectant powder by pigs up to 7% and 2% of the daily ration. Hence, a transfer of contained toxic metals into the food chain might occur. Although current maximum levels for toxic elements in animal tissues were not exceeded due to dietary inclusion of peat or disinfectant powder, dietary exposure through food of animal origin should be reduced to a possible minimum. This applies specifically for elements, where no health-based guidance values for humans could have been derived (e.g. arsenic). Thus, labelling guidelines for enrichment and bedding materials can be a perspective to limit the entry of toxic metals and trace elements into the environment.

Sampling Volatile Organic Compound Emissions from Consumer Products: A Review.

Volatile organic compounds (VOCs) are common constituents of many consumer products. Although many VOCs are generally considered harmless at low concentrations, some compound classes represent substances of concern in relation to human (inhalation) exposure and can elicit adverse health effects, especially when concentrations build up, such as in indoor settings. Determining VOC emissions from consumer products, such as toys, utensils or decorative articles, is of utmost importance to enable the assessment of inhalation exposure under real-world scenarios with respect to consumer safety. Due to the diverse sizes and shapes of such products, as well as their differing uses, a one-size-fits-all approach for measuring VOC emissions is not possible, thus, sampling procedures must be chosen carefully to best suit the sample under investigation. This review outlines the different sampling approaches for characterizing VOC emissions from consumer products, including headspace and emission test chamber methods. The advantages and disadvantages of each sampling technique are discussed in relation to their time and cost efficiency, as well as their suitability to realistically assess VOC inhalation exposures.

Commensal-Related Changes in the Epidermal Barrier Function Lead to Alterations in the Benzo[a]Pyrene Metabolite Profile and Its Distribution in 3D Skin.

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (B[a]P) are among the most abundant environmental pollutants, resulting in continuous exposure of human skin and its microbiota. However, effects of the latter on B[a]P toxicity, absorption, metabolism, and distribution in humans remain unclear. Here, we demonstrate that the skin microbiota does metabolize B[a]P on and in human skin in situ, using a recently developed commensal skin model. In this model, microbial metabolism leads to high concentrations of known microbial B[a]P metabolites on the surface as well as in the epidermal layers. In contrast to what was observed for uncolonized skin, B[a]P and its metabolites were subject to altered rates of skin penetration and diffusion, resulting in up to 58% reduction of metabolites recovered from basal culture medium. The results indicate the reason for this altered behavior to be a microbially induced strengthening of the epidermal barrier. Concomitantly, colonized models showed decreased formation and penetration of the ultimate carcinogen B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), leading, in consequence, to fewer BPDE-DNA adducts being formed. Befittingly, transcript and expression levels of key proteins for repairing environmentally induced DNA damage such as xeroderma pigmentosum complementation group C (XPC) were also found to be reduced in the commensal models, as was expression of B[a]P-associated cytochrome P450-dependent monooxygenases (CYPs). The results show that the microbiome can have significant effects on the toxicology of external chemical impacts. The respective effects rely on a complex interplay between microbial and host metabolism and microbe-host interactions, all of which cannot be adequately assessed using single-system studies. IMPORTANCE Exposure to xenobiotics has repeatedly been associated with adverse health effects. While the majority of reported cases relate to direct substance effects, there is increasing evidence that microbiome-dependent metabolism of xenobiotic substances likewise has direct adverse effects on the host. This can be due to microbial biotransformation of compounds, interaction between the microbiota and the host's endogenous detoxification enzymes, or altered xenobiotic bioavailability. However, there are hardly any studies addressing the complex interplay of such interactions in situ and less so in human test systems. Using a recently developed microbially competent three-dimensional (3D) skin model, we show here for the first time how commensal influence on skin physiology and gene transcription paradoxically modulates PAH toxicity.

Exposure Assessment of Toxicologically Relevant Volatile Organic Compounds Emitted from Polymer-Based Costume Masks.

Plastic costume masks regularly exhibit unpleasant odors that may be associated with the emissions of volatile organic compounds (VOCs). Upon inhalation, VOCs might adversely affect the wearer's health if the exposure exceeds regulatory threshold values. The VOCs emitted from a selection of costume masks (n = 12) were characterized semiquantitatively with a screening method based on GC/MS measurements in dynamic headspace sampling mode. Furthermore, odors associated with the masks were evaluated by a sensory panel. Two masks emitted particularly high concentrations of ethylbenzene, xylenes, and cyclohexanone and exhibited the most intense and unpleasant odors, which were described as rubber-like, pungent, and leather-like. To simulate and assess the inhalation exposures for wearers of these masks, an innovative experimental setup based on a doll's head was developed, with sampling of emitted volatiles on adsorption material and subsequent analysis by thermal desorption-GC/MS. The measured inhalable concentrations of cyclohexanone exceeded the derived no-effect level (DNEL) for systemic effects on the general population over several hours of wearing, and also after repeated use. Importantly, the cyclohexanone DNEL was reevaluated in relation to a recent study on inhalation toxicity in rodents and was found to be significantly lower (1.4 mg·m-3) compared to the industry-derived values (10-20 mg·m-3), thus aggravating the health risks associated with inhalation exposure from some of the costume masks tested. Finally, a comparison of the inhalable concentrations derived from the simulated exposure assessments with those derived from measurements in miniaturized emission test chambers indicate that microchambers represent a useful tool for high-throughput analysis. The influences of temperature and inhalation/exhalation flow rates on VOC exposures were also studied.

Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores.

Aldehyde moieties on 2D-supports or micro- and nanoparticles can function as anchor groups for the attachment of biomolecules or as reversible binding sites for proteins on cell surfaces. The use of aldehyde-based materials in bioanalytical and medical settings calls for reliable methods to detect and quantify this functionality. We report here on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and nonfluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymer microparticles with different aldehyde labeling densities. Excess reporter molecules can be easily removed by washing, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of hydrazones at acidic pH assisted by a carbonyl trap releases the fluorescent reporters rapidly and quasi-quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface. This circumvents common issues associated with light scattering and signal distortions that are caused by binding-induced changes in reporter fluorescence as well as quenching dye-dye interactions on crowded particle surfaces. In addition, we demonstrate that the release of a nonfluorescent chromophore via disulfide cleavage and subsequent quantification by absorption spectroscopy gives comparable results, verifying that both assays are capable of rapid and sensitive quantification of aldehydes on microbead surfaces. These strategies enable a quantitative comparison of bead batches with different functionalization densities, and a qualitative prediction of their coupling efficiencies in bioconjugations, as demonstrated in reductive amination reactions with Streptavidin.

Influence of compatible solute ectoine on distinct DNA structures: thermodynamic insights into molecular binding mechanisms and destabilization effects.

In nature, the cellular environment of DNA includes not only water and ions, but also other components and co-solutes, which can exert both stabilizing and destabilizing effects on particular oligonucleotide conformations. Among them, ectoine, known as an important osmoprotectant organic co-solute in a broad range of pharmaceutical products, turns out to be of particular relevance. In this article, we study the influence of ectoine on a short single-stranded DNA fragment and on double-stranded helical B-DNA in aqueous solution by means of atomistic molecular dynamics (MD) simulations in combination with molecular theories of solution. Our results demonstrate a conformation-dependent binding behavior of ectoine, which favors the unfolded state of DNA by a combination of electrostatic and dispersion interactions. In conjunction with the Kirkwood-Buff theory, we introduce a simple framework to compute the influence of ectoine on the DNA melting temperature. Our findings reveal a significant linear decrease of the melting temperature with increasing ectoine concentration, which is found to be in qualitative agreement with results from denaturation experiments. The outcomes of our computer simulations provide a detailed mechanistic rationale for the surprising destabilizing influence of ectoine on distinct DNA structures.

Self-Transfecting Micellar RNA: Modulating Nanoparticle Cell Interactions via High Density Display of Small Molecule Ligands on Micelle Coronas.

The intracellular delivery of synthetic nucleic acids represents a major challenge in biotechnology and in biomedicine. Methods to deliver short, double-stranded RNA to living cells are of particular interest because of the potential to engage the RNA interference machinery and to regulate mRNA expression. In this work, we describe novel RNA-polymer amphiphiles that assemble into spherical micellar nanoparticles with diameters of ca. 15-30 nm and efficiently enter live cells without transfection reagents. Each micelle consists of approximately 100 RNA strands forming a densely packed corona around a polymeric core. Importantly, the surface-displayed RNA remains accessible for hybridization with complementary RNA. Chemical modification of the termini of hybridized RNA strands enabled the display of small organic moieties on the outer surface of the micelle corona. We found that some of these modifications can have a tremendous impact on cellular internalization efficiencies. The display of hydrophobic dabcyl or stilbene units dramatically increased cell uptake, whereas hydrophilic neutral hydroxy or anionic phosphate residues were ineffective. Interestingly, neither of these modifications mediated noticeable uptake of free RNA oligonucleotides. We infer that their high density display on micellar nanoparticle surfaces is key for the observed effect; achieved with local effective surface concentrations in the millimolar range. We speculate that weak interactions with cell surface receptors that are amplified by the multivalent presentation of such modifications may be responsible. The installation of small molecule ligands on nanomaterial surfaces via hybridization of chemically modified oligonucleotides offers a simple and straightforward way to modulate cellular uptake of nanoparticles. Biological functionality of micellar RNA was demonstrated through the sequence-specific regulation of mRNA expression in HeLa cells.

Micellar Thrombin-Binding Aptamers: Reversible Nanoscale Anticoagulants.

Aptamers are nucleic acid-based ligands that exhibit promising features including specific and reversible target binding and inhibition. Aptamers can function as anticoagulants if they are directed against enzymes of the coagulation cascade. However, they typically suffer from nucleolytic digestion and fast clearance from the bloodstream. We present thrombin-binding aptamer amphiphiles that self-assemble into nanoscale polymeric micelles with a densely functionalized aptamer-displaying corona. We show that these micellar aptamers retain their native secondary structure in a crowded environment and are stabilized against degradation by nucleases in human serum. Moreover, they are effective inhibitors of human plasma clotting in vitro. The inhibitory effect can be rapidly reversed by complementary nucleic acids that break the aptamers' secondary structure upon hybridization. Compared to free aptamers, the increased molecular weight and size of the overall assembly promotes extended blood circulation times in vivo.

Amplification by nucleic acid-templated reactions.

Nucleic acid-templated reactions enable the design of conditional reaction systems, in which bond formation occurs only when a particular DNA or RNA molecule is present. Such reaction systems are currently being explored for applications in DNA/RNA diagnosis, drug screening and as a means to design gene expression specific therapy. However, biological nucleic acid templates usually have low abundance. Therefore, either the targeted nucleic acid template has to be multiplied by means of an amplification step or the template itself has to act as a catalyst which amplifies product formation. This critical review highlights the recent advancements in nucleic acid-templated reactions that proceed with turnover in template and thereby provide a means of amplification. Improvements in reaction engineering and the development of new chemistries have pushed the limits from 10(1) to 10(2)-10(3) turnovers. This includes reaction systems that lead to the ligation of oligonucleotides or to the interconversion of appended functional groups beyond ligation as well as templated chemistries that enable the activation of catalysts for subsequent triggering of reactions between non-nucleotidic substrates. The present limitations and future opportunities are discussed.

DNA-templated native chemical ligation of functionalized peptide nucleic acids: a versatile tool for single base-specific detection of nucleic acids.

Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.

Reducing product inhibition in nucleic acid-templated ligation reactions: DNA-templated cycligation.

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA-triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid-programmed ligation reactions. A DNA-triggered ligation-cyclization sequence ("cycligation") of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA-thioesters and isocysteinyl-PNA-cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product-template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur-halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2-3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25-0.01 equiv). We believe that cyclization of products from DNA-templated reactions could ultimately afford systems that completely overcome product inhibition.

The role of reactivity in DNA templated native chemical PNA ligation during PCR.

DNA templated fluorogenic reactions have been used as a diagnostic tool for the sequence specific detection of nucleic acids; and it has been shown that the native chemical ligation between thioester- and 1,2-aminothiol-modified PNA probes is amongst the most selective DNA detection methods reported. We explored whether a DNA templated reaction can be interfaced with the polymerase chain reaction (PCR). This endeavor posed a significant challenge. The reactive groups involved must be sufficiently stable to tolerate the high temperature applied in the PCR process. Nevertheless, the ligation reaction must proceed very rapidly and sequence specifically within the short time available in the annealing and primer extension steps before denaturation is used after approx. 1 min to commence the next PCR cycle. This required a careful optimization of the ternary complex architecture as well as adjustments of probe length and probe reactivities. Our results point to the prime importance of the ligation architecture. We show that once suitable annealing sites have been identified less reactive probe sets may be preferable if sequence specificity is of major concern. The reactivity tuning enabled the development of an in-PCR ligation, which was used for the single nucleotide specific typing of the V600E (T1799A) point mutation in the human BRaf gene. Showcasing the efficiency and sequence specificity of native chemical PNA ligation, attomolar template proofed sufficient to trigger signal while a 1000-fold higher load of single mismatched template failed to induce appreciable signal.

Evolution of synthetic polymers.

A strategy for the enrichment of a DNA template that encodes a functionalized PNA oligomer is discussed. The method relies on iterated cycles of chemical translation (of the template into PNA), selection (for function), and amplification (of the survivors). Potential restrictions and future perspectives are considered.