Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

The state-of-the-art in the area of modelling of organisations is based on fixed metamodels. Due to rapid changing business requirements the complexity in developing applications which deliver business solutions is continually growing. To manage this complexity, environments providing flexible metamodelling capabilities instead of fixed metamodels has shown to be helpful. The main characteristic of such environments is that the formalism of modelling -the metamodel -can be freely defined and therefore be adapted to the problem under consideration. This paper gives an introduction into metamodelling concepts and presents a generic architecture for metamodelling platforms. Three best practice examples from industry projects applying metamodelling concepts in the area of business process modelling for e-business, e-learning, and knowledge management are presented. Finally, an outlook to future developments and research directions in the area of metamodelling is given.

The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations caused by the oxidatively damaged lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) by removal of misincorporated adenine residues in OG:A mismatched base pairs using N-glycosylase activity. MutY also has glycosylase activity toward adenine in the mismatched base-pairs G:A and C:A. We have investigated the interaction of MutY with DNA duplexes containing the 2′-deoxyadenosine (A) analogs 2′-deoxytubercidin (7-deaza-2′-deoxyadenosine, Z) and 2′-deoxyformycin A (F). Both F and Z should effectively mimic the recognition properties of A but be resistant to the glycosylase activity of MutY, owing to their structural properties. Thus, these derivatives will provide a method for forming a stable MutY-substrate analog complex amenable to structural and biochemical investigation. We find that oligonucleotide duplexes containing OG/G:F and OG/G:Z base-pairs are not substrates for MutY as expected. Using a gel retardation method to measure relevant K d values, we determined that MutY has an increased association with duplexes containing OG/G:F and OG/G:Z base-pairs over their OG/G:C counterparts. Interestingly, MutY has a higher affinity for the F-containing duplexes than the Z counterparts. Additionally, MutY binds to the OG:F and G:F duplexes with a similar, albeit lower, affinity as the substrate OG:A and G:A duplexes. In footprinting experiments using methidiumpropyl-EDTA-Fe(II), a region of the duplex surrounding the OG:F base-pair is observed which is protected by MutY from hydroxyl radical cleavage. These results provide additional evidence for specific recognition of the OG:F base-pair within the DNA duplex. Furthermore, these results also illustrate the utility of OG:F duplexes for providing information regarding the MutY-mismatched DNA complex which could not be obtained with the normal OG:A substrate since a footprint on both strands of the duplex could only be observed with the OG:Fcontaining duplex. These substrate analog duplexes will provide avenues for structural analysis of the MutYmismatched DNA complex and for investigating the properties of the unusual [4Fe-4S] center in MutY.

We demonstrate a purely electrical method for single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes, in order to achieve high affinity and sequence-specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics, as well as in pathogen detection for fighting infectious diseases. KeywordsPNA; invasion; sequence detection; nanopore; DNA Numerous techniques in life sciences, biotechnology, medicine, and forensics are based on nucleic acid hybridization. The invention of nucleic acid analogs with improved hybridization affinity, hybridization rate, and/or mismatch discrimination as compared to natural nucleic acids, has significantly extended the diagnostic utilities of these applications. Peptide nucleic acids (PNAs), a prominent class of artificial nucleic acid analogs, are neutral, oligomers with peptide-like backbone onto which nucleobases are grafted in a designed sequence. Moreover, bis-PNA molecules, consisting of two PNA oligomers connected by a flexible linker, spontaneously invade double-stranded DNA (dsDNA) molecules, binding to one of the two dsDNA strands with high affinity and sequence-specificity, owing to the simultaneous formation of Watson-Crick and Hoogsteen base-pairs 1-3 . This high affinity and sequencespecificity makes bis-PNA, and other synthetic variants (e.g., pseudocomplementary PNA 2, 4 and γ-PNA 5, 6 ) extremely promising sequence-tagging candidates for analysis of individual dsDNA fragments. Single-molecule mapping methods, which detect and localize PNA/DNA hybridization on minute quantities of dsDNA can lead to cheaper and faster pathogen and mutation diagnostics platforms. Low-cost and high speed platforms are essential for effective response to emerging threats of infection and will ultimately result in more accurate treatment, as well as an overall decrease in morbidity and mortality. While a

Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.