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The demand for sensitive and cost-effective diagnostics Analysis is steadily increasing.
Addressable immunoarrays based on matching oligo-deoxynucleotide (ODN) strands have been described before 1,2. Short ODNs (e.g. 10 mers) could be used for addressing but are not stable enough to resist washing steps which lead to a sensitivity reduction.
The demand for sensitive and cost-effective diagnostic tools is steadily increasing. Short ODNs (e.g. 10mers) could be used for addressable immunoarrays 1,2 but are not stable enough to resist washing steps which lead to a sensitivity reduction. Our aim is to establish a new platform for addressable immuno-arrays using shorter ODNs by increasing their stability via covalent linking. The main strategy consisted in the use of furan-modified ODNs that can be selectively oxidized leading to the formation of a site-specific interstrand cross-link (ICL)
Comparison of the surface plasmon resonance (SPR) response shows successful ICL of short DNA duplex on surface. After ICL formation only the binding of the cross-linked DNA duplex with the recognition antibody is stable after regeneration using Na2CO3 (Fig. 1).
Figure1. Method for detection of DNA ICL formation using an FITC labeled ODN and an anti-FITC antibody as model system a) Hybridized ODNs are efficiently separated after using Na2CO3 for regeneration b) Oxidation of the furan using 1O2 results in the formation of a stable ICL, which makes the short ODN duplex stable after regeneration
Quality control requirements imposed on assays used in clinical diagnostics and point-of-care-diagnostic testing (POCT), utilizing amplification reactions performed at elevated temperatures of 35 to 95 °C are very stringent. As the temperature of a reaction vessel has a large impact on the specificity and sensitivity of the amplification reaction, simple tools for local in situ temperature sensing and monitoring are required for reaction and assay control. We describe here a platform of stem-and-loop structured DNA hairpins (molecular beacons, MBs), absorbing and emitting in the visible and red spectral region, rationally designed for precise temperature measurements in microfluidic assays for POCT, and their ap-plication for temperature measurements in a common DNA-based molecular biological assay utilizing thermophilic helicase-dependent amplification (tHDA). Spectroscopic studies of these MBs, rationally designed from DNA se-quences of different thermal stabilities, chosen not to interact with the DNA probes applied in the nucleic acid amplification assay, and temperature-dependent fluorescence measurements of MB-assay mixtures revealed the suitability of these MBs for temperature measurements directly in such an assay with a temperature resolution of about 0.5 °C without interferences from assay components. Combining two spectrally distinguishable MBs provides a broader response range and an increase in temperature sensitivity up to 0.1 °C. This approach will find future application for temperature monitoring and quality control in commercialized diagnostics assays using dried reagents and microfluidic chips as well as assays read out with tube and microplate readers and PCR detection systems for temperature measurements in the range of 35 to 95 °C.
Multiply negatively charged DNA oligonucleotides of small sizes (n=15-40) have been subjected to a comprehensive tandem mass spectrometric study (MS/MS). Collision induced dissociation (CID) mass spectrometry (MS) was applied as a tool to break down isolated DNA oligonucleotides with a defined number of charges. Various lenghts and sequences were analyzed and all of them showed a comparable direct correlation regarding the threshold collision energy for fragmentation and dependence on number of charges on the precursor ions. It was also revealed that the increase in charges and thus Coulomb repulsion results in the transition from a folded, compact form to an elongated structure of the precursor ions.
Multiply negatively charged DNA oligonucleotides of small sizes (n=15-40 bases) have been subjected to a comprehensive tandem mass spectrometric study (MS/MS). Collision induced dissociation (CID) mass spectrometry (MS) was applied as a tool to break down isolated DNA oligonucleotides with a defined number of charges. Various lenghts and sequences were analyzed unravelling a comparable direct correlation between the threshold collision energy for fragmentation and the charge-per-base density in the precusor ion. It was also revealed that the increase in charges and thus Coulomb repulsion results in the transition from a folded, compact form to an elongated structure of the precusor ions.
The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine geant4 particle-scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E1/2=6±4 eV. The presented method is applicable for ionizing radiation (e.g., γ rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells.
Electron beam therpy is used to treat tumor cells by irradiation with high energy electrons (HEE). The irradiation is performed by medical linear accelerators.
By interaction of the HEE with the irradiated tissue a broad spectra of secondary electrons and water dissoziation products is generated.
These products interact with biomolecules, especially DNA, by various processes. The disentanglement of the resulting direct- and indirect damage to DNA and other cellular components is still under debate.
To increase the efficiency of future therapies a better understanding of the microscopic damaging processes is highly important.
Especially the various contributions of the secondary species produced, such as low energy electrons (LEE) and radicals is far from understood and quantified.
To increase the understanding we present a combination of a microdosimetric simulations and experiments to quantify the damage by means of electron scattering and diffusion simulations within the Geant4-DNA framework.
In combination with in-liquid irradiation of plasmid DNA it possible to determine microdosimetric quantities for biomolecules in liquid environment.
The presented method was applied to the irradiation of plasmid DNA (pUC19) in water.
This opens up new possibilities in radiation research to quantify the dosage-damage relationship for microscopic plasmid volumes under well defined physiological condition.
The damage caused by ionizing radiation to biomolecules, especially DNA, is the reason to treat cancer via radiation therapy. A better understanding of the molecular processes and the quantification of the various damaging mechanisms is the prerequisit to develope efficient therapies. Hereby the understanding of the processes involved in the damage to DNA are of key interest due to its central role in reproduction and mutation. Due to the high amount of water in biological tissue, most of the damage is caused by the secondary particles produced by the interaction of the IR with water. Thereby a multitude of species are produced, e.g. kinetic low energy electrons, prehydrated electrons, OH-radicals and ions. The quantification of the contribution to DNA damage of the various species is of interest. Here we present an experimental approach to disentangle their relative DNA strand break yield. Plasmid DNA is irradiated in water with electrons under the presence of different scavengers. With the presented method it will be possible to reveal the relative contributions of OH-radicals, low energy electrons and prehydrated electrons to the DNA single and double strand break yield.