TY - JOUR A1 - Kienzler, Andrea Altevogt Nee A1 - Flehr, Roman A1 - Gehne, Sören A1 - Kumke, Michael Uwe A1 - Bannwarth, Willi T1 - Verification and biophysical characterization of a New Three-Color Forster Resonance-Energy-Transfer (FRET) System in DNA JF - Helvetica chimica acta N2 - We report on a new three-color FRET system consisting of three fluorescent dyes, i.e., of a carbostyril (=quinolin-2(1H)-one)-derived donor D, a (bathophenanthroline)ruthenium complex as a relay chromophore A1, and a Cy dye as A2 (FRET=Forster resonance-energy-transfer) (cf. Fig. 1). With their widely matching spectroscopic properties (cf. Fig. 2), the combination of these dyes yielded excellent FRET efficiencies. Furthermore, fluorescence lifetime measurements revealed that the long fluorescence lifetime of the Ru complex was transferred to the Cy dye offering the possibility to measure the whole system in a time-resolved mode. The FRET system was established on double-stranded DNA (cf. Fig. 3) but it should also be generally applicable to other biomolecules. KW - Forster resonance energy transfer (FRET) system KW - DNA KW - Fluorescence KW - Ruthenium complexes Y1 - 2012 U6 - https://doi.org/10.1002/hlca.201100460 SN - 0018-019X VL - 95 IS - 4 SP - 543 EP - 555 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Abouzar, Maryam H. A1 - Poghossian, Arshak A1 - Cherstvy, Andrey G. A1 - Pedraza, Angela M. A1 - Ingebrandt, Sven A1 - Schöning, Michael J. T1 - Label-free electrical detection of DNA by means of field-effect nanoplate capacitors experiments and modeling JF - Physica status solidi : A, Applications and materials science N2 - Label-free electrical detection of consecutive deoxyribonucleic acid (DNA) hybridization/denaturation by means of an array of individually addressable field-effect-based nanoplate silicon-on-insulator (SOI) capacitors modified with gold nanoparticles (Au-NP) is investigated. The proposed device detects charge changes on Au-NP/DNA hybrids induced by the hybridization or denaturation event. DNA hybridization was performed in a high ionic-strength solution to provide a high hybridization efficiency. On the other hand, to reduce the screening of the DNA charge by counter ions and to achieve a high sensitivity, the sensor signal induced by the hybridization and denaturation events was measured in a low ionic-strength solution. High sensor signals of about 120, 90, and 80 mV were registered after the DNA hybridization, denaturation, and re-hybridization events, respectively. Fluorescence microscopy has been applied as reference method to verify the DNA immobilization, hybridization, and denaturation processes. An electrostatic charge-plane model for potential changes at the gate surface of a nanoplate field-effect sensor induced by the DNA hybridization has been developed taking into account both the Debye length and the distance of the DNA charge from the gate surface. KW - DNA KW - field-effect KW - gold nanoparticle KW - label-free detection KW - nanoplate capacitor Y1 - 2012 U6 - https://doi.org/10.1002/pssa.201100710 SN - 1862-6300 VL - 209 IS - 5 SP - 925 EP - 934 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Cherstvy, Andrey G. A1 - Teif, Vladimir B. T1 - Electrostatic effect of H1-histone protein binding on nucleosome repeat length JF - Physical biology : a journal for the fundamental understanding of biological systems N2 - Within a simple biophysical model we describe the effect of electrostatic binding of H1 histone proteins on the nucleosome repeat length in chromatin. The length of wrapped DNA optimizes its binding energy to the histone core and the elastic energy penalty of DNA wrapping. The magnitude of the effect predicted from our model is in agreement with the systematic experimental data on the linear variation of nucleosome repeat lengths with H1/nucleosome ratio (Woodcock C L et al 2006 Chromos. Res. 14 17-25). We compare our model to the data for different cell types and organisms, with a widely varying ratio of bound H1 histones per nucleosome. We underline the importance of this non-specific histone-DNA charge-balance mechanism in regulating the positioning of nucleosomes and the degree of compaction of chromatin fibers in eukaryotic cells. KW - electrostatics KW - DNA KW - nucleosome Y1 - 2014 U6 - https://doi.org/10.1088/1478-3975/11/4/044001 SN - 1478-3967 SN - 1478-3975 VL - 11 IS - 4 PB - IOP Publ. Ltd. CY - Bristol ER - TY - JOUR A1 - Li, Chenhong A1 - Corrigan, Shannon A1 - Yang, Lei A1 - Straube, Nicolas A1 - Harris, Mark A1 - Hofreiter, Michael A1 - White, William T. A1 - Naylor, Gavin J. P. T1 - DNA capture reveals transoceanic gene flow in endangered river sharks JF - Proceedings of the National Academy of Sciences of the United States of America N2 - For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks. KW - freshwater sharks KW - DNA KW - museum specimens Y1 - 2015 U6 - https://doi.org/10.1073/pnas.1508735112 SN - 0027-8424 VL - 112 IS - 43 SP - 13302 EP - 13307 PB - National Acad. of Sciences CY - Washington ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Ermilova, Elena A1 - Pannwitz, Daniel A1 - Gibbons, Jessica A1 - Hölzel, Ralph A1 - Bier, Frank Fabian T1 - Dielectric Spectroscopy of Biomolecules up to 110 GHz JF - Frequenz N2 - Radio-frequency fields in the GHz range are increasingly applied in biotechnology and medicine. In order to fully exploit both their potential and their risks detailed information about the dielectric properties of biological material is needed. For this purpose a measuring system is presented that allows the acquisition of complex dielectric spectra over 4 frequency decade up to 110 GHz. Routines for calibration and for data evaluation according to physicochemical interaction models have been developed. The frequency dependent permittivity and dielectric loss of some proteins and nucleic acids, the main classes of biomolecules, and of their sub-units have been determined. Dielectric spectra are presented for the amino acid alanine, the proteins lysozyme and haemoglobin, the nucleotides AMP and ATP, and for the plasmid pET-21, which has been produced by bacterial culture. Characterisation of a variety of biomolecules is envisaged, as is the application to studies on protein structure and function. KW - dielectric KW - spectroscopy KW - permittivity KW - protein KW - DNA KW - amino acid KW - plasmid Y1 - 2018 U6 - https://doi.org/10.1515/freq-2018-0010 SN - 0016-1136 SN - 2191-6349 VL - 72 IS - 3-4 SP - 135 EP - 140 PB - De Gruyter CY - Berlin ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - Dielectrophoretic Stretching of DNA JF - DNA Nanotechnology N2 - The spatial control of DNA and of self-assembled DNA constructs is a prerequisite for the preparation of DNA-based nanostructures and microstructures and a useful tool for studies on single DNA molecules. Here we describe a protocol for the accumulation of dissolved lambda-DNA molecules between planar microelectrodes by the action of inhomogeneous radiofrequency electric fields. The resulting AC electrokinetic forces stretch the DNA molecules and align them parallel to the electric field. The electrode preparation from off-the-shelf electronic components is explained, and a detailed description of the electronic setup is given. The experimental procedure is controlled in real-time by fluorescence microscopy. KW - Alignment KW - Dielectrophoresis KW - DNA KW - Electrokinetics KW - Interdigitated electrodes KW - Stretching Y1 - 2018 SN - 978-1-4939-8582-1 SN - 978-1-4939-8581-4 U6 - https://doi.org/10.1007/978-1-4939-8582-1_14 SN - 1064-3745 SN - 1940-6029 SP - 199 EP - 208 PB - Humana Press Inc. CY - New York ET - 2 ER - TY - JOUR A1 - Kasyanenko, Nina A1 - Unksov, Ivan A1 - Bakulev, Vladimir A1 - Santer, Svetlana T1 - DNA interaction with head-to-tail associates of cationic surfactants prevents formation of compact particles JF - Molecules N2 - Cationic azobenzene-containing surfactants are capable of condensing DNA in solution with formation of nanosized particles that can be employed in gene delivery. The ratio of surfactant/DNA concentration and solution ionic strength determines the result of DNA-surfactant interaction: Complexes with a micelle-like surfactant associates on DNA, which induces DNA shrinkage, DNA precipitation or DNA condensation with the emergence of nanosized particles. UV and fluorescence spectroscopy, low gradient viscometry and flow birefringence methods were employed to investigate DNA-surfactant and surfactant-surfactant interaction at different NaCl concentrations, [NaCl]. It was observed that [NaCl] (or the Debye screening radius) determines the surfactant-surfactant interaction in solutions without DNA. Monomers, micelles and non-micellar associates of azobenzene-containing surfactants with head-to-tail orientation of molecules were distinguished due to the features of their absorption spectra. The novel data enabled us to conclude that exactly the type of associates (together with the concentration of components) determines the result of DNA-surfactant interaction. Predomination of head-to-tail associates at 0.01 M < [NaCl] < 0.5 M induces DNA aggregation and in some cases DNA precipitation. High NaCl concentration (higher than 0.8 M) prevents electrostatic attraction of surfactants to DNA phosphates for complex formation. DAPI dye luminescence in solutions with DNA-surfactant complexes shows that surfactant tails overlap the DNA minor groove. The addition of di- and trivalent metal ions before and after the surfactant binding to DNA indicate that the bound surfactant molecules are located on DNA in islets. KW - azobenzene trimethylammonium bromide KW - head-to-tail surfactant associates KW - DNA KW - ionic strength KW - multivalent ions Y1 - 2018 U6 - https://doi.org/10.3390/molecules23071576 SN - 1420-3049 VL - 23 IS - 7 PB - MDPI CY - Basel ER - TY - JOUR A1 - Fudickar, Werner A1 - Bauch, Marcel A1 - Ihmels, Heiko A1 - Linker, Torsten T1 - DNA-triggered enhancement of singlet oxygen production by pyridinium alkynylanthracenes JF - Chemistry - a European journal N2 - There is an ongoing interest in O-1(2) sensitizers, whose activity is selectively controlled by their interaction with DNA. To this end, we synthesized three isomeric pyridinium alkynylanthracenes 2 o-p and a water-soluble trapping reagent for O-1(2). In water and in the absence of DNA, these dyes show a poor efficiency to sensitize the photooxygenation of the trapping reagent as they decompose due to electron transfer processes. In contrast, in the presence of DNA O-1(2) is generated from the excited DNA-bound ligand. The interactions of 2 o-p with DNA were investigated by thermal DNA melting studies, UV/vis and fluorescence spectroscopy, and linear and circular dichroism spectroscopy. Our studies revealed an intercalative binding with an orientation of the long pyridyl-alkynyl axis parallel to the main axis of the DNA base pairs. In the presence of poly(dA : dT), all three isomers show an enhanced formation of singlet oxygen, as indicated by the reaction of the latter with the trapping reagent. With green light irradiation of isomer 2 o in poly(dA : dT), the conversion rate of the trapping reagent is enhanced by a factor >10. The formation of O-1(2) was confirmed by control experiments under anaerobic conditions, in deuterated solvents, or by addition of O-1(2) quenchers. When bound to poly(dG : dC), the opposite effect was observed only for isomers 2 o and 2 m, namely the trapping reagent reacted significantly slower. Overall, we showed that pyridinium alkynylanthracenes are very useful intercalators, that exhibit an enhanced photochemical O-1(2) generation in the DNA-bound state. KW - Anthracene KW - DNA KW - intercalations KW - photochemistry KW - singlet oxygen Y1 - 2021 U6 - https://doi.org/10.1002/chem.202101918 SN - 1521-3765 VL - 27 IS - 54 SP - 13591 EP - 13604 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Heinsohn, Natascha Katharina A1 - Niedl, Robert Raimund A1 - Anielski, Alexander A1 - Lisdat, Fred A1 - Beta, Carsten T1 - Electrophoretic mu PAD for purification and analysis of DNA samples JF - Biosensors : open access journal N2 - In this work, the fabrication and characterization of a simple, inexpensive, and effective microfluidic paper analytic device (mu PAD) for monitoring DNA samples is reported. The glass microfiber-based chip has been fabricated by a new wax-based transfer-printing technique and an electrode printing process. It is capable of moving DNA effectively in a time-dependent fashion. The nucleic acid sample is not damaged by this process and is accumulated in front of the anode, but not directly on the electrode. Thus, further DNA processing is feasible. The system allows the DNA to be purified by separating it from other components in sample mixtures such as proteins. Furthermore, it is demonstrated that DNA can be moved through several layers of the glass fiber material. This proof of concept will provide the basis for the development of rapid test systems, e.g., for the detection of pathogens in water samples. KW - microfluidic paper analytic device (mu PAD) KW - patterning glass microfiber KW - fiber-electrophoresis chip KW - DNA KW - imprinted electrodes KW - cross layer chip KW - polymerase chain reaction (PCR) KW - purification Y1 - 2022 U6 - https://doi.org/10.3390/bios12020062 SN - 2079-6374 VL - 12 IS - 2 PB - MDPI CY - Basel ER - TY - THES A1 - Gonzalez Duran, Enrique T1 - Genetic control of intracellular gene transfer by DNA repair in N. tabacum N2 - Mitochondria and plastids are organelles with an endosymbiotic origin. During evolution, many genes are lost from the organellar genomes and get integrated in the nuclear genome, in what is known as intracellular/endosymbiotic gene transfer (IGT/EGT). IGT has been reproduced experimentally in Nicotiana tabacum at a gene transfer rate (GTR) of 1 event in 5 million cells, but, despite its centrality to eukaryotic evolution, there are no genetic factors known to influence the frequency of IGT in higher eukaryotes. The focus of this work was to determine the role of different DNA repair pathways of double strand break repair (DSBR) in the integration step of organellar DNA in the nuclear genome during IGT. Here, a CRISPR/Cas9 mutagenesis strategy was implemented in N. tabacum, with the aim of generating mutants in nuclear genes without expected visible phenotypes. This strategy led to the generation of a collection of independent mutants in the LIG4 (necessary for non-homologous end joining, NHEJ) and POLQ genes (necessary for microhomology mediated end joining, MMEJ). Targeting of other DSBR genes (KU70, KU80, RPA1C) generated mutants with unexpectedly strong developmental phenotypes.. These factors have telomeric roles, hinting towards a possible relationship between telomere length, and strength of developmental disruption upon loss of telomere structure in plants. The mutants were made in a genetic background encoding a plastid-encoded IGT reporter, that confers kanamycin resistance upon transfer to the nucleus. Through large scale independent experiments, increased IGT from the chloroplast to the nucleus was observed in lig4 mutants, as well as lines encoding a POLQ gene with a defective polymerase domain (polqΔPol). This shows that NHEJ or MMEJ have a double-sided relationship with IGT: while transferred genes may integrate using either pathway, the presence of both pathways suppresses IGT in wild-type somatic cells, thus demonstrating for the first time the extent on which nuclear genes control IGT frequency in plants. The IGT frequency increases in the mutants are likely mediated by increased availability of double strand breaks for integration. Additionally, kinetic analysis reveals that gene transfer (GT) events accumulate linearly as a function of time spent under antibiotic selection in the experiment, demonstrating that, contrary to what was previously thought, there is no such thing as a single GTR in somatic IGT experiments. Furthermore, IGT in tissue culture experiments appears to be the result of a "race against the clock" for integration in the nuclear genome, that starts when the organellar DNA arrives to the nucleus granting transient antibiotic resistance. GT events and escapes of kanamycin selection may be two possible outcomes from this race: those instances where the organellar DNA gets to integrate are recovered as GT events, and in those cases where timely integration fails, antibiotic resistance cannot be sustained, and end up considered as escapes. In the mutants, increased opportunities for integration in the nuclear genome change the overall ratio between IGT and escape events. The resources generated here are promising starting points for future research: (1) the mutant collection, for the further study of processes that depend on DNA repair in plants (2) the collection of GT lines obtained from these experiments, for the study of the effect of DSBR pathways over integration patterns and stability of transferred genes and (3) the developed CRISPR/Cas9 workflow for mutant generation, to make N. tabacum meet its potential as an attractive model for answering complex biological questions. N2 - Mitochondrien und Plastiden sind beides Organellen endosymbiotischen Ursprungs. Im Laufe der Evolution gehen viele Gene aus den Organellengenomen verloren und werden in das Kerngenom integriert, was als intrazellulärer/endosymbiotischer Gentransfer (IGT/EGT) bezeichnet wird. IGT konnte experimentell in Nicotiana tabacum mit einer Gentransferrate (GTR) von einem Ereignis in fünf Millionen Zellen nachgestellt werden, aber trotz seiner zentralen Bedeutung für die eukaryotische Evolution sind keine genetischen Faktoren bekannt, die die Häufigkeit von IGT in höheren Eukaryoten beeinflussen. Der Schwerpunkt dieser Arbeit lag auf der Bestimmung der Rolle verschiedener DNA-Reparaturwege der Doppelstrangbruchreparatur (DSBR) bei der Integration von Organellen-DNA in das Kerngenom während des IGT. Dazu wurde in N. tabacum eine CRISPR/Cas9-basierte Mutagenesestrategie angewandt, mit dem Ziel, Mutanten in Kerngenen zu erzeugen, für die keine sichtbaren Phänotypen zu erwarten sind. Diese Strategie führte zur Erzeugung einer Reihe unabhängiger Mutanten im LIG4-Gen (notwendig für „non-homologous end joining“, die nicht-homologe Verbindung von Enden, NHEJ) und POLQ (notwendig für „microhomology mediated end joining“, die Mikrohomologie-vermittelte Verbindung von Enden, MMEJ). Die gezielte Beeinflussung anderer DSBR-Gene (KU70, KU80, RPA1C) führte zu Mutanten mit unerwartet starken Entwicklungsphänotypen. Diese Gene spielen eine Rolle beim Erhalt der Telomere, was auf einen möglichen Zusammenhang zwischen der Telomerlänge und der Stärke der Entwicklungsstörung bei Verlust der Telomerstruktur in Pflanzen hindeutet. Die Mutanten wurden in einem genetischen Hintergrund erzeugt, der über einen in den Plastiden lokalisierten IGT-Reporter verfügt, der nach Übertragung in den Zellkern Kanamycin-Resistenz vermittelt. In groß angelegten unabhängigen Experimenten wurde in lig4-Mutanten sowie in Linien, die für ein POLQ-Gen mit einer defekten Polymerase-Domäne (polqΔPol) kodieren, eine erhöhte GTR vom Chloroplasten zum Zellkern beobachtet. Dies zeigt, dass NHEJ oder MMEJ eine zweischneidige Beziehung zum IGT haben: Während übertragene Gene folglich über jeden der beiden Mechanismen integriert werden können, unterdrückt das gleichzeitige Vorhandensein beider Wege IGT in somatischen Wildtyp-Zellen, wodurch zum ersten Mal gezeigt wird, in welchem Ausmaß Kerngene die IGT-Häufigkeit in Pflanzen kontrollieren. Die erhöhte Verfügbarkeit von Doppelstrangbrüchen für die Integration könnte für die erhöhte IGT-Häufigkeit in den Mutanten verantwortlich sein. Darüber hinaus zeigt die Analyse des Zeitverlaufs, dass Gentransferereignisse (GT) in Abhängigkeit von der Zeit, die im Experiment unter Antibiotikaselektion verbracht wurde, linear akkumulieren, was beweist, dass es, anders als bisher angenommen, in somatischen IGT-Experimenten keine statische GTR gibt. Darüber hinaus scheint IGT in Gewebekulturexperimenten das Ergebnis eines Wettlaufs mit der Zeit um die Integration in das Kerngenom zu sein, der beginnt, wenn die Organellen-DNA in den Zellkern gelangt und eine vorübergehende Antibiotikaresistenz gewährt. Echte GT-Ereignisse und „Escapes“ (scheinbare Resistenz, ein vorläufiges Ausweichen vor der Kanamycin-Selektion) können zwei mögliche Ergebnisse dieses Wettlaufs sein: Die Fälle, in denen die Organellen-DNA integriert wird, werden als GT-Ereignisse gewertet, und in den Fällen, in denen die rechtzeitige Integration scheitert, kann die Antibiotikaresistenz nicht aufrechterhalten werden und sie werden als „Escape“ betrachtet. In den Mutanten verändern sich die Möglichkeiten zur Integration in das Kerngenom, wodurch sich das Gesamtverhältnis zwischen IGT- und „Escape“-Ereignissen ändert. Die hier erzeugten Ressourcen sind vielversprechende Ausgangspunkte für künftige Forschungen: (1) die Mutantensammlung, für die Untersuchung von weiteren Prozessen, die von der DNA-Reparatur in Pflanzen abhängen, (2) die Sammlung von GT-Linien, die aus den hier beschriebenen Experimenten gewonnen wurden, für die Untersuchung der Auswirkungen von DSBR-Mechanismen auf Integrationsmuster und Stabilität übertragener Gene und (3) der hier entwickelte Arbeitsablauf für die Mutantenerzeugung mittels CRISPR/Cas9, damit N. tabacum sein Potenzial als attraktives Modell für die Beantwortung komplexer biologischer Fragestellungen erfüllen kann. T2 - Genetische Kontrolle des intrazellulären Gentransfers durch DNA-Reparatur in N. tabacum KW - endosymbiosis KW - organelles KW - gene KW - transfer KW - DNA KW - repair KW - genome KW - editing KW - evolution KW - plant Y1 - 2023 ER -