Specificity of DNA triple helix formation analyzed by a FRET assay

BACKGROUND: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis. RESULTS: We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 × 10(5) and 2.3 × 10(6) M(-1). Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg(2+)ions whereas it is inhibited by K(+)ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 × 10(4) and 8.1 × 10(4) min(-1) M(-1). Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation. CONCLUSION: We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA

A solid‐phase transfection platform for arrayed CRISPR screens [Corrigendum]

Since the publication of this study, it has come to our attention that a citation to the study by Bulkescher et al (2017) was omitted from the Introduction. The following sentence should have been included in the introduction: “A previously reported solid‐phase reverse transfection method for proteins (Bulkescher et al , 2017) was used for the delivery of RNPs for three endogenous genes suggesting the potential of solid‐phase reverse transfection for CRISPR/Cas9‐based gene editing, despite its low efficiency”. We apologise for any inconvenience this omission may have caused

A solid-phase transfection platform for arrayed CRISPR screens

Arrayed CRISPR‐based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid‐phase transfection platform that enables CRISPR‐based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high‐throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies

Automatisierte Beurteilung von Korrosion-Inhibitoren und Festkörper Batterien dotierter Garnet Typ Electrolyte Li7La3Zr2O12

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheDiese Arbeit ist an der TU Wien und an der ETH Zürich entstanden und besteht daher aus zwei Teilen. Der erste Teil wurde an der TU Wien durchgeführt und beschäftigt sich mit der Methodenverbesserung der Zyklovoltammetrie. Diese Methode wird für Tests der Inhibitorwirkung gegen Korrosion angewandt, um das Korrosionspotential und den Korrosionsstrom zu berechnen. Dazu müssen die gewonnenen Daten transformiert werden um den Tafel Plot zu erhalten und lineare Regressionen müssen an die Kurve gelegt werden. Dieser Prozess ist oft nicht einheitlich durchgeführt, da die Kriterien für die Regression nicht genau festgelegt sind. Im Rahmen dieser Arbeit wurde daher ein Computerprogramm basierend auf MATLAB entwickelt, weches die einheitliche Auswertung übernimmt. Ein großer Vorteil dieser Automatsierung ist die dadurch erbrachte Zeitersparnis. Die Auswertung einer Messreihe mit zwei Zyklen konnte in weniger als einer Minute ausgewertet werden. Das erstellte Programm konnte sogar stark rauschende Signale verwerten. Der zweite Teil der Arbeit wurde an der ETH Zürich durchgeführt und beschäftigte sich mit Li7La3Zr2O12 (LLZO), einem Feststoffelektrolyt mit Granat_Struktur. Durch Einbringen geringer Mengen an bestimmten Fremdelementen wie z.B. Ga kann die Struktur von tetragonal auf kubisch geändert werden. Die kubische Phase ist als Elektrolyt für Li-Ionen-Batterien sehr interessant, da sie Li_Ionen gut leitet. Im Rahmen dieser Arbeit wurde an neuen Dotierungen geforscht und die erste funktionierende Batterie mit Feststoffelektrolyt hergestellt. Mit dieser Batterie wurde auch ein LED-Lämpchen zum Leuchten gebracht.This work was executed at Vienna University of Technology and ETH Zürich. Therefore, it is split in two parts. The first part was done at Vienna University of Technology and was dedicated to the method refinement of cyclic voltammetry. This method is used for corrosion inhibitor testing, especially for finding the corrosion potential and the corrosion current. First, the data needs to be transformed to get the Tafel plot. Next, the linear regression needs to be done. There does not exist an uniform method, therefore the regression might differ with the operator. In this work a computer program based on MATLAB for data evaluation was developed. The big advantage is the saving of evaluation-time. A measurement with two cycles was evaluated in less than a minute. Even noisy data could be processed. The second part of this work was done at ETH Zürich and was dedicated to the garnet-type electrolyte Li7La3Zr2O12 (LLZO). With the help of small amounts of doping, e.g. Ga, the structure could be changed from tetragonal to cubic. This cubic structure is interesting because of it high ionic conductivity for Li_ions. This phase can be used for the development of new all solid state Li_ion-batteries. In this work new dopings were investigated. Furthermore, the first solid state battery was built and operated. This battery was also able to light a LED.11

Reprogramming of 5-methyl-cytosin in mammals

Die Methylierung von DNA spielt in vielen essentiellen Prozessen, so auch in der Entwicklung eines Lebewesens, eine entscheidende Rolle. Sie wird zumindest nach der Befruchtung und während der Entwicklung der Keimzellen schnell, effizient und replikationsunabhängig reprogrammiert. Ich konnte einen Versuchsansatz entwickeln, mit dessen Hilfe man den biochemischen Mechanismus, und die an diesem Prozess beteiligten Komponenten studieren kann. Außerdem war ich in der Lage zu zeigen, dass die Erhaltung der Methylierung von drei verschiedenen repetitiven DNA-Elementen des Mausgenoms während der Entwicklung der Keimzellen unterschiedlich effizient ist. Dazu habe ich ein Modell entworfen, das zeigt, wie der Histonkode im Zusammenspiel mit den Methyltransferasen zu dem jeweiligen Verlust oder Erhalt der Methylierung in diesen Regionen beitragen kann. Die Untersuchung einer Familie mit männlichen monozygoten Zwillingen, diskordant für BWS, ergab, dass eine Hypomethylierung im IC2 der BWS Region nicht exklusiv bei weiblichen monozygoten Zwillingen, die diesen Phänotyp zeigen, auftritt. In diesem Zusammenhang konnte ich feststellen, dass in dem Promotorbereich des humanen Lit1 Gens des gesunden Zwillings zwei CpGs in den unmethylierten Klonen methyliert sind. Dies ist ein Hinweis auf einen möglichen Regulationsmechanismus dieser Region. Zudem habe ich ein Ungleichgewicht in der Expression von Igf2 und CDKN1C detektiert, was evtl. mit dem Auftreten von BWS in Zusammenhang steht.In mammals methylation is an important developmental modification of DNA. During development this modification has to be reprogrammed in a fast, efficient and replication independent manner. To date it is not known how this is facilitated especially in a genome wide range after fertilization or during PGC development. In my thesis I was able to develop a suitable assay to study the basic biochemistry of this process and its participating compounds. In addition I studied the maintenance of methylation in repetitive elements in the mouse genome during PGC development and designed a model how PGCs keep or lose their methylation in these regions. A thorough analysis of a family with male monozygotic twins discordant for BWS revealed that aberrant methylation of IC2 is not exclusive for female monozygotic twins who are discordant for BWS. In addition I found two distinct CpGs in the promoter region of Lit1 which showed methylation on the unmethylated sequences especially in the healthy twin of a male monozygotic twin pair discordant for BWS, which might be correlated with allelic repression in this IC. At the end I was able to show, that an imbalance of the expression between Igf2 and Cdkn1C might be a cause for BWS

BigDataProcessor2 : A free and open-source Fiji plugin for inspection and processing of TB sized image data

SUMMARY: Modern bioimaging and related areas such as sensor technology have undergone tremendous development over the last few years. As a result, contemporary imaging techniques, particularly electron microscopy (EM) and light sheet microscopy, can frequently generate datasets attaining sizes of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2-a Fiji plugin facilitating processing workflows for TB sized image datasets.AVAILABILITY AND IMPLEMENTATION: BigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2)

BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing

SUMMARY: Manual processing of DNA methylation data from bisulfite sequencing is a tedious and error-prone task. Here we present an interactive software tool that provides start-to-end support for this process. In an easy-to-use manner, the tool helps the user to import the sequence files from the sequencer, to align them, to exclude or correct critical sequences, to document the experiment, to perform basic statistics and to produce publication-quality diagrams.Emphasis is put on quality control: The program automatically assesses data quality and provides warnings and suggestions for dealing with critical sequences. The BiQ Analyzer program is implemented in the Java programming language and runs on any platform for which a recent Java virtual machine is available. AVAILABILITY: The program is available without charge for non-commercial users and can be downloaded from http://biq-analyzer.bioinf.mpi-inf.mpg.de/ CONTACT: [email protected]