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Methylierung von Cytosin zu 5-Methylcytosin (5mC) dient der epigenetischen Kontrolle der Gentranskription in Saugetieren und hat direkte Auswirkungen auf die Entwicklung und Pathogenese des Menschen. Insbesondere die Diagnose und Prognostik von Krebspatienten profitierte in den letzten Jahren enorm von der Entwicklung neuer Methoden zur Analyse von DNA-Methylierungsmustern. Wir zeigen hier, dass zwei hitzestabile DNA-Polymerasen – die DNA-Polymerase KlenTaq aus Thermus aquaticus und die KOD-DNA-Polymerase aus Thermococcus kodakaraensis – 3′-fehlgepaarte Oligonukleotid-Primer gegenuber 5mC effektiver verlangern als gegenuber nichtmethyliertem C. Wir konnten auserdem eine DNA-Polymerase-Mutante mit weiter verbesserter 5mC/C-Diskriminierung erzeugen. Diese Mutante wurde erfolgreich in einer neuartigen 5mC-Detektierungsmethode eingesetzt, die es ermoglicht, den Methylierungszustand eines Cytosins direkt durch PCR von unbehandelter genomischer DNA zu bestimmen.

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

„Untreu“: Eine DNA-abhängige DNA-Polymerase, deren Wildtyp-Vorläufer keine signifikante Reverse-Transkriptase-Aktivität zeigte, wurde beim Einbau eines Nukleotids gegenüber einem RNA-Templat abgefangen und erstmals strukturell charakterisiert. Die signifikant erhöhte Reverse-Transkriptase-Aktivität der DNA- Polymerase-Variante ermöglicht ihre Anwendung in PCR-basierten molekular- diagnostischen Prozessen. published

The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

thesis is based on bringing small-molecule reactants in close proximity through DNA conjugation and hybridization, and thereby increasing the effective molarity, which significantly A rates of chemical reactions. Several applications of this approach, including compound discovery from synthetic libraries, reaction discovery, and nucleic-acid sensing, have been described. [1] All of the depicted approaches have in common the fact that the two reactants or a reactant and a catalyst are covalently linked to two DNA or PNA strands and are subsequently brought to react by self hybridization or aligned through hybridization. Roelfes and Feringa reported a Diels– Alder reaction mediated by a catalyst intercalated into a DNA duplex. Binding to the duplex resulted in good enantioselectivity of the reaction. [2] Poulin-Kerstien and Dervan employed binding of two polyamides to double-stranded DNA (dsDNA) to promote a 1,3-dipolar cycloaddition; this resulted in linked polyamides. [2] Previously, we found that proline-modified DNA acts as a catalyst in the aldol reaction between a complementary DNA-tethered aldehyde and various nontethered ketones. [3] The formation of a Watson–Crick duplex between the

Light-dependent gene regulation systems are advantageous as they allow for precise spatio-temporal control of target gene expression. In this paper, we present a novel UV-A and blue-light-inducible gene control system that is based on the light-dependent heterodimerization of the CRY2 and C1BN domains. Upon their interaction, a transcription factor is released from the cell membrane and initiates target gene expression. Capitalizing on that, sun cream UV-A protection properties were measured intracellularly.

Unfaithful: The structure of a DNA-dependent DNA polymerase, whose parental ancestor has no significant reverse transcriptase activity, was obtained for the first time incorporating a nucleotide against an RNA template and then structurally characterized. The DNA polymerase variant has significant reverse transcriptase activity that allows its application in PCR-based molecular diagnostics.

In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors.

Artificial RNA riboswitches – apart from protein-based gene regulation systems, which have been known about for a long time – have become increasingly important in biotechnology and synthetic biology. Aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. Since aptazymes are cis-acting elements that are located in the untranslated regions of a gene of interest, their application does not need any further protein co-factor. This presents the opportunity to simplify complex gene networks while simultaneously expanding the repertoire of available parts. Nevertheless, the generation of novel aptazymes requires a functional aptamer–ribozyme connection, which can be difficult to engineer. This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional aptazymes in bacteria and their subsequent transfer into mammalian cells.

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks., Nucleic Acids Research, 39 (22), ISSN:1362-4962, ISSN:0301-5610

SummaryDeveloping artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria.

SummaryRNA-based modules such as riboswitches represent straightforward and simplified approaches for the regulation of gene expression, as no additional proteins are needed. G-rich sequences are known to adopt stable four-stranded structures, and such quadruplexes have been suspected to play important roles in key functions such as the control of gene expression. Here we demonstrate that RNA quadruplexes readily form in vivo. We have constructed mRNA-based G-rich elements that mask the ribosome binding site by folding into four-stranded structures. The suppression of gene expression correlates with the stability of inserted G quadruplexes. Moreover, quadruplexes with moderate stability respond to changes in temperature, thus behaving as artificial RNA thermometers. In conclusion, we introduce tuneable mRNA-based devices that enable modulation of gene expression by a predictable but thus far unknown mechanism.