TY - GEN A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 983 KW - cohesive ends KW - DNA cleavage KW - genetic vectors KW - modified primers KW - molecular methods KW - polymerase chain reaction KW - recombinant Escherichia coli KW - restriction enzymes Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431085 SN - 1866-8372 IS - 983 ER - TY - JOUR A1 - Feiner, Rebecca Christine A1 - Teschner, Julian A1 - Teschner, Kathrin E. A1 - Radukic, Marco T. A1 - Baumann, Tobias A1 - Hagen, Sven A1 - Hannappel, Yvonne A1 - Biere, Niklas A1 - Anselmetti, Dario A1 - Arndt, Katja Maren A1 - Müller, Kristian Mark T1 - rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations JF - International journal of molecular sciences N2 - Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme beta-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with beta-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology. KW - adeno-associated-virus KW - beta-lactamase KW - inverted terminal repeat (ITR) KW - loop modification KW - capsid stability Y1 - 2019 U6 - https://doi.org/10.3390/ijms20225702 SN - 1422-0067 VL - 20 IS - 22 PB - MDPI CY - Basel ER - TY - JOUR A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V JF - BMC biotechnology N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. KW - Cohesive ends KW - DNA cleavage KW - Genetic vectors KW - Modified primers KW - Molecular methods KW - Polymerase chain reaction KW - Recombinant Escherichia coli KW - Restriction enzymes Y1 - 2013 U6 - https://doi.org/10.1186/1472-6750-13-81 SN - 1472-6750 VL - 13 IS - 10 PB - BioMed Central CY - London ER - TY - JOUR A1 - Jedrusik-Bode, Monika A1 - Studencka, Maja A1 - Smolka, Christian A1 - Baumann, Tobias A1 - Schmidt, Henning A1 - Kampf, Jan A1 - Paap, Franziska A1 - Martin, Sophie A1 - Tazi, Jamal A1 - Müller, Kristian M. A1 - Krüger, Marcus A1 - Braun, Thomas A1 - Bober, Eva T1 - The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals JF - Journal of cell science N2 - SIRT6 is a NAD(+)-dependent deacetylase that modulates chromatin structure and safeguards genomic stability. Until now, SIRT6 has been assigned to the nucleus and only nuclear targets of SIRT6 are known. Here, we demonstrate that in response to stress, C. elegans SIR-2.4 and its mammalian orthologue SIRT6 localize to cytoplasmic stress granules, interact with various stress granule components and induce their assembly. Loss of SIRT6 or inhibition of its catalytic activity in mouse embryonic fibroblasts impairs stress granule formation and delays disassembly during recovery, whereas deficiency of SIR-2.4 diminishes maintenance of P granules and decreases survival of C. elegans under stress conditions. Our findings uncover a novel, evolutionary conserved function of SIRT6 in the maintenance of stress granules in response to stress. KW - C. elegans KW - G3BP KW - SIRT6 KW - Sirtuins KW - Stress KW - Stress granules Y1 - 2013 U6 - https://doi.org/10.1242/jcs.130708 SN - 0021-9533 SN - 1477-9137 VL - 126 IS - 22 SP - 5166 EP - + PB - Company of Biologists Limited CY - Cambridge ER - TY - THES A1 - Baumann, Tobias T1 - Stability and Interconnected protein properties studied with TEM ß-lactamase Y1 - 2013 CY - Potsdam ER - TY - JOUR A1 - Hagen, Sven A1 - Baumann, Tobias A1 - Wagner, Hanna J. A1 - Morath, Volker A1 - Kaufmann, Beate A1 - Fischer, Adrian A1 - Bergmann, Stefan A1 - Schindler, Patrick A1 - Arndt, Katja Maren A1 - Mueller, Kristian M. T1 - Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy JF - Scientific reports N2 - The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). Y1 - 2014 U6 - https://doi.org/10.1038/srep03759 SN - 2045-2322 VL - 4 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Kuekenshoener, Tim A1 - Hagemann, Urs B. A1 - Wohlwend, Daniel A1 - Raeuber, Christina A1 - Baumann, Tobias A1 - Keller, Sandro A1 - Einsle, Oliver A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - Analysis of Selected and Designed Chimeric D- and L-alpha-Helix Assemblies JF - Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences N2 - D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base. Y1 - 2014 U6 - https://doi.org/10.1021/bm5006883 SN - 1525-7797 SN - 1526-4602 VL - 15 IS - 9 SP - 3296 EP - 3305 PB - American Chemical Society CY - Washington ER -