2016
BioNEC MiniSymposium on Biomolecular Synthesis
and Nanotechnology
PROGRAM AND ABSTRACTS
Symposium Organizers: Knud J. Jensen and Jesper Wengel
BIOMOLECULAR NANOSCALE ENGINEERING CENTER,
UNIVERSITY OF COPENHAGEN AND UNIVERSITY OF
SOUTHERN DENMARK
November 18, 2016
Welcome
It is a pleasure to welcome all participants of the Biomolecular Synthesis and Nanotechnology
Mini-Symposium.
The synposium is supported by BioNEC (Biomolecular Nanoscale Engineering Center), a joint
research center of excellence – funded by THE VILLUM FOUNDATION – involving the three
Danish universities – University of Southern Denmark, University of Copenhagen and
University of Aarhus.
We wish all participants a scientifically stimulating meeting.
The Organizing Committee
BioNEC Mini-Symposium on Biomolecular Synthesis
and Nanotechnology
Friday November 18, 2016, Festsalen, Frederiksberg Campus, University of Copenhagen,
Bülowsvej 17, Frederiksberg C
Program
12:45 – 13:00
Registration outside Festsalen
13:00 – 13:05
Welcome and Opening remarks by Knud J. Jensen
13:05 – 13:50
Chairman: Knud J. Jensen
Ralf Jungmann, Ludwig Maximilian University, Munich, Germany:
DNA-PAINT: Super-resolution microscopy with DNA molecules
13:50 – 14:35
Thomas Hoeg-Jensen, Novo Nordisk A/S, Denmark: Design of insulin
degludec and co-formulations with insulin aspart and liraglutide
14:35 – 15:00
- Coffee/tea break -
15:00 – 15:45
Chairman: Stefan Vogel
Ulf Diederichsen, Georg-August Universität Göttingen, Germany:
SNARE protein mimicking peptides as membrane fusion mediators
15:45 – 16:30
Oliver Seitz, Humboldt University Berlin, Germany: Responsive probes
and conditional reactions for biological interrogation
16:30 – 16:40
Concluding remarks by Jesper Wengel
October 2016
Biographical Sketch: Professor Ralf Jungmann
Ralf Jungmann studied Physics at Saarland University, followed by a
one-year diploma research stay with Paul Hansma at UC Santa Barbara
where he worked on functional imaging of bone ultrastructure using
Atomic Force Microscopy and High-Speed-Photography.
In 2007, Jungmann joined Prof. Friedrich C. Simmel’s lab at TU
München as a Ph.D. student and was among the first researchers in
Germany to apply and extend the DNA origami technique. During his
Ph.D., Jungmann applied single-molecule fluorescence techniques to
DNA Nanotechnology, constructing the first nanoscopic DNA origami
“rulers” for super-resolution microscopy. He also pioneered a novel type of super-resolution
microscopy, termed DNA-PAINT, that uses programmable DNA molecules as imaging probes.
After receiving his Ph.D. at TUM, he moved to the labs of Prof. Peng Yin and Prof. William M.
Shih at the Wyss Institute for Biologically Inspired Engineering at Harvard University as an
Alexander von Humboldt fellow. At Harvard, Jungmann worked on applications of DNAPAINT for multiplexed cellular imaging.
In 2014, Jungmann received an Emmy Noether Fellowship from the German Research
Foundation (DFG) and since then heads the research group “Molecular Imaging and
Bionanotechnology” at the MPI of Biochemistry and the LMU Munich. In 2015, Jungmann cofounded Ultivue, a US-based company commercializing imaging reagents for DNA-PAINT
super-resolution microscopy.
In 2016 he received an ERC Starting Grant to bring DNA-based super-resolution imaging from
single molecules to whole cells and tissues. Since August 2016, he is Professor of Physics at the
LMU Munich.
DNA-PAINT: Super-Resolution Microscopy with DNA Molecules
Ralf Jungmann 1 , 2
1
Department of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539
Munich, Germany
2
Max Planck Institute of Biochemistry, 82152 Martinsried near Munich, Germany
Super-resolution fluorescence microscopy is a powerful tool for biological research, but
obtaining multiplexed images for a large number of distinct target species in whole cells and
beyond remains challenging. Here we use the transient binding of short fluorescently labeled
oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale
topography) for simple and easy-to-implement multiplexed super-resolution imaging that
achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures.
We report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of
multiple targets using only a single dye and a single laser source. We experimentally
demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as
four-color two-dimensional imaging and three-color 3D imaging of proteins in fixed cells.
Finally, we demonstrate whole cell imaging using DNA- and Exchange-PAINT and optical
sectioning, now allowing DNA-based super-resolution imaging deep inside cells, away from the
glass coverslip.
1. T. Schlichthaerle, M.T. Strauss, F. Schueder, J.B. Woehrstein, R. Jungmann, Current Opinion in Biotechnology (2016)
2. M. Dai, R. Jungmann, P. Yin, Nature Nanotechnology (2016)
3. R. Jungmann*, M.S. Avendaño*, M. Dai, J.B. Woehrstein, S.S. Agasti, Z. Feiger, A. Rodal, P. Yin, Nature Methods (2016)
4. R. Jungmann*, M.S. Avendaño*, J.B. Woehrstein*, M. Dai, W.M. Shih, P. Yin, Nature Methods (2014)
5. R. Iinuma*, Y. Ke*, R. Jungmann*, T. Schlichthaerle., J.B. Woehrstein, P. Yin, Science (2014)
6. N.D. Derr*, B.S. Goodman*, R. Jungmann, A.E. Leschziner, W.M. Shih, S.L. Reck-Peterson. Science (2012)
7. C. Lin, R. Jungmann, A.M. Leifer, C. Li, D. Levner, G.M. Church, W.M. Shih, and P. Yin. Nature Chemistry (2012)
8. R. Jungmann*, C. Steinhauer*, M. Scheible, A. Kuzyk, P. Tinnefeld, and F.C. Simmel. Nano Letters (2010)
October 2016
Biographical Sketch: Adjunct Professor Thomas Hoeg-Jensen
Thomas Hoeg-Jensen has 21 years of experience in design and
preparation of diabetes-related peptide and protein drug candidates, and
Thomas is co-inventor of insulin degludec, which is approved in most
parts of the world under the brand name Tresiba®, along with the basalbolus co-formulation with insulin aspart (Ryzodeg®). Thomas is adjunct
professor at University of Copenhagen, and apart from drug discovery
his research has included protein ligation methods, glucose binders, and
development of chemoinformatics systems for better handling of
chemically modified proteins.
Design of insulin degludec and co-formulations with insulin
aspart and liraglutide
Thomas Hoeg-Jensen
Novo Nordisk A/S, Denmark
Insulin degludec (Tresiba®) is a basal insulin analogue that forms a depot of soluble multihexamers upon subcutaneous injection, from which monomers are slowly and continuously
absorbed into the circulation resulting in a half-life longer than 24 hours in humans and low
hypoglycaemia risk. The unique ability of insulin degludec to form soluble and stable
dihexamers in the presence of zinc and phenol in the pharmaceutical formulation allows
degludec to be co-formulated with rapid-acting insulin aspart for simultaneous basal/meal
coverage. Under the right formulation conditions, insulin degludec and insulin aspart retain their
dihexameric and hexameric structures respectively, so no formation of mixed hexamers takes
place, neither in the pharmaceutical preparation nor in vivo upon subcutaneous injection. The
combination product (Ryzodeg®) is therefore able to retain the distinct pharmacokinetic
properties of the individual components. Finally, insulin degludec can also be co-formulated
with liraglutide, a long-acting GLP-1 receptor agonist, to give Ideglira (Xultophy®).
October 2016
Biographical Sketch: Professor Ulf Diederichsen
Ulf Diederichsen, born 1963 in Germany, studied chemistry in Freiburg,
Germany, with a diploma work in organic synthesis and completed his
Ph.D. in 1993 under the supervision of Albert Eschenmoser at the ETH
Zürich working on homo and glucose-DNA. After postdoctoral work on
radical chemistry at the University of Pittsburgh with Dennis Curran, he
gained his habilitation at the Technical University Munich. In 1999, he
was appointed professor of Organic Chemistry at the University
Würzburg, until joining 2001 the Georg-August-University Göttingen as
full professor of Organic Chemistry. He was visiting professor at the
LMU Munich and Goering Visiting Professor at the University of Wisconsin and is member of
Göttingen Academy of Sciences and Humanities. Currently he is serving as vice president for
research at the University Göttingen. His research interests focus on the modification of peptide,
protein, nucleic acid, and lipid biomolecules by organic synthesis allowing for new functions,
specific recognition, or molecular architecture.
SNARE protein mimicking peptides as membrane fusion mediators
Ulf Diederichsen, Samit Guha, Barbara Hubrich, Pawan Kumar, Antonina Lygina, Karsten
Meyenberg, Muheb Sadek, Jan-Dirk Wehland
Georg-August Universität Göttingen, Institut für Organische und Biomolekulare Chemie,
Tammannstraße 2, D-37077 Göttingen, Germany, [email protected]
SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) proteins are highly
conserved proteins mediating synaptic membrane fusion.[1] Whereas transmembrane domains are
anchoring in the respective membranes, the formation of a four α-helix bundle, known as
SNARE motif, brings the membranes in proximity, thereby initiating the fusion process. The
mechanistical hypothesis for the formation of the SNARE recognition motif is based on a
zippering process that is even extended to the linker/transmembrane peptides facilitating the
reorganization of lipids in membrane fusion.[2] In order to mechanistically investigate the
SNARE mediated fusion process artificial SNARE-analogues were synthesized based on various
peptide nucleic acid (PNA) oligomers replacing the SNARE recognition motif.[3] The PNA
recognition motifs vary with respect to their backbone topology considering aminoethyl glycine
PNA as well as β-peptide or alanyl PNA; a significant influence on fusion efficiency and
mechanism is indicated.[4,5] Variation of the C-terminal charge on the transmembrane helices also
turned out to significantly influence the fusion process.[6] Furthermore, the zippering mechanism
is investigated using nucleobase caged PNA influencing the directionality of the recognition
process.[7] PNA with 14-helical β-peptide backbone serves as recognition unit mediating vesicle
fusion processes with the option of an arrest in the intermediate docking state.[8]
Figure. SNARE-mediated membrane fusion (left), PNA-SNARE analog (center) and β-peptide
PNA based membrane fusion analog (right).
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
R. Jahn, R. H. Scheller, Nature Rev. 2006, 7, 631.
A. N. Ngatchou, K. Kisler, Q. Fang, A. M. Walter, Y. Zhao, D. Bruns, J. B. Sørensen, M. Lindau, PNAS 2010, 107, 18463.
P. Kumar, S. Guha, U. Diederichsen, J. Pept. Sci. 2015, 21, 621.
A. S. Lygina, K. Meyenberg, R. Jahn, U. Diederichsen, Angew. Chem. Int. Ed. 2011, 50, 8597.
K. Meyenberg, A. Lygina, G. van den Bogaart, R. Jahn, U. Diederichsen, Chem. Commun. 2011, 47, 9405.
J.-D. Wehland, A. S. Lygina, P. Kumar, S. Guha, B. E. Hubrich, R. Jahn, U. Diederichsen, Mol. BioSyst. 2016, 12, 2770.
S. Guha, J. Graf, B. Göricke, U. Diederichsen, J. Pept. Sci. 2013, 19, 415.
M. Sadek, D. Berndt, D. Milovanovic, R. Jahn, U. Diederichsen, ChemBioChem 2016, 17, 479.
October 2016
Biographical Sketch: Professor Oliver Seitz
Oliver Seitz was born 1966 in Frankfurt a.M. , Germany and studied
chemistry at the Johannes-Gutenberg University Mainz, Germany.
There he obtained his Ph.D. in Organic Chemistry with Horst Kunz
(1995). After postdoctoral research with Chi-Huey Wong from 1997
to 1997 at the Scripps Research Institute in La Jolla, USA he moved
in 1997 to the Technical University Karlsruhe. In 2000 he became
group leader in the Department of Chemical Biology at the MaxPlanck Institute for Molecular Physiology and obtained the venia
legendi in Organic Chemistry from the Technical University
Dortmund. In 2003 he was appointed Full Professor at the
Humboldt-Universität zu Berlin. His broad interests cover nucleic
acid and protein sciences. His group develops new probes for live cell imaging and perturbation
of RNA and protein molecules. Recently, he is exploring RNA-directed chemistry as a means to
design molecules (molecular doctors) that read and translate RNA information into a drug-like
output. His activities in the protein sciences include the development of methods for facilitating
synthetic access and functional analysis of posttranslationally modified proteins. Oliver Seitz has
published 125 papers and is the recipient of an ERC Advanced Grant.
Responsive Probes and Conditional Reactions for Biological
Interrogation
Oliver Seitz
Department of Chemistry, Humboldt University Berlin, Brook-Taylor-Str. 2, D-12489 Berlin,
Germany
Email: [email protected]
DNA and DNA analogues are versatile scaffolds and provide unique opportunities for the
controlled presentation of functional units. We demonstrate that nucleic acid hybridization can
be used to control the photophysical properties of appended dye molecules or dye aggregates.
Such responsive molecules allow the detection of complementary DNA or RNA targets. A
current challenge in the field is the imaging and quantification of specific RNA molecules in live
cells. We have introduced the FIT-Probes,1 i.e. quencher-free hybridization probes which contain
a single asymmetric cyanine dye that serves as a fluorescent base surrogate. The ‘dye base’ acts
as a local intercalator probe and reports hybridization. The combination with additional dyes2 or
the use of LNA constraints3 provide exceptional brightness and quantitative imaging of RNA
inside live cells.
DNA-based self-assemblies can be used to control/explore the localization, structure and the
bioactivity of proteins and protein ligands. Nucleic acid-constrained hairpin peptide beacons4
fluoresce upon interaction with target proteins. We have introduced a technique, which we
termed DNA-programmed spatial screening. Here, the affinity enhancement induced by
sequence-programmed multivalency provides opportunities to probe the spatial arrangement of
the binding sites of a protein receptor of interest. We demonstrated the spatial screening of
lectins, tandem-SH2 domains of kinases, the estrogen receptor and adaptor proteins involved in
clathrin-dependent endocytosis.5
Nucleic acids can be used as templates that trigger chemical reactions. We explore whether RNA
templated reactions can be used for the design of molecular doctors, which initiate the synthesis
of drug-like compounds only in those cells expressing particular RNA sequences.6 Templated
reactions are not bound to nucleic acid-based recognition. Currently, we are exploring peptidetemplated reactions that allow ultrafast labeling of membrane proteins on live cells.7
1. a) F. Hövelmann, O.Seitz, Acc. Chem. Res. 2016 (DOI: 10.1021/acs.accounts.5b00546; b) O. Köhler, D. V. Jarikote, O. Seitz,
ChemBioChem 2005, 6, 69-77; c) S. Kummer, A. Knoll, E. Socher, L. Bethge, A. Herrmann, O. Seitz; Angew. Chem. Int. Ed.
2011, 50, 1931-1934.
2. F. Hövelmann, I. Gaspar, A. Ephrussi, O. Seitz, J. Am. Chem. Soc. 2013, 135, 19025-19032.
3. a) F. Hövelmann, I. Gaspar, S. Loibl, E.A. Ermilov, B. Röder, J. Wengel, A. Ephrussi, O. Seitz, Angew. Chem. Int. Ed. 2014,
53, 11370-11375; b) F. Hövelmann, I. Gaspar, J. Chamiolo, M. Kasper, J. Steffen, A. Ephrussi, O. Seitz, Chem. Sci. 2016, 7,
128-135.
4. M. Fischbach, U. Resch-Genger, O.Seitz, Angew. Chem. Int. Ed. 2014, 53, 11955-11959.
5. For example: a) H. Eberhard, F. Diezmann, O. Seitz, Angew. Chem. Int. Ed. 2011, 50, 4146-4150; b) F. Abendroth, A.
Bujotzek, M. Shan, R. Haag, M. Weber, O. Seitz, Angew. Chem. Int. Ed. 2011, 50, 8592-8596; c) F. Abendroth, O. Seitz,
Angew. Chem. Int. Ed. 2014, 53, 10504-10509.
6. a) A. Erben, T. N. Grossmann, O. Seitz, Angew. Chem. Int. Ed. 2011, 50, 2828-2832M; b) O. Vázquez, O.Seitz, Chem.Sci.
2014, 5, 2850-2854.
7. U. Reinhardt, J. Lotze, S. Zernia, K. Mörl, A.G. Beck-Sickinger, O. Seitz, Angew. Chem. Int. Ed. 2014, 53, 10237-10241.
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