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Metabolic labeling of cellular and viral DNA

English title Metabolic labeling of cellular and viral DNA
Applicant Luedtke Nathan W.
Number 146754
Funding scheme Project funding (Div. I-III)
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Organic Chemistry
Start/End 01.04.2013 - 31.03.2016
Approved amount 520'000.00
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All Disciplines (2)

Discipline
Organic Chemistry
Cellular Biology, Cytology

Keywords (10)

antiviral drugs; G-quadruplex; i-motif; nucleic acids; developmental biology; organic chemistry; virology; nucleosides; cell biology; fluorescent probes

Lay Summary (German)

Lead
Sonden mit fluoreszierende Eigenschaften ermöglichen es Faltung, Funktion und Lokalisierung von Biomolekülen zu untersuchen. Um wichtige Fragen der Entwicklungsbiologie und Virologie klären zu können, basiert dieses Projekt auf der Synthese und Evaluation neuer fluoreszierender Marker. Diese sollen genutzt werden, um die Struktur, die Funktion und den Fluss von Nukleinsäuren in vivo zu charakterisieren.
Lay summary

Das Ziel ist die Entwicklung neuartiger DNA Marker, um grundlegende, bislang ungeklärte biologische Fragstellungen wie beispielsweise die „Immortal Strand“ Hypothese zu  untersuchen, welche erstmals im Jahre 1975 von Cairns aufgestellt wurde. Diese Theorie besagt, dass Stammzellen Mutationen in ihrem Genom minimieren können, indem sie ihre DNA asymmetisch verteilen.Durch die Bewahrung dieser sogenannten „Ur-DNA“ könnte ein kleiner Teil der Stammzellpopulation über eine ursprüngliche Kopie dieses codogener Strangs verfügen, der dadurch von Mutationsanreicherung durch DNA Replikation geschützt ist. Unser Ziel ist es, diese Hypothese mit Hilfe von DNA Markern neu zu evaluieren. Diese würden es erlauben, den Fluss und das Verbleiben der embryonaler Chromosomen in vivo zu detektieren. Stammzellen, die kontinuierlich replizieren, aber ein kompletes Set an Chromosomen mit genau 50 % der embryonalen Marker besitzen, wären ein direkter Beweis für die Theorie von Cairns. Sollte sich diese Hypothese bewahrheiten, könnte unsere Methode die erste universelle Technik zur Lokalisierung von Stammzellen im gesamten Modellorganismus sein.

 

Dies würde das Verständniss der Stammzellbiologie fördern und könnte die Entwicklung neuer medizinischer Behandlungsmethoden gegen Krebs, Diabetes, Parkinson oder vielen anderen Krankheiten erleichtern.      
Des Weiteren arbeiten wir auch an neuen Vernetzungs- und Einfang Techniken, die eine eindeutige Charakterisierung markierter Nukleinsäuren und deren assozierten Proteine erlauben sollen. Beispielsweise könnte mit Hilfe von chemisch gekennzeichneten Virusgenomen ein Katalog von zellulären Faktoren erstellt werden, welche mit dem viralen Genom während der frühen und späten Phase der Herpes Virus Replikation interagieren. Diese Studien würden neues Licht in den Bereich der Virus-Wirt Interaktionen bringen und könnten neue Möglichkeiten therapeutischer Ansätze eröffnen.

Direct link to Lay Summary Last update: 27.03.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
A fluorescent surrogate of thymidine in duplex DNA
Mata Guillaume, Schmidt Olivia P., Luedtke Nathan W. (2016), A fluorescent surrogate of thymidine in duplex DNA, in Chem. Commun., 52(25), 4718-4721.
Molecular Design and Synthesis of a Planar Telomestatin Analogue
Seyfried Martin S., Alzeer Jawad, Luedtke Nathan W. (2016), Molecular Design and Synthesis of a Planar Telomestatin Analogue, in European Journal of Organic Chemistry, 2016(2), 367-372.
A Bioorthogonal Chemical Reporter of Viral Infection
Neef Anne B., Pernot Lucile, Schreier Verena N., Scapozza Leonardo, Luedtke Nathan W. (2015), A Bioorthogonal Chemical Reporter of Viral Infection, in Angewandte Chemie International Edition, 54(27), 7911-7914.
Fluorescent Probe for Proton-Coupled DNA Folding Revealing Slow Exchange of i-Motif and Duplex Structures
Mata Guillaume, Luedtke Nathan W. (2015), Fluorescent Probe for Proton-Coupled DNA Folding Revealing Slow Exchange of i-Motif and Duplex Structures, in Journal of the American Chemical Society, 137(2), 699-707.
Alkene-Tetrazine Ligation for Imaging Cellular DNA
Rieder Ulrike, Luedtke Nathan W. (2014), Alkene-Tetrazine Ligation for Imaging Cellular DNA, in Angewandte Chemie International Edition, 53(35), 9168-9172.
An Azide-Modified Nucleoside for Metabolic Labeling of DNA
Neef Anne B., Luedtke Nathan W. (2014), An Azide-Modified Nucleoside for Metabolic Labeling of DNA, in ChemBioChem, 15(6), 789-793.
Photodynamic Agents with Anti-metastatic Activities
Vummidi Balayeshwanth R., Noreen Faiza, Alzeer Jawad, Moelling Karin, Luedtke Nathan W. (2013), Photodynamic Agents with Anti-metastatic Activities, in ACS Chemical Biology, 8(8), 1737-1746.
Synthesis and Solvatochromic Fluorescence of Biaryl Pyrimidine Nucleosides
Mata Guillaume, Luedtke Nathan W. (2013), Synthesis and Solvatochromic Fluorescence of Biaryl Pyrimidine Nucleosides, in Organic Letters, 15(10), 2462-2465.
Tracking Viral Genomes in Host Cells at Single-Molecule Resolution
Wang I-Hsuan, Suomalainen Maarit, Andriasyan Vardan, Kilcher Samuel, Mercer Jason, Neef Anne, Luedtke Nathan W., Greber Urs F. (2013), Tracking Viral Genomes in Host Cells at Single-Molecule Resolution, in Cell Host & Microbe, 14(4), 468-480.

Collaboration

Group / person Country
Types of collaboration
Prof. Stephan Neuhauss Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Urs Greber Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof Leonardo Scapozza Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
University of Toronto, Organic Chemistry Seminar Series Individual talk Chemical Biology of Nucleic Acids: Probing DNA In Vivo 22.01.2016 University of Toronto, Canada Luedtke Nathan W.;
McMaster University, Chemistry Seminar Series Individual talk Chemical Biology of Nucleic Acids: Probing DNA In Vivo 21.01.2016 McMaster University, Toronto, Canada Luedtke Nathan W.;
Centre for Research on Biomolecular Interactions Seminar Series Individual talk Chemical Biology of Nucleic Acids: Probing DNA In Vivo 20.01.2016 York University, Toronto, Canada Luedtke Nathan W.;
University of Konstanz Graduate School in Chemical Biology Summer Retreat Talk given at a conference Chemical Biology of Fluorescent Nucleobase Analogs 01.09.2015 Gültstein, Germany Luedtke Nathan W.;
Gordon Research Conference on Nucleosides, Nucleotides & Oligonucleotides Poster Chemical Biology of Fluorescent Nucleobase Analogs 28.06.2015 Newport, Rhode Island, United States of America Luedtke Nathan W.;
ISACS16: Challenges in Chemical Biology Talk given at a conference Fluorescent DNA in Chemical Biology 15.06.2015 ETH Zurich, Switzerland Luedtke Nathan W.;
Karlsruhe Institute of Technology Organic Chemistry Seminar Series Individual talk Chemical Biology of Fluorescent Nucleobase Analogs 12.05.2015 Karlsruhe Institute of Technology, Germany Luedtke Nathan W.;
University of Rome Chemistry Seminar Series Individual talk Chemical Biology of Fluorescent Nucleobase Analogs 30.01.2015 University of Rome, Italy Luedtke Nathan W.;
King’s College London Chemistry Seminar Series Individual talk Fluorescent Nucleosides and Nucleotides in Chemical Biology 12.12.2014 King’s College London, Great Britain and Northern Ireland Luedtke Nathan W.;
University of Basel, Organic Chemistry Seminar Series Individual talk Synthetic Probes of DNA Folding and Biology 24.10.2014 University of Basel, Switzerland Luedtke Nathan W.;
Swiss-Japanese Chemical Biology Symposium 2014 Talk given at a conference Bioorthogonal chemical reactions on cellular DNA 02.10.2014 University of Bern, Switzerland Luedtke Nathan W.;
Clinical Research Priority Program (CRPP) Tumor Oxygenation Seminar Individual talk Photodynamic Agents with Anti-Metastatic Activities 12.12.2013 University Hospital, Zurich, Switzerland Luedtke Nathan W.;


Knowledge transfer events



Self-organised

Title Date Place
Swiss Summer School 2014 in Chemical Biology 01.09.2014 Eurotel Victoria, Villars sur Ollon, Switzerland, Switzerland

Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Scientifica 2015 German-speaking Switzerland 2015

Associated projects

Number Title Start Funding scheme
165949 Live-cell imaging of DNA conformation and metabolism 01.04.2016 Project funding (Div. I-III)
130074 Fluorescent probes for quadruplex and viral nucleic acids 01.04.2010 Project funding (Div. I-III)

Abstract

As compared to the extensive methodologies developed for proteins, relatively few imaging techniques are available for nucleic acids - all of which are limited by their low sensitivity to alternatively folded structures, large perturbations to native systems, and/or inability to be applied in unmodified cells and organisms. To address unresolved questions in developmental biology and virology, we propose the synthesis and evaluation of new fluorescent probes for characterizing the structure, function, and flow of nucleic acids in vivo. In this proposal, synthetic nucleosides are used to deliver bioorthogonal functional groups (azide, alkyne, vinyl, or aryl halide) into cellular or viral genomes by metabolic incorporation. Subsequent bioorthogonal chemical reactions will be used to further modify the DNA to introduce fluorophores, affinity tags, and/or photocrosslinking groups. In addition to the development of these chemical tools, we propose important biological experiments that have been difficult or even impossible to accomplish using existing technologies, for example: (1) Azide-alkyne “click” reactions that furnish highly fluorescent nucleobase analogs will be used in FRET experiments to probe for non-canonical DNA structures like G-quadruplexes in vivo. (2) Modified nucleosides that exhibit non-toxic labeling of cellular genomes will be used in DNA “birth dating” experiments to evaluate the controversial “immortal DNA strand” hypothesis in Zebrafish. (3) Virus-specific genomic labeling will be conducted using modified nucleotides that are incorporated into virus genomes by error-prone viral polymerases, but rejected by high-fidelity cellular polymerases. This way, the newly synthesized viruses can be selectively labeled and visualized in whole animals without the need for specific antibodies or genetically modified viruses. In addition to visualization, we also propose new crosslinking and capture techniques that will enable unambiguous characterization of the labeled nucleic acids and their associated proteins. By using purified viruses that contain chemically-labeled genomes to infect cells, we will generate a catalog of all cellular factors that interact with the viral genomes during early- and late-phase herpes virus replication. Together these studies will shed new light on virus-host interactions, and may reveal new opportunities for therapeutic intervention.
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