Synthesis and Characterisation of NAD+ Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation

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2017
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Within the last decade, research on poly(ADP-ribose) experienced a renaissance and various studies unveiled the importance of this biopolymer as a complex, functionally diverse protein modification and signalling molecule. Fundamental processes such as DNA repair and transcription are coordinated by poly(ADP-ribose) and its binding to proteins. Thus, different pathophysiological conditions and disease states, e.g. in cancer and inflammation, have been associated with this protein modification and motivated its targeting in clinical trials.
ADP-ribose chains are tightly regulated in a posttranslational manner by ADP-ribosyl transferases and hydrolases in response to external stimuli and thus cellular levels fluctuate rapidly. Built from NAD+, single or multiple ADP-ribose units are covalently attached onto arginines, glutamates, aspartates or lysines of acceptor proteins. Thereby, nicotinamide is released and mono(ADP-ribos)ylated or poly(ADP-ribos)ylated proteins are formed. Due to these dynamics and complexity, the visualisation of poly(ADP-ribos)ylation remains not only a challenging task, but would also be of high importance to understand these processes on a cellular level.
The aim of this PhD project was to explore the applicability of chemically modified NAD+ analogues for the detection of DNA damage induced poly(ADP-ribos)ylation. Thus, a perfect analogue should be an efficient substrate of ADP-ribosyl transferases in vitro and in cellula. It should be able to specifically label cellular formation of poly(ADP-ribose) in dependency of extrinsic stimuli. Ideally, the new analogue enables to monitor poly(ADP-ribos)ylation in realtime and in a dynamic fashion.
The challenge was met by synthesising NAD+ analogues that can be built into poly(ADPribose) by ARTD1, the major poly(ADP-ribos)ylating enzyme in DNA repair. Thus, the positions as well as the types of NAD+ modifications were investigated, and analogues substituted in adenine position 2 were found to be best-suited for this purpose. Using bioorthogonal reporter groups, the intracellular visualisation of poly(ADP-ribose) was demonstrated simultaneously in two colours, e.g. as required in time dependent or pulsechase experiments. Moreover, a fluorophore-modified NAD+ enabled the direct monitoring of poly(ADP-ribos)ylation inside of a living cell. Thereby, a full turnover of poly(ADP-ribose) was observed after laser-induced DNA damage in real-time. Additionally, protein-specific interaction with poly(ADP-ribose) was detected in intact cells using a powerful FLIM-FRET technique and the GFP-tagged protein of interest. Finally, the substrate scopes of other poly(ADP-ribose) synthesising enzymes like ARTD2, ARTD5 and ARTD6 were explored to broaden the applicability of the developed NAD+ analogues.
In summary, the chemical biology approaches developed in here proved powerful for biological applications and the novel tools will help to elucidate PAR biology by studying the polymer in its natural environment.

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540 Chemie
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ARTD; click chemistry; NAD+; poly(ADP-ribose); posttranslational modification
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ISO 690WALLRODT, Sarah, 2017. Synthesis and Characterisation of NAD+ Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Wallrodt2017Synth-38571,
  year={2017},
  title={Synthesis and Characterisation of NAD<sup>+</sup> Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation},
  author={Wallrodt, Sarah},
  address={Konstanz},
  school={Universität Konstanz}
}
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March 31, 2017
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Konstanz, Univ., Diss., 2017
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