Development of Zinc-Finger-Based Artificial Restriction Endonucleases and Fluorescent Peptidyl Metal Sensors

by Florian Czerny
Doctoral thesis
Date of Examination:2016-08-08
Date of issue:2016-11-17
Advisor:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Franc Meyer
Referee:Prof. Dr. Sofi Elmroth
Persistent Address: http://dx.doi.org/10.53846/goediss-5976

 

 

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Abstract

English

Zinc finger proteins are ubiquitous in the cellular environment having an excessive diversity and performing variable tasks. Their outstanding capability to develop a sequence-specific bond to deoxyribonucleic acid (DNA) makes them one of the most abundant DNA-binding domains present in many eukaryotic and prokaryotic transcription factors.  The first part of this thesis describes the modification of the zinc finger protein from Zif268 by introducing artificial dinuclear amino acids to establish novel and substantially downsized artificial restriction endonucleases. Furthermore, a phosphoserine residue was incorporated in the protein to study its recruiting capacity for an external Ce(IV)/EDTA complex representing the hydrolytically active species.  In the second part, the zinc finger’s overwhelming zinc coordination properties were used to develop fluorescent peptidyl metal sensors. This was achieved by exchanging a histidine residue, which is part of the metal coordination site of the protein, for a propargylglycine residue and subsequent attachment of the fluorophore by the means of a copper-induced azide alkyne cycloaddition.
Keywords: Zinc finger; Zif268
 
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