Photocontrol of artificial transcription factors

Chanapai, Seni 2013. Photocontrol of artificial transcription factors. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The design of a photoswitchable homeodomain artificial transcription factor (PATF), modelled on an engrailed homeodomain, for the purpose of controlling DNA binding affinity and controlling the transcription process in cells using light has been investigated. This study was conducted using a 3,3’-bis(sulfo)- 4,4’bis(chloroacetamino)azobenzene crosslinker, alkylated between two cysteine residues with different spacings (i, i+4, i, i+7 and i, i+11) and either a rigid or flexible linker domain. In previous studies, basic leucine zipper transcription activators have been photocontrolled in living cells by incorporating a photoswitchable azobenzene crosslinker. Circular dichroism spectroscopy showed the conformation of crosslinked PATF (XLPATF) peptides (i, i+11 spacing) containing rigid and flexible linkers could be controlled reversibly by light. Fluorescence anisotropy experiments using labelled DNA confirmed the in vitro DNA binding affinity of PATF was considerably higher with the crosslinker in the trans (ground state) configuration than in the cis (photoexcited state) configuration. Further studies of peptides with i, i+4 and i, i+7 spacings with a semirigid and rigid linker domains showed increased binding affinity with the crosslinker in the cis configuration. Initiation of transcription was investigated by an in vitro transcription assay to measure the ability of PATF molecules to moderate the production of RNA by irradiation with UV light. PATF molecules with i, i+11 spacings showed increased transcriptional activation with the crosslinker in the ground state configuration and i, i+4 and i, i+7 spacings resulted in increased transcription activation with the crosslinker in the excited state conformation. Control of 50% of transcriptional activity was achieved for i, i+11 v spacings, and PATFs with a rigid linker domain were more effective switches than those with flexible linkers. Using i, i+4 and i, i+7 spacings in PATFs resulted in a lower degree of control but, as anticipated, transcriptional activation was increased after irradiation.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:36
URI:	https://orca.cardiff.ac.uk/id/eprint/58014

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