Design and Applications of Split-Luciferase Systems in Vitro and in Cellulo
type: CreativeWork
description: Split-protein reassembly methods allow for the detection of a variety of macromolecular interactions in vitro, in cellulo and in vivo. Spilt-protein reporters, such as split-firefly luciferase (Fluc), depend upon the conditional reassembly of the genetically fragmented enzyme. The two fragments of Fluc conditionally reassemble and regain activity when protein domains appended to the N-terminal and C-terminal Fluc fragments interact. Our laboratory has previously reported the use of the split-Fluc system in the detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions. Herein, I describe further developments in the split-Fluc system as applied to the specific detection of DNA, DNA modifications, and small molecule kinase inhibitors both in vitro and in cellulo. We have previously developed one of the first direct methods to detect any double stranded DNA sequence by attaching DNA binding domains, specifically designed Cys₂-His₂ zinc finger domains, to split-Fluc fragments. We previously discovered that designed Cys₂-His₂ zinc fingers were not specific for targeting a variety of promoter sequences. In order to improve DNA detection, I have designed and tested split-Fluc sensors against several relevant DNA targets using both designed zinc fingers and transcription activator-like effectors (TALEs). The results demonstrate that TALEs allow for significantly higher specificity than zinc fingers and provide a significant improvement for designing sequence specific split-Fluc systems for a variety of applications such as the detection of DNA methylation. The methylation at cytosine-guanine (CpG) dinucleotides in DNA is associated with transcriptional repression in normal cells. Aberrant changes in the mCpG levels on the promoter regions of tumor suppressor genes have been linked to cancer. We have previously shown that a sequence-specific mCpG split-Fluc sensor can be designed by attaching one fragment of the split-Fluc to a zinc finger an…
abstract: Split-protein reassembly methods allow for the detection of a variety of macromolecular interactions in vitro, in cellulo and in vivo. Spilt-protein reporters, such as split-firefly luciferase (Fluc), depend upon the conditional reassembly of the genetically fragmented enzyme. The two fragments of Fluc conditionally reassemble and regain activity when protein domains appended to the N-terminal and C-terminal Fluc fragments interact. Our laboratory has previously reported the use of the split-Fluc system in the detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions. Herein, I describe further developments in the split-Fluc system as applied to the specific detection of DNA, DNA modifications, and small molecule kinase inhibitors both in vitro and in cellulo. We have previously developed one of the first direct methods to detect any double stranded DNA sequence by attaching DNA binding domains, specifically designed Cys₂-His₂ zinc finger domains, to split-Fluc fragments. We previously discovered that designed Cys₂-His₂ zinc fingers were not specific for targeting a variety of promoter sequences. In order to improve DNA detection, I have designed and tested split-Fluc sensors against several relevant DNA targets using both designed zinc fingers and transcription activator-like effectors (TALEs). The results demonstrate that TALEs allow for significantly higher specificity than zinc fingers and provide a significant improvement for designing sequence specific split-Fluc systems for a variety of applications such as the detection of DNA methylation. The methylation at cytosine-guanine (CpG) dinucleotides in DNA is associated with transcriptional repression in normal cells. Aberrant changes in the mCpG levels on the promoter regions of tumor suppressor genes have been linked to cancer. We have previously shown that a sequence-specific mCpG split-Fluc sensor can be designed by attaching one fragment of the split-Fluc to a zinc finger an…
author: Mok, Pui-Wing
language: en_US
keywords: Chemistry
date published: 2014
date modified: 2026-07-24T00:57:10Z
provider: University of Arizona
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: The University of Arizona.

PropertyValue
property ID: Handle
value: http://hdl.handle.net/10150/333354