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Title:Study and engineering of nucleic acid-binding repeat proteins
Author(s):Abil, Zhanar
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Schroeder, Charles M.; Huang, Raven H.; Chen, Lin-Feng
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):transcription activator-like effector (TALE)
Pumilio
Pumilio/fem-3 binding factor (PUF)
DNA-binding protein
RNA-binding protein
Abstract:From DNA replication and repair to transcription and translation, all aspects of molecular biology are governed by interactions between proteins and nucleic acids. Some of the nucleic acid-binding proteins have evolved repetitive structures and a modular sequence recognition mode. In particular, DNA-binding transcription activator-like effectors (TALE) and RNA-binding Pumilio/fem-3 binding factor homology (PUF) proteins were shown to bind their target sequences in a single repeat/base fashion. This modular recognition nature allowed researchers to rationally design these proteins for novel specificities and utilize them in diverse biotechnological applications such as genome engineering, gene expression regulation, and imaging of specific nucleic acids. The first part of my dissertation was dedicated to the study and engineering of TALE. Using the published specificity code, a pair of TALE nucleases was engineered to site-specifically cut the Cystic Fibrosis Transmembrane Regulator gene (CFTR) carrying the Δ508F mutation responsible for the most cases of cystic fibrosis. Coexpression of these proteins in human cell lines allowed homologous recombination-mediated repair of the episomal and chromosomal reporter gene interrupted with the sequence fragment from CFTR. In the next project, the binding dynamics of TALE on DNA substrates was investigated using single molecule fluorescence microscopy. For the first time, the binding dynamics and 1-D diffusion of TALE proteins along DNA were directly visualized. The data strongly suggest that TALE searches its target using the facilitated diffusion mechanism, with a combination of 1-D and 3-D diffusion before it reaches its target sequence. This study can contribute to improved rational design of these proteins for biomedical applications. The aim of the second part of my dissertation was to engineer new PUF-based biosynthetic tools. First, the Golden Gate cloning method was implemented for an efficient one-step assembly of designer PUF proteins. To this end, a repeat module library that is potentially capable of generating a PUF domain with any desired specificity was created. The assembled novel PUF variants exhibited high in vitro binding efficiencies to cognate RNA sequences, corroborating the applicability of the modular assembly approach for PUF engineering. Next, the PUF domain was fused to a post-transcriptional regulator, which allowed for a sequence-specific reporter and endogenous gene repression in human cell lines. This work was a demonstration of the efficacy of a synthetic PUF-based gene expression regulator. In the last project, a PUF-based system for intracellular directional transport of mRNA was developed in mammalian cells. The biosynthetic device consists of a “motor”, which provides a directional movement, and the PUF protein that dimerizes with the “motor” in a ligand-dependent manner. The system allowed RNA-sequence and motor-specific transport and colocalization of PUF and its RNA cargo. Currently, this PUF-motor system is being implemented for the transport as well as axonal and dendritic local protein translation of reporter and endogenous genes in rat neurons. This prototypical synthetic device should allow easy and controlled intracellular mRNA transport regulation in eukaryotes for applications in basic science and therapeutics.
Issue Date:2015-04-13
Type:Thesis
URI:http://hdl.handle.net/2142/78603
Rights Information:Copyright 2015 Zhanar Abil
Date Available in IDEALS:2015-07-22
Date Deposited:May 2015


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