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Title:Developing high-resolution methods to study DNA and RNA dynamics
Author(s):Zhang, Jichuan
Director of Research:Ha, Taekjip
Doctoral Committee Chair(s):Cheng, Jianjun
Doctoral Committee Member(s):Kilian, Kristopher; Kuhlman, Thomas E; Leal, Cecilia
Department / Program:Materials Science & Engineerng
Discipline:Materials Science & Engr
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):single-molecule imaging
FRET
DNA-protein interaction
RNA labeling
bacterial small RNA
fluorescence-activated cell sorting
high-throughput sequencing
Abstract:Nucleic acids, including DNA and RNA, are one of the most important biomacromolecules inside the cell. DNA stores genetic information, while RNA has more versatile roles including conveying and deciphering genetic information, catalyzing biological reactions, conducting post- transcriptional regulation, and even storing genetic information. To understand the functioning principles of cells and living organisms, it is very important to study DNA- and RNA-involved biochemical reactions as well as the molecular mechanisms behind them, which is quite challenging due to the small size and high metabolism rate of cells. Thanks to the technology advancement in the past several decades, we are able to develop research methods to achieve high-rate, high-resolution and high-throughput study on DNA and RNA. Here we developed several fluorescence-based high-resolution assays to study DNA and RNA dynamics in vitro and in vivo. First, we established a single molecule FRET assay to study homodimeric single-stranded binding protein (Thermus thermophilus SSB) specifically binding and protecting single-stranded DNA during DNA metabolism. With the help of specifically designed DNA constructs and total internal reflection microscopy, we discovered that homodimeric SSB showed similar one- dimensional diffusion and salt-dependent binding mode transition behavior which was confirmed for intensively studied homotetrameric SSB, suggesting that those behaviors might be universal among SSB homologues from different organisms. Second, we developed a live cell RNA labeling and imaging method based on an aptamer- fluorogen system called “Spinach”, which contains an RNA sequence that binds a fluorogen DFHBI and induces its fluorescence. We constructed a Spinach array with tandem Spinach repeats and it greatly enhanced the cellular fluorescence signal, and we could easily visualize mRNAs in the cell. We further characterized the Spinach RNA imaging method and found that either single Spinach or Spinach arrays do not affect RNA transcription, protein translation or RNA degradation. Therefore we proposed that aptamer/fluorogen imaging and aptamer array construction could be a generalizable strategy for high performance and low perturbation live cell RNA imaging. Finally, we expanded the research to gene expression regulation. Our research target, sgrS, is a bacterial small RNA that regulates several target genes at post-transcriptional level in response to sugar-phosphate stress. It is known that sgrS anneals target transcripts via basepairing interaction with the guide of Hfq protein. Nevertheless, how individual nucleotides within sgrS sequence contribute to the regulation process is not clear. Here we utilized the recently developed Sort-Seq method combining fluorescence-activated cell sorting (FACS) and high-throughput DNA sequencing to study sequence-dependent sgrS regulation on its primary target, ptsG. We constructed a target-reporter system with ptsG 5’ UTR, which is responsible for sgrS annealing, fused to GFP, whose expression level is indicated by fluorescence level and is capable of being regulated by sgrS. By introducing an sgrS random mutation library, cells show various fluorescence levels due to diverse regulation capabilities of sgrS mutants. By sorting the cells into different groups based on its fluorescence signal followed by extracting the sgrS mutation distribution among sorted groups, we could find out which mutations totally abolished sgrS function. Results showed that 2 nucleotides involved in ptsG/sgrS annealing, G176 and G178, are extremely significant; other 24 nucleotides are equally significant, but they are involved in sgrS binding by Hfq. The study suggested important nucleotides within the sgrS/ptsG annealing region, and emphasized the significance of Hfq in maintaining sgrS function.
Issue Date:2016-11-16
Type:Thesis
URI:http://hdl.handle.net/2142/95570
Rights Information:Copyright 2016 by Jichuan Zhang
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12


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