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Title:Mechanical manipulation and visualization of single biomolecules using fluorescence spectroscopy
Author(s):Mitra, Jaba
Director of Research:Ha, Taekjip
Doctoral Committee Chair(s):Cheng, Jianjun
Doctoral Committee Member(s):Braun, Paul; Leal, Cecilia; Chemla, Yann R.
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):mechanical manipulation, single molecule
Abstract:Interplay between mechanical forces and chemical transformations is fundamental to the strength of materials, polymer chemistry and biological processes including, cellular locomotion, segregation of chromosomes, enzymatic catalysis etc. Many major biological processes, such as translation, transcription etc. are regulated by forces in the range of a few picoNewtons (pNs). Hence, understanding the conformational dynamics of nucleic acids and proteins under physiological levels of tension is integral to interpreting their structure and function relationship in vivo. To address this, we employed an integrated force-FRET (Fluorescence resonance energy transfer) assay to interrogate single biomolecules under applied forces between ~ 0.3 and 28 pN. In human cells, the ends of chromosomes are capped by 100-200 nt long telomeric DNA, consisting of tandem hexanucleotide guanine (G)-rich repeats. Four of such telomeric, G-rich motifs can spontaneously associate and fold into thermodynamically stable structures, known as G-Quadruplexes (GQs). GQs are widely studied as negative regulators of cancer-associated physiological machinery. Using our single molecule force-FRET assay, we demonstrated unprecedented heterogeneity in GQs, with at least six interconvertible species which differ in mechanical responses, but are largely resistant to unfolding by cellular motor proteins implicated in transcription, replication etc. We further demonstrated polarized GQ formation, initiated at the 3’ end of longer telomeric sequences. Our force-FRET studies revealed direct conformational regulation of a GQ-core in presence of unassociated G-rich motifs. The extreme conformational and mechanical diversity of GQs may induce differential protein binding in cells and serve as cornerstones for engineering novel anti-cancer therapeutics. In view of the structural and functional versatility of GQs in the eukaryotic genome, they have been harnessed for development of molecular tools to bind to a myriad of ligands. For example, “light-up” aptamers used for visualization and localization of RNA in cells, consist of an integral GQ-core. Such aptamers are of intricate geometry and hence susceptible to inadvertent misfolding or unfolding in cells. We evaluated the mechanical stabilities of two classes of such RNA aptamers, known as Mango and Spinach. Our results revealed that Spinach is mechanically weaker and hence vulnerable to unraveling via helicases and polymerases. Furthermore, we demonstrated, for the first time, high fidelity folding of the aptamers using a vectorial assay, mimicking co-transcriptional RNA folding. We next extended our assay to demonstrate linear spring-like mechanical response of a malarial circumsporozoite protein (CSP). CSP being a surface protein, this explains the high motility of the sporozoites through vasculature with varying diameters. Moreover, as the repeat peptides are highly sensitive to forces up to ~ 28 pN, they can be used for engineering cellular tension sensors, for reporting force values within a wide range between ~ 1 and 28 pN. Macromolecular complexes of proteins are ubiquitous in the cellular milieu. Their composition, stoichiometry, order of assembly etc. are intrinsic to their underlying functions. We combined the principles of conventional immunoprecipitation assays with single molecule fluorescence microscopy into a single-molecule pull-down (SiMPull) assay, to probe native macromolecular complexes. With this assay, we investigated the mechanism of disruption of complexes of mechanistic target of Rapamycin (mTOR), underlying neuronal signaling and -synuclein mediated neurodegeneration.
Issue Date:2019-12-18
Type:Thesis
URI:http://hdl.handle.net/2142/108076
Rights Information:Copyright 2020 Jaba Mitra
Date Available in IDEALS:2020-08-26
Date Deposited:2020-05


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