Development and application of NMR spectroscopy in G-quadruplexes : structure, dynamics and formation in biofilm

• Main Author: Winnerdy, Fernaldo Richtia
• Other Authors: Phan Anh Tuan
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2019
• Subjects: DRNTU::Science::Biological sciences::Biophysics
• Online Access: https://hdl.handle.net/10356/89640; http://hdl.handle.net/10220/47708
• Institution: Nanyang Technological University

G-quadruplex is a secondary non-canonical structure of nucleic acid which gained popularity due to increasing evidence of its biological functions, potential therapeutic prospects and nanotechnology applications. Encouraged for a better comprehension of G-quadruplex as an alternative DNA structures, we address multiple angles of G-quadruplex properties including its dynamics, ligand binding kinetics, numerous novel topologies and potential role in biofilm networking. This research was accomplished through a combination of biophysical techniques, primarily Nuclear Magnetic Resonance (NMR) spectroscopy. In this dissertation, G-quadruplex conformational and ligand-interaction dynamics are investigated with NMR spectroscopy. In particular, we developed two specific NMR methodologies for the measurements of the rotation rates of amino protons in guanines of G-quadruplexes via lineshape analysis and the determination of the residence time of ligands bound to G-quadruplex receptors via NOESY exchange analysis. Applications of NMR spectroscopy in the investigation of novel G-quadruplex folding topologies and the analysis of G-quadruplex formation in biofilm are demonstrated. Multiple high-resolution structures are resolved using NMR spectroscopy: (1) Complex structures of 11-guanine G-quadruplex (D4 ) bound to cell-signaling metabolite cyclic guanosine-adenosine monophosphate (cGAMP), (2) a fold-back G-quadruplex structure featuring a triad and base-pair formation on an anti-parallel G-quadruplex core (AT21S ), and (3) a left and right-handed G-quadruplex hybrid formation from multiple trinucleotide repeats (GGT ) 8 . Additionally, we de-termined that extracellular nucleic acid is the main contributor towards polymer networking property of P. aeruginosa biofilm (extracellular matrix). We proved the existence of non-canonical RNA-DNA complexes and potential formation of G-quadruplexes are responsible for the gel-networking characteristic of biofilms. The methodologies presented in this thesis — lineshape analysis and NOESY exchange analysis — contribute as effective options in measuring G-quadruplex dynamics. Furthermore, the applications of NMR spectroscopy explored here provide insights on: (1) Vacancy-bearing G-quadruplex as metabolite binder, (2) fundamentals of G-quadruplex structural elements of fold-back diagonal loops, (3) expansion of G-quadruplex diversity based on the existence of left-handed right-handed junction, and (4) potential biological importance of G-quadruplexes in biofilm network.