The studies of structure and physical properties of DNA triplex with a tight-turn on sugar-phosphate backbone and with imperfect base-triads

緊彎DNA三螺旋與不完整配對DNA三螺旋的結構和物理性質研究

魏明財(Ming-Tsai Wey)

指導教授 ： 呂平江 ； 甘魯生

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2009年)

摘要

Triple-stranded nucleic acid helix is well recognized since its discovery in 1957. Ever since, there has been growing interest in DNA triplexes due to their potential roles in diagnostics and/or therapeutics as antigenes. DNA triplex formation is known to involve loops which are stabilized by the base pairs contained within, similar to the case of double helixes. However, we have found novel stable DNA triplexes formed by folding the chains without loops, i.e., in a tight-turn structure. It is the aim of this thesis to study the formation and the physical properties of this particular kind of triple DNAs. Chapters 2, 3, and 4 of this dissertation contain: structural elucidation of tight-turn triplexes by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation; formation confirmation by native gel electrophoresis, ultraviolet (UV) thermal melting curve and circular dichroism (CD) spectroscopy; and determination of association constant (Ka) and thermodynamic parameters (ΔH, ΔS, and ΔG) by fluorescence resonance energy transfer (FRET). The structure and shape of the tight-turn triplex can be observed directly at the single molecular level with atomic force microscopy (AFM). Chapter 5 describes the discovery of an imperfect but stable tri-molecular triplex containing two non-pyrimidine/purine/pyrimidine base triads in the middle. Native gel electrophoresis, UV absorption, and CD spectroscopy were used to monitor the formation reaction, at each stage, of the imperfect triplex; isothermal titration calorimetry (ITC) and surface Plasmon resonance (SPR) were used to acquire thermodynamic and kinetic information, respectively.

關鍵字

緊彎DNA三螺旋 ； 不完整配對DNA三螺旋

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