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

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研究生：	魏明財 Wey, Ming-Tsai
論文名稱：	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
指導教授：	呂平江 Lyu, Ping Chiang 甘魯生 Kan, Lou-Sing
口試委員:
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2009
畢業學年度：	97
語文別：	英文
論文頁數：	160
中文關鍵詞：	緊彎DNA三螺旋、不完整配對DNA三螺旋
相關次數：	點閱：2 下載：0
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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.

Table of content

Abstract ……………………………………………………………………… 1
Abbreviations ………………………………………………………..…..... 2
Table of content ………………………………………………………..…. 4
List of tables …………………………………………………………....… 10
List of figures ………………………………………………………...…... 11

Chapter One:  Preface ………………………………………….…..… 14
References …………………………………………………………….……….. 19

Chapter Two:  The paperclip triplex: understanding the role of apex residues in tight-turns ………………………………………………... 20
2.1  Introduction ………………………………………………………………. 21
2.2  Results ……………………………………………………………………. 24
2.2.1  A complete pypy/pu apex triad is required for a paperclip type triplex … 24
2.2.2  Requirement for a complete pypy/pu is independent of base identity ….. 25
2.2.3  Triplex formation is independent of the location of the CC/G pypy/pu turn ………………………………………………………………... 26
2.2.4  Triplexes are more stable than duplexes ……………………………... 27
2.2.5  Formation of a bimolecular triplex cc+ag ……………………………. 27
2.2.6  Native gel electrophoresis …….…………………...………………... 28
2.2.7  NMR assignment and conformation of cc+ag ………………………... 28
2.2.8  Structure analysis …………………………………………………... 32
2.3  Discussion ………………………………………………………………... 34
Tables ……………………………………………………………………….…. 39
Figures ………………………………………………………………………… 42
References …………………………………………………………….……….. 51

Chapter Three:  The formation and detection of tight-turn in pyrimidine/purine/pyrimidine type DNA triplexes – new findings on motifs CC and TT by fluorescence spectroscopy at pH 7 ……………. 55
3.1  Introduction …………………………………………………..…………... 56
3.2  Results and discussion ………………………………………………….... 58
3.2.1  The confirmation of triplex formation of probe + target by native gel electrophoresis …………………………………………………….. 58
3.2.2  The confirmation of triplex formation by fluorescent intensity reduction of the probe ………………………………………………………….. 59
3.2.3  The fluorescent emission of probes as a function of the concentration of the targets ……………………………………………………………... 60
3.2.4  The determination of the equilibrium constant of triplexes ………….... 61
3.2.5  The thermodynamic analysis of the DNA triplex formation …………... 61
3.3  Conclusion ………………………………………………………………... 63 Tables …………………………………………………………………………... 64
Figures ……………………………………………………………………….… 66
References ……………………………………………………………………... 72

Chapter Four:  Direct observation of single molecule conformational change of tight-turn paperclip DNA triplex in solution ………………………………………………………………….……. 74
4.1  Introduction ……………………………………………………………… 75
4.2  Results and discussion ……………………………………….….……….. 77
4.2.1  Direct visualization of DNA triplexes and duplexes in solution by AFM ……………………………………………………….……… 77
4.2.2  Paperclip type triplex and duplex or single-strand structure of TTGA36, CCAG36, TTGA54, and CCAG54 and the duplex CDC25 by CD spectra ………..…...................................................................……. 79
4.2.3  Similar sequences may regulate gene expression in biological systems ... 80
4.3  Conclusion ……………………………………………………………….. 82
Figures ………………………………………………………………………… 83
References …………………………………………………………………….. 87

Chapter Five:  Thermodynamic and kinetic studies of a stable imperfect DNA triplex by spectroscopic and calorimetric methods ... 90
5.1  Introduction ………………………………………………………………. 91
5.2  Results and discussion …………………………………………….……. 94
5.2.1  Formation of WCH+ triplex …………………………………………. 94
5.2.2  Confirmation of the WCH+ triplex formation using CD spectra ………. 95
5.2.3  Stability studies of WCH+ triplex in UV melting experiment …………. 96
5.2.4  ITC analysis for WCH+ triplex formation ……………………………. 96
5.2.5  Real time analysis and kinetic rate constant measurements of WCH+ triplex formation by SPR spectroscopy …………………………………….. 98
5.3  Conclusion ……………………………………………………….…....... 100
Table ……………………………………………………………………..…… 102
Figures ……………………………………………………………………..… 103
References ………………………………………………………………...…. 110

Chapter Six:  Summary and perspectives …………………..…..… 115

Chapter Seven:  Materials and methods ………………..……....... 120
7.1   Synthesis of oligodeoxyribonucleotides …………………………....…. 121
7.2   Melting temperature (Tm) measurements by UV spectrophotometry .... 122
7.3   Circular Dichroism spectropolarimetry (CD) ……………………..…... 122
7.4   Native gel electrophoresis …………………………………………..…. 123
7.5   Fluorescence spectroscopic measurements ……………………..……... 123
7.6   Calculation of equilibrium dissociation constant and thermodynamic parameters of hairpin (and tight-turn) triplex formation …………….... 124
7.7   Thermodynamic analysis of hairpin triplex association …………..…... 125
7.8   Pretreatment of mica with NiCl2 and APTES ………………..………... 126
7.9   AFM imaging ………………………………………………………….. 126
7.10  Isothermal titration calorimetry (ITC) ……………………………….... 127
7.11  Surface Plasmon Resonance (SPR) experiment ……………………...... 128
7.12  NMR spectroscopy ………………………………………………..…… 129
7.13  Constraint determination …………………………………….……..….. 132
7.14  Molecular Dynamic Simulation ………………………………..……… 132
Tables …………………………………………………………………..…….. 134
References ………………………………………………………………....…. 138

Appendix ………………………………………………………….....…… 140
Appendix 2.1  Amino proton region on cytosines of NOESY spectrum of cc+ag in H2O at pH 6.0 acquired at 500 MHz at 20℃ ……....…...... 141
Appendix 2.2  Region of NOESY spectrum acquired at 600 MHz of cc+ag in D2O at pH 6.0 depicting the H1'-H3'/H4' correlations …….... 142
Appendix 2.3  Sequential assignments of 1H-31P correlation spectrum of cc+ag in D2O at pH 6.0, 35℃, acquired at 600 MHz …....…….…... 143
Appendix 2.4  Cross-section through each triad in the cc+ag structure ....…. 144
Appendix 3.1  Fluorescence emission spectra of probe (50 nM) in the presence of different concentrations of specific target at pH6 and 7 in a temperature 20ºC …...........................................................…. 145
Appendix 3.2  The plots of fluorescent intensities at 520 nm (I520) of probes versus the concentration of targets at various temperatures and pH 6 or 7 ………………………………………………...…. 152
Appendix 4.1  AFM images showing the pH dependence in formation of
paperclip triplexes and duplexes in the dry state …….…..…. 158
Appendix 5.1  Native polyacrylamide gels of the three oligomer components in the imperfect triplex WCH+ at 10℃ and 45℃ …................... 159
Appendix 5.2  A comparison of DNA melting curve for the single strand C  and CH, duplex WC and WH, and triplex WCH+ at pH=4.5 ... 160

                                

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