臺灣博碩士論文加值系統

DNA 三螺旋在自然態基因體中是一個動態的結構。本論文之研究標的為一穩定
17mer 寡核酸鏈組成之DNA 三螺旋結構。針對DNA 雙螺旋解離能與拉開雙股所需之力
的大小，我們是以自製之雙光束單光光鉗作為介觀尺度研究工具。由研究所得水溶液中，
可將17 個鹼基對之雙股螺旋拉開所需之力的大小約為30pN，而其解離能約是
3.1*10-18J。同時我們亦經由分子信標標定在雙股螺旋寡核酸鏈的3’端鍵結了一個
quencher，在Hoogsteen 寡核酸鏈的3’端接了一個螢光標定物，經由位置相關性之分子
信標之螢光亮滅來觀察DNA 配對形成。由實驗得知Hoogsteen 與Watson 兩股之方向
為同一極性。而DNA 三螺旋在酸性溶液中，即可形成穩定之三螺旋結構亦可由分子信
標証明。
在蛋白質分子操作上，我們經由過臨界折疊路徑來置換金屬硫蛋白中金屬的種類、
比例，得到了Mn,Cd-MT-2。Mn,Cd-MT-2 為一具有鐵磁性質的金屬結合蛋白質，包含了
2 個錳離子與5 個鎘離子，為一已摺疊蛋白質，且在277K-330K 都還有磁性。同時，由
DLS 量測出Mn,Cd-MT-2 有一均勻的尺寸分布，這個結果指出每一個Mn,Cd-MT-2 分子
皆為單分子磁體。而尺寸適中的Mn,Cd-MT-2 磁矩可能是因電子自旋雙交換模型中，硫
在兩個Mn2+，一個Cd2+的β 金屬結合的團簇中，如同一硫橋一樣，形成閃鋅礦結構。
而Mn,Cd-MT-2 的胜肽骨架提供了像是配對一樣的硫橋讓磁矩以接近完美的結構排列
著。這分子磁體工程不僅確認為單分子層級的電子自旋雙交換機制，且其分子磁性為獨
一無二的特性和生物相容性，使得Mn,Cd-MT-2 為一生物影像廣大應用的對象與生物奈
米元件之重要材料。

DNA triplex is a kinetic structure in native gene system. In this study, the rupture force
and dissociation energy of double strand DNA was measured by a home-made optical tweezer.
The magnitude of rupture force of ds-DNA (17 mer) is around 30 pN and the dissociation
energy is around 3.1*10-18 J for water environment . Meanwhile, the pair of positional
dependent molecular beacon those were labeled on each oligonucleotide strand inducted that
both Hoogsteen and Watson strands had same polarity in DNA triplex. Meanwhile, the CW
and H can self-assembly and formed triplex structure in an acidic environment.
Magnetic properties of metal binding protein, metallothionein (MT), can be engineered
by replacing the ratio and species of metal ions containing of MT via an over-critical refolding
process. A ferromagnetic MT containing two Mn and five Cd (Mn,Cd-MT-2) has been
refolded and its magnetization persists from 277 to 330 K. Meanwhile, the uniform size
distribution as tested by dynamic light scattering indicated that each MT molecule is a single
molecule magnet. The sizable magnetic moment of Mn,Cd-MT-2 may be attributable to the
electrons spin double exchange model among two Mn2+ and one Cd2+ via sulfur bridges of its
　-metal binding cluster, a Zinc Blende structure, of MT. Meanwhile, the peptide backbone
of Mn,Cd-MT-2 serves as a bridging ligand to align these magnetic moments near perfection.
This magnetic engineering process not only verifies the mechanism of electron double
exchange model in single molecular level, and the unique features of its molecular magnetism
and bio-compatibility make it a good candidate for biological applications and sensing sources
of new nano-devices.

摘要……………………………………………………………………………….…Ⅲ
Abstract …………………………………………………………………………………Ⅳ
第I 章緒論..................................................................................................................... 1
I-1 前言.......................................................................................................................... 1
I-2 DNA 簡介............................................................................................................... 1
i DNA 三螺旋結構.................................................................................................... 2
I-3 奈米粒子應用......................................................................................................... 4
i 光學之應用......................................................................................................... 4
ii 催化性質及應用................................................................................................. 4
I-4 本論文研究動機..................................................................................................... 6
第II 章介觀尺度下雙股螺旋DNA 形成與解離之能量解析.................................... 7
II-1 論文回顧............................................................................................................. 7
i 光鉗的源起......................................................................................................... 7
ii 光鉗理論模型..................................................................................................... 9
iii 幾何光學模型推導........................................................................................... 10
iv 整道光束的作用力推導................................................................................... 13
II-2 實驗原理與設計............................................................................................... 18
i 光鉗基本架設................................................................................................... 18
ii 三軸可移動雙光束單光光鉗設計................................................................... 18
iii 系統元件原理................................................................................................... 24
iv 倒立式螢光顯微鏡........................................................................................... 26
v 分子信標(Molecular beacons).......................................................................... 38
II-3 實驗方法........................................................................................................... 39
i 樣品配製........................................................................................................... 39
ii 核酸雙螺旋解離能........................................................................................... 40
iii laser power 對應的熱動能............................................................................... 41
iv 分子信標實驗................................................................................................... 41
II-4 結果與討論....................................................................................................... 42
i 光鉗架設........................................................................................................... 42
ii 實驗遇到問題與解決方法............................................................................... 43
iii 分子信標........................................................................................................... 44
iv 核酸雙螺旋解離能........................................................................................... 46
第III 章磁性蛋白質....................................................................................................... 58
III-1 論文回顧........................................................................................................... 58
II
i 細菌：............................................................................................................... 59
ii 蜜蜂：............................................................................................................... 59
iii 海龜：............................................................................................................... 61
iv 龍蝦：............................................................................................................... 62
v 金屬蛋白(metalloproteins) ............................................................................... 64
III-2 實驗原理........................................................................................................... 65
i 量子磁學理論................................................................................................... 65
ii 電子自旋磁矩................................................................................................... 65
iii 電子軌道磁矩................................................................................................... 66
iv 原子磁矩........................................................................................................... 66

v 物質磁性分類................................................................................................... 68
vi 磁滯現象........................................................................................................... 73
vii 超交換作用模型(Super-exchange model)........................................................ 75
viii 雙交換作用模型(Double-exchange model) ..................................................... 75
ix 蛋白質似一階態轉變模型(First-order-like state transition) .......................... 78
III-3 實驗方法........................................................................................................... 80
i 研究樣品介紹................................................................................................... 80
ii 熱平衡透析法(Thermal equilibrium dialysis,TED) ......................................... 81
iii 超導量子干涉元件簡介................................................................................... 84
III-4 結果與討論....................................................................................................... 86
i 圓二色光譜....................................................................................................... 86
ii 動態光散射儀................................................................................................... 86
iii 紫外線吸收光譜............................................................................................... 86
iv 磁滯區線分析................................................................................................... 86
v 磁性蛋白質磁性分析....................................................................................... 92
第IV 章結論................................................................................................................... 94

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