本論文的第一部分主要是探討有關DNA-胜?網路異位交互作用的能量變化之研究。根據先前研究設計，十種以XPRK及XHypRK為根據並結合N-甲基?咯（Py）的新型態胜?，觀察與DNA股間的交互作用。 足跡法實驗發現，經過兩種不同類型的網路異位協同模式，胜?通過交互作用並互補到DNA鏈上的多個結合位置。在溫度變化實驗中使用十二?RY-12結果顯示，在較低溫度的環境下（25℃）展現出網路協同模式（circuit type），而在較高的溫度（31℃及37℃）則表現出局部協同模式（partial-circuit type）。在圓二色光譜的研究方面，胜?明顯的誘導DNA結構變化並以二聚體的方式結合於DNA的小凹槽內。 等溫滴定微卡計使用了十二個胜?來做能量上的相互比較，實驗結果發現與短片段DNA雙股相互結合後產生強烈的放熱反應，焓的範圍由-14.7至-74.4千卡莫耳，熵能變化範圍則為-6.5至-65.9千卡莫耳。熵-焓補償作用（EEC）關係可以經由12個胜?以焓及熵能變化做圖，斜率趨近於1，而y截距則對應平均的自由能（ΔG）為-8.5千卡莫耳，而所觀察到的自由能範圍則為-8.2至-9.1千卡莫耳。另外RY-12變溫的能量變化，同樣符合熵-焓補償作用，使自由能能夠維持在恆定的範圍內，由三維圖可以更明顯的見到相互間的關係。此篇論文的第二部分，聚丙烯胺凝膠電泳和MALDI-TOF兩項實驗表明，苯丁酸氮芥-胜?綴合物:CLB-HyM-10和CLB-HyQ-10可以容易且有效率的在沒有加熱、化學輔助劑及UV輻射的幫忙下進行選擇性的切割DNA雙股序列。 圓二色光譜研究發現，CLB-HyM-10和CLB-HyQ-10可能以二聚體的形式結合至DNA小凹槽並產生結構上的變化。DNA-胜?的結合通過等溫滴定微卡計測得各項能量參數，顯示結合模式是屬於焓驅動的。且在DNA-胜?的相互作用中，維持恆定的自由能並符合熵-焓補償現象。最終推論出苯丁酸氮芥-胜?以強而有力的焓驅動結合至DNA 並產生類核酸?序列選擇性切割。 The first part of this thesis reports the study of the energetic basis of complex DNA–peptide interactions relating to allosteric interactions. In common with other designed peptides, ten new conjugates incorporating the XPRK or XHypRK motif (Hyp = hydroxyproline) attached to a N-methylpyrrole (Py) tract with a basic tail have been found to display cooperative binding to DNA involving multiple monodentate as well as interstrand bidentate interactions. Quantitative DNase I footprinting show that allosteric communication via cooperative binding to multiple sites on complementary DNA strands corresponds to two different types of DNA–peptide interaction network. Temperature variation experiments using a dodecapeptide RY-12 show that lower temperature (25 °C) favors a circuit type of allosteric interaction network, whereas higher temperatures (31 and 37 °C) afford only a partial-circuit type of network. Circular dichroism studies show that the peptides induce significant local conformational changes in DNA via the minor groove, with apparently dimeric binding stoichiometry. Isothermal titration calorimetry reveals that the peptides are strongly exothermic upon binding to a model 13-mer DNA duplex, as characterized by ΔH ranging from ?14.7 to ?74.4 kcal mol?1, and also high TΔS ranging from ?6.5 to ?65.9 kcal mol?1. Distinctive enthalpy–entropy compensation (EEC) relationships are demonstrated for the interaction of all twelve designed peptides with DNA, affording a straight line of slope close to unity when ΔH is plotted versus TΔS, with a y-axis intercept (average ΔG) corresponding to ?8.5 kcal mol?1, while the observed ΔG ranges from ?8.2 to ?9.1 kcal mol?1 for the peptides. The EEC seen with peptide RY-12 binding to the model duplex persists throughout various incubation temperatures. The net compensation of energy between the favorable negative ΔH and unfavorable negative ΔS components thus constrains the value of net binding free energy ΔG within a remarkably constant range, as is clearly visible in a 3-dimensional energetic plot. For the second part of this thesis, both polyacrylamide gel electrophoresis and MALDI-TOF experiments showed that chlorambucil-peptides conjugates CLB-HyM-10 and CLB-HyQ-10 can readily cleave DNA duplexes very effectively and sequence-selectively in the absence of heat, chemical additives and UV irradiation.Circular dichroism studies show that the conjugates CLB–HyM-10 and CLB–HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven. The maintenance of a conserved negative binding free energy in DNA–conjugate interactions is a crucial feature of the universal enthalpy–entropy compensation phenomenon. It is concluded that the strongly enthalpy-driven binding of CLB–peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.
