為探討aptamer與其ligand之交互作用機制，本研究選擇文獻上以L-tyrosinamide為ligand，且利用SELEX程序篩選所得的DNA aptamer（pe35 aptamer）作為研究之目標。本文利用恆溫滴定微卡計（ITC）與圓二色光譜儀（CD），設計不同的實驗條件，包含不同鹽濃度、金屬離子種類（一價（K+、Na+）、二價（Mg2+））、溫度、緩衝液pH、緩衝液種類以及結構相似物(analogues)等。期望藉由熱力學（ITC）與二級結構（CD）分析，來獲得aptamers與ligands辨識機制之相關資訊。 在熱力學分析上，pe35 aptamer與L-tyrNH2結合反應的發生是enthalpy driven，同時結合過程中伴隨著induced-fit程序。在二級結構探討上，pe35 aptamer具有自我形成hairpin之結構而stem區域所形成之helix為B-DNA form。當反應發生後其二級結構將由B-DNA form轉變成具有A-like特性之helix結構。在結合作用力的探討上，反應過程中包含：（1）靜電作用力且L-tyrNH2至少有一個NH３＋參與反應，（2）鹼基堆疊之疏水作用力，（3）質子化耦合效應以及（4）氫鍵等。在構型相似物的比較，研究發現認為L-tyrNH2上的phenolic hydroxyl是pe35 aptamer辨識L-tyrNH2的重要因素之ㄧ，影響因素可能包含提供氫鍵作用力以及參與誘導構型轉變使複合物更加緊密來提高結合常數。在金屬離子對結合行為影響的研究上，不同一價金屬離子（K+、Na+）對於pe35 aptamer與L-tyrNH2結合行為無明顯差異之影響，當提高鹽濃度，均造成結合常數下降。而在二價金屬離子（Mg2+）的研究中，實驗結果發現，Mg2+的存在對於結合行為是不可或缺的，且Mg2+本身即具有誘導pe35 aptamer構型轉變之能力。在熱力學部份，增加Mg2+濃度，則需要提供更多熱量來調整aptamer構型，並由減少entropy cost來驅動反應的進行。 本研究利用ITC所得之結合常數（KA）、熱力學參數（ΔH、ΔS），配合CD結構上的說明，已清楚說明DNA aptamer與其ligand辨識行為之作用力、熱力學以及結構等相關資訊。 Aptamers are macromolecules composed of nucleic acids, such as RNA or DNA, that bind tightly to a specific molecular target. In this study, we used Isothermal Titration Microcalorimetry (ITC) and Circular Dichroism (CD) to study the binding mechanism between a DNA aptamer and L-tyrosinamide. In order to gain further insights into the binding driven force in the recognizing behavior and the thermodynamic discrepancy, binding enthalpy measurements at different system parameters such as salt ion temperature pH value and analogues were carried out. Noteworthily, stabilizing the aptamer structure and enhanced target-aptamer complex formation by magnesium cation was also demonstrated in this study. ITC results indicate that the binding behavior is an enthalpy driven and entropy cost process. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding behavior is an induced-fit process and the conformation of DNA aptamer changes from B-form to A-from like in the binding process. In addition, binding mechanism analysis suggest that the interaction driven force in the binding process may include electrostatic interactions, hydrophobic interactions, hydrogen bonding and binding-linked protonation process. Furthermore, Mg2+ could not only help the forming of the complex by stable the conformation of the DNA aptamer but also change the structure of DNA aptamer.
