鑭系金屬與大環多胺多酸基配位錯合物的動力學研究及其在DNA及RNA水解上的應用

Abstract

自從1994年第一個由人工合成的核酸酶被發表以來，原本半衰期極長的核糖核酸及去氧核糖核酸，也能夠在短暫的時間內經由人造的模擬酵素所催化分解，因此這引起了許多科學家們的注意和研究，並且加速RNA及DNA的水解速率便成為許多生化學家的挑戰，原因即在於人造的限制酶可能應用於基因工程及可能提供許多疾病一個更有效率、較低傷害的治療方式。 在此篇論文中，我們先從鑭系金屬與多胺多酸基大環配位子所形成的錯合物其動力學開始探討，由酸解反應速率常數，我們可得知吾人所設計的大環配位子和鑭系金屬具有很強的鍵結（binding）能力，也藉此證實了這系列的金屬錯合物具有很高的穩定性。 接著我們也利用此系列穩定性高之錯合物中的Ce(DO2A)+及Eu(DO2A)+在37℃，pH=8.0的條件下來切割雙股螺旋DNA（pUC19），並求出其個別的水解反應速率常數，得到的擬一級催化反應速率常數（kcat）分別為1.0601 ×10-3, 3.426 ×10-4 (s-1)，以及KM值為1.7846 ×10-3, 9.132 ×10-4 (M)。另外我們更進一步以Eu(DO2A)+來水解mRNA上帽體結構中的三磷酸鍵，同樣也求出其水解反應速率常數值（kcat）為1.746 ×10-4 (s-1) 及KM值為1.199 ×103 (M) 。從這些數據與其他文獻相較之後可證實此系列鑭系金屬錯合物不論在水解DNA及RNA上的確都擁有了高的水解能力。

ENGLISH ABSTRACT Since the first wholly synthetic artificial ribozyme was published in 1994, DNA and RNA whose half-life is very long can also be degradation in the short time. Therefore, it took scientists’ attention and researches, and is to promote the DNA’s or RNA’s hydrolysis rate became a challenge for many biochemists. Because the artificial restriction enzyme may be applied for gene engineering and probably provided a more effective and harmless cure. In the beginning of this thesis, we are using macrocyclic ligand 1,7-bis(carboxymethyl)1,4,7,10-tetraazacyclododecane (DO2A) and lanthanide ions to from complexes, and study their dissociation kinetics. Based on the obtained rate constants, we have found that, DO2A have strong binding affinity with lanthanide ions. Moreover, we proved that these complexes have very high stabilities again. In the other side, we use Ce(DO2A)+ and Eu(DO2A)+ to cleavage double-strand DNA(pUC19) and obtained the value of kcat are 1.0601×10-3, 3.426×10-4(s-1) and Km are 1.785×10-3,9.13210×-4(M). Further, we use Eu(DO2A)+ to hydrolysis the phosphate triester bonds at the cap structure of the mRNA. Also, we achieved the kcat which is 1.746×10-4(s-1). Compared with other’s data, it proved that the complexes have the excellent capability to cleave DNA and RNA.