經由TW17核酸酶in trans 催化系統設計之應用於該磷酸硫酯鍵水解反應機制探討

中文摘要本實驗室先前經人為演化篩選，成功的發現一個具有鋅二價離子催化相依性之RNA 分子，該RNA 我們命名為TW17 核酸酶 (ribozyme)，並對TW17 核酸酶的結構與催化機理已有深入的研究；因此本論文承襲先前TW17 核酸酶的基礎研究成果，對於TW17 核酸酶的催化機理及其催化效能之改善，進行更深入的探討。之前研究結果，顯示TW17 核酸酶具有催化水解磷酸硫酯鍵功能之專一性，鋅二價離子在TW17 核酸酶的催化反應裏，扮演不可或缺的角色。為了進一步證實TW17 核酸酶的催化機制，我們純化出TW17 核酸酶的 cis-acting 及trans-acting 催化產物，並經由利用MALDI-TOF MS 分析這些產物，以期驗證TW17 RNA 的確是水解磷酸硫酯鍵之核酸酶，並且證實其催化產物之一為5’端具有GMP (guanosine-5’-monophosphate)的RNA。另外為了改善TW17 核酸酶的 trans-acting 催化系統之效能，我們改變受質端與催化端的鹼基配對的長度，藉由古典的Langmuir binding isotherm 分析，探討是否能達到降低該催化系統吸附解離常數(Kd)之目標。我們實驗結果顯示原始的 TW17 核酸酶的trans-acting 催化系統，已有相當的吸附能力(Kd = 0.18 ?b 0.06 ?嵱；?孱binding = -9.19 kcal/mol)，而且改變受質端與催化端的鹼基配對長度，並無法降低Kd 值;這些結果顯現在TW17 核酸酶的 VI trans-acting 催化裏，受質端與催化端的鹼基配對過程，有可能並非催化反應的速率決定步驟。本論文研究，不僅將能驗證TW17 核酸酶催化水解磷酸硫酯鍵之專一性，也提供更深入了解其催化機制的重要資訊。

關鍵字

核酸酶催化；磷酸硫酯鍵

並列摘要

英文摘要 The TW17 RNA is a unique Zn2+-dependent ribozyme capable of performing cis-acting catalysis to hydrolyze a phosphorothiolate thiolester bond between the RNA molecule and its substrate. The objectives of the study are to further decipher the reaction mechanism of TW17 ribozyme catalysis and improve its catalytic efficiency. We previously dissected the ribozyme into a trans-acting ribozyme system containing two RNA fragments: a smaller substrate-bearing RNA (18-mer) and a larger RNA containing the catalytic core (69-mer), and demonstrated catalytic efficiency of the trans-acting ribozyme system. We first purified the reaction product of the trans-acting TW17 ribozyme system and analyzed the acquired RNA by MALDI-TOF MS to unravel the substrate specificity of the ribozyme. The MALDI-TOF analysis intends to provide evidence that the reaction product is a GMP-primed 18-mer RNA and support the TW17 ribozyme as a phosphorothiolate thiolesterase RNA. We also reasoned that further enhancing adsorption of the substrate-bearing RNA on the catalytic RNA moiety could improve catalytic efficiency of the original trans-acting TW17 ribozyme system. Our studies indicated that the original trans-acting TW17 ribozyme system had satisfactory binding affinity with Kd of 0.18 ?b 0.06 ?嵱. When strengthening the binding stem between the two RNA moieties or extending their binding stem length, these structural-modified trans-acting TW17 ribozyme systems showed similar or inferior binding affinity (similar or higher Kd values) to that of the original one. Adsorption of the substrate RNA on the catalytic core RNA is thus unlikely the rate-determining step during catalysis. This study sheds light on the mechanism of TW17 ribozyme catalysis.