DNAzyme核心催化區域序列分析

Analysis of the Sequences of DNAzyme in the Catalytic Core Region

曾麒升(Chi-Sheng Tseng)

指導教授 ： 尤禎祥

國立交通大學/生物科技學院/生物資訊及系統生物研究所/碩士(2020年)

摘要

一般的DNA為雙股結構且被當成生物的遺傳物質，但DNAzyme為單股DNA組成的巨分子且非遺傳物質。DNAzyme首先於1994年以人工合成的方式被發現，不同於核酶（ribozymes）廣泛存在於生物體中，DNAzymes截至目前為止尚未被發現存在於自然界的生物之中，但卻與核酶一樣具有催化功能。除此之外，組成的DNAzymes的DNA比起組成核酶的RNA要更加穩定，故被認為是有潛力的酵素。近二十多年來的研究透過體外選擇（in vitro selection）合成越來越多的DNAzymes，而這些酵素分別能催化多種生化反應，諸如：RNA裂解反應、RNA接合反應等等。然而體外選擇屬於隨機生成的方法以篩選出具有催化能力的DNA片段，雖然隨著研究DNAzyme的文獻與日俱增，DNAzyme的數量也隨之上升，但這些不同DNAzymes其序列之間的關聯性至今仍不甚明瞭；DNAzyme催化生化反應的反應機制等相關研究也尚在研究初期。

本論文蒐集並統整出142段DNAzyme序列，包含38段核心反應區域，依照DNAzyme之功能分類排列製表，有利於後續研究者更有效率的檢索。為了研究DNAzyme其序列彼此的關聯，本論文亦使用多種多重序列比對工具來分析38段核心反應區域是否具有同源性，並使用序列模體工具分析38段核心反應區域序列中是否具有獨特且重要的序列模體。鑑於DNAzyme的研究偏向個別案例發掘與研究，本論文盡可能地進行論文獻回顧並蒐集1994年至今發現的所有具功能之DNAzyme序列，以期達到對DNAzyme統整性的研究並試圖找出致使DNAzyme擁有生化催化功能的序列以釐清DNAzyme在催化反應上運作的關鍵機制。

經由多重序列比對的結果，本論文發現即使DNAzyme的生成較具隨機性，仍有些許核心反應區域具有高度的相似性，藉由分析這些類似的核心反應區域，我們得到兩段顯著的序列模體M1以及M2。將該二序列模體比對具結晶結構的DNAzyme後，發現本文所得到的M1及M2確實位於核心反應區域內。透過M1以及M2可以確立部分DNAzyme的核心反應區域並且建立指標，未來推廣到其他DNAzyme結晶結構及其反應機制的分子模擬將有所助益。

關鍵字

DNAzyme ； 多重序列比對 ； 序列模體

並列摘要

DNA is recognized as genetic material generally and stabilized with double helix structure, however, DNA also has the catalytic ability with single strand form -- DNAzyme. DNAzymes were discovered in 1994 by Breaker and Joyce. Unlike ribozymes existing in natural, DNAzymes are not found in natural so far. After the research, DNAzymes became popular theme of research in recently two decades. A DNAzyme is a single strand DNA similar with ribozyme, but the structure of DNAzyme is more stable than ribozyme. DNAzymes are able to catalyze many kinds of reactions including ligation of RNA and cleavage of RNA. By in vitro selection, there are more and more DNAzymes that have been synthesized. Due to the randomness of in vitro selection, the relevance among DNAzymes are still hard to identify. The researches in mechanism of DNAzyme just start and still remain unclear nowadays.
Because most research of DNAzymes focus on specific cases, this thesis would collect sequences of DNAzymes and give a systematic list of DNAzymes according to their functions. Through this thesis, we would like to clarify the key reactions of DNAzymes and organize the research of DNAzyme sequences collected in previous studies. There are 142 DNAzyme sequences in our list, including 38 sequences of core reaction regions. These DNAzymes are classified in this thesis by functions for easily searching. Besides organizing the information of DNAzymes, various multiple sequence alignment (MSA) algorithms were used to analyze the similarity of all core reaction regions of DNAzymes. After analyzing the core reaction regions by MSA, the high similarity sequences of core reaction regions were used to find the sequence motif.
As the result of MSA, different DNAzymes with high similarity are found even under randomness of in vitro selection. Moreover, two sequence motif, M1 and M2, are found in the sequence motif analysis. Comparing the sequence motif with the crystal structures of DNAzymes, both motif M1 and M2 are located in the core reaction regions of crystal structure. The result showing the sequence motif we found is meaningful. With M1 and M2, we make a benchmark for core reaction regions of some DNAzymes and provide the clue for the future simulation with those crystal structure.

並列關鍵字

DNAzyme ； multiple sequences alignment ； sequence motif

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