Kandadai Srinivas A, Li Yingfu
Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
Nucleic Acids Res. 2006 Jan 3;33(22):7164-75. doi: 10.1093/nar/gki1013. Print 2005.
We previously demonstrated--through the isolation of RNA-cleaving deoxyribozymes by in vitro selection that are catalytically active in highly acidic solutions--that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a k(obs) of approximately 1 min(-1) at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indicated that pH4DZ1 was a considerably large deoxyribozyme, requiring approximately 80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyribozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experimentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of approximately 10(6)-fold. This study provides valuable information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA.
我们之前通过体外筛选分离出在高酸性溶液中具有催化活性的RNA切割脱氧核酶，证明了尽管DNA化学结构简单，但在具有挑战性的化学条件下仍能进行催化作用[Liu, Z., Mei, S.H., Brennan, J.D. 和Li, Y. (2003) J. Am. Chem. Soc. 125, 7539 - 7545]。其中一个引人注目的DNA分子是pH4DZ1，它是一种自我切割的脱氧核酶，在pH 3.8时表现出约1 min⁻¹的表观速率常数(k(obs))。在本研究中，我们进行了一系列实验来研究这种酸性脱氧核酶的序列和催化特性。广泛的核苷酸截短实验表明，pH4DZ1是一种相当大的脱氧核酶，最初的123个核苷酸中约80个对最佳催化活性是必需的。一项重新筛选实验确定了分布在三个催化关键序列元件中的10个绝对保守的核苷酸。此外，成功设计了一种反式脱氧核酶。将pH4DZ1的观测速率常数与未催化反应的实验测定速率常数进行比较，发现pH4DZ1实现了约10⁶倍的速率增强。本研究为这种低pH功能的脱氧核酶提供了有价值的信息，并为进一步对这种独特的催化DNA进行结构和机制表征铺平了道路。
Zotero 插件
