Secondary-structure characterization of two proficient kinase deoxyribozymes
Secondary-structure characterization of two proficient kinase deoxyribozymes
复制标题
DOI：
10.1021/bi0483054
复制
发表时间：
2005-03-15
期刊：
影响因子：
2.9
通讯作者：
Li, YF
中科院分区：
文献类型：
--
作者：
Achenbach, JC;Jeffries, GA;Li, YF
DK1和DK2是两个催化熟练的、依赖于锰的、富含鸟嘌呤的脱氧核酶，以前分离出来用于DNA磷酸化。在本研究中，我们开展了一系列实验，旨在了解DK1和DK2的结构特性，并比较这两种具有相似催化功能的不同脱氧核酶的结构异同。首先，我们在原始DK1和DK2序列的基础上，从两个部分随机化的DNA文库中进行重选，以分离催化活性序列变体，并鉴定不变的、保守的或高度诱变的核苷酸。在二级结构预测的辅助下，对这些变异体的序列分析导致了每个脱氧核酶中可能的Watson-Crick碱基配对区域的鉴定。序列截断和碱基对伙伴交换实验证实或排除了预测的二级结构元件的存在。最后，应用甲基化干扰实验来鉴定对脱氧核酶的三级结构折叠具有潜在重要性的核苷酸。我们的数据表明，尽管DK1和DK2的一级序列和NTP要求不同，但它们使用类似的茎环元件锚定一个具有实质性三级相互作用的结构域来执行其催化功能。
Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.