Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores
Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores
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DOI：
10.1016/j.jmb.2006.01.036
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发表时间：
2006-03-31
影响因子：
5.6
通讯作者：
Li, YF
中科院分区：
文献类型：
--
作者：
Chiuman, W;Li, YF
包含多达 10(16) 个候选核酸的文库通常用于在体外选择实验中分离人工核酶和脱氧核酶 (DNAzyme)，只有少数序列能够在多轮严格的选择步骤中幸存下来。这些获胜物种通常是人们关注的焦点，而竞争力较弱的竞争者通常不会受到审查。然而，在选择过程中被遗弃的分子种类可能仍然代表着丰富的催化基序库，这些基序对于检查 DNA 固有的催化能力以及设计实际应用的分子工具很有用。在这里，我们报告了一项对六种 RNA 切割、荧光信号脱氧核酶的研究，这些脱氧核酶出现在先前体外选择实验的早期世代中，使用了重选、合理结构分析和反应条件优化的组合方法。发现所有六种脱氧核酶都使用三路连接作为催化的共同结构框架。然而，在保守核苷酸分配、甲基化干扰模式和金属离子选择性方面观察到的差异表明不同的催化核心。优化后的脱氧核酶的速率常数在0.2至1.6min(-1)范围内，与类似核酶的速率常数相当。我们的研究结果表明，通过严格的选择标准消除的脱氧核酶是结构简单的分子，可以定制成有效的催化剂。 (c) 2006 Elsevier Ltd. 保留所有权利。
A library containing as many as 10(16) nucleic acid candidates is typically used to isolate artificial ribozymes and deoxyribozymes (DNAzymes) in an in vitro selection experiment, with only a handful of sequences surviving many rounds of stringent selection steps. These winning species are generally the focus of interest whereas the less competitive contenders are usually not examined. Nevertheless, molecular species abandoned during the selection process might still represent a rich pool of catalytic motifs that are useful for the examination of DNA's inherent catalytic ability, and for the design of molecular tools for practical applications. Here we report a study of six RNA-cleaving, fluorescence-signa ling deoxyribozymes that appeared in the early generations of a previous in vitro selection experiment, using the combined approaches of reselection, rational structural analysis, and reaction condition optimization. All six deoxyribozymes were found to use a three-way junction as a common structural framework for catalysis. However, disparities observed in the conserved nucleotide allocations, methylation interference patterns and metal-ion selectivities, pointed to distinct catalytic cores. The rate constants of the optimized deoxyribozymes fell in the range of similar to 0.2 to 1.6 min(-1), which are comparable to those of similar ribozymes. Our findings indicate that deoxyribozymes eliminated by harsh selection criteria are structurally simple molecules that can be tailored into efficient catalysts. (c) 2006 Elsevier Ltd. All rights reserved.