Characterization of non-8-17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes.
Characterization of non-8-17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes.
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DOI：
10.1039/c1mb05034f
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发表时间：
2011-01
影响因子：
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通讯作者：
Jeffrey C. F. Lam;Samantha O. Kwan;Yingfu Li
中科院分区：
文献类型：
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作者：
Jeffrey C. F. Lam;Samantha O. Kwan;Yingfu Li
RNA裂解脱氧核酶(DNAzyme)可以通过体外选择的方法从随机序列的DNA池中分离出来。然而，小而简单的催化基序，如8-17DNAzyme，通常在序列空间中观察到，这给发现大而复杂的DNAzyme带来了挑战。为了研究来自体外选择的不具代表性的分子物种，在本研究中，我们试图表征从先前的体外选择实验中获得的非8-17序列，其中8-17脱氧核酶是主要基序。我们通过对21个基序的9个序列家族的结构和功能特征的分析，研究了它们的结构和功能。我们利用三向或四向连接结构框架发现了9个具有大催化结构域(>；40个核苷酸)的新型脱氧核酶。动力学研究表明，与其他已知的裂解核糖核酸的核酶相比，这些脱氧核酶表现出中等到优秀的催化速率(k(Obs)从0.003到1min(-1))。虽然化学探测实验、定点突变分析和金属辅因子依赖性测试表明每个脱氧核酶都有独特的催化核心，但具有相似二级结构特征的DNAzyme之间存在共同的二核苷酸连接选择性。总之，我们的发现表明，尽管序列空间由更小、结构更简单的催化剂主导，但更大、结构更复杂和多样化的催化基序能够在体外选择的过程中存活下来。
RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.