Large Scale Mutational and Kinetic Analysis of a Self-Hydrolyzing Deoxyribozyme
Large Scale Mutational and Kinetic Analysis of a Self-Hydrolyzing Deoxyribozyme
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DOI：
10.1021/acschembio.7b00621
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发表时间：
2017-12-01
影响因子：
4
通讯作者：
Yokobayashi, Yohei
中科院分区：
文献类型：
--
作者：
Dhamodharan, V.;Kobori, Shungo;Yokobayashi, Yohei
脱氧核酶是催化DNA序列，其原子结构通常难以阐明。突变分析仍然是理解和设计具有不同催化活性的脱氧核酶的主要方法。然而，单个序列的费力制备和生化表征严重限制了可以进行生化研究的突变体的数量。在这里，我们应用深度测序直接测量高通量的自水解脱氧核酶序列的活性。首先，测定脱氧核酶I-R3的15个碱基催化核心内的所有单突变体和双突变体，以明确确定每个位置处的耐受和不耐受突变。随后，4096脱氧核酶变异体与耐受的碱基取代在7个位置进行了动力学平行测定。我们确定了533个活性突变体的一阶速率常数和活化能的测定。结果表明，在脱氧核酶序列空间中的一个孤立的和狭窄的峰，并首次提供了多个突变对脱氧核酶活性的影响的定量视图。
Deoxyribozyrnes are catalytic DNA sequences whose atomic structures are generally difficult to elucidate. Mutational analysis remains a principal approach for understanding and engineering deoxyribozymes with diverse catalytic activities. However, laborious preparation and biochemical characterization of individual sequences severely limit the number of mutants that can be studied biochemically. Here, we applied deep sequencing to directly measure the activities of self-hydrolyzing deoxyribozyme sequences in high throughput. First, all single and double mutants within the 15-base catalytic core of the deoxyribozyme I-R3 were assayed to unambiguously determine the tolerated and untolerated mutations at each position. Subsequently, 4096 deoxyribozyme variants with tolerated base substitutions at seven positions were kinetically assayed in parallel. We identified 533 active mutants whose first-order rate constants and activation energies were determined. The results indicate an isolated and narrow peak in the deoxyribozyme sequence space and provide a quantitative view of the effects of multiple mutations on the deoxyribozyme activity for the first time.