Enhancement of deoxyribozyme activity by cationic copolymers.
Enhancement of deoxyribozyme activity by cationic copolymers.
复制标题
阳离子共聚物增强脱氧核酶活性。
作者：
Jueyuan Gao;Naohiko Shimada;A. Maruyama
脱氧核酶或DNA酶是具有酶活性的DNA分子。具有核糖核酸酶活性的DNA酶在生物医学和生物分析领域具有各种潜在的应用，然而，尽管优化了反应条件和DNA酶结构，但大多数构建体的周转率有限。阳离子梳型共聚物加速DNA杂交和链交换速率，我们假设该共聚物将通过促进营业额提高脱氧核酶活性。该共聚物没有改变DNAzyme活性在单营业额条件下，这表明该共聚物既不影响DNAzyme的折叠结构，也不影响二价阳离子，催化辅因子，DNAzyme的关联。该共聚物增强活性的评价DNAzyme在一个很宽的温度范围内，在多个营业额的条件下。该共聚物在50 °C下使DNAzyme kcat/KM增加了50倍，50 ° C是在不存在该共聚物的情况下DNAzyme的最佳温度。当反应温度略高于酶/底物复合物的熔融温度时，加速效应最显著;观察到两个数量级的加速。我们的结论是，共聚物加速周转步骤，而不影响化学裂解步骤。相反，共聚物，阳离子表面活性剂，CTAB，强烈抑制DNAzyme活性下，无论是单或多营业额条件。
Deoxyribozymes, or DNAzymes, are DNA molecules with enzymatic activity. DNAzymes with ribonuclease activity have various potential applications in biomedical and bioanalytical fields; however, most constructs have limited turnover despite optimization of reaction conditions and DNAzyme structures. A cationic comb-type copolymer accelerates DNA hybridization and strand exchange rates, and we hypothesized that the copolymer would enhance deoxyribozyme activity by promoting turnover. The copolymer did not change DNAzyme activity under single-turnover conditions, suggesting that the copolymer affects neither the folding structure of DNAzyme nor the association of a divalent cation, a catalytic cofactor, to DNAzyme. The copolymer enhanced activity of the evaluated DNAzyme over a wide temperature range under multiple-turnover conditions. The copolymer increased the DNAzyme kcat/KM by fifty-fold at 50 °C, the optimal temperature for the DNAzyme in the absence of the copolymer. The acceleration effect was most significant when the reaction temperature was slightly higher than the melting temperature of the enzyme/substrate complex; acceleration of two orders of magnitude was observed. We concluded that the copolymer accelerated the turnover step without influencing the chemical cleavage step. In contrast to the copolymer, a cationic surfactant, CTAB, strongly inhibited the DNAzyme activity under either single- or multiple-turnover conditions.
DOI：
10.1073/pnas.88.18.8237
发表时间：
1991-09-01
影响因子：
11.1
作者：
PONTIUS, BW;BERG, P
通讯作者：
BERG, P
影响因子：
2.9
作者：
Santoro, SW;Joyce, GF
通讯作者：
Joyce, GF