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首页> 外文期刊>Biochemistry >In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage
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In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage

机译：脱氧核糖核酸酶的合作选择结合两个镧系离子的RNA切割。

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Trivalent lanthanide ions (Ln(3+)) were recently employed to select RNA-cleaving DNAzymes, and three new DNAzymes have been reported so far. In this work, dysprosium (Dy3+) was used with a library containing 50 random nucleotides. After six rounds of in vitro selection, a new DNAzyme named Dy10a was obtained and characterized. Dy10a has a bulged hairpin structure cleaving a RNA/DNA chimeric substrate. Dy10a is highly active in the presence of the five Ln(3+) ions in the middle of the lanthanide series (Sm3+, Eu3+, Gd3+, Tb3+, and Dy3+), while its activity descends on the two sides. The cleavage rate reaches 0.6 min(-1) at pH 6 with just 200 nM Sm3+, which is the fastest among all known Ln(3+)-dependent enzymes. Dy10a binds two Ln(3+) ions cooperatively. When a phosphorothioate (PS) modification is introduced at the cleavage junction, the activity decreases by >2500-fold for both the R-p and S-p diastereomers, and thiophilic Cd2+ cannot rescue the activity. The pH-rate profile has a slope of 0.37 between pH 4.2 and 5.2, and the slope was even lower at higher pH. On the basis of these data, a model of metal binding is proposed. Finally, a catalytic beacon sensor that can detect Ho3+ down to 1.7 nM is constructed.
机译：三价镧系离子（Ln（3+））最近被用于选择可裂解RNA的DNAzyme，到目前为止，已经报道了三种新的DNAzyme。在这项工作中，s（Dy3 +）与包含50个随机核苷酸的文库一起使用。经过六轮体外选择后，获得并鉴定了一种新的名为Dy10a的DNA酶。 Dy10a具有凸出的发夹结构，可切割RNA / DNA嵌合底物。 Dy10a在镧系系列中的五个Ln（3+）离子（Sm3 +，Eu3 +，Gd3 +，Tb3 +和Dy3 +）的中间存在时具有很高的活性，而其活性却在两侧下降。在pH 6下，仅200 nM Sm3 +的裂解速率达到0.6 min（-1），这是所有已知的Ln（3+）依赖酶中最快的。 Dy10a协同结合两个Ln（3+）离子。当在裂解连接处引入硫代磷酸酯（PS）修饰时，R-p和S-p非对映异构体的活性降低> 2500倍，并且亲硫Cd2 +无法挽救该活性。 pH速率分布在pH 4.2和5.2之间具有0.37的斜率，并且在较高的pH下斜率甚至更低。基于这些数据，提出了金属结合的模型。最后，构建了一个催化信标传感器，可以检测低至1.7 nM的Ho3 +。

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