Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization
Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization
复制标题
DOI：
10.1073/pnas.0503733102
复制
发表时间：
2005-11-08
影响因子：
11.1
通讯作者：
Griffiths, AD
中科院分区：
文献类型：
--
作者：
Agresti, JJ;Kelly, BT;Griffiths, AD
体外区室化（IVC）以前曾被用来进化蛋白质酶。在这里，我们演示了如何应用 IVC 来选择 RNA 酶（核酶），以获得以前无法选择的特性：真正的分子间催化。含有 10(11) 个核酶基因的文库被划分在油包水乳液的水滴中，使得大多数水滴含有不超过一个基因，并进行原位转录。通过共封装基因、RNA 和催化反应的底物/产物，可以选择核酶用于所有酶促反应。特性：底物识别、产物形成、速率加速和周转。在这里，我们利用 IVC 与指数富集配体系统进化 (SELEX) 的互补性，允许选择较大的文库 (>= 10(15)) 和非常小的速率加速 (k(cat)/k(uncat))，但仅选择分子内单周转反应。我们选择大约 10(14) 个随机 RNA 进行 Diels-Alderase 活性，进行五轮 SELEX，然后进行六到九轮 IVC。所有选定的核酶都以真正的双分子方式和多次周转来催化狄尔斯-阿尔德反应。几乎所有仅使用 11 轮 SELEX 选出的核酶都含有共同的催化基序。然后用SELEX进行选择，然后IVC给出了在该催化基序中具有显着序列变化的核酶和具有全新基序的核酶。有趣的是，所有选定的核酶的催化特性都非常相似。核酶受到强烈的产物抑制，与与产物非常相似的狄尔斯-阿尔德过渡态一致。更有效的狄尔斯-阿尔德酶可能需要催化第二个反应，以转化产物并防止产物抑制。
In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 10(11) ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By coencapsulating the gene, RNA, and the substrates/products of the catalyzed reaction, ribozymes can be selected for all enzymatic. properties: substrate recognition, product formation, rate acceleration, and turnover. Here we exploit the complementarity of IVC with systematic evolution of ligands by exponential enrichment (SELEX), which allows selection of larger libraries (>= 10(15)) and for very small rate accelerations (k(cat)/k(uncat)) but only selects for intrannolecular single-turnover reactions. We selected approximate to 10(14) random RNAs for Diels-Alderase activity with five rounds of SELEX, then six to nine rounds with IVC. All selected ribozymes catalyzed the Diels-Alder reaction in a truly bimolecular fashion and with multiple turnover. Nearly all ribozymes selected by using eleven rounds of SELEX alone contain a common catalytic motif. Selecting with SELEX then IVC gave ribozymes with significant sequence variations in this catalytic motif and ribozymes with completely novel motifs. Interestingly, the catalytic properties of all of the selected ribozymes were quite similar. The ribozymes are strongly product inhibited, consistent with the Diels-Alder transition state closely resembling the product. More efficient Diels-Alderases may need to catalyze a second reaction that transforms the product and prevents product inhibition.