Rojas Stütz Jan A, Richert Clemens
Institut für Organische Chemie, Universität Karlsruhe TH, 76131 Karlsruhe, Germany.
Chemistry. 2006 Mar 8;12(9):2472-81. doi: 10.1002/chem.200501008.
The replication of genetic information relies on the template-directed extension of DNA primers catalyzed by polymerases. The active sites of polymerases accept four different substrates and ensure fidelity and processivity for each of them. Because of the pivotal role of catalyzed primer extension for life, it is important to better understand this reaction on a molecular level. Here we present results from primer-extension reactions performed with chemical systems that show high reactivity in the absence of polymerases. Small molecular caps linked to the 5'-terminus of templates are shown to enhance the rate and selectivity of primer extension driven by 2-methylimidazolides as activated monomers for any of the four different templating bases (A, C, G, and T). The most consistent effect is provided by a stilbene carboxamide residue, rather than larger aromatic or aliphatic substituents. Up to 20-fold rate enhancements were achieved for the reactions at the terminus of the template. The preference for a medium size cap can be explained by competing interactions with both the oligonucleotides and the incoming deoxynucleotide. The data also show that there is no particularly intractable problem in combining promiscuity with fidelity. Exploratory experiments involving a longer template and a downstream-binding strand with a 5'-cap show up to 38-fold rate acceleration over the same reaction templated by a single overhanging nucleotide.
遗传信息的复制依赖于聚合酶催化的DNA引物的模板导向延伸。聚合酶的活性位点接受四种不同的底物，并确保对每种底物的保真度和持续合成能力。由于催化引物延伸对生命起着关键作用，因此在分子水平上更好地理解这一反应非常重要。在此，我们展示了在无聚合酶情况下具有高反应活性的化学体系进行引物延伸反应的结果。与模板5'-末端相连的小分子帽被证明可以提高由2-甲基咪唑鎓盐作为四种不同模板碱基（A、C、G和T）的活化单体驱动的引物延伸的速率和选择性。芪甲酰胺残基提供了最一致的效果，而不是更大的芳香族或脂肪族取代基。模板末端的反应速率提高了20倍。对中等大小帽的偏好可以通过与寡核苷酸和进入的脱氧核苷酸的竞争相互作用来解释。数据还表明，在将通用性与保真度相结合方面没有特别棘手的问题。涉及更长模板和带有5'-帽的下游结合链的探索性实验表明，与由单个突出核苷酸作为模板的相同反应相比，反应速率加快了38倍。
