Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Org Biomol Chem. 2010 Jun 21;8(12):2704-10. doi: 10.1039/c002766a. Epub 2010 Apr 21.
The development of alternative architectures for genetic information-encoding systems offers the possibility of new biotechnological tools as well as basic insights into the function of the natural system. In order to examine the potential of benzo-expanded DNA (xDNA) to encode and transfer biochemical information, we carried out a study of the processing of single xDNA pairs by DNA Polymerase I Klenow fragment (Kf, an A-family sterically rigid enzyme) and by the Sulfolobus solfataricus polymerase Dpo4 (a flexible Y-family polymerase). Steady-state kinetics were measured and compared for enzymatic synthesis of the four correct xDNA pairs and twelve mismatched pairs, by incorporation of dNTPs opposite single xDNA bases. Results showed that, like Kf, Dpo4 in most cases selected the correctly paired partner for each xDNA base, but with efficiency lowered by the enlarged pair size. We also evaluated kinetics for extension by these polymerases beyond xDNA pairs and mismatches, and for exonuclease editing by the Klenow exo+ polymerase. Interestingly, the two enzymes were markedly different: Dpo4 extended pairs with relatively high efficiencies (within 18-200-fold of natural DNA), whereas Kf essentially failed at extension. The favorable extension by Dpo4 was tested further by stepwise synthesis of up to four successive xDNA pairs on an xDNA template.
替代遗传信息编码系统的架构的发展为新的生物技术工具以及对自然系统功能的基本了解提供了可能性。为了研究苯并扩展 DNA（xDNA）编码和传递生化信息的潜力，我们研究了 DNA 聚合酶 I Klenow 片段（Kf，一种结构刚性的 A 家族酶）和 Sulfolobus solfataricus 聚合酶 Dpo4（一种灵活的 Y 家族聚合酶）对单个 xDNA 对的处理。通过在单个 xDNA 碱基上掺入 dNTP，测量并比较了 Kf 和 Dpo4 酶促合成四种正确的 xDNA 对和十二种错配的 xDNA 对的稳态动力学。结果表明，与 Kf 一样，Dpo4 在大多数情况下为每个 xDNA 碱基选择了正确配对的碱基对，但由于碱基对尺寸增大，效率降低。我们还评估了这些聚合酶在 xDNA 碱基对和错配之外的延伸动力学，以及 Klenow exo+ 聚合酶的外切核酸酶编辑动力学。有趣的是，这两种酶有明显的不同：Dpo4 以相对较高的效率（在天然 DNA 的 18-200 倍范围内）延伸碱基对，而 Kf 基本上无法延伸。通过在 xDNA 模板上逐步合成多达四个连续的 xDNA 对进一步测试了 Dpo4 的有利延伸。
