Fa Ming, Radeghieri Annalisa, Henry Allison A, Romesberg Floyd E
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037-1000, USA.
J Am Chem Soc. 2004 Feb 18;126(6):1748-54. doi: 10.1021/ja038525p.
Nucleic acid polymerases are the most important reagents in biotechnology. Unfortunately, their high substrate specificity severely limits their applications. Polymerases with tailored substrate repertoires would significantly expand their potential and allow enzymatic synthesis of unnatural polymers for in vivo and in vitro applications. For example, the ability to synthesize 2'-O-methyl-modified polymers would provide access to materials possessing properties that make them attractive for biotechnology and therapeutic applications, but unfortunately, no known polymerases are capable of efficiently accepting these modified substrates. To evolve such enzymes, we have developed an activity-based selection method which isolates polymerase mutants with the desired property from libraries of the enzyme displayed on phage. In this report, mutants that could efficiently synthesize an unnatural polymer from 2'-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of their activity-dependent modification of a DNA oligonucleotide primer attached to the same phage particle. In each case, directed evolution resulted in relocating a critical side chain to a different position in the polypeptide, thus re-engineering the overall active site while preserving critical protein-DNA interactions. Remarkably, one evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.
核酸聚合酶是生物技术中最重要的试剂。不幸的是，它们高度的底物特异性严重限制了其应用。具有定制底物库的聚合酶将显著扩展其潜力，并允许酶促合成用于体内和体外应用的非天然聚合物。例如，合成2'-O-甲基修饰聚合物的能力将提供获得具有使其对生物技术和治疗应用具有吸引力的特性的材料的途径，但不幸的是，目前已知的聚合酶都不能有效接受这些修饰的底物。为了改造这类酶，我们开发了一种基于活性的筛选方法，该方法从展示在噬菌体上的酶库中分离出具有所需特性的聚合酶突变体。在本报告中，能够从2'-O-甲基核糖核苷三磷酸高效合成非天然聚合物的突变体通过其对附着在同一噬菌体颗粒上的DNA寡核苷酸引物的活性依赖性修饰而被固定并分离出来。在每种情况下，定向进化导致一个关键侧链重新定位到多肽中的不同位置，从而在保留关键的蛋白质-DNA相互作用的同时对整个活性位点进行重新设计。值得注意的是，一种进化后的聚合酶被证明能够以与野生型酶对天然底物相当的效率和保真度掺入修饰后的底物。
