Ooi Aik T, Stains Cliff I, Ghosh Indraneel, Segal David J
Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 2006 Mar 21;45(11):3620-5. doi: 10.1021/bi0517032.
This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 beta-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences.
这项工作描述了一种检测特定双链DNA序列的新方法的开发。我们之前表明，绿色荧光蛋白的两个无活性片段，每个片段都与工程化锌指DNA结合蛋白偶联，在存在预定义DNA序列的情况下能够重新组装成一个活性报告复合物。这个系统，称为序列驱动重组（SEER），在体外被证明能产生DNA浓度依赖性信号。在这里，我们使用报告酶TEM-1β-内酰胺酶赋予SEER系统催化能力。该系统能够在不到5分钟的时间内区分目标DNA和非目标DNA，比我们之前的SEER设计有超过1000倍的改进。DNA结合序列中的单个碱基对替换将信号降低到几乎背景水平。替换不同的定制锌指DNA结合结构域仅在新的同源靶标上产生信号。当基因组DNA与目标DNA质量相等时，信号强度不受其影响。这些结果表明SEER是一种快速、灵敏的双链DNA序列检测方法。
