SEER - a novel method for the detection of genetic sequences

描述（由申请人提供）：本提案描述了用于特定DNA序列成像的新技术的开发。这些试剂将由具有识别特定DNA序列能力的信号产生酶的两个无活性部分组成。当合适的DNA序列存在时，这两个部分将相互结合并产生荧光信号。如果序列缺失或突变，则不会产生信号。因此，这些组件将充当"开启"传感器，在不存在序列定向重组的情况下基本上没有背景。这个系统被命名为SEER（Sequence-Enabled Enzyme Reactivation），能够“看到”或检测遗传信息。它应该提供一种灵敏而廉价的测定，可用作临床诊断剂。SEER可以检测病原体特有的特定核酸序列，例如用于检测食源性病原体或生物恐怖剂。它可用于检测基因组重排或指示端粒长度，这可以是癌症或年龄相关疾病的标志物。它还可以允许检测DNA可及性，不寻常的DNA结构和DNA修饰，这是目前无法通过类似的方法检测。这种方法最新颖的方面是它能够识别双链DNA，而不是变性的单链DNA。这一功能提供了报告单个活细胞内基因组信息的可能性，这是任何现有技术都无法提供的能力。该系统可以通过重新激活细胞毒性酶来杀死细胞，产生序列依赖性细胞死亡。因此，这项研究有可能影响疾病检测和治疗的研究。两个原型的SEER系统已被构建，基于GFP（SEER-GFP）和b-内酰胺酶（SEER-LAC）的重组。在这里，我们建议优化系统（目标1），并检查其检测能力的具体方面，在体外和活细胞中使用生物相关的目标（目标2 - 5）。

DESCRIPTION (provided by applicant): This proposal describes the development of a new technology for the imaging of specific DNA sequences. These agents will consist of two inactive parts of a signal-generating enzyme that have the ability to recognize specific DNA sequences. When the appropriate DNA sequence is present, the two parts will bind near each other and generate a fluorescent signal. If the sequence is absent or mutated, no signal will be generated. The components will therefore act as "turn-on" sensors, with essentially no background in the absence of sequence-directed reassembly. This system, designated SEER (Sequence-Enabled Enzyme Reactivation), is able to "see" or detect genetic information. It should provide a sensitive yet inexpensive assay that may be useful as a clinical diagnostic agent. SEER could detect specific nucleic acid sequences that are unique to a pathogen, such as for detecting food-borne pathogens or bio terror agents. It could be used to detect genomic rearrangements or indicate telomere length, which can be markers for cancer or age related diseases. It could also allow for the detection of DNA accessibility, unusual DNA structures and DNA modifications, which are presently undetectable by similar methods. The most novel aspect of this method is its ability to recognize double-stranded DNA, rather than denatured single-strand DNA. This feature presents the possibility to report on genomic information within individual living cells, an ability not provided by any existing technology. The system could be reconfigured to kill cells through reactivation of a cytotoxic enzyme, producing sequence-dependent cell death. This research therefore has the potential to impact studies of disease detection and treatment. Two prototype SEER systems have been constructed, based on the reassembly of GFP (SEER-GFP) and b-lactamase (SEER-LAC). Here we propose to optimize the systems (Aim 1) and examine specific aspects of their detection capabilities using biologically relevant targets in vitro and in living cells (Aims 2-5).