COMPLEMENTATION ASSAY FOR FUNCTIONAL GENOMICS

这项拟议的研究将证明一种新的、均一的核酸酶检测方法的可行性，该方法可用于核酸酶抑制剂的药物发现和核酸杂交检测。该方法利用大肠杆菌β-半乳糖苷酶失活酶片段的互补作为信号放大机制：将构建独特的环状分子，以包含β-半乳糖苷酶互补所需的多肽，以及核酸酶的寡核苷酸识别位点。寡核苷酸的切割将允许形成活性酶，从而提供核酸酶切割事件的催化放大。核酸酶检测方法非常灵敏，因为环状酶片段的本底活性低，切割时活性酶形成得很快，并且β-半乳糖苷酶的周转率很高。这种新的方法可以用作特定核酸酶的检测，以高通量筛选抑制具有关键细胞调控作用的核酸酶的潜在药物的化合物文库，如核酸复制、错配修复和细胞凋亡。此外，核酸酶裂解可以与核酸杂交的检测相结合，包括检测代表突变或序列多态(例如，SNPs)的序列错配位置。β-半乳糖苷酶互补法的高灵敏度将简化SNPs的检测，并有可能消除靶序列扩增。拟议的商业应用：功能基因组学领域是一个价值数十亿美元的市场。我们的技术将极大地降低对基因组序列进行突变分析的成本，为我们提供了相对于任何其他功能基因组学公司的竞争优势。这将是一个价值数亿美元的市场。此外，核酸酶分析将是HTS的第一次此类分析，并可能开辟药物发现的新领域，获得更多资金。

The proposed research will demonstrate feasibility for a novel, homogeneous method for detection of nucleases; which can be used for drug discovery of nuclease inhibitors and for detection of nucleic acid hybridization. This method uses complementation of inactive enzyme fragments of E. coli beta-galactosidase as a signal amplification mechanism: Unique,cyclic molecules will be constructed to contain a peptide required for beta-galactosidase complementation, along with an oligonucleotide recognition site for nuclease. Cleavage of the oligonucleotide will allow formation of active enzyme, thus providing catalytic amplification of the nuclease cleavage event. The nuclease detection method is extremely sensitive, due to the low background activity of the cyclic enzyme fragment, the rapid formation of active enzyme upon cleavage, and the high turnover rate of beta-galactosidase. This novel method can be used as an assay for specific nuclease enzymes, to enable high throughput screening of compound libraries for potential drugs which inhibit nucleases with key cellular regulatory roles, such as nucleic acid replication, mismatch repair and apoptosis. In addition, nuclease cleavage can be coupled to detection of nucleic acid hybridization, including detecting sites of sequence mismatch which represent mutations or sequence polymorphisms (e.g., SNPs). The high sensitivity of the beta-galactosidase complementation method will simplify detection of SNPs, with the potential for eliminating target sequence amplification. PROPOSED COMMERCIAL APPLICATIONS: The functional genomics field is a multiple billion dollar market. Our technology will greatly reduce the costs of doing mutation analysis of genomic sequences providing us with a competitive advantage over any other functional genomics company. This would be a market worth hundreds of millions of dollars. Furthermore, the nuclease assays would be the first such assays for HTS and could open up a new field of drug discovery, garnering even more funds.