Two-dimensional Capillary Electrophoresis - SELEX

描述（由申请人提供）：摘要。需要高通量产生用于诊断和基础研究的适体。目前的技术是基于SELEX，这是非常难以自动化，不适合高通量适体的产生。最近，明尼苏达大学的鲍泽和约克大学的克雷洛夫开发了一种基于毛细管电泳的适体产生的高速方法。他们的技术，鲍泽称之为毛细管电泳-SELEX（CE-SELEX），是基于观察到单链DNA在游离溶液中的迁移率与寡核苷酸的序列无关。在CE-SELEX中，靶分子与寡核苷酸文库一起孵育。未复合的DNA文库在毛细管电泳过程中以紧密条带的形式迁移。相反，DNA-靶复合物以不同的迁移率迁移。通过丢弃未复合的DNA并扩增与靶标复合的DNA，可以在一至四个选择循环中产生高亲和力适体。我们建议开发一种仪器，将CE-SELEX的速度提高两个数量级。我们的系统基于二维毛细管电泳，其中蛋白质样品在第一毛细管中分离。在第一个毛细管中可以分离多达100种组分。将级分自动转移到孵育器中，在那里它们与寡核苷酸文库混合并可以与寡核苷酸文库复合。然后将每个馏分转移到第二个毛细管中，在那里内容物进行CE-SELEX。丢弃未复合的DNA，并扩增蛋白质复合物中的DNA。将来自二维系统的每个循环的扩增的DNA合并并用作随后的SELEX步骤的DNA文库。SELEX完成后，产物DNA被克隆和测序，相应的靶蛋白被消化并通过质谱鉴定。 公共卫生相关性：（由申请人提供）：叙述。适体是紧密结合特定靶标的合成DNA片段。它们可用于分析靶点，并已发现作为治疗剂的应用。使用当前的技术，它们的生成是缓慢的。该提议将使适体的生成速度提高两个数量级。

DESCRIPTION (provided by applicant): Abstract. There is a need for the high throughput generation of aptamers for diagnostic and basic research. Current technology is based on SELEX, which is very difficult to automate and is not suitable for high-throughput aptamer generation. Recently, Bowser at the University of Minnesota and Krylov at York University developed a high-speed method for aptamer generation based on capillary electrophoresis. Their technology, which Bowser calls capillary electrophoresis-SELEX (CE-SELEX), is based on the observation that the mobility of single stranded DNA in free solution is independent of the oligonucleotide's sequence. In CE-SELEX, a target molecule is incubated with an oligonucleotide library. The uncomplexed DNA library migrates as a tight band during capillary electrophoresis. In contrast, DNA-target complexes migrate with a different mobility. By discarding the uncomplexed DNA and amplifying the DNA that is complexed with the target, high affinity aptamers can be generated in one to four selection cycles. We propose to develop an instrument that will increase the speed of CE- SELEX by two orders of magnitude. Our system is based on two-dimensional capillary electrophoresis, where a protein sample is separated in the first capillary. Up to 100 components can be separated in the first capillary. Fractions are automatically transferred to an incubator, where they are mixed with and can complex with an oligonucleotide library. Each fraction is then transferred to a second capillary, where the contents undergo CE-SELEX. Uncomplexed DNA is discarded and the DNA in the protein-complexes is amplified. The amplified DNA from each cycle of the two-dimensional system is pooled and used as the DNA library for subsequent SELEX steps. After SELEX is complete, the product DNA is cloned and sequenced, and the corresponding target protein is digested and identified by mass spectrometry. PUBLIC HEALTH RELEVANCE: (provided by applicant): Narrative. Aptamers are synthetic pieces of DNA that tightly bind to a specific target. They are useful in the analysis of the target and have found application as therapeutics. Using current technology, their generation is slow. This proposal will speed aptamer generation by two orders of magnitude.