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首页> 外文学位 >Microfluidic selection of DNA aptamers using capillary electrophoresis and micro free flow electrophoresis systematic evolution of ligands by exponential enrichment.
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Microfluidic selection of DNA aptamers using capillary electrophoresis and micro free flow electrophoresis systematic evolution of ligands by exponential enrichment.

机译：DNA适体的微流选择使用毛细管电泳和微自由流动电泳通过指数富集系统地配体进化。

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摘要

SELEX is the process used to generate aptamers, which are ssDNA or RNA molecules that can bind specific targets with high affinity. Although aptamers show great potential in clinical applications, the generation process is currently tedious and inefficient, being a limiting step. Thus, understanding, developing, and applying advanced partitioning platform are pivotal. CE is an advanced separation method in SELEX and has been successfully used to generate aptamers toward multiple targets. However, there are still interesting questions unanswered, making our understanding in CE-SELEX lag behind its applications. We applied high-throughput sequencing on CE-SELEX selected pools against rhVEGF and obtained sequencing information of more than 104 sequences per pool, which allowed characterization on diversity of individual pools. This study revealed the coexistence of high diversity and fast enrichment rate of CE-SELEX. To further improve the separation platform, we integrated a &mgr;FFE device into the SELEX process. Using this device, 1014~1015 sequences were introduced and analyzed within 30 min, which was a 370 fold improvement compared to CE. As a proof of concept, four cycles of selection were performed to the target human IgE, and high affinity ligands were generated even after the first round of selection, proving the feasibility and high efficiency of &mgr;FFE in SELEX. Later, &mgr;FFE-SELEX was applied to generate aptamers for a membrane protein SERCA, whose selection has never been achieved in conventional SELEX due to the technical difficulty in target immobilization. High nM Kd pool was generated toward SERCA solubilized in C12E8 at the fifth round of selection, demonstrating that this separation strategy is more compatible with membrane proteins due to the free solution based separation, lower electric field, faster separation speed, and straightforward fraction collection. The success of this application opens a door for high-throughput generation of aptamers toward complex targets in the future. Besides SELEX, the function and activity of the ATPase SERCA were also explored in this thesis. It was discovered that nonspecific ssDNA sequences can bind to the endogenous regulator of SERCA, PLN, in a length dependent way. A highly sensitive and reproducible SERCA activity assay, which cut the use of SERCA by 2,000 folds, was developed to directly detect the product ADP via TR-FRET.
机译：SELEX是用于生成适体的过程，所述适体是可以高亲和力结合特定靶标的ssDNA或RNA分子。尽管适体在临床应用中显示出巨大的潜力，但生成过程目前繁琐且效率低下，是限制步骤。因此，了解，开发和应用高级分区平台至关重要。 CE是SELEX中的一种高级分离方法，已成功用于生成针对多个靶标的适体。但是，仍然有一些有趣的问题尚未解决，这使我们对CE-SELEX的理解落后于其应用。我们在针对rhVEGF的CE-SELEX选定池上应用了高通量测序，并获得了每个池超过104个序列的测序信息，从而可以表征各个池的多样性。这项研究揭示了CE-SELEX高多样性和快速富集率的共存。为了进一步改善分离平台，我们将＆FFR设备集成到SELEX过程中。使用该装置，在30分钟内引入并分析了1014〜1015个序列，与CE相比，提高了370倍。作为概念证明，对目标人IgE进行了四个选择周期，即使在第一轮选择后仍产生了高亲和力配体，证明了FFE在SELEX中的可行性和高效率。后来，＆nbsp; FFE-SELEX被用于产生膜蛋白SERCA的适体，由于靶标固定技术上的困难，其选择在常规SELEX中从未实现。在第五轮选择中，向溶解在C12E8中的SERCA生成了高nM Kd库，这表明由于基于游离溶液的分离，较低的电场，更快的分离速度和简单的馏分收集，该分离策略与膜蛋白更兼容。该应用程序的成功为将来向复杂目标物产生高通量的适体打开了大门。除了SELEX，本文还探讨了ATPase SERCA的功能和活性。发现非特异性ssDNA序列可以以长度依赖的方式结合到SERCA，PLN的内源调节子。开发了一种高度灵敏且可重现的SERCA活性测定方法，该方法将SERCA的使用减少了2000倍，从而可以通过TR-FRET直接检测ADP产物。

著录项

  • 作者

    Jing, Meng.;

  • 作者单位

    University of Minnesota.;

  • 授予单位 University of Minnesota.;
  • 学科 Analytical chemistry.;Bioinformatics.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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