Browsing by Subject "Micro free flow electrophoresis"

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    Microfluidic selection of DNA aptamers using capillary electrophoresis and micro free flow electrophoresis systematic evolution of ligands by exponential enrichment
    (2012-08) Jing, Meng
    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 μFFE 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 μFFE in SELEX. Later, μFFE-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.