Browsing by Subject "Binding affinity"

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    Beyond the cycle: investigating the sequencing, binding affinity, and utility of aptamers selected with CE-SELEX.
    (2023-02) Brinza, Nicholas
    Aptamers are single stranded DNA or RNA sequences that bind with high affinity to a target molecule. The discovery of sequences that can bind to a target of interest starts with a mixture of random sequences which are refined through repeated incubation with the target followed by amplification of binding sequences in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers produced by multiple SELEX rounds can assist bioanalytical scientific research through detecting molecules of interest in biosensors and have the potential to improve drug delivery to specific cells in humans. However, developing aptamers capable of useful application is difficult due to multiple unique challenges. Quantifying the ability for a pool of aptamers produced by SELEX to bind to a target has no single perfect method. In the field of aptamer research, scientists employ a variety of binding affinity tests which contain specific advantages and disadvantages. Reported values for binding affinity vary between different methods. When a pool does show evidence of most sequences binding, identifying the best sequences in a mixture of more than thousands of unique base pair arrangements provides a separate challenge. Finally, even after finding a sequence with evidence of strong binding to a desired target, there remains uncertainty if the aptamer will bind to the target as well in a real-world application as it does in a carefully controlled lab environment. This study examines aptamers at three distinct stages of development for separate capillary electrophoresis SELEX (CE-SELEX) experiments. For an aptamer already selected against transferrin receptor 1 (TfR1) with high affinity, a combination of flow cytometry and confocal microscopy provided evidence the aptamer did not significantly internalize into cancerous liver cells used to model human drug delivery. A separate aptamer pool, previously developed over 4 rounds of selection against low-density lipoprotein receptor (LDLR), is analyzed with preexisting bioinformatic tools as well as custom Python code on next generation sequencing (NGS) data, revealing a primer dimer sequence from polymerase chain reaction (PCR) which impacted the selection of aptamer length candidates but remained at a lower frequency than one possible aptamer candidate. After performing 4 rounds of CE-SELEX against mouse leptin, testing the binding affinity of rounds 1-3 with CE showed weak evidence of binding in early rounds which was lost in later rounds after correcting for an earlier false positive result. Documenting CE problems and optimization, even for an unsuccessful aptamer selection, may help improve future CE research, while detailing the experimental set up, discovery, and correction of the false positive result is important to help prevent future misleading conclusions.
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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.