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Browsing by Subject "SELEX"

Now showing 1 - 4 of 4
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    Aptamer selection using capillary electrophoresis-SELEX
    (2008-10) Mosing, Renee Karen
    SELEX is a method used for the combinatorial selection of aptamers, or single stranded nucleic acid sequences that bind with high affinity and specificity to target molecules. Although successful, SELEX is a very time consuming, laborious process. We introduced a modification to this process called capillary electrophoresis-SELEX. This protocol proved to be significantly more efficient, which greatly decreased the time requirement of the process from weeks to days. This improvement was observed despite the lower loading capacity and resolution limited injections on CE which introduced approximately 1013 sequences to the separation instead of the 1015 sequences introduced in more traditional selections protocols. In fact, ssDNA aptamers with picomolar affinity for HIV-1 RT were identified in 4 rounds. Further sequence/structure characterization of these sequences demonstrated no homology, indicating that several sequences can bind with high affinity to the target. Interestingly, the aptamers were 10 fold more selective for the original target (HIV-1 RT) than other reverse transcriptases. Despite this result, the aptamers did not demonstrate inhibition of reverse transcriptase activity. The success of these collections prompted investigation of more challenging targets such as mitochondria and bacteria. These targets are difficult to purify and have surface chemistries that are constantly changing. Aptamers for these targets must identify a feature on the surface that is consistent in order to be conserved throughout the process. Our experiments indicated that the aptamers may have bound to features on the surface that are not very abundant, making affinity characterization cumbersome. Future experiments aim to determine if the aptamers are becoming more refined for specific features rather than just focusing on increase in affinity. Finally, initial experiments were performed for a model that will drive selections toward a specific binding site on the surface of the HIV assembly protein, capsid. Further experiments are proposed to allow aptamer binding to specific sites, and for aptamer characterization.
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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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    Fabrication of a PDMS microfluidic device for size selective DNA transport and single-stranded DNA generation for SELEX
    (2012-07) Sheng, Yixiao
    An innovative nanopore based microfluidic device for SELEX has been developed for single-stranded DNA generation and sizing. The objective of my research is to design, fabricate, test and model this device and make this device applicable for SELEX. The nanopores of the membrane controls fluidic transport between different planes in the device. It adds more functionality and flexibility to the microfluidic device. This device consists of two polydimethylsiloxane (PDMS) channels separated by a polycarbonate membrane. Channels were designed in a CAD software and a master was fabricated using rapid prototyping of PDMS. Multiple PDMS replicas were then cast from this master. The membrane was sandwiched between two channels. Oxygen plasma treatment was applied to bond two PDMS layers and glass substrates were used to support the whole device. Recovery of fluorescein across the membrane was compared with 10 and 80 nucleotide (nt) single stranded DNA(ssDNA) to characterize the device. Recovery of analytes improved with decreasing flow rate. Size selectivity was observed. Two mathematical models which were built based on conservation of mass and constitutive relationships described the process of DNA transportation in the microfluidic device. Trends in recovery measured at various flow rates were consistent with the trends predicted in the two models which support the premise that diffusion dominated the molecular transport in this device. In addition to that, Model 2 demonstrated recovery was affected by the ionic strength of the buffer as well. One application of this device was to automate the process to make double stranded DNA(dsDNA) single stranded which can be integrated into an automatic SELEX system. Streptavidin-coated polystyrene beads were immobilized with dual-biotin labeled dsDNA and alkaline treatment was adopted to denature dsDNA and release the non-biotinylated ssDNA. 25mM sodium hydroxide (NaOH) was optimized to achieve the best purity. 95.7% of the strands collected across the membrane were the non-biotinylated ssDNA. Capillary Electrophoresis (CE) results confirmed that the non-biotinylated ssDNA was the major component across the membrane.
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    Identification of compounds inhibiting a Leishmania RNA editing reaction.
    (2010-05) Liang, Shuang
    Several species of Leishmania are human pathogens that afflict more than 12 million people worldwide, and the current treatment options are limited. An RNA editing reaction that is both essential and specific to the parasites is an attractive target for new drug development. The editing reaction involves the post-transcriptional modification of specific mitochondrial mRNAs through the precise deletion or insertion of uridylates. Many aspects of the editing mechanism are still unclear, and the lack of specific inhibitors to probe the reaction has hindered the field. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify inhibitors, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A novel editing assay was developed in this thesis that overcame previous limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter for the assay consists of an RNA editing substrate linked to a streptavidin-binding domain that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding domain is only activated by an editing-induced conformational change. The reporter RNA is labeled with a ruthenium complex, and an electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis and binding affinity measurements were used to characterize the reporter. This highly sensitive assay was optimized and validated for use in high-throughput screening, and a pilot screen of a 1280 compound library identified compounds that are the first specific inhibitors of the editing reaction. Some of the identified inhibitors will have value as probes of the editing reaction and have already provided insights into possible regulatory mechanisms. The identification of novel drugs through screens of large chemical libraries is now possible with the new assay.