Status : Verified
| Personal Name | Molejon, Nerissa A. |
|---|---|
| Resource Title | Generation of single-stranded DNA aptamer against cholera toxin by proximity ligation selection : potential for environmental water surveillance |
| Date Issued | 22 June 2021 |
| Abstract | Cholera toxin (CT) is a key biomarker for toxigenic strains of Vibrio cholerae O1 and O139 which causes acute diarrheal infection. Detection of CT in isolates from environmental water samples is important in containing outbreaks or preventing epidemic cholera. Existing CT detection methods require expensive equipment and expert personnel to perform the assay. This study pursued a simpler and less-expensive detection method using single-stranded DNA aptamers. Aptamers are highly specific recognition molecules generated through an iterative process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). CT aptamers reported in journals were selected for the B subunit of CT only, not on the whole protein (CTX). This study aims to develop dual aptamers against CTX to be used in sandwich assays. Dual aptamers are obtained through Proximity Ligation Selection (PLS) where two independently-binding aptamers in close proximity are ligated by a DNA ligase through hybridization with an oligo connector. In this work, a modified PLS was performed using the sequences at the penultimate round of selection done separately in two ssDNA libraries (F2 and R2). No PLS method using two pre-enriched libraries had been reported in literatures. At the end, 5 CTX aptamer candidates from F2 library and 4 from R2 were selected through Next Generation Sequencing (NGS). One aptamer candidate was also selected for the counter-target, heat-labile enterotoxin LT from Escherichia coli (ETEC). To date, there is no reported aptamer against this protein. The selected sequences showed rich guanine content with high possibility of G-quadruplex formation, a stable aptamer structure. In-depth characterization is needed for the identification of aptamers with the best binding properties. Also, screening of different combinations of aptamers is required in order to identify a pair of aptamer for sandwich-type assays. This dual aptamer can then be used to construct low-cost, simple, and ra |
| Degree Course | MS Environmental Engineering |
| Language | English |
| Keyword | Aptamer; SELEX; proximity ligation selection; vibrio cholerae cholera toxin; Escherichia coli heat-labile enterotoxin LT; environmental surveillance for epidemic cholera |
| Material Type | Thesis/Dissertation |
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