Generation And Characterisation Of RNA Aptamers Against ESAT6 Protein From Mycobacterium Tuberculosis
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Date
2017-07
Authors
Bukari, Bakhtiar Affendi
Journal Title
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Publisher
Universiti Sains Malaysia
Abstract
Aptamers are chemical ligands made up of short nucleotides sequences that are able to bind to target proteins with high affinity and specificity. They are generated using a process called Systemic Evolution of Ligands via Exponential Enrichment (SELEX). Due to their chemical stability and high specificity against the target, aptamers have the potential to become very useful biological tools. The 6 kDa Early Secretory Antigenic Target, or ESAT6, is a secretory protein produced by Mycobacterium tuberculosis and is thought to be a major player in mycobacterial pathogenesis. The protein is found on a locus known as Region of Difference 1 (RD1) and the loss of this locus has been shown to render the Mycobacterium sp. unable to cause severe TB. Following this line of evidence, ESAT6 could potentially be a good biomarker for TB infection. As such, producing an aptamer against this protein could prove valuable in the attempt to create a novel and economical TB diagnostic assay with the potential in treating the disease. The objective of this study is to generate RNA aptamers that can bind specifically and with high affinity to ESAT6 protein. Eleven SELEX cycles were carried out using the N40-randomised RNA pool. Stringency of the binding reaction in each SELEX cycles was increased gradually by varying the amounts of protein, RNA pool and the competitor. The resulting RNA pool from the 8th and 11th cycle of SELEX was subjected to filter binding assay to assess its binding against ESAT-6 protein. Filter binding assay against the target protein confirmed that binding enrichment of the RNA pool has indeed occurred. Nitrocellulose Filter Membrane Binding assay suggested the presence of potential binders in the RNA pool.
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Keywords
Characterisation of RNA aptamers against ESAT6 protein , from mycobacterium tuberculosis