Mohammed, Nurul Adila
(2020)
Isolation of RNA aptamers specific toward 16 KDA mycobacterium tuberculosis antigen protein.
Masters thesis, Universiti Sains Malaysia.
Abstract
Tuberculosis (TB) is an old, infectious disease scourge caused by
Mycobacterium tuberculosis (M.tb) that affects human. There are several laboratories
diagnostic methods available for TB screening such as sputum smear microscopy,
culture and nucleic acid amplification that can diagnose patients with active TB.
However, the methods are not for detection of latent TB infection (LTBI). Despite
the advent of Interferon Gamma Release Assay (IGRAs) for diagnosis of LTBI, the
test is expensive and laborious. The test is also not accessible for poor countries that
have high TB burden due to cost and lack of rapid point-of-care setting. Thus, a new
point-of-care technology is urgently needed to improve the current LTBI diagnosis to
more economical and easily implemented technology especially for low resource
settings countries. So far, aptamers have received considerable attention due to its
properties that could imitate function of antibody against the target with high
specificity and affinity. The 16 kDa protein of M.tb. was selected as a prime
candidate for this study due to its importance in immunodominant property and
tuberculosis latency. Thus, this study was aimed to isolate and characterise the RNA
aptamers against the 16 kDa protein. By performing Systematic Evolution of Ligands
by Exponential Enrichment (SELEX) method using nitrocellulose membrane, the
RNA aptamers against 16 kDa protein were successfully isolated and characterised.
The isolated aptamers were then clustered together based on their nucleotide
homology sequences, while the secondary structure, motif sequence and Gquadruplex
analysis were identified using free online software. Electrophoretic
Mobility Shift Assay (EMSA) was performed using agarose gel electrophoresis to
determine the binding and the dissociation constant (Kd) value for each isolated
RNA aptamer. Out of five isolated clusters of RNA aptamers, the cluster
(TB_APG01) was had the nucleotide sequences with the highest frequency clones
(14/104) and Kd value 6.428±4.97 μM, however the cluster TB_APG04 was
recognized as the strongest aptamer with the lowest Kd value (3.935±1.60μM)
although only have 2/104 clones. As a conclusion, this study was successfully
isolated the RNA aptamers that bound to the 16 kDa protein and maybe useful for
direct LTBI diagnosis or as imaging tool.
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