Isolation of RNA aptamers specific toward 16 KDA mycobacterium tuberculosis antigen protein

Tuberculosis (TB) is an old, infectious disease scourge caused by
\nMycobacterium tuberculosis (M.tb) that affects human. There are several laboratories
\ndiagnostic methods available for TB screening such as sputum smear microscopy,
\nculture and nucleic acid amplification that can diagnose patients with active TB.
\nHowever, the methods are not for detection of latent TB infection (LTBI). Despite
\nthe advent of Interferon Gamma Release Assay (IGRAs) for diagnosis of LTBI, the
\ntest is expensive and laborious. The test is also not accessible for poor countries that
\nhave high TB burden due to cost and lack of rapid point-of-care setting. Thus, a new
\npoint-of-care technology is urgently needed to improve the current LTBI diagnosis to
\nmore economical and easily implemented technology especially for low resource
\nsettings countries. So far, aptamers have received considerable attention due to its
\nproperties that could imitate function of antibody against the target with high
\nspecificity and affinity. The 16 kDa protein of M.tb. was selected as a prime
\ncandidate for this study due to its importance in immunodominant property and
\ntuberculosis latency. Thus, this study was aimed to isolate and characterise the RNA
\naptamers against the 16 kDa protein. By performing Systematic Evolution of Ligands
\nby Exponential Enrichment (SELEX) method using nitrocellulose membrane, the
\nRNA aptamers against 16 kDa protein were successfully isolated and characterised.
\nThe isolated aptamers were then clustered together based on their nucleotide
\nhomology sequences, while the secondary structure, motif sequence and Gquadruplex
\nanalysis were identified using free online software. Electrophoretic
\nMobility Shift Assay (EMSA) was performed using agarose gel electrophoresis to
\ndetermine the binding and the dissociation constant (Kd) value for each isolated
\nRNA aptamer. Out of five isolated clusters of RNA aptamers, the cluster
\n(TB_APG01) was had the nucleotide sequences with the highest frequency clones
\n(14/104) and Kd value 6.428±4.97 μM, however the cluster TB_APG04 was
\nrecognized as the strongest aptamer with the lowest Kd value (3.935±1.60μM)
\nalthough only have 2/104 clones. As a conclusion, this study was successfully
\nisolated the RNA aptamers that bound to the 16 kDa protein and maybe useful for
\ndirect LTBI diagnosis or as imaging tool.