Generation And Characterization Of Rna Aptamer Against rHuEPO-a By Selex Technology

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dc.contributor.authorMarimuthu, Citartan
dc.date.accessioned2019-07-12T03:07:25Z
dc.date.available2019-07-12T03:07:25Z
dc.date.issued2014-03
dc.description.abstractSystematic Evolution of Ligands by Exponential Enrichment (SELEX) enables the isolation of aptamer (ssDNA or RNA that possesses high binding affinity and specificity against virtually any target molecules). In this study, a RNA aptamer against recombinant human EPO-alpha (rHuEPO-α) was successfully isolated by SELEX technology. After 11 cycles of SELEX, cloning and sequence analysis showed an appearance of a single major clone constituting the putative RNA aptamer, termed REPORA-6, with a dissociation constant of 25±1 nM. Dissociation constant of REPORA-6 against deglycosylated rHuEPO-α was 24.6±2 nM. REPORA-6 could possibly act as 'universal probe' against all rHuEPOs. REPORA-6 and anti-EPO monoclonal antibody bind at different binding sites on the surface of rHuEPO-α, facilitating the design of Sandwich Enzyme-Linked Aptamer Assay (ELAA). The detection limit achieved was 0.29 nM, suggesting that this assay has the potential to be an aptamer-based diagnostic (aptanostic) test for detection of rHuEPO. REPORA-6 was used in aptamer-based capture assay, which is a better method than pH-based elution of the target protein that can incur irreversible degradation on the protein. REPORA-6 could be also be used as a potential agent for replacing both primary/secondary antibodies in isoelectric-focusing (IEF)-double immunoblotting. Gel shift assay revealed that REPORA-6 has therapeutic potential through its ability to block the interaction between EPO and EPO receptor (EPO-R); one of the interactions of which mediates tumor growth/angiogenesis. REPORA-6 is resistant against nuclease degradation action and can be truncated via rational truncation approach (aided by mapping analyses) down to 58-nt. This miniaturized aptamer (REPORA-6b) has better transfection efficiency and is less susceptible to the immune system, maintaining its binding affinity against the target (22±2 nM).en_US
dc.identifier.urihttp://hdl.handle.net/123456789/8471
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectRNA aptamer against recombinant human EPO-alphaen_US
dc.subjectsuccessfully isolated by SELEX technologyen_US
dc.titleGeneration And Characterization Of Rna Aptamer Against rHuEPO-a By Selex Technologyen_US
dc.typeThesisen_US
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