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In vitro selection of DNA aptamers that neutralise autoantibodies to cytosolic 5’-nucleotidase-1A

Slater, Nataliya (2019) In vitro selection of DNA aptamers that neutralise autoantibodies to cytosolic 5’-nucleotidase-1A. Honours thesis, Murdoch University.

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Abstract

Background. Inclusion Body Myositis (IBM) is the most commonly diagnosed inflammatory muscle disease associated with aging. It’s characterised by degeneration of skeletal muscles that inevitably leads to the loss of mobility. The mechanisms that drive IBM pathogenicity remain poorly understood. However, studies reveal that on average 50% of IBM sera contains circulating autoantibodies targeting cytosolic 5’-nucleotidase 1A (cN1A) – a metabolic enzyme that is highly expressed in skeletal muscles. Accumulating evidence suggests that anti-cN1A antibodies may play a role in IBM pathogenesis making them an attractive target for aptamer development.

Problem. IBM remains refractive to current immunosuppressive treatments. Preventing anti-cN1A antibodies binding to their target protein may reduce the severity of IBM symptoms and halt the disease progression.

Aim. Within this project, we aim to select DNA aptamers that conjugate with anti-cN1A binding sites and prevent their interaction with cN1A protein.

Results. Sera of forty-eight IBM patients were screened with enzyme-linked immunosorbent assay (ELISA) against purified cN1A protein. Eighteen patients (38%) were found seropositive. 6.5 mg of polyclonal anti-cN1A were purified out of 90 ml of a selected IBM patient’s blood. Their biological activity against cN1A was confirmed by ELISA.

Purified anti-cN1A were used as a target for aptamer selection. Initial library of single-stranded DNA fragments (aptamers) was synthesised with a 40-nucleotide randomised region flanked by constant primer-binding sites. Neutralising aptamers were selected through the process of systematic evolution of ligands by exponential enrichment (SELEX). Two complete cycles of selection against anti-cN1A were performed. Selected aptamers were assayed for their neutralising capacity by ELISA. Anti-cN1A were pre-incubated with the aptamers at various concentrations. Following a one-hour incubation, the ELISA-obtained absorbance remained unchanged or increased in a dose-dependent manner. While upon an extended overnight incubation, a 25% decrease in absorbance values corresponding to a 35% decrease in anti-cN1A binding was observed.

Conclusions. The methodology developed during this project resulted in the generation of aptamers that conjugate with anti-cN1A binding sites. Following two SELEX cycles, the selected aptamers demonstrate 35% neutralisation capacity upon prolonged incubation with the immunoglobulins. Our results strongly suggest that the aptamers target monomeric IgG and IgA while having low or no affinity for IgM anti-cN1A.

Item Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor(s): Coudert, Jerome and Veedu, Rakesh
URI: http://researchrepository.murdoch.edu.au/id/eprint/54117
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