Development and identification of candidate aptamers for growth differentiation factor 9
Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted growth factors that are essential for fertility and ovarian development in many mammalian species. It is hypothesised that the ratios of these growth factors within ovarian follicles are unique to each species and determine litter size in mammals. The current detection method for these proteins is by Western blotting with monoclonal antibodies, however this methodology generates semi-quantitative results at best. Systematic evolution via exponential enrichment (SELEX) is a method involving iterative rounds of affinity maturation. It has been used for generating synthetic DNA- and RNA-based sequences (aptamers) that are capable of recognising a target molecule with high sensitivity. To understand more about the biological functions of BMP15 and GDF9, reliable detection methods need to be developed so these proteins can be measured in biological samples. The generation of aptamers that recognise BMP15 and GDF9 of multiple species could enable a novel detection system to be developed to further explore the role of BMP15 and GDF9 in determining litter size in mammals. In this study, recombinant BMP15 and GDF9 proteins from species that vary in litter size were produced and purified by IMAC and HPLC for aptamer validation. Membrane-SELEX was used to generate candidate aptamers against recombinant human GDF9. After ten rounds of SELEX, seven candidate aptamers were identified and sequenced. Alignment of the sequences revealed a conserved region of 29bp in four of the seven candidate aptamers. These sequences also showed a high (85-90%) guanine/thymine content consistent with G-quadruplex forming aptamers, a tertiary structure that increases specificity to the target molecule. These four sequences also showed homology with that of a previously published aptamer for BMP15. This is interesting as BMP15 and GDF9 are both members of the transforming growth factor beta superfamily and as such, share a high peptide homology suggesting convergent evolution occurred in two unrelated SELEX experiments against related proteins. Whilst time constraints did not allow for characterisation of the resultant GDF9 aptamer candidates, nor their validation using recombinant ovine and mouse GDF9 produced herein, this study showed that GDF9 aptamer candidates were generated by SELEX that exhibited sequence similarities to an aptamer generated against a structurally-related protein.