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SoCoBio (Universities of Southampton, Kent, Sussex, Portsmouth and NIAB EMR)

Using phosphorothioate reactivity to analyse and control aptamers for protein binding


Dr Christopher Serpell (PI) – University of Kent
Helen Lavender (CoI) – Centauri Therapeutics
Prof Michelle Garrett (CoI) – University of Kent


Project Summary:

Aptamers are single stranded nucleic acids which fold, giving selective binding to substrates such as proteins. As cheaper and more chemically tractable alternative to antibodies, aptamers are emerging as powerful tools in biotechnology, chemical biology, biosensors, and medicine. However, information about their structure and binding mode is often lacking. NMR and crystallography have been used but require much work and expense. We propose a faster method to obtain information about aptamer structure and target binding, and identify locations for modifications which would improve biostability and/or binding strength. The phosphate group of nucleic acids can be replaced by a phosphorothioate using established chemistry in-line with automated synthesis. Phosphorothioates have little effect upon nucleic acid 3D structure, but are nucleophilic, allowing alkylation. We will apply this alkylation to aptamers bound to their targets initially using a phosphorothioate version of a known aptamer for the epidermal growth factor receptor (EGFR) as a model system. EGFR is overexpressed in NSCLC and a drug target for this disease. Only phosphorothioate sites which are solvent-exposed, and thus not involved in binding will be alkylated. The result of the experiment will be read-out using mass spectrometry and gel electrophoresis. Using the information obtained, we will then chemically synthesis modified forms of the aptamer which are modified in one of two ways. Firstly, we will attach polyethylene glycol units on the solvent-exposed phosphates and assess the extent to which stability in serum can be prolonged without affecting binding. Secondly, we will neutralise non-solvent exposed phosphates with methyl groups to increase hydrophobicity which should provide efficient increases in binding strength in a sequence-specific manner. Aptamers are on the verge of breakthrough for many applications in biology, but are held back by their modest affinity, biostability, and analytical tractability. This work will provide improvements and insights on all three fronts.