<i>In silico</i> and <i>in vitro</i> characterisation and affinity maturation of human red blood cell binding aptamers

Aptamers are short, single-stranded DNA or RNA oligonucleotides that can specifically bind to their target with high affinity and specificity. Aptamers have gained widespread attention in recent years as possible replacements for antibodies within many analytical fields, due to their high chemical and thermal stability and relative low cost of production. Red blood cells are of interest within not only the medical field, but also are of interest within forensic science. Few aptamers have been reported that can specifically detect human red blood cells, or surface proteins of, but they have great potential for use as biorecognition elements within immunoassays or biosensors. Three aptamers have been identified from recent literature that have been designed to bind to human red blood cells as a whole cell target or glycophorin A as a protein-based target. However, they are yet to be fully characterised for their binding affinity to red blood cells, and no sequence optimisation has been conducted. Within this work, a comprehensive characterisation of three reported aptamers has been conducted. <i>In silico</i> modelling has been explored as a means to better understand the 3D structures and the target ligand of each aptamer. The 3D structures of these aptamers have been reported and utilised within the HDOCK server to predict the docking of the aptamers to red blood cell-specific surface proteins. Both enzyme-linked oligonucleotide assays and microscale thermophoresis have been used to characterise aptamer-target biding, with dissociation constants being predicted in the nanomolar to low micromolar range for each aptamer. Additionally, sequence optimisation has been conducted to enhance the binding of the sequences to human red blood cells through sequence truncation mechanisms. To the best of our knowledge, this work represents the first characterisation of these aptamers and will guide future use of these aptamers as analytical probes.