Aptamer affinity maturation by resampling of aptamer libraries based on an aptamer family consensus motif

Abstract

When selecting for aptamers the probability of selecting the fittest aptamer is extremely low due to the stochastic nature of the selection and insufficient sequence space coverage of the initial library. However, aptamers related to the fittest aptamer are likely to be selected. Herein we describe ‘Resample’, a computer program which takes an aptamer family motif and produces a library of sequences representing every aptamer permutation of that family motif. Taking aptamers previously isolated against the malarial antigen plasmodium falciparum lactate dehydrogenase, we used sequence alignment to create the likely aptamer family motif of the highest affinity aptamer. The motif was then be used by ‘Resample’ to combinatorially generate an aptamer library containing every aptamer represented by the aptamer family motif. This library was then synthesised onto high density DNA microarray and binding of all library members measures simultaneously by incubating with fluorescent target protein and array scanning. Three lead candidates from the microarray were analyses using microscale thermophoresis and found a 10-fold increase in binding affinity of a novel aptamer sequence. Increasing aptamer affinity using this method has the potential for reducing the limit of detection of aptamer based diagnostic tests and increasing efficacy of aptamer based therapeutics.