Kinetic screening of nucleic acid ligand libraries for hit identification, binding mode characterization, and sequence optimization

Binding and unbinding rates are important characteristics of molecular interactions as they dictate binding affinities and complex lifetimes. Particularly, the engineering and maturation of nucleic acid-based ligands for therapeutic or diagnostic purposes will greatly benefit from adopting kinetic characterization in screening efforts. Here we introduce a kinetic screening setup for nucleic acid ligands based on a fluorescence biosensor platform, allowing rapid selection and kinetic characterization of aptamer sequences from random or rationally designed mutant libraries. The modular, non-covalent approach allows for a rapid and cost-effective exchange of nucleic acid ligands and automated measurements of the association and dissociation of various analytes. We screened 846 unique biomolecular interactions covered by 1774 kinetic measurements, enabling us to map essential and non-essential nucleotides in two independent aptamers, evaluate the effects of sequence variations on binding kinetics, and provide lead sequences to introduce specificity in an inherently promiscuous small molecule-binding aptamer. • Screening of aptamer mutant libraries that bind proteins and small molecules. • Characterization of individual base exchanges and their influence on kinetic rates. • Essential bases for aptamer function are determined. • Analyte selectivity of aptamers is achieved via mutation library screening.