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Kinetic and Stoichiometric Characterisation of Streptavidin‐Binding Aptamers
Author(s) -
Ruigrok Vincent J. B.,
van Duijn Esther,
Barendregt Arjan,
Dyer Kevin,
Tainer John A.,
Stoltenburg Regina,
Strehlitz Beate,
Levisson Mark,
Smidt Hauke,
van der Oost John
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100774
Subject(s) - aptamer , streptavidin , surface plasmon resonance , receptor–ligand kinetics , oligonucleotide , systematic evolution of ligands by exponential enrichment , kinetics , chemistry , selex aptamer technique , biotinylation , biophysics , computational biology , biochemistry , biotin , biology , nanotechnology , dna , microbiology and biotechnology , materials science , nanoparticle , gene , rna , receptor , physics , quantum mechanics
Aptamers are oligonucleotide ligands that are selected for high‐affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer–target interactions is available. To this end, streptavidin‐binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35–375 n M with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin‐binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post‐selection characterisation, through the use of complementary approaches as described in this study.