title: Kinetic and stoichiometric characterisation of streptavidin-binding aptamers.
abstract: 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 nM 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.
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands. Vincent.Ruigrok@wur.nl
authors.forename: Vincent J B
authors.initials: VJ
authors.lastname: Ruigrok
authors.forename: Esther
authors.initials: E
authors.lastname: van Duijn
authors.forename: Arjan
authors.initials: A
authors.lastname: Barendregt
authors.forename: Kevin
authors.initials: K
authors.lastname: Dyer
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.forename: Regina
authors.initials: R
authors.lastname: Stoltenburg
authors.forename: Beate
authors.initials: B
authors.lastname: Strehlitz
authors.forename: Mark
authors.initials: M
authors.lastname: Levisson
authors.forename: Hauke
authors.initials: H
authors.lastname: Smidt
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D009841:Oligonucleotides; D019809:Streptavidin