title: Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo.
abstract: L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting.
  
  
  BACKGROUND
  During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address: sebastian.riese@charite.de.
authors.forename: Sebastian B
authors.initials: SB
authors.lastname: Riese
authors.affiliation: Institute for Physiological Chemistry Pathobiochemistry, University of Muenster, Muenster, Germany; Department of Nephrology and Rheumatology, University Hospital Muenster, Muenster, Germany. Electronic address: kbuscher@uni-muenster.de.
authors.forename: Konrad
authors.initials: K
authors.lastname: Buscher
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address: sven.enders@gmx.de.
authors.forename: Sven
authors.initials: S
authors.lastname: Enders
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address: christian.kuehne@charite.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Kuehne
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address: rudolf.tauber@charite.de.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address: jens.dernedde@charite.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
mesh_terms: D052157:Aptamers, Nucleotide / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000627:therapeutic use*; D058621:Blood Buffy Coat / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D064368:Healthy Volunteers; D006801:Humans; D007249:Inflammation / Q000188:drug therapy* / Q000473:pathology; D019041:L-Selectin / Q000008:administration & dosage* / Q000737:chemistry; D007962:Leukocytes / Q000187:drug effects*; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D009841:Oligonucleotides; D019041:L-Selectin
keywords: Aptamer; Extravasation; Flow chamber; L-selectin; Multivalency