title: A flexible nanoarray approach for the assembly and probing of molecular complexes.
abstract: Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers' rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska; Department of Physics, University of Nebraska Omaha, Omaha, Nebraska.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Krasnoslobodtsev
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Cepheid, Bothell, Washington.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Viazovkina
authors.affiliation: Cepheid, Bothell, Washington.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gall
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska; Department of Biology and Chemistry, Morningside College, Sioux City, Iowa.
authors.forename: Chad
authors.initials: C
authors.lastname: Bertagni
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska. Electronic address: ylyubchenko@unmc.edu.
authors.forename: Yuri L
authors.initials: YL
authors.lastname: Lyubchenko
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry; D018625:Microscopy, Atomic Force; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D016229:Amyloid beta-Peptides; D055767:Immobilized Proteins