title: Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance.
abstract: Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference-nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid-nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to L-arginine-bisoxyran-Sepharose and L-lysine-Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid-nucleotide interactions and determine the binding affinities of the complexes.
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. carlacruz@fcsaude.ubi.pt
authors.forename: Carla
authors.initials: C
authors.lastname: Cruz
authors.forename: Eurico J
authors.initials: EJ
authors.lastname: Cabrita
authors.forename: João A
authors.initials: JA
authors.lastname: Queiroz
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013499:Surface Properties
chemical_list: D000596:Amino Acids; D009711:Nucleotides; D012685:Sepharose