title: Application of NMR methods to identify detection reagents for use in development of robust nanosensors.
abstract: Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying bimolecular interactions at the atomic scale. Our NMR laboratory is involved in the identification of small molecules, or ligands, that bind to target protein receptors such as tetanus neurotoxin (TeNT) and botulinum neurotoxin, anthrax proteins, and HLA-DR10 receptors on non-Hodgkin lymphoma cancer cells. Once low-affinity binders are identified, they can be linked together to produce multidentate synthetic high-affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors or biochips for the detection of pathogen proteins in environmental samples or body fluids. Here we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known "marker" ligand, doxorubicin. Using this assay, one can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.
authors.affiliation: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA, USA.
authors.forename: Monique
authors.initials: M
authors.lastname: Cosman
authors.forename: Viswanathan V
authors.initials: VV
authors.lastname: Krishnan
authors.forename: Rod
authors.initials: R
authors.lastname: Balhorn
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D015374:Biosensing Techniques; D003627:Data Interpretation, Statistical; D004317:Doxorubicin / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000031:analogs & derivatives*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D036103:Nanotechnology; D012097:Repressor Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D012097:Repressor Proteins; C107173:TetC protein, Bacteria; D004317:Doxorubicin; C013411:biocytin; D008239:Lysine