title: Biosensor-based fragment screening using FastStep injections.
abstract: We have developed a novel analyte injection method for the SensíQ Pioneer surface plasmon resonance-based biosensor referred to as "FastStep." By merging buffer and sample streams immediately prior to the reaction flow cells, the instrument is capable of automatically generating a two- or threefold dilution series (of seven or five concentrations, respectively) from a single analyte sample. Using sucrose injections, we demonstrate that the production of each concentration within the step gradient is highly reproducible. For kinetic studies, we developed analysis software that utilizes the sucrose responses to automatically define the concentration of analyte at any point during the association phase. To validate this new approach, we compared the results of standard and FastStep injections for ADP binding to a target kinase and a panel of compounds binding to carbonic anhydrase II. Finally, we illustrate how FastStep can be used in a primary screening mode to obtain a full concentration series of each compound in a fragment library.
authors.affiliation: Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Rich
authors.forename: John G
authors.initials: JG
authors.lastname: Quinn
authors.forename: Tom
authors.initials: T
authors.lastname: Morton
authors.forename: J David
authors.initials: JD
authors.lastname: Stepp
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D013395:Sucrose / Q000737:chemistry; D013449:Sulfonamides / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D013449:Sulfonamides; D013395:Sucrose; D000244:Adenosine Diphosphate; D024402:Carbonic Anhydrase II
references.citation: Anal Biochem. 2010 Jul 15;402(2):170-8
references.pmid: 20371220
references.citation: Anal Biochem. 2004 Jul 1;330(1):98-113
references.pmid: 15183767
references.citation: J Mol Recognit. 1999 Sep-Oct;12(5):279-84
references.pmid: 10556875
references.citation: Anal Biochem. 2004 Jun 15;329(2):316-23
references.pmid: 15158493
references.citation: Pharm Sci Technol Today. 2000 Sep 1;3(9):310-317
references.pmid: 10996572
references.citation: Protein Sci. 2002 May;11(5):1017-25
references.pmid: 11967359
references.citation: Anal Biochem. 2006 Feb 1;349(1):136-47
references.pmid: 16337141
references.citation: J Biomol Tech. 2003 Dec;14(4):247-69
references.pmid: 14715884
references.citation: Anal Biochem. 2006 Dec 1;359(1):94-105
references.pmid: 17007806
references.citation: Anal Biochem. 2010 Aug;403(1-2):30-5
references.pmid: 20371356
references.citation: Anal Biochem. 2007 May 1;364(1):67-77
references.pmid: 17362870
references.citation: Anal Biochem. 2006 Nov 15;358(2):281-8
references.pmid: 16962556