Biosensor-based fragment screening using FastStep injections.
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.
Rich
Rebecca L
RL
Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
Quinn
John G
JG
Morton
Tom
T
Stepp
J David
JD
Myszka
David G
DG
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
D002257:Carbonic Anhydrase Inhibitors; D013449:Sulfonamides; D013395:Sucrose; D000244:Adenosine Diphosphate; D024402:Carbonic Anhydrase II
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