Quantitation of Affinity, Avidity, and Binding Kinetics of Protein Analytes with a Dynamically Switchable Biosurface

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• 作者：Jelena Knezevic ; Andreas Langer ; Paul A. Hampel ; Wolfgang Kaiser ; Ralf Strasser ; Ulrich Rant
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：September 19, 2012
• 年：2012
• 卷：134
• 期：37
• 页码：15225-15228
• 全文大小：264K
• 年卷期：v.134,no.37(September 19, 2012)
• ISSN：1520-5126

文摘

A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers鈥?dynamic motion. We demonstrate the quantitation of binding kinetics (k_on, k_off rate constants), dissociation constants (K_D in the pM regime), and the influence of competitive binders (EC₅₀ values). Moreover, the 鈥渟witchSENSE鈥?method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA₃) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins鈥?individual chemical environments significantly influence the His₆鈥揘TA₃ interaction.