Label-Free Solution-Based Kinetic Study of Aptamer–Small Molecule Interactions by Kinetic Capillary Electrophoresis with UV Detection Revealing How Kinetics Control Equilibrium

Bao, Jiayin; Krylova, Svetlana M.; Reinstein, Oren; Johnson, Phyllis E.; Krylov, Sergey N.

doi:10.1021/ac2026699

Cited by 46 publications

(49 citation statements)

References 30 publications

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“…The effect of fluorescent labels has been investigated by mass spectrometry(Salih and Zenobi 1998), X-ray crystallography(Ianeselli, Zhang et al 2010), electrophoresis(Bao, Krylova et al 2011) and molecular dynamics simulation(Unruh, Gokulrangan et al 2005). These methods are suitable for evaluating the effect of the labels on the structural or other static properties of the molecules, but they do not provide direct kinetic information that is necessary for quantifying interactions between molecules.…”

Section: Introductionmentioning

confidence: 99%

How does fluorescent labeling affect the binding kinetics of proteins with intact cells?

Yin

Wang

et al. 2015

Biosensors and Bioelectronics

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Fluorescent labeling is a mainstream technology for detecting molecular binding. Despite the importance, few studies have been devoted to quantitatively examine the effect of labeling on the molecular binding processes. Here we present a quantitative study on the binding kinetics of fluorescent-labeled and un-labeled molecules (lectin proteins) with glycoproteins on the membrane of cells using surface plasmon resonance imaging (SPRi) technique. The study shows that fluorescent labeling has a significant influence on the binding behaviors of proteins, especially the association processes, and the influence depends sensitively on the charge of fluorescent labels. It further shows that the labels also affect the local distribution of probe proteins, due to the inhomogeneous surface charge distribution of the cell membrane. Our work indicates that fluorescent labeling in general affects the binding behaviors, but proper design of the label will help to minimize its effect.

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Section: Introductionmentioning

confidence: 99%

How does fluorescent labeling affect the binding kinetics of proteins with intact cells?

Yin

Wang

et al. 2015

Biosensors and Bioelectronics

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“…KCE has been utilized to provide binding strengths and rate constants for several systems, including protein-oligonucleotide, protein-peptide, protein-small molecule, and oligonucleotide-small molecule interactions [20,159,160,162,164–171]. These methods have been used to measure dissociation rate constants that have ranged from 10 −4 to 1 s −1 [159,160,166,172] and association rate constants that have ranged from 10 1 to 10 7 M −1 s −1 [164,165,167,168,169–172]. …”

Section: Capillary Electrophoresismentioning

confidence: 99%

“…For instance, the overall range of dissociation rate constants that have been measured by CE is 10 −6 to 1 s −1 [149–151,159,160,166,172], and the association rate constants have spanned from 1 to 10 7 M −1 s −1 [164,165,167,168,169–172]. …”

Section: Capillary Electrophoresismentioning

confidence: 99%

Analytical methods for kinetic studies of biological interactions: A review

Zheng

Zhao

et al. 2015

Journal of Pharmaceutical and Biomedical Analysis

142

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The rates at which biological interactions occur can provide important information concerning the mechanism and behavior of these processes in living systems. This review discusses several analytical methods that can be used to examine the kinetics of biological interactions. These techniques include common or traditional methods such as stopped-flow analysis and surface plasmon resonance spectroscopy, as well as alternative methods based on affinity chromatography and capillary electrophoresis. The general principles and theory behind these approaches are examined, and it is shown how each technique can be utilized to provide information on the kinetics of biological interactions. Examples of applications are also given for each method. In addition, a discussion is provided on the relative advantages or potential limitations of each technique regarding its use in kinetic studies.

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“…In-capillary technique, firstly described by Bao and Regniner, integrated multiple functions "injection, mixing, reaction, separation and detection" inside a capillary [22]. It has already been applied in many analyses, such as aptamersmall molecule interactions [23], enzymatic reaction [24] and QDs-biomolecule interactions [25,26]. To understand the interaction mechanism and to develop practical affinity assays, it is really important to obtain all the kinetic parameters (K d , K on , K off ) of QDs interacting with biomolecules [23].…”

Section: Introductionmentioning

confidence: 99%

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti‐HA antibody in single in‐capillary assay

Wang

Qin

et al. 2016

Electrophoresis

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Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions.

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Label-Free Solution-Based Kinetic Study of Aptamer–Small Molecule Interactions by Kinetic Capillary Electrophoresis with UV Detection Revealing How Kinetics Control Equilibrium

Cited by 46 publications

References 30 publications

How does fluorescent labeling affect the binding kinetics of proteins with intact cells?

How does fluorescent labeling affect the binding kinetics of proteins with intact cells?

Analytical methods for kinetic studies of biological interactions: A review

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti‐HA antibody in single in‐capillary assay

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