Analysis of Ligand Binding by Two-Colour Fluorescence Cross-Correlation Spectroscopy

Weidemann, Thomas; Wachsmuth, Malte; Tewes, M.; Rippe, Karsten; Langowski, Jörg

doi:10.1002/1438-5171(200204)3:1<49::aid-simo49>3.0.co;2-t

Cited by 79 publications

(59 citation statements)

References 39 publications

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“…Measurement of this parameter can be obscured by a variety of artifacts that can both raise or lower the measured cross-correlation amplitude. 55 Using control samples that establish the upper and lower bounds of the cross-correlation measurement enables calibration of the cross-correlation signal and quantification of the amount of heterodimer formed (SI and Figure S5). 52 Performing this analysis of the data shown in Figure 2c provides an estimate of 52–60% yield of assembled heterodimer.…”

Section: Resultsmentioning

confidence: 99%

DNA-Mediated Assembly of Protein Heterodimers on Membrane Surfaces

Coyle

Chiang³

et al. 2013

J. Am. Chem. Soc.

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We present a method based on self-assembling oligonucleotides to anchor proteins to a supported membrane surface. This anchoring method allows control of the surface density of multiple proteins. By incorporating additional recognition sequences into the DNA linkers, defined heterodimers can be produced upon the addition of a heterospecific DNA cross-linking strand. Characterization by fluorescence cross-correlation spectroscopy (FCCS) confirmed lateral mobility and the formation of specific heterodimers. We further demonstrate that proteins linked in this manner as either monomers or dimers can form functional interfaces with living cells.

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Section: Resultsmentioning

confidence: 99%

DNA-Mediated Assembly of Protein Heterodimers on Membrane Surfaces

Coyle

Chiang³

et al. 2013

J. Am. Chem. Soc.

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“…However, there are limitations to using a very low and a very high laser power. A low laser power means a low particle brightness, which directly affects the quality of the correlation curves 9, 25. Moreover, low count rates result in a greater uncertainty in the count-rate ratio f and thus in less reliability of the cross-talk correction [Eq.…”

Section: Discussionmentioning

confidence: 99%

Correcting for Spectral Cross‐Talk in Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Bacia¹,

Petrášek

Schwille

2012

ChemPhysChem

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Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored.

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“…Here, the device‐dependent values were between 41% and 45 %. Due to the wavelength dependence of diffraction, this value can maximally amount to 54% in a perfectly adjusted microscope (Weidemann et al ., 2002).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous detection of intracellular target and off‐target binding of small molecule cancer drugs at nanomolar concentrations

Glauner

Ruttekolk

Hansen³

et al. 2010

British J Pharmacology

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Background and purpose:In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. Experimental approach: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. Key results: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. Conclusions and implications:The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.British Journal of Pharmacology (2010) 160, 958-970; doi:10.1111/j.1476-5381.2010.00732.x Keywords: fluorescence correlation spectroscopy; fluorescence cross-correlation spectroscopy; anti-cancer drug; cellular pharmacology; drug-target interaction; drug development Abbreviations: Abl 1, Abelson kinase 1; cpm, counts per molecule; DHFR, dihydrofolate reductase; FCS, fluorescence correlation spectroscopy; FCCS, fluorescence cross-correlation spectroscopy; FRET, fluorescence resonance energy transfer; Hek, human embryonic kidney; MTX, methotrexate; RP-HPLC, reversed-phase high-performance liquid chromatography; PBS, phosphate-buffered saline; PD, Park Davis; PEG, polyethylene glycol; Pur, purvalanol; RT, room temperature IntroductionCellular assays are gaining significance in drug development (Perlman et al., 2004;Lang et al., 2006;Korn and Krausz, 2007). While providing less information on the interaction of a drug candidate towards a specific molecular target, such assays reveal off-target effects and cytotoxicity. In addition, many compounds fail to show any biological effect, even if activity was detected towards an isolated target molecule in an in vitro screen.For compounds that exert their activity inside the cell, such failure is typically attributed to poor cellular uptake. Howeve...

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Analysis of Ligand Binding by Two-Colour Fluorescence Cross-Correlation Spectroscopy

Cited by 79 publications

References 39 publications

DNA-Mediated Assembly of Protein Heterodimers on Membrane Surfaces

DNA-Mediated Assembly of Protein Heterodimers on Membrane Surfaces

Correcting for Spectral Cross‐Talk in Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Simultaneous detection of intracellular target and off‐target binding of small molecule cancer drugs at nanomolar concentrations

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