Practical guidelines for dual-color fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.     

荧光相关谱技术及其应用

基于对处于平衡态少量荧光分子集合的强度涨落进行时间平均的技术,荧光相关谱fluoreswceance correlation spectroscopy,FCS)技术最近已经应用于细胞环境过程的研究。FCS优秀的灵敏特性为我们实时测量许多参数提供了途径,而且具有快速的时间特性和高空间分辨率。测量的参数包括扩散速率、局部浓度、聚合状态和分子间的相互作用。荧光互相关谱(fluorescence cross-correlation spectroscopy,FCCS)进一步扩展了FCS技术的应用,包括在活细胞中的广泛应用。本文介绍了FCS技术的原理、实验装置及其应用。     

Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques to measure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrP(Sc)) was comparable to that of enzyme-linked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis.     

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches

Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes, a key metabolic regulator in Gram-positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is twofold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that control catabolite protein of gluconeogenic genes forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation coupled with fluorescence anisotropy and hydrodynamic modeling allowed unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which analytical ultracentrifugation is precluded, due to protein stability, solubility, or availability, or, more obviously, when the studies are carried out in live cells. If information concerning the self-association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity, and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.     

Pulsed interleaved excitation

In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.     

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method

Fluorescence cross-correlation spectroscopy (FCCS) is used as a powerful technique to analyze molecular interactions both in vitro and in vivo. This method basically requires two laser excitations for two target molecules labeled with fluorophores of different colors. Their coincidence in a microscopic detection volume is analyzed using two detectors. Any overlap of emission spectra of the two fluorophores, however, gives rise to false-positive data about their interaction. To overcome this problem, we have developed a new FCCS system, in which two excitation lasers are switched alternately by modulation using an acousto-optic tunable filter (AOTF). In this report, we demonstrate the feasibility of switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) with a cleavage site of caspase-3 inserted was expressed in HeLa cells, and proteolysis assay was performed during apoptotic cell death. Due to the absence of cross-talk signals, the FCCS measurement with the switching function gave a large change in relative cross-correlation amplitude after protein cleavage. Hence, switching FCCS enables more reliable measurement of molecular interactions than conventional FCCS.     

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

Toward the detection of single virus particle in serum

There is a grand challenge for the detection of target molecules at single molecule sensitivity in a bulk body fluid for the early diagnosis of diseases. We report our progress on tackling this challenge via the combination of fluorescence cross-correlation spectroscopy (FCCS) and micro fabricated devices toward highly sensitive detection of the dengue virus. We demonstrate that by using a dengue-specific antibody, we can probe the individual dengue virus in a nanomolar bulk solution by following the specific association of dengue antibody using FCCS. Consequently, we designed and fabricated a microfluidic chamber array structure and were able to compartmentalize the bulk aqueous dengue sample into femtoliter volumes using such a device. More importantly we demonstrate that we can differentiate between the compartments containing the dengue virus and the virus-free compartments. Our experiment suggests that by expanding the throughput using microfluidic devices integrated with FCCS, both of which can be achieved practically, we should be able to detect single virus particle in human body fluids in the near future.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

Direct detection of caspase-3 activation in single live cells by cross-correlation analysis

Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells.     

荧光相关光谱在生物化学领域中的应用

荧光相关光谱(fluorescence correlation spectroscopy,FCS)是一种通过监测荧光涨落从而获得单分子水平的分子扩散行为信息的技术。FCS高灵敏度的优点使得它已发展成为一种可以在活体外与活体内检测分子浓度、扩散系数、结合和解离常数等参数的有力工具。荧光互相关光谱(fluorescence cross-correlation spectroscopy,FCCS)是FCS技术的进一步发展,其大大扩展了FCS技术的应用范围。本文介绍了FCS及其衍生技术的原理及其在生物化学领域的应用。     

In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy

We employed dual color Fluorescence Cross Correlation Spectroscopy (FCCS) to measure the interaction between PKA regulatory (RII) and catalytic subunits (CAT) in living cells. Elevation of intracellular cAMP with forskolin decreased the cross-correlation amplitude between RFP-fused RII (RII-mRFP) and GFP-fused CAT (CAT-EGFP) by 50%, indicating that cAMP elevation leads to dissociation of RII-CAT complexes. Moreover, diffusion coefficient analysis showed that the diffusion rate of CAT-EGFP was significantly increased, suggesting that the decreased RII-CAT association caused by cAMP generated free CAT subunits. Our study demonstrates that in vivo FCCS measurements and their quantitative analysis permit one not only to directly quantify protein-protein interactions but also to estimate changes in the intracellular cAMP concentration.     

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.     

Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K_ds) of biomolecules. The determination of a K_d depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ～0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K_d.     

Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.     

Homogeneous time-resolved G protein-coupled receptor–ligand binding assay based on fluorescence cross-correlation spectroscopy

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor–ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.     

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Escherichia coli ribosomal protein L20 binds as a single monomer to its own mRNA bearing two potential binding sites

Ribosomal protein L20 is crucial for the assembly of the large ribosomal subunit and represses the translation of its own mRNA. L20 mRNA carries two L20-binding sites, the first folding into a pseudoknot and the second into an imperfect stem and loop. These two sites and the L20-binding site on 23S ribosomal RNA are recognized similarly using a single RNA-binding site located on one face of L20. In this work, using gel filtration and fluorescence cross-correlation spectroscopy (FCCS) experiments, we first exclude the possibility that L20 forms a dimer, which would allow each monomer to bind one site of the mRNA. Secondly we show, using affinity purification and FCCS experiments, that only one molecule of L20 binds to the L20 mRNA despite the presence of two potential binding sites. Thirdly, using RNA chemical probing, we show that the two L20-binding sites are in interaction. This interaction provides an explanation for the single occupancy of the mRNA. The two interacting sites could form a single hybrid site or the binding of L20 to a first site may inhibit binding to the second. Models of regulation compatible with our data are discussed.