A strategy is devised for the conversion of cellulose nanofibrils (CNF) into fluorescently labele... more A strategy is devised for the conversion of cellulose nanofibrils (CNF) into fluorescently labeled probes involving the synthesis of CNF-based macroinitiators that initiate radical polymerization of methyl acrylate and acrylic acid N-hydroxysuccinimide ester producing a graft block copolymer modified CNF. Finally, a luminescent probe (Lucifer yellow derivative) was labeled onto the modified CNF through an amidation reaction. The surface modification steps were verified with solid-state (13)C nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy. Fluorescence correlation spectroscopy (FCS) confirmed the successful labeling of the CNF; the CNF have a hydrodynamic radius of about 700 nm with an average number of dye molecules per fibril of at least 6600. The modified CNF was also imaged with confocal laser scanning microscopy. Luminescent CNF proved to be viable biomarkers and allow for fluorescence-based optical detection of CNF uptake and distribution in organisms such as crustaceans. The luminescent CNF were exposed to live juvenile daphnids and microscopy analysis revealed the presence of the luminescent CNF all over D. magna's alimentary canal tissues without any toxicity effect leading to the death of the specimen.
933-Plat Measuring interactions between the Antennapedia Homeodomain and DNA using Fluorescence C... more 933-Plat Measuring interactions between the Antennapedia Homeodomain and DNA using Fluorescence Cross-Correlation Spectroscopy with a biarsenical ligand Per Rigler1, Marina Hugot-Beaufils1, Yoshi Adachi1, Stefan Wennmalm2, Rudolf Rigler3, Walter J. Gehring1 1 University of Basel, Basel, Switzerland 2 Royal Institute of Technology, Stockholm, Sweden 3 Karolinska Institutet, Stockholm, Sweden. Recombinant proteins containing a tetracysteine motif CCPGCC on C-terminus can be covalently labelled with a biarsenical fluorescein derivate called FlAsH. Here we use this approach to label the homeodomain of the transcription factor Antennapedia from Drosophila. In this study, we use Fluorescence Cross-Correlation Spectroscopy (FCCS) to analyse the DNA binding properties of this transcription factor with different DNA fragments. We developed, for the first time, a reproducible in vitro fluorescence assay, based on the FlAsH technology, at the single molecular level. The obtained dissociation c...
ABSTRACT Fluorescence correlation spectroscopy (FCS) was introduced in the early seventies for th... more ABSTRACT Fluorescence correlation spectroscopy (FCS) was introduced in the early seventies for the analysis of thermodynamic fluctuations of chemical systems [15, 18, 28] in an attempt to complement chemical relaxation spectroscopy as introduced by Eigen and de Meyer for the analysis of ultrafast kinetics. Chemical relaxation refers to the adjustment of chemical reactions into a new equilibrium state after an instantaneous change of intensive parameters such as temperature, pressure, or electric field [16]. Chemical fluctuations depend on the spontaneous change in number density of chemical systems due to processes involving transitions into the excited state [15], Brownian motion [15, 28] as well as chemical kinetics [18, 29].
Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimer's disease. Increased kn... more Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimer's disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aβ oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Fö rster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aβ, FRET-FCS detected oligomers consisting of less than 10 Aβ molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aβ oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.
Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the abs... more Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 ± 12 nm diameter, and 40 nm domains were estimated to 65 ± 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.
A central goal in bioanalytics is to determine the concentration of and interactions between biom... more A central goal in bioanalytics is to determine the concentration of and interactions between biomolecules. Nanotechnology allows performing such analyses in a highly parallel, low-cost, and miniaturized fashion. Here we report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.
A novel fluctuation spectroscopy technique based on interferometry is described. The technique, t... more A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward-scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate K d from a single binding measurement.
The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which ha... more The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.
Conformational fluctuations in single nucleic acid molecules have recently been observed through ... more Conformational fluctuations in single nucleic acid molecules have recently been observed through excited state lifetime measurements. Immobilisation of the sample molecule has also enabled direct observation of the fluorescence intensity fluctuations generated as the molecule switches between two conformations. As a probe for conformational fluctuations we use tetramethylrhodamine linked to a 217-bp DNA oligonucleotide. The measurements on this and similar systems reveal the existence of a distribution of reaction rates between the conformations. Here we report 37 detected single-molecule conformational fluctuations collected with the same immobilisation method as described earlier. Within the time window of observation the reaction rates differ between the molecules, but stay constant within a single molecule. The distribution of the relaxation rates between the molecules correspond to the distribution seen in a bulk measurement on a similar system. We therefore conclude that within the observation time window the single DNA molecules behave in a non-ergodic way.
In fluorescence measurements on the single molecule level the photochemical stability of the fluo... more In fluorescence measurements on the single molecule level the photochemical stability of the fluorophore is an important factor. We measured in 102 cases the photodestruction of individual tetramethylrhodamine molecules linked to a 217-bp DNA sequence which was attached to a streptavidin-coated glass surface. The fluorophores were excited by a green HeNe laser at 543.5 nm. For each molecule the survival time (time during which fluorescence can be observed), the fluorescence intensity, and the death number (number of detected photons before decomposition) were registered. On the basis of the analysis of single molecule observations, an exponential distribution of the death numbers and survival times of the 102 molecules was found.
Measurement of f luorescent lifetimes of dye-tagged DNA molecules reveal the existence of differe... more Measurement of f luorescent lifetimes of dye-tagged DNA molecules reveal the existence of different con-formations. Conformational f luctuations observed by f luo-rescence correlation spectroscopy give rise to a relaxation behavior that is described by ''stretched'' exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomoge-neous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethyl-rhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational f luctuations. Fluorescence f luctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational f luctuations typical for single molecules are similar to those seen in single ion channels in cell membranes. Fluorescence correlation spectroscopy (FCS) allows the analysis of single-molecule events and their time correlations in solution (1–6). Since the introduction of confocal excitation in extremely small volume elements (2), single molecules can be detected almost background-free in solution (3, 4). FCS has opened the possibility to analyze the behavior of single molecules in relation to their ensemble averages. In particular information can be obtained from the analysis of single molecules that cannot be obtained from the ensemble average alone. As has been pointed out by Wang and Wolynes (7), properties found for the molecular ensemble such as the distribution of states can also be a property of a single molecule (the homogenous case). Alternatively, the ensemble behavior can be caused by a collection of individual molecules, each representing a different state (the inhomogeneous case). Such situations are likely to be found in biological systems as has been put forward recently by Frauenfelder (8). We have earlier been able to demonstrate the existence of different conformational states in single molecules of M13 phage DNA (9) from the analysis of the exited state of tetramethylrhodamine (TMR) linked to the DNA by a 6-atom carbon linker and serving as a sensor for different conforma-tional states of the DNA molecule. The redox potential between aromatic dye molecules and purine as well as pyrim-idine bases (10) leads in the case of rhodamine dyes to an electron transfer from guanine to the dye that competes with the photon emission from the excited singlet state of TMR (11, 12). The electron transfer is characteristic for guanine and indicates that in one conformation electron transfer takes place but not in the other. The time range of the observed conformational transitions is in the millisecond region, pointing to the involvement of intercalative processes (13, 14). The rates as measured in a molecular ensemble by FCS exhibited a nonexponential behavior that could be best represented by a ''stretch'' parameter (0.44) and indicates a distribution of rates (9). We show herein the results obtained by the measurement of conformational transitions in a single DNA molecule that is attached to a surface by a biotin–streptavidin interaction and carries the TMR sensor. For this purpose a 217-bp DNA piece was prepared by PCR. We are able to show fluorescence intensity fluctuations in single DNA molecules in the range of hundreds of milliseconds that are due to conformational fluctuations. They are analogous to the single ion channel fluctuations that have been observed by Neher and Sakmann (15). We have been able to analyze the transition rates for individual DNA molecules and compare their calculated relaxation rates with the situation found for the ensemble. Within the time limit of observation set by the photochemical lifetime of the TMR sensor (a few seconds), we find different relaxation rates for different DNA molecules, supporting the existence of an inhomogeneous distribution. An important result of this study is the demonstration that molecular transitions in single biomolecules can be observed by optical spectroscopy, in particular by FCS.
A strategy is devised for the conversion of cellulose nanofibrils (CNF) into fluorescently labele... more A strategy is devised for the conversion of cellulose nanofibrils (CNF) into fluorescently labeled probes involving the synthesis of CNF-based macroinitiators that initiate radical polymerization of methyl acrylate and acrylic acid N-hydroxysuccinimide ester producing a graft block copolymer modified CNF. Finally, a luminescent probe (Lucifer yellow derivative) was labeled onto the modified CNF through an amidation reaction. The surface modification steps were verified with solid-state (13)C nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy. Fluorescence correlation spectroscopy (FCS) confirmed the successful labeling of the CNF; the CNF have a hydrodynamic radius of about 700 nm with an average number of dye molecules per fibril of at least 6600. The modified CNF was also imaged with confocal laser scanning microscopy. Luminescent CNF proved to be viable biomarkers and allow for fluorescence-based optical detection of CNF uptake and distribution in organisms such as crustaceans. The luminescent CNF were exposed to live juvenile daphnids and microscopy analysis revealed the presence of the luminescent CNF all over D. magna's alimentary canal tissues without any toxicity effect leading to the death of the specimen.
933-Plat Measuring interactions between the Antennapedia Homeodomain and DNA using Fluorescence C... more 933-Plat Measuring interactions between the Antennapedia Homeodomain and DNA using Fluorescence Cross-Correlation Spectroscopy with a biarsenical ligand Per Rigler1, Marina Hugot-Beaufils1, Yoshi Adachi1, Stefan Wennmalm2, Rudolf Rigler3, Walter J. Gehring1 1 University of Basel, Basel, Switzerland 2 Royal Institute of Technology, Stockholm, Sweden 3 Karolinska Institutet, Stockholm, Sweden. Recombinant proteins containing a tetracysteine motif CCPGCC on C-terminus can be covalently labelled with a biarsenical fluorescein derivate called FlAsH. Here we use this approach to label the homeodomain of the transcription factor Antennapedia from Drosophila. In this study, we use Fluorescence Cross-Correlation Spectroscopy (FCCS) to analyse the DNA binding properties of this transcription factor with different DNA fragments. We developed, for the first time, a reproducible in vitro fluorescence assay, based on the FlAsH technology, at the single molecular level. The obtained dissociation c...
ABSTRACT Fluorescence correlation spectroscopy (FCS) was introduced in the early seventies for th... more ABSTRACT Fluorescence correlation spectroscopy (FCS) was introduced in the early seventies for the analysis of thermodynamic fluctuations of chemical systems [15, 18, 28] in an attempt to complement chemical relaxation spectroscopy as introduced by Eigen and de Meyer for the analysis of ultrafast kinetics. Chemical relaxation refers to the adjustment of chemical reactions into a new equilibrium state after an instantaneous change of intensive parameters such as temperature, pressure, or electric field [16]. Chemical fluctuations depend on the spontaneous change in number density of chemical systems due to processes involving transitions into the excited state [15], Brownian motion [15, 28] as well as chemical kinetics [18, 29].
Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimer's disease. Increased kn... more Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimer's disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aβ oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Fö rster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aβ, FRET-FCS detected oligomers consisting of less than 10 Aβ molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aβ oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.
Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the abs... more Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 ± 12 nm diameter, and 40 nm domains were estimated to 65 ± 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.
A central goal in bioanalytics is to determine the concentration of and interactions between biom... more A central goal in bioanalytics is to determine the concentration of and interactions between biomolecules. Nanotechnology allows performing such analyses in a highly parallel, low-cost, and miniaturized fashion. Here we report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.
A novel fluctuation spectroscopy technique based on interferometry is described. The technique, t... more A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward-scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate K d from a single binding measurement.
The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which ha... more The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.
Conformational fluctuations in single nucleic acid molecules have recently been observed through ... more Conformational fluctuations in single nucleic acid molecules have recently been observed through excited state lifetime measurements. Immobilisation of the sample molecule has also enabled direct observation of the fluorescence intensity fluctuations generated as the molecule switches between two conformations. As a probe for conformational fluctuations we use tetramethylrhodamine linked to a 217-bp DNA oligonucleotide. The measurements on this and similar systems reveal the existence of a distribution of reaction rates between the conformations. Here we report 37 detected single-molecule conformational fluctuations collected with the same immobilisation method as described earlier. Within the time window of observation the reaction rates differ between the molecules, but stay constant within a single molecule. The distribution of the relaxation rates between the molecules correspond to the distribution seen in a bulk measurement on a similar system. We therefore conclude that within the observation time window the single DNA molecules behave in a non-ergodic way.
In fluorescence measurements on the single molecule level the photochemical stability of the fluo... more In fluorescence measurements on the single molecule level the photochemical stability of the fluorophore is an important factor. We measured in 102 cases the photodestruction of individual tetramethylrhodamine molecules linked to a 217-bp DNA sequence which was attached to a streptavidin-coated glass surface. The fluorophores were excited by a green HeNe laser at 543.5 nm. For each molecule the survival time (time during which fluorescence can be observed), the fluorescence intensity, and the death number (number of detected photons before decomposition) were registered. On the basis of the analysis of single molecule observations, an exponential distribution of the death numbers and survival times of the 102 molecules was found.
Measurement of f luorescent lifetimes of dye-tagged DNA molecules reveal the existence of differe... more Measurement of f luorescent lifetimes of dye-tagged DNA molecules reveal the existence of different con-formations. Conformational f luctuations observed by f luo-rescence correlation spectroscopy give rise to a relaxation behavior that is described by ''stretched'' exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomoge-neous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethyl-rhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational f luctuations. Fluorescence f luctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational f luctuations typical for single molecules are similar to those seen in single ion channels in cell membranes. Fluorescence correlation spectroscopy (FCS) allows the analysis of single-molecule events and their time correlations in solution (1–6). Since the introduction of confocal excitation in extremely small volume elements (2), single molecules can be detected almost background-free in solution (3, 4). FCS has opened the possibility to analyze the behavior of single molecules in relation to their ensemble averages. In particular information can be obtained from the analysis of single molecules that cannot be obtained from the ensemble average alone. As has been pointed out by Wang and Wolynes (7), properties found for the molecular ensemble such as the distribution of states can also be a property of a single molecule (the homogenous case). Alternatively, the ensemble behavior can be caused by a collection of individual molecules, each representing a different state (the inhomogeneous case). Such situations are likely to be found in biological systems as has been put forward recently by Frauenfelder (8). We have earlier been able to demonstrate the existence of different conformational states in single molecules of M13 phage DNA (9) from the analysis of the exited state of tetramethylrhodamine (TMR) linked to the DNA by a 6-atom carbon linker and serving as a sensor for different conforma-tional states of the DNA molecule. The redox potential between aromatic dye molecules and purine as well as pyrim-idine bases (10) leads in the case of rhodamine dyes to an electron transfer from guanine to the dye that competes with the photon emission from the excited singlet state of TMR (11, 12). The electron transfer is characteristic for guanine and indicates that in one conformation electron transfer takes place but not in the other. The time range of the observed conformational transitions is in the millisecond region, pointing to the involvement of intercalative processes (13, 14). The rates as measured in a molecular ensemble by FCS exhibited a nonexponential behavior that could be best represented by a ''stretch'' parameter (0.44) and indicates a distribution of rates (9). We show herein the results obtained by the measurement of conformational transitions in a single DNA molecule that is attached to a surface by a biotin–streptavidin interaction and carries the TMR sensor. For this purpose a 217-bp DNA piece was prepared by PCR. We are able to show fluorescence intensity fluctuations in single DNA molecules in the range of hundreds of milliseconds that are due to conformational fluctuations. They are analogous to the single ion channel fluctuations that have been observed by Neher and Sakmann (15). We have been able to analyze the transition rates for individual DNA molecules and compare their calculated relaxation rates with the situation found for the ensemble. Within the time limit of observation set by the photochemical lifetime of the TMR sensor (a few seconds), we find different relaxation rates for different DNA molecules, supporting the existence of an inhomogeneous distribution. An important result of this study is the demonstration that molecular transitions in single biomolecules can be observed by optical spectroscopy, in particular by FCS.
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