Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series**

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.     

Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics

Single-molecule F?rster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.     

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy.     

Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity

In Fo?rster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 ? in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 ? is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 ?). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.     

Light-Harvesting Nanoparticle Probes for FRET-Based Detection of Oligonucleotides with Single-Molecule Sensitivity

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20-nm light-harvesting nanoantenna particles, built of a tailor-made hydrophobic charged polymer poly(ethyl methacrylate-co-methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87-fold brighter than quantum dots QDots 605 in single-particle microscopy (with 550-nm excitation), these DNA-functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250-fold signal amplification. The obtained FRET nanoprobes enable single-molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi-fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.     

Determination of cellulase colocalization on cellulose fiber with quantitative FRET measured by acceptor photobleaching and spectrally unmixing fluorescence microscopy

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.     

The effect of Brownian motion of fluorescent probes on measuring nanoscale distances by Förster resonance energy transfer

F?rster resonance energy transfer (FRET) is a powerful optical technique to determine intra-molecular distances. However, the dye rotational motion and the linker flexibility complicate the relationship between the measured energy transfer efficiency and the distance between the anchoring points of the dyes. In this study, we present a simple model that describes the linker and dye dynamics as diffusion on a sphere. Single-pair energy transfer was treated in the weak excitation limit, photon statistics and scaffold flexibility were ignored, and different time-averaging regimes were considered. Despite the approximations, our model provides new insights for experimental designs and results interpretation in single-molecule FRET. Monte Carlo simulations produced distributions of the inter-dye distance, the dipole orientation factor, κ(2), and the transfer efficiency, E, which were in perfect agreement with independently derived theoretical functions. Contrary to common perceptions, our data show that longer linkers will actually restrict the motion of dye dipoles and hence worsen the isotropic 2∕3 approximation of κ(2). It is also found that the thermal motions of the dye-linker system cause fast and large efficiency fluctuations, as shown by the simulated FRET time-trajectories binned on a microsecond time scale. A fundamental resolution limit of single-molecule FRET measurements emerges around 1-10 μs, which should be considered for the interpretation of data recorded on such fast time scales.     

Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis

Proteins are highly complex biopolymers, exhibiting a substantial degree of structural variability in their properly folded, native state. In the presence of denaturants, this heterogeneity is greatly enhanced, and fluctuations take place among vast numbers of folded and unfolded conformations via many different pathways. To better understand protein folding it is necessary to explore the structural and energetic properties of the folded and unfolded polypeptide chain, as well as the trajectories along which the chain navigates through its multi-dimensional conformational energy landscape. In recent years, single-molecule fluorescence spectroscopy has been established as a powerful tool in this research area, as it allows one to monitor the structure and dynamics of individual polypeptide chains in real time with atomic scale resolution using F?rster resonance energy transfer (FRET). Consequently, time trajectories of folding transitions can be directly observed, including transient intermediates that may exist along these pathways. Here we illustrate the power of single-molecule fluorescence with our recent work on the structure and dynamics of the small enzyme RNase H in the presence of the chemical denaturant guanidinium chloride (GdmCl). For FRET analysis, a pair of fluorescent dyes was attached to the enzyme at specific locations. In order to observe conformational changes of individual protein molecules for up to several hundred seconds, the proteins were immobilized on nanostructured, polymer coated glass surfaces specially developed to have negligible interactions with folded and unfolded proteins. The single-molecule FRET analysis gave insight into structural changes of the unfolded polypeptide chain in response to varying the denaturant concentration, and the time traces revealed stepwise transitions in the FRET levels, reflecting conformational dynamics. Barriers in the free energy landscape of RNase H were estimated from the kinetics of the transitions.     

Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics

We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.     

Long-Term,Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.     

The rylene colorant family--tailored nanoemitters for photonics research and applications

This Review summarizes the latest advances in the field of rylene dyes and rylene nanoemitters for applications in photonics, and describes the influence of the dye design on the optical properties, the self-assembly, the molecular interactions, as well as the labeling specificity of the compounds. The interplay between tailored (macro)molecular design and bulk/single-molecule spectroscopy enables complex processes to be explained, for example, the kinetics of energy-transfer processes or (bio)catalysis. Such investigations are essential for the ultimate design of optimized nanoemitters, and require a close cooperation between spectroscopists and preparative organic chemists.     

Bi-analyte SERS with isotopically edited dyes

Isotopically substituted rhodamine dyes provide ideal probes for the study of single-molecule surface enhanced Raman scattering (SM-SERS) events through multiple-analyte techniques. Isotopic editing should, in principle, provide probes that have identical chemical properties (and surface chemistries); while exhibiting at the same time distinct Raman features which enable us to identify single-molecule SERS events. We present here a specific example of two-analyte SM-SERS based on the isotopic substitution of a methyl ester rhodamine dye. The dyes are carefully characterized (in both standard and SERS conditions) to confirm experimentally their similar chemical properties. We then demonstrate their utility for bi-analyte SERS (BiASERS) experiments and, as an example, highlight the transition from a single, to a few, to many molecules in the statistics of SM-SERS signals.     

Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ～10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (～100 μs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.     

Bleaching-resistant,Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.     

High‐Performance Förster Resonance Energy Transfer (FRET)‐Based Dye‐Sensitized Solar Cells: Rational Design of Quantum Dots for Wide Solar‐Spectrum Utilization

High‐performance Förster resonance energy transfer (FRET)‐based dye‐sensitized solar cells (DSSCs) have been successfully fabricated through the optimized design of a CdSe/CdS quantum‐dot (QD) donor and a dye acceptor. This simple approach enables quantum dots and dyes to simultaneously utilize the wide solar spectrum, thereby resulting in high conversion efficiency over a wide wavelength range. In addition, major parameters that affect the FRET interaction between donor and acceptor have been investigated including the fluorescent emission spectrum of QD, and the content of deposited QDs into the TiO₂ matrix. By judicious control of these parameters, the FRET interaction can be readily optimized for high photovoltaic performance. In addition, the as‐synthesized water‐soluble quantum dots were highly dispersed in a nanoporous TiO₂ matrix, thereby resulting in excellent contact between donors and acceptors. Importantly, high‐performance FRET‐based DSSCs can be prepared without any infrared (IR) dye synthetic procedures. This novel strategy offers great potential for applications of dye‐sensitized solar cells.     

Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer

Intramolecular distances in proteins and other biomolecules can be studied in living cells by means of fluorescence resonance energy transfer (FRET) in steady-state or pulsed-excitation experiments. The major uncertainty originates from the unknown orientation between the optical dipole moments of the fluorescent markers, especially when the molecule undergoes thermal fluctuations in physiological conditions. We introduce a statistical method based on the von Mises-Fisher distribution for the interpretation of fluorescence decay dynamics in donor-acceptor FRET pairs that allows us to retrieve both the orientation and the extent of directional fluctuations of the involved dipole moments. We verify the method by applying it to donor-acceptor pairs controllably attached to DNA helices and find that common assumptions such as complete rotational freedom or fully hindered rotation of the dipoles fail a physical interpretation of the fluorescence decay dynamics. This methodology is applicable in single-molecule and ensemble measurements of FRET to derive more accurate distance estimates from optical experiments, without the need for more complex and expensive NMR studies.     

Design and synthesis of an enzyme activity-based labeling molecule with fluorescence spectral change

Methods of covalent labeling of a specific tag protein with small-molecular dyes play an important role in studying dynamic behaviors of proteins in living cells. On the basis of quinone methide chemistry, we designed and synthesized a beta-galactosidase labeling probe, CMFbeta-gal, which shows a fluorescence wavelength change accompanying the labeling reaction, owing to fluorescence resonance energy transfer (FRET). Since the FRET efficiency changes accompanying the labeling reaction, fluorescence of labeled protein can be observed separately from that of the unreacted probe, so immediate detection of the target protein is possible. This is the first report of a protein labeling probe which features a change of fluorescence wavelength upon reaction, allowing the labeled protein to be detected even in the presence of unreacted probe.     

Unfolded protein and peptide dynamics investigated with single-molecule FRET and correlation spectroscopy from picoseconds to seconds

Single-molecule fluorescence spectroscopy and correlation methods are finding increasing applications in the investigation of biomolecular dynamics, especially together with F?rster resonance energy transfer (FRET). Here, we use the combination of start-stop experiments and classical fluorescence correlation spectroscopy (FCS) to obtain complete intensity auto- and cross-correlation functions from picoseconds to seconds for investigating the dynamics of unfolded proteins and peptides. In combination with distance information from single-molecule transfer efficiency histograms, we can analyze the data in terms of a diffusive process on a potential of mean force to obtain intramolecular diffusion coefficients. This allows us to extend our previous analysis of the time scales of chain dynamics into the low nanosecond range for peptides and into the microsecond range for a small cold shock protein (Csp). Dynamics in short unstructured peptides can be detected down to a time scale of about 10 ns, placing a lower limit on the time scales accessible with correlation methods and currently used dye pairs. We find no evidence for microsecond fluctuations in unfolded Csp, suggesting that its global chain dynamics occur predominantly in the tens of nanosecond range. We further investigate the position dependence of these dynamics by placing donor and acceptor dyes at different positions within the chain and find a decrease in the intramolecular diffusion coefficient by a factor of 3 upon moving one of the dyes toward the center of the polypeptide. Obtaining dynamic information on a wide range of time scales from single-molecule photon statistics will be of increasing importance for the study of unfolded proteins and for biomolecules in general.     

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and β-arrestin-1 (βarr1). Using this strategy, we monitored the subtle but functionally important conformational change of βarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

A facile bioconjugation reaction for site-specific protein modification was developed for smFRET measurement, which detected the subtle but important conformational change of the β-arrestin/GPCR complex for the first time.