Orientation of cyanine fluorophores terminally attached to DNA via long, flexible tethers

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5′-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5′-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15°C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.     

The structure of cyanine 5 terminally attached to double-stranded DNA: implications for FRET studies

Fluorescence resonance energy transfer, FRET, can be used to obtain long-range distance information for macromolecules and is particularly powerful when used in single-molecule studies. The determination of accurate distances requires knowledge of the fluorophore position with respect to the macromolecule. In this study we have used NMR to determine the structure of the commonly used fluorophore indocarbocyanine-5 (Cy5) covalently attached to the 5'-terminus of double-helical DNA. We find that Cy5 is predominantly stacked onto the end of the duplex, in a manner similar to an additional base pair. This is very similar to the behavior of Cy3 terminally attached to DNA and suggests that the efficiency of energy transfer between Cy3 and Cy5, that are attached to nucleic acids in this way, will exhibit significant dependence on fluorophore orientation.     

Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic F?rster length for this pair on DNA is 56 A, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.     

Cy3-DNA stacking interactions strongly depend on the identity of the terminal basepair

We characterized the effect of the first basepair on the conformational dynamics of the fluorescent dye Cy3 attached to the 5′ end of double-stranded DNA using Gaussian-mixture adaptive umbrella sampling simulations. In the simulations, the sampling of all five dihedral angles along the linker was enhanced, so that both stacked and unstacked states were sampled. The affinity of Cy3 for a T·A basepair (with the dye attached to T) was found to be significantly less than for the other basepairs. This was verified experimentally by measuring the activation energies for cis-trans isomerization of the dye. The simulation and experimental results indicate the existence of partially unstacked conformations amenable to photoisomerization. The simulations also showed that stacking of Cy3 straightens the DNA while stabilizing the first basepair. Our findings indicate that fluorescence is modulated by Cy3-DNA interactions in a sequence-dependent manner.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the?Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.     

Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study

The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5' 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 degrees C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from approximately 15 to 100 A). Subsequent integration assays at 37 degrees C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (deltaF) show that IN binds to a particular set of 3' OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to approximately 20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower deltaF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as "nonspecific" binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower deltaF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.     

Analysis of oligonucleotide microarrays by 3' end labeling using fluorescent nucleotides and terminal transferase

A simple enzymatic labeling procedure is described to determine spot quality in oligonucleotide microarrays. By using fluorescently labeled dideoxynucleotides or ribonucleotides as substrate for terminal deoxynucleotidyl transferase (TdT), a single fluorophore can be covalently attached at the 3' end of each oligonucleotide probe molecule in the spot. Fluorescein-12-ddUTP CyTM3-ddUTP Cy5-UTP, and Cy3-UTP were compared as TdT substrates for 3' end labeling an array of 1273 hexamer probes. Cy5-UTP was found to show minimal bias toward probe base composition and is therefore well suited for quantitative analysis of microarray spots where the oligonucleotide probes are coupled via a 5' end linkage to the solid phase.     

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.     

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07–0.2 (on a scale of 0–1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.     

Optical properties of Alexa 488 and Cy5 immobilized on a glass surface

The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret these red shifts in terms of a change in the polarizability and polarity of the effective solvent. A formula is given that can be used to estimate expected shifts in absorption and emission maxima for surface-immobilized fluorophores. Spectroscopic ellipsometry measurements provide identification of the fluorophores confined on a glass surface. These results suggest that the design of microarray detection systems should be based on the optical properties of fluorophores attached to the surface and not on the optical properties of fluorophores in solution.     

Nonblinking and long-lasting single-molecule fluorescence imaging

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.     

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.     

Electroporation and Microinjection Successfully Deliver Single-Stranded and Duplex DNA into Live Cells as Detected by FRET Measurements

Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.     

Global and local dynamics of the U1A polyadenylation inhibition element (PIE) RNA and PIE RNA-U1A complexes

The structure and dynamics of the polyadenylation inhibition element (PIE) RNA, free and bound to the U1A protein, have been examined using time-resolved FRET and 2-aminopurine (2AP) fluorescence. This regulatory RNA, located at the 3' end of the U1A pre-mRNA, adopts a U-shaped structure, with binding sites for a single U1A protein at each bend (box 1 and box 2). The distance between the termini of the arms of the RNA is sensitive to its three-dimensional structure. Using Cy3/Cy5 FRET efficiency to monitor binding of Mg(2+), we show that the PIE RNA binds two Mg(2+) ions, which results in a restriction of its distance distribution of conformations. Local RNA structure probing using 2AP fluorescence shows that the structure of box 2 changes in response to Mg(2+) binding, thus tentatively locating the ion binding sites. Steady-state FRET data show that the distance R between the termini of the PIE RNA stems decreases from 66 A in the free RNA, to 58 A when N-terminal RNA binding domains (RBD1) of U1A are bound, and to 53 A when U1A proteins bind. However, anisotropy measurements indicate that both Cy3 and Cy5 stack on the ends of the RNA. To examine the consequences of the restricted motion of the fluorophores, FRET data are analyzed using two different models of motion and then compared to analogous data from the Cy3/fluorescein FRET pair. We conclude that the error introduced into distance calculations by stacking of the dyes is within the error of our measurements. Distance distributions of the RNA structures show that the intramolecular distance between the arms of the PIE RNA varies on the time scale of the fluorescence measurements; the mean distance is dependent on protein binding, but the breadth of the distributions indicates that the RNA retains structural heterogeneity.     

Single-molecule three-color FRET

Fluorescence resonance energy transfer (FRET) measured at the single-molecule level can reveal conformational changes of biomolecules and intermolecular interactions in physiologically relevant conditions. Thus far single-molecule FRET has been measured only between two fluorophores. However, for many complex systems, the ability to observe changes in more than one distance is desired and FRET measured between three spectrally distinct fluorophores can provide a more complete picture. We have extended the single-molecule FRET technique to three colors, using the DNA four-way (Holliday) junction as a model system that undergoes two-state conformational fluctuations. By labeling three arms of the junction with Cy3 (donor), Cy5 (acceptor 1), and Cy5.5 (acceptor 2), distance changes between the donor and acceptor 1, and between the donor and acceptor 2, can be measured simultaneously. Thus we are able to show that the acceptor 1 arm moves away from the donor arm at the same time as the acceptor 2 arm approaches the donor arm, and vice versa, marking the first example of observing correlated movements of two different segments of a single molecule. Our data further suggest that Holliday junction does not spend measurable time with any of the helices unstacked, and that the parallel conformations are not populated to a detectable degree.     

Quantitative time domain analysis of lifetime‐based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions

Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime‐based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.      

Preparation and characterization of calibration beads for sorting cells expressing a beta-lactamase gene reporter.

BACKGROUND: Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate. METHODS: To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer. RESULTS: We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate. CONCLUSION: We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.     

Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the mothManduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMR Famide and SCPb, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.