Fluorescence resonance energy transfer in dye-labeled DNA

We present the results of molecular modeling of dye-labeled, double-stranded DNA. The structural information obtained from the simulations are used as input to an analysis of energy transfer in this system. The simulations reveal the nature of the interaction between a pair of fluorophores and DNA. The donor, tetramethylrhodamine, TMR, attached to the 5′-end of DNA with a six-carbon tether, interacts primarily with DNA's minor groove, but occasionally stacks against the DNA base pairs. The acceptor, Cy5, attached to the opposite strand at positions n (n = 7, 12, 14, 16, 19, 24, 27), binds in the major groove in two distinct locations on the upper and lower part of the groove. We analyzed in detail the dye-to-dye distances, dipole orientation factors and fluorescence resonance energy transfer (FRET) rates. Tests of the validity of the Förster model were conducted using the transition density cube (TDC) method, which provides the exact Coulombic interaction within a certain model chemistry. Our studies show that the use of long tethers does not guarantee rotational freedom of the dyes, as intended in the experiments. Instead, the tethers allow Cy5 to bind in two different geometries, which causes a large uncertainty in the dye-to-dye distances. Our results also show significant fluctuation in the orientation factor, κ², which, together with uncertainty in dye-to-dye distances, cause considerable uncertainty in interpreting FRET measurements. We suggest that molecular modeling, combined with the TDC method, provides a useful tool in designing and interpreting FRET experiments.     

Single lanthanide-doped oxide nanoparticles as donors in fluorescence resonance energy transfer experiments

We used lanthanide-ion doped oxide nanoparticles, Y(0.6)Eu(0.4)VO(4), as donors in fluorescent resonance energy transfer (FRET) experiments. The choice of these nanoparticles allows us to combine the advantages of the lanthanide-ion emission, in particular the long lifetime and the large Stokes shift between absorption and emission, with the detectability of the nanoparticles at the single-particle level. Using cyanine 5 (Cy5) organic molecules as acceptors, we demonstrated FRET down to the single-nanoparticle level. We showed that, due to the long donor lifetime, unambiguous and precise FRET measurements can be performed in solution even in the presence of large free acceptor concentrations. Highly efficient energy transfer was obtained for a large number of acceptor molecules per donor nanoparticle. We determined FRET efficiencies as a function of Cy5 concentration which are in good agreement with a multiple acceptor-multiple donor calculation. On the basis of the donor emission recovery due to acceptor photobleaching, we demonstrated energy transfer from single-nanoparticle donors in fluorescence microscopy experiments.     

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.     

The photoluminescent graphene oxide serves as an acceptor rather than a donor in the fluorescence resonance energy transfer pair of Cy3.5-graphene oxide

We have systematically studied the fluorescence resonance energy transfer (FRET) efficiency between the photoluminescent graphene oxide (GO) and Cy3.5 dye by controlling the donor-acceptor distance with a double stranded DNA and demonstrated that the GO serves as an acceptor rather than a donor in this FRET system.     

Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors

We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 A and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dye-labeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding F?rster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.     

Towards multi-colour strategies for the detection of oligonucleotide hybridization using quantum dots as energy donors in fluorescence resonance energy transfer (FRET)

The potential for a simultaneous two-colour diagnostic scheme for nucleic acids operating on the basis of fluorescence resonance energy transfer (FRET) has been demonstrated. Upon ultraviolet excitation, two-colours of CdSe/ZnS quantum dots with conjugated oligonucleotide probes act as energy donors yielding FRET-sensitized acceptor emission upon hybridization with fluorophore (Cy3 and Alexa647) labeled target oligonucleotides. Energy transfer efficiencies, Förster distances, changes in quantum yield and lifetime, and signal-to-noise with respect to non-specific adsorption have been investigated. The dynamic range and limit-of-detection are tunable with the concentration of QD-DNA conjugate. The Cy3 and Alexa647 acceptor schemes can detect target from 4 to 100% or 10 to 100% of the QD-DNA conjugate concentration, respectively. Nanomolar limits of detection have been demonstrated in this paper, however, results indicate that picomolar detection limits can be achieved with standard instrumentation. The use of an intercalating dye (ethidium bromide) as an acceptor to alleviate non-specific adsorption is also described and increases signal-to-noise from S/N < 2 to S/N = 9-10. The ethidium bromide system had a dynamic range from 8 to 100% of the QD-DNA conjugate concentration and could detect target in a matrix containing an excess of non-complementary nucleic acid.     

Intramolecular electronic energy transfer via exchange interaction in bichromophoric molecules

A theory is presented for intramolecular electronic energy transfer in bichromophoric molecules. Expressions are given for the donor moiety fluorescence (phosphorescence) decay and for its fluorescence (phosphorescence) quantum yield in terms of the average distance between the donor and acceptor moieties and the donor—acceptor bridge flexibility. Comparison with available experimental data supports the predictions of the analysis.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.     

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.     

Can luminescent quantum dots be efficient energy acceptors with organic dye donors?

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.     

Diheteroarylethenes as thermally stable photoswitchable acceptors in photochromic fluorescence resonance energy transfer (pcFRET)

We have employed diheteroarylethenes as acceptors for photochromic FRET (pcFRET), a technique introduced for the quantitative determination of fluorescence resonance energy transfer (FRET). In pcFRET, the fluorescent emission of the donor is modulated by cyclical transformations of a photochromic acceptor. Light induces a reversible change in the structure and, concomitantly, in the absorption properties of the acceptor. Only the closed forms of the selected diheteroarylethenes 2a and 2b have an absorption band overlapping the emission band of the donor, 1. The corresponding variation in the overlap integral (and thus critical transfer distance R(o)) between the two states provides the means for reversibly switching the process of FRET on and off, allowing direct and repeated evaluation of the relative changes in the donor fluorescence quantum yield. The diheteroarylethenes demonstrate excellent stability in aqueous media, an absence of thermal back reactions, and negligible fatigue. The equilibration of these systems after exposure to near-UV or visible light follows simple monoexponential kinetics. We developed a general conceptual scheme for such coupled photochromic-FRET reactions, allowing quantitative interpretations of the photostationary and kinetic data, from which the quantum yields for the cyclization and cycloreversion reactions of the photochromic acceptor were calculated.     

Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on Förster resonance energy transfer

The blinking kinetics of individual Cy5 fluorophores conjugated to DNA are directly measured using single-molecule spectroscopy. Under deoxygenated aqueous conditions, Cy5 fluorescence exhibits spontaneous and reversible on/off fluctuations with a period lasting seconds. This blinking is observed when directly exciting Cy5 with 640 nm light and by Forster resonance energy transfer (FRET). We find that Cy5 blinking is influenced by the proximity of the donor, the structure of the donor, the presence of 514 nm excitation, and FRET. In the context of single-molecule FRET, blinking of the acceptor produces anticorrelated donor-acceptor intensity fluctuations, which can be difficult to discern from variations in the interdye distance. Slow blinking is, in particular, problematic because it overlaps with biologically relevant time scales. By employing an alternating 514640 nm laser excitation scheme, we show that the dark states can be readily resolved and discriminated from FRET distance fluctuations.     

Influence of quantum dot's quantum yield to chemiluminescent resonance energy transfer

The resonance energy transfer between chemiluminescence donor (luminol-H₂O₂ system) and quantum dots (QDs, emission at 593 nm) acceptors (CRET) was investigated. The resonance energy transfer efficiencies were compared while the oil soluble QDs, water soluble QDs (modified with thioglycolate) and QD-HRP conjugates were used as acceptor. The fluorescence of QD can be observed in the three cases, indicating that the CRET occurs while QD acceptor in different status was used. The highest CRET efficiency (10.7%) was obtained in the case of oil soluble QDs, and the lowest CRET efficiency (2.7%) was observed in the QD-HRP conjugates case. This result is coincident with the quantum yields of the acceptors (18.3% and 0.4%). The same result was observed in another similar set of experiment, in which the amphiphilic polymer modified QDs (emission at 675 nm) were used. It suggests that the quantum yield of the QD in different status is the crucial factor to the CRET efficiency. Furthermore, the multiplexed CRET between luminol donor and three different sizes QD acceptors was observed simultaneously. This work will offer useful support for improving the CRET studies based on quantum dots.     

Nonclassical SNAPFL analogue as a Cy5 resonance energy transfer partner

We have synthesized a new SNAPFL analogue (1) that exhibits a large Stokes shift (78 nm) (abs. 542 nm, em. 620 nm) and a good quantum yield. Because of the large overlap between the emission spectrum of 1 and the absorption spectrum of Cy5, 1 functions well as a fluorescence donor to Cy5 and has been used in FRET-based experiments using estrogen receptor site-specifically labeled with Cy5 and a receptor ligand conjugated to SNAPFL.     

A new method for the detection of ATP using a quantum-dot-tagged aptamer

Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection. Zhang Chen and Guang Li contributed equally to this work.     

Long distance energy transfer in a polymer matrix doped with a perylene dye

Exciton migration over long distances is a key issue for various applications in organic electronics. We investigate a disordered material system which has the potential for long exciton diffusion lengths in combination with a high versatility. The perylene bisimide dye Perylene Red is incorporated in a polymer matrix with a high concentration. The dye molecules represent active sites with a narrow energy distribution for the electronically excited states. Excitons can be efficiently exchanged between them by F?rster resonance energy transfer (FRET). The narrow energy distribution reduces drastically the trapping probability of the excitons compared to polymers and allows for long transfer distances. To characterize the mobility of the excitons and their diffusion length the dye Oxazine 1 is added as an acceptor in low concentration and the transfer probability to the acceptor is determined by measuring the reduction of Perylene Red fluorescence. The quenched quantum yield is measured for dye concentrations varying from 0.05?M to 0.15 M for Perylene Red and from 0.3 mM to 3 mM for Oxazine 1. The experimental results are compared to a model which assumes that excitons can diffuse through the material by FRET between Perylene Red sites and are trapped at an acceptor with a final hetero FRET step. We find a quite good match between theory and experiment though the observed diffusion constant is about two times smaller than the calculated one. The exciton diffusion length extracted from the data is 30 nm for a Perylene Red concentration of 0.1 M and demonstrates that long distance energy transfer is possible in this disordered material system.     

Rate of excitation energy transfer between fluorescent dyes and nanoparticles

Because of the sensitivity of the rate of Coulomb interaction induced long range resonance energy transfer (RET) on the distance between the donor (D) and the acceptor (A) molecules, the technique of FRET (fluorescence resonance energy transfer) is popularly termed as “spectroscopic ruler” and is increasingly being used in many areas of biological and material science. For example, the phenomenon is used to monitor the in vivo separation between different (bio)polymers/units of (bio)polymers and hence the dynamics of various biomolecular processes. In this work, we examine the distance and the orientation dependence of RET in three different systems: (i) between a conjugated polymer and a fluorescent dye, (ii) between a nanometal particle (NMP) and a fluorescent dye and (iii) between two NMP. We show that in all the three cases, the rate of RET follows a distance dependence of d^−σ where exponent σ approaches 6 at large distance d (Förster type dependence) but has a value varying from 3–4 at short to intermediate distances.     

Orientation control of fluorescence resonance energy transfer using DNA as a helical scaffold

The efficiency of fluorescence resonance energy transfer (FRET) between two chromophores positioned at opposite ends of DNA base pair domains has been investigated. The base pair domain serves as a helical scaffold which defines both the distance between chromophores and the dihedral angle between their electronic transition dipole moments, each incremental base pair increasing the distance and stepping the dihedral angle. Fluorescence quantum yields and lifetimes have been determined for both the donor and acceptor chromophores. The experimental data are found to be in excellent accord with an oriented dipole model, rather than with the averaged dipole model conventionally assumed for FRET.     

Thermal profiles of förster energy transfer preliminary studies of luminescent probes of protein dynamics in transferrin and calmodulin

Fluorescence energy transfer, the transfer of energy from a donor to an acceptor via a dipole/induced dipole mechanism, has long been used to measure distances between donors and acceptors in proteins and other macromolecules. Because the transfer can occur over time scales larger than protein bending and breathing modes, multiple conformational states can be sampled. The analysis of these states is weighted by the donor-acceptor distance; shorter distances carry more weight, because the energy transfer depends on the inverse sixth power of the distance. The usefulness of fluorecence energy transfer in probing these large amplitude protein motions is studied here. The method involves measuring the nergy transfer efficiency while perturbing the protein conformation with heat. As the temperature increases, the amplitudes of vibrations increase, and fluorescence energy transfer should also increase if the donor and acceptor are in flexible region of the protein. This hypothesis was tested in two different protein systems; calmodulin, a calcium- activated regulatory protein, and transferrin, a blood serum iron shuttle. The preliminary studies show a differential sensitivity of the transfer efficiency to heat for the systems. Normalized energy transfer over 10 Å in calmodiulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 297 to 322 K. Normalized energy transfer over 42 Å in transferrin from a Tb(III) donor to an Fe(III) acceptor increase 35% over the same temperature range. In marked contrast to these systems, energy transfer from tyrosine to a chelated Tb(III) shows anomalously high temperature- dependence.     

Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1:1 complex formation has been established and the binding constant (K) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (R ₀) for FRET and the Stern-Volmer constant (K _sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, K _SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.