Jozef Kusba

Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles

We examined the effects of metallic silver particles on resonance energy transfer (RET) between fluorophores covalently bound to DNA. A coumarin donor and a Cy3 acceptor were positioned at opposite ends of a 23-bp double helical DNA oligomer. In the absence of silver particles the extent of RET is near 9%, consistent with a Forster distance R(0) near 50 A and a donor to acceptor distance near 75 A. The transfer efficiency increased when the solution of AMCA-DNA-Cy3 was placed between two quartz plates coated with silver island films to near 64%, as determined by both steady-state and time-resolved measurements. The apparent R(0) in the presence of silver island films increases to about 110 A. These values of the transfer efficiency and R(0) represent weighted averages for donor-acceptor pairs near and distant from the metallic surfaces, so that the values at an optimal distance are likely to be larger. The increased energy transfer is observed only between two sandwiched silvered slides. When we replaced one silvered slide with a quartz plate the effect vanished. Also, the increased energy transfer was not observed for silvered slides separated more than a few micrometers. These results suggest the use of metal-enhanced RET in PCR, hybridization, and other DNA assays, and the possibility of controlling energy transfer by the distance between silver surfaces.

Photostability of Cy3 and Cy5-Labeled DNA in the Presence of Metallic Silver Particles

We examined the photostability of a double-stranded DNA oligomer, covalently labeled with Cy3 or Cy5 on one strand, in the presence of metallic silver island films. In our experimental configuration a minor fraction of the labeled DNA was close to the silver particles and the remainder was distant from the particles. Proximity of the fluorophores to silver island films resulted in increased intensity. Upon continuous illumination we found a fraction of the emission that was resistant to the photobleaching. The emission spectra of the residual fractions were identical to the initial spectra. The frequency-domain lifetime measurements of this fraction revealed greatly shortened decay times. These results are consistent with the photostable fraction being close to the silver particles. This results suggest that the number of photons detected per fluorophore, prior to photobleaching, can be increased 5-fold or more by proximity to silver particles. Localization at an optimal distance from the silver surface may result in larger enhancements.

Effects of metallic silver particles on resonance energy transfer in labeled bovine serum albumin

Resonance energy transfer (RET) is widely used to detect proximity between biomolecules. In transparent solution the maximum donor-to-acceptor distance for RET is about 70 A. We measured the effects of metallic silver island films on RET from the intrinsic tryptophan of a protein to a bound probe as the acceptor. These preliminary experiments revealed a dramatic increase in the apparent Förster distance increasing from 28.6 to 63 A. These results suggest the use of silver island films for detecting long range proximity between biomolecules and for biotechnology applications based on RET.

Effects of Light Quenching on the Emission Spectra and Intensity Decays of Fluorophore Mixtures

We examined a series of fluorophore mixtures to determine the wavelength selectivity of light quenching and the effects of light quenching on the emission spectra and intensity decays. Light quenching can be accomplished using a single excitation pulse train and quenching wavelength (one-beam) or with longer-wavelength quenching pulses time-delayed relative to the excitation pulses (two-beam). Both one-beam and two-beam light quenching were found to alter the intensity decays of the mixtures. The frequency-domain intensity decay data were analyzed to reveal the fractional intensity of each fluorophore in the mixture and the effects of light quenching on the fractional contribution of each fluorophore to the total intensity. Fluorophores were selected to provide a range of decay times and emission wavelengths. The extent of quenching in the mixtures was dependent on which fluorophore had the higher radiative decay rate and emission intensity at the quenching wavelength. A general theory is presented which describes the intensity decays in terms of the extent of quenching of each fluorophore and the time delay between excitation and quenching pulses. The effects of light quenching on the fractional intensities of each fluorophore in the mixture, recovered from the intensity decay analysis, were found to be in quantitative agreement with that predicted from steady-state measurements of light quenching and from the spectral properties of the fluorophores. The data on light quenching of mixtures demonstrate that light quenching may be used for selective quenching of fluorophores and thus of potential value for studies of multichromophoric systems.

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10−6, 0.44×10−6, and 4.3×10−6 cm2/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.

Wavelength-selective light quenching of biochemical fluorophores

The use of light quenching to selectively eliminate the emission of biochemical fluorophores based on the emission wavelength is described. To demonstrate the possibility of wavelength-selective light quenching, a mixture of two fluorophores, 4-(dimethylamino)-48-cyanostillene (DCS) and Prodan, emitting at different wavelengths was examined first. The emission spectrum and intensity decay were altered by the 570-nm quenching pulse due to selective quenching of the longer wavelength emission of DCS. Quenching of the solvent-sensitive fluorophore partially bound to human serum albumin and partially in the aqueous phase was then examined. Light quenching with a long wavelength (570 nm) time-delayed pulse selectively quenched the Prodan fluorophore in the aqueous phase while in the presence of the Prodan bound to human serum albumin, which emitted at shorter wavelengths, was not quenched. Using one-beam short wavelength excitation and quenching, a selective quenching of the blue-shifted emission of ribonuclease T1 in the presence of the red-shifted emission of the tryptophan residue in adrenocorticotropic hormone was observed. In both systems wavelength-selective light quenching was demonstrated by a shift in the emission spectra, and by changes in the intensity decay consistent with preferential quenching of one species. Light quenching is instantly reversible by blocking or defocusing of the quenching beam. It can occur for inaccessible residues or in viscous solvents, and thus can be of wide applicability for resolving the complex emission of biological macromolecules.

Effect of Molecular Ordering On Distance Distributions of Flexible Donor-Acceptor Pairs

Measurements of time-resolved intramolecular energy transfer in progressively stretched poly(vinyl alcohol) films were performed. The donor (tryptophan) and acceptor (dansyl) were linked with flexible polymethylene chain. Distance distributions were recovered from frequency-domain measurements of the donor decay. In isotropic PVA (in solution) a wide range of distances were detected (Gaussian full width at half maximum of about 16 angstroms) with an average distance of 13 angstroms. The donor-acceptor distance distribution became progressively more narrow when the PVA films were stretched. Four-fold or more stretching results in a single donor-acceptor distance. The maximal measured donor-acceptor distance of 23 angstroms is in excellent agreement with the computed distance for the fully stretched conformation. The possibility of partial and/or full ordering of polymethylene chains by stretching the PVA films can also be useful in the study of other distance-dependent interactions, such as electron transfer or distance-dependent quenching.

Site-to-site diffusion in proteins as observed by energy transfer and frequency-domain fluorometry

We report measurements of site-to-site diffusion in proteins, using frequency-domain measurements of time-dependent energy transfer. The possibility of such measurements is shown from simulations which demonstrate that donor-to-acceptor (D-to-A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of distances. For decay times typical of tryptophan fluorescence, the simulations indicate D-to-A diffusion coefficients can be measured ranging from 10-7 to 10-5 cm2/s. This possibility was verified by studies of a methylene-chain linked D-A pairs in solutions of varying viscosity. D-to-A diffusion was also measured for acceptor-labeled melittin in the random coil and (alpha) -helical states. Unfolding of troponin I results in increased D-A diffusion. Surprisingly, more rapid diffusion was observed for melittin in the (alpha) -helical state, but over a limited range of distances.

Dimensionality of energy transfer between intercalated and surface-bound donor-acceptor pairs in DNA

The energy transfer among several fluorophores when bound to linear DNA has been studied. The intercalation and groove binding of the fluorophores and relatively large persistent length of DNA makes it a good model for one dimensional energy transfer. In this case, as predicted by Foersters theory, the donor intensity decayed with a t1/6 time dependence. The presence of a finite volume with a restricted geometry leads to significantly different donor intensity decays from that predicted by Foersters model. We used intercalated/groove-bound fluorophores as donors and transition metal ion complexes which only bind on the outside surface of the DNA as acceptors, to characterize energy transfer in a cylindrical geometry. Two models were used: a hard cylinder with a donor on the z-axis and acceptors on the surface, and a soft-boundary cylinder where a distribution of acceptors within a cylindrical volume was allowed. The energy transfer among intercalated/groove-bound donors and surface-bound acceptors in DNA can be described with soft-boundary cylindrical geometry with reasonable parameters.

Detection of a distance-dependent rate of quenching by frequency-domain fluorometry

The effect of the collisional quenching on the fluorescence intensity decays has been studied by frequency-domain fluorometry. We used an efficient (CBr4) and/or inefficient (CCl4 quencher to quench the fluorescence of 1,2-benzanthracene (1,2-BA). The wide range of diffusion has been obtained by using propylene glycol at different temperatures (-40 degree(s)C to 40 degree(s)C). The measured intensity decays cannot be satisfactorily fitted either to the Smoluchowski or Collins-Kimball (RBC) model, except the case of inefficient quencher in the presence of high diffusion. In particular, we observed quenching in diffusionless conditions (-40 degree(s)C). To describe the collisional quenching of the fluorescence more correctly we propose a new model which includes a distance-dependent quenching rate (DDQ model). The DDQ simulations show that the local concentration of quencher surrounding the excited fluorophore cannot be approximated by using the RBC model, except in the case of high diffusion and low quenching rate. The DDQ model describes well all measured intensity decays of 1,2-benzanthracene in the presence of CBr4 and/or CCl4. Also, the DDQ model more correctly predicts the curvature of Stern-Volmer plots and activation energies obtained from the temperature dependent rate of quenching.