Differences in the red fluorescence of acridine orange bound to single-stranded RNA and DNA.

Fluorescence of acridine orange bound to RNA or DNA in the single-stranded form including single-stranded synthetic polyribo- or polydeoxyribonucleotides was measured in the expectation that some distinct structural characteristic between single-stranded RNA and DNA might be reflected by a specific fluorescent behaviour of bound dyes. It was found that the complex of the dye with single-stranded RNA emits a weaker red fluorescence around 650 nm than the complex with single-stranded DNA at low phosphate-to-dye ratios. The fact could be explained neither by a direct interaction of bound dyes with the 2′-hydroxyl group of ribose in RNA nor by the difference in the G-C content of the nucleic acids. On the basis of the character of dye molecules emitting the red fluorescence, it was suggested that the bases in single-stranded RNA might be buried in some hydrophobic environment that would make the dyes less likely to interact with them, compared with the bases in single-stranded DNA. It was further inferred that some conformational rigidity of single-stranded RNA may partially be responsible for the weaker red fluorescence.     

The metachromasia of fluorescence of acridine dyes

Summary 1. Fluorescence- and absorption spectra of a number of acridine dyes were measured at several concentrations, in different solvents and at various pH and temperatures.2. In aqueous solutions metachromatic shifts are visible with all dyes — except acridine-yellow — with increasing concentrations, even when the dye is present as di- or trivalent ions in strongly acid solutions.3. Under conditions where reabsorption of the fluorescent light is excluded: completely separated fluorescence and absorption spectra, or measurement of fluorescence in capillaries with 0.8 mm internal bore, metachromatic effects are absent.4. Reasons are given to consider the hypothesis of specific aggregation (formation of dimeric dye-particles) as doubtful. In the case of the acridine dyes the optical properties of the monomeric dye ions are sufficient to explain the metachromatic shifts.With 10 Figures in the Text     

Polarized fluorescence resonance energy transfer microscopy

Current methods for fluorescence resonance energy transfer (FRET) microscopy of living cells involve taking a series of images with alternating excitation colors in separate camera exposures. Here we present a new FRET method based on polarization that requires only one camera exposure and thereby offers the possibility for better time resolution of dynamic associations among subcellular components. Polarized FRET (p-FRET) uses a simultaneous combination of excitation wavelengths from two orthogonally polarized sources, along with an emission channel tri-image splitter outfitted with appropriate polarizers, to concurrently excite and collect fluorescence from free donors, free acceptors, and FRET pairs. Based upon the throughput in each emission channel as premeasured on pure samples of each of the three species, decoupling of an unknown sample's three polarized fluorescence images can be performed to calculate the pixel-by-pixel concentrations of donor, acceptor, and FRET pairs. The theory of this approach is presented here, and its feasibility is experimentally confirmed by measurements on mixtures of cyan fluorescent protein (CFP), citrine ((Cit) a yellow fluorescent protein variant), and linked fusion proteins (CFP-L16-Cit, CFP-L7-Cit, CFP-L54-Cit) in living cells. The effects of shot noise, acceptor polarization, and FRET efficiency on the statistical accuracy of p-FRET experimental results are investigated by a noise-simulation program.     

A phenomenon of photoenhancement of acridine orange-induced fluorescence.

It is a common experience that, with exposure to exciting radiation of the fluorescence microscope, the acridine orange-induced red fluorescence of the nucleus, produced by Feulgen hydrolysis, fades with a concomitant shift to green. The present investigation reports a phenomenon of photoenhancement observed in the hydrolyzed cytoplasm where pale green fluorescence increases in intensity with exposure to exciting radiation. The phenomenon has been noticed in Rhizobium, Oscillatoria, tomato root tip cells and human buccal epithelial cells. It is tentatively concluded that the gain in fluorescence yield is due to certain conformational changes of the acridine orange-protein complex induced by ultraviolet light flux.     

Polarized fluorescence measurements on ordered photosynthetic antenna complexes: Chlorosomes of Chloroflexus aurantiacus and B800-B850 antenna complexes of Rhodobacter sphaeroides

We have used a new and relatively easy approach to study the pigment-organization in chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus and in B800-850 antenna complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides. These particles were embedded in compressed and uncompressed gels and the polarized fluorescence was determined in a 90° setup. Assuming both a rotational symmetric distribution of the particles in the gel and of the transition dipole moments in the particles, the order parameters <P₂> and <P₄>, describing the orientation of the symmetry axis of the particles with respect to the direction of gel expansion can be determined. Moreover, the direction parameters, describing the orientation of the absorption and emission dipole moments with respect to the symmetry axis of the particles can be obtained.

The value of <P₂> is essential for quantitative interpretation of linear dichroism measurements and usually it is estimated from theoretical approaches, which may lead to incorrect results. For the rod-like chlorosomes the value of <P₂> appears to be the same as predicted by the theoretical approach of Ganago, A. O., M. V. Fok, I. A. Abdourakhmanov, A. A. Solov'ev, and Yu. E. Erokhin (1980. Mol. Biol. [Mosc.]. 14:381-389). The agreement with linear dichroism results, analyzed with this theoretical approach shows that the transition dipole moments are indeed in good approximation distributed in a rotationally symmetric way around the long axis of the chlorosomes. Moreover, it appears those BChl c molecules, which fluoresce, are oriented in the same way with respect to the symmetry axis as the rest of these pigments, with the dipole moments close to parallel to the long axis.

The B800-850 complexes appear to orient like discs, whereas the transition dipoles of the BChl a 800- and 850-nm bands are oriented almost perpendicular to the symmetry axis. These findings are in agreement with the minimal model for these complexes proposed by Kramer, H. J. M., R. van Grondelle, C. N. Hunter, W. H. J. Westerhuis, and J. Amesz (1984. Biochim. Biophys. Acta. 156-165).

The amount of orientation of the particles appears to vary for different gels and it is lower than predicted by the theory of Ganago et al., showing that application of their approach for these particles leads to incorrect interpretations.

The approach that is used in this study allows determination of orientations of those dipole moments, which transfer their excitation energy to the fluorescing species, in contrast to linear dichroism measurements, where the orientations of all absorbing dipole moments are studied. For the polarized fluorescence measurements, the amount of orientation of the particles is determined experimentally, whereas for linear dichroism this amount has to be estimated from theoretical models. The value of <P₂> that can be determined from the fluorescence measurements can, however, also be used for a quantitative interpretation of the linear dichroism results.

     

Polarized fluorescence studies of electrically oriented DNA-dye solutions

Transient changes have been recorded in each of the four polarized components of fluorescence, when dilute solutions of dye-tagged DNA are subjected to short electric pulses. The directions of the absorption and emission transition moments, and hence of the plane of the dye molecules, relative to the DNA geometry have been estimated for eleven dyes. Data obtained for ethidium bromide and five acridine derivatives are consistent with the intercalation model generally accepted for these dyes. In addition, it is shown that neutral red, acridine red and probably auramine O also bind with their molecular planes essentially perpendicular to the long helical axis. The remaining two, hydroxystilbamidine and the bibenzimidazole derivative Hoechst 33258, give rise to effects which indicate that these molecules bind in such a manner that the absorption and emission transitions are closely associated with the grooves of the DNA helix.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Analysis of glycosaminoglycans in urine by using acridine orange fluorescence.

The fluorescence technique described here utilizes the electrostatic interaction between the polyanionic sites of glycosaminoglycans and the cationic dye Acridine Orange to analyse urinary glycosaminoglycans from patients suffering from mucopolysaccharidoses. The basis of the titration is the decrease in the fluorescence of free Acridine Orange that occurs when it is bound to polyanions. The effect of the presence of possible interfering materials such as salt, proteins and trace materials in urine was evaluated. This fluorescence technique is technically simple.     

Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA

Interactions of several acridine dyes with DNA from different species were studied by measuring fluorescence lifetimes in the 2–30-nsec range, using the single-photon counting technique, and by measuring fluorescence quantum yields in the steady state. The results confirm the existence of two principal site classes, one in which the dye fluorescence is quenched by interaction with guanine and another in which fluorescence results from the hydrophobic environment of the A·T base pairs. The emitting sites are found, in some cases, to exhibit fluorescent decay curves which can be resolved into two exponential components corresponding to a short and to a long lifetime. The deviation from one exponential component is particularly clear with rivanol, 9-aminoacridine, and quinacrine, with which one component is two or three times longer than the other. The relative proportion of these two components depends only slightly on the DNA base composition and does not depend on the nature of the acridine derivatives. We postulate that this lifetime heterogeneity corresponds to the two discrete steps in the complex formation elucidated by kinetic studies: the first step corresponds to a semi-intercalated, or “external,” dye with a short fluorescence lifetime and the second step corresponds to a totally intercalated dye with a long lifetime. In this model, we assumed that a transient opening of the site near a semi-intercalated dye induces solvent diffusion which in turn is responsible for its short-lived fluorescence.