Recent advances in FRET: distance determination in protein-DNA complexes

Fluorescence resonance energy transfer (FRET) provides information on the distance between a donor and an acceptor dye in the range 10 to 100 A. Knowledge of the exact positions of some dyes with respect to nucleic acids now enables us to translate these data into precise structural information using molecular modeling. Advances in the preparation of dye-labeled nucleic acid molecules and in new techniques, such as the measurement of FRET in polyacrylamide gels or in vivo, will lead to an increasingly important role of FRET in structural and molecular biology.     

The improved method in agarose gel electrophoresis of nucleic acid

In most molecular experiments, nucleic acids are subjected to agarose gel electrophoresis to determine the size of the molecule. The addition of a nucleic acid dye allows the nucleic acid to be detected under the UV image system after running the gel, so the nucleic acid dye is an integral part of the electrophoresis experiment. But when considering the mutagenicity and toxicity of nucleic acid dyes, one must be careful to insure the proper disposal of experimental waste. In this article, a new usage of nucleic acid dye in agarose gel electrophoresis is described where the nucleic acid dyes were added to the loading buffer and nucleic acid marker buffer. The results show that this method has advantages as: a smaller amount of dye can be used, there is less time in contact with the dye, and its operation is easier and reduces toxicity damage. Also the bands showed a much clearer image, having a lower background value. The improved method shows better results with lower toxicity and is superior to the traditional method.     

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.     

Polyvinylpyrrolidone-Agarose Gel Electrophoresis Purification of Polymerase Chain Reaction-Amplifiable DNA from Soils

This communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase chain reaction-amplifiable DNA from soil. This modification is to add polyvinylpyrrolidone to the agarose gel. The polyvinylpyrrolidone addition retards the electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids.     

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.     

Reagents specific for cell surface components.

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.     

Strategy for efficient site-specific FRET-dye labeling of ubiquitin

To study conformational changes within a single protein molecule, sp-FRET (single pair fluorescence resonance energy transfer) is an important technique to provide distance information. However, incorporating donor and acceptor dyes into the same protein molecule is not an easy task. Here, we report a strategy for the efficient double-labeling of a protein on a solid support. An ubiquitin mutant with two Cys mutations, one with high solvent accessibility and the other with low solvent accessibility, was constructed. The protein was bound to magnetic beads and reacted with the dyes. The first dye reacted with the side-chain of the Cys with the high solvent accessibility and the second with the other Cys under partially denaturing conditions. Using this method, we can easily label two dyes in a site-specific way on ubiquitin with a satisfied yield. The labeling sites for donor and acceptor dyes can be easily swapped.     

Native and activated properdin: interconvertibility and identity of amino- and carboxy-terminal sequences

The development of a two-step purification procedure of native properdin with good yield has allowed the physical and chemical comparison of native and activated properdin. The two forms of properdin have identical electrophoretic mobility, subunit size, and amino- as well as carboxyl-terminal amino acid sequences. The two forms of properdin can be interconverted by using mild denaturing agents, indicating that the change in biologic activity is conformational. Circular dichroism analysis of properdin reveals a significant variability in the tertiary structure. However, the differences are a result of the method of purification and do not correspond to the biologic activity of the protein, because the spectra of the interconverted forms of properdin do not change. This indicates that the conformational transition that causes biologic activity changes is small, relative to the conformational variations produced by other conditions that do not alter the biologic activity.     

Denaturation of Nucleic Acids Induced by Intercalating Agents. Biochemical and Biophysical Properties of Acridine Orange-DNA Complexes

Abstract

At high binding denstities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar-and base-composition) of nucleic acids.     

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.     

New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA-specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X-100. Dye VL772 exhibits yellow fluorescence and little DNA-RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue-fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.     

Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography

SYBR Green 1 is an asymmetrical cyanine DNA-binding dye that provides an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with gel electrophoresis. In this paper, we summarize the general properties and specific uses of SYBR green 1 in ion-pair reversed-phase denaturing high-performance liquid chromatography (IP DHPLC). We describe several applications for the WAVE DHPLC platform that illustrate the generic potential of such intercalating dyes in mutation detection and gene expression profiling. We show that SYBR Green 1 obviates the need to use end-labeled oligodeoxynucleotides for the sensitive detection of nucleic acids during chromatography. Moreover the incorporation of SYBR Green 1 into samples and elution buffers does not impair resolution and has no significant effect on the retention times of DNA fragments compared with dye-free DHPLC.     

Hydration of biological molecules: lipids versus nucleic acids

We used FTIR spectroscopy to comparatively study the hydration of films prepared from nucleic acids (DNA and double-stranded RNA) and lipids (phosphatidylcholines and phosphatidylethanolamines chosen as the most abundant ones) at room temperature by varying the ambient relative humidity in terms of solvent-induced structural changes. The nucleic acids and phospholipids both display examples of polymorphism on the one hand and structural conservatism on the other; even closely related representatives behave differently in this respect. DNA undergoes a hydration-driven A-B conformational transition, but RNA maintains an A-like structure independently of the water activity. Similarly, a main transition between the solid and liquid-crystalline phases can be induced lyotropically in certain phosphatidylcholines, while their phosphatidylethanolamine counterparts do not exhibit chain melting under the same conditions. A principal difference concerning the structural changes that occur in the studied biomolecules is given by the relevant water-substrate stoichiometries. These are rather high in DNA and often low in phospholipids, suggesting different mechanisms of action of the hydration water that appears to induce structural changes on global- and local-mode levels, respectively.     

FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.     

The Mechanism of Staining Explained on A Chemical Basis. I. The Reaction Between Dyes,Proteins and Nucleic Acid

Basic dyes cause an increase in hydrogen-ion concentration when added to a solution containing nucleic acid, the both solutions were originally at the same pH. Acid dyes have no effect on nucleic acid solutions. Basic dyes show the same behavior when treated with solutions of typical proteins. Acid dyes when treated with proteins show an analogous effect but in the opposite direction. The only adequate explanation found is that there is a definite reaction between the dye ions and the oppositely charged ions of protein or nucleic acid. The bearing of these results on the theory of staining is discussed. The growing recognition of the dominance of chemical forces in colloidal adsorption behavior is indicated, and certain of the experimental bases for this recognition are pointed out and discussed.     

The Mechanism of Staining Explained on A Chemical Basis. I. The Reaction Between Dyes, Proteins and Nucleic Acid

Basic dyes cause an increase in hydrogen-ion concentration when added to a solution containing nucleic acid, the both solutions were originally at the same pH. Acid dyes have no effect on nucleic acid solutions. Basic dyes show the same behavior when treated with solutions of typical proteins. Acid dyes when treated with proteins show an analogous effect but in the opposite direction. The only adequate explanation found is that there is a definite reaction between the dye ions and the oppositely charged ions of protein or nucleic acid. The bearing of these results on the theory of staining is discussed. The growing recognition of the dominance of chemical forces in colloidal adsorption behavior is indicated, and certain of the experimental bases for this recognition are pointed out and discussed.     

Gallocyanin-Chrome Alum: I. Technique and Specificity

Certain technical aspects of gallocyanin-chrome alum were examined relative to its supposed specificity for nucleic acids. Five different lake formulae were prepared using four different batches of gallocyanin. Spectrophotometric curves were made of each lake and of each dye in a simple water solution. Paraffin sections 6-8 μ thick of spinal cords from albino rats and from cats fixed in CaCl₂-formalin or plain formalin were stained 10 min to 48 hr with gallocyanin lakes made with chrome alum, ferric alum, strontium chloride and copper nitrate. Similar sections were treated with ribonuclease or perochloric acid and stained in the same manner. The spectrophotometric data indicates considerable variation in dye content between different batches and different lakes. Chrome alum was the best of the 4 mordants and a 12-15 hr staining time with Einarson's 1932 preparation was optimal. Neither perchloric acid nor ribonuclease destroyed cytoplasmic basophilia as revealed by gallocyaninchrome alum. Staining was more intense after CaCl₂-formalin fixation than after plain formalin. Variation of the dye content in the different batches of dyes, the poorly understood role of boiling in preparing the lakes, and the inability of ribonuclease or perchloric acid to destroy cytoplasmic basophilia indicates that we are not dealing with a histochemically specific reagent for nucleic acid, but only a desirable nuclear stain.     

The role of water structure in conformational changes of nucleic acids in ambient and high-pressure conditions.

This review describes and summarizes data on the structure and properties of water under normal conditions, at high salt concentration and under high pressure. We correlate the observed conformational changes in nucleic acids with changes in water structure and activity, and suggest a mechanism of conformational transitions of nucleic acids which accounts for changes in the water structure. From the biophysical, biochemical and crystallographic data we conclude that the Z-DNA form can be induced only at low water activity produced by high salt concentrations or high pressure, and accompanied by the stabilizing conjugative effect of the cytidine O4' electrons of the CG base pairs.     

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.