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.     

A fluorimetric method for the determination of nucleic acids using Ce(IV) and sodium triphosphate.

A novel and stable fluorimetric method was established for the determination of nucleic acids. The proposed method is based on the reduction by nucleic acids of Ce(IV) to fluorescent Ce(III). The fluorescence intensity can be greatly increased by sodium triphosphate. The enhanced fluorescence intensity is proportional to the concentration of nucleic acids in the range 4.2 x 10(-8)-4.2 x 10(-6) g/mL for fish sperm DNA and 5.0 x 10(-8)-6.5 x 10(-6) g/mL for yeast RNA, and the detection limits (S/N = 3) are 13.5 ng/mL and 45 ng/mL, respectively. The reaction mechanism of the hydrolytic scission of nucleic acids by Ce(IV) is discussed.     

Synthesis, photophysical properties, and nucleic acid binding of phenanthridinium derivatives based on ethidium

A series of substituted phenanthridine derivatives has been synthesized by converting the amines at the 3- and 8-positions of ethidium bromide into guanidine, pyrrole, urea, and various substituted ureas. The resulting derivatives exhibit unique spectral properties that change upon binding nucleic acids. The compounds were analyzed for their ability to inhibit the HIV-1 Rev-Rev Response Element (RRE) interaction, as well as for their affinity to calf thymus DNA. One derivative (3,8-bis-urea-ethylenediamine-5-ethyl-6-phenylphenanthridinium trifuroracetate) has an enhanced affinity and specificity for HIV-1 RRE as compared to ethidium bromide. These results indicate that the nucleic acid affinity and specificity of an intercalating agent can be tuned by synthetic modification of its exocyclic amines.     

Fiuorimetric assessment of Pseudomonas fluorescens viability after freeze-thawing using ethidium bromide

E.O. PUCHKOV AND A.N. MELKOZERNOV. 1995. The relationship between impairment of the Pseudomonas fluorescens cell envelope's permeability barrier for ethidium cation, the fluorescent moiety of ethidium bromide, and viability after freeze-thawing was investigated. Ethidium fluorescence in the suspension of intact bacteria did not change. Disruption of the bacterial permeability barrier by cetyltrimethylammonium bromide (CTAB) led to ethidium fluorescence increase due to interaction of the fluorochrome with intracellular nucleic acids. In the suspension of freeze-thawed cells, ethidium fluorescence increased and the subsequent treatment by CTAB resulted in further fluorescence increase up to the final level corresponding to that in CTAB-treated intact bacteria. For bacteria exposed to different freeze-thawing regimes, the relative ethidium fluorescence increase closely correlated with the relative number of fluorescing cells revealed microscopically. In the suspension of freeze-thawed cells, the relative additional ethidium fluorescence increase after CTAB treatment closely correlated with viability evaluated by plate counts. It is concluded that the fluorimetric approach may be used as a means of rapidly evaluating bacterial viability after freeze-thawing.     

Binding of quinoline analogues of echinomycin to deoxyribonucleic acid. Role of the chromophores.

Two novel antibiotics were isolated, designated compounds 1QN and 2QN respectively, having quinoline rings in place of one or both of the quinoxaline chromophores of echinomycin. Each removes and reverses the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs. For compound 1QN, the unwinding angle at I0.01 is almost twice that of ethidium, whereas for compound 2QN the value is indistinguishable from that of ethidium. Binding of both analogues produced changes in the viscosity of sonicated rod-like DNA fragments corresponding to double the helix extension found with ethidium, a feature characteristic of bifunctional intercalation by quinoxaline antibiotics. These results suggest that both compounds 1QN and 2QN behave as bifunctional intercalators but that compound 2QN produces only half the helix unwinding seen with compound 1QN and the natural quinoxalines. Binding curves for the interaction of both analogues with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis. Values for compound 2QN were also obtained by a fluorimetric method and found to agree well with the solvent-partition measurements. Compound 1QN bound most tightly to Micrococcus lysodeikticus DNA and, like echinomycin, exhibited a broad preference for (G + C)-rich DNA species. For compound 2QN no marked (G + C) preference was indicated, and the tightest binding among the natural DNA species studied was found with DNA from Escherichia coli. The two analogues also displayed different patterns of specificity in their interaction with synthetic nucleic acids. Compound 2QN bound to poly(dA-dT) slightly more tightly than to poly-(dG-dC), whereas compound 1QN displayed a large (approx. 11-fold) preference in the opposite sense. There was evidence of co-operativity in the binding to poly(dA-dT). It may be concluded that the chromophore moieties play an active role in determining the capacity of quinomycin antibiotics to recognize and bind selectively to specific sequences in DNA.     

Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids.

The non-covalent binding of ethidium monoazide to nucleic acids is entirely analogous to that of ethidium (binding constant approximately 2-3 X 10(5) M). The ethidium monoazide can be photochemically covalently linked to nucleic acids in high yield, up to 75%, by long wavelength light. The fluorescence of ethidium monoazide and ethidium crosslinked to nucleic acids show the same environmental sensitivity as does the fluorescence of ethidium. These properties of ethidium monoazide indicate its use as a fluorescent photoaffinity label for nucleic acids. Ethidium diazide can be photochemically linked to nucleic acids but appears to have properties substantially different from those of ethidium.     

Molecular determinants for DNA minor groove recognition: design of a bis-guanidinium derivative of ethidium that is highly selective for AT-rich DNA sequences

The phenanthridinium dye ethidium bromide is a prototypical DNA intercalating agent. For decades, this anti-trypanosomal agent has been known to intercalate into nucleic acids, with little preference for particular sequences. Only polydA-polydT tracts are relatively refractory to ethidium intercalation. In an effort to tune the sequence selectivity of known DNA binding agents, we report here the synthesis and detailed characterization of the mode of binding to DNA of a novel ethidium derivative possessing two guanidinium groups at positions 3 and 8. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties. The study of the mode of binding to DNA by means of circular and electric linear dichroism revealed that, unlike ethidium, DB950 forms minor groove complexes with AT sequences. Accurate quantification of binding affinities by surface plasmon resonance using A(n)T(n) hairpin oligomer indicated that the interaction of DB950 is over 10-50 times stronger than that of ethidium and comparable to that of the known minor groove binder furamidine. DB950 interacts weakly with GC sites by intercalation. DNase I footprinting experiments performed with different DNA fragments established that DB950 presents a pronounced selectivity for AT-rich sites, identical with that of furamidine. The replacement of the amino groups of ethidium with guanidinium groups has resulted in a marked gain of both affinity and sequence selectivity. DB950 provides protection against DNase I cleavage at AT-containing sites which frequently correspond to regions of enhanced cleavage in the presence of ethidium. Although DB950 maintains a planar phenanthridinium chromophore, the compound no longer intercalates at AT sites. The guanidinium groups of DB950, just like the amidinium group of furamidine (DB75), are the critical determinants for recognition of AT binding sites in DNA. The chemical modulation of the ethidium exocyclic amines is a profitable option to tune the nucleic acid recognition properties of phenylphenanthridinium dyes.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

A continuous kinetic assay for RNA-cleaving deoxyribozymes,exploiting ethidium bromide as an extrinsic fluorescent probe

We describe a rapid and inexpensive method to monitor the kinetics of small RNA-cleaving deoxyribozymes, based on the exogenous fluorophore ethidium bromide. Ethidium binds preferentially to double-stranded nucleic acids, and its fluorescence emission increases dramatically upon intercalation. Thus, ethidium can be used in single-turnover experiments to measure both annealing of the deoxyribozyme to its substrate and release of the products. Under conditions in which dissociation of the product is fast compared with cleavage, the apparent rate of product release reflects the cleavage step. The method was developed for characterizing the so-called 8-17 catalytic DNA, but its general applicability in the deoxyribozyme field was verified using the 10-23 RNA-cleaving construct. Catalysis by both deoxyribozymes was not inhibited in the presence of substoichiometric amounts of ethidium, and the rates obtained through the ethidium assay were virtually identical to the rates determined using radiolabeled substrates. In contrast, the assay cannot be applied to the large, structured ribozymes, and its use to study the kinetics of the small hammerhead ribozyme was hampered by the presence on the catalyst of at least one high-affinity ethidium binding site.     

A fiber optic biosensor for fluorimetric detection of triple-helical DNA.

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.     

The EcoRI restriction endonuclease, covalently closed DNA and ethidium bromide.

The reactions of the EcoRI restriction endonuclease on the covalently closed DNA of plasmid pMB9 were studied in the presence of ethidium bromide. At the concentrations of ethidium bromide tested, which covered the range over which the DNA is changed from negatively to positively supercoiled, the dye caused no alteration to the rate at which this enzyme cleaved the covalently closed DNA to yield the open-circle form, but the rate at which these open circles were cleaved to the linear product could be inhibited. The fluorescence change, caused by ethidium bromide binding with different stoichiometries to covalently closed and open-circle DNA, provided a direct and sensitive signal for monitoring the cleavage of DNA by this enzyme. This method was used for a steady-state kinetic analysis of the reaction catalysed by the EcoRI restriction enzyme. Reaction mechanisms where a complex between DNA and Mg2+ is the substrate for this enzyme were eliminated, and instead DNA and Mg2+ must bind to the enzyme in separate stages. The requisite controls for this fluorimetric assay in both steady-state and transient kinetics studies, and its application to other enzymes that alter the structure of covalently closed DNA, are described.     

Rayleigh light scattering study on the reaction of nucleic acids and methyl violet

A new quantitative determination method for nucleic acids in aqueous solutions, based on the enhancement of Rayleigh light scattering of methyl violet by nucleic acids, has been developed. The sensitivity of the assay allows amounts of nucleic acids as little as 100 ng/ml to be quantitated reliably. In addition to its high sensitivity, this method has other advantages: rapidity of reaction (<5 min), simplicity of operation (one-step assay), commonality of spectrofluorimeter and reagents, stability of mixtures formed, and reproducibility. Under the experimental conditions, there is little or no interference from proteins, nucleosides, and most metal ions. Interference by a few metal ions, detergents, and some salts can be minimized by dilution. The method can also be used to determine the total amount of nucleic acids without the arduous choice of standard and difficult separation of DNA and RNA.     

Selective staining by 4'', 6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels.

4', 6-Diamidine-2-phenylindole.2HCl (DAPI) forms fluorescent complexes with double-stranded (ds) DNA but not with ds RNA as shown by fluorescence titration. The widely used dye ethidium bromide (EB) forms fluorescent complexes with both types of nucleic acids. Also, in contrast to EB, DAPI forms much weaker fluorescent complexes with single-stranded DNA than with ds DNA. These observations were utilized to develop staining procedures for the selective visualization of ds DNA on gels. The use of DAPI in addition to EB for staining makes possible the localization of ds DNA and other species of nucleic acids on a single gel.     

Nucleotide sequence of genes coding for dicyclohexylcarbodiimide-binding protein and the alpha subunit of proton-translocating ATPase of Escherichia coli

A liquid membrane electrode has been made which is selective for ethidium ion. The membrane is formed in a capillary by a 3-nitro-o-xylene solution of an ethidium-tetraphenyl borate complex. The electrode emf (vs saturated KCl-calomel reference) has a linear dependence upon the logarithm of ethidium concentration from 2 μM to 0.5 mM. The electrode is used here to measure free ethidium ion in mixtures with calf thymus DNA. The binding isotherms obtained are in general agreement with a control photometric titration and with literature results. Direct measurement of free ethidium concentration by convenient potentiometric methods is useful in the study of ligand binding to nucleic acids and to related compounds.     

Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures

A simple method for the detection of sequence- and structural-selective ligand binding to nucleic acids is described. The method is based on the commonly used thermal denaturation method in which ligand binding is registered as an elevation in the nucleic acid melting temperature (T_m). The method can be extended to yield a new, higher -throughput, assay by the simple expediency of melting designed mixtures of polynucleotides (or oligonucleotides) with different sequences or structures of interest. Upon addition of ligand to such mixtures at low molar ratios, the T_m is shifted only for the nucleic acid containing the preferred sequence or structure. Proof of principle of the assay is provided using first a mixture of polynucleotides with different sequences and, second, with a mixture containing DNA, RNA and two types of DNA:RNA hybrid structures. Netropsin, ethidium, daunorubicin and actinomycin, ligands with known sequence preferences, were used to illustrate the method. The applicability of the approach to oligonucleotide systems is illustrated by the use of simple ternary and binary mixtures of defined sequence deoxyoligonucleotides challenged by the bisanthracycline WP631. The simple mixtures described here provide proof of principle of the assay and pave the way for the development of more sophisticated mixtures for rapidly screening the selectivity of new nucleic acid binding compounds.     

Association of endonuclease activity with serotypes belonging to the three subgroups of human adenoviruses.

Endonuclease activity has been detected in association with highly purified virions, pentons, and/or dodecons of adenovirus types 2, 3, 5, 9, 12, 15, and 16. Only single-strand scissions in substrate DNA were detected. The nuclease activity was detected by a highly sensitive ethidium bromide fluorimetric assay procedure.     

Fluorescence-detected circular dichroism of ethidium in vivo and bound to deoxyribonucleic acid in vitro

Fluorescence-detected circular dichroism (FDCD) spectra are reported for ethidium in Escherichia coli cells and bound to E. coli DNA in vitro. FDCD bands are observed at 325 and 385 nm. These bands change amplitude as the ethidium to DNA ratio changes. Spectra are similar for in vivo and in vitro measurements. However, the bands at 325 and 385 nm disappear when ethidium binds to macromolecules without intercalating between base pairs. The results demonstrate that FDCD spectra can be measured in cell suspensions and indicate that ethidium binds to nucleic acids in E. coli cells by intercalation.     

The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium bromide

Eilatin-containing ruthenium complexes bind to a broad range of different nucleic acids including: calf thymus (CT) DNA, tRNA(Phe), polymeric RNAs and DNAs, and viral RNAs including the HIV-1 RRE and TAR. The nucleic acid specificity of Lambda- and Delta-[Ru(bpy)2eilatin]2+ have been compared to that of the 'free' eilatin ligand, and to the classic intercalating agent ethidium bromide. Interestingly, all four compounds appear to bind to nucleic acids by intercalation, but the trends in nucleic acid binding specificity are highly diverse. Unlike ethidium bromide, both eilatin and the eilatin-containing coordination complexes bind to certain single-stranded RNAs with high affinity (K(d) < or = 1 microM). Eilatin itself is selective for electron-poor polymeric purines, while the eilatin-coordination complexes exhibit preference for the polypyrimidine r(U). These results show how the binding specificity of an intercalating ligand can change upon its incorporation into an octahedral metal complex.     

Assessment of the in vitro growth of Plasmodium falciparum using fluorometry

The growth of Plasmodium falciparum in vitro was quantitatively assessed by applying fluorometry using ethidium bromide. The fluorescence intensity of parasites stained with this dye was found to parallel the uptake of 3H-hypoxanthine into nucleic acids during one growth cycle of development. The assay system can be used as a substitute of morphological and radiometric methods in drug-sensitivity tests and for the screening of antimalarials.     

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.