Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.     

Polarization of fluorescence of an oriented solution of rodlike particles bearing a fluorescent label

The variation of the polarized components of fluorescence of a rodlike particle bearing a fluorescent label upon partial orientation is calculated for some special geometry of the dye macromolecules complexes. Explicit expressions are given for the case where the energy of the molecule in the field depends only on one angle θ, showing that the result is a function of both 〈sin²θ〉 and 〈sin⁴θ〉. For the case of orientation in an electric field through an anisotropic induced moment, the expressions allow the calculation of this anisotropy of polarizability. The method is applied to the measurement of the polarizability of rodlike fragments of DNA labeled by intercalated molecules of Acridine Orange.     

Left-Handed Intercalated DNA Double Helix: Rendezvous of Ethidium and Actinomycin D in the Z-Helical Conformation Space

Abstract

It is now very well recognized that the DNA double helix is conformationally pluralistic and that this flexibility is derived from internal motions due to backbone torsions. But what is less apparent is that such internal motions can occur in a correlated fashion and express themselves in a wide variety of structural motifs and phenomena. For example, flexibility inherent in the DNA molecule can lead to a family of Z-DNA, LZ1 and LZ2 being the two extremes and correlated internal motion can cause LZ1?LZ2 transition. More interestingly, such motions manifest themselves as breathing modes on the DNA lattice resulting in the sequence specific intercalation sites. Following a detailed stereochemical analyses we observed that the intercalation site for ethidium is located at the dCpdG sequence of the intercalated LZ1 helix (LZ1*) while that for actinomycin D is located at the dGpdC sequence of the intercalated LZ2 helix (LZ2*). From the stereochemistry of the drug binding we make experimentally testable predictions which are in fact supported by a few recent experimental studies. These studies also show that a left-handed intercalated B-DNA model is a viable intermediate in the Z to B transition which can hold the drug with binding energy comparable to that of the intercalated right-handed B-DNA.     

Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO.

Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO), its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been investigated as a function of binding ratio. On visible illumination, both YO and YOYO cause single-strand cleavage, with an efficiency that varies with the dye/DNA binding ratio in a manner which can be rationalized in terms of free dye being an inefficient photocleavage reagent and externally bound dye being more efficient than intercalated dye. Moreover, the photocleavage mechanism changes with binding mode. Photocleavage by externally bound dye is, at least partly, oxygen dependent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate. Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibited by moderate concentrations of beta- mercaptoethanol--direct attack on the phosphoribose backbone is a possible mechanism. TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand breaks (ssb) are observed to accumulate to eventually produce double-strand cleavage. With intercalated YO the accumulation occurs in a manner consistent with random generation of strand lesions, while with bisintercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fold higher. A contributing factor is the slow dissociation of the bis-intercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considerably lower for externally bound than for bis-intercalated YOYO at low dye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, double-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event to occur adjacent to the first (i.e. to be induced by the same YOYO molecule). With TOTO the efficiency of the ssb-accumulation is comparable to that observed with YOYO.     

CD of ethidium bromide complexes with normal and electrophoretically anomalous DNA restriction fragments

The equilibrium binding of ethidium bromide (EB) to two small 147 base-pair (bp) DNA restriction fragments, which exhibit different mobilities in polyacrylamide gels, was investigated by CD. Two larger DNA restriction fragments and calf thymus DNA were also studied for comparison. Difference spectra were calculated by subtracting the spectrum of the pure DNA from the spectra of its DNA–EB complexes. The D/P ratios ranged from 0.03 to 1.0. The difference CD spectra of all fragments are characterized by bands with maxima near 310, 275, and 207 nm, and minima near 290, 253, 225, and 190 nm. The band near 310 nm, which has a shoulder at about 335 nm, has zero intensity at D/P ≤ 0.05, and rises to a plateau value, different for each fragment, at D/P ? 0.3 for large fragments (≥ 1400 bp), and D/P ～ 0.7 for the two small 147 bp fragments. The minimum near 290 nm is markedly blue shifted with increasing D/P, the wavelength of the extremum corresponding approximately to the wavelength of the uv absorption maximum of the DNA–EB complex. The negative amplitude of this band at D/P = 1.0 depends on the molecular weight of the DNA. The difference CD maximum near 275 nm is positive at low D/P ratios, increases and goes through a maximum at D/P = 0.06–0.1, and then becomes increasingly negative with increasing D/P. The amplitude of the negative ellipticity per added dye is constant at high D/P ratios, suggesting that the transition can be attributed to outside-bound EB molecules. The ellipticities at 310, 290, and 253 nm increase in absolute magnitude with increasing D/P at approximately the same rate, suggesting that all three bands are associated with the same optical and/or conformational transition. For the two small 147 bp fragments the fractional increases in amplitude of these bands parallel the fractional increase in length of the DNA upon binding EB, determined by electric birefringence measurements. The titration of the restriction fragments with EB was also followed by optical absorption. Two end points are observed, the first at a D/P ratio of ～ 0.1, reflecting the transition between intercalated and outside-bound dye molecules, and the second at D/P ? 1.0, the equivalence point of the titration.     

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.     

Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides.     

Competitive interaction of serotonin and the dye acridine orange with DNA

The interaction of serotonin and acridine orange dye with DNA isolated from bacterium Escherichia coli and the yeast Candida utilis has been analysed by spectrofluorimetric method. Using data on competitive binding to DNA of serotonin and acridine orange, known as DNA intercalator, a conclusion concerning the formation of intercalated complex between serotonin and DNA has been made. It is shown that for yeast DNA the constant of intercalated binding of serotonin is 3,5-fold smaller than for the bacterial one.     

Changes of the DNA packaging mode during boar sperm maturation

Changes in the mode of DNA packaging in nuclei during spermatogenesis were studied by measuring of the fluorescence anisotropy decay of an ethidium dye intercalated in the DNA in whole nuclei. The nuclei were isolated from boar spermatid or sperm cells at three distinct stages of spermatogenesis: just before the completion of a maturation process in the testis (late spermatid), immediately after a subsequent transformation into spermatozoa (caput spermatozoon), and after full maturation (cauda spermatozoon). Although these three kinds of nucleus were morphologically indistinguishable from each other, the anisotropy decay detected a clear difference. In the late spermatid nuclei, in which the replacement of histones by protamine was still in progress, the anisotropy decayed extensively. The decay suggested that the DNA in the spermatid nuclei contained very flexible regions, in which the interaction of the DNA and proteins may be weak. The rapid and extensive anisotropy decay was absent in the caput and cauda nuclei. The flexible portions must have turned into very rigid structure during transformation from the late spermatid into the caput spermatozoon.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates

DNA-protein condensates that give positive and negative psi-type circular dichroism (CD) spectra (psi condensates) bind intercalative and nonintercalative dyes. CD depends both on circular differential scattering and on circular differential absorption; scattering-corrected CD measurements are approximations to circular differential absorption. The circular differential scattering and scattering-corrected CD patterns observed in the DNA absorption band of psi condensates are mimicked in the induced CD band of intercalators bound to psi condensates. The induced scattering-corrected CD and circular differential scattering patterns of the groove-binding dye Hoechst 33342 bound to psi condensates are the inverse of the patterns seen with intercalative dyes, whereas the groove-binding dye manganese(III) meso-tetrakis(4-N-methylpyridyl)porphine [MnIIITMpyP-4] shows no significant induced CD patterns. The large circular differential scattering and scattering-corrected CD bands are interpreted as resulting from long-range chiral packing, rather than near-neighbor short-range interactions. Dyes intercalated into the DNA of the psi condensates have the same type of long-range chiral packing as the DNA bases. Therefore, the psi-type CD spectra seen in the UV spectra originating from the long-range packing of the DNA bases are also observed in the visible spectra when dyes are intercalated in the DNA of the psi condensates. Our interpretation comes from the observation that the induced circular differential scattering and circular differential absorption of the dye bound to the psi condensates depend only upon the sign of the circular differential absorption and the pattern of the circular differential scattering of the psi condensates without bound dye.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the optical properties of the proflavine–DNA complex

We report studies of the optical properties of the proflavine–DNA complex, using absorbance and circular dichroism spectroscopy. From comparison of the absorption spectra of proflavine complexed with calf thymus and T2 DNA, we conclude that stacking of the dyes external to the double helix is comparatively much weaker with T2 DXA, probably because of its glucosylation. Several sources are found for the circular dichroism induced in proflavine when it is complexed with DNA. There is a relatively weak circular dichroism induced when the dye is infinitely dilute on the DNA lattice; this presumably arises from the environmental asymmetry of the binding site. Stronger circular dichroism effects are induced by interaction of intercalated and stacked dyes; studies with T2 DNA, for which stacking seems to be blocked, permit a tentative resolution of effects due to the two modes of binding. One recurring theme of these studies is the observation that the optical properties are quite dependent on environment. The most dramatic example is a strong variation with salt concentration of the amplitude of the circular dichroism induced in the isolated (intercalated) monomer by the surrounding DNA. This suggests that the structure of the intercalated complex is quite sensitive to external conditions.     

Frequency-domain measurements of the rotational dynamics of the tyrosine groups of calmodulin

We used frequency-domain fluorometry to determine the intensity and anisotropy decay kinetics of tyrosine residues in calmodulin and its fragments. Excitation was provided by a continuous ultraviolet laser source, a frequency-doubled rhodamine 6G ring dye laser, whose output was externally modulated to 200 MHz. Both the intensity and anisotropy decays were found to be multiexponential and dependent upon temperature and solution conditions. By examination of calmodulin fragments we determined that energy transfer between the two tyrosine residues reduces the steady-state anisotropy values by about 20%. Additionally, the frequency-domain anisotropy decays indicate local torsional motions of the tyrosine residues, as well as significant individual motions of the two domains of calmodulin.     

In situ measurement of circular dichroism of DNA adsorbing onto a solid surface

The adsorption process of DNA dissolved in aqueous solutions onto the surface of polyethyleneimine (PEI) has been examined by the observation of in situ circular dichroism (CD), including time-resolved measurements, to elucidate the conformation of DNA at the liquid/solid interface. The adsorption process can be characterized by two stages that are characterized in terms of CD. In the first stage, time (t)<700 s, a slight change in the time-resolved CD spectra of DNA is observed, whereas the value of the induced CD of the dye intercalated in DNA is constant. This result can be explained by the interaction between DNA and PEI during the adsorption at the liquid/solid interface. The weakness of the interaction is attributed to the geometrical restriction of this interface. In the second stage, t>700 s, where no further adsorption occurs, a change in the induced CD as well as in the CD of DNA is observed. This change in the induced CD can be interpreted as a significant conformational change of DNA for stabilizing the ion complex with PEI.     

Kinetic and hydrodynamic studies of the complex of proflavine with poly A·poly U

Relaxation kinetic experiments reveal general similarity between the mechanism of binding of proflavine to poly A·poly U and DNA. There are differences in detail, however. For example, the rate constants are roughly an order of magnitude smaller for the former, and the thermodynamic parameters of the individual steps are also different. The total heat and free energy for intercalation of free dye are quite similar in the two cases. As was the case with DNA, considerable dye (up to 25% of the bound form) is attached externally to the double helix, even in the strong binding region of the isotherm. Sedimentation measurements on small, rodlike fragments of poly A·poly U reveal a length increase on binding proflavine of a magnitude similar to that found with DNA. This length increase seems to become smaller under conditions (high temperature) where the relaxation measurements indicate a higher fraction of externally bound dye.     

Effects of chloroquine on the torsional dynamics and rigidities of linear and supercoiled DNAs at low ionic strength

The magnitude and uniformity of the torsion elastic constant (alpha) of linear and supercoiled pBR322 DNAs are measured in 3 mM Tris as a function of added chloroquine/basepair ratio (chl/bp) by studying the fluorescence polarization anisotropy of intercalated ethidium dye. The time-resolved FPA is measured using a picosecond dye-laser for excitation and time-correlated single-photon counting detection. For both linear and supercoiled DNAs, alpha remains uniform except at the very highest chl/bp ratio examined. For the linear DNA, alpha decreases from 5.0 x 10(-12) dyne-cm at chl/bp = 0 to about 3.5 x 10(-12) dyne-cm at chl/bp = 0.5, and remains at that value up to chl/bp = 5, whereupon it increases back up to its original value. For the supercoiled DNA, alpha remains constant at about 5.2 x 10(-12) dyne-cm from chl/bp = 0 up to chl/bp = 5, whereupon it increases in parallel with the linear DNA. The effect of chloroquine on the secondary structure, torsion constant, and torsional dynamics evidently differs substantially between linear and supercoiled DNAs, even under conditions where the supercoiled DNA is completely relaxed and both DNAs bind the same amount of dye. This strongly contradicts any notion that the local structures of linear and relaxed supercoiled DNA/dye complexes with the same binding ratio are identical. The increase in apparent alpha at chl/bp = 5 for both DNAs may be due to stacking of the chloroquine in the major groove and consequent stiffening of the filament.     

RigidFinder: A fast and sensitive method to detect rigid blocks in large macromolecular complexes

Advances in structure determination have made possible the analysis of large macromolecular complexes (some with nearly 10,000 residues, such as GroEL). The large‐scale conformational changes associated with these complexes require new approaches. Historically, a crucial component of motion analysis has been the identification of moving rigid blocks from the comparison of different conformations. However, existing tools do not allow consistent block identification in very large structures. Here, we describe a novel method, RigidFinder, for such identification of rigid blocks from different conformations—across many scales, from large complexes to small loops. RigidFinder defines rigidity in terms of blocks, where inter‐residue distances are conserved across conformations. Distance conservation, unlike the averaged values (e.g., RMSD) used by many other methods, allows for sensitive identification of motions. A further distinguishing feature of our method, is that, it is capable of finding blocks made from nonconsecutive fragments of multiple polypeptide chains. In our implementation, we utilize an efficient quasi‐dynamic programming search algorithm that allows for real‐time application to very large structures. RigidFinder can be used at a dedicated web server ( http://rigidfinder.molmovdb.org ). The server also provides links to examples at various scales such as loop closure, domain motions, partial refolding, and subunit shifts. Moreover, here we describe the detailed application of RigidFinder to four large structures: Pyruvate Phosphate Dikinase, T7 RNA polymerase, RNA polymerase II, and GroEL. The results of the method are in excellent agreement with the expert‐described rigid blocks. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.

Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given.     

Effect of ethidium on the torsion constants of linear and supercoiled DNAs.

The torsion elastic constants (alpha) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266-278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the torsion constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the torsion constant is independent of binding ratio up to r = 0.20 dye/bp. If alpha d denotes the torsion constant between ethidium and a base-pair, and alpha 0 that between two base-pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The torsion constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r* = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the torsion constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r* should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAs.     

Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting.

The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.