Structural implications of electric-field induced dichroism of nucleic acids: Studies of alternating purine-pyrimidines

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1 Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 3 & 4, August 1985, pp Printed in India. Structural implications of electric-field induced dichroism of nucleic acids: Studies of alternating purine-pyrimidines ELLIOT CHARNEY Laboratory of Chemical Physics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205, USA Abstract. Transient and steady-state electric dichroism measurements of double helical poly(dg-dc) and its 5-methyl guanosine analogue and transient electric dichroism measurements of double helical poly(da-dt) are shown to give the following information on the structural and dynamical properties of these molecular systems: (i) the Z form structure of the alternating guanosine-cytidine moieties has the same inter base-pair separation in solution as it does in the fibre and crystalline forms; (ii) the mean normal to the base pairs (and thus their average tilt and twist) of the B and Z forms of the guanosine-cytidine moieties is very nearly the same despite the large difference in their secondary structure; (iii) the alternating adenosinethymidine nucleic acid is at least twice as flexible as random-sequence DNA. Keywords. Electric dichroism; transient electric dichroism; nucleic acids; flexibility of poly(dg-dc) structure of poly(da-dt). Introduction In 1875 Kerr reported the optical effect induced in materials subject to an electric field stress to which his name is now attached, and in the early part of this century classical theories were developed relating the experimental observations to the optical and electric polarizability tensors of the molecules subjected to the electric field stress (Langevin, 1910; Born, 1918; Raman and Krishnan, 1927). When we started our studies of the nucleic acids in 1965, it was clear nevertheless that the interpretation of the optical information in terms of physical and structural molecular parameters would be anything but straightforward. We will show here, that while this is still to some extent true, there is information that can be accurately and precisely obtained from the measurements. Except, however, for referring briefly to some of the theoretical work (Mandel, 1961; Fixman, 1979; Rau and Charney, 1982, 1983) on the relation between the orientation forces and the dielectric properties of polyelectrolytes, this paper will be concerned with the results of recent measurements on two nucleic acid systems, poly(dg-dc) and poly(da-dt) in their double-stranded helical configurations. Before discussing these directly, a few remarks are desirable on their polyelectrolyte properties, as it was as much the lack of realization of the special significance of their high density of charge (Kikushi and Yoshioka, 1976; Tricot and Houssier, 1976; Sokorov and Weill, 1979; Charney, 1980; Charney et al., 1980), as anything else that led to the long gestation time in the development of sound interpretations of the observations on the nucleic acids. 517

2 518 Charney Spaced along the surface of the DNA double helix at nearly regular intervals are phosphate groups which, in aqueous solution at or near hydrogen ion neutrality, are negatively charged. Under these conditions DNA is an archetype macromolecular polyion. In a way that depends on the charge separation on the macromolecule and on its dimensions, counterions condense on it in a more or less uniform sheath providing partial charge compensation (Manning, 1978). In solution at moderate and low salt concentrations, the distribution of the remaining ions, both counter and coions, can be theoretically described and the theories used to demonstrate that electric fields induce moments that are strong enough to orient the polyion sufficiently to measure its optical birefringence and dichroism. Electric-linear dichroism especially, is peculiarly well suited for obtaining structural information on polymer or polymer like substances in solution, provided only that their structural integrity remains unperturbed or is perturbed in an analytically determinable way by the imposition of the electric field. The measurements are made by applying the field to the sample between parallel metallic electrodes of sufficient size to insure that the field is uniform across the sample. These fields are applied in pulse lengths, long enough to assure that the molecules attain orientation equilibrium. Under these conditions, the system attains uniaxial symmetry with respect to rotation about the field direction, and all the available optical information is obtainable from two measurements made with orthogonally polarized light propagating perpendicular to the field direction. The last decade has seen a proliferation of these measurements and theoretical developments as a result of a coincidence of interest in the biological and hydrodynamic (and mechanical) properties of DNA and in the dielectric properties of polyelectrolytes. For example, it has been clear for some time that the hydrodynamic and other transport properties of polyelectrolytes are strongly dependent on the ionicity of the solvent. One only has to recall the early work on the sedimentation of DNA and the work of Harrington, Eisenberg, Mandel, and others on the effect of the ionic strength on light scattering of DNA to realize that the linear polymer like molecule is subject to substantive variations in overall tertiary structure, to say nothing of possible variations in secondary structural parameters. It has now, in fact, become part of the conventional wisdom, as the result of the discovery of new structures for DNA and polynucleotide sequences (Arnott et al., 1980, 1983a; Gupta et al., 1983; Sasisekharan, 1981) that polymorphism in DNA with its accompanying changes in structural parameters is ubiquitous, and that transitions between the various structures are both sequence and environmentally dependent, viz. the salt dependence of the transition of the poly(dgdc) double helical B and Z forms at high salt concentrations (Pohl and Jovin, 1972) or in the presence of small amounts of multivalent ions (Behe and Felsenfeld, 1981). In this paper we summarize the TED measurements on poly(dg-m 5 dc) from which the change in inter base-pair separation between the B and Z conformers in solution was obtained (Chen et al., 1982) and report on the results of the transient decay measurements on poly(da-dt) from which its flexibility relative to that of random sequence DNA is determined. We also report steady-state ED measurements on the poly(dg-dc) system which show that the mean effective angles between the base plane normals and the helix axis are almost identical in the B and Z structures.

3 Electric dichroism of nucleic acids 519 Methods and materials The measurements are of two types: transient measurements of the rise or decay of the dichroism when the field is turned on or off rapidly compared to the molecular orientation time (transient electric dichroism, TED) and, equilibrium or static measurements of the dichroism when the molecules have come to steady-state orientation in the field during the pulse (electric dichroism, ED). The dichroism at any time, t, and wavelength, λ, is given by where A and A are absorbances for light polarized respectively parallel and perpendicular to the field direction. It is possible to define a reduced linear dichroism, ρ, which is characteristic of the sample under the conditions of the measurement: where A 0 is the isotropic absorbance of the sample. The relationship between these measured optical quantities and the parameters are determined by: (i) the degree of orientation of the molecule in the field and (ii) the orientation of the vector transition moment responsible for the absorbance at the wavelength of the measuring light with respect to the molecular axis of orientation. If the molecule or complex is uniaxial with the shape of a cylinder or a prolate ellipsoid of revolution, then the orientation axis is almost of necessity coincident with the molecular axis. A full exploration of these parameters and the techniques for measuring them may be found in the literature (Fredericq and Houssier, 1973; Charney, 1971). It is necessary here to point out only that if, as is customarily the case, complete orientation is not achieved at any practical field strength, recourse must be made to data extrapolation for the interpretation of structural information from steady-state data. This procedure is somewhat hazardous in the absence of a firm theoretical foundation (Rau and Charney, 1983). For the interpretation of transient data from the decay of the dichroism at the end of a field pulse, the situation is more straightforward as these measurements are made at lownon-perturbing field strengths and no data extrapolation is required if the solutions are sufficiently dilute (Hagerman and Zimm, 1981; Elias and Eden, 1981; Charney and Milstien, 1978). At low field strengths, the field free, exponential relaxation of the induced linear dichroism is directly related to the rotational diffusion coefficient, D θ' of the macromolecule. The observed relaxation time, τ (= l/6d θ ) is a very sensitive function of the end-to-end distance, L, of the molecule; for rigid rods, τ ~ L 2 5, and is given by the modified and empirically well-established Broersma equation (Broersma, 1960; Newman et al., 1977): where η 0 is the solvent viscosity, p = L/D where D is the diameter of the rod. For a fully extended rod, the length L is obtained by fitting this equation to the measured relaxation time, and used to determine the inter base-pair separation from the number of base-pairs in the measured sample. If the molecule is flexible under the conditions of

4 520 Charney the experiment and therefore not fully extended, then L is a measure of the length of the equivalent relaxing cylinder. In this case theoretical models relating the conformational distribution to the length and diameter of the equivalent cylinder may be used in combination with the TED measurement to obtain the persistance length, P, which is a measure of the flexibility of the molecule (Hagerman and Zimm, 1981). Values for the rise per base pair (RPB) calculated from the measured TED relaxation are in good agreement with X-ray fiber diffraction values when the measurements are made on fully extended rod-like fragments. If they are not fully extended, then the difference between the end-to-end extension of a rigid rod and of a flexible rod of the same contour length is sensitively reflected in the difference between the observed relaxation time of the latter and the calculated relaxation time of the former. This is the basis for persistence length measurements using TED or transient electric birefringence (TEB). Details of the properties of poly(da-dt) and poly(dg-dc) used in the experiments reported here, in addition to conditions cited in this text may be found respectively in the references by Chen, Rau and Charney (1984) and Chen, Charney and Rau (1982). The only other significant fact is that all measurements of the dichroism reported here were made at 2 C. Results and discussion Transient electric dichroism of Poly(dG-m 5 dc) As the results of these measurements have already been reported in detail (Chen et al., 1982), only those aspects necessary to the general concept of this paper will be reported and discussed here. Poly(dG-dC) and poly(dg-m 5 dc) are known to undergo a conformational transition (Pohl and Jovin, 1972; Behe and Felsenfeld, 1981). The righthanded double-helical form of B-DNA with a helical repeat of approximately 10 base pairs and an interbase-pair spacing of 3 4A, can be transformed to a left-hand form designated Z-DNA with an inter base-pair spacing of 3 7A as determined by X-ray diffraction of fibers of poly(dg-dc) and of crystals of dg-dc hexamers (Arnott et al., 1980; Wang et al., 1981). The length increase (~ 11 %) expected from the solid state X- ray diffraction data for the B Z transition is readily calculated and compared to the values obtained from the TED measurements (Broersma, 1960; De la Torre and Bloomfield, 1981). In this case at the very low ionic strength at which the measurements were made, the 146 base-pair lengths of DNA and poly(dg-m 5 dc) that were used are essentially fully extended and the hydrodynamic relations are adequately applied to their contour length. The data for poly(dg-m 5 dc) in the presence of (very small quantities of)co(nh 3 ) which converts the right- to the left-handed form are shown in figure 1. The calculated values of the inter base-pair separation based on these measurements are given in table 1. It is clear that the inter base-pair separation in the absence of Co(NH 3 ), is the same as in the canonical structure of B-DNA and, that in the presence of Co(NH 3 ), the structure must be substantially the same as in the fiber at high salt and the crystal of the dg-dc hexamer, i. e. the Z structure with an inter basepair separation of 3 7A.

5 Electric dichroism of nucleic acids 521 Figure 1. The orientation relaxation of 146 base pair fragments measured by the decay of the parallel dichroism, poly(dg-m 5 dc) with Co(NH 3 ) ; O ply(dg-m 5 dc) without Co(NH 3 ) ; Δ--random sequence DNA without Co(NH 3 ). All in ph = 7 0 phospate buffer. The corresponding values of the relaxation times, τ, are given in table 1. Table 1. Base-pair separation in B and Z poly(dg-m 5 dc); *The X-ray diffraction determined values are taken from the review by Zimmerman (1982). Transient electric dichroism of Poly(dA-dT) Evidence from light scattering and fluorescence anisotropy decay (Thomas and Bloomfield, 1983; Hogan et al., 1983) that the alternating and non-alternating G-C helical complexes are, if anything less flexible than that of random sequence DNA prompted us to examine the TED of the other common alternating purine-pyrimidine pair, poly (da-dt). If, as might be expected from the relative stiffness of the CG pair, the AT pair turned out to be more flexible than "random" DNA, it could be significant in explaining the frequent occurrence of both short and long (as many as 100 base pairs or more) runs of adenine and thymine bases in DNA. For this purpose we have measured the orientational relaxation time τ of samples of alternating poly (da-dt) of different molecular weight and calculated the end-to-end distances of the equivalent rods using

6 522 Charney Figure 2. The orientation relaxation of 136 ± 25 base pair fragments of poly (da-dt). The orientation relaxation is theoretically claculated (solid line) for 142 base pairs which is effective relation length for the distribution of ± 25 base pairs as the longer fragments contribute more heavily than the shorter to the mean relaxation time. The fit corresponds to the observed relaxation if the base-pair separation is 2 85 A see text. the same technique that was used for the 146 base-pair fragments of (dg-m 5 dc). The data for a 136 ± 25 base pair sample are plotted as solid points in figure 2. The dichroism decay appears as essentially a single exponential in time over two orders of magnitude of the dichroism; the slight curvature is accurately accounted for by the size distribution of the sample. The inter base-pair separation calculated from these data under the assumption that the poly (da-dt) is a rigid rod is 2 85A which is 0 15A shorter than that of the most compact structure that has been proposed for poly (da-dt) by Arnott et al. (1983b) and 0 43A shorter than observed in fibers of poly (da-dt) in B- form (Davies and Baldwin, 1963). Clearly either the structure in solution is different from any of the structures that have been proposed or this sequence is very flexible and even fragments this short are not on the average fully extended in the 1 mm ionic strength range. Measurements of τ for a range of sizes from 136 to 270 base pairs were used to obtain the inter base-pair separation from a rigid rod interpretation of the data and, using the method by which Hagerman and Zimm (1981) treated the transient electric birefringence data to obtain the persistence length of DNA. The results for poly (da-dt) are shown in figure 3 where the filled dots, based on a rigid rod length interpretation infer that the base-pair separation varies strongly with molecular length, an interpret-

7 Electric dichroism of nucleic acids 523 Figure 3. O The apparent inter base-pair separati on of four different size fragments of double helical poly(da-dt) calculated from the experimental relaxation data assuming the fragments are non-flexible rods; -the persistence length of the same fragments calculated using the Hagerman-Zimm (1981) treatment of the orientation relaxation of semi-flexible rods. ation wholly incompatable with any reasonable physical intuition. By comparison, the data treated to obtain the persistence length (empty dots) are nearly constant with molecular length, the slight upward curvature at short lengths being consistent with the increasing effectiveness of repulsion of the ends as the molecule decreases in length. Most significantly, the persistence length of poly(da-dt) in these solution is of the order of 250 A, about one-half that of DNA at the same ionic strength and reasonably independent of ionic strength over a rather wide range. It is of interest to note that a classical calculation of the bending energy (Landau and Lifshitz, 1958) based on these values of the persistence lengths indicates that bending would be favoured at alternating AT sequences by about one Kcal per helical turn. Steady-state dichroism of poly (dg-dc) The conventional wisdom has been that extrapolation of steady-state dichroism values from low and moderate fields where % of the orientation is achieved, to infinite field strength defines the average orientation of the absorbing chromophore at the wavelength of the measuring light with respect to the dipole axis. With the added assumption that for nucleic acids the dipole axis is the long molecular axis direct structural conclusions about the tilt or twist of the nucleotide base planes have been drawn. There has, albeit, been significant differences in the way in which the extrapolation has been made. Fortuitously for some systems, for example that of unravelled chromation, the extrapolation method is of little consequence. For the bare polynucleotides, there can be significant differences in the inferred orientation of the bases that depend among other things on the way in which the extrapolation is done, for example whether from plots of ρ against E 2 or against E 1. Thus early studies of calf thymus DNA in which extrapolations were made from plots of ρ against E 2, E 2, or

8 524 Charney E 1 led to values of ρ ranging from about 0 7 to 1 4 (Soda and Yoshioka, 1965; Charney and Yamaoka, 1971; Ding et al., 1972; Fredericq and Houssier, 1973). Under the assumptions (1) that these values reflect the values expected for a transition of the optical chromophore at 270 nm polarized in the plane of the bases, and (2) that the molecular axis is a straight rod, these values could be, and occassionally were interpreted as indicating that the bases were tilted about 25 (ρ = 0 7) to 8 (ρ = 1 4). The possibility of other interpretations were discussed in a paper by Charney and Milstein (1978) but no definitive conclusion drawn. More recently at least two interpretations of the low values of ρ have been made in terms of the existence of non-rodlike tertiary stable structures for DNA (Lee and Charney, 1982; Diekmann et al., 1982). With the recognition that special characteristics of nucleic acids are associated with their high density of charge, attempts have been made to develop a valid theoretical basis for the dependence of the orientation on the field strength in order to establish the validity of the data extrapolation (e.g. Sokorov and Weill, 1979). It has now been demonstrated that the experimentally observed variation of the values of ρ with molecular length could have its origin in the fact that the orienting dipole in the free ion-atmosphere of polyelectrolytes in high electric fields goes through a maximum in the field strength dependence, rather than saturating to a constant value and, in the dependence of the location of this maximum on the molecular length (Rau and Charney, 1983). This result does not preclude other structural effects from affecting the observed values, but makes it important, to consider to what extent the extrapolated values are influenced by the orientational forces. As one step in a series designed to evaluate the importance of this phenomenon, and as well for intrinsic structural interest, we have measured the steady state dichroism of double helical poly(dg-dc) with the results shown in table 2 where the values are obtained by linear extrapolation of the data from plots of ρ against E 1. We have Table 2. Steady-state dichroism, ρ. a The experimental values are obtained from a plot of ρ against 1/E by extrapolation to infinite field strength. b The calculated data are computed from the magnitudes of the electric dipole transition components parallel and perpendicular to the helix axis, We thank Dr. Eric Henry for help with this computer assisted calculation using structures generated from GC base pair coordinates of the dodecamer d(cpgpcpgpapaptptpcpgpcpgp) (Drew et al., 1981) and spectroscopic assignments (Dougherty et al., 1983) of the guanosine and cytidine transitions at 260 nm.

9 Electric dichroism of nucleic acids 525 shown that these plots should be meaningful so far as the current theory of polyelectrolyte to polarization in an electric field is concerned and mimic the experimental data over a wide range at high fields as well or better than any other available treatment (Charney, E., Chen, H. H. and Rau, D. C., unpublished results). The value, 1 1, of ρ for poly(dg-dc) is compatable with the value, 1 2 ± 0 1, obtained for DNA of comparable length (~ 2100 A) by interpolation from the data of Lee and Charney (1982); Ding et al. (1972); Hogan et al. (1978), and of Diekmann et al. (1982). Both the DNA and poly(dg-dc) values are less than that expected for a straight rod canonical B-form DNA. Taken together all the data (for DNA of this and other molecular weights and for poly(dg-dc) are consistent with theoretical treatment of the high field polarization (Rau and Charney, 1983). Because of the molecular weight dependence that results in more negative values of the dichroism for higher molecular weights (Lee and Charney, 1982; Diekmann et al., 1982), they are also consistent with, but do not prove that the base planes are nearly perpendicular, ± 80 82, to the principal helical axes of these nucleic acids. The observation (table 2) that the dichroism of poly(dg-m 5 dc) is almost identical in the B and Z forms is also consistent with the expected relationship between the two calculated from commonly accepted coordinates for these forms (Arnott et al., 1980; Wang et al., 1981) and from known assignments of the optical transition moments of guanosine and cytidine at 270 (Dougherty et al., 1983). Conclusions The flexibility of poly(da-dt) is shown to be considerably higher than that of random sequence DNA by TED measurements from which it is determined that the intrinsic persistence length of the former is about 250 A compared to about 550 A for the latter. TED measurements also show that the change in inter base-pair separation between the B and Z forms of poly(dg-dc) in solution is the same as in the solid state. Measurements of the steady-state dichroism of poly(dg-dc) are consistent with its expected polyelectrolyte polarization properties and indicate, but do not prove, that the bases in both the B and Z forms are not highly tilted or twisted with respect to the helix axis. Acknowledgements The work described here on alternating purine-pyrimidines has been carried out in full collaboration with Drs. Donald C. Rau and Holly Ho Chen. References Arnott, S., Chandrasekaran, R., Birdsall, D. L., Leslie, A. G. W., Ratliff, R. L. (1980) Nature (London), 283, 743. Arnott, S., Chandrasekaran, R., Banerjee, A. K., He, R. and Walker, J. K. (1983a) J. Biomol. Struct. Dyn., 1, 437. Arnott, S., Chandrasekaran, R, Duigjaner, L. C, Walker, J. K., Hall, I. H. and Birdsall, D. L. (1983b) Nucleic Acids Res., 11, 1457.

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