Optical Rotatory Dispersion of DNA in Concentrated Salt Solutions

Transcription

1 1218 COMhfUNICATIONS TO THE EDITORS Optical Rotatory Dispersion of DNA in Concentrated Salt Solutions The fact that the structure of DNA in fibers is sensitive to the relative humidity of the atmosphere suggests that it may be interesting to study the structure in solution under conditions of low water activity. There is considerable evidence that high concentrations of salt have large effects on the structure and stability of the DNA helix.213 Optical rotatory dispersion (OR])) is very sensitive to secondary structure. In an attempt to gain more insight into the nature of the structural changes, the ORD of DNA was measured as a function of salt concentration in several salts. Materials and Methods The DNA used in these experiments was calf thymus DNA, supplied by Nutritional Biochemicals. It was dissolved in DSC buffer (0.015M NaC1, M Na citrate, ph 7.0) to a concentration of about 7.5 OD, by gentle stirring at 5 C for 48 hr. In the DSC buffer, it melted over a 10 C range with a midpoint at 70 C. Its absorption spectrum showed it to be native and undegraded, by the analysis proposed by Hirschman and Fel~enfeld.~ NaCl, NaBr, LiBr, and LiCl were analytical grade reagents; CsCI, optical grade, and Cs formate were supplied by Harshaw Chemical Co., CsBr by A. B. MacKay, Inc. Stock solutions of these salts were prepared by dissolving them in DSC buffer and adjusting the ph to The Cs trifluoroacetate (CsTFA) stock solution was prepared by neutralizing Cs2C03 (K and K Laboratories, 99.8% assay) with trifluoroacetic acid. Solutions were clarified by filtering through a sintered glass filter. ph measurements were uncertain in these highly concentrated salt solutions, but no significant changes in the ORD were observed as a result of deliberate variation of the ph in this range. Solution samples were prepared by dilution of the stock salt solutions with DSC, and addition of the DNA stock to a final DNA concentration of about 0.75 OD. Salt concentrations were determined by measuring the refractive index of the solution in an Abbe refractometer and comparing with tabulated nd versus c data.6,6 DNA concentrations were determined spectrophotometrically, assuming Epmax = 6.5 X lo3. The tendency observed by Emanuelz for some salts to bring about changes in the extinction coefficient of the DNA complicates the calculation of specific rotation. For the most part this effect was ignored. At most it makes a difference of the order of 7% at the highest salt concentrations, which is a very small difference compared with the magnitude of the effect on the ORD. Corrections of the data on the basis of Emanuel s observations has no effect on the trends observed. Samples were run in cells of 1 cm path length in a Cary Model 60 recording spectropolarimeter at room temperature. Measurements were begun at 350 mp and continued to decreasing wavelength until solution absorption caused noise levels to become too high for meaningful data. Results and Discussion The primary effect of the presence of high concentrations of salt on the ORD of DNA appears to be a general loss of rotation at the higher wavelengths. The rotation in the vicinity of 290 mp becomes less positive and that in the vicinity of 260 mp less negative. In some cases it appeared that these changes were associated with the appearance of a peak or shoulder in the vicinity of 270 mp. This was particularly obvious in concentrated solutions of LiCI, where the depression of rotation produced by the presence of

2 COMMUNICATIONS TO THE EDITORS 1219 salt WM the greatest (Fig. 1). The peak near 230 mp is independent of these changes and appears to be relatively unaffected by the presence of salt. The bromides and KTFA absorb too strongly in this wavelength region to observe this peak, but in the presence of high concentrations of NaCI, CsCl, or LiCl, there is no tendency of this peak to decrease. There may even be a slight increase in rotation in this region. That the changes in the ORD brought about by high salt concentrations are quite different from those accompanying denaturation is seen in Figure 2. Denaturation in -I0 t V -Buffer 1.1 M LiCl --5.4M 6.8 M x Fig. 1. Effect of high salt concentrations on OR11 of calf thymus DNA. \ \ 10 a Y -I 0 -native in buffer (250) denatured in buffer (90 ) - - native in CsCl 6M (25O) denatured incscl 6M (25O) Fig A Effect of high salt concentrations on ORD of native and denatured calf thymus DNA.

3 1220 COh'IMUNTCATIONS TO TTIE EDITORS dilute buffcr causes a large decrease in rotation at the 290 mp peak with a much smaller change in the 260 mp region. The peak nrar 230 mp appcws to be very sensitive to long-range order iri the helix, and the large decrease in rotation brought about by denaturation shows less revcmal on cooling than the chaiiges at higher wavelengths. The spectrum in CsC1 is quite differont, the changcs in the 260 mp rcagion being much greater than those accompanying denaturation, while those in the vicinity of 230 mp are very small. The optical rotation of denatured DNA in CsCl is intermediate between that for the native form in CsCI, and that for the denatured form in dilute buffer. The effects of all the salts are similar, hut quantitativc.ly there arc significant differences from one sdt to another. Figure 3 shows the rotat,ion plotted against molar concentration of the various salts. The order of effectiveness of the ions in decreasing the rotation is: Ti+ > Cs+ > Na+ for the cations, and Cl- > Br- for the anions. Isolated experiments in CsBr, Cs formate, and CsTFA r,onfirm that t,he effect of Br- is slightly smaller than that of CI- and indicates that formate and trifliioroacet.ate both have a larger effect than chloride. There is no correlation with water act,ivity (aw). A plot, of rotation versus aw does not look very different, than when plotted versus molarity of salt. The effect appears to be reversible. One experiment was done measuring the rotation in 7.8M LiCI, then dialyzing the I)NA hack into 1)SC. The rotat,ion of the dialyxed DNA was within experimental error of that of 1)NA unexposed to high salt concentrations. The degree to whirh these changes are due to vicinal effects of the solvent is uncertain, but it is unlikely that this factor is of primary importance. The changes in the shape of the spectrum, with large effects at some wavelengths and none at others, makes it seem more likely that there are actual changes in the structure of the molecule. hloreover, there is evidence that the rotation spectra of several kinds of single-stranded RNA and the double-stranded DNA-RNA hybrid from F2 bacteriophage are relatively c (moles/l salt) Fig. 3. Comparison of effectiveness of various salts in decreasing optical rotation of calf thymus DNA at the 290 mp peak and the 260 mp trough.

4 COA4MUNICATIONS TO THE EDITORS 1221 unuffected by the presence of high salt conceiitratio~is,~ and one would expect the vicinal solvent effects to be very similar for these macroniolocules. It is thercfore most likely that the spectral changes in the presence of high xalt concentrations are a result of some structural change in the DNA. This is suggestive of the B + '4 transition in fibers, which also occura on dehydration. Thus it is appropriate to ask whether the presence of salt causes a change in helical structure to the A form in solution. To answer this qilcstion we need information on the ORD of the A strueture. The x-ray diffraction patterns of double-stranded RNA are quite similar to those of the A form of DNA,*,g although the detaiied structure is probably somewhat different from both the A and B forms of I)NA.lOJL Recent studies of Milmari et al12 indicate that the structure of the DNA-RNA hybrid is very close to that of the A form of DNA, and shows very little change as the relatively humidity is varied. They concludc that the Y-OH group precludes the existence of thc B form in polyribonucleotides for steric rcasons. It is thcwfore interesting to look at the ORD of these structures in solutioii and see what one can predict about that of the A form of DNA. Figure 4 shows a comparison of the rotation spectra of DNA, double-strandcd RNA, and the I)NA-RKA hybrid. The RXA curve was taken from the curve published by Oriel and Wilderla for MS2 ILNA. The spectrum of the hybrid is that of F2 bacteriophage; the sample was generously provided by M. Chamberlin and is the same material as that on which the x-ray diffraction was done. The ORD spectrum of the hybrid is more similar to doublc-stranded RNA than to DNA. Let us assume that both these structures are in the A form. The most conspicuous differences from the prcsumed B form of DNA are in the larger absolute magnitude of the rotation at the maxima near 285 mp and the minima near 250 nip, and a smaller pcak near 230. (However, it is difficult to predict what oiic might expect of the A form of 1)NA at 230 mp, because of the tcwlency of the hybrid to be more positive there than HNA). These differencw are roughly the opposite of the changes obwrved in the DNA spectra. on adding salt. Any predictions one could make about the spectrum of the A form of DNA from these data would be yuit.e differcnt from t.hc: spcctrum actually observed on the addition of salt. Thus we would conclude that a B+A transition docs not occur in c,oiicciitrated salt solutions as the 1)NA moleculc is dehydrated. Whatever structural change is induwd by the presence of salt is quite different both from this transition and from denaturation. I Fig. 4. Coinparisoil of OlW of double-stranded nuclcic acid helices.

5 1222 COMMUNICATIONS TO THE EDITORS The ORD of the hybrid was also measured in the presence of 3M CsCl. One can see from Figure 5 that the effect of the salt here is very small compared to the effect on DNA. The salt effect, then, acts fairly specifically on the B structure. The changes in the ORD of DNA show some resemblance to changes in ORD observed by Chengl4 in the presence of very low concentrations of some divalent cations. The possibility cannot be excluded that part of the changes observed here are due to the presence of heavy ions. However, it seems unlikely that heavy ion contamination could account for the major part of the effects observed, since the citrate in the buffer should keep the concentrations of divalent impurities below the level used by Cheng, except possibly at the highest concentrations of salt used. It seems more likely that Effect of salt on DNA buffer CsCl 2.4 M A

6 COMMUNICATIONS TO THE EDITORS 1223 both low concentrations of divalent ions and higher concentrations of monovalent ions are capable of bringing about structural changes in the DNA responsible for the alterations in its ORD spectrum. The order of effectiveness of the various ions in bringing about the ORD changes cannot be correlated either with effects on water structure or with the ability of the salts to lower the melting temperature of DNA. Moreover, although the loss of rotation appears to follow roughly the loss of hydration with water activity,16 the large specific ion effect suggests that whatever structural changes are taking place depend on more than just the quantity of water bound to the helix. What kind of structural change actually does occur is uncertaiii. One possibility is a change in the number of base-pairs per turn in the Watson-Crick helix. There is evidence from the work of Wang et a1.16 that this parameter is sensitive to the ionic environment of the DNA. It seems likely that the nature of the specific interaction of the DNA with its counterions causes significant differences in the fine structure of the DNA in solution. We would like to acknowledge the contribution of Dr. R. Davis, who originally observed and told us of the effect of high salt concentration on the ORD of DNA, and to thank Dr. M. Maestre for many helpful discussions while this work was in progress. We are very grateful to Dr. M. Chamberlin for material and help with the experiments on the DNA-RNA hybrid. The irivcstigation was supported in part by a U.S. Public Health Service Grant (GM 11180) and fellowship (F1-6M 17791) from thc National Institute of General Medical Scieiices. References 1. R. Franklin and R. G. Gosliiig, Acta Cryst., 6, 673 (1953). 2. C. F. Emanuel, Biochim. Biophys. Acta, 42, 91 (1960). 3. K. Hamaguchi and E. P. Geiduschek, J. Am. Chem. Soc., 84, 1329 (1962). 4. S. Z. Hirschman and G. Felsenfeld, J. hlol. Biol., 16, 347 (1966). 5. J. Timmermans, The Physico-chemical Constants of Binary Systems in Concentrated Solutions, Vol. 3, Interscience, NEW York, J. Vinograd and J. E. Hearst, Progr. Chem. Orq. Nat. Prod., 20, 372 (1962). 7. M. Maestre, private communication (1967). 8. R. Langridge and P. J. Gomatos, Science, 141, 694 (19K3). 9. M. Spencer, W. Fuller, M. H. F. Wilkins, and G. L. Brown, Xature, 194, 1014 (1962). 10. T. Sato, Y. Kyogoku, S. Higuchi, Y. Mitsui, Y. Iitaka, and M. Tsuboi, J. MoZ. BioZ., 16, 180 (1966). 11. S. Arnott, M. H. F. Wilkiiis, W. Fuller, J. H. Veiiablc, niid R. Langridge, J. Mol. Bid, 27, 549 (1967). 12. G. Milman, R. Laiigridgc, and M. Chamberliti, Proc. Nat. dcad. Sci. U.S., 57, 1804 (1967). 13. P. J. Oricl and J. Wilder, Nature, 214, 702 (1967). 14. P. Cheng, Biochim. Biophys. Acta, 102, 314 (1965). 15. J. E. Hearst and J. Vinograd, Proc. Nat. Acad. Sci. U.S., 47,825, 999, 1005, 1015 (1961). 16. J. C. Wang, D. Baumgarten, and B. M. Olivera, Proc. Nat. Acad. Sci. U.S., (1967). MARY-JANE B. TUNIS JOHN E. HEARST Group in Biophysics and Departmelit of Chemistry University of California Berkeley, Califronia Itcccived Fcbrunry 5, 1968