Nucleic Acids Research Volurne3 no.10 October'1976 Vlue3n.0Otbr96NcecAisRsah Interaction of ethidium bromide with DNA: effect of LiCl and ethylene glycol

James S. Balcerski* and E. S. Pysh Departnent of Chemistry, Brown University, Providence, RI 02912, USA

Received 7 April 1976

ABSTRACT The interaction of the intercalating dye ethidium bromide with several native and synthetic polydeoxyribonucleic acids has been studied by means of circular dichroic spectra. The CD of DNA-ethidium bromide complexes in the 290-360 nm region is characterized, especially at high salt and at high ethylene glycol content, by positive and negative bands near 308 run and 295 run, respectively. These dye associated CD bands are unaffected by the addition of LiCl or ethylene glycol, suggesting that the relative conformation of dye and neighboring base pairs does not change when the conformation of the rest of the DNA changes.

INTRODUCTION The circular dichroism of DNA-ethidium bromide (EB) complexes is characterized by dye assoicated bands in the 290360 nm region. The CD of these complexes consists of a positive 308 nm band and a positive shoulder of the same band near 330 nm. However at high ratios of [EB]/[DNA] (1,2) or at high salt concentrations (2), alterations occur in the dye associated spectra. The most dramatic change occurs in the sign of the dichroism in the 295 nm region. Complexes,in buffer solution display a positive trough near 295 nm, while in concentrated salt solutions (2) and in solutions containing high [EB]/[DNA] ratios (R) (1,2) a negative 295 nm band is observed. Since both the presence of high ethidium bromide concentrations and high salt concentrations alter the conformation of DNA (3-11), the presence of the negative 295 nm CD band may reflect a conformational change in the DNA-EB complex. Recently we have completed an extensive investigation of the interaction of ethidium bromide with DNA in both LiCl and ethylene glycol (EG). We show that for all native DNA-EB complexes examined, a negative 295 nm CD band appears C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research in both 7.5 M LiCl and in 60% EG. Furthermore we show that the dye associated CD bands do not change as LiCl or EG is added. Therefore the regions where dye molecules are inserted between adjacent base pairs remain unaltered in their relative conformation while the rest of the DNA is undergoing a conformational transition. EXPERIMENTAL SECTION Six native DNA polymers, Micrococcus Lysodeikticus (Miles Biochemicals), Escherichia Coli and Salmon Sperm (Calbiochemicals), Salmon Testes, Chicken Blood, and Calf Thymus (P-L Biochemicals), plus two synthetic polynucleotides, poly [d(A-T)].poly [d(A-T)] and poly [d(G-C)]jpoly [d(G-C)] (P-L Biochemicals), were all used without further purification. The absorption extinction coefficients were taken from experimental data, if available, otherwise they were calculated from the empirical relation developed by Pysh and Richards (12). All DNA solutions were dialyzed against the appropriate solvent and adjusted to have a final absorbance of 1 at 260 rum. LiCl solutions of 0.01 M and 7.5 M were prepared by dissolving the weighed quantity of salt in doubly distilled water. DNA solutions in ethylene glycol were prepared by making a 60:40 (v/v) solution of ethylene glycol with 0.01 M LiCl. All solutions were buffered with Tris-HCl to keep the pH range between 7-8. Ethidium bromide solutions were prepared fresh before each experiment. The EB concentrations were determined spectrometrically from the molar extinction coefficient of 5600 lit/mol-cm at 480 nm (13). Variations in the [EB]/[DNA] ratio were made by adding small aliquots of a concentrated ethidium bromide (Sigma Chemicals) stock solution (usually 1.8 x 10 3 M) to the polynucleotide solution in the cell. The concentrations of bound dye were calculated from the shift in the absorption maximum of ethidium bromide upon binding to DNA (14). CD measurements were performed at room temperature The on a Cary 60 spectropolarimeter with a 6001 attachment. are reDNA-EB solutions measured ellipticities, 08, of the lated to molar ellipticities by [o]= 100 O0/lc where 1 is the 2402

Nucleic Acids Research pathlength in centimeters and c is the bound concentration of ethidium bromide, unless otherwise noted. Quartz cells with pathlengths between 0.1-1.0 cm were used. RESULTS Figure 1 displays the CD spectra of M. Lysodeikticus 4 V

E

0

M. Ly sodeiktic us

3 2 00

*0

0

00o -j I~

~

*0.OI M Li CI(R 0. 16) ~~~(m A60% EG (R=0.081) o 7.5 M Li CI(R =0.045)

-2

I

290

310

33.0 X(nm)

I

350

370

Figure 1. The circular dichroic spectra of M. Lysodeikticus DNA-ethidium bromide complexes. DNA-EB complexes in LiCl and in EG solutions. These results are typical of the data obtained for native DNA-EB complexes over a large range of base composition (29%-60% A-T). In all spectra, there are dye associated CD bands near 308 nm and a positive shoulder of this band near 330 nm. As the LiCl concentration is increased to 7.5 M or the EG concentration increased to 60%, the positive 308 nm band remains relatively unchanged in magnitude; the position of this band shifts slightly to higher wavelengths. The 330 nm positive shoulder does not change significantly for complexes in LiCl or in EG. Similar results are obtained for synthetic polynucleotides complexed with EB (Figure 2). The spectral region that is affected dramatically by the presence of concentrated LiCl or EG is near 295 rm. The effect of salt and ethylene glycol on the dye associated CD near 295 rnm, for both native and synthetic polynucleotides, is 2403

Nucleic Acids Research

7610 -JMb 12

0-

0

C

X .__

2

*0.O IM Li CI(R=0.15) 060%

EG

0 7.5M LiCI

290

(R=0.80)

310

(R=0.048)\\ 330

350

370

X (nm)

The circular dichroic spectra of poly d(A-T).poly d(A-T)-ethidium bromide complexes.

Figure 2.

summarized in Table I. All polymers in 0.01 M LiCl exhibit positive dichroism in the 295 nm region. As the LiCl concentration is increased to 7.5 M or the ethylene glycol increased to 60%, the 295 nm CD band becomes negative. However for both poly [d(G-C)].poly [d(G-C)] and poly [d(A-T)].poly [d(A-T)], this band remains positive throughout, although reduced in magnitude. Therefore these results demonstrate that both LiCl and EG alter the sign of the dichroism of the ethidium bromide band at 295 nm in a similar manner. The CD of the positive 308 nm band and the negative 295 nm band for DNA-EB complexes in 60% EG is dependent on the ratio of [EB]/[DNA]. Figure 3 displays the results obtained for M. Lysodeikticus in 60% EG at different R values. An increase in R leads to an increase in magnitude of both the 308 nm and 295 nm CD bands. Similar results have been obtained for DNA-EB complexes in 5.0 M NaCl (2). As in the case of complexes in NaCl, the overall shape of the ethidium bromide CD bands remains relatively unchanged as the LiCl or the EG content is increased. This is an indication that the nature of DNA-EB interactions is not significantly modified as the DNA conformation is altered. Furthermore, since the 308 nm band for DNA-EB complexes in 60% EG shows the same dependence on 2404

Nucleic Acids Research Table I

Variation of ellipticity a. near 295 rnm for DNA-ethidium bromide complexes in LiCl and in EG

%A-T

DNA M. Lysodeikticus E. Coli Salmon Testes Chicken Blood Calf Thymus Salmon Sperm poly dGC:dGC poly dATtdAT

0.01 M

(tO]

29 50

54 56 57 60 0 100

Calculated from bound ratio of

a.

LiCl

x 10

7.5 M

LiCl

60 % EG

4deg-cm3/decimole)

0.5

-1.4

1.0 1.0 1.0 0.5 0.8

-2.1

1.8

1.2

-0.7 -0.9 -1.0 -1.5 -2.1 -2.3 1.3

7.0

4.9

5.6

-2.6 -3.8 -4.4 -5.0

[EB]/IDNA].

0

E U 0 CQ

E 0c as M

0 It L__

290

310

330 X (nm)

350

370

Figure 3. The circular dichroic spectra of DNA-ethidium bromide in 60% ethylene glycol for various molar ratios of DNAbound ethidium bromide to DNA phosphate. R as for complexes in 0.01 M LiCl, the interactions that occur between DNA and ethidium bromide must be virtually unchanged. The CD spectra in the 230-360 nm region for Calf Thymus DNA-EB complexes are displayed in Figure 4. These results are typical of those obtained for all native DNA-EB com2405

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0

E U O

_10

O2

0

complexed wit

M Lin 0.01 Calf DA Thymus A60% hEG xrse o ~~~07.5 M Li CI C

240

280

320

litct

360

X (n m)

Figure 4. The circular dichroic spectra of ethidium bromide complexed with Calf Thymus DNA. The expressed ellipticity is based upon the nucleotide phosphate concentration. plexes in 0.01 M and 7.5 M LiCl and in 60% EG. [e] values were calculated using the nucleotide concentration. As the LiCl and EG concentration increases, the negative 295 nm band appears concurrent with the decrease in the positive 275 nm band. The 275 nm DNA band decreases in magnitude (0 30-50%) while the negative component of the same transition at 245 nm remains relatively unaffected. This behavior is analogous to the observed CD changes for uncomplexed native polynucleotides in 7.5 M LiCl and in 60% EG (Balcerski and Pysh, unpublished results). DISCUSSION

Polynucleotides complexed with ethidium bromide in 0.01 M LiCl exhibit dye associated CD bands in the 290-360 nm region that are independent of the DNA base composition. This is in agreement with previously published results (1,15). Complexes in 7.5 M LiCl and in 60% ethylene glycol also display CD bands that are invariant with base composition. Additionally, all native DNA-EB complexes in 0.01 M LiCl, in 7.5 M LiCl and in 60% EG display ethidium bromide CD bands that are highly dependent on the bound dye concentration. This dependence for ethidium bromide complexes in 60% EG is identical to that observed for Calf Thymus DNA-EB complexes in concentrated 2406

Nucleic Acids Research salt solutions (2).

That is, increasing the ratio of [EB]/ [DNA] monotonically increases the magnitude of all ethidium bromide bands. Therefore not only do ethylene glycol and LiCl interact with ethidium bromide complexes in a similar manner, but also the DNA-EB interaction in 60% ethylene glycol and in 7.5 M LiCl are unchanged from that in low ionic strength solutions. Since the dye associated bands at 295 rnm and 308 rm have the same dependence on the bound concentration of ethidium bromide and since they are located in the same spectral region, it is probable that these bands are the exciton components of the same optical transition (2). The negative component of this transition is not observed for DNA-EB complexes in buffer because the strong positive 275 nm DNA CD band washes out the much weaker 295 nm band. However as the salt or the EG content is increased or if the bound concentration of ethidium bromide is increased, the large positive 275 nm DNA band decreases in magnitude. Concurrent with this decrease in the 275 nm DNA band is the appearance of the negative 295 rm EB band. Since synthetic polynucleotides possess much larger CD band magnitudes than the native DNA polymers, the decrease in the 275 nm band is not sufficient to result in the appearance of the negative 295 nm band. Therefore it is the conformational change in the DNA that reduces the magnitude of the 275 rm CD band and subsequently urmasks the much weaker 295 nm band. When ethidium bromide is added to DNA solutions, there is an alteration in the DNA conformation due to the intercalation of dye molecules. While this conformational change is smaller for complexes in salt or in ethylene glycol than for complexes in buffer (16), the exact nature of this dye induced alteration is still undecided (4,17,18). Recent X-ray data (5) indicate that the intercalation of ethidium bromide between adjacent base pairs unwinds the backbone by 260. Winding of the helix occurs for polynucleotides in concentrated salt (6-11) or ethylene glycol (11,19,20) solutions. However the amount of winding associated with this conformational transition is less than 30. Therefore it 2407

Nucleic Acids Research appears that the conformational change in the DNA helix that results when ethylene glycol or LiCl is added to DNA-EB solutions does not affect the helix conformation in the regions where ethidium bromide molecules are intercalated. In summary, we show that the appearance of the negative ethidium bromide CD band at 295 nm results from the B-C conformational transition in DNA. This transition decreases the magnitude of the 275 rm DNA CD band and subsequently unmasks the weak ethidium bromide CD band at 295 nm. Furthermore we find that the ethidium bromide bands are relatively unaffected as the DNA conformation is altered. A model that is compatible with this finding suggests that the conformational transition that occurs when ethylene glycol or salt is added to DNA-EB solutions does not perturb the relative conformation between the dye molecules and adjacent base pairs. This study was supported by a grant from the

National Science Foundation (GB-40426). *

Present Address:

Department of Biochemical Science Princeton University 08540 Princeton, New Jersey

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