Vol. 148, No. 2, 1987 October 29, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 790-794

BASE STACKING AND MOLECULAR POLARIZABILITY:

EFFECT OF A METHYL GROUP IN THE 5-POSITION OF PYRIMIDINES

Lawrence C. Sowersl, Barbara Ramsay Shaw and W. David Sedwick’

Departments of Medicine and Chemistry, Duke University, Durham, N.C.

Received August 26, 1987

Substitution of a methyl group in the 5-position of pyrimidines increases melting temperatures and modifies biological properties of DNA. Increased DNA stability is often attributed to hydrophobic interactions between water and the methyl group. However, we present evidence that the major effect of methyl substitution is to increase the molecular polarizability of the pyrimidine, thereby increasing the base stacking. Experimentally determined base stacking interaction constants for free bases in water are shown to correlate well with calculated molecular polarizability and DNA melting temperatures. 0 1987 Academic Press, Inc.

Chemical substitution at the 5-position of pyrimidines affects both physical and

biological properties of DNA. Replacement of T by U in DNA may occur via

incorporation of dUTP, particularly in cells treated with antifolate chemotherapy agents

(1). The presence of uracil in DNA reduces template-primer activity for DNA

polymerases and uracil containing oligonucleotides have significantly reduced melting

temperatures (2-4).

Cytosine residues in specific DNA sequences may be enzymatically methylated in

the 5-position in vivo (5). Cytosine methylation in some DNA sequences is correlated

with supressed gene activity (5) and 5-methylcytosine (m5C) containing oligonucleotides

have significantly increased melting temperatures (6,7). Poly(dG)-poly(m’C) is the most

thermodynamically stable of all known oligodeoxynucleotides (7).

What is the physical basis for the enhanced stability of methylated DNA? In water,

DNA helix stability is due predominantly to base stacking interactions (8-11). Because

base stacking occurs only in aqueous solution, and base residues stack within the interior

of the DNA helix, base stacking has often been considered largely “hydrophobic” in

nature (11-14). It has been suggested that the increased DNA stability conferred by alkyl

substituents is due to an increase in “hydrophobic forces”, in analogy to the stabilizing

effect of neighboring apolar side chains in polypeptides (4,6). Alkylated bases in solution

’ Correspondence should be addressed to L.C.S., Molecular Biology Division, University of Southern California, Los Angeles, CA, 90089-1483 2 Present address: Division of Hematology and Oncology, Case Western Reserve University Medical School, Cleveland, OH 44106.

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved. 790

Vol. 148, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

also show increased stacking associations which have been attributed to “hydrophobic

interactions” (15,16).

There are several lines of evidence which suggest that hydrophobicity may not

adequately explain the relative stability of methylated and unmethylated DNA. First,

the methyl group in the 5-position of pyrimidines is on the surface of the DNA molecule,

projecting into the major groove and not into the interior of the molecule. Second, DNA

melting temperatures show a strong correlation with calculated stacking interaction

energy (10,17). While a hydrophobic contribution was observed, it was independent of

base composition (lo), and thus is unlikely to explain the methyl group effect. Third, the

relative water solubility of the pyrimidines is contrary to expectation. One would

predict that methylation would decrease water solubility. However, substitution of a

hydrcgen atom with a methyl group INCREASES water solubility: T is twice as soluble as

U, and m5C is ten times more soluble than C (Table 1).

We propose that increased water solubility, increased base stacking in solution and

increased DNA melting temperatures resulting from methyl substitution may be

explained by molecular polarizability. Molecular polarizability is a measure of the ease

with which a dipole moment may be induced in the molecule. The importance of

polarization forces in base stacking interactions has been suggested in several studies

(18-23). Since molecular polarizability increases upon substitution with a methyl group

(24, 25), the increased polarizability of methylated bases could enhance base stacking. In

this study, we demonstrate that 1) base stacking interactions in water show a strong

correlation with molecular polarizability and 2) stacking energy differences between

methylated and unmethylated pyrimidines in water are the same as those determined

from DNA melting temperatures.

MATERIALS AND METHODS: All pyrimidines and adenine were purchased from Sigma Chemical Co. Compounds were chromatographically pure and were used without further purification. Concentrations of single component nucleobase solutions were spectrophotometrically determined on a Cary 219 spectrophotometer. Concentrations of nucleobases in complex solutions were measured by UV absorbance after separation of components by HPLC. Separations were obtained with a Partisil-10 SCX cation exchange column eluting with 0.045 M phosphate buffer, pH 2.3. Stock solutions of pyrimidines were prepared in 0.045 M phosphate buffer, pH 7.2, containing 10 mM cacodylate as a bacteriostatic agent. Complex mixtures were prepared by serial dilution of stock pyrimidine solutions and mixtures with a fixed amount of excess adenine. Complex mixtures were shaken at room temperature for three days, and then centrifuged, millipore filtered, and analyzed.

Solubility diagrams were constructed by plotting the increase in adenine concentration as a function of pyrimidine concentration. Stacking association constants were calculated from the slopes of the lines in the phase solubility diagrams as previously

described (21,26,27).

RESULTS: Stacking association constants (K 1:1) of the pyrimidines with adenine

were determined by measuring the ability of the pyrimidine to increase the solubility of

adenine. Stacking association constants are reported in Table 1. The order of association with adenine is m5C > T > C > U. The values of K1:l determined here for the

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Vol. 148, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1

Physical properties of the naturally occurring pyrimidines

m5C T C IJ

El3 6.38 6.07 4.72 4.16

12,12+ 11.48* 10.27* 9.63+

water solubility (g/lOOml)*

UV absobance max. (nm)

350 69.3 31.7 32.1

275 267 265 260

* Calbiochem document No. 4955 (1976) SR. No. 7816-0058 + See text * From reference 24

A/C and A/U interactions are similar to values reported previously by Nakano and

Igarashi (21), however, our value for the A/T interaction is somewhat lower. The parallel

study with G could not be done due to the low solubility of G in water.

Miller and Savchik (24) have calculated the molecular polarizability of C and T to

be 10.27 and 11.48 pi3, respectively. The change in molecular poiarizability upon

substitution of a hydrogen atom for a methyl group is + 1.85 a3, and this value was used

in order to estimate the molecular polarizability of U and m5C (Table 1). The order of

calculated molecular polarizability for the pyrimidines is the same as that observed for

the stacking interaction constants. The relationship between these parameters is shown

graphically in Fig. 1.

2.0 -

m5C /’ 1.9 -

T +

/

t

/ 1.8 - /

/ v- / ;-‘1.7- ,

Y /

= 1.6- c /’ /

1.5 - u / 4

t

/

1.4- / /

/ 1.3 - I I I 1

9 10 11 12 13

molecular polarizability h3

Fig. 1. Relationship between calculated molecular polarizability and the natural

logarithm of experimentally determined stacking interaction constants (Klzl) for

pyrimidine-adenine interactions in aqueous solution: U, uracil; C, cytosine; T, thymine;

and 5-m5C, 5-methylcytosine.

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Vol. 148, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION: In aqueous solution, bases associate with one another by vertical

stacking and not by hydrogen bonding (28). We have reinvestigated base stacking

interactions between pyrimidines and adenine in aqueous solution with the solubility

enhancement technique. Previously, radioisotopes have been required in such studies (21,26); however, here we have utilized HPLC which is ideally suited for quantitative

analysis of complex mixtures. An advantage of the solubility enhancement technique is

that it allows examination of the base-base component of the stacking interaction in the

absence of sugar and phosphate considerations.

Theoretically, the magnitude of base-water and base-base interactions are the sum

of several components, including electrostatic, polarization and dispersion forces

(9,19,23). The magnitudes of the polarization and dispersion interactions are directly

proportional to molecular polarizability (9). An increase in molecular polarizability of a

pyrimidine, due to methyl substitution, will increase the magnitude of the polarization

(dipole-induced dipole) interaction of the pyrimidine with water. This may explain why

substitution with a methyl group INCREASES water solubility of pyrimidines.

Similarly, an increase in molecular polarizability increases both dipole-induced

dipole and dispersion components of base-base stacking interactions (18-23). If these components dominate DIFFERENCES in the magnitude of the stacking interaction

between pyrimidines, a linear correlation should exist between the natural log of the

stacking equilibrium constant and the molecular polarizability. Such a correlation was

found and is presented in Fig. 1. Not only can the increase in base stacking due to

methyl-group substitution be attributed to increased molecular polarizability, but the

stacking tendency of all the naturally occurring pyrimidines was found to increase

linearly with increasing molecular polarizability. Molecular polarizability for

pyrimidines is also related to UV absorbance maxima (Table 1) and thus UV spectra may

give an estimate of relative molecular polarizability in cases where the value has not

been calculated.

Is there a relationship between stacking interaction constants for free bases in

solution and melting temperatures of oligonucleotides? Qualitatively, it is known that

methylated duplexes melt at higher temperatures than the corresponding unmethylated

oligonucleotides, (3,4,6,7) consistent with the trends presented here. Quantitatively, the

relationship also holds. The stacking interaction constants given in TABLE 1 may be

expressed as free energy differences at 25’C. We find that, in the uracii series, the

difference in stacking energy between T and U is 0.22 kcal/mol and between C and m5C

is 0.18 kcal/mol. Free energy differences have also been determined from

oligonucleotide melting temperatures (10). The difference between T and U containing

oligos is 0.24 kcal/mol and for C vs m5C containing oligos, the difference is 0.19

kcal/mol, the methylated oligos (T and 5-MeC) being the more stable in each case. Thus,

for both free bases and DNA, the same relative order of stability and the same energy

differences were obtained. This suggests that the increased stability of oligos arises from the same forces which increase the stability of the free bases. Because

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Vol. 148, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

methylation leads to an increase in base stacking for free bases, which in turn correlates

with an increase in molecular polarizability, we suggest that the major effect of

methylation is to increase the stability of oligos by increasing the molecular

polarizability of the bases.

1.

2. 3.

4.

5. 6. 7.

a. 9. 10.

11. 12. 13. 14.

15. 16. 17. la. 19. 20. 21. 22.

23.

24. 25. 26. 27. 28.

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