Biochemistry 1991, 30, 4042-4047
4042
Thermodynamic Characterization of Deoxyribooligonucleotide Duplexes Containing Bulges? Darryl A. LeBlanc and Kathleen M. Morden* Department of Biochemistry, Louisiana State University, Baton Rouge, Louisiana 70803 Received October 22, 1990; Revised Manuscript Received January 16, I991
Ultraviolet absorption techniques were used to study the thermodynamics of duplex formation for a D N A decamer, d(GCGAAAAGCG)-d(CGCTTTTCGC), and a series of related duplexes, each of
ABSTRACT:
which contains a bulged base centered in the A-T tract. Thermodynamic parameters were obtained from nonlinear least-squares fits of the melting curves and the concentration dependences of the melting temperatures. Duplexes containing a localized single-base bulge were found to be 3.5-4.6 kcal/mol less stable than the decamer at 37 OC. These results indicate that both the identity of the bulged base and the strand in which it is located may influence the amount by which the duplex is destabilized. Bulged bases located in the T-strand, d(CGCTTYTTCGC), in position Y, were observed to be slightly more destabilizing than those located in the A-strand, d(GCGAAXAAGCG), in position X. Bulged purines may be more destabilizing than bulged pyrimidines.
x e availability of synthetic oligonucleotides and the advancement of techniques applicable to the study of nucleic acid structure have led to a wealth of detailed structural information on the various families of nucleic acid helices. With this foundation, we can begin to explore the effects of various helical perturbations. Of particular interest are perturbations in nucleic acid helices where only one of the two strands contains an unpaired base or bases. This perturbation is referred to as a bulge, and the unpaired base is commonly known as a bulged base. A clear understanding of the factors which modulate the stability of helices with bulged bases would greatly facilitate research in several areas. Such knowledge is vital to testing Streisinger's model for the mechanism of frameshift mutation (Streisinger et al., 1966). It will also aid in the design of oligonucleotide probes by allowing the identification of alternative binding sites generated through the incorporation of one or more bulged bases. Many investigations aimed at bulges in DNA have utilized nuclear magnetic resonance (NMR) (Haasnoot et al., 1980; Pate1 et al., 1982; Morden et al., 1983, 1990; Hare et al., 1986; Roy et al., 1987; Woodson & Crothers, 1987, 1988a,b, 1989; van den Hoogen et al., 1988; Kalnik et al., 1989a,b, 1990; Nikonowicz et al., 1989) or X-ray diffraction (Joshua-Tor et al., 1988; Miller et al., 1988) techniques to obtain highly detailed structural information about the helical deformations introduced by the presence of the bulged base. However, a complete characterization of the effects that a bulged base has on duplex stability also requires an investigation of the global thermodynamic properties of duplex formation. Ultraviolet absorption techniques can be utilized to obtain thermodynamic parameters for the process of duplex formation (Marky & Breslauer, 1987). The stability of DNA duplexes has been investigated in this manner with the goal of predicting duplex stability based upon nucleotide sequence (Breslauer et al., 1986). UV absorption techniques have also been used to estimate the destabilizing free energies of bulges in both DNA (Morden et al., 1983; Woodson & Crothers, 1987) and 'This work was supported by National Institutes of Health Grant GM 38 I37 and Louisiana Education Quality Support Fund LEQSF(8689)-RD-A-12. D.A.L. was supported by a fellowship from the US. Department of Agriculture (Grant 87-GRAD-9-0091).
0006-296019 110430-4042$02.50/0
Scheme I
decamer
5 G C G m G C G 31 3'CGCTTTTCGCsI
GCGAAXAAGCG3 A-strand bulges, 3iCGCTT-TTCGC5D X = C, Tor G
5
GCGAA-AAGCG3 T-strand bulges, 3iCGCTTYTTCGC5i Y = C, A or G
5
1
1
RNA (Fink & Crothers, 1972; Longfellow et al., 1990). We describe below the thermodynamic characterization of a duplex DNA decamer and a series of related oligomers, each of which contains a bulged base centered in an A-T tract as shown in Scheme I. The melting characteristics of two individual single strands are also briefly discussed. EXPERIMENTAL PROCEDURES
Materials Synthetic DNA oligomers purified by semipreparative anion-exchange HPLC were purchased from the Midland Certified Reagent Co. (Midland, TX) as lyophilized single strands. Purity was confirmed in our laboratory by analytical strong anion-exchange HPLC. Oligomers were suspended in deionized, glass-distilled H 2 0 and used without additional purification. Methods Melting Curves. UV absorbance at 260 nm was monitored as a function of temperature from 5 to 95 OC for the thermal denaturation of the DNA oligomers. Experiments were performed using a Gilford Response I1 spectrophotometer. Absorbance readings were taken every 0.5 OC. The temperature of the cuvettes was controlled electronically by dual Peltier devices within the cuvette holder. The spectral bandwidth was 0.5 nm. All experiments were performed in 10 m M phosphate buffer, pH 7, containing 1.O M NaCl and 0.1 mM ethylenediaminetetraacetic acid (EDTA).' Appropriate individual strands in distilled H 2 0 at concentrations of 5-10 pM were mixed to form a dilute solution of the desired duplex, which EDTA, ethylenediaminetetraacetic acid; NLS, I Abbreviations: nonlinear least squares.
0 1991 American Chemical Society
Thermodynamics of Duplexes Containing Bulges
Biochemistry, Vol. 30, No. 16, 1991 4043
was then lyophilized and resuspended to generate a concentrated DNA stock solution. Samples for each melt were prepared by using the DNA stock, a concentrated buffer stock, and degassed, deionized, glass-distilled H20.Each duplex was studied at seven different concentrations ranging from 10 pM to 1 mM single strand. Each single strand was also studied independently, in the absence of the complementary strand, at several concentrations ranging from 5 to 500 pM. Measurements were made by using cuvettes with path lengths ranging from 0.01 to 1 .O cm, so that measured absorbances were between 0.6 and 1.8. The experiments were conducted according to the procedures described by Nelson et al. (1981). Absorbances obtained at 5 and 25 OC before and after each experiment were compared; samples which showed a greater than 2% increase in absorption (due to evaporation) were rejected and repeated. Extinction coefficients at 25 OC were calculated by nearest-neighbor approximation (Fasman, 1975). Extinction coefficients in units of mM-' cm-' at 25 OC are as follows: dGCGAAAAGCG, 104; dGCGAACAAGCG, 1 12; dGCGAATAAGCG, 114; dGCGAAGAAGCG, 116; dCGCTTTTCGC, 80.4; dCGCTTATTCGC, 94.4; dCGCTTGTTCGC, 9 1.1 ; dCGCTTCTTCGC, 87.6. Concentrations of mixtures of complementary strands were calculated from absorbances measured at 85 OC, where all strands exist only in the single-stranded state. Extinction coefficients at 85 OC were calculated by t(85 "C) = e(25 OC)[A(85 OC)/A(25 "C)] (Albergo et al., 1981). Data Analysis. The absorbance versus temperature plots were normalized to an absorbance of 1.OO at 85 OC and were fit by a nonlinear least-squares (NLS) routine using the method of Marquardt (Bevington, 1969) on a MicroVAX I1 computer. The extinction coefficients for the double- and single-stranded states (eQ and e,, respectively) are described by the equations: d T ) = 66, + m&T t,,(T)
= b,, + m,T
+ C, + C2T3
(1)
(2)
where T is the temperature in degrees kelvin and b and m represent the intercepts and slopes of the lines describing the temperature dependencies of e& and E , (the lower and upper base lines, respectively). The constants C , and C2are introduced to account for the nonlinear nature of the temperature dependence of the single-strand extinction coefficient, e,. Equation 2, in effect, describes the temperature dependence of the combined extinction coefficients of the two single strands in solution in the absence of duplex formation. Because our oligomers are non-self-complementary, the temperature dependence of the extinction coefficient for each strand can be determined experimentally. The upper base line required for the NLS fits of the melting curves for a particular duplex is obtained by summing the melting curves of the appropriate two individual strands and then normalizing to an absorbance of 1.00 at 85 OC. Upper base lines for all duplexes were derived in this manner. However, two of the single strands (dGCGAAGAAGCG and dGCGAATAAGCG) exhibit concentration-dependent hypochromism. The lowest concentrations were used to derive the singlestrand (upper) base lines, and hypochromism was observed only below 35 "C. A similar oligomer, dGCGAACAAGCG, does not display hypochromism under these conditions. Therefore, the behavior of dGCGAAGAAGCG and dGCGAATAAGCG at temperatures below 35 "C in the absence of hypochromism was approximated based on the melting curve of dGCGAACAAGCG. Each of the corrected curves was combined with the melting curve of dCGCTTTTCGC to produce the upper
base lines for the NLS fits of the melting curves of the Astrand G-bulge and A-strand T-bulge. The approximation resulted in only slight changes (- 1%) in the thermodynamic parameters obtained, probably because the melting temperatures of the heteroduplexes are well above 35 OC. The extinction coefficient ( e ) and absorbance ( A ) of a solution at any temperature T can be calculated as c ( T ) = A(T)/LCT = - a[%s(T)- eds(n1 (3) where L is the path length of the cell, CTis the total singlestrand concentration, (Y is the fraction of strands in the duplex state, and e, and tQ are given in eq 1 and 2. The term a is related to the equilibrium constant K for the association of two non-self-complementary strands to form a dimer by
'%s(n
+
(Y
= exp(-AHo/RT ASo/R) (4) (cT/2)(1 - a)* where, again, CTis the total single-strand concentration with each strand present in an equal concentration of cT/2, and (Y is the fraction of single strands in the duplex state. The parameters AHo and ASo are the changes in enthalpy and entropy, respectively, for the process of duplex formation (Marky & Breslauer, 1987). The six adjustable parameters for the nonlinear-least squares fits to eq 1-4 were AHO, ASo, m, b,, m&, and bds. Although m, and b, were variable parameters, significant variation from the experimental values was never observed, probably because the curvature of the single-strand base lines was maintained by the constants C1 and C, (eq 2). The thermodynamic parameters AHo and ASo for a particular duplex were calculated by averaging the values obtained from the fits of the seven experimental melting curves. The free energies of duplex formation (AGO)were calculated by using the standard thermodynamic relationship described by eq 5 . In cases such as this, when a variable is calculated AGO = AHo - T U o (5) from two highly correlated parameters, it can be determined with a high degree of accuracy, even if errors in the correlated parameters are large (Bevington, 1969). Errors in AHo and ASofrom the NLS fits can be as large as 1596, but because variations in these parameters are highly correlated, AGO can be calculated quite accurately near the T,, in most cases to within less than &5%. An alternative method for determining AHo and ASo is from the concentration dependence of the melting temperature, T, which is defined as the point at which half of the strands exist in the duplex state. A plot of Tm-I vs In (cT/4) is used to determine the thermodynamic parameters according to the equation: Tm-' = ( R / A H o ) In ( C T / ~ ) ASo/AHo (6)
K=
+
where R is the ideal gas constant and the other variables are as described above for eq 4. Melting temperatures were obtained from the NLS fits. In this case, errors in A W and ASo reflect errors in the slope and intercept and are less than or equal to 5%. The free energy of duplex formation at a specific T , can also be obtained (indirectly) from eq 6, by using the slope, intercept, and the chosen temperature to calculate the term In (CT/4). At the T,, the equilibrium constant K is equal to 4/CT (Marky & Breslauer, 1987), and AGO can be obtained from AGo(T,,,) = -RT, In K (7)
RESULTS Melting Curves and Thermodynamics. Experimental melting curves for the A-strand C-bulge at seven different
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Biochemistry, Vol. 30, No. 16, 1991
LeBlanc and Morden
Table I: Thermodynamic Parameters for Helix Formation -AHo (kcal/mol) -ASo (eu) -AG0(37 OC) (kcal/mol) Tm( 0 ~ at ) NLS" Tm-' vs In (cT/4)b NLS Tm-' vs In (cT/4) NLS Tm-lvs In (cT/4) CT = 100 pM perfect duplex 76 f 5 74 f 2 207 f 16 201 f 7 11.9 f 0.3 11.7 f 0.4 60.7 f 0.4 calcula ted' 86.7 215 15.1 A-strand 8.4 f 0.5 8.1 0.4 46.3 f 0.4 176 i 23 157 f 6 57 f 2 T-bulge 63 f 7 C-bulge 64 f 9 55 f 1 179 f 28 151 f 3 8.4 f 0.6 8.0 f 0.2 45.7 f 0.2 7.9 f 0.6 7.8 0.2 44.5 f 0.3 162 f 17 150 f 4 54 f 2 G-bulge 58 6 T-strand 8.0 f 0.2 7.9 0.1 44.6 f 0.1 155 i 23 I54 f 2 C-bulge 56 i 7 56 f 1 G-bulge 52 f 7 48 f 1 145 f 22 132 f 3 7.5 f 0.1 7.3 f 0.2 42.6 f 0.3 A-bulge 54 f 6 48 f 1 149 19 131 f 3 7.3 f 0.4 7.2 f 0.2 41.2 & 0.2 'Nonlinear least-squares fits. Standard errors from the NLS fits are 5 1 5 % for AH' and ASoand
