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Department of Chemistry and Biochemistry, Seton Hall University, South Orange, New Jersey 07079
Correspondence: Address reprint requests to Richard D. Sheardy at his present address, Dept. of Chemistry and Physics, Texas Woman's University, PO Box 425859, Denton, TX 76204. E-mail: rsheardy{at}twu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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700 cal/mol basepair for the enthalpy of the B-Z transition compares favorably with previously published results obtained by different techniques. DSC was used to monitor the duplex to single strand transitions for both oligomers under the different concentrations. These results indicated that methylation of the cytidine destabilizes (dm5C-dG)4 relative to (dC-dG)4. Coupling the DSC data with the ITC data allowed construction of a thermodynamic cycle which gives insight into the influence of both temperature and ionic strength on the heat content of the two DNA systems studied. Further, this study reveals the utility of using ITC for determinations of transition enthalpies with the appropriate choice of control. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) were the first to propose a right-handed double-helical conformation for DNA, now known as B-DNA from x-ray diffraction studies. Since then, it has been shown that under certain physiological conditions, double-helical DNA is highly polymorphic. In fact, nearly twenty other slight variations of DNA conformations exist, such as A-DNA, C-DNA, and Z-DNA. Perhaps the most radical change in conformation occurs when the B-DNA double helix undergoes a conformational transition to a left-handed conformation with the elimination of the major groove apparent in B-DNA. Pohl and Jovin (2) were the first to propose the existence of a left-handed double helix by observing an inverted circular dichroism spectrum of poly(dC-dG) under high salt concentrations. In 1979, Rich et al. (3) determined the structure of an alternating (GC) oligomer in the presence of Mg+2 and [Co(NH3)6]3+ by x-ray crystallography to be a left-handed helical structure, which maintains its Watson-Crick basepairing. Due to the zig-zag nature of the sugar-phosphate backbone, this conformation was designated as Z-DNA. Behe and Felsenfeld (4) studied the effects on the B-DNA to Z-DNA transition upon methylation of the cytidine residues at the 5 position in poly(dG-dC) using circular dichroism. They found that the transition occurred at a much lower ionic strength for poly(dm5C-dG) than for the unmethylated polymer, poly(dC-dG), when using Na+, Mg2+, or trivalent cobalt hexamine [Co(NH3)6]3+ as the Z-conformation inducer.
The importance in understanding the B-to-Z transition has led our group and others to study the thermodynamics involved in the transition. The Na+ induced B-to-Z transition of poly(dC-dG) as reported by Pohl and Jovin (2) was found to be independent of temperature over the range of 30–50°C. Their van't Hoff analysis yielded, under their conditions, an enthalpy near zero (0.0 ± 1 kcal/mol basepair) for the B-to-Z transition. On the contrary, Chaires and Sturtevant (5) reported a reversible thermally-driven B-to-Z transition for poly(m5dG-dC) upon raising the temperature from 18 to 50°C and determined an enthalpy of 
H = 0.61 ± 0.07 kcal/mol basepair by differential scanning calorimetry.
Our research entails observing conformational changes and their thermodynamic parameters associated with two self-complementary eight-basepair DNA oligomers, known as Z8A, (dC-dG)4, and Z8M, (dm5C-dG)4. Both double-stranded DNA oligomers are studied under low and high concentrations of Na+ and [Co(NH3)6]3+ to observe any conformational differences. Circular dichroism (CD) studies were used to determine the conformation of each oligomer under all conditions and isothermal titration calorimetry (ITC) was used to determine the enthalpy of the B-to-Z conformational transition of Z8M using Z8A as a control. Using differential scanning calorimetry (DSC) to determine the enthalpy of the duplex to single strand transition under various solution conditions allowed the construction of a thermodynamic cycle.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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,7). The ß-cyanoethylphosphoramidites and other DNA synthesis reagents were obtained from Glen Research, Inc. After detritylation, the purified DNA oligomers were lyophilized to dryness, reconstituted in water and subjected to exhaustive dialysis versus water and then lyophilized to dryness. Purity and size analysis of the oligomers was accomplished through a combination of analytical RPHPLC and/or denaturing polyacrylamide gel electrophoresis. The [Co(NH)3)6]Cl3 was obtained from Kodak (Rochester, NY) and used without further purification. The lyophilized samples were reconstituted in a 10 mM phosphate buffer (pH 7.0), 0.1 mM EDTA with NaCl or [Co(NH)3)6]Cl3 complex added to vary their concentrations. Samples were then heated to 90°C for 2 min followed by slowly cooling and equilibration for 48 h at 4°C.
Ultraviolet/visible spectroscopy
DNA concentrations were determined with a Varian CARY 100E (Varian, Mulgrave, Victoria, Australia) ultraviolet/visible spectrophotometer interfaced to Windows-based computer with Cary/Varian WinUVBio version 2.0 software. This spectrometer was equipped with a Peltier thermoelectric heating/cooling block, a multicell-transport device and a nitrogen-purged sample compartment. The DNA concentrations were determined by spectrophotometric absorbance using extinction coefficients, 
(L mol–1 cm–1 in basepairs), of 13,000 for Z8A and 13,620 for Z8M at 255 nm.
Circular dichroism spectropolarimetry
The conformation of each oligomer in the presence of low and high concentrations of Na+ or [Co(NH3)6]3+ was determined using an AVIV 60DS circular dichroism spectropolarimeter equipped with a multiple cell turret and Peltier heating/cooling device. The CD spectrum of each DNA oligomer ([DNA] = 0.5 – 1.0 µM) in standard phosphate buffer with 115 mM Na+, 2.0 M Na+ or 115 mM Na+ with 200 µM [Co(NH3)6]3+ were determined. All CD data, reported as the average of triplicate scans, were blank corrected and analyzed with Aviv DOS-based software and exported into a graph-plotting software.
Isothermal titration calorimetry
Isothermal Titration Calorimetry measurements were carried out using the isothermal titration module of CSC Model 4200 ITC (Calorimetry Sciences Lindon, UT) interfaced to a Gateway 2000 Pentium II MMX PC. CSC Run, Bindwork and Origin 4.0 software were used for data acquisition and analysis. The calorimeter was calibrated using the Tris base reaction with HCl, for which H = –11.38 kcal/mol for acid neutralization. Each experiment was set up such that 10 µl of 800 µM [Co(NH)3)6]Cl3 or 10 µl of 4 M NaCl was titrated into the sample cell containing either Z8A or Z8M at 115 µM duplex for up to a total of 25 injections.
Each titration experiment was performed so that the unitless parameter C (C = KbMt(0), where Kb is the binding constant and Mt(0) is the initial DNA concentration) had values between 1 and 200. The lag time between subsequent injections allowed equilibration such that the heat exchange between the sample and reference cell was no more than ±5 µW.
The heat absorbed or released due to the addition of aliquots of the salt solution to the DNA solution was measured by a thermoelectric device between the sample and reference cells. The heat associated with each injection was observed as a peak which corresponds to the power required to maintain the sample and the reference cells at identical temperatures. The peaks produced over the course of a titration can be converted to heat output per injection by integration and correcting for cell volume and sample concentration. The heat released for the ith injection, Q(i), is given by:
 | (1) |
where dVi is the volume of salt titrant added to the DNA solution and V is the cell volume. The total heat absorbed or released (Qt) was fit by a nonlinear least-square minimization method to the total DNA concentration (Xt) using the following equation:
 | (2) |
where n is the number of binding sites per monomer. Control experiments were carried out to determine the contributions to the enthalpy from the heat of dilution for both the Na+ and [Co(NH)3)6]+ into buffer or water alone. The net enthalpy for each injection was determined by subtraction of the component heats of dilution.
Differential scanning calorimeter
All heat capacity measurements were made with a Nano-II power-compensation differential scanning calorimeter from Calorimetric Sciences (Lindon, Utah). The calorimeter was interfaced directly to a Windows-based computer with "DSCRun" version 2.1.1 software (Calorimetric Sciences) for both data collection and DSC control. The reference solution for the reference side of the calorimetric measurement was derived from the buffer used to equilibrate the sample. Calorimetric measurements were performed as the average of two independent samples. Each DNA ([DNA] = 1.0 mM in basepairs in standard phosphate buffer plus added salts) sample was thermally scanned from 20°C to 95°C at 0.5°C/min over a minimum of 4 forward and 4 reverse scans. The samples were equilibrated for ten minutes at the upper (reverse scan) or lower (forward scan) set points between thermal scans. The raw thermographic data were blank corrected and integrated with "CpCalc" version 2.1 (Calorimetric Sciences) software. For most samples, the nature of the pretransition and posttransition baselines enabled integration of the calorimetric enthalpy with the use of a linear baseline.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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,7) has shown that the duplex formed from (dm5C – dG)4 is a right-handed double helical structure in 115 mM Na+ but undergoes a conformation transition to a left-handed double helical structure upon the addition of Na+ or [Co(NH3)6]3+. This DNA oligomer, designated here as Z8M, served as a control sequence for studies focusing on the formation of B-Z junctions in 16 basepair oligonucleotides (8–11). Fig. 1 (lower panel) shows the CD spectra of this oligonucleotide under different conditions. The CD spectrum under conditions of 115 mM Na+ is indicative of a right-handed conformation (i.e., B-DNA) with the characteristic peak at 280 nm and trough at 255 nm. However, in the presence of either 2 M Na+ or 200 µM [Co(NH3)6]3+, each CD spectrum is characterized by a trough at 295 nm and a peak at 278 nm, characteristic of a left-handed conformation (i.e., Z-DNA). However, the unmethylated analog, Z8A, remains in a right-handed conformation under these same conditions (Fig. 1, upper panel). Hence, Z8A does not undergo the B-to-Z transition in the presence of 2 M Na+ nor 200 µM [Co(NH3)6]3+. Behe and Felsenfeld (4) demonstrated that methylation of the cytidine residues dramatically enhances the facility of the B-to-Z transition in the alternating GC polymer as evidenced by much lower concentrations of Z-inducing agent required to attain the transition midpoint. For short oligomers, the number and order of GC repeats is also crucial for the ease of the transition (12,13). Thus, the observation of the transition for Z8M and not for Z8A at the concentrations of Z-inducer used (i.e., 2 M Na+ or 200 µM [Co(NH3)6]3+) is consistent with these findings. At concentrations >4 M Na+, Z8A did appear to undergo the B-to-Z transition; however, even at concentrations >200 mM [Co(NH3)6]3+, the oligomer did not undergo the transition (data not shown).
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,5,14–20) have reported the enthalpy of the B-to-Z transition obtained by a variety of experimental techniques. One of the main goals of this project was to determine the enthalpy of the transition using ITC based on the following premise. We have demonstrated by the CD studies above that Z8M will undergo the B-to-Z transition using either Na+ or [Co(NH3)6]3+ but Z8A does not undergo the transition at [Na+] <4.0 M or cobalt hexammine concentrations <200 mM. Hence, any enthalpy differences between Z8A and Z8M in the thermograms obtained from their titrations with these inducers must be due to conformational transitions.
Examination of the raw calorimetric data in the upper panels of Fig. 2 reveal that the titrations of both Z8A and Z8M with Na+ are endothermic, whereas those with [Co(NH3)6]3+ are exothermic at 25°C, as are the respective heats of dilution (data not shown). Subtraction of the heats of dilution from the respective raw data, followed by integration results in the isotherms shown in the lower panels of Fig. 2. Titration of Z8A with either Na+ or [Co(NH3)6]3+ results in a fairly broad isotherm whereas the titration of Z8M with either inducer appears to have a sharp, cooperative transition. In addition, titration of Z8M with Na+ is more endothermic than titration of Z8A, whereas titration of Z8A with [Co(NH3)6]3+ is more exothermic than titration of Z8M. The fitting parameters for these isotherms, Kb and n, are given in Table 1 and the resultant enthalpies (Hobs,Z8A and Hobs,Z8M) for these titrations in Table 2.
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,7). Our previously published work also reported that the Na+ induced transition for Z8M is best described as three state (via deconvolution of the CD titration data). We have also determined by the same approach that the [Co(NH3)6]3+ induced B-to-Z transition is also three state (data not shown). The formation of the intermediate state is likely due to dehydration before the conformational transition. The apparent thermodynamic transition observed here with Z8A most likely corresponds to the formation of a semidehydrated state. Hence, a transition for Z8A is observed calorimetrically that is not observed optically. Regardless of any type of transition, increasing the concentration of Na+ in a DNA solution should be enthalpically unfavorable as observed here due to the release of water from the duplex. Increasing the concentration of [Co(NH3)6]3+ will also induce the release of water from the duplex. However, this enthalpically unfavorable process apparently is compensated by the enthalpically favorable binding of the [Co(NH3)6]3+ to the DNA backbone as indicated by the ITC determined enthalpy.
Although the B-to-Z transition is three state, we are only interested in the initial and final states. Thus, we assume that the difference in total enthalpy between Z8A and Z8M upon titration with either Na+ or [Co(NH3)6]3+ is the transition enthalpy. In other words,
 | (3) |
where Hobs is the observed calorimetric enthalpy for Z8M, Hbz is the enthalpy for the transition itself and Hpe is the enthalpy for all other possible processes such as uptake or loss of Na+, uptake or loss of water, and the binding of [Co(NH3)6]3+ when titrating with that inducer. The assumption is that Hpe is the same for Z8A and Z8M. In other words,
 | (4) |
As seen in Table 2, the enthalpy of the transition is 700 cal/mol basepair when inducing with Na+ and 720 cal/mol basepair when inducing with [Co(NH3)6]3+. Adding validity to our approach is the comparison of our results of previously published work (Table 3). Our transition enthalpy of 0.70 kcal/mol basepair obtained at 25°C using either Na+ or [Co(NH3)6]3+ compares quite favorably with the results of Chaires and Sturtevant (5) and is close in agreement in sign and magnitude with the other studies, although obtained by different techniques (i.e., ITC versus CD, DSC, or NMR).
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Regardless of the inducer used, the B-to-Z transition is accompanied by the release of both water, as noted above, and Na+, due to the lower charge density of Z-DNA, from the duplex into the bulk solution. Such release is entropically favorable. We have previously shown that the free energy for the Na+ induced B-to-Z transition for Z8M can be calculated for any [Na+]. As noted above, SVD (singular value decomposition) of the CD spectra obtained in the titration of Z8M with Na+ indicated that the transition from B-to-Z is best described as three state:
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The free energy for each transition can be calculated from the respective equilibria constants, which are obtainable from the fit of the data in the fraction of transition versus concentration of cation plot. The total free energy of the B-to-Z transition is simply the sum of the free energies for the individual steps (6). Analysis of the transition profile induced by [Co(NH3)6]3+ also indicates a three state transition.
Using the titration end point of 2.0 M Na+, the free energy of the B-to-Z transition is calculated as –2.6 kcal/mol bp. Upon application of the Gibbs relationship, TS = H – G, we obtain the TS contribution of +3.5 kcal/mol bp at 298K. Proceeding in a similar fashion, one obtains the free energy of transition for Z8M induced by [Co(NH3)6]3+ to be –4.3 kcal/mol bp at 200 µM [Co(NH3)6]3+ (the end point of the titration) giving a TS contribution of –5.0 kcal/mol bp at 298K. Thus, the B-to-Z transition is entropically driven for both Na+ and [Co(NH3)6]3+ induced transitions. The complete thermodynamic values for the B-to-Z transition are reported in Table 4.
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Hls-melt for the low salt transitions and Hhs-melt for the high salt transitions. The enthalpies for isothermally changing the conditions from low salt to high salt are designated as Hlt-conf for the low temperature transitions (experimentally determined) and Hht-conf (calculated by difference) for the high temperature transitions. Several observations can be made from the data in Table 5. First, methylation of the cytidine base enthalpically destabilizes the duplex. This destabilization can be attributed to the presence of the methyl group in the major groove giving rise to a less hydrated groove. Second, both duplexes are more enthalpically stable in the higher salt concentrations, as expected (22). Finally, the change in the state of the system from low salt to high salt is more endothermic for Z8M than for Z8A when using just Na+, regardless of temperature. However, when using [Co(NH3)6]3+, the change in the state of the system from low salt to high salt is less exothermic for Z8M than for Z8A at low temperature but more exothermic at high temperature. Thus, the binding of the cobalt complex is influencing the thermodynamic state of both the duplex and single strands in different ways. Finally, the last column in Table 5 indicates the difference in enthalpy between the low and high salt conditions at temperatures where the oligos are single stranded. Thus, the application of the thermodynamic cycle allows determination of enthalpy differences that may not be directly obtainable experimentally.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Submitted on March 23, 2006; accepted for publication July 28, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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2. Pohl, F. M., and T. M. Jovin. 1972. Salt-induced transitions between two double-helical forms of oligo(dC-dG). J. Mol. Biol. 67:375–396.[CrossRef][Medline]
3. Wang, A. H., G. J. Quigley, F. J. Kolpak, J. L. Crawford, J. H. van Boom, G. van der Marel, and A. Rich. 1979. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature. 282:680–686.[CrossRef][Medline]
4. Behe, M., and G. Felsenfeld. 1981. Effects of methylation on a synthetic polynucleotide: the B-Z transition in poly(dG-m5C) poly(dG-m5C). Proc. Natl. Acad. Sci. USA. 78:1619–1623.
5. Chaires, J. B., and J. M. Sturtevant. 1986. Thermodynamics of the B-to-Z transition in poly(dG-m5dC). Proc. Natl. Acad. Sci. USA. 83:5479–5483.
6. Sheardy, R. D., D. Suh, R. Kurzinsky, M. J. Dotycz, A. S. Benight, and J. B. Chaires. 1993. Sequence dependence of the free energy of BZ junction formation in deoxyoligonucleotides. J. Mol. Biol. 231:475–488.[CrossRef][Medline]
7. Calderone, D. M., E. J. Mantilla, M. Hicks, D. H. Huchital, W. R. Murphy, Jr., and R. D. Sheardy. 1995. Binding of Co(III) to a DNA oligomer via reaction of [Co(NH3)5(OH2)]3+ with (5medC-dG)4. Biochemistry. 34:13841–13846.[CrossRef][Medline]
8. Sheardy, R. D. 1988. Preliminary spectroscopic characterization of a synthetic DNA oligomer containing a B-Z junction at high salt. Nucleic Acids Res. 16:1153–1167.
9. Sheardy, R. D., and S. A. Winkle. 1989. Temperature dependent CD and NMR studies on a synthetic DNA oligomer containing a B-Z junction at high salt. Biochemistry. 28:720–725.[CrossRef][Medline]
10. Sheardy, R. D., N. Levine, S. Marotta, D. Suh, and J. B. Chaires. 1994. A thermodynamic investigation of the melting of B-Z junction forming DNA oligomers. Biochemistry. 33:1385–1391.[CrossRef][Medline]
11. Otokiti, E. O., and R. D. Sheardy. 1997. Sequence effects on the relative thermodynamic stabilities of B-Z junction forming DNA oligomers. Biophys. J. 73:3135–3141.
12. Manzini, G., L. E. Xodo, F. Quadriffolglio, J. H. van Boom, and G. A. van der Marel. 1987. dC-dG alternating oligonucleotides: thermodynamic and kinetic aspects of the B-Z transition. J. Biomol. Struct. Dyn. 4:651–662.[Medline]
13. Chen, F.-M. 1988. Effects of A:T basepairs on the B-Z conformational transition of DNA. Nucleic Acids Res. 16:2269–2281.
14. Klump, H. H. 1986. Thermodynamics of left-handed helix formation. FEBS Lett. 196:175–196.[CrossRef][Medline]
15. Holak, T. A., P. N. Borer, G. C. Levy, J. H. van Boom, and A. H.-J. Wang. 1984. 31P NMR analysis of the B-to-Z transition in double stranded (dG-dC)3 and (dC-dG)4 in high salt solution. Nucleic Acids Res. 12:4625–4635.
16. O'Connor, T. R., D. S. Kang, and R. D. Wells. 1986. Thermodynamic parameters are sequence dependent for the supercoil induced B-to-Z transition in recombinant plasmids. J. Biol. Chem. 261:13302–13308.
17. Feigon, J., A. H.-J. Wang, G. A. van der Marel, J. H. van Boom, and A. Rich. 1984. A one- and two-dimensional NMR study of the B-to-Z transition of (m5dC-dG)3 in methanolic solution. Nucleic Acids Res. 12:1243–1263.
18. Klump, H. H., E. Schmid, and M. Wosgien. 1993. Energetics of Z-DNA formation in poly d(A-T), poly d(G-C) and poly d(A-C) poly d(G-T). Nucleic Acids Res. 21:2343–2348.
19. Tran-Dinh, S., J. Taboury, J.-M. Neumann, T. Huynh-Dinh, B. Genissel, B. Langlois d'Estaintot, and J. Igolen. 1984. 1H NMR and circular dichroism studies of the B and Z conformations of the self-complementary deoxyhexanucleotide d(m5C-G-C-G-m5C-G): mechanism of the Z-B-coil transitions. Biochemistry. 23:1362–1371.[CrossRef][Medline]
20. Chaires, J. B., and J. M. Sturtevant. 1988. Thermodynamics of the B-to-Z transition in poly(dG-dC). Biopolymers. 27:1375–1387.[CrossRef][Medline]
21. Matulis, D., I. Rouzina, and V. A. Bloomfield. 2000. Thermodynamics of DNA binding and condensation: isothermal titration calorimetry and electrostatic mechanism. J. Mol. Biol. 296:1053–1063.[CrossRef][Medline]
22. Record, M. T., Jr., S. J. Mazur, P. Melacon, J.-H. Roe, S. L. Shaner, and L. Unger. 1981. Double helical DNA: conformations, physical properties, and interactions with ligands. Annu. Rev. Biochem. 50:997–1024.[CrossRef][Medline]
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