Microcalorimetric Investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)]

Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

http://www.jbsdonline.com

Thermodynamic parameters of melting process (∆Hm, Tm, ∆Tm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A‑C)).poly(d(G‑T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A‑C))poly(d(G‑T)). It was shown that TOEpyp(4) revealed GC specificity, it in‑creased Tm of satellite fraction by 24 ºC, but ZnTOEpyp(4), on the contrary, predomi‑nately bound with AT‑rich sites and increased DNA main stage Tm by 18 ºC, and Tm of poly(dA)poly(dT) increased by 40 ºC, in comparison with the same polymers without por‑phyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes – strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode – strong binding – took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by ~ 30‑40 ºC. Invariability of ∆Hm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01‑0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four‑fold decrease of calf thymus DNA melting range width ∆Tm caused by increase of added ZnTO‑Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well‑known dependence, according to which ∆T~TGC‑TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank‑Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193‑205 (1974)). Poly (d(A‑C))poly(d(G‑T)) in the pres‑ence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.

Introduction

In the past decade a significant progress has been achieved in the understanding of molecular interaction mechanisms of water soluble cationic porphyrins with nucleic acids (1‑6). The increased interest is connected with unique properties of these porphyrins. Being small flat molecules, which are able to selectively bind with membrane surface of tumor cells and to penetrate into cellular nucleus, they invade chromatin DNA (7). The binding of cationic porphyrins with DNA double helix has two modes (8, 9) – strong one (intercalation) and weak one (external binding). The latter occurs inside of a minor groove and causes oxidizing and free‑radical attack on deoxiribous sugars, in particular, at C1’ position and induces DNA strand cleavage. In case of non‑planar metalloporphyrins, due to close neighbor‑hood of metal ion (coordinated in the center of porphyrin) to C1’ and O3 atoms of sugar skeleton, the potential of DNA cleavage is increased (10). The increase of DNA cleavage is also reached by daylight irradiation of DNA‑metalloporphyrin complex due to photosensible properties of porphyrines and at present this effect

Journal of Biomolecular Structure &Dynamics, ISSN 0739-1102Volume 25, Issue Number 4, (2007)©Adenine Press (2007)

Monaselidze, J.1,*

Majagaladze, G.1Barbakadze, Sh.1Khachidze, D.1Gorgoshidze, M.1Kalandadze, Y.2Haroutiunian, S.3Dalyan, Y.3Vardanyan, V.3

1E. Andronikashvili Institute of Physics6, Tamarashvili StreetTbilisi, 0177, Georgia2Tbilisi State University1, Chavchavadze Ave.Georgia3Yerevan state University1, Alex Manoogian StreetArmenia

419

Phone: (+995 32) 326858Fax: (+995 32) 391494Email: mon@iphac.ge

420

Monaselidze et al.

is successfully used in treatment of various diseases (11). The intercalation mainly occurs between GC pairs. It was shown that water soluble cationic metalloporphy‑rin TMPyP(4) intercalated in guanine quadruplexes in human telomere sequences, resulting in inhibition of telomerase activity and this causes prolong survival and inhibits tumor growth rate in vivo (12).

The given work is devoted to determination of thermodynamic characteristics of melting processes of the complexes: DNA‑TOEPyP(4), DNA‑ZnTOEPyP(4), poly(dA)poly(dT)‑ZnTOEPyP(4), and poly[d(A‑C)].poly[d(G‑T)]‑ZnTOEPyP(4) in wide range of porphyrin concentration. A goal of this work is a deeper under‑standing a mechanism of the complex formation of TOEPyP(4) and its Zn complex with DNA, in particular, to find out: if a formation of complex occurs, mainly, at AT and GC sites, as it is affirmed in the work (13) and if the binding has a se‑quence‑specific character; if porphyrins under studied cause rapprochement of AT and GC pairs thermostability; and how they influence on the double helix DNA structural organization and what is an energetic contribution of the complex for‑mation in DNA melting total energy (∆Hm). For fulfillment of our task we chose objects, complexes of which were investigated with optical methods and it was shown that physical and chemical characteristics of TOEPyP(4) are identical to the same characteristics of TMPyP(4) (3).

Materials and Methods

The preparations of calf thymus DNA were kindly presented by Professor D. Lan‑do. The DNA was isolated in his laboratory (Institute of Bioorganic Chemistry BAS, Minsk). Molecular weight: 19 MDa; protein content: ~0.5%; RNA: <0.2%; hyper chromium effect: ~39%. Preparations were prepared in 20 mM phosphate buffer, pH 7.02. Porphyrin concentrations in solutions range from 2 × 105 – 10‑4, DNA concentration is from 0.18 to 0.09%, poly(dA)poly(dT) and poly[d(A‑C)].poly[d(G‑T)], 0.03‑0.08.

The measurements were performed using a differential scanning microcalorimeter with sensitivity of 0.1 μW (14‑16). The volume of measuring vessel was 0.30 cm3, the heating rate was 0.55 ºC/min, and the temperature range of measurements, 25‑140 ºC. The exactness of the temperature measurements was not less than 0.05 ºC. The error in determination of melting enthalpy (∆Hm) was not more than 6%. The microcalirimeter (DSC) processor was equipped with all software needed for determination of the thermodynamic parameters of investigated solutions and de‑convulation of calorimetric curves. The synthetic polymers poly(dA)poly(dT) and poly[d(A‑C)].poly[d(G‑T)] were purchased from Serva chemical company. The water soluble cationic meso‑tetra‑(4‑N‑oxyethylpyridyl) porphyrin (MW = 980 D) and its Zn complex (MW = 1003 D) were synthesized by Dr. R. Ghazaryan in De‑partment of Pharmaceutical Chemistry of Yerevan State Medical University (3).

Results

Melting of DNA-TOEPyP(4) Complex

Figure 1 presents the dependences of dQ/dT on temperature for DNA solutions without porphyrins (a) and in presence of different concentrations of TOEPyP(4). It is known that the melting curve has a complex character and it is connected with a block type building of calf thymus DNA (17). The transition of helix‑coil DNA of higher eukaryotes at low concentrations of neutral salts is carried out by successive melting of DNA sites with various content of GC b.p., and the higher is the percent‑age of GC b.p., the higher is Tm of this block. It’s known that the melting of calf thymus DNA main stage, which contains less GC‑pairs than its satellite fraction, occurs at Td = 76.5 ºC, and the satellite fractions enriched with GC‑pairs melt at Tm = 84.8 ºC and 86.7 ºC, accordingly (17).

Figure 1: Microcalorimetric melting curves of calf thymus DNA‑TOEPyP(4) solutions, calculated per gram of dry mass (20 mM phosphate buffer, pH 7.02, CDNA = 0.18%). (1) Native DNA, (2) r = 0.02, (3) r = 0.08, (4) r = 0.22.

421Micocalorimetry on

DNA-Porphyrin Complexes

As it is seen from Figure 1 a change of the melting curve profile and shift of the curve to higher temperatures are observed already at r = 0.02, where r is molar ratio of porphyrin to DNA b.p. At r = 0.08 a clear peak at 84.8 ºC and a shoulder at 86.7 ºC, which correspond to melting of the DNA satellite fraction, are joined into one wide peak with Tm = 96.5 ºC. The shift of the satellite fraction Tm to higher temperatures is more significant than shift of the main one, in the range 0.01 < r < 0.1, and it equals to 15 and 4 for DNA without porphyrin, accordingly (Fig. 1, 2). The further increase of porphyrin concentration where 0.1 ≤ r ≤ 0.2 – causes a smoothing of the main stage profile and a sharp increase of its Tm in comparison with satellite fraction Tm. In particular, Tm of the main stage increases by 17 ºC and Tm of satellite fraction increases only by 7 ºC (Fig. 1, curve 4 and Fig. 2), Fig‑ure 2 presents the dependences Tm = f(r) only for the main stage and satellite peak which are clearly expressed on DNA melting curve with Tm = 76.5 ºC and 84.8 ºC, accordingly. The total melting enthalpy (∆Hm) of the complex DNA‑TOEPyP(4) (for 0.01 < r < 0.22), calculated from the area under the curves are presented in the Figure 3. As it is seen from figure ∆Hm of DNA‑TOEPyP(4) complex increases from 11.6 ± 0.6 to 13.0 ± 0.6 cal/g when r increases from 0.01 to 0.1, the further increase of r up‑to 0.22 does not cause the increase of ∆Hm. We observe complex aggregation at higher values of r.

Melting of the Complex DNA-ZnTOEpyp(4)

The addition of ZnTOEPyP(4) in DNA solution caused additional stabilization in comparison with the complex without Zn(II) ions, as well as more serious change of the melting curve profile, where the peaks in the satellite DNA disappeared on the melting curve of the complex already at r = 0.0135.

The melting process has two stage of transition in the range 0.01 < r < 0.165. As the concentration of ZnTOEpyp(4) increases together with increase of both stages Tm, the rearrangement of heat between them is observed and the heat lost in the first low‑temperature stage is added to the second high‑temperature stage. The integral heat is increased insignificantly in this range of r (Fig. 3). The melting curve is characterized by asymmetry in the range 0.18 < r < 0.22 (Fig. 4, curve 5). The transition parameters at r = 0.25 are equal to ∆H = 12 ± 0.6 cal/g, Tm = 99 ± 1.0 ºC, ∆T = 2.9 ± 0.1 (Fig. 4.).

Figure 2: Dependence of the main stage and satellite fraction Tm of calf thymus DNA on r.

Figure 3: Dependences of calf thymus DNA ∆H on r; l, DNA with ZnTOEPyP(4); p, DNA with TOEPyP(4).

Figure 4: Microcalorimetric melting curves of calf thy‑mus DNA‑ZnTOEPyP(4) solutions, calculated per gram of dry mass (20 μM phosphate buffer, pH 7.02, CDNA = 0.16%). (1) Native DNA, (2) r = 0.0135, (3) r = 0.16, (4) r = 0.21. Insertion‑deconvulation of the curve 3.

Figure 5: Dependence of Tm and ∆Hm of I and II transition stages of calf thymus DNA on r. ° and r, Tm(I) and Tm(II); l and p, ∆H(I) and ∆H(II).

422

Monaselidze et al.

Melting of the Complex polyd(A)polyd(T) and poly[d(A-C)]poly[d(G-T)] with ZnTOEPyP(4)

The microcalorimetric studying was carried out to elucidate a binding character of ZnTOEPyP(4) with different sequences of base pairs in DNA double helix syn‑thetic polydeoxyribonucleases poly(dA)poly(dT) and poly[d(A‑C)].poly[d(G‑T)]. The melting parameters of these polymers, according to our studies, are the follow‑ing: ∆H = 11.5 ± 0.6 cal/g, ∆Tm = 1.1 ± 0.1º, Tm = 60.5 ± 0.5 ºC, and ∆Hm = 11.9 ± 1.0 cal/g, ∆Tm = 2.2 ± 0.1º, Tm = 82.4 ± 0.5 ºC, accordingly. The values obtained coincide with data (18, 19) with exactness of experimental error. The addition of small quantities of ZnTOEPyP(4) into poly(dA)poly(dT) solution (r > 0.01) causes a shift of the melting curve to higher temperatures and a division of curve profile in two independent transition stages. For example, at r ‑ 0.02 parameters of transi‑tion are Tm(I) = 61.2 ± 0.5 ºC, ∆Tm(I) = 1.2 ± 0.1º, ∆Hm(I) = 10.1 ± 0.5 cal/g; and Tm(II) = 76.2 ºC, ∆Tm(II) = 2.5 ± 0.1º, ∆Hm(II) = 1.5 ± 0.1 cal/g. The increase of ZnTOEPyP(4) concentration causes the further shift to higher temperature of both transition stages with simultaneous redistribution of heat between these stages, and besides, the heat ∆Hm(I) coming on low temperature stage decreases by the value, which is added to high temperature stage with ∆Hm(II) (Fig. 6).

In the case r = 0.2 transition parameters are equal to ∆Hm = 12.5 cal/g, Tm = 101.5 ºC, ∆T = 1.8º (Fig. 6). Independence of ∆Hm and Tm of poly(dA)poly(dT) complex on various values of r are presented Figure 7.

Two‑stage character of ZnTOEPyP(4)‑poly(dA)poly(dT) complex transition and the heat redistribution between them are explained on the basis of experimental and theoretic data (3, 6, 20).

The different picture is observed in case of complex ZnTOEPyP(4) with poly[d(A‑C)] poly[d(G‑T)]. Here we see only a small shift of curve to higher temperatures in the range of r = 0.01 ÷ 0.2 (∆Tm ~ 3.0) without a noticeable change of melting profile. So we conclude that ZnTOEPyP(4) primary binding to tandemly locatedsites but not with ‑sites.

Discussion

It is known that TOEPyP(4) has three modes of binding with DNA double helix: (i) a strong outside regulated binding “outside stacking”, when porphyrins are packed in stacks on outside surface of double helix AT‑rich sites, and the porphyrin plane is perpendicular to helix axis; (ii) a strong inside binding “inside stacking”, in this case porphyrin molecules preferable intercalate between G‑C pairs; (iii) a weak outside irregular binding “face‑on” – here porphyrins are located flat, accidentally in double helix groove (3, 8, 9, 18, 19). Consequently, the increase of Tm of GC rich DNA satellite fraction by 16 ºC, and the main stage (which contains less GC‑pairs than satellite fraction) by 5 ºC shows that two modes of binding exist simultane‑ously when the concentration of added TOEpyp(4) for r ≤ 0.11 – a strong binding – inside stacking and a weak one – “face on”. Two modes of binding is also fulfilled

Figure 6: Microcalorimetric melting curves of poly‑mer polyd(A)polyd(T) solution calculated per gram of dry mass in 2 mM phosphate buffer, pH 7.02, C = 0.065, at various values of r, where r is molar ratio of ZnTOEPyP(4) per polymer base pair. (1) r = 0, (2) r = 0.02, (3) r = 0.06, (4) r = 0.15, (5) r = 0.2. The melting process of the complex at < 0.1 r < 0.2 is characterized by one asymmetric peak.

Figure 7: Dependence of total melting en‑thalpy of poly(dA)poly(dT) and ∆H and Tm of the main stage (peak), and a newly formed peak on r (r = mole ZnTOEPyP(4)/mole b.p.). Dashed line, total enthalpy; ∆H (l) and Tm (p), enthalpy and melting tempera‑ture of the main stage. ∆H (°) and Tm (r), enthalpy and melting temperature of newly formed stage due to ZnTOEPyP(4) addition.

Figure 8: Microcalorimetric melting curves of poly‑mer poly[d(A‑C)]poly[d(G‑T)] solution, calculated per gram of dry mass in 20 mM phosphate buffer, pH 7.02, C = 0.075, at various values of r, where r is molar ratio of ZnTOEPyP(4) per base pair. (solid line) r = 0, (dash line) r = 0.02, (dot line) r = 0.07.

↓↑ TT

AA

↓↑ CT

GA

423Micocalorimetry on

DNA-Porphyrin Complexes

for r > 0.1 – outside and inside stacking. This conclusion is made on the basis of data that shows a sharp increase of main stage Tm by 18º and a satellite one – only by 7º in comparison with the same parameters for r = 0.1 (Fig. 2 ).

The increase of total interval of melting temperature and separation of the heat absorption peak corresponding to satellite fraction from the main stage of peak heat absorption (Fig. 1) means that the melting temperature of GC pairs (TGC) increases more significantly than melting temperature of AT pairs (TAT) and it is in agreement with a well‑known dependence, ∆T ~ TGC – TAC for DNA extracted from higher or‑ganisms (21). The significant increase of half‑width melting range of satellite DNA fraction (∆Tm) is an obvious result of DNA duplex morphology change due to the binding of porphyrin to GC‑pairs.

As ZnTOEPyP(4) has axial ligand and it is not a flat compound, it realizes only two modes of binding‑“face‑on” and outside stacking with DNA and poly(dA)poly(dT). Therefore, two‑stage character of thymus DNA and poly(dA)poly(dT) melting process in the presence of ZnTOEPyP(4) says that ZnTOEPyP(4) carries out two modes of binding at low and moderate concentrations (Fig. 4). We suppose that the main role in stabilization of structure DNA and poly(dA)poly(dT) with ZnTO‑EPyP(4) plays “face‑on” binding with AT pairstill porphyrin concentration is low (r ≤ 0,05, r ≤ 0,08). In this case, Tm of low temperature DNA stage (peak), which is richer with AT pairs than the higher temperature peak, increases by 9 ºC, and the high‑temperature one increases only by 5 ºC (Fig. 4). The increase of Zn porphyrin concentration r ≥ 0,14 sharply changes the profile of melting curve, it becomes asymmetric and the melting at r ≥ 0,2 occurs in a narrow temperature range (∆Tm = 2.4 ± 0.1 ºC). Besides, what is the most important, the melting enthalpy, coming to this stage, is practically equal to the integral one (∆Hm).

These observed changes we interpret as formation of outside stacking of ZnTOEP‑yP with AT rich sites of DNA. Similar situation is observed with poly(dA)poly(dT), but in this case the effect of the outside stacking is more intensive and it is expressed in Tm increase of poly(dA)poly(dT) by 40 ºC. We explain the sharp narrowing of interval melting temperature of complex DNA‑ZnTOEPyP for r ≥ 0,1 by the rap‑prochement of AT and GC pairs stability that agrees with data (21). A small shift of calorimetric curve of poly[d(A‑C)]poly[d(G‑T)] in the presence of ZnTOEPyP(4), in comparison with poly dA poly dT is caused by primary binding of Zn porphyrin to tandemly located sites but not with sites. Hence, we can con‑clude that binding of ZnTOEPyP(4) with DNA has a sequence‑specific character.

Dependences of ∆Hm=ƒ(r) and Tm=ƒ(r) for DNA and poly(dA)poly(dT), in the pres‑ence of TOEpyp(4) and its Zn complex ZnTOEPyP(4), showed that ∆Hm of these complexes were not practically changed for r≥0.10, while Tm of these complexes were sharply increased, in particular, it reaches 40 0C for poly poly(dA)poly(dT). According to the known dependence Tm=∆Hm/∆Sm, the increase of macromol‑ecules melting temperature initiated by external factors at constant melting enthalpy is possible only at increase of transition entropy (∆Sm). This means that the binding of Zn TOEPyP(4) with AT sites occurs according to the “outside stacking” mecha‑nism, it has entropic character, and is obviously connected with supplanting of H2O molecule from the connection sites.

References and Footnotes

↓↑ TT

AA ↓↑ C

TGA

1.2.

3.

4.5.

R. I. Fiel. J Biomol Struct Dyn 6, 1259‑1274 (1989).U. Sehlstedt, S. K. Kim, P. Carter, J. Goodisman, J. F. Vollano, B. Nordén, J. C. Dabrowiak. Biochemistry 33, 417‑426 (1994).Y. Dalyan, S. Haroutiunian, G. Ananyan, V. Vardanyan, V. Madakyan, R. Kazaryan, L. Saa‑kyan, L. Messory, P. Orioli, A. Benight. J Biomol Struct Dyn 18, 677‑687 (2001).Y. B. Dalyan. Biophysics 47, 253‑258 (2002). (in Russian).A. Ghazaryan, Y. Dalyan, S. Haroutiunian, A. Tikhomirova, N. Taulier, J. W. Wells, T. Chali‑kian. J Am Chem Soc 128, 1914‑1921 (2006).

424

Monaselidze et al.

6.

7.8.

9.10.

11.

12.

13.14.

15.

16.

17.

18.

19.20.

21.

A. A. Ghazaryan, Y. B. Dalyan, S. G. Haroutiunian, V. I. Vardanyan, R. K. Ghazaryan, T. V. Chalikian. Journal of Biomol Structure & Dynamics 24, 67‑74 (2006).B. Mestre, A. Jacobs, G. Pratviel, B. Meunier. Biochemistry 35, 9146‑9149 (1996).Y. Dalyan, N. Karapetyan, S. Haroutiunian, G. Ananyan, L. Aloyan, V. Vardanyan, T. Chali‑kian. Molecular & Cellular Proteomics 2, 972 (2003).L. R. Aloyan, V. I. Vardanyan, Y. B. Dalyan. European Biophysics Journal 34, 667 (2005).M. Benett, A. Krah, F. Wien, F. Carman, R. Mekenna, M. Sanderson, S. Neidle. PNAS 97, 9476‑9481 (2000).T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan, Q. Peng. Journal of the National Cancer Institute 90, 889‑905 (1998).E. Izbicka, R. T. Wheelhouse, E. Raymond, K. K. Davidson, R. A. Lawrence, D. Sun, B. E. Windle, L. H. Hurley, D. D. Von Hoff. Cancer Research 59, 639‑644 (1999).S. D. Bromley, B. W. Ward, J. C. Dabrowiak. Nucleic Acid Research 14, 9133‑9148 (1986)J. Monaselidze, S. Barbakadze, S. Kvirikashvili, G. Majagaladze, D. Khachidze, L. Topch‑ishvili. Biomacromolecules 3, 783‑786 (2002).J. Monaselidze, M. Abuladze, N. Asatiani, E. Kiziria, S. Barbakadze, G. Majagaladze, M. Io‑badze, L. Tabatadze, H.‑Y. Holman, N. Sapojnikova. Thermochemia Acta 441, 8–15 (2006).J. Monaselidze, M. Kiladze, D. Tananashvili, S. Barbakadze, A. Naskidashvili, A. Khiza‑nishvili, R. Kvavadze, G. Majagaladze. Journal of Thermal Analysis and Calorimetry 84, 613‑618 (2006).A. D. Voskoboinik, J. R. Monaselidze, G. N. Mgeladze, Z. I. Chanchalashvili, Y. S. Lazurkin, M. D. Frank‑Kamenetski. Molecular Biology 9, 783‑789 (1975).E. Andronikashvili, G. Mgeladze, J. Monaselidze, A. G. Lezius. Biopolymers 13, 1751‑1756 (1974).J. R. Monaselidze, G. N. Mgeladze. Biofizika (Russian) 5, 950‑958 (1977).M. Chkhaidze, J. Monaselidze, S. G. Haroutiunian, S. G. Gevorkian, V. I. Vardanyan. Bull Georg Acad Sci 174, 143‑145 (2006).L. V. Berestetskaya, M. D. Frank‑Kamenetskii, Y. S. Lazurkin. Biopolymers 13, 193‑205 (1974).

Date Received: September 24, 2007

Communicated by the Editor Valery Ivanov