Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and their metallocomplexes with duplex DNA.

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.

Role of small loops in DNA melting.

Short melted regions less than 100 base pairs (bp) in length are rarely found in the differential melting curves (DMC) of natural DNAs. Therefore, it is supposed that their characteristics do not affect DNA melting behavior. However, in our previous study, a strong influence of the form of the entropy factor of small loops on melting of cross-linked DNAs was established (D. Y. Lando, A. S. Fridman et al., Journal of Biomolecular Structure and Dynamics, 1997, Vol. 15, pp. 141-150; Journal of Biomolecular Structure and Dynamics, 1998, Vol. 16, pp. 59-67). Quite different dependencies of the melting temperature on the relative concentration of interstrand cross-links were obtained for the loop entropy factors given by the Fixman-Freire (Jacobson-Stockmayer) and Wartell-Benight relations. In the present study, the influence of the entropy factor of small loops on the melting of natural DNAs, cross-linked DNAs and periodical double-stranded polynucleotides is compared using computer simulation. A fast combined computational method for calculating DNA melting curves was developed for this investigation. It allows us to assign an arbitrary dependence of the loop entropy factor on the length of melted regions for the terms corresponding to small loops (less than tau bp in length). These terms are calculated using Poland's approach. The Fixman-Freire approach is used for long loops. Our calculations have shown that the temperature dependence of the average length of interior melted regions (loops) has a maximum at T approximately T(m) (T(m) is the DNA melting temperature) in contrast to the dependence of the total average length of melted regions, which increases almost monotonously. Computer modeling demonstrates that prohibition of formation of loops less than tau base pairs in length does not markedly change the DMC for tau < 150 bp. However, the same prohibition strongly affects the average length of internal melted regions for much smaller tau's. The effect is already noticeable for tau = 1 bp and increases with tau. A tenfold increase in the entropy factor of all loops with length less than tau bp causes a noticeable alteration of the DMC for tau > or = 30 bp. It is shown that DMCs are identical for the Wartell-Benight and for the Fixman-Freire (Jacobson-Stockmayer) form of the loop entropy factor. However, for low degree of denaturation, the average length of internal melted regions is 40% lower for the Wartell-Benight form due to the fluctuational opening of short AT-rich regions less than 10 bp in length. The same calculations carried out for periodical polynucleotides demonstrate a much stronger difference in melting behavior for different forms of entropy factors of short loops. The strongest difference occurs if the length of stable GC-rich and unstable AT-rich stretches is equal to 30 bp. However, the comparison carried out in this work demonstrates that the entropy factor of short loops influences melting behavior of cross-linked DNA much stronger than of unmodified DNA with random or periodical sequences.

Long-range interactions between ligands bound to a DNA molecule give rise to adsorption with the character of phase transition of the first kind.

Influence of long-range interactions between ligands bound to DNA molecule on the character of their adsorption is studied using computer modeling. For this investigation, two calculation procedures are developed. They are based upon the method of the free energy minimum and on the partition function method. The both procedures demonstrate that in the case of a strong enough attraction between all the bound ligands their binding to DNA has the character of phase transition of the first kind. There is a break in the binding curve c(c0) where c - relative concentration of bound ligands, c0 - molar concentration of free ligands. The break occurs because there is an interval of central degrees of binding (approximately 50% of the maximum c value) that are prohibited for individual DNA molecules. Such a transition might be caused by some types of DNA condensation. Attraction between the neighboring ligands only, adjacent or/and separated by double helix regions, does not cause this effect.

Melting of cross-linked DNA IV. Methods for computer modeling of total influence on DNA melting of monofunctional adducts, intrastrand and interstrand cross-links formed by molecules of an antitumor drug.

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.

Melting of cross-linked DNA. III. Calculation of differential melting curves.

In our previous papers I and II (D. Y. Lando et al, J. Biomol. Struct. Dynam. (1997) v. 15, N1, p. 129-140, p. 141-150), two methods were developed for calculation of melting curves of cross-linked DNA. One of them is based on Poland's and another on the Fixman-Freire approach. In the present communication, III, a new theoretical method is developed for computation of differential melting curves of DNAs cross-linked by anticancer drugs and their inactive analogs. As Poland's approach, the method allows study of the influence of the loop entropy factor, delta(n), on melting behavior (n is the length of a loop in base pairs). However the method is much faster and requires computer time that inherent for the most rapid Fixman-Freire calculation approach. In contrast to the computation procedures described before in communications I and II, the method is suitable for computation of differential melting curves in the case of long DNA chains, arbitrary loop entropy factors of melted regions and arbitrary degree of cross-linking including very low values that occur in vivo after administration of antitumor drugs. The method is also appropriate for DNAs without cross-links. The results of calculation demonstrate that even very low degree of cross-linking alters the DNA differential melting curve. Cross-linking also markedly strengthens the influence of particular function delta(n) upon melting behavior.

Melting of cross-linked DNA: I. Model and theoretical methods.

Covalent and strong coordination binding to DNA of a large number of antitumour drugs and other compounds leads to interstrand cross-link formation. To investigate cross-link influence on double helix stability, two methods are developed for the calculation of melting curves. The first method is based on Poland's approach. It requires computer time proportional to u.N, where u is the average distance (in base pairs) between neighboring cross-links and N is the number of base pairs in the DNA chain. The method is more suitable when u is not large, and small loops formed by interstrand cross-links in melted regions strongly affect DNA melting. The computer time for the second method, based on the Fixman-Freire approach, does not depend on the number of cross-links and is proportional to I.N (I is the number of exponential functions used for a decomposition of the loop entropy factor). It is more appropriate when N and u are large, and therefore particular values of the entropy factors of small loops do not influence DNA melting behavior.

Melting of cross-linked DNA: II. Influence of interstrand linking on DNA stability.

In the previous paper (D.Y. Lando, J. Biomol. Struct. Dynam, 15, 129-140 (1997)) the melting of cross-linked DNA with N base pairs and omega interstrand cross-links has been considered theoretically. In the present study on the basis of these results, two simple schemes are developed for the computation of melting curves of cross-linked DNA. The investigation of influence of interstrand linking on DNA stability has been carried out by computer simulation. It is shown that the relative concentration of cross-links, CCT = omega/N, their distribution along a DNA molecule, and particular values of the entropy factors of small loops formed by cross-links in melted regions strongly affect the DNA melting temperature, Tm. On the contrary, for DNA without cross-links, a ten-fold increase or decrease in the entropy factors of small loops does not cause the Tm variation. The comparison of the results of calculation with experimental data suggests that the majority of types of cross-link neither maintain ordered parallel orientation of bases in melted regions nor increase considerably the thermostability of cross-linked base pairs. Four different ways of influence of interstrand cross-linking on the DNA double helix stability are considered. It is shown that cross-linking significantly enhances the influence of single strand stiffness in melted regions on DNA melting behavior.