Thermodynamics of DNA binding and condensation: isothermal titration calorimetry and electrostatic mechanism

The thermodynamics of binding of the trivalent cations cobalt hexammine and spermidine to plasmid DNA was studied by isothermal titration calorimetry. Two stages were observed in the course of titration, the first attributed to cation binding and the second to DNA condensation. A standard calorimetric data analysis was extended by applying an electrostatic binding model, which accounted for most of the observed data. Both the binding and condensation reactions were entropically driven (TDeltaS approximately +10 kcal/mol cation) and enthalpically opposed (DeltaH approximately +1 kcal/mol cation). As predicted from their relative sizes, the binding constants of the cations were indistinguishable, but cobalt hexammine had a much greater DNA condensing capacity because it is more compact than spermidine. The dependence of both the free energy of cobalt hexammine binding and the critical cobalt hexammine concentration for DNA condensation on temperature and monovalent cation concentration followed the electrostatic model quite precisely. The heat capacity changes of both stages were positive, perhaps reflecting both the temperature dependence of the dielectric constant of water and the burial of polar surfaces. DNA condensation occurred when about 67 % of the DNA phosphate charge was neutralized by cobalt hexammine and 87 % by spermidine. During condensation, the remaining DNA charge was neutralized.     

Condensation of DNA by trivalent cations. 2. Effects of cation structure

Electron microscopy is employed to examine DNA aggregates produced by three tripositively charged condensing agents. Spermidine, hexammine cobalt (III), and me8spermidine (in which the amine groups of spermidine are exhaustively methylated) all produce condensates. The predominant form of condensate observed is toroidal; however, me8spermidine produces a large fraction of rodlike condensates. Distributions of toroidal radii and estimated volumes suggest that the size of condensates depends on the condensing agent employed, its concentration, and the time elapsed after addition of condensing agent. While ligand charge seems to be the major factor in predicting condensing power, ligand structure influences the morphology and dimensions of the particles produced. The ability to form hydrogen bonds is not required to promote condensation, since me8spermidine has no NHs. There may be a kinetic barrier to condensation at low me8spermidine concentrations. The relative proportions of toroids and rods may depend on the energetic compensation between bending and binding in cyclic structures, or on rate-limiting formation of sharply bent or kinked regions in rods.     

The function of zinc in gene 32 protein from T4

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II) bound in a tetrahedral complex to -S- ligands, proposed on spectral evidence to include Cys-77, Cys-87, and Cys-90 [Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., & Coleman, J. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452]. The Zn(II) can be completely removed by treatment with the mercurial reagent p-(hydroxymercuri)benzenesulfonate and ethylenediaminetetraacetic acid. The resultant apo-g32P is rapidly digested by trypsin in contrast to the zinc protein which undergoes specific limited proteolysis to yield a resistant DNA-binding core. Rebinding of Zn(II) to the apoprotein restores the same limited susceptibility to proteolysis displayed by the native Zn(II) protein. In the presence of 150 mM NaCl, Zn(II) g32P reduces the melting temperature Tm of poly[d(A-T)] by 47 degrees C, while apo-g32P is unable to melt poly[d(A-T)] at this salt concentration, as the protein thermally unfolds before melting can take place. At 25 mM NaCl, however, apo-g32P lowers the Tm of poly[d(A-T)] by 36 degrees C, but the melting curve is broad compared to the steep cooperative melting induced by Zn(II) g32P. Association constants Ka calculated from the poly[d(A-T)] melting curves for Zn(II) and apo-g32P differ by 3 orders of magnitude, 4.8 X 10(10) M-1 and 4.3 X 10(7) M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation.

The nature of binding of Ru(phen) 2+ (I), Ru(bipy) 2+ (II), Ru(terpy) 2+ (III) (phen = 1,10-phenanthroline, bipy 3 = 2,2'-bipyridyl, 3 terpy = 2,2'2," - 2 terpyridyl) to DNA, poly[d(G-C)] and poly[d(A-T)] has been compared by absorption, fluorescence, DNA melting and DNA unwinding techniques. I binds intercalatively to DNA in low ionic strength solutions. Topoisomerisation shows that it unwinds DNA by 22 degrees +/- 1 per residue and that it thermally stabilizes poly[d(A-T)] in a manner closely resembling ethidium. Poly[d(A-T)] induces greater spectral changes on I than poly[d(G-C)] and a preference for A-T rich regions is indicated. I binding is very sensitive to Mg2+ concentration. In contrast to I the binding of II and III appears to be mainly electrostatic in nature, and causes no unwinding. There is no evidence for the binding of the neutral Ru(phen)2 (CN)2 or Ru(bipy)2 (CN)2 complexes. DNA is cleaved, upon visible irradiation of aerated solutions, in the presence of either I or II.     

Effect of ligands with selective type of binding on the DNA helix-coil transition

The influence of the extended interacted under adsorption ligands with a selective binding on the DNA helix-coil transition has been theoretically studied. It was found that contact interaction between ligands or/and their extent give rise to a marked non-linearity of the GC-content dependence of the melting temperature. This non-linearity causes a few features of the dependence of the melting range width on ligand concentration [delta T(C0)]. Such as a non-monotony of the delta T (C0) increase in the presence of ligands increasing the difference between the thermostabilities of poly(d(A-T)] and poly[d(G-C)] polymers. The degree of a non-linearity defines the character of changes of the form of the differential melting curves in the presence of ligands.     

A unified theory of the B-Z transition of DNA in high and low concentrations of multivalent ions

We showed recently that the high-salt transition of poly[d(G-C)]. poly[d(G-C)] between B-DNA and Z-DNA (at [NaCl] = 2.25 M or [MgCl(2)] = 0.7 M) can be ascribed to the lesser electrostatic free energy of the B form, due to better immersion of the phosphates in the solution. This property was incorporated in cylindrical DNA models that were analyzed by Poisson-Boltzmann theory. The results are insensitive to details of the models, and in fair agreement with experiment. In contrast, the Z form of the poly[d(G-m5C)] duplex is stabilized by very small concentrations of magnesium. We now show that this striking difference is accommodated quantitatively by the same electrostatic theory, without any adjustable parameter. The different responses to magnesium of the methylated and nonmethylated polymers do not come from stereospecific cation-DNA interactions: they stem from an experimentally derived, modest difference in the nonelectrostatic component of the free energy difference (or NFED) between the Z and B forms. The NFED is derived from circular DNA measurements. The differences between alkaline earth and transition metal ions are explained by weak coordination of the latter. The theory also explains the induction of the transition by micromolar concentrations of cobalt hexammine, again without specific binding or adjustable parameters. Hence, in the case of the B-Z transition as in others (e.g., the folding of tRNA and of ribozymes), the effect of multivalent cations on nucleic acid structure is mediated primarily by nonspecific ion-polyelectrolyte interactions. We propose this as a general rule for which convincing counter-examples are lacking.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Synthesis, characterization and DNA binding of magnesium-ciprofloxacin (cfH) complex [Mg(cf)2] * 2.5H2O

Interactions of the tested systems (title compound [Mg(cf)(2)] * 2.5H(2)O (1), ciprofloxacin (cfH) and ciprofloxacin in the mixture with MgCl(2)), with single and double stranded calf thymus DNA, poly[d(AT)] * poly[d(AT)] and poly[d(GC)] * poly[d(GC)] were studied by UV-spectrophotometric (melting curves) and fluorescence emission measurements. Pronounced quenching of ciprofloxacin's fluorescence intensity has been observed for all the tested compounds after titration with various GC containing DNA molecules. It seems probable that quenching originates in the electron transfer from guanine to the photo-excited fluoroquinolone. The UV-spectrophotometric results obtained for 1 are substantially different from the other solutions and the biggest differences were observed for GC containing DNAs. Solution of 1 provokes a large thermal destabilization of poly[d(GC)] * poly[d(GC)]. This process is irreversible which suggests that the species present in solution of 1 alone inhibit re-annealing by associating irreversibly with the single strands. We have realized that aqueous solutions of 1 are colloidal and we propose that colloidal particles are involved in specific binding to GC containing sequences, most probably in the major groove of DNA.     

Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein

The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).     

Intercalative and nonintercalative binding of large cationic porphyrin ligands to polynucleotides

The interactions of two positional isomers and one analogue of meso-tetra (4-N-methylpyridyl) porphine, with the synthetic polynucleotides poly[d(A-T)] . poly[d(A-T)] and poly[d(G-C)] . poly[d(G-C)] have been investigated by circular dichroism. All four porphyrins were found to bind to the polynucleotides as shown by the induction of circular dichroism in their Soret bands. Furthermore, the sign of the induced ellipticity reflects selective occupation of binding sites by the porphyrin ligands. The conformational lability of poly[d(A-T)] X poly[d(A-T)] was found to be appreciable as micromolar amounts of meso-substituted 4-N-methylpyridyl, 3-N-methylpyridyl, and p-N-trimethylanilinium porphines induced a CD spectrum similar but not identical to that of DNA in the Z-form, i.e. a negative band at 280 nm and a positive band at 259 nm. The effect of porphyrin binding to poly[d(G-C)] X poly[d(G-C)] was less pronounced and dissimilar to that seen in the AT polymer.     

Characterization of parazoanthoxanthin A binding to a series of natural and synthetic host DNA duplexes

Parazoanthoxanthin A is a fluorescent yellow nitrogenous pigment of the group of zoanthoxanthins, which show a broad range of biological activity. These include, among others, the ability to bind to DNA. In this study we have used a variety of spectroscopic (intrinsic fluorescence emission and UV-spectroscopy) and hydrodynamic techniques (viscometry) to characterize in more detail the binding of parazoanthoxanthin A to a variety of natural and synthetic DNA duplexes in different buffer conditions. Our results reveal the following five significant features: (i) Parazoanthoxanthin A exhibits two modes of DNA binding: One binding mode exhibits properties of intercalation, while the second binding mode is predominantly electrostatic in origin. (ii) The apparent binding "site size" for parazoanthoxanthin A near physiological salt concentration (100 mM NaCl) is in the range of 7 +/- 1 base pairs for natural genomic DNA duplexes (calf thymus and salmon testes DNA) and alternating synthetic polynucleotides (poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)]). A slightly larger apparent binding site size of 9 +/- 1 bp was obtained for parazoanthoxanthin A binding to the synthetic homopolymer poly[d(A)]. poly[d(T)]. (iii) Near physiological salt concentration (100 mM NaCl) parazoanthoxanthin A binds with the same approximate binding affinity of 2-5 x 10(5) M(-1) to all DNA polymers studied. (iv) At low salt concentration, parazoanthoxanthin A preferentially binds alternating poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)] host duplexes. (v) Parazoanthoxanthin A inhibits DNA polymerase in vitro.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Circular dichroism studies of the interaction of a limited hydrolysate of T4 gene 32 protein with T4 DNA and poly[d(A-T)].poly[d(A-T)].

gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation.     

Synthesis,characterization and binding studies of chromium(III) complex containing an intercalating ligand with DNA

A chromium(III) complex [Cr(DPPZ)(2)Cl(2)](+), where DPPZ is a planar bidentate ligand with an extended aromatic system, has been found to bind strongly to CT DNA with an apparent binding constant of (1.8+/-0.5)x10(7) M(-1). The effects of [Cr(DPPZ)(2)Cl(2)](+) on the melting temperature and the viscosity of DNA clearly show that the chromium(III) complex interacts with DNA intercalatively. Competitive binding study shows that the enhancement in emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(DPPZ)(2)Cl(2)](+) indicating that the Cr(III) complex displaces EthBr from its binding site in DNA. The binding of this complex has been found to bring about B to Z conformational transition in CT DNA as well as poly(dG-dC).poly(dG-dC). Molecular modeling study also shows that binding energy of the complex with d(GC)(12) is much higher than Dickerson model and d(AT)(12). Modeling studies show that [Cr(DPPZ)(2)Cl(2)](+) brings about twist in the DNA base pairs as well as phosphate ester backbone resulting in conformational transition in DNA.     

Structural and functional heterogeneity of single-stranded DNA-binding proteins from calf thymus

A new purification technique for ‘single-stranded DNA-binding proteins’ from calf thymus permits the demonstration of a considerable heterogeneity within these proteins. Several molecular species are obtained with M_r between 24·10³ and 30·10³ and pI values between 6 and 8, showing significant differences with regard to the following functional properties: (1) strength of binding to single-stranded DNA; (2) lowering of melting temperature of poly[d(A-T)]; (3) stimulation of DNA polymerase α on a poly[d(A-T)] template. Analysis of trypsin digestion products demonstrates that the different molecular species share extensive primary sequence homology. Experiments with antibodies show that the different molecular species are antigenically related and that a 31 kDa protein present in low amounts in our preparations is very cross-reactive.     

Design, synthesis, and DNA binding properties of photoisomerizable azobenzene-distamycin conjugates: an experimental and computational study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene-distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.     

Theoretical calculations of the helix–coil transition of DNA in the presence of large,cooperatively binding ligands

Theoretical calculations are conducted on the helix–coil transition of DNA, in the presence of large, cooperatively binding ligands modeled after the DNA-binding proteins of current biological interest. The ligands are allowed to bind both to helx and to coil, to cover up any number of bases or base pairs in the complex, and to interact cooperatively with their nearest neighbors. The DNA is treated in the infinite homogeneous Ising model approximation, and all calculations are done by Lifson's method of sequence-generating functions. DNA melting curves are calculated by computer in order to expolore the effects on the transition of ligand size, binding constant, free activity, and ligand–ligand cooperativity. The calculations indicate that (1) at the same intrinsic free energy change per base pair of the complexes, small ligands, for purely entropic reasons, are more effective than are large ligands in shifting the DNA melting temperature; (2) the response of the DNA melting temperature to increased ligand binding constant K and/or free ligand activity L is adequately represented at high values of KL (but not at low KL) by a simple independent site model; (3) if curves are calculated with the total amount of added ligand remaining constant and the free ligand activity allowed to vary throughout the transition, biphasic melting curves can be obtained in the complete absence of ligand–ligand cooperativity. In an Appendix, the denaturation of poly[d(A-T)] in the presence of the drug, netropsin, is used to verify some features of the theory and to illustrate how the theory can be used to obtain numerical estimates of the ligand binding parameters from the experimental melting curves.     

Bis(pyrrolecarboxamide) linked to intercalating chromophore oxazolopyridocarbazole (OPC): selective binding to DNA and polynucleotides.

We have investigated some properties related to interaction with DNA and recognition of AT-rich sequences of netropsin-oxazolopyridocarbazole (Net-OPC) (Mrani et al., 1990), which is a hybrid groove-binder-intercalator. The hybrid molecule Net-OPC binds to poly[d(A-T)] at two different sites with Kapp values close to 7 x 10(6) and 6 x 10(8) M-1 (100 mM NaCl, pH 7.0). Data obtained from melting experiments are in agreement with these values and indicate that Net-OPC displays a higher binding constant to poly[d(A-T)] than does netropsin. On the basis of viscometric and energy transfer data, the binding of Net-OPC to poly[d(A-T)] is suggested to involve both intercalation and external binding of the OPC chromophore. In contrast, on poly[d(G-C)], Net-OPC binds to a single type of site composed of two base pairs in which the OPC chromophore appears to be mainly intercalated. The binding constant of Net-OPC to poly[d(G-C)] was found to be about 350-fold lower than that of the high-affinity binding site in poly[d(A-T)]. As evidenced by footprinting data, Net-OPC selectively recognizes TTAA and CTT sequences and strongly protects the 10-bp AT-rich DNA region 3'-TTAAGAACTT-5' containing the EcoRI site. The binding of Net-OPC to this sequence results in a strong and selective inhibition of the activity of the restriction endonuclease EcoRI on the plasmid pBR322 as substrate. The extent of inhibition of the rate constant of the first strand break catalyzed by the enzyme is about 100-fold higher than the one observed in the presence of netropsin under similar experimental conditions.     

Hairpin Polyamides That use Parallel and Antiparallel Side-by-Side Peptide Motifs in Binding to DNA

Abstract

Pt-bis-netropsin is a synthetic sequence-specific DNA-binding ligand comprizing two netropsin-like fragments which are linked in a tail-to-tail manner via a cis-diammineplat-inum (II) residue. The CD studies and thermodynamic characterization of the DNA-binding properties exhibited by this compound reveal that it forms two types of complexes with poly[d(AT)]?poly[d(AT)] and DNA oligomers containing nucleotide sequences 5′-CC (TA)_nCC-3′, with n = 4, 5 and 6. The first type corresponds to the binding of Pt-bis-netropsin in the extended conformation and is characterized by the saturating ratio of one bound Pt-bis-netropsin molecule per 9 AT-base pairs. The second type of the complex corresponds to the binding of Pt-bis-netropsin to DNA in the folded hairpin form. The binding approaches saturation level when one Pt-bis-netropsin molecule is bound per four or five AT-base pairs. The hairpin form of Pt-bis-netropsin complex is built on the basis of parallel side-by-side peptide motif which is inserted in the minor DNA groove. The CD spectral profiles reflecting the binding of Pt-bis-netropsin in the hairpin form are different from those observed for binding of another bis-netropsin with the sequence Lys-Gly-Py-Py-Gly-Gly-Gly-Py-Py-Dp, where Py is a N-propylpyrrole amino acid residue and Dp is a dimethylaminopropylamino residue. The hairpin form of this bis-netropsin is formed on the basis of antiparallel side- by-side peptide motif. The CD spectra obtained for complexes of this polyamide in the hairpin form with poly[dAT)]?poly[d(AT)] exhibit positive CD band with a peak at 325 nm, whereas the CD spectral profiles for the second complex of Pt-bis-Nt with poly[d(AT)] ?poly[d(AT)] and short DNA oligomers have two intense positive CD bands near 290 nm and 328 nm. This reflects the fact that two bis-netropsins use different structural motifs on binding to DNA in the hairpin form.