Binding of meso-tetrakis(N-methylpyridiniumyl)porphyrin isomers to DNA: quantitative comparison of the influence of charge distribution and copper(II) derivatization

Factors influencing the binding of tetracationic porphyrin derivatives to DNA have been comprehensively evaluated by equilibrium dialysis, stopped-flow kinetics, etc., for mesotetrakis (4-N-methylpyridiniumyl)porphyrin [TMpyP (4)]. Technical difficulties have previously precluded a comprehensive study of metalloporphyrins. Since electrostatic interactions with the DNA and metal derivatization of the porphyrins have important consequences, we have investigated in greater detail two isomers of TMpyP (4) (meso-tetrakis(3-N-methylpyridiniumyl)porphyrin, [TMpyP (3)] and meso-tetrakis(2-N-methylpyridiniumyl)porphyrin [TMpyP (2)]) in which the position of the charged centers has been varied. A comprehensive study of the Cu(II) derivatives, e.g., CuTMpyP (4), was possible since the difficulties encountered previously with Ni(II) compounds were not a problem with Cu(II) porphyrins [J. A. Strickland, L. G. Marzilli, M. K. Gay, and W. D. Wilson (1988) Biochemistry 27, 8870-8878]. At 25 degrees C, the apparent equilibrium constants [Kobs] decreased with increasing [Na+] for all porphyrins. The Kobs values were comparable for TMpyP (4) and TMpyP (3) binding to either polyd(G-C).polyd(G-C) [poly[d(G-C)2]] or poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]]. For the copper(II) porphyrins, the Kobs values were about fivefold greater. The Kobs value for CuTMpyP (2) binding to poly[d(G-C)2] was too small to measure under typical salt conditions; however, Kobs for binding to poly[d(A-T)2] was about two orders of magnitude smaller than those found for CuTMpyP (4) or CuTMpyP (3). Application of the condensation theory for polyelectrolytes suggests about three charge interactions when CuTMpyP (4), CuTMpyP (3), and TMpyP (3) bind to poly[d(G-C)2] or poly[d(A-T)2], a result comparable to that reported for TMpyP (4). At 20 degrees C and 0.115 M [Na+], incorporation of copper decreased the rates of dissociation from poly[d(A-T)2] by a 100-fold compared to those reported for TMpyP (4) but had little effect on the rates of dissociation from poly[d(G-C)2]. Also, movement of the H3CN+ group from the fourth to the third position of the pyridinium ring enhanced the rates of dissociation from poly[d(A-T)2] but decreased the rates of dissociation from poly[d(G-C)2]. From polyelectrolyte theory, the [Na+] dependence of the dissociation rates from poly[d(G-C)2] is consistent with intercalative binding, while that for poly[d(A-T)2] is consistent with an outside binding model. For calf thymus [CT] DNA at 20 degrees C, a greater decrease in the AT than in the GC imino 1H-nmr signal was observed upon addition of CuTMpyP (2), suggesting selective outside binding to the AT regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Equilibrium binding of benzo[a]pyrene tetrol to synthetic polynucleotides: sequence selectivity, thermodynamic properties, and ionic strength dependence

We have investigated the equilibrium binding of racemic 7r,8t,9t,10c-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene to the double-stranded, synthetic polynucleotides poly[d(A-T)], poly[d(G-C)], and poly[d(G-m5C)] at low binding ratios. Difference absorption spectroscopy shows a 10-nm red shift for binding to poly[d(A-T)] and an 11-nm red shift for binding to either poly[d(G-C)] or poly[d(G-m5C)]. The value of delta epsilon for binding is approximately the same for all three hydrocarbon-polynucleotide complexes. Binding of this neutral polycyclic aromatic hydrocarbon derivative to these polynucleotides is dependent upon ionic strength and temperature. Analysis of complex formation employing polyelectrolyte theory shows a greater release of counterions associated with binding to poly[d(A-T)] than with the other two polynucleotides (0.5 and ca. 0.36, respectively). Thus, sequence-selective binding of this hydrocarbon in DNA would be expected to change depending on salt concentration. The temperature dependence of binding was studied at 100 mM Na+ where the equilibrium binding constants for poly[d(A-T)] and poly[d(G-m5C)] are roughly equivalent and 6-fold greater than the binding affinity for poly[d(G-C)]. The binding to poly[d(A-T)] and poly[d(G-C)] is characterized by a delta H omicron = -7.0 kcal/mol, and the large difference in affinity constants arises from differences in negative entropic contributions. Formation of hydrocarbon-poly[d(G-m5C)] complexes is accompanied by a delta H = -9.1 kcal/mol. However, the affinity for poly[d-(G-m5C)] is the same as that for poly[d(A-T)] due to the much more negative entropy associated with binding to poly[d(G-m5C)].     

Kinetic and equilibrium analysis of a threading intercalation mode: DNA sequence and ion effects

The interaction of a symmetric naphthalene diimide with alkylamino substituents at each imide position was investigated with the alternating sequence polymers, poly[d(A-T)]2 and poly[d(G-C)]2. Spectrophotometric binding studies indicate strong binding of the diimide to both sequences although the GC binding constant is 20-25 times larger than the AT binding constant. Analysis of the effects of salt concentration on the binding equilibria shows that the diimide forms two ion pairs in its complex with both polymers as expected for a simple dication. Stopped-flow kinetics experiments demonstrate that the diimide both associates and dissociates from DNA more slowly than classical intercalators with similar binding constants. Analysis of salt concentration effects on dissociation kinetics rate constants (kd) reveals that slopes in log kd versus log [Na+] plots are only approximately half the value obtained for classical dicationic intercalators that have both charged groups in the same groove. These kinetics results support a threading intercalation model, with one charged diimide substituent in each of the DNA grooves rather than with both side chains in the same groove, for the diimide complex with DNA. In the rate-determining step of the mechanism for dissociation of a threading complex only one ion pair is broken; the free side chain can then slide between base pairs to put both diimide side chains in the same groove, and this is followed by rapid full dissociation of the diimide. This sequential release of ion pairs makes the dissociation slope for dicationic threading intercalators more similar to the slope for classical monocationic intercalating ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

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.     

Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy

A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly[d(A-T)].poly[d(A-T)] and poly[d-(G-C)].poly[d(G-C)]. As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature. An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C. A decrease was observed in the case of poly[d(A-T)].poly[d(A-T)] at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly[d(G-C)].poly[d(G-C)]. Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C. Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin. The interaction between pyrenyl RNA polymerase and poly[d-(A-T)].poly[d(A-T)] at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Right- and left-handed helixes of poly[d(A-T)].poly[d(A-T)] investigated by infrared spectroscopy

The secondary structures of double-stranded poly[d(A-T)].poly[d(A-T)] in films have been studied by IR spectroscopy with three different counterions (Na+, Cs+, and Ni2+) and a wide variety of water content conditions (relative humidity between 100 and 47%). In addition to the A-, B-, C-, and D-form spectra, a new IR spectrum has been obtained in the presence of nickel ions. The IR spectra of Ni2+-poly[d(A-T)].poly[d(A-T)] films are analyzed by comparison with previously assigned IR spectra of left-handed poly[d(G-C)].poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)], and it is possible to conclude that they reflect a Z-type structure for poly[d(A-T)].poly[d(A-T)]. The Z conformation has been favored by the high polynucleotide concentration, by the low water content of the films, and by specific interactions of the transition metal ions with the purine bases stabilized in a syn conformation. A structuration of the water hydration molecules around the double-stranded Ni2+-poly[d(A-T)].poly[d(A-T)] is shown by the presence of a strong sharp water band at 1615 cm-1.     

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.     

Rotation of periphery methylpyridine of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) and its selective binding to native and synthetic DNAs

By utilizing circular and linear dichroism, the binding mode of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) to various DNAs was studied in this work. 2-N-(methylpyridiniumyl)porphyrin(o-TMPyP), in which rotation of the periphery pyridinium ring is prevented, exhibits similar spectral properties when bound to DNA, poly[d(G-C)(2)] and poly[d(A-T)(2)], suggesting a similar binding mode. Close analysis of the spectral properties led us to conclude that o-TMPyP sits in the major groove. However, both 3-N- and 4-N-(methylpyridiniumyl)porphyrin (m- and p-TMPyP), of which the periphery pyridinium ring is free to rotate, intercalate between the basepairs of DNA and poly[d(G-C)(2)]. In the presence of poly[d(A-T)(2)], m-TMPyP exhibits a typical bisignate excitonic CD spectrum in the Soret band, while p-TMPyP shows two positive CD bands. The excitonic CD spectrum of the m-TMPyP-poly[d(A-T)(2)] complex and the positive CD band of the o-TMPyP-poly[d(A-T)(2)] complex were not affected by the presence of the minor groove binding drug, 4',6-diamidino-2-phenylindole (DAPI), indicating that this porphyrin is bound in the major groove. In contrast, two positive CD bands of the p-TMPyP-poly[d(A-T)(2)] complex altered in the presence of DAPI. From the changes in CD spectrum and other spectral properties, a few possible binding modes for p-TMPyP to poly[d(A-T)(2)] are suggested.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Effect of N-alkyl substituents on the DNA binding properties of meso-tetrakis (4-N-alkylpyridinium-4-yl)porphyrins and their nickel derivatives

Resonance Raman, NMR, and visible spectroscopies, as well as viscosity and equilibrium dialysis studies were used to assess the effect of the N-alkyl substituent of meso-tetrakis(4-N-alkylpyridinium-4-yl)porphyrin cations on DNA binding. The DNAs studied include the native DNA, calf thymus DNA (CT DNA), the synthetic polynucleotides [poly(dGdC)]2 and [poly(dAdT)]2, and the oligonucleotide d(TATACGTATA)2. Both the porphyrins and the metalloporphyrins containing Ni(II) were examined with the N-alkyl = propyl (TPrpyP(4) and NiTPrpyP(4)) and 2-hydroxyethyl (TEtOHpyP(4) and NiTEtOHpyP(4)). The results were compared to those from the parent porphyrins with the N-methyl substituent (TMpyP(4) and NiTMpyP(4)). For almost all the comparisons made, the new porphyrin cations gave results very similar to those for the TMpyP(4) species. The resonance Raman study indicated that for the three DNA polymers all the Ni species were in the four-coordinate form when bound to all three polymers. It is suggested that both TPrpyP(4) and TEtOHpyP(4) bind to GC regions of DNA in the same intercalative manner as TMpyP(4) with the N-alkyl substituent extended into the solvent. For AT regions of DNA, the binding of TPrpyP(4) and TEtOHpyP(4) is nonintercalative, as found previously for TMpyP(4). The NiPrpy(4) and NiTEtOHpyP(4) cations bind to these polymers in a similar manner to the apo-porphyrins. The similar Raman spectral changes for the three Ni porphyrins upon addition of [poly(dAdT)]2 suggest that partial intercalation is not occurring because models indicate that it would be difficult to accommodate the bulkier N-alkyl substituents.     

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.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.     

Lac repressor binding to poly (d(A-T)). Conformational changes

The binding of $\text{lac}$ repressor to poly [d(A-T)] at low ionic strength has been investigated by circular dichroism, fluorescence and light scattering. Poly [d(A-T)] undergoes an important conformational change upon binding to $\text{lac}$ repressor. The maximum number of binding sites corresponds to about one tetrameric repressor per 11 base pairs of poly[d(A-T)]. The inducer isopropyl β-D-thiogalactoside (IPTG) does not affect the binding of $\text{lac}$ repressor to poly[d(A-T)]. It binds equally well to free and poly[d(A-T)] -bound repressor.     

Stopped-flow kinetic studies of actinomycin binding to DNAs.

Stopped-flow kinetic studies of the association of actinomycins with narural and synthetic DNA duplexes are presented. The actinomycins examined were D (C1), D lactam (in which the pentapeptide rings are closed by lactam instead of lactone linkages), X2, XObeta, and actinomine. The DNAs used included claf-thymus DNA, PM2, DNA, and two synthetic d(A-T)-lide copolymers containing 2,6-diaminopurine (DAP) in place of adenine residues, poly[d(DAP-T)]-poly[d(DAP-T)] and poly[d(DAP-A-T]-poly[d(DAP-A-T)]. Apparent equilibrium constants indicate that the DAP-containing polynucleotides bind actinomycin strongly. Comples formation of actinomycins D, D lactam, X2 and XObeta with these DNAs can be deconvoluted into five rate processes. These steps do not necessarily proceed to completion. The rates of two of these steps display a firstorder dependence on DNA concentration. The large negative entropies of activation of these steps suggest a high degree of restriction to freedom of motion on the respective transition states. The rates of the remaining three steps are independent of DNA concentration. Kinetic parameters of actinimycin binding to DNAs are presented and suggestions are made about some of the molecular evente believed to be responsible for the appearance of the five rate processes. For example, for DNA, poly[d(DAP-A-T)], and poly[d(DAP-T)], the observed order of apparent second-order rate constants, normalized to the concentration of actinomycin binding sites, suggests that binding of the antibiotic occurs most rapidly at binding sites (G-C of d DAT-T) near d(A-T) base pairs, where weakening of the double-helical conformation requires the least energy. Results obtained from studies of actinomycin D binding to heat-denatured poly[d(DAP-A-T)] and of actinomine and actinomycin D lactam binding to DNA suggest that the slow rate processes are related to an actinomycyl-pentapeptide-induced unwinding of the sugar-phosphate backbone of DNA accompanying insertion of the cyclic peptides into DNA.     

Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities.

Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.     

The influence of ionic strength on the binding of a water soluble porphyrin to nucleic acids.

The ionic strength dependences of the binding of tetrakis (4-N-methylpyridyl)porphine (H2TMpyP) to poly(dG-dC) and calf thymus DNA have been determined. For the former system the results are typical of other intercalators, i.e., a plot of log K vs log [Na+] is linear albeit with a slope which suggests that the "effective charge" of the porphyrin is closer to two than the formal charge of +4. For calf thymus DNA, the binding profile is not completely compatible with the predictions of condensation theory. Whereas the avidity of binding does decrease with increasing [Na+] as predicted, of greater interest is the relocation of the porphyrin from GC-rich regions to AT-rich regions as the ionic strength increases.     

Amine group of guanine enhances the binding of norfloxacin antibiotics to DNA.

The binding mode of norfloxacin, a quinolone antibacterial agent, in the synthetic polynucleotides poly[d(G-C)2], poly[d(I-C)2] and poly[d(A-T)2] was studied using polarized light spectroscopy, fluorescence spectroscopy and melting profiles. The absorption, circular and linear dichroism properties of norfloxacin are essentially the same for all the complexes, and the angle of electric transition dipole moment I and II of norfloxacin relative to the DNA helix axis is measured as 68-75 degrees for all complexes. These similarities indicate that the binding mode of norfloxacin is similar for all the polynucleotides. The decrease in the linear dichroism (LD) magnitude at 260 nm upon binding norfloxacin, which is strongest for the norfloxacin-poly[d(G-C)2] complex, and the identical melting temperature of poly[d(A-T)2] and poly[d(I-C)2] in the presence and absence of norfloxacin rule out the possibility of classic intercalation and minor groove binding. However, the characteristics of the fluorescence emission spectra of norfloxacin bound to poly[d(A-T)2] and to poly[d(I-C)2] are similar but are different to that of norfloxacin bound to poly[d(G-C)2]. As the amine group of the guanine base protrudes to the minor groove, this result strongly suggests that norfloxacin binds in the minor groove of B-form DNA in a nonclassic manner.     

Comparison of the effects of cationic porphyrins on DNA properties: influence of GC content of native and synthetic polymers

Interactions of meso-tetra(4-N-methylpyridyl)porphyrin [TMpyP(4)], meso-tetra(2-N-methylpyridyl)porphyrin [TMpyP(2)], and meso-tetra(para-N-trimethylanilinium)porphyrin (TMAP) with several native and synthetic DNAs were studied by a variety of physical techniques: nmr (³¹P and ¹H), absorption spectroscopy, viscosity, and flow dichroism (FD). Of the three porphyrins studied, only the interaction of TMpyP(4) with poly [d(G-C)₂] was fully consistent with intercalation. In particular, a large increase in viscosity, a downfield ³¹P-nmr signal (ca. -1 ppm), and upfield imino proton signals (11 to 12 ppm range) were observed. Comparison of the effects of TMpyP(4) on DNAs of different GC contents revealed larger changes in solution viscosity with increased GC content. However, the characteristic changes in ³¹P- and ¹H-nmr spectra were not observed. The viscosity increases observed in studies with poly[d(A-C)(G-T)] and C. Perf. DNA were much lower than with poly[d(G-C)₂], M. Lys. DNA, and calf thymus DNA. Thus, GC sequence and content are clearly important. The principal change in the ³¹P-nmr signal of native DNA is the appearance of a very broad shoulder centered at ca. -2.0 ppm, which is larger in M. Lys. DNA than in C. Perf. DNA. FD studies indicate highly ordered TMpyP(4) cations arranged perpendicular to the DNA axis of calf thymus DNA. Together, these results suggest the major effects of TMpyP(4) on DNA properties are due to strong GC-binding interactions that influence DNA structure. The data are consistent with combined intercalative and outside binding interactions of TMpyP(4) with GC regions of DNA. In contrast, similar studies with TMAP suggest that it influences AT regions of DNA by an outside binding mode. On the other hand, TMpyP(2) effects on DNA properties are consistent with nonselective outside binding.