RNA triplex-to-duplex and duplex-to-triplex conversion induced by coralyne.

Spectrophotometric, circular dichroism, calorimetric, displacement assay and kinetic analyses of the binding of the fluorescent dye coralyne to poly(A)2poly(U) have served to enlighten the ability of the dye to produce dramatic changes in the RNA structure. The sets of data assembled convey that coralyne is able to induce the triplex-to-duplex conversion and also the duplex-to-triplex conversion according to a non-reversible cycle governed by temperature, provided that the [dye]/[polymer] ratio (CD/CP) is maintained constant above unity. Alternatively, at room temperature the triplex is formed at (roughly) CD/CP < 1 and the duplex at CD/CP > 1.

The mode of binding ACMA-DNA relies on the base-pair nature.

A thermodynamic and kinetic study on the mode of binding of 9-amino-6-chloro-2-methoxi-acridine (ACMA) to poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) has been undertaken at pH = 7.0 and I = 0.1 M. The spectrophotometric, kinetic (T-jump), circular dichroism, viscometric and calorimetric information gathered point to formation of a fully intercalated ACMA complex with poly(dA-dT)·poly(dA-dT) and another one only partially intercalated (7%) with poly(dG-dC)·poly(dG-dC). The ACMA affinity with the A-T bases was higher than with the G-C bases. The two polynucleotide sequences give rise to external complexes when the ACMA concentration is raised, namely, the electrostatic complex poly(dA-dT)·poly(dA-dT)-ACMA and the major groove binding complex poly(dG-dC)·poly(dG-dC)-ACMA. A considerable quenching effect of the ACMA fluorescence is observed with poly(dA-dT)·poly(dA-dT), ascribable to face-to-face location in the intercalated A-T-ACMA base-pairs. The even stronger effect observed in the presence of poly(dG-dC)·poly(dG-dC) is related to the guanine residue from on- and off-slot ACMA positions.

New aspects of the interaction of the antibiotic coralyne with RNA: coralyne induces triple helix formation in poly(rA)*poly(rU).

The interaction of coralyne with poly(A)*poly(U), poly(A)*2poly(U), poly(A) and poly(A)*poly(A) is analysed using spectrophotometric, spectrofluorometric, circular dichroism (CD), viscometric, stopped-flow and temperature-jump techniques. It is shown for the first time that coralyne induces disproportionation of poly(A)*poly(U) to triplex poly(A)*2poly(U) and single-stranded poly(A) under suitable values of the [dye]/[polymer] ratio (C(D)/C(P)). Kinetic, CD and spectrofluorometric experiments reveal that this process requires that coralyne (D) binds to duplex. The resulting complex (AUD) reacts with free duplex giving triplex (UAUD) and free poly(A); moreover, ligand exchange between duplex and triplex occurs. A reaction mechanism is proposed and the reaction parameters are evaluated. For C(D)/C(P)> 0.8 poly(A)*poly(U) does not disproportionate at 25 degrees C and dye intercalation into AU to give AUD is the only observed process. Melting experiments as well show that coralyne induces the duplex disproportionation. Effects of temperature, ionic strength and ethanol content are investigated. One concludes that triplex formation requires coralyne be only partially intercalated into AUD. Under suitable concentration conditions, this feature favours the interaction of free AU with AUD to give the AUDAU intermediate which evolves into triplex UAUD and single-stranded poly(A). Duplex poly(A)*poly(A) undergoes aggregation as well, but only at much higher polymer concentrations compared to poly(A)*poly(U).

Route to metallacrowns: the mechanism of formation of a dinuclear iron(III)-salicylhydroxamate complex.

The equilibria and the kinetics of the binding of Iron(III) to salicylhydroxamic (SHA) and benzohydroxamic (BHA) acids have been investigated in aqueous solution (I = 1 M (HClO(4)/NaClO(4)), T = 298 K) using spectrophotometric and stopped-flow methods. Whereas Iron(III) forms a 1:1 complex (ML) with BHA, it forms both ML and M(2)L complexes with SHA. The presence of M(2)L in aqueous medium is corroborated by FTIR measurements. The reactive form of Iron(III) is the hydrolyzed species FeOH(2+), which binds to the O,O site in ML and to the O,O and O(P),N (P = phenolate) sites in M(2)L, inducing full deprotonation of the latter. The reaction pathway is discussed in terms of a multistep mechanistic scheme in which the metal-ligand interaction is coupled to hydrolysis and self-aggregation steps of Iron(III). The observation and characterization of M(2)L as a stable species is important because it contains the -Fe-O-N-Fe- sequence, which constitutes the repetitive motif of the SHA-based metallacrown ring and provides the rationale for 12-MC-4 metallacrowns. In the framework of this study, the kinetics of the Iron(III) dimerization and trimerization have also been investigated using the stopped-flow method to perform dilution jumps. The reaction scheme put forward involves two parallel steps (FeOH(2+) + FeOH(2+) and Fe(3+) + FeOH(2+)) that lead to formation of the Fe(2)(OH)(2)(4+) dimer and a slower step (FeOH(2+) + Fe(2)(OH)(2)(4+)) to form the trimer species. The kinetics of the last step have been investigated here for the first time, and the results deduced indicate that, of the two possible trimer structures reported in the literature, Fe(3)(OH)(3)(6+) and Fe(3)(OH)(4)(5+), the latter prevails by far.

7-Aminoactinomycin binding to DNA sequences lacking GpC sites: a thermodynamic and kinetic study.

The interaction of 7-aminoactinomycin (7AAMD) with selected DNA sequences (TAGTTA, R5, HP5, and HP1) of different lengths and secondary structures, all containing a 5'-TAGT-3' block, was studied at an ionic strength of 0.02 M and pH 7.7 by means of fluorescence equilibrium and kinetic (stopped-flow) measurements. Both approaches indicated that the antibiotic binds strongly to both the single-stranded and hairpin (HP1) structures, although the sequences lacked the canonical GpC sites favored by actinomycin. Binding isotherms and initial rate analyses revealed that the binding stoichiometry was 1:1 in all cases. While the single-stranded sequences displayed a simple monoexponential kinetic behavior, the binding of 7AAMD to HP1 at <30 degrees C was biphasic and could be rationalized in terms of a sequential formation of two isomeric bound forms or alternatively in terms of an ssHP1-hpHP1 equilibrium, with both HP1 forms reacting with 7AAMD. The rates of complex dissociation induced by the detergent SDS were also measured. After correction of the kinetic traces for spurious effects that can be attributed to the SDS, monoexponential traces were obtained, with relaxation times in agreement with the kinetics of complex formation.

Kinetics and equilibria of the interaction of 8-hydroxyquinoline with gallium(III) in water and sodium dodecyl sulfate solution.

The kinetics and equilibria of binding of gallium(III) to the 8-hydroxyquinoline (HQ) have been investigated in water and in the presence of sodium dodecyl sulfate (SDS) micelles. Moreover, the pKA1 and pKA2 of HQ and first hydrolysis constant of Ga3+ ion have been measured in water and SDS solution. The analysis of the kinetic and thermodynamic behavior reveals that the reactive form of Ga(III) is GaOH2+ in both cases. Although in water the only bound form of Ga(III) appears to be the deprotonated complex GaL, evidence for stabilization of the protonated form, GaHL on the micelle surface and stabilization of Ga3+ with respect to GaOH2+ is provided by the kinetic behavior in SDS. The addition of SDS at concentrations around the critical micellar concentration, results in a large enhancement of the rate of complex formation. The large catalytic effect produced by the SDS micellar solution provides a promising basis for the extraction of gallium from water using the HQ/SDS system. A procedure for gallium(III) extraction and recovery based on ligand modified-micellar enhanced ultrafiltration method, using the HQ/SDS system, is described.

Solvent effects on the thermodynamics and kinetics of coralyne self-aggregation.

The role of solvent effects on the thermodynamics and kinetics of the coralyne self-aggregation process has been investigated in ethanol-water mixtures of different compositions. The changes in the UV/visible spectra of coralyne and FAB/LSIMS mass spectrometry agreed well with the formation of a dimer species. 1D and 2D 1H experiments have allowed one to look into the features of the self-aggregation process and to determine the equilibrium constant and the deltaH0 and deltaS0 values for the aggregate formation in 0-50% ethanol-water mixtures. The kinetics of self-aggregation has been investigated by the T-jump chemical relaxation method, and the results have been interpreted in terms of dimer formation. The dependence of the relative viscosity of coralyne solutions on the dye concentration was studied in different ethanol-water mixtures. Finally, it was found that coralyne behaves as a solvatochromic indicator which is preferentially solvated according to the sequence ethanol > ethanol-water > water. All of the results concur in elucidating the relevant role of the hydrophobic interaction process of coralyne stack formation.

Polyamine-polycarboxylate metal complexes with different biological effectiveness as nitric oxide scavengers. Clues for drug design.

The synthesis of the Fe(III), Co(II), Mn(II), and Ru(III) complexes with two polyamine-polycarboxylate ligands, N-(2-hydroxyethyl)ethylenediamine-N, N', N'-triacetic acid (H3L1) and ethylene bisglycol tetraacetic acid (H4L2) is reported. Potentiometric studies showed that these ligands form stable complexes in aqueous solution and no metal release occurs, thus accounting for their low toxicity in cultured RAW 264.7 macrophages. X-ray characterization of the [Co(L1)](-) complex showed that binding sites are available at the metal for NO binding. Efficiency of these compounds to bind NO was studied by UV-vis spectrophotometry. Then their NO-scavenging properties were assayed in a cell-free system under physiological conditions, using S-nitroso-N-acetyl-D,L-penicillamine (SNAP) as NO source. The L1 complexes caused the most effective reduction of free NO, [Mn(L1)](-) being the most efficient. Conversely, in NOS II induced RAW 264.7 macrophages, the Ru(III) and Co(II) complexes with L2 were the most effective compounds. [Ru(L2)](-) also afforded significant protection against lipopolysaccharide-induced endotoxic shock in the mouse in vivo.

Role of the third strand in the binding of proflavine and pt-proflavine to poly(rA).2poly(rU): a thermodynamic and kinetic study.

The interactions of triple strands of poly(rA).2poly(rU) with proflavine (PR) and the proflavine cis-platinum derivative [{PtCl (tmen)} 2{NC 13H 7(NCH 2CH 2) 2}] (+) (PRPt) are examined at pH 7.0, T = 25 degrees C, and 0.2 M ionic strength by spectrophotometry, spectrofluorometry, circular dichroism, viscosimetry, stopped-flow, and T-jump relaxation techniques. The melting experiments demonstrate that both drugs tend to destabilize the triplex structure, although the PRPt effect is more relevant. By contrast, both drugs tend to slightly stabilize the duplex structure. The viscosity and circular dichroism measurements show that, at a low dye-to-polymer ratio ( C D/ C P), the binding is intercalative, whereas at high C D/ C P values, the external binding dominates. The binding kinetics and equilibria have been investigated over the C D/ C P region, where intercalation is operative. Both drugs bind to the RNA triplex according to the excluded site model. With PR, two kinetic effects have been observed, whereas with PRPt, only one has been observed. The results are interpreted according to the reaction schemes D + S right arrow over left arrow DS I, with PRPt, and D + S right arrow over left arrow DS I right arrow over left arrow DS II, with PR. The electrostatic contribution to the formation activation energy for DS I is similar (40%) for both systems. The results suggest that DS I is a partially intercalated species. Absence of the second step with PRPt is put down to groove interaction of the Pt-containing moiety, which prevents the PR residue from further penetration through the base pairs to form the fully intercalated complex, DS II. Comparison with the binding of the same drugs to the duplex reveals that the occupation of the major groove in poly(rA).2poly(rU) by the third strand plays a critical role in the kinetic behavior.

Mechanism of indium(III) exchange between NTA and transferrin: a kinetic approach.

The equilibria and kinetics for the process of In(3+) exchange between nitrilotriacetic acid (NTA) and bovine serum transferrin (T) have been investigated in aqueous solution containing sodium bicarbonate. The metal exchange equilibria have been measured by difference ultraviolet spectroscopy at 25 degrees C, pH=7.4, and I=0.2 M (NaClO4). The acid dissociation constants of NTA and the binding constants of In(III) to NTA have also been measured. Kinetic experiments revealed that the process of In(3+) uptake by transferrin from [In(NTA)2](3-) is biphasic, the fast phase being completed in a few seconds, the slow phase lasting for hours. The fast phase has been investigated by the stopped-flow method and results in monoexponential kinetics. It involves rapid interaction of the 1:1 complex ML (M=In, L=NTA) with TB (T=transferrin, B=CO3(2-)) to give a quaternary intermediate MLTB which then evolves to an "open" MTB* ternary complex complex with expulsion of L. In turn, this complex interconverts to a "closed", more stable, form MTB. Neither the prevailing complex M2L nor the TB2 form of transferrin are directly involved in the exchange process but act as metal and protein reservoirs. The pH dependence of the reaction has been also investigated. The slow phase has not been investigated in detail; it takes several hours to go to the completeness, its slowness being ascribed to metal redistribution between the C-site and N-site of the protein, and/or metal release from polynuclear In(III) species.

The two modes of binding of Ru(phen)(2)dppz(2+) to DNA: thermodynamic evidence and kinetic studies.

The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A).

Mg(II) and Ni(II) induce aggregation of poly(rA)poly(rU) to either tetra-aggregate or triplex depending on the metal ion concentration.

The ability of magnesium(II) and nickel(II) to induce dramatic conformational changes in the synthetic RNA poly(rA)poly(rU) has been investigated. Kinetic experiments, spectrofluorometric titrations, melting experiments and DSC measurements contribute in shedding light on a complex behaviour where the action of metal ions (Na(+), Mg(2+), Ni(2+)), in synergism with other operators as the intercalating dye coralyne and temperature, all concur in stabilising a peculiar RNA form. Mg(2+) and Ni(2+) (M) bind rapidly and almost quantitatively to the duplex (AU) to give a RNA/metal ion complex (AUM). Then, by the union of two AUM units, an unstable tetra-aggregate (UAUA(M2)*) is formed which, in the presence of a relatively modest excess of metal, evolves to the UAUM triplex by releasing a single AM strand. On the other hand, under conditions of high metal content, the UAUA(M2)* intermediate rearranges to give a more stable tetra-aggregate (UAUA(M2)). As concerns the role of coralyne (D), it is found that D strongly interacts with UAUA(M2). Also, in the presence of coralyne, the ability of divalent ions to promote the transition of AUD into UAUD is enhanced, according to the efficiency sequence [Ni(2+)]≫[Mg(2+)]≫[Na(+)].

Aggregation features and fluorescence of Hoechst 33258.

The functionality of the bisbenzimide Hoechst 33258 in solution has been largely exploited in the quantification of DNA. Understanding of its behavior is essential to promote its widespread application and learning of biological processes. A detailed study of the dimerization process of the fluorescent blue dye Hoechst 33258 is carried out by isothermal titration calorimetry, absorbance, fluorescence, differential scanning calorimetry and T-jump kinetic measurements. The dimer/monomer ratio depends on the dye concentration and the ionic strength. The dimerization constant determined under physiological conditions (pH = 7.0; I = 0.10 M), KD = 3 × 10(4) M(-1), conveys that only micromolar concentrations of the dye can ensure reasonably high amounts of the monomer species in solution. For instance, for 10 µM dye content, the dimer prevails for I > 0.08 M, whereas the monomer is observed at low ionic strength, a key issue to be elucidated as long as the dimer species is more fluorescent than the monomer and the fluorescence intensity strongly relies on the ionic strength and the dye concentration.

Kinetics and equilibria for the formation of a new DNA metal-intercalator: the cyclic polyamine Neotrien/copper(II) complex.

A study has been performed of the kinetics and equilibria involved in complex formation between the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) and Cu(II) in acidic aqueous solution and ionic strength 0.5 M (NaCl), by means of the stopped-flow method and UV spectrophotometry. Spectrophotometric titrations and kinetic experiments revealed that the binding of Cu(II) to Neotrien gives rise to several 1:1 complexes differing in their degree of protonation. Under the experimental hydrogen ion concentration range investigated, complexation occurs by two parallel paths: (a) M2+ + (H4L)4+ <==> (MH4L)6+ and (b) M2+ + (H3L)3+ <==> (MH3L)5+. The rate constants values found for complex formation, by paths (a) and (b), are much lower than the values expected from water exchange at copper(II) and other amine/Cu(II) complexation kinetic constants. Kinetic experiments at different NaCl concentrations indicated that this finding was not due to chloride ion competition in complex formation with Neotrien, but it was related to a ring rigidity effect. As the phenanthroline moiety could, in principle, interact with nucleic acids by intercalation or external binding, some preliminary measurements concerned with the possible interactions occurring between the Cu(II)/Neotrien complex and calf thymus DNA (CT-DNA) have also been carried out. The absorption spectra of the Cu(II)/Neotrien complex change upon addition of CT-DNA at pH 7.0, revealing the occurrence of complex-nucleic acid interactions. Moreover, fluorescence titrations, carried out by adding the Cu(II)/Neotrien complex to CT-DNA, previously saturated with ethidium bromide (EB), show that the Cu(II)/Neotrien complex is able to displace EB from DNA, suggesting the complex is able to intercalate into the polynucleotide and then to cleave the phosphodiester bond of DNA.

Intercalation of Zn(II) and Cu(II) complexes of the cyclic polyamine Neotrien into DNA: equilibria and kinetics.

The equilibria and kinetics of the interaction of the Zn(II) and Cu(II) complexes of the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) with calf thymus DNA have been investigated at pH=7.0 and T=25 degrees C by spectrophotometry, spectrofluorimetry and stopped-flow method. At low dye/polymer ratios both complexes bind to DNA according to the excluded site model. At high dye/polymer ratios the binding displays cooperative features. The logarithm of the binding constant depends linearly on -log[NaCl]. The kinetic results suggest the D + S <==> D, S <==> DS mechanism where the metal complexes (D) react with the DNA sites (S) leading to fast formation of an externally bound form (D,S) which, in turn, is converted into internally bound complex (DS) by intercalation. The binding constants, evaluated as ratios of rate constants, agree with those obtained from equilibrium binding experiments, thus confirming the validity of the proposed model. Fluorescence titrations, where the metal-Neotrien complexes were added to DNA previously saturated with ethidium bromide (EB), show that both complexes displace EB from the DNA cavities. The reverse process, i.e. the addition of excess ethidium to the DNA/metal Neotrien systems, leads to fluorescence recovery for DNA/ZnNeotrien but not for DNA/CuNeotrien. This observation suggests that the binding of CuNeotrien induces deep alterations in the DNA structure. Experiments with Poly(dA-dT)*Poly(dA-dT) and Poly(dG-dC)*Poly(dG-dC) reveal that CuNeotrien mainly affects the structure of the latter polynucleotide.

Mechanism of Ni2+ and NiOH+ interaction with hydroxamic acids in SDS: evaluation of the contributions to the equilibrium and rate parameters in the aqueous and micellar phase.

The equilibria and kinetics (stopped-flow) of the binding of Ni(II) to salicylhydroxamic acid (SHA) and phenylbenzohydroxamic acid (PBHA) have been investigated in aqueous solutions containing SDS micelles. The two ligands are fairly distributed between the two pseudophases present, so the binding reaction occurs in both phases. The contributions to the total reaction from each phase has been evaluated, following a procedure where use is made of the experimentally determined partition coefficients of the reactants involved. The mechanism of the reaction occurring on the micelle surface has been derived and comparison with the mechanism in water shows that the step Ni(2+) + HL ⇄ NiHL(2+) is operative in both pseudophases, whereas the step Ni(2+) + L(-)⇄ NiL(+), which is operative in water, is replaced in SDS by the step NiOH(+) + HL ⇄ NiL(+). The analysis of the equilibrium and of the kinetic data enabled the evaluation of the equilibrium and the rate constants of the individual steps taking part in the binding process over the micelle surface. Interestingly, the first hydrolysis constant of the Ni(H(2)O)(6)(2+) ion in SDS is more than two orders of magnitude higher than in water. The agreement between the equilibrium constants derived from kinetics and those obtained by static measurements confirms the validity of the proposed mechanism.

The fluorophore 4',6-diamidino-2-phenylindole (DAPI) induces DNA folding in long double-stranded DNA.

DAPI (4',6-diamidino-2-phenylindole) is a widely used fluorescent dye, whose complicated binding features to DNAs and RNAs have been the object of debates and are still not fully understood. In this study, different approaches were employed, including binding equilibrium measurements (spectrofluorometry), melting experiments (spectrophotometry), viscometric measurements, circular dichroism, and T-jump kinetic analyses; all data concur in shedding light on the complex mechanistic aspects of the binding mode of DAPI to natural DNA. Conditions are found that induce the mode of the DAPI/DNA interaction to change from groove binding to intercalation. Moreover, it is observed, for the first time, that DAPI is able to induce the formation of a rather compact polymer-dye adduct under particular conditions. The results suggest that this form is a folded or coiled DNA structure stabilized by DAPI dye bridges.

Synthesis, characterization, DNA interaction and potential applications of gold nanoparticles functionalized with Acridine Orange fluorophores.

Two new water-soluble gold nanoparticles (AO-TEG-Au and AO-PEG-Au NPs) are prepared and characterized. They are stabilized by thioalkylated oligoethylene glycols and functionalized with fluorescent Acridine Orange (AO) derivatives. Despite the different core sizes (11.8 and 3.9 nm respectively) and shell composition, they are both well dispersed and are stable in water, even if some self-aggregation is observed in the case of AO-TEG-Au NPs. However, AO-PEG-Au NPs show much lower emission efficiency with respect to AO-TEG-Au NPs. Spectrophotometric and spectrofluorometric experiments indicate that both types of nanoparticle are able to bind to calf thymus DNA, either by external binding or partial intercalation. Preliminary FACS flow cytometry tests seem to indicate that the AO-TEG-Au nanoparticle is able to cross the cell membrane where it is absorbed by Chinese hamster ovary (CHO) cells at the picomolar concentration level.

DNA binding of a proflavine derivative bearing a platinum hanging residue.

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I=0.1M and 25°C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides.

Solvent effects on the kinetics of the interaction of 1-pyrenecarboxaldehyde with calf thymus DNA.

The kinetics of the interaction of a fluorescent probe, 1-pyrenecarboxaldehyde, with calf thymus DNA has been studied in different water/alcohol mixtures (ethanol, 2-propanol, and ter-butanol) at 25 degrees C, by using the stopped flow technique. The kinetic curves are biexponential and reveal the presence of two processes whose rates differ by about 1 order of magnitude on the time scale. The dependence of the reciprocal fast relaxation time on the DNA concentration is linear, whereas the concentration dependence of the reciprocal slow relaxation time tends to a plateau at high DNA concentrations. The simplest mechanism consistent with the kinetic results involves a simple two-step series mechanism reaction scheme. The first step corresponds to the formation of a precursor complex, (DNA/Py)(I), while the second one corresponds to full intercalation of the pyrene dye between the DNA base pairs. The values of the rate constants of both steps decrease as water activity decreases. The results have been discussed in terms of solvation of the species and changes in the viscosity of the solution.