Doxorubicin binds to duplex RNA with higher affinity than ctDNA and favours the isothermal denaturation of triplex RNA

Abstract

The interaction of doxorubicin (DOX) with triplex, duplex and single RNA helices has been studied by fluorimetric, circular dichroism, viscometry, DSC, ITC, T-jump kinetic relaxation technique and fluorescence lifetime measurements in near physiological conditions (pH = 7, I = 0.1 M and 25 °C). DOX binds to the groove of poly(rA)·2poly(rU), while it intercalates into poly(rA)·poly(rU) and forms external complex with poly(rA) and poly(rU). Fluorescence lifetime measurements have shown that all the RNA/DOX complexes are non-fluorescent. The affinity with the duplex is some 15 times greater than with the triplex; this behaviour favours the isothermal denaturation of the triplex/DOX complex according to reaction poly(rA)·2poly(rU)/DOX + DOX ⇌ poly(rA)·poly(rU)/DOX + poly(rU)/DOX, that is, the reaction shifts to right upon increasing the DOX content. ITC measurements have revealed that, under the same conditions, the affinity of DOX with the RNA duplex is higher than with ctDNA, a striking outcome as long as the binding of DOX to DNA seems to be the origin of its biological action.

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Introduction

Doxorubicin (DOX), also known as hydroxydaunorubicin, is the trade name of the antibiotic Adriamycin discovered in 1969.¹ This anthracycline ranks among the most effective anticancer drugs used in the treatment of a wide variety of cancers such as breast, ovarian, prostrate, lung, neuroblastoma, gastric, liver, sarcomas and hematological.² However, the clinical use of DOX is limited due to the appearance of chemo resistance and side effects, especially cardiotoxicity.^3,4 Doxorubicin intercalates into DNA and inhibits topoisomerase II, causing the inhibition of DNA and RNA syntheses.⁵ Concerning its metabolism, harmful free radicals such as reactive oxygen species (ROS) can be released. DNA oxidative damage, DNA adducts and crosslinking, lipids peroxidation and perturbation of cellular membranes are also caused by DOX.^6,7 Three main functional moieties of DOX (Fig. 1) have been identified: the intercalative region (A, B, C rings), an anchoring function (ring D) and the amino sugar moiety.

Fig. 1 Structure of doxorubicin.

Much is known about DOX/DNA interaction. Recently, we have reported the formation of two different complexes at low ionic strength as a function of the drug/ctDNA ratio.⁸ However, less is known regarding the interaction of DOX with RNA, a key process involved in a number of biological events, ranging from gene expression regulation to synthesis of proteins, and plays an important role in a variety of diseases such as myotonic muscular dystrophy, HIV, AIDS, SARS and hepatitis C viral infections.⁹ Fluorescent synthetic probes capable of binding to RNA structures have been used as a powerful tool for the study of RNA functions. These probes can, in principle, recognize single-stranded regions of target RNAs, accompanying the fluorescence light-up response.¹⁰ Meanwhile, targeting double stranded RNA structures by fluorescent probes has been a challenging task.

RNA molecules can adopt a number of structures and conformations able to modulate the cell functions,^11,12 thus rendering RNA an attractive target for the design of drugs.¹³ In addition to duplex hybridization and sequence-specific protein binding, formation of triplex helices represents a plausible mechanism for the cell to target nucleic acid sequences. Importantly, it enables the single-stranded nucleic acid (presumably RNA) to bind the targeted duplex structure (RNA or DNA) without prior unwinding. This peculiar ability meets considerable biotechnological potential and has been extensively studied in applications such as modulation of transcription and site-directed recombination as well as mutagen delivery.¹⁴

Abundant literature on the interaction of small molecules with triplex DNA is available, however reliable information on the interaction with triplex RNA is scarce and ample consensus on the real influence of the type of interaction is lacking. Noncovalent interactions can either stabilize,^15,16 destabilize^17,18 the triplex structure or remain the triplex unaltered¹⁹ compared to the duplex. Even isothermal denaturation and disproportionation are also feasible, as observed in the presence of thionine²⁰ and coralyne,^21,22 respectively. These features reveal that small molecules that may affect the stabilization of triplex RNA is an issue more complicated than initially thought.

Anthracyclines have been shown capable of binding to the iron responsive element mRNAs²³ and reducing the mRNA-binding activity of the iron regulatory proteins in tumour cells.²⁴ In addition, the translational inhibition activity exerted by DOX seems to be due to its interaction with rRNA.²⁵ Moreover, the binding of DOX to the tRNA²⁶ and RNA aptamers²⁷ have been reported. Bulges seem to be key for DOX intercalation, even if there are also weakly interactions with the HIV-1 RNA helical termini.²⁴ However, the binding mode of DOX with single, double and triple stranded RNAs and the thermodynamic aspects of the interaction still remain to be elucidated.

In this work, we address the interaction of DOX with single poly(rA) and poly(rU), double poly(rA)·poly(rU) and triplex poly(rA)·2poly(rU) of RNA. The homopolynucleotide poly(rA)·poly(rU) consists of two chains, poly(rA) and poly(rU), attached to yield a secondary A-duplex structure. The poly(rA)·2poly(rU) triple helix is formed by binding of poly(rA)·poly(rU), joined through Watson–Crick base pairing, with another poly(rU) chain, joined through Hoogsteen base pairing, running parallel the major groove.²⁸ The thermodynamics of the interaction of DOX with poly(rA), poly(rU), poly(rA)·poly(rU) and poly(rA)·2poly(rU) (from now on A, U, AU and UAU, respectively), is studied by fluorescence, Circular Dichroism (CD), Differential Scanning Calorimetry (DSC), Isothermal Titration Calorimetry (ITC), viscosity, T-jump kinetic relaxation technique and fluorescence lifetime measurements.

Experimental

Materials

Doxorubicin hydrochloride was used from Sigma Aldrich without further purification. Stock DOX solutions were prepared by dissolving weighed amounts in 0.1 M NaCl, using 2.5 × 10⁻³ M sodium cacodylate ((CH₃)₂AsO₂Na) to maintain the pH constant to 7.0. The AU, A and U samples used were commercially available and, according to the supplier, the average length of the nucleotides is ca. 700 base-pairs, the distribution remaining unaltered from sample to sample. UAU stock solutions were prepared by mixing equimolar amounts of AU and U at pH = 7.0, leaving the mixture stay overnight. At this pH, A and U are single strands.²⁹

The polynucleotide concentration is denoted as C_P, where C_P is expressed in molarity of single bases for single stranded (A and U), molarity of base pairs for AU and molarity of base triplets for UAU. The molar concentration of DOX is denoted as C_D. Doubly distilled water from a Puranity TU System (VWR) was used to prepare aqueous solutions. Stock solutions of the polynucleotides were standardized spectrophotometrically at λ = 260 nm, using ε = 14 900 M⁻¹ cm⁻¹ for AU and ε = 8900 M⁻¹ cm⁻¹ for U, and at λ = 257 nm using ε = 10 100 M⁻¹ cm⁻¹ for A.³⁰ UAU was prepared by incubating for 24 h at 25 °C an equimolar mixture of AU and U, a time range in which the triple helix is formed.

Methods

pH measurements were carried out with a Metrohm 16 DMS Titrino pH meter, fitted out with a combined glass electrode with a 3 M KCl solution as a liquid junction. Spectrophotometric measurements were performed with a Hewlett-Packard 8453A (Agilent Technologies, Palo Alto, CA) photodiode array spectrophotometer with a Peltier temperature control system. Titrations were carried out at 25 °C by adding increasing amounts of polynucleotide solutions to a DOX solution. The sample was prevented from light during the equilibration period.

Fluorescence titrations were performed on a Shimadzu Corporation RF-5301PC spectrofluorometer (Duisburg, Germany) at λ_ex = 490 nm. The ITC experiments were performed at 25 °C using a Nano ITC Instrument (TA, Waters LLC, New Castle, USA). To prevent formation of air bubbles, all solutions were degassed in a degassing station (TA, Waters LLC, New Castle, USA). Drug solutions were placed in a 50 μL syringe and continuously stirred. Twenty five additions of 2 μL in 300 s intervals were injected into the sample cell containing the buffer or the polynucleotide solution. The integration of these peaks, corrected by the dilution effect, gave the binding isotherms (heat change versus C_D/C_P mole ratio). All the data were analyzed using the NanoAnalyze software. CD measurements were performed with a MOS-450 biological spectrophotometer (Bio-Logic SAS, Claix, France) fitted out with 1.0 cm path length cells. Titrations were carried out at 25 °C by adding increasing amounts of DOX to the polynucleotide solution. Spectrograms were obtained in the 200–800 nm range at 2 nm s⁻¹ speed. Molar ellipticity (Deg M⁻¹ cm⁻¹) was calculated using [θ] = 100θ/C_Pl, where C_P is the polynucleotide concentration and l is the cell light path (cm).

Thermal denaturation studies were performed by DSC readings in a nano DSC (TA Instruments, Newcastle, USA). Cells were 300 μL platinum capillary tubes. Measurements were performed by heating the dye/polynucleotide system from 20 to 90 °C, at 1 °C min⁻¹ scan rate and 3 atm pressure. To reduce to a minimum the formation of bubbles upon heating, the reference and the sample solutions were previously degassed for 30 min in a degassing station (TA Instruments, Newcastle, USA). The thermograms recorded were analysed with the NanoAnalyze 2.0 software. The buffer–buffer baseline was run at least five heating/cooling cycles, until the heating was reproducible and then it was subtracted from the sample data.

Viscosity measurements were taken with a Micro-Ubbelohde viscometer with external control of temperature (25 ± 0.1 °C). The viscosity data were analysed using η/η₀ = (t − t₀)/(t_RNA − t₀), where t₀ and t_RNA are the buffer and polynucleotide solution flow times, respectively, whereas t is the flow time of the DOX/RNA mixture. Mean values of triplicated measurements were adopted to evaluate the RNA viscosity in the absence (η₀) and in the presence, (η) of DOX.³¹

Time correlated single photon counting (TCSPC) measurements were carried out for the fluorescence decay of DOX in the absence and in the presence of increasing concentrations of RNA and DNA. The equipment used was FLS980. Photo excitation was made at 490 nm using a EPL 375 laser. The data were collected and analysed by FAST 3.4.0 software using the following equation,

F(t) = ∑α_ie^−(1/τ_i) (1)

where, α_i and τ_i are the i^th preexponential factor and decay time of the excited species, respectively. Kinetic measurements were performed with a T-jump apparatus built up according to the Rigler et al. prototype,³² working in the absorbance mode. The kinetic curves were stored in an Agilent 54622A oscilloscope (Santa Clara, CA), transferred to a PC and evaluated with the Table Curve program of the Jandel Scientific package (AISN software, Richmond, CA).

Results and discussion

Equilibria

Spectrofluorometric titration. Fig. 2A, C, E and G plot the fluorescence spectra recorded for the AU/DOX, UAU/DOX, A/DOX and U/DOX systems, showing the quenching of the fluorescence of DOX upon interaction with the duplex, triplex and single strands.

Fig. 2 Fluorometric spectra and binding isotherms for the titration of DOX with AU (A and B), UAU (C and D), A (E and F) and U (G and H). C⁰_D = 4.6 × 10⁻⁶ M, λ = 558 nm, I = 0.1 M (NaCl), pH = 7.0 and T = 25 °C. Insets: linear analysis according to eqn (3).

The binding of DOX to RNA can be represented by the apparent reaction:

where P stands for the polynucleotide, D denotes the free DOX and PD is the complex formed. The apparent binding constants (K_app) are evaluated using the McGhee and von Hippel equation:³³where ΔF = F − ϕ_DC_D, Δφ = ϕ_PD − ϕ_D and [P] stands for the equilibrium polynucleotide concentration calculated from the expression [P] = C_Pf(r), where f(r) = (1 − nr)ⁿ/[1 − (n − 1) − r]⁽¹⁻ⁿ⁾. In this expression, r is the ratio between the bound and the free polynucleotide and n, the site size, that is, the number of base pairs occupied by a single dye molecule upon binding. Isotherms at 558 nm and data analysis according to eqn (3) are shown in figures and insets 2B, 2D, 2F and 2H. The values obtained are collected in Table 1.

Table 1 Apparent binding constants, K_app, obtained from fluorescence measurements for RNAs/DOX systems. Thermodynamic constant, K, and molar enthalpy, ΔH, obtained for ctDNA/DOX and RNAs/DOX from ITC measurements. I = 0.1 M (NaCl), pH = 7.0 and T = 25 °C

	10^−5 Kapp (M^−1)	10^−5 K (M^−1)	ΔH (kJ mol^−1)
	Fluorescence	ITC
ctDNA/DOX		3.40 ± 0.61	−21.6 ± 0.4
AU/DOX	12.1 ± 0.2	11.4 ± 2.1	−9.40 ± 0.12
UAU/DOX	1.22 ± 0.05	1.05 ± 0.27	−10.16 ± 0.31
A/DOX	2.37 ± 0.10	2.34 ± 0.61	−11.65 ± 0.64
U/DOX	0.46 ± 0.08	0.58 ± 0.09	−15.30 ± 2.73

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Isothermal calorimetry titrations. The equilibrium interaction of DOX with RNAs was characterized thermodynamically by ITC. RNA solutions were titrated at I = 0.1 M with increasing amounts of DOX. Fig. 3 shows the binding isotherms of the systems studied. The values of thermodynamic molar enthalpy (ΔH) obtained for these systems are listed in Table 1. The binding constant obtained from fluorescence and ITC were very close.

Fig. 3 ITC titration of DOX with (A) AU, (B) UAU, (C) A, (D) U and (E) ctDNA and the corresponding dilution heats. C⁰_P between 0.4 and 0.8 mM, I = 0.1 M (NaCl), pH = 7.0 and T = 25 °C.

To stand fair comparison, Fig. 3 also shows the binding isotherm and Table 1 the K and ΔH values for the DOX/ctDNA system. Under similar conditions, DOX displays greater affinity with the duplex AU than with ctDNA; the latter is a strongly exothermic reaction, indicating that the entropy term is related to hydration effects. In pure water, the counterions in the vicinity of AU retain only (34 ± 21)% solvation sphere, whereas those in the vicinity of ctDNA are fully solvated³⁴ and, in consequence, ΔS_RNA > ΔS_ctDNA follows. The greater affinity with AU relative to ctDNA is quite an interesting outcome, in view that DOX is a very effective type of anticancer drug currently in use.^2,5,7 Our results with AU are in fairly good agreement with the binding constants reported by Marcheschi et al. with the bulge of HIV-1 RNA²⁴ and the K value is one order higher than that for tRNA/DOX.²⁵ Regarding RNA, the binding constants show greater affinity of DOX with AU, followed by A, UAU and U.

CD measurements

CD titrations were performed by adding increasing amounts of DOX to AU, UAU, A and U solutions. Fig. 4 shows the CD spectra recorded. Different behaviours can be distinguished for the duplex, triplex and single strands. For AU (Fig. 4A), the intensity of the characteristic negative DOX band at 300 nm increased and the positive band at 270 nm underwent a slight decrease. A new band in the induced circular dichroism (ICD) region rose at 460 nm. The same bands (though with lower intensity) are observed with UAU (Fig. 4C). A new negative ICD band emerged at 570 nm for the RNA/DOX single stranded systems. The positive band at 270 nm decreased, showing hypsochromic shift for A, whereas for U it increased and shifted to blue (Fig. 4E and G). The CD variation observed with U was larger.

Fig. 4 CD spectrograms recorded between C_D/C_P 0 and 1.5, for AU/DOX (A), UAU/DOX (B), A/DOX (C) and U/DOX (D) C_D = 0.9 mM, I = 0.1 M (NaCl), pH = 7.0 and T = 25 °C.

Thermal stability

The effect of DOX on the thermal stability of AU and UAU was evaluated by DSC measurements. In the presence of single helices (A and U), no transitions were observed. Fig. 5A and C show the evolution of the calorimetric curves for AU/DOX and UAU/DOX systems at C_D/C_P ratios from 0.0 to 0.6. For AU/DOX, only one peak corresponding to the double-to-single helix transition was observed for low C_D/C_P ratio. However, as the C_D/C_P ratio was raised (C_D/C_P ≥ 0.5), the denaturation process yielded two peaks corresponding to two different thermal transitions, whose maxima temperatures were T_m0 and T_mD, with T_m0 < T_mD (Fig. 5A). Also is observed that T_m0 is very close to T_m for AU alone (Fig. 5B), therefore, according to eqn (4) and (5), T_m0 and T_mD are assigned to the denaturation reaction to free duplex and the bound AU/DOX complex transitions, respectively. The T_mD value increased by 4 °C between C_D/C_P 0 and 0.4 and reached a plateau, which is a typical feature of intercalation reactions that entail thermal stabilization.¹⁷

Fig. 5 DSC melting curves for AU/DOX (A), T_m versus C_D/C_P ratio for AU/DOX (B), DSC melting curves for UAU/DOX (C) and T_m versus C_D/C_P ratio for UAU/DOX system (D). C_P = 5 × 10⁻⁴ M, scan rate = 1 °C min⁻¹; P = 3 atm; I = 0.1 M (NaCl), pH = 7.0 and T = 20–95 °C.

Regarding the UAU/DOX system, Fig. 5C and D show the denaturation transition and T_m values, respectively, obtained as a function of the C_D/C_P ratio; these figures reveal quite a different behaviour compared to the AU/DOX. At C_D/C_P = 0.5 and 0.6, three peaks are observed (T_mT, T_m0 and T_mD), in sequence T_mT < T_m0 < T_mD. Fig. 5D shows that T_m0,T is close to the denaturation of UAU at C_D/C_P = 0, thereby such transition is assigned to denaturation of the free UAU present in the solution of UAU/DOX. The difference between the T_m0 for free UAU (C_D/C_P = 0) and UAU at equilibrium (eqn (2)) can be due to a concentration effect. By contrast, T_mT should correspond to denaturation of UAU/DOX, according to reactions (6), whose reaction product, AU/DOX, undergoes the following up denaturation process at T_mD, according to reaction (5). Inspection of Fig. 5B and D reveals that the values of T_mD at C_D/C_P ≥ 0.1 from AU/DOX and from UAU/DOX are nearly identical; this outcome indicates that the two temperatures correspond to denaturation of the duplex. From the slopes of T_mD (and T_mT) versus C_D/C_P it follows that DOX does stabilize AU (positive slope) and strongly destabilizes UAU (negative slope). The different behaviours observed reveal intercalation of DOX into AU and formation of an external complex in UAU, most likely linked to the groove.¹⁷

Viscosity measurements

Viscosity measurements were carried out for the AU and UAU systems at different C_D/C_P concentrations. The relative viscosity η/η₀ is related to the polynucleotide elongation by:³⁵where L is the contour length of the polynucleotide/drug system, L₀ is that of the free polynucleotide, and β is the slope. For the A/DOX and U/DOX systems, no viscosity variations were observed. Fig. 6A and B show the L/L₀ versus C_D/C_P ratio plot for AU/DOX and UAU/DOX systems, respectively.

Fig. 6 Relative polynucleotide elongation L/L₀ versus C_D/C_P ratio for: (A) AU/DOX and (B) UAU/DOX systems. C⁰_P = 2.1 × 10⁻⁴ M, I = 0.1 M (NaCl), pH = 7.0, T = 25 °C.

The binding isotherm described for AU/DOX plot, consists of a sharp increase in the relative contour length when the drug content was raised up to ∼0.12, followed by a plateau. The slope of the first linear stretch is β = 0.9. This behaviour is characteristic of intercalation binding, concurrent with the DSC results. However, for the UAU/DOX system, L/L₀ exhibits a minimum with further increase up to a plateau for values close to those of AU/DOX. In both cases, precipitation occurs for high C_D/C_P ratio. For C_D > 0.02, the viscosity increased, an effect that could be ascribed, in principle, to formation of an intercalated UAU/DOX complex. However, this explanation is not fully reasonable as long as the amount of DOX bound to the groove so far would prevent new DOX units from intercalation. The observed viscosity enhancement can be ascribed to formation of the intercalated complex UA/DOX as a consequence of the isothermal denaturation of groove binder UAU/DOX in the presence of DOX. In other words, for each C_D/C_P ratio the L/L₀ ratio is the outcome of two opposed effects: first, a negative effect related to the groove binder UAU/DOX complex and, secondly, a positive effect related to the intercalated AU/DOX complex. This behaviour is consistent with the DSC observations for the UAU/DOX system, which shows simultaneous decreasing T_mT and increasing T_mD melting temperature profiles (Fig. 5D).

The viscosity and DSC measurements performed at 25 °C show that in the UAU/DOX solution, depending on the DOX concentration, predominates either UAU/DOX or AU/DOX according to isothermal denaturation process (eqn (8)),

where K_D stands for the equilibrium constant. This equilibrium shifts to right upon increasing the DOX content. This behaviour is supported by the rather large formation constant obtained for AU/DOX relative to UAU/DOX (Table 1); the binding of DOX to the groove of UAU can favour the rupture of Hoogsteen-type binding, promoting further intercalation of DOX into AU. From the formation constants of the different complexes (Table 1), K₁ = 7.7 × 10⁴ M⁻¹ for UAU/DOX, K₂ = 1.3 × 10⁶ M⁻¹ for AU/DOX and K₃ = 3.6 × 10⁴ M⁻¹ for U/DOX, it follows that K_D = 460, calculated as (K₂ × K₃)/(K₁ × K_f,T), the formation constant of UAU from AU and U being K_f,T = 1.3 × 10³ M⁻¹. The K_f,T value deduced from kinetic measurements³⁶ at 0.011 M (NaCl) ionic strength was corrected for 0.1 M (NaCl).³⁷ The denaturation process at constant temperature, also has been reported for the RNA/thionine system.³⁸

The behavior of the DOX system with RNA (AU and UAU) is quite different from that displayed by ethidium bromide (EB) with DNA (duplex poly(dA)*poly(dT) and triplex poly(dA)*2poly(dT))³⁹ (1) the DOX fluorescence decreases upon interaction with RNA, whereas the EB fluorescence increases when either the duplex or the triplex form is present; (2) the binding of EB to the triple helical form is substantially stronger than that of the duplex, in contrast to the observations with DOX/RNA; (3) EB intercalates into the duplex and triplex DNA, similar to the observations with berberine derivatives with RNA⁴⁰ while DOX intercalates only into the duplex and binds to the groove with triplex RNA; (4) EB does stabilize the DNA triple helix, while DOX destabilizes the RNA triplex helices. Destabilization of triple RNA and stabilization of its Watson–Crick strands has also been observed with aristololactam-β-D-glucoside,⁴¹ whereas triplex RNA is stabilized by 9-O-(vamino) Alkyl Ether Berberine Analogs.⁴² In summary, a general type of behaviour cannot be established when comparing helix multiplicity (duplex and triplex), because this issue also depends on the particular ligand and the polynucleotide (DNA or RNA).

Kinetic reactions (slow and fast)

The formation of AU/DOX from UAU/DOX according to reaction (8) is corroborated kinetically. Fig. 7A shows the variation of the spectra of freshly prepared UAU/DOX and UA/DOX solutions at same concentration, C_DOX/C_RNA = 0.1. The kinetic trace indicates slow isothermal denaturation (Fig. 7B) whose mechanism is not straightforward because denaturation implies a number of steps, such as dissociation of DOX from the groove of UAU/DOX system, unwinding, release of U from UAU by a cooperative process and intercalation of DOX into AU. Though dissociation and intercalation are fast processes, this is not the case for unwinding and denaturation by rupture of Hoogsteen binding.

Fig. 7 (A) Variation of absorbance of UAU/DOX (red) and AU/DOX (black) at same concentration ratio, C_DOX/C_RNA = 0.1. (B) Kinetic trace recorded at λ = 359 nm. I = 0.1 M (NaCl), pH = 7.0, T = 25 °C.

Fig. 8A shows the T-jump fast kinetic trace corresponding to intercalation of DOX into AU; the data pairs were fitted by a monoexponential function, yielding rate constant value independent of the DOX concentration, the average value being k = 5.8 × 10⁴ s⁻¹ (Fig. 8B). This outcome can be interpreted if the intercalation is preceded by an even faster step, not observable by T-jump. In a similar way as in the presence of DNA,⁸ we can surmise here two types of complexes. Firstly, the sugar moiety of DOX binds to the groove faster than microseconds to give the (AU/DOX)’ complex and, from this groove complex, the aromatic moiety of DOX intercalates monomolecularly to yield the intercalated AU/DOX (eqn (9)).

AU + DOX ⇌ (AU/DOX)′ ⇌ AU/DOX (9)

Fig. 8 (A) T-Jump kinetic curve and fitting of the data-pairs by a monoexponential function (continuous line) for the AU/DOX system; (B) variation of the time constant, 1/τ, with the sum of DOX and AU concentrations in equilibrium. (C) T-Jump curve and fitting of the data-pairs by a monoexponential function (continuous line) for the UAU/DOX system after 6 h incubation time. C_D = 2.14 × 10⁻⁵ M. C_D/C_P = 1.0.

The formation of UAU/DOX or U/DOX (for which the binding to the groove and external binding have been put forward above) cannot be observed by T-jump measurements from freshly prepared solutions; both types of interaction are faster than intercalation. However, when the triplex helix is incubated for 6 h, a kinetic trace is observed just alike that of AU/DOX (Fig. 8C), that is, the rate constant is the same when one starts with either AU (Fig. 8A) or UAU (Fig. 8C), which corroborates the denaturation of UAU/DOX according to reaction (8).

Fluorescence lifetime measurements

TCSPC measurements were carried out for fluorescence decay of DOX in the absence and in the presence of increasing concentration of U, AU and UAU. Within the 0.05 < C_DOX/C_RNA < 0.6 concentration range, only a single, identical lifetime was observed (τ = 1.01 ns) in all cases, corresponding to free DOX (Fig. 9A–D); this feature indicates that the RNA/DOX complexes are non-fluorescent. By contrast, the kinetic trace of DOX/DNA for C_DOX/C_DNA = 0.6 fulfils biexponential pattern (eqn (1)), with τ₁ = 1.21 ns and τ₂ = 2.28 ns (Fig. 9E).

Fig. 9 TCSPC measurements for: AU/DOX (A), UAU/DOX (B), U/DOX (C) and A/DOX (D). In all systems 0 < C_DOX/C_RNA < 0.6. C_D = 4.6 × 10⁻⁶ M. DNA/DOX: black line C_DOX/C_DNA = 0 and red line C_DOX/C_DNA = 0.6 (E).

Conclusions

Thermodynamic and kinetic experiments have shown that interaction of DOX with AU gives rise to an intercalated complex, whereas with UAU it gives a groove binding complex and external complexes with U and A. Similar behaviour was observed in the presence of thionine,¹⁹ in both cases followed by isothermal denaturation of UAU/ligand. Common features in both types of interaction are: (1) intercalation of the ligand into the AU and binding to the groove in the UAU, (2) greater affinity with the AU than with UAU, (3) reaction of the ligand with the U and A single strands. It follows that the binding of the ligand to the groove favours dissociation of the uracil strand bound by Hoogsteen-type binding. The affinity of DOX with RNA in double and triple helices differs by a factor of nearly 15. The higher affinity of DOX with AU reinforces the assumption of disproportionation of the UAU/DOX to give rise to the AU/DOX and the U/DOX complexes at 25 °C. Moreover, in near physiological conditions, the affinity of DOX with the AU is 3 times greater than with ctDNA, a relevant feature in view of the interest aroused by DOX as antitumour and inhibitor agent of topoisomerases I and II.

Acknowledgements

Thanks are due to Prof. F. Secco (University of Pisa, Italy) and C. Pérez-Arnaiz (University of Burgos, Spain) for their kind help and discussion. This work was supported by Obra Social “la Caixa” (project OSLC-2012-007), MINECO (CTQ2014-58812-C2-2-R, FEDER Funds) and Junta de Castilla y Leon, Spain (BU042U16). A. R. R. is grateful for the grant from Junta de Castilla y Leon, Spain (Cofinanced by Fondo Social Europeo) forward to Consejería de Educación.

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