From Nucleobases to Nucleolipids: An ITC Approach on the

Article pubs.acs.org/JPCB

From Nucleobases to Nucleolipids: An ITC Approach on the Thermodynamics of Their Interactions in Aqueous Solutions Angelos Thanassoulas,*,†,§ Philippe Barthélémy,‡ Laurence Navailles,† and Gilles Sigaud† †

Centre de Recherche Paul Pascal, 33600 Pessac, France Univ. Bordeaux, ARNA Laboratory and INSERM, U869, ARNA Laboratory F-33000 Bordeaux, France

‡

ABSTRACT: Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have become an extremely interesting class of molecules, especially for their potential biomedical applications. In this matter, it seemed important to define the nature and estimate the strength of their interaction with polynucleotides by different ways. We report in this work a systematic investigation through isothermal titration calorimetry of the thermodynamics of the association and dissociation of adenine and thymine derivatives, not previously performed. Then we use the results obtained on these simple systems as a basis for comparison with the binding of phospholipids functionalized with adenosine and thymidine to polyadenylic or polyuridylic acids applying the same experimental technique.

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INTRODUCTION

This approach was initially investigated by Yanagawa and colleagues in 19881 and developed since by different groups.2 Owing to their affinity to DNA and RNA, over the past decade, nucleolipids have fostered extensive works both in vitro and in vivo, with tentative application to protecting and conveying genetic materials together with their incidental cytotoxic behavior.3−5 Thus, investigating the interplay of the occurring interactions in these systems can prove valuable for the optimal design of novel biocompatible self-assembling materials4−8 and synthesis of supramolecular materials and sensors.7,9,10 This paper aims at describing the equilibrium thermodynamics of the complex formation among supramolecular nucleotide-based lipid assemblies and nucleic acid counterparts using the isothermal titration calorimetry technique. Obviously, the thermodynamic characterization of these associations is not trivial, considering the interrelatedness of the numerous forces involved, the variety of interfacial morphologies that can be produced (micelles, lamellae, vesicles, tubules, liquid crystalline phases, etc.), and the size of the resulting mesoscopic formations, as they extend to the scale of typical biological structures. Noncovalent interactions, which bring together different kinds of building components into supramolecular entities, are “weak” (some tens of kJ/mol for the strongest hydrogen bonding, in contrast to the covalent interactions that dominate classical chemistry typically a few hundreds of kJ/mol).

The natural world has always been a formidable source of inspiration for chemists and material scientists. Astonished by the complexity and efficiency of the biological machinery, scientists take advantage of billions of years of evolution by mimicking life’s answer to fundamental problems, such as selfassembly and molecular recognition, for the development of novel nanostructures with a wide spectrum of applications. Bioinspired hybrid molecules are a product of this school of thought, rationally designed with the potential to form sophisticated mesoscopic architectures with specific functions, placement, and orientation. A typical approach for designing such a molecule is to couple one bulk moiety, which will play the role of a scaffold, to a second moiety with the desired functional properties. The resulting compound is expected at least to inherit the characteristics of both parental components; nevertheless, cases were synergistic phenomena enhance these properties or even lead to original behavior are not uncommon. Nucleoside and/or nucleotide-based lipids, also known as nucleolipids, are an intriguing class of hybrid molecules, composed of a nucleoside or a nucleotide and two aliphatic chains sharing a common phosphatidyl linking group, which completes the structure. In this architecture, the nucleic acid part plays the role of the “functional” moiety providing the design with selective interaction capacity, while the bulkier amphiphile acts as the “scaffold” moiety with the potential ability to create assemblies of characteristic geometries and orientations. The nucleolipid structure retains the fundamental property of amphiphiles to self-assemble in aqueous solutions. © 2014 American Chemical Society

Received: November 21, 2013 Revised: June 1, 2014 Published: June 2, 2014 6570

dx.doi.org/10.1021/jp411459w | J. Phys. Chem. B 2014, 118, 6570−6585

The Journal of Physical Chemistry B

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A wide range of attractive and repulsive forces is described under the term noncovalent: (i) Hydrophobic/hydrophilic balance, monitored by the aliphatic and aromatic contents of the molecule and its nucleoside end, is the major driving force for the assemblage of the monomeric amphiphiles into ordered forms. The sum of these interactions with water “pays” the entropic cost for the association process (ii) Then hydrogen bonding capability and stacking tendency through π−π interactions are expectedly inherited properties of the nucleobases: these selective short-range interactions are likely to take part, enhancing functional specificity and shaping the building blocks to an energy-minimum configuration. Interactions among nucleobases via hydrogen bonds have been evidenced in the gas phase11 as well as in aprotic solvents like chloroform and DMSO in which they do not compete with water hydrogen bonding.12−16 On the opposite side, π−π stacking is primarily responsible for nucleobase associations in aqueous solutions.17−21 (iii) Furthermore, one should take into account the electrostatic forces due to the presence of negatively charged phosphoryl groups and counterions and, finally, the omnipresent van der Waals forces, generated by myriads of dipole− dipole interactions. Isothermal titration calorimetry (ITC), like every other calorimetric technique, is unable to discriminate among these simultaneous contributions of the participating association forces to the enthalpy. In hopes of breaking down this problem, we have undertaken an extensive ITC investigation of systems containing at least a nucleobase unit, starting from the simplest one (an aqueous solution of thymine injected into an aqueous solution of adenine), and proceeding step by step with increasing structural complexity to end with a solution of a polynucleotide injected into a nucleolipid in a buffer.

Chart 1. Molecular Structure of the Two Nucleolipids That Have Been Used in This Study, Labeled as diC16-dA and diC16-dT

allows the determination of the “microscopic” binding constant (Kassoc), reaction stoichiometry (N), and molar enthalpy (ΔrH) from which the molar entropy (ΔrS) is deduced, thus providing a complete thermodynamic profile of the molecular interaction in a single experiment. In ITC, there are two methods to run titration experiments, both employed in the present study. In the conventional way, the solution in the syringe is introduced into the cell as multiple discrete injections (the number of injections corresponds to the number of experimental points). In the second lesser used approach, the so-called single-injection method (SIM), the whole content of the syringe enters the cell as one large injection over a given period of time, producing a continuous binding isotherm. In both cases, the syringe is loaded with a concentrated ligand solution (typically 2−5 mM), while the calorimetric cell contains only the solution of the host at concentrations 10−30 times less than that of the ligand (as for polynucleic acids, the concentrations are given in monomer equivalent units). The addition of the ligand to the cell provides data for the total heat released by the ligand-acceptor binding but also by the heats of hydration associated with dilution. The contribution from the heat of dilution of the ligand is accounted for in separate experiments by injecting the same solution in a plain buffer solution. These blank data are subsequently subtracted from the titration data to obtain the net binding isotherm as a function of overall ligand concentration in the cell. All ITC measurements were carried out on an ITC200 titration calorimeter (Microcal Inc., Northampton, MA/GE Healthcare). All the titrations presented were repeated at least three times. All the experiments were run at the same temperature 298.15 K, unless the influence of temperature was worth exploring. In conventional titration mode, the ligand solution is added sequentially in 4 μL aliquots (for a total of 10 injections, each at 8 s duration) at 400 s intervals to give the system enough time to recover the experimental baseline and at a syringe rotating speed of 600 rpm, which ensures the homogeneity of the calorimetric cell solution. The filtering time used was 1 s. In a typical SIM experiment, 40 μL of ligand

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EXPERIMENTAL METHODS Compounds and Buffers. Following multiple assays, the buffer chosen for this study is 50 mM HEPES, pH 7.2 (SigmaAldrich, CAS Number: 7365-45-9). HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is one of the Good’s buffers,22 known for its limited effect on biochemical reactions. The very low ionic strength (0.014 M at pH 7.2, 25 °C) and pH stability upon heating together with unnoticeable interference with the systems studied in this work were the decisive factors for the choice. Adenine (CAS 73-24-5), adenosine (CAS 58-61-7), adenosine 5′-monophosphate, monohydrate form (CAS 18422-05-4), thymine (CAS 65-71-4), thymidine (CAS 5089-5), disodium thymidine-5′-monophosphate (CAS 33430-625), polyadenylic acid, potassium salt form (CAS 26763-19-9), and polyuridylic acid, potassium salt form (CAS 27416-86-0) were obtained from Sigma-Aldrich at ∼99% purity. Disodium adenosine 5′-phosphate (CAS 4578-31-8) was purchased from Fluka Analytical. Two nucleolipids have been used in this study, labeled as diC16-dA and diC16-dT (Chart 1). Both were obtained from Prof P. Barthélémy’s laboratory and prepared according to a synthetic route previously reported elsewhere.5 Isothermal Titration Calorimetry. ITC has become over the years the method of choice for the determination of the thermodynamic parameters associated with biomolecular noncovalent interactions. The direct measurement of the heat generated or absorbed by a binding (or dissociation) event 6571



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Table 1. Solubility Data in Water for Some of the Compounds Used in ITC Experiments, at Various Temperaturesa compd

solubility at 293.15 K (mM)



refs

adenine adenosine AMP thymine thymidine

7.0

7.6 19.2 21.5b 27.8 206.4

10.9

96−98 61 99 61, 96, 98 100

18.2b 22.0

31.2b 35.0

a

No data are available for TMP; nevertheless, it is considered more soluble than thymidine. bValue from linear regression of data reported within reference.

titrated into 200 μL of 4.9 mM adenine at a speed of 0.1 μL/s. The reason for selecting thymine as the titrant is that it shows higher solubility in water than adenine (see Table 1). The resulting calorimetric data and the corresponding reference experiments are presented in Figure 1.

solution is injected into the cell in a period lasting 400 s at a syringe stirring speed of 1500 rpm using a 5 s filtering time. Two different sets of experiments were performed for each system studied: (i) dilution experiments to detect the dissociation of homoassociations, if any, among ligands and acceptors as well and (ii) titration experiments to probe the interaction between the acceptor and the ligand. Origin 7.0 software, including calorimetric analysis routines, is used to fit the ITC experimental data to the appropriate thermodynamic model (either dissociation or association) to get the enthalpy ΔrH, the equilibrium constant K, and the stoichiometry N of the reaction. Regarding the data analysis of such binding isotherms, Wiseman et al. noted23 that the overall shape of the curve depends on both the association constant (Kassoc) and the concentration of binding sites (the product N[Mt]). Equation 1 describes this correlation, where c is a dimensionless quantity known as the Wiseman c-parameter. c = N[M t]K assoc

(1)

Extensive analysis of the Wiseman isotherm revealed that a cvalue between 1 and 1000, corresponding to a neat S-shaped curve, is the optimum “window” for the most reliable determination of ΔrH, Kassoc, and N.23−26 However, for some systems, following these guidelines is a hopeless task, especially when poor solubility is accompanied by a low affinity for the ligand. Nonetheless, several studies have reported with confidence thermodynamic parameters for systems that fail to meet this c-value optimal analysis criterion.27−32 Theoretical and experimental statistical error analysis of low c-value systems showed that meaningful thermodynamic information can be extracted from systems well outside the desirable window of 1 < c < 1000, even for c < 10−3 in some cases.33,34 The c-value for most of the ITC experiments presented in this study is in the region of ∼0.01 due to the limited solubility of the materials in aqueous environment (Table 1) and the innate weakness of their homo- and heteromolecular interactions.

Figure 1. Concentration-normalized ITC data for the single-injection titration of a 20 mM thymine solution (syringe) into 4.9 mM adenine (cell) and the corresponding reference experiments at 298.15 K. Red line: Adenine (cell)−thymine (syringe) titration data. Black line: Dilution of the 20 mM thymine solution into cell containing only buffer. Green line: Injection of buffer into the cell containing 4.9 mM adenine solution. Blue line: Injection of buffer to buffer.

As expected, the buffer-to-buffer injection shows a flat thermogram. The thymine injection results in an endothermic trace, and a similar behavior is observed following the injection of buffer into adenine. In contrast, injecting the thymine solution in adenine solution provides a weak, though clearly exothermic, trace. These facts can only be interpreted as (i) self-associations of both adenine and thymine exhibited by the endothermic signal upon dilution of either one; (ii) some binding of the two nucleobases since the net signal of the titration experiment is opposite, that is, exothermic. To our knowledge, this is the first time that an experimental evidence of this interaction in bulk aqueous solution is given. Unfortunately, the concentrations used for these experiments, dictated by poor sample solubility, could not result in a fully saturated system. In addition, the greater heats involved in the blank runs compared to the heat released during the titration lead to a large uncertainty on the subtracted signal.

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RESULTS AND DISCUSSION The Thermodynamic Behavior of the Nucleobases (Adenine and Thymine). There is a plethora of in silico studies focusing on the interactions among nucleobases,35−40 but the experimental work in the field is far less extensive. Since the pioneering work of Ts’o and colleagues in the 1960s,19,21 the behavior of nucleobases in aqueous solutions has been described by solubility studies,18 ultracentrifugation, ultraviolet spectroscopy, and osmotic pressure experiments.20 Furthermore, NMR experiments provide evidence for the association of adenine rings in water by π−π stacking.17 Our approach is to probe the interaction of adenine and thymine in water by ITC. In a SIM experiment, 40 μL of 20 mM thymine solution was 6572



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A subsequent multiple-injections experiment, using the same arrangement and concentrations as the SIM titration (10 injections of thymine, 4 μL each), confirmed the A-T exothermic interaction but also its weakness. The signal intensity is 1 order of magnitude smaller than that of the dissociation of adenine, the enthalpy change between consecutive injections lying at the limits of the instrument’s resolution, thus impeding again a reliable quantitative analysis (Figure 2).

Figure 3. Normalized enthalpies for the incremental dilution of three solutions of adenine (syringe) at different concentrations into 50 mM HEPES, pH 7.2 (cell) at 298.15 K. Red circles, 4.9 mM adenine; blue down triangles, 3.6 mM adenine; green up triangles, 2.4 mM adenine.

a stacked conformation, upon adenine dimer hydration. It is worth mentioning that a similar behavior has also been established for cytosine, which is a pyrimidine base.44 Second, according to Cantor and Schimmel,45 purine−purine and purine−pyrimidine stacking in aqueous media is a noncooperative or even, in some cases, an anticooperative process, a limitation which would rule out extensive aggregation beyond the dimeric form. Assuming thus that adenine dissociation involves only dimers, we applied the simplest model

Figure 2. Concentration-normalized ITC profiles at 288.15 K, vertically translated for reasons of clarity: Injection of a 4.9 mM solution of adenine (green line) and a 20 mM solution of thymine (black line) into the buffer; titration of a 20 mM thymine solution [syringe] into a 4.9 mM solution of adenine [cell] (red line), and a buffer to buffer titration (blue line) as a reference experiment.

K dssoc

(A 2 XoooooY A + A) K assoc

to extract the thermodynamic parameters (Kdissoc = 1/Kassoc, ΔrH°, ΔrS°) of the process from these ITC data (Table 2). The results from a typical adenine ITC dissociation experiment at 298.15 K are presented in Figure 4. Table 2 presents the values of the curve fittings from a series of multi-injection dilutions in buffer of a 3.6 mM solution at different temperatures ranging from 278.15 to 308.15 K. The dissociation of adenine dimers upon dilution is an entropydriven phenomenon opposed by an unfavorable (positive) enthalpy change. It is noticeable that the molar enthalpy of the dissociation decreases with temperature, indicative of a negative value for the molar heat capacity of dissociation. This parameter can be derived from the plot of molar dissociation enthalpy vs temperature, which shows a linear dependence (Figure 5). Negative changes in heat capacity are frequently observed in DNA−ligand interactions.46−48 In cases where complementary high-resolution structural information was available, the measured negative Δ rCp values for DNA intercalation correlated well with corresponding values calculated based on the binding-induced burial of solvent-accessible surfaces. However, osmotic stress studies revealed that the DNA intercalation reactions were accompanied neither by a net change in hydration nor by a net uptake of water which is inconsistent with the predicted hydration changes for the burial of solvent-accessible surfaces. Thus, it is unlikely that the observed negative ΔrCp values for the DNA intercalation

These experiments suggest also a smaller self-association constant for the thymine in comparison to adenine, which is in agreement with the purine−purine > purine−pyrimidine > pyrimidine−pyrimidine scheme of stacking preference proposed by Ts’o et al.21 based on experiments performed on other, more soluble, members of these chemical families. On the other hand, the adenine dilution is energetic enough to attempt to elucidate further the nature of its endothermic trace. First, dilution experiments were performed varying the concentration of adenine in HEPES buffer in the syringe. The concentration-normalized calorimetric data are presented in Figure 3 for three of these experiments. It is straightforward from this figure that the normalized enthalpies for the progressive injections of adenine do not overlap as one would expect for a simple dilution phenomenon. Thus, it is most probable that this behavior is connected to the dissociation upon dilution of aggregates of stacked adenine stabilized by π−π interactions. First, in aqueous solution, the predominant water will obviously overcome the formation of hydrogen bonds among nucleobase solutes, rendering stacking the likeliest driving force for nucleobase association. This assumption is supported by NMR spectra for the selfassociation of nucleobase analogues41,42 and by a series of ab initio molecular dynamics calculations and Monte Carlo simulations,36,43 which provide strong theoretical evidence of a rapid transition from a planar H-bonded geometry in vacuo to 6573



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Table 2. Thermodynamic Parameters at Various Temperatures for the Interactions Discussed, Derived from Nonlinear Least Square Fit of ITC Data T (K)

stoichiometry [N]

Kdissoc (mM) 7.8 10.0 12.9 13.8

± ± ± ±

0.4 0.5 1.8 1.8

molar enthalpy [ΔdissocH] (kJ/mol)

n/a n/a n/a n/a

298.15

2.4 ± 0.2

278.15 288.15 298.15 308.15

n/a n/a n/a n/a

298.15

1.1 ± 0.2

6.8 ± 2.6

1.4 ±

298.15

0.88 ± 0.1

4.2 ± 0.4

2.2 ±

298.15

0.5 ± 0.05

0.15 ± 0.01

298.15

0.2 ± 0.05

1.2 ± 0.1

6.4 ± 1.0 6.6 10.3 12.3 15.7

± ± ± ±

0.8 1.5 1.8 1.4

2.6 2.4 2.3 2.3

± ± ± ±

278.15 288.15 298.15 308.15

0.8 ± 3.4 2.9 2.1 1.3

± ± ± ±

−5.6 ± 2.1 ±

molar entropy [ΔdissocS] (J/mol K)

adenine dissociation −31.2 ± 0.5 −29.9 ± 0.5 −28.3 ± 1.2 −28.0 ± 1.1 thymine to adenine SIM titration 0.1 −39.4 ± 1.3 adenosine dissociation 0.1 −29.5 ± 1.0 0.1 −27.8 ± 1.2 0.1 −29.4 ± 1.1 0.1 −30.4 ± 0.8 thymidine to adenosine SIM titration 0.1 −36.7 ± 3.2 TMP to AMP titration 0.1 −38.1 ± 0.7 Poly-A to diC16dT 0.3 −92.3 ± 1.0 Poly-U to diC16dT 0.2 −49.0 ± 1.0 0.1 0.1 0.1 0.1

molar entropic term [−TΔdissocS] (kJ/mol) 8.7 8.6 8.4 8.6

± ± ± ±

0.2 0.2 0.4 0.4

11.8 ± 0.4 8.2 8.0 8.8 9.4

± ± ± ±

0.3 0.4 0.4 0.3

molar free energy [ΔdissocG] (kJ/mol) 11.3 11.0 10.7 10.9

± ± ± ±

0.2 0.2 0.4 0.4

12.6 ± 0.4 11.6 10.9 10.9 10.7

± ± ± ±

0.3 0.4 0.4 0.3

10.9 ± 1.0

12.3 ± 1.0

11.4 ± 0.2

13.6 ± 0.2

−27.5 ± 0.4

21.9 ± 0.3

−14.6 ± 0.4

16.7 ± 0.3

Figure 5. Molar enthalpies of reaction versus temperature plot for the dissociation of adenine. The slope of the linear fit represents the molar heat capacity of dissociation (ΔrCp= −9.7 ± 0.9 J/mol K).

reactions reflect surface burial alone, if at all. Studies attempting to solve this inconsistency suggested that this phenomenon might reflect contributions from the base pair destacking equilibrium,49,50 a hypothesis that is now supported by our data on heat capacity changes upon adenine dissociation. A significant number of theoretical and experimental studies have been published over the years for the self-association of nucleic acid bases, nucleosides, nucleotides, and their derivatives in aqueous media.18,20,21,43,51−60 An inspection of the reported results, measured by different groups for the same reaction, reveals a strong variance. For example, the free energy change for the adenine−adenine (AA) dimer formation in water is reported between −5.155 and −24 kJ/mol.61 This can

Figure 4. ITC report for the endothermic dissociation of adenine dimers. Upper panel: Raw data for the injection of a 3.6 mM solution of adenine into the buffer at 298.15 K. Lower panel: Normalized injection heats, corrected for control heats and fitting curve to the simple dimer dissociation model (red solid line). The dissociation constant is Kdissoc = 12.9 mM, and the dissociation enthalpy is 2.3 kJ/ mol.

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be attributed to the diversity of techniques and approaches used, each measuring a dependent variable with different sensitivity to the phenomenon in question. Our estimate of the free energy change for the AA dimer formation (ΔrGAA = −10.8 ± 0.4 kJ/mol, at 298.15 K) is in good agreement with the values reported by Nakano and Igarashi,18 and Cieplak and Kollman52 at room temperature (ΔrGAA = −7.5 kJ/mol and ΔrGAA = −9.0 kJ/mol respectively). Related studies, focusing on the self-association in aqueous solutions of moieties structurally similar to adenine, also report free energy changes upon homodimer formation in the region of −10 kJ/mol.42,62,63 From our analysis, one must conclude that the selfassociation of adenine in stacked dimer form is an entropydriven process, supported by a favorable enthalpy; at 298.15 K, the enthalpy contribution is approximately 3.5 times less than the entropic term (Table 2). Base stacking implies an extensive overlap of the ring systems of adjacent bases. Following the thermodynamic considerations of Kauzmann for the burial of nonpolar groups in proteins,64 it was widely believed that the nucleobase stacking in aqueous media was hydrophobic in nature. Typical hydrophobic interactions have a thermodynamic signature of both positive molar enthalpy and molar entropy of reaction,65 in contrast to our experimental findings and others, directly obtained by calorimetric measurements of several similar systems. The different thermodynamic signature of the interactions of nucleobases with water, as compared to those of the nonpolar side chains, has been commented in a number of studies;66,67 in all cases, the authors concluded that the interactions of the bases with water are of different type from those described by Kauzmann for the hydrocarbons. As mentioned above, the accurate thermodynamic analysis of the SIM data for the thymine to adenine titration is problematic, considering the small heat signal, the failure to reach a fully saturated system by the end of the titration due to concentration limitations, and the interrelatedeness of the interactions (Figure 6). The interaction of A and T to form a heterodimer is not a single-step mechanism; it does not involve only free nucleobases in the bulk solvent, but also includes destacking of AA and TT homodimers, in a delicate balance between monomeric and dimeric forms governed by thermodynamic equilibrium. Nevertheless, we can exploit these data to get a rough thermodynamical description of the process, by reducing it to a single-step reaction of the form:

Figure 6. SIM thymine to adenine titration. Upper panel: Calorimetric trace for the SIM titration of 20 mM thymine (syringe) into 4.9 mM adenine (cell) at 298.15 K. Lower panel: Normalized enthalpies (open circles) for the interaction fitted with a single set of sites thermodynamic model (red line).

that of AA and TT, for a negative-enthalpy ITC trace to occur in the first place. The fact that the calculated complexation stoichiometry (N = 2.4) diverges largely from the expected N = 1 value, reflects on the poor quality of the data due to the intrinsic experimental limitations described earlier. The Thermodynamic Behavior of the Nucleosides (Adenosine and Thymidine). The study of nucleosides solutions revealed a behavior very similar to that of the nucleobases. SIM experiments show endothermic traces for the dilution of adenosine and thymidine, again related to a dissociation process, while a SIM titration of adenosine with thymidine reveals an exothermic signal, proof of the association between these two species (Figure 7). Multiple injections experiments were also performed for dilutions of adenosine and thymidine at various temperatures to determine the thermodynamic parameters for the dissociation of their aggregates. NMR and vapor pressure osmometry measurements led to the conclusion that these homoassemblies are mainly stacked dimers with coexisting populations of higher-order oligomers.51,55,68 Typical results for adenosine ITC dissociation at 298.15 K are presented in Figure 8. The thermodynamic parameters determined from these dilution experiments are presented in Table 2. Experimental results analysis shows that the addition of a ribose sugar moiety to adenine does not significantly affect the monomer−dimer equilibrium. In both cases, the dissociation

K assoc

adeninemonomer + thymine monomer XooooooY adenine−thyminecomplex K dissoc

The binding constant of this interaction reflects in all the intercoupling reactions masked under this simple chemical equation. The results from the nonlinear least-square fit of the data to this model are presented in Table 2. The experimental data suggest that the overall AT complexation in aqueous solutions is almost exclusively entropy-driven, with a minuscule favorable enthalpy of approximately −0.8 kJ/ mol. This is not very surprising, considering the fact that the endothermic heterodimer formation is coupled to the exothermic destacking of both homodimeric populations, leading to a cancel-out effect. The dissociation constant of the overall reaction is two times smaller than the one describing the AA dissociation (6.4 and 12.9 mM, respectively). Given that the heterodimer and homodimer formations are antagonistic processes, both claiming unbound nucleobases from the bulk solution, it is to be expected for the AT affinity to be larger than 6575



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constants are almost identical over the temperature range studied, while the dissociation enthalpy of adenosine compared to adenine is higher at 278.15 K and smaller at 308.15 K. This increased temperature factor for the enthalpy is an indication of a larger-than-adenine heat capacity change upon adenosine dilution. This parameter can be derived from the plot of molar dissociation enthalpy versus temperature by linear fitting (Figure 9). Indeed, the heat capacity change for adenosine is

Figure 7. Concentration-normalized ITC data for the SIM titration of a 22.5 mM thymidine solution (syringe) into 15 mM adenosine (cell) and the corresponding reference experiments at 298.15 K. Red line: Adenosine (cell)−thymidine (syringe) titration data. Green line: Dilution of the 22.5 mM thymidine solution into cell containing only buffer. Black line: Injection of buffer into the cell containing 15 mM of adenosine solution. Blue line: Injection of buffer to buffer.

Figure 9. Molar enthalpy of reaction versus temperature plot for the dissociation of adenosine. The slope of the linear fit represents the molar heat capacity of dissociation (ΔrCp= −63.3 ± 4.5 J/mol K).

6 times larger than the one evaluated for adenine within the same temperature range (ΔrCp = −63.3 ± 4.5 J/mol K and ΔrCp = −9.7 ± 0.9 J/mol K, respectively). The molecular change, which provides the structure with additional rotational and vibrational modes, could be the reason behind this substantial increase. The thermodynamic parameters for the aggregational equilibrium of adenosine and adenosine derivatives in aqueous solutions vary significantly between studies,10,55,63,69,70 with the dissociation constant lying in the range from 67 to 4550 mM at room temperature. Our data suggest an even smaller value (Kdissoc = 12.3 ± 1.8 mM, at 298.15 K) for the dissociation constant, which practically means that adenine and adenosine have the same affinity for stacking. The ribose moiety could not possibly participate in this kind of interaction, as it is facing away from the nucleobase−nucleobase interface and lacks aromaticity; in this context, since the total surface area between adenines remains the same, it is reasonable to expect similar Kdissoc values for the adenine and adenosine dimers. The ribose moiety neither favors the stacking, which seems reasonable considering the lack of aromaticity, nor structurally hinders the specific interaction of the nucleobase moieties. Thymidine dilution shows a very weak endothermic signal, which was impossible to analyze further; assuming a similar to adenosine dissociation enthalpy, this is indicative of a dissociation constant at least an order of magnitude larger than its purine counterpart. This hypothesis is in favorable agreement with earlier NMR observations by Ts’o et al. for the thymidine dissociation constant (Kdissoc = 909 mM). Adopting the same assumptions as for the case of adenine− thymine interaction, we analyzed the SIM exothermic trace for

Figure 8. Calorimetric report for the endothermic dissociation of adenosine dimers. Upper panel: Raw data for the injection of a 15 mM solution of adenosine into the buffer at 298.15 K. Lower panel: integrated injection heats corrected for control heats (open circles) and fitting curve to the simple dimer dissociation model (red solid line). The dissociation constant is Kdissoc = 12.3 mM, and the dissociation enthalpy is 2.1 kJ/mol.

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enthalpic terms are both favorable in direct contradiction with the hydrophobic mechanism as suggested by Kauzmann. The Thermodynamic Behavior of the Nucleotides (Adenosine-5′-monophosphate and Thymidine-5′monophosphate). Adding a phosphate group to nucleosides marks a major leap in system complexity. In this case, to describe accurately the occurring interactions, we have to account for the effect of the electrostatic interactions emerging not only between nucleotides themselves, but also among the dynamic “atmosphere” of screening counterions that is formed around them. The question arises whether the negatively charged nucleotides (at pH 7.4) associate in water as the nonionic homologues do, or are impeached by the electrostatic repulsion. To clear the uncertainty, we carried out a series of multiple injection experiments for the introduction of nucleotides (to detect possible oligomerization) into the buffer and for the titration of TMP to AMP (to detect possible interaction). The results from a typical nucleotide dilution experiment are illustrated in Figure 11. From the calorimetric traces of both adenosine-5′-monophosphate (AMP) and thymidine-5′-monophosphate (TMP) dilutions, it is unquestionable that nucleotides do self-associate in water to some extent. Thus, the presence of counterions organized around the charged phosphate groups appear to provide a sufficient electrostatic screening for stacking to occur. However, the

the adenosine−thymidine titration using a single set of sites binding model. The results of the fit are presented in Table 2 and depicted in Figure 10. Again, the accuracy of the data is

Figure 10. SIM thymidine to adenosine titration. Upper panel: Calorimetric trace for the SIM titration of 22.5 mM of thymine (syringe) into 15 mM of adenosine (cell) at 298.15 K. Lower panel: Normalized enthalpies (open circles) for the interaction fitted with a single set of sites thermodynamic model (red line).

affected by the inability to reach a fully saturated system at the end of the titration; nevertheless, a crude assessment of the interaction thermodynamics is still possible. The apparent stoichiometry is close to 1:1 (N = 1.11 ± 0.16), pointing out that after the final injection, the homodimer populations of the system are relatively small, with most of the nucleosides being either in a heterodimeric state or free in the bulk solvent. The dissociation constant for the adenosine-thymidine complexation at 298.15 K is about two times smaller than the corresponding one for adenosine dimer (6.8 mM and 12.3 mM respectively), and almost the same as for the adenine−thymine binding (6.4 mM). Bearing in mind that the π-stacking interface remains largely unaltered by the transition from nucleobases to nucleoside moieties, it is logical to expect roughly the same binding affinity for these interactions. Similar to the adenine− thymine thermodynamic behavior, the entropic contribution to the free enthalpy is dominant for the association of adenosine and thymidine (−10.9 kJ/mol), accompanied by a weak though favorable enthalpy change of −1.4 kJ/mol. As discussed previously for the adenine−thymine interaction, entropic and

Figure 11. Upper panel: Concentration-normalized ITC profiles at 298.15 K, vertically translated for reasons of clarity. Injection of an 18 mM solution of AMP (black line) and a 22 mM solution of TMP (blue line) into the buffer. Lower panel: Normalized enthalpies for the dilutions of AMP (black circles) and TMP (blue triangles). 6577



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4.2 mM. A favorable entropic change is once again the driving force for the interaction, contributing five times more than the enthalpy term to the total free energy change (11.4 and 2.2 kJ/ mol, respectively). Interestingly enough, AMP−TMP association is characterized by a smaller dissociation constant thus more efficient than the association of the nucleoside analogues despite the presence of electrostatic repulsions. This counterintuitive result probably reflects on a significant rearrangement of the hydration layer and reorganization of the surrounding ion “atmosphere” upon complex formation, to the new global energy minimum of the system (solvent, interacting moieties and counterions). Unfortunately, ITC lacks the ability to discriminate among concurring thermal events, and the collected data could not provide any further information on this puzzling issue. The Thermodynamic Behavior of the NucleosideDecorated Lipids and Their Interactions with Nucleic Acid Counterparts. It is well-known that amphipathic lipids spontaneously form a variety of structures in aqueous solution depending on their molecular structure and concentration. This self-assembly efficiency and versatility for are well-desired for the fabrication of novel supramolecular assemblies; however, simple amphiphilic molecules fall short in terms of molecular recognition capacity. On the other hand, nucleobase moieties are known for their ability to recognize their counterparts with high affinity and specificity by hydrogen bonding, yet this ability disappears in aqueous environments. This is the key concept that gave birth to nucleolipids: by anchoring a nucleoside to the specifically oriented and solvent-confined interface of amphiphile aggregates, it was expected to re-enable the molecular recognition. The geometry of the resulting mesoscopic structure of amphiphilic lipids hinges on factors such as the length of their hydrophobic chains, their degree of saturation, and the size and hydrophilicity of the headgroup. Accordingly, it is not surprising that nucleolipids display a great variety of morphologies like vesicles, ribbons, wormlike micelles, hexasomes, cubosomes, and so forth.9,74−78 The fact that the modified lipids diC16dT and diC16dA retain the ability to form mesoscopic aggregates after the anchoring of the nucleosides can be evidenced by dilution assays. A typical dilution experiment follows the same concept as the dissociation titrations of the nucleobase moieties; a concentrated solution of the lipid is progressively injected into the buffer. If aggregates are present in the syringe solution, they dismember upon injection and a corresponding heat of disaggregation is measured. After a few injections, the concentration of the amphiphile in the cell reaches the value known as the critical aggregate concentration (CAC), at which the single amphiphile molecules dispersed in the solvent are at equilibrium with the molecules organized into higher-order structures. Consequently, there is no more destruction of micelles upon further injections and the heat signal disappears except for a simple solvation effect. The CAC can be easily determined by calculating the first derivative of the concentration-normalized measured heats with respect to the total concentration of amphiphile monomers in the cell.79,80 This assay is a direct and convenient method for determining the CAC of the system in question, and has been utilized in many studies over the years.81−84 Typical ITC dilution assays for the nucleoside-decorated lipids diC16dA and diC16dT at 298.15 K are presented in Figures 13 and 14, respectively. In both cases, the calorimeter

normalized enthalpies are extremely small ( 130 mM) than the one determined for the homologous nucleobases and nucleosides. This conclusion is in agreement with previous findings on AMP self-association,19,56,57,60 which place the dissociation constant somewhere in the region of 0.5−5 M. Interestingly, a number of NMR studies suggest that the self-stacking of AMP proceeds beyond the dimeric state.19,55,71−73 It appears that the presence of counterions organized around the charged phosphate groups provide a sufficient electrostatic screening for stacking to occur, although diminished in comparison with the uncharged analogues. The association between AMP and TMP has been conducted through multiple injections experiments only. Parallel to the homologous nucleobase and nucleoside processes, the interaction is exothermic and strong enough to be modeled as a single set of sites binding event. A representative ITC profile for the titration of TMP into a solution of AMP is presented in Figure 12, and the results from the nonlinear least-square fit of the data are summarized in Table 2. The stoichiometry of the reaction is close to 1:1, attesting a simple heterodimer formation, while the dissociation constant of the complex is

Figure 12. Calorimetric data for the exothermic interaction of AMP with TMP. Upper panel: Raw data for the injection of an 18 mM solution of TMP into 6 mM of AMP at 298.15 K. Lower panel: Integrated injection heats, corrected for control heats and fitting curve to the single set of sites model (red solid line). The dissociation constant is Kdissoc = 4.22 mM, and the dissociation enthalpy is 2.2 kJ/ mol. 6578



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Figure 13. Determination of the critical aggregate concentration (CAC) of diC16dA. A 0.27 mM solution of diC16dA is injected in HEPES buffer at 298.15 K. Upper panel: Heat flow versus time. Middle panel: concentration-normalized integrated heat per injection versus the equivalent monomer concentration of diC16dA injected in the cell. Lower panel: The CAC is defined as the concentration where the first derivative of the middle panel curve reaches an extremum. For diC16dA, the CAC is 27 μM.

Figure 14. Determination of the critical aggregate concentration (CAC) of diC16dT. A 0.8 mM solution of diC16dT is injected in HEPES buffer at 298.15 K. Upper panel: Heat flow versus time. Middle panel: concentration-normalized integrated heat per injection versus the equivalent monomer concentration of diC16dT injected in the cell. Lower panel: The CAC is defined as the concentration where the first derivative of the middle panel curve reaches an extremum. For diC16dT, the CAC is 56 μM.

records endothermic peaks (ΔrHdiss > 0) which decrease progressively to the baseline level, marking the end of disaggregation events. This is clear evidence that the nucleoside anchoring does not affect the spontaneous self-assembly of the nucleolipids. The apparent endothermicity is a balancing act of several contributions: the disruption of chain−chain attractions in hydrocarbon tails and π-stacking and/or hydrogen bonding between the nucleoside headgroups have significant enthalpic penalties, while, on the other hand, increased solvation give rise to negative enthalpy change. From these measurements the CAC of diC16dA is almost half that of diC16dT (27 and 56 μM respectively): there is a higher self-association propensity for the adenosine-containing derivative than the thymidine one.

The mesoscopic morphology of these aggregates cannot be determined directly from ITC data. However, ellipsometry measurements suggest that both diC16dA and diC16dT form bilayers at room temperature.85 Nucleolipids with comparable molecular packing parameters have also been reported to form bilayers under analogous conditions.86 Following these assays, we have carried out the titration experiments. To minimize any unwanted heat from dissociation events, the nucleolipid at a concentration well above its CAC 6579



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As expected, the interaction is entropy-driven with a favorable entropy contribution (−27.5 kJ/mol) which is five times larger than the enthalpic penalty (−5.6 kJ/mol). The apparent stoichiometry of the process is 1:2 (two adenine nucleobases of poly-A interact with one thymine nucleobase of diC16dT). ITC, per se, is unable to provide structural information for any binding event; however, some interesting conclusions can be deducted from the association energetics. Regardless of whether the reacting species are in nucleobase, nucleoside, or nucleotide form, binding events between complementary bases are exothermic in aqueous solutions. Thus, the endothermic nature of the poly-A−diC16dT is indicative for the coexistence of additional phenomena. It is known that, at room temperature and polar environments, poly-A molecules form helical and/or coiled domains extending 20−50 adenine bases long, stabilized by π-stacking interactions.87−89 The disruption of this extensive adenine-stacking network could provide the molecular basis for the endothermic signature of the interaction. Such disturbances can arise either from significant poly-A conformational changes upon binding or by the intervention of thymine headgroups between neighboring adenine bases in a “zipper” pattern. A completely different picture emerges from the poly-U− diC16dT interaction. Figure 16 shows a typical ITC profile for the titration of 22 mM of poly-U into a 5.57 mM diC16dT solution at 298.15 K; the parameters of the process derived from fitting the data to a single set of sites model are presented in Table 2. The interaction now is clearly exothermic and remains entropy-controlled with a dissociation constant of 1.2 M. U−U interactions should not differ from T−T interactions from a thermodynamic point of view, due to physicochemical similarities of the molecules. We already demonstrated that the self-association of thymine moieties in aqueous solutions is characterized by very small affinities and could not give rise to extensive secondary structure formation as in the case of polyA. In the absence of substantial π-stacking disruptions, the thermodynamics of the poly-U−diC16dT interaction resembles that of simple nucleobase association in water, enhanced by cooperative effects. The interaction of poly-U with the diC16dA nucleolipid is more complex than that of diC16dT. Typical ITC profiles for the titration of poly-U into a solution of diC16dA, along with the appropriate reference experiments, are presented in Figure 17. The interaction is clearly exothermic with a normalizedenthalpy curve that diverges from the conventional sigmoidal shape of a simple binding event sigmoidal in the best case (parameter c within optimum range), or continuously decreasing curve (value of c below optimum range); for that reason, we were unable to fit the ITC data using a simple single set of sites model. This V-shaped thermogram is characteristic for the coexistence of two enthalpically opposite processes.90−92 The favorable enthalpy contribution would most certainly arise from the interaction of the complementary bases. The most probable mechanism for the parallel endothermic process, in our opinion, is the disruption of the adenine headgroups stacking in the nucleolipid−polymer interface upon association, following a mechanism similar to that of poly-A−diC16dT interaction. In the absence of further evidence, this remains a hypothesis to be tested. Interestingly, Berti et al. have observed analogous structural rearrangement upon nucleolipid−nucleic acid complexation; globular micelles of diC8P-adenosine interacting with single-stranded poly-U, eventually form

was put in the calorimetric cell and the nucleic acid solution, which is not known to self-associate, was put in the syringe. We carried to completion a series of multiple injection ITC experiments for the interactions between diC16dA, diC16dT, polyadenylic (poly-A), and polyuridylic (poly-U) acid, in an attempt to evaluate whether the hybrid designs at least keep the affinity showed by the bare purine and pyrimidine bases or at best enhance their recognition properties. A typical ITC profile for the titration of poly-A into a solution of diC16dT is presented in Figure 15, along with the

Figure 15. Calorimetric data for the interaction of poly-A with diC16dT. Upper panel: Raw data for the titration of a 6 mM solution of poly-A [syringe] into 1 mM solution of diC16dT [cell] (black line), the injection of a 6 mM solution of poly-A into buffer (red line), and the injection of buffer into 1 mM solution of diC16dT (blue line) at 298.15 K, vertically translated for reasons of clarity. Lower panel: Normalized injection heats, corrected for control heats and fitting curve to the single set of sites model (red solid line). The dissociation constant is Kdissoc = 150 μM, and the dissociation enthalpy is −5.6 kJ/ mol.

appropriate reference experiments. While injecting a 6 mM solution of poly-A (all concentrations refer to the amount of repeat unit of the polymers) into buffer or injecting buffer to 1 mM solution of diC16dT results in negligible exothermic peaks, the titration of poly-A into diC16dT produces a sequence of endothermic peaks. As in previous titration experiments, an estimation of the parameters of the interaction was extrapolated by a nonlinear least-square fit of the ITC data using a single set of sites thermodynamic model (Figure 15). The results are summarized in Table 2. 6580



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Figure 17. Calorimetric data for the interaction of poly-U with diC16dA; Upper panel: Raw data for titration of a 6 mM solution of poly-U [syringe] into 1 mM solution of diC16dA [cell] (black line), the injection of a 6 mM solution of poly-U into buffer (red line), and the injection of buffer into 1 mM solution of diC16dA (blue line) at 298.15 K, vertically translated for reasons of clarity. Lower panel: Integrated injection heats, corrected for control heats.

Figure 16. Calorimetric data for the interaction of poly-U with diC16dT. Upper panel: Raw data for the injection of a 22 mM solution of poly-U into 5.57 mM diC16dT at 298.15 K. Lower panel: Normalized injection heats, corrected for control heats and fitting curve to the single set of sites model (red solid line). The dissociation constant is Kdissoc = 1.2 M, and the dissociation enthalpy is 2.1 kJ/mol.

hexagonal superstructures, where the biopolymer is confined between cylindrical micelles.93 Finally, we investigated the possible interaction between poly-A and diC16dA by titrating a 6 mM solution of poly-A into 1 mM solution of diC16dA, at 298.15 K. Unfortunately, the resulting ITC trace was undistinguishable from that of the reference experiments, signifying that under these conditions diC16dA is unable to recognize poly-A (Figure 18). This result is compatible with the findings so far, as the excess of adenine moieties in the nucleolipid−polymer interface is tilting the thermodynamic balance toward intramolecular rather than intermolecular π-stacking.

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CONCLUSIONS In its first part, this paper provides a clear evidence of the presence of weak interactions among adenine and thymine nucleobases, adenosine and thymidine nucleosides, as well as AMP and TMP nucleotides in aqueous solution. These interactions involve both homo- and heteroassociations, according to the general purine−purine > purine−pyrimidine > pyrimidine−pyrimidine order of affinity.

Figure 18. Concentration-normalized ITC profiles at 298.15 K, vertically translated for reasons of clarity: Injection of a 6 mM solution of poly-A into buffer (red line); injection of buffer into 1 mM solution of diC16dA (blue line); titration of a 6 mM solution of poly-A [syringe] into 1 mM solution of diC16dA [cell] (black line).

ITC dilution experiments conducted on these nucleobasecontaining species exhibit a weak to very weak positive heat of 6581



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dissociation (from a few kJ mol−1 to one tenth of kJ mol−1). These small enthalpies are described better by a π−π destacking interaction (as described by various in silico studies) rather than the disruption of the more energetic H-bonds in the highly competing aqueous environment. The estimated values of the constants for these dissociations are in the range of Kd ≈ 2 × 10−2 to 10−2 M, meaning that for a millimolar solution the dimer population is between 15% to 25%, but falls to negligible values for concentrations less than 10−5 M. This order of magnitude is in agreement with the in silico calculations. The titration experiments of adenine opposed to thymine and of adenosine opposed to thymidine show in both cases, and despite the endothermic dilution of both compounds which occurs at each injection, an exothermic net reaction which we believe is also the first direct evidence of specific pair associations between nucleobases in aqueous solution, not related to the commonly described “molecular recognition” through H-bonding but more likely to a heterodimers of π−π type in planar geometry. Despite the electrostatic repulsion, the behavior of AMP and TMP nucleotides does not appear basically different from their uncharged counterparts. On the other hand, the thermodynamic behavior of the A or T nucleolipids and poly-A or poly-U nucleic acids in aqueous solutions appears far less uniform, with the only common characteristic being the weak free energy changes involved. The dilution experiments conducted on nucleolipids give evidence of an endothermic disaggregation, indicating that the intrinsic self-assembly properties of the lipidic moiety are preserved. For the nucleolipid concentrations used in this work, the polynucleic acids (poly A and poly U) interact at the solventexposed surface of the nucleolipid assemblies. As shown by the less than 1 stoichiometry of the titration experiments, due to steric restrictions, only a fraction of the nucleolipid molecules are able to interact with the nucleic acid analogues. Observing such a variety is of interest, for entropy driven interactions with such low enthalpies are usually nonspecific. Specificity appears as an advantage for the future of the nucleolipids as gene carriers, but the strength of the interaction appears insufficient. However, one can imagine a large number of chemical variations around these hybrid molecules that would increase their binding efficiency. A recent publication from our group explores further that approach by incorporating locked nucleic acid−based lipids.94 From this work, it is evident that even small alterations to the initial solution conditions (such as different counterions, concentrations, or even the average size of the reacting species), while producing the same qualitative results, can significantly alter the thermodynamics of these weak interactions. To that direction, we believe it is valuable to perform experiments disrespectful of the Wiseman parameter, knowing from the beginning that the uncertainties in the fit would be large, provided that one conducts a systematic investigation of parent systems varying their chemical structure and is interested in their comparison. The present work, certainly not comprehensive, is part of a wider project aiming to describe further the interaction of these nucleobase derivatives with nucleic acids.85,94,95

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Present Address §

A.T.: Biomolecular Physics Laboratory, National Centre for Scientific Research “Demokritos”, 15310 Aghia Paraskevi, Greece. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

Angelos Thanassoulas thanks the University of Bordeaux 1 for financial support of his fellowship.

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ABBREVIATIONS ITC, isothermal titration calorimetry; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SIM, single-injection method; AMP, adenosine-5′-monophosphate; TMP, thymidine-5′-monophosphate; CAC, critical aggregate concentration

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