Differential repair enzyme-substrate selection within dynamic DNA energy landscapes.

We demonstrate that reshaping of the dynamic, bulged-loop energy landscape of DNA triplet repeat ensembles by the presence of an abasic site alters repair outcomes by the APE1 enzyme. This phenomenon depends on the structural context of the lesion, despite the abasic site always having the same neighbors in sequence space. We employ this lesion-induced redistribution of DNA states and a kinetic trap to monitor different occupancies of the DNA bulge loop states. We show how such dynamic redistribution and associated differential occupancies of DNA states impact APE1 repair outcomes and APE1 induced interconversions. We correlate the differential biophysical properties of the dynamic, DNA ensemble states, with their ability to be recognized and processed as substrates by the APE1 DNA repair enzyme. Enzymatic digestions and biophysical characterizations reveal that APE1 cuts a fraction (10-12%) of the dynamic, rollameric substrates within the initial kinetic distribution. APE1 interactions also 'induce' rollamer redistribution from a kinetically trapped distribution to an equilibrium distribution, the latter not containing viable APE1 substrates. We distinguish between kinetically controlled ensemble (re)distributions of potential DNA substrates, versus thermodynamically controlled ensemble (re)distribution; features of importance to DNA regulation. We conclude that APE1 activity catalyzes/induces ensembles that represent the thermodynamically optimal loop distribution, yet which also are nonviable substrate states for abasic site cleavage by APE1. We propose that by inducing substrate redistributions in a dynamic energy landscape, the enzyme actually reduces the available substrate competent species for it to process, reflective of a regulatory mechanism for enzymatic self-repression. If this is a general phenomenon, such a consequence would have a profound impact on slowing down and/or misdirecting DNA repair within dynamic energy landscapes, as exemplified here within triplet repeat domains. In short, APE1-instigated redistribution of potential substrates induces a preferred pathway to an equilibrium ensemble of enzymatically incompetent states.

Tetraplex formation of a guanine-containing nonameric DNA fragment.

A combination of spectroscopic and calorimetric techniques has been used to characterize the structures formed by a family of short, guanine-containing DNA single strands of the form d[GGTTXTTGG], X = A, C, G, T. In 1 molar NaCl at low temperatures, these molecules do not behave like single strands, but rather exhibit properties consistent with tetraplex formation. The standard state enthalpies, entropies, and free energies for formation of each tetraplex have been measured, as have preliminary nuclear magnetic resonance (NMR) spectra. In 1 molar KCl, the melting behavior of the structure or structures is more complex than in 1 molar NaCl. This observation may be related to the recently proposed "sodium-potassium switch."

Thermodynamic analysis of biomolecules: a volumetric approach.

Fundamental thermodynamic relationships reveal that volumetric studies on molecules of interest can yield useful new information. In particular, appropriately designed volumetric studies can characterize the properties of molecules as a function of solution conditions, including the role of solvation. Until recently, such studies on biologically interesting molecules have been limited because of the lack of readily available instrumentation with the requisite sensitivity; however, during the past decade, advances in the development of highly sensitive, small-volume densimetric, acoustic and high-pressure spectroscopic instrumentation have enabled biological molecules to be subjected to a wide range of volumetric studies. In fact, the volumetric methods used in these studies have already provided unique insights into the molecular origins of the intramolecular and intermolecular recognition events that modulate biomolecular processes. Of particular note are recent volumetric studies on globular proteins and nucleic acid duplexes. These studies have provided unique insights into the role of hydration in modulating the stabilities of these biopolymers, as well as their conformational transitions and ligand-binding properties.

Calorimetry of proteins and nucleic acids.

The availability of sensitive calorimetric instrumentation has led to a considerable increase in thermodynamic studies of proteins, nucleic acids, and their interactions. This article reviews some of the recent contributions of calorimetry to characterizing the thermodynamic origins of protein and nucleic acid stability and conformational preferences, as well as the interactions of proteins with each other, with small molecules, and with nucleic acids.

The thermodynamics of DNA structures that contain lesions or guanine tetrads.

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data.

Thermodynamics of an intramolecular DNA triple helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally induced structural transitions.

We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5'-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3') which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5 loop. The 3'-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5 loop, form Hoogsteen interactions in the major groove of the Watson-Crick hairpin. From ultraviolet (UV) melting studies we find that the 31-mer exhibits either one or two transitions, depending on solution conditions. We use pH- and temperature-dependent circular dichroism (CD) to assign the initial and final states associated with each transition. We find that the disruption of the Hoogsteen hairpin is accompanied by a release of protons and an uptake of sodium ions while the disruption of the Watson-Crick hairpin is accompanied by a release of sodium ions with no change in protonation state. From these data, we construct a phase diagram for this intramolecular DNA triple helix as a function of pH, sodium ion concentration, and temperature. We characterize the energetics of each transition using a van't Hoff analysis and differential scanning calorimetry (DSC). Significantly, the DSC data provide a model-independent thermodynamic characterization of the thermally induced transitions of this triplex. By combining the spectroscopic and calorimetric data, we develop a semi-empirical model which describes the state of the 31-mer as a function of pH, sodium ion concentration, and temperature. With this model we successfully predict characteristics of the 31-mer, which are beyond the data which are used in establishing the model (for example, the salt dependence of the apparent pKa of the Hoogsteen strand). This semi-empirical model may serve as a prototype for developing a method to predict the phase diagrams of intramolecular triple helix systems.

The native and the heat-induced denatured states of alpha-chymotrypsinogen A: thermodynamic and spectroscopic studies.

We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy to detect and to characterize the conformational transitions of alpha-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements. At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly "softens" at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less "tight". This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, kM, of the protein "native" state. Our volumetric data also allow us to conclude that the heat-induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible core of the native state. By contrast, our volumetric data are consistent with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated.

The hydration of globular proteins as derived from volume and compressibility measurements: cross correlating thermodynamic and structural data.

We report the first thermodynamic characterization of protein hydration that does not depend on model compound data but rather is based exclusively on macroscopic (volumetric) and microscopic (X-ray) measurements on protein molecules themselves. By combining these macroscopic and microscopic characterizations, we describe a quantitative model that allows one for the first time to predict the partial specific volumes, v(zero), and the partial specific adiabatic compressibilities, ks(zero), of globular proteins from the crystallographic coordinates of the constituent atoms, without using data derived from studies on low-molecular-mass model compounds. Specifically, we have used acoustic and densimetric techniques to determine v(zero) and ks(zero) for 15 globular proteins over a temperature range from 18 to 55 degrees C. For the subset of the 12 proteins with known three-dimensional structures, we calculated the molecular volumes as well as the solvent-accessible surface areas of the constituent charged, polar and nonpolar atomic groups. By combining these measured and calculated properties and applying linear regression analysis, we determined, as a function of temperature, the average hydration contributions to v(zero) and ks(zero) of 1 A2 of the charged, polar, and nonpolar solvent-accessible protein surfaces. We compared these results with those derived from studies on low-molecular-mass compounds to assess the validity of existing models of protein hydration based on small molecule data. This comparison revealed the following features: the hydration contributions to v(zero) and ks(zero) of charged protein surface groups are similar to those of charged groups in small organic molecules. By contrast, the hydration contributions to v(zero) and ks(zero) of polar protein surface groups are qualitatively different from those of polar groups in low-molecular-mass compounds. We suggest that this disparity may reflect the presence of networks of water molecules adjacent to polar protein surface areas, with these networks involving waters from second and third coordination spheres. For nonpolar protein surface groups, we find the ability of low-molecular-mass compounds to model successfully protein properties depends on the temperature domain being examined. Specifically, at room temperatures and below, the hydration contribution to ks(zero) of protein nonpolar surface atomic groups is close to that of nonpolar groups in small organic molecules. By contrast, at higher temperatures, the hydration contribution to ks(zero) of protein nonpolar surface groups becomes more negative than that of nonpolar groups in small organic molecules. We suggest that this behaviour may reflect nonpolar groups on protein surfaces being hydrated independently at low temperatures, while at higher temperatures some of the solvating waters become influenced by neighboring polar groups. We discuss the implications of our aggregate results in terms of various approaches currently being used to describe the hydration properties of globular proteins, particularly focusing on the limitations of existing additive models based on small molecule data.

Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH.

Cytochrome c can exist in a native (N), a molten globule (MG) or an unfolded (U) state depending on solution conditions. We have used high-precision ultrasonic and densimetric techniques to measure volume and compressibility changes accompanying the N to MG, N to U and U to MG transitions of the protein. For the N to MG transition (induced by lowering the pH to 2 in the presence of 200 mM CsCl), we measure a volume increase of 0.014 cm3g-1 and a compressibility increase of 3.8 x 10(-6) cm3g-1bar-1. For the N to U transition (induced by lowering the pH to 2 in the absence of salt), we measure a volume increase of 0.010 cm3 g-1 and a compressibility decrease of 2.0 x 10(-6) cm3 g-1 bar-1. For the U to MG transition at pH 2 (induced by adding CsCl up to 200 mM), we measure a volume increase of 0.006 cm3 g-1 and a compressibility increase of 6.8 x 10(-6) cm3 g-1 bar-1. We interpret these data to reach the following conclusions about the three states of cytochrome c. (1) A solvent-inaccessible core is preserved in the molten globule state, with the volume of this core being about 40% of the intrinsic volume of native cytochrome c. (2) The coefficient of the adiabatic compressibility of this preserved molten globule core is 61 x 10(-6) bar-1, a value that is over four times higher than that of the interior of the native protein. This result is consistent with the interior of the preserved MG core being liquid-like in contrast to the more tightly packed, solid-like interior of the native state. (3) In the unfolded state of cytochrome c, only 70 to 80% of the surface area of a fully unfolded conformation is exposed to the solvent, a result that reflects some level of order in the "denatured" state. (4) The relative volume fluctuations of the solvent-inaccessible interiors of the native, molten globule and unfolded states are equal to 0.6%, 2.0% and 2.9%, respectively. These data are consistent with the solvent-inaccessible core of the molten globule state being much more loosely packed than the core of the native state. In fact, the fluctuations in the molten globule and unfolded states are so high that one cannot exclude the possibility that formally buried atomic groups transiently contact solvent molecules. To the best of our knowledge, the data reported here provide the first characterizations of the intrinsic volume and compressibility properties of the native, molten globule and unfolded states of a single protein. We discuss in terms of the current protein literature the new insights that can be derived from these data.

Counterion association with native and denatured nucleic acids: an experimental approach.

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA.

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-¿Pt(NH(3))(2)¿(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins.

Tyrosine-PEG-derived poly(ether carbonate)s as new biomaterials. Part II: study of inverse temperature transitions.

Tyrosine-poly(alkylene oxide)-derived poly(ether carbonate)s represent a new group of degradable biomaterials that exhibit inverse temperature transitions. Poly(DTE co 70%PEG,1000 carbonate) was chosen as an example to study this special phase transition behavior of the polymers. The observed transition temperature varied slightly depending on the technique used, e.g. CD always gave a lower temperature than UV/Vis. CD and UV/Vis studies indicated that the transition temperature was both heating rate and concentration dependent. Thermodynamic parameters of the transition (enthalpy, entropy, and free energy) were determined by DSC. The molecularity of the transition was 2.6, as calculated from UV and DSC data. The transition temperature could be varied from 18 to 58 degrees C by changing the polymer structure. The new poly(ether carbonate)s may be used in medical applications such as injectable drug delivery formulations and bioresorbable barriers for the prevention of surgical adhesions.

A calorimetric investigation of single stranded base stacking in the ribo-oligonucleotide A7.

Differential scanning calorimetry has been employed to determine the energy change associated with single stranded base stacking in the ribo-oligonucleotide A7. A total enthalpy change of 20.3 kcal (mole of heptamer)-1 was measured. This corresponds to 2.9 kcal (mole of adenine)-1 or 3.4 kcal (mole of A-A stack)-1 if one assumes that all six stacking interactions are energetically equivalent. These results represent the first direct determination of this important parameter for a ribo-oligonucleotide. It is noted that the calorimetrically determined value reported here is considerably lower than any of the previously published van't Hoff enthalpies but is consistent with values that can be derived from other calorimetric data.

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7.

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A7U7 are reported over a concentration range of 6.9 micrometer to 19.6 micrometer in single strand in 1 M NaCl. The rate constants for helix formation are about 2 X 10(6) M-1 s-1 and decrease with increasing temperature yielding an activation enthalpy of -6 kcal/mole. The rate constants for helix dissociation range from 3 to 250 s-1 and increase with increasing temperature yielding an activation enthalpy of +45 kcal/mole. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.

Calorimetric and spectroscopic investigation of the helix-to-coil transition of the self-complementary deoxyribonucleotide ATGCAT.

Differential scanning calorimetry and temperature-dependent uv spectroscopy are used to thermodynamically characterize the double-strand to single-strand transition of the self-complementary deoxyribo-oligonucleotide ATGCAT. The calorimetric experiments provide a value of 33.6 kcal (mol of double strand)-1 for the transition between 10 and 90 degree C. In conjunction with available temperature-dependent nmr data (which reveals terminal base pair fraying), we attempt to define specifically those interactions to which the calorimetrically measured enthalpy change refers. Values of delta HV.H. (van 't Hoff enthalpy change) are derived from the spectroscopic and calorimetric data and compared with the delta H obtained directly from the calorimetric experiment. This comparison reveals that the part of the thermally-induced transition that occurs between 10 and 90 degree C is well represented by a two-state process. It is noted that is assessing the applicability of the two-state model it is best to compare the delta with delta HV.H. Hcal. obtained from the calorimetric rather than the spectroscopic data.

Hydration of diglycyl tripeptides with non-polar side chains: a volumetric study.

We have determined the apparent molar volumes and the apparent molar adiabatic compressibilities at 25 degrees C of 10 X-Gly-Gly and Gly-Gly-X tripeptides in which X represents a residue with a non-polar side chain. We also have determined the changes in volume and compressibility which accompany neutralization of the amino and carboxyl termini in these tripeptides. The mutual influence of the non-polar side chain of the X residue and the terminal amino and carboxyl groups on the hydration of each other depends on the chemical nature of the side chain and the state of ionization of the termini. We interpret our data in terms of the hydration of the component aliphatic, aromatic, and charged atomic groups, as well as the mutual interactions between these groups.

Hydration and partial compressibility of biological compounds.

We review the results of compressibility studies on proteins, nucleic acids, and systematically altered low molecular weight compounds that model the constituents of these biopolymers. The model compound data allow one to define the compressibility properties of water surrounding charged, polar, and nonpolar groups. These results, in conjunction with compressibility data on proteins and nucleic acids, were used to define the properties of water that is perturbed by the presence of these biopolymers in aqueous solutions. Throughout this review, we emphasize the importance of compressibility data for characterizing the hydration properties of solutes (particularly, proteins, nucleic acids, and their constituents), while describing how such data can be interpreted to gain insight into role that hydration can play in modulating the stability of and recognition between biologically important compounds.

The role of solvents in the stabilization of helical structure: the low pH ribo A8 and A10 double helices in mixed solvents.

The order-disorder transitions of the double helices formed by the ribo-oligoadenylic acids rA8 and rA10 at pH 4.2 have been investigated in a series of organic/aqueous mixed solvents. Melting temperature data, Tm, derived from the uv melting curves were used to define the stability of the double helices in the different mixed solvent systems. It was found that the extent of helix destabilization depended in a predictable fashion on both the quantity and the nature of the added organic solvents. For the C1 through C4 aliphatic alcohols, the longer, less branched alcohols proved to be more effective destabilizers of the helical structure. Significantly, the amides proved to be more powerful destabilizers than the alcohols. Analysis of the melting curves provided the Van't Hoff enthalpy change for each transition. The data are interpreted in terms of the role of solvent in the stabilization of ribonucleic acid structure.

Base stacking in the dinucleoside phosphate rapa.

The pH-induced unstacking of rRpA has been investigated by batch calorimetry and uv spectroscopy. Equilibrium uv melting curves confirmed that the adenine bases in rApA are stacked at pH7 but unstacked at pH 1.5. The enthalpy change accompanying this pH-induced unstacking is +2.65 kcal (mole of A-A stack)-1 as measured by batch calorimetry. This represents the first direct determination of this important parameter for a dinucleoside phosphate. It is noted that the calorimetrically determined value reported here is considerably lower than published van't Hoff enthalpies but is consistent with values that can be derived from calorimetric data on polymers.

The thermodynamics of drug-DNA interactions: ethidium bromide and propidium iodide.

We report the first calorimetrically-derived characterization of the thermodynamics of ethidium bromide (EB) and propidium iodide (PI) binding to a series of nucleic acid host duplexes. Our spectroscopic and calorimetric measurements yield the following results: 1) At low salt (16mM Na+) and 25 degrees C. PI binds more strongly than EB to a given host duplex. The magnitude of this PI preference depends only marginally on base sequence, with AT base pairs showing a greater PI preference than GC base pairs. 2) The enhanced binding of PI relative to EB at low salt and 25 degrees C reflects a more favorable entropic driving force for PI binding. 3) The PI binding preference diminishes at higher salt concentrations (216mM). In other words, the binding preference is electrostatic in origin. 4) The salt dependence of the binding constants (delta lnKb/delta ln[Na+]) reveal that PI binds as a dication while EB binds as a monocation. 5) PI and EB both exhibit impressive enthalpy-entropy compensations when they bind to the deoxy homopolymers poly dA.poly dT and poly dA.poly dU. We have observed a similar enthalpy-entropy compensation for netropsin binding to the poly dA.poly dT homopolymer duplex. We therefore conclude that the compensation phenomenon is an intrinsic property of the host duplex rather than reflecting a property of the binding ligand. 6) When either PI or EB bind to the corresponding ribo homopolymer (poly rA.poy rU) we do not observe the enthalpy-entropy compensation that characterizes the binding to the deoxy homopolymer. 7) EB and PI both bind more strongly to poly d(AT).poly d(AT) than to poly d(AU).poly d(AU). Specifically, the absence of the thymine methyl group in poly d(AU).poly d(AU) reduces the binding constant of both drugs by a factor of four. This reduction in binding is due to a less favorable entropy change. In this paper we present and discuss possible molecular origins for our observed thermodynamic and extra-thermodynamic data. In particular, we evoke solvent effects involving both the drugs and the host duplexes when we propose molecular interpretations which are consistent with our thermodynamic data.