Complete Thermodynamic Profiles Research Articles

Novel Solution and Membrane-Based Assays Reveal Lack of Interaction Between Murine FVIIa and Murine EPCR

Abstract 531Generation of anticoagulant activated protein C (aPC) is enhanced by the presentation of protein C (PC) on the endothelial protein C receptor (EPCR) to the thrombin/thrombomodulin complex. Apart from its role in anticoagulation, EPCR also binds human coagulation Factor VIIa (FVIIa). However, the physiological consequences of this interaction either in vitro or in vivo are still unclear. In lieu of the widespread use of high-dose human FVIIa in hemophilic patients with anti Factor VIII or FIX antibodies as well as off-label applications, in vivo experimentation in appropriate mouse models is necessary to further define of the role of the FVIIa-EPCR interaction. Towards this, the aim of this study was to characterize the interaction of murine FVIIa with murine EPCR (mEPCR) in vitro. We used two novel assays that we have developed to study this interaction either in solution (by isothermal titration calorimetry [ITC]) or on the cell surface of mEPCR-expressing cells. The choice of ITC was based on its ability to provide a complete thermodynamic profile of protein-protein interaction in a single experiment: binding constant (K), stoichiometry (n), enthalpy (δH) and entropy (δS), parameters that cannot be determined with other methodologies in a single experiment. We first generated stable cells lines expressing recombinant murine soluble EPCR (msEPCR) or recombinant mFVIIa. The latter was generated as we have previously shown by insertion of a furin intracellular cleavage site between the light and heavy chains of murine FVII (mFVII). This resulted in a molecule secreted and purified in the activated form (mFVIIa) with ∼100% extrinsic activity vs. human FVIIa. Recombinant purified msEPCR had an apparent molecular weight of ∼46 kDa (vs. a predicted ∼26kDa size) which was consistent with extensive carbohydrate modifications. This was confirmed by expressing msEPCR in CHO cells modified to limit the size of the attached N-linked glycan chains: expressed msEPCR from such cells showed a reduction in its apparent molecular size (∼ 38kDa). Next, to validate ITC for the study the FVIIa-EPCR interaction, we used msEPCR and plasma-derived human PC (pd-hPC), a known msEPCR binder, that interacted with msEPCR with a Kd of ∼200nM, a stoichiometry of ∼1:1 and a ΔH and ΔS of −2733 ± 62 cal/mol and 21.3 cal/mol/deg, respectively. Initial ITC experiments with purified recombinant murine PC (mPC) also confirmed its binding to msEPCR. In contrast, we did not observe an interaction between recombinant mFVIIa and msEPCR. This finding was not due to protein degradation, as confirmed by Coomassie protein staining prior to and after ITC. Subsequently, to investigate the mFVIIa-msEPCR interaction in the context of a membrane-anchored murine EPCR, we generated a CHO-K1 cell line that expressed surface-exposed, full-length mEPCR, as verified by flow cytometry. These cells were incubated with pd-hPC or recombinant mFVIIa (50nM) in the presence of physiological Ca2+ (1.6mM) and Mg2+ (0.6mM) ion concentration. The bound protein was eluted with 10mM EDTA, electrophoresed and detected by western blotting. Despite the low femtomolar sensitivity of this assay (corresponding to 0.4% and 0.16% of total mFVIIa and pd-hPC used, respectively), we did not observe mFVIIa binding on mEPCR expressing CHO-K1 cells. Corroborating the ITC data, pd-hPC exhibited detectable binding to mEPCR expressing CHO-K1 cells. As a control, neither pd-hPC nor mFVIIa bound to naïve CHO-K1 cells (no mEPCR expression). In conclusion, we have established two novel assays to study the putative mFVIIa-mEPCR binding and clearly documented the lack of such interaction either in solution or on the cell-membrane. This is in good agreement with recently-published data obtained with surface plasmon resonance. In contrast to human FVIIa-EPCR, our results suggest that extrapolation of certain EPCR-dependent pathologic/physiologic processes using the mouse may be biased by species-specific effects. However, if the mEPCR binding capacity can be engineered to mFVIIa (as seen for human FVIIa-EPCR binding), such mFVIIa molecule(s) may facilitate the dissection of processes involving FVIIa and EPCR using the mouse as a human surrogate in vivo system. The assays developed here can easily assess such possibilities. Disclosures:Pavani:Bayer Healthcare: Research Funding. Margaritis:Bayer Healthcare: Research Funding.

Interaction of berberine, palmatine, coralyne, and sanguinarine to quadruplex DNA: A comparative spectroscopic and calorimetric study

BackgroundInteraction of isoquinoline alkaloids berberine, palmatine, coralyne and sanguinarine with human telomeric quadruplex DNA, dAGGG(TTAGGG)3, has been investigated and compared with ethidium. MethodsBiophysical techniques such as absorption, fluorescence, circular dichroism, optical melting and microcalorimetry have been used. ResultsAbsorption and fluorescence studies revealed noncooperative 1:1 binding for all the molecules. Coralyne showed highest affinity (106 M−1) and for others it was ~105 M−1. The binding affinity varied as coralyne>sanguinarine>berberine>palmatine. Ethidium showed affinity close to sanguinarine. Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of coralyne and sanguinarine compared to berberine and palmatine. Circular dichroic spectral perturbations were similar in all the cases, but a strong induced circular dichroism for the bound molecules was observed only for coralyne and sanguinarine. The interaction of all the alkaloids was exothermic. Binding of coralyne and sanguinarine was predominantly enthalpy driven while that of berberine and palmatine was entropy driven. Heat capacity values of −169, −198, −105 and −95cal/molK, respectively, for coralyne, sanguinarine, berberine, and palmatine suggested significant differences in the hydrophobic contribution to the binding. ConclusionsThis study presents a complete structural and thermodynamic profile of the binding of isoquinoline alkaloids with G-quadruplex. General significanceThese results suggest strong and specific binding of these molecules to the G-quadruplex and highlight the differences in their structure in the interaction profile.

Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

Accurately predicting binding affinity constant (KA) is highly required to determine the binding energetics of the driving forces in drug–DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining KA for PD153035, where KA is determined from the changes in B-form contour length (L) of PD153035–DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1mM sodium cacodylate was exhibited. Furthermore, our results showed that KA=1.18(±0.09)×104 (1/M) at 23±0.5°C and the minimum distance between adjacent bound PD153035≈11bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.

Recent Developments in Isothermal Titration Calorimetry Label Free Screening

Isothermal titration calorimetry (ITC) is a label free technique used for direct detection of biological interactions by measurement of the heat given off or taken up during the reaction. In this article we will introduce the ITC technique and review two applications of ITC in drug discovery; small molecule/protein interactions and enzyme kinetics. We will also describe the characteristics of a new miniaturized, ultrasensitive calorimeter. This new microcalorimetry system reduces the quantity of protein (or other macromolecule sample) required to obtain a complete thermodynamic profile (n, K, DeltaH and DeltaS) by up to 7-fold. The reduction in required sample quantities allows ITC to be effectively utilized at earlier stages of the drug discovery and development process.

Small molecule–RNA interaction: Spectroscopic and calorimetric studies on the binding by the cytotoxic protoberberine alkaloid coralyne to single stranded polyribonucleotides

Background RNA has attracted recent attention for its key role in gene expression and hence targeting by small molecules for therapeutic intervention. This study is aimed to elucidate the specificity of the alkaloid coralyne to poly(G), poly(C), poly(I) and poly(U) in the light of its ability in inducing self-structure in poly(A). Methods Multifaceted experimental techniques like competition dialysis, absorption, fluorescence, circular dichroism and calorimetry were employed. Salt dependence and temperature dependence of the binding was also elucidated. Results Results of competition dialysis, absorption and fluorescence studies revealed that coralyne binds strongly to the polypurines, poly(G) and poly(I) compared to the polypyrimidines, poly(U) and poly(C). Partial intercalative binding due to the stacking of the molecules between the bases was envisaged. The binding was predominantly enthalpy driven with favourable entropy term with a large favourable non-electrostatic contribution revealed from salt dependent data and the dissection of the free energy. The heat capacity change of − 125 and − 119 cal/mol K − 1 respectively for poly(G) and poly(I) and the partial enthalpy–entropy compensation phenomenon observed confirmed the involvement of multiple weak noncovalent interactions. Circular dichroism studies provided evidence for significant perturbation of the conformation of the RNAs, but no self-structure induction was evident in any of the polymers under the condition of the study. Conclusions This study presents a complete structural and thermodynamic profile of coralyne interaction to four single stranded RNA polymers. General significance The study for the first time elucidates the base specificity of coralyne–RNA complexation at the single stranded level.

RNA targeting through binding of small molecules: Studies on t-RNA binding by the cytotoxic protoberberine alkaloidcoralyne

Interaction of the protoberberine alkaloid coralyne with t-RNA(phe) was investigated using various biophysical techniques. Results of absorption and fluorescence studies revealed that the alkaloid binds to t-RNA exhibiting positive cooperativity. Isothermal titration calorimetry results suggested that the binding of the alkaloid was predominantly enthalpy driven with a smaller favourable entropy term. A surprisingly large favourable component for non-electrostatic contribution to the binding of coralyne to t-RNA was revealed from salt dependence data and the dissection of the free energy. The alkaloid enhanced the thermal stability of t-RNA and the binding affinity values obtained from optical thermal melting data was in agreement with that from calorimetry. The heat capacity change of -125 cal mol(-1) K(-1) and the observed significant enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak noncovalent interactions. Circular dichroism studies provided evidence for significant perturbation of the t-RNA structure with concomitant induction of optical activity in the bound achiral alkaloid molecules. Binding isotherms generated from circular dichroic data confirmed the cooperative binding mode of the alkaloid as deduced from spectroscopic data. Docking studies provided further insights into the partially intercalated state of coralyne inside the t-RNA structure. This study presents a complete binding and thermodynamic profile of coralyne interaction to t-RNA.

Binding of berberine to human telomeric quadruplex – spectroscopic, calorimetric and molecular modeling studies

This study examines the characteristics of binding of berberine to the human telomeric d[AG(3)(T(2)AG(3))(3)] quadruplex. By employing UV-visible spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry, we found that the binding affinity of berberine to the human telomeric quadruplex is 10(6). The complete thermodynamic profile for berberine binding to the quadruplex, at 25 degrees C, shows a small negative enthalpy (DeltaH) of -1.7 kcal.mol(-1), an entropy change with TDeltaS of +6.5 kcal.mol(-1), and an overall favorable free energy (DeltaG) of -8.2 kcal.mol(-1) . Through the temperature dependence of DeltaH, we obtained a heat capacity (DeltaC(p)) of -94 (+/- 5) cal.mol(-1).K(-1). The osmotic stress method revealed that there is an uptake of 13 water molecules in the complex relative to the free reactants. Furthermore, the molecular modeling studies on different quadruplex-berberine complexes show that berberine stacking at the external G-quartet is mainly aided by the pi-pi interaction and the stabilization of the high negative charge density of O6 of guanines by the positively charged N7 of berberine. The theoretical heat capacity (DeltaC(p)) values for quadruplex-berberine models are -89 and -156 cal.mol(-1).K(-1).

Calorimetry and Thermodynamics in Drug Design

Modern instrumentation for calorimetry permits direct determination of enthalpy values for binding reactions and conformational transitions in biomolecules. Complete thermodynamic profiles consisting of free energy, enthalpy, and entropy may be obtained for reactions of interest in a relatively straightforward manner. Such profiles are of enormous value in drug design because they provide information about the balance of driving forces that cannot be obtained from structural or computational methods alone. This perspective shows several examples of the insight provided by thermodynamic data in drug design.

Berberine–DNA complexation: New insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA–dT).poly(dA–dT) and poly(dG–dC).poly(dG–dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K a versus [Na +] for poly(dA).poly(dT) and poly(dA–dT).poly(dA–dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (ΔCp o) of berberine binding to poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), Clostridium perfringens and calf thymus DNA were − 98, − 140, − 120 and − 110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.

Effect of Cholesterol and Ergosterol on the Compressibility and Volume Fluctuations of Phospholipid-Sterol Bilayers in the Critical Point Region: A Molecular Acoustic and Calorimetric Study

Although sterol-phospholipid interactions have been of interest for many years now, a complete thermodynamic profile of these systems is still missing. To contribute to a better understanding of the thermodynamic functions of these systems, we determined isothermal compressibility coefficient data for dipalmitoylphosphocholine (DPPC) and DPPC-containing cholesterol and ergosterol vesicles by means of molecular acoustics (ultrasound velocimetry and densimetry) and differential scanning and pressure perturbation calorimetric techniques. A particular focus was on the influence of the differential structural properties of the two sterols on the thermodynamic properties of lipid bilayers, and on the nature of the critical point region of phospholipid-sterol systems by determining thermodynamic fluctuation parameters. Contrary to significant changes in conformational and dynamical properties of the DPPC-sterol membranes, no marked differences were found in the various thermodynamic properties studied, including the adiabatic ( β S lipid ) and isothermal ( β T lipid ) compressibility, as well as the volume fluctuations. Differences in β T lipid and β S lipid become dramatic in the gel-fluid transition region only, due to a significant degree of slow relaxational processes in the microsecond time range in the transition region. Our data show no evidence for the existence of a typical critical point phenomenon in the concentration and temperature range where a critical point in the DPPC-sterol phase diagram is expected to appear. Hence, on a macroscopic level, it seems more appropriate to describe the sterol-phospholipid binary mixtures in the liquid-ordered/liquid-disordered coexistence region as a phase region consisting essentially of small nanodomains only. Such small-domain dimensions, with a series of particular properties such as increased line energy, spontaneous curvature, and limited lifetime, seem also to be typical of raftlike domains in cell membranes.

Thermodynamics of DNA−RNA Heteroduplex Formation: Effects of Locked Nucleic Acid Nucleotides Incorporated into the DNA Strand

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue exhibiting enhanced hybridization efficiency toward complementary strand. The potential of LNA-based oligonucleotides has been sought to improve the selectivity and specificity of probe sets employed in detection and specific targeting of nucleic acids. We have evaluated the influence of "locked nucleic acid" residues on hybridization thermodynamics, counterions and hydration of DNA.RNA heteroduplex using spectroscopic and calorimetric techniques. One to three LNA substitutions have been introduced either at the adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) residues of the DNA strand. A complete thermodynamic profile for heteroduplex formation suggested that LNA-induced stabilization results from a favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, DeltaCp, accompanying the heteroduplex formation. Isothermal titration calorimetry measurements indicated an increase in binding affinity of the two strands as the LNA content of the heteroduplex is increased. Overall our result demonstrated that the effect of LNA-substitution at the thymine residue is more pronounced compared to the adenine residue. Furthermore, optical melting studies showed that, compared to an unmodified duplex, the formation of LNA-modified duplex is accompanied by a higher uptake of counterions and a lower uptake of water molecules. Our result, thus, presents a preliminary attempt toward the characterization of hybridization thermodynamics of the LNA-based probe-target sets, which will in turn aid in the selection of optimal conditions for hybridization experiments, and evaluation of the minimum probe-length required for hybridization and cloning experiments.

Advances in the analysis of isothermal titration calorimetry data for ligand–DNA interactions

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1: n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand–DNA interactions.

Thermodynamic, Counterion, and Hydration Effects for the Incorporation of Locked Nucleic Acid Nucleotides into DNA Duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.

Unfolding of G-Quadruplexes: Energetic, and Ion and Water Contributions of G-Quartet Stacking

It has been shown that DNA oligonucleotides composed, in part, of G repeat sequences can adopt G-quadruplex structures in the presence of specific metal ions. In this work, we use a combination of spectroscopic and calorimetric techniques to determine the spectral and thermodynamic characteristics of two DNA aptamers, d(G2T2G2TGTG2T2G2), G2, and d(G3T2G3TGTG3T2G3), G3; a sequence in the promoter region of the c-MYC oncogene, d(TG4AG3TG4AG3TG4A2G2), NHE-III; and the human telomere sequence d(AG3T2AG3T2AG3T2AG3), 22GG. The circular dichroism spectra of these oligonucleotides in the presence of K+ indicate that all form G-quadruplexes with G-quartets in an antiparallel arrangement (G2), in a parallel arrangement (NHE-III and 22GG), or in a mixed parallel and antiparallel G-quartet arrangement (G3). Melting profiles show transition temperatures, TM, above 45 degrees C that are independent of strand concentration, consistent with the formation of very stable intramolecular G-quadruplexes. We used differential scanning calorimetry to obtain complete thermodynamic profiles for the unfolding of each quadruplex. Subtracting the thermodynamic folding profiles of G2 from those of G3 yielded the following thermodynamic profile for the formation of a G-quartet stack: DeltaG degrees 20 = -2.2 kcal/mol, DeltaHcal = -14.6 kcal/mol, TDeltaScal = -12.4 kcal/mol, DeltanK+ = -0.3 mol of K+/mol, and DeltanW = 13 mol of H2O/mol. Furthermore, we used this profile to estimate the thermodynamic contributions of the loops and/or extra base sequences of each oligonucleotide in the G-quadruplex state. The average free energy contributions of the latter indicate that the incorporation of loops and base overhangs stabilizes quadruplex structures. This stabilization is enthalpy-driven and is due to base-stacking contributions.

Complexes of Poly(ethylene glycol)-Based Cationic Random Copolymer and Calf Thymus DNA: A Complete Biophysical Characterization

Complete biophysical characterization of complexes (polyplexes) of cationic polymers and DNA is needed to understand the mechanism underlying nonviral therapeutic gene transfer. In this article, we propose a new series of synthesized random cationic polymers (RCPs) from methoxy poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride with different mole ratios (32:68, 11:89, and 6:94) which could be used as a model system to address and answer the basic questions relating to the mechanism of the interaction of calf thymus DNA (CT-DNA) and cationic polymers. The solubility of the complexes of CT-DNA and RCP was followed by turbidity measurements. It has been observed that complexes of RCP with 68 mol % MePEGMA precipitate near the charge neutralization point, whereas complexes of the other two polymers are water-soluble and stable at all compositions. Dnase 1 digestion experiments show that DNA is inaccessible when it forms complexes with RCP. Ethidium bromide exclusion and gel electrophoretic mobility show that both polymers are capable of binding with CT-DNA. Atomic force microscopy images in conjunction with light scattering experiments showed that the complexes are spherical in nature and 75-100 nm in diameter. Circular dichroism spectroscopy studies indicated that the secondary structure of DNA in the complexes is not perturbed due to the presence of poly(ethylene glycol) segments in the polymer. Furthermore, we used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the helix-coil transition of CT-DNA in the complexes. UV and differential scanning calorimetry melting experiments revealed that DNA in the complexes is more stable than in the free state and the extent of stability depends on the polymer composition. Isothermal titration calorimetry experiments showed that the binding of these RCPs to CT-DNA is associated with small exothermic enthalpy changes. A complete thermodynamic profile showed that the RCP/DNA complex formation is entropically favorable. Much broader opportunities to vary the architecture of the polymers studied here make these systems promising in addressing various basic and practical problems in gene delivery systems.

Energetics of echinomycin binding to DNA.

Differential scanning calorimetry and UV thermal denaturation have been used to determine a complete thermodynamic profile for the bis-intercalative interaction of the peptide antibiotic echinomycin with DNA. The new calorimetric data are consistent with all previously published binding data, and afford the most rigorous and direct determination of the binding enthalpy possible. For the association of echinomycin with DNA, we found DeltaG degrees = -7.6 kcal mol(-1), DeltaH = +3.8 kcal mol(-1) and DeltaS = +38.9 cal mol(-1) K(-1) at 20 degrees C. The binding reaction is clearly entropically driven, a hallmark of a process that is predominantly stabilized by hydrophobic interactions, though a deeper analysis of the free energy contributions suggests that direct molecular recognition between echinomycin and DNA, mediated by hydrogen bonding and van der Waals contacts, also plays an important role in stabilizing the complex.

DAUNOMYCIN BINDING TO DEOXYPOLYNUCLEOTIDES WITH ALTERNATING SEQUENCES: COMPLETE THERMODYNAMIC PROFILES OF HETEROGENEOUS BINDING SITES

ABSTRACT Complete thermodynamic binding profiles for the interaction of the anticancer drug, daunomycin with natural DNA and synthetic deoxypolynucleotides were described. Fluorescence titration method was used to estimate the equilibrium binding constants. Binding isotherms were found to be surprisingly complex in some cases, presumably because there were heterogeneous sites even in simple deoxypolynucleotides of repeating sequence. Some polynucleotides consisting of alternating sequence contain at least two different binding sites for daunomycin. The binding affinity of the primary binding sites of alternating and non-alternating sequences was found to differ by two orders of magnitude. An isothermal microtitration calorimeter was used to directly measure the binding enthalpy at 25°C with a high sensitivity. The binding enthalpy of poly[d(A-T)] was found to be −5.5 Kcal/mol, which was much lower than any other polynucleotides, while the binding constant of the high affinity sites, was similar. In this report, the complete thermodynamic profiles of daunomycin binding to deoxypolynucleotides were reliably shown for the first time.

Energetic contributions for the formation of TAT/TAT, TAT/CGC(+), and CGC(+)/CGC(+) base triplet stacks.

We used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the folding of a set of triple helices and control duplexes. Specifically, we studied the sequences: d(A(7)C(5)T(7)C(5)T(7)), d(A(6)C(5)T(6)C(5)T(6)), d(A(6)C(5)T(6)), d(AGAGAGAC(5)TCTCTCTC(5)TCTCTCT), d(AGAGAC(5)TCTCTC(5)TCTCT), d(AGAGAC(5)TCTCTC(2)), d(AAGGAC(5)TCCTTC(5)TTCCT), d(AGGAAC(5)TTCCTC(5)TCCTT), and d(GAAAGC(5)CTTTCC(5)CTTTC). Circular dichroism spectroscopy indicated that all triplexes and duplexes are in the "B" conformation. DSC melting experiments revealed that the formation of triplexes is accompanied by a favorable free energy change, which arises from the compensation of a large and favorable enthalpic contribution with an unfavorable entropic contribution. Comparison of the thermodynamic profiles of these triplexes yielded enthalpic contributions of -24 kcal/mol, -23 kcal/mol, and -22 kcal/mol for the formation of TAT/TAT, TAT/CGC(+), and CGC(+)/CGC(+) base triplet stacks, respectively. UV melts as a function of sodium concentration show sodium ions stabilize the triplexes that contain only TAT triplets but destabilize the triplexes that contain CGC(+) triplets. UV melts as a function of pH indicate that the protonation of the third strand and loop cytosines stabilizes the triplexes that contain CGC(+) and TAT triplets, respectively. Our overall results suggest that the triplex to duplex transition of triplexes that contain CGC(+) triplets is accompanied by a release of protons and an uptake of sodium, while their duplex to random coil transition is accompanied by a release of sodium ions. A consequence of this opposite sodium dependence is that their coupled transitions are nearly independent of sodium concentration but are dependent on the experimental pH.

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(omega-aminoalkyl)-2'-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2'-deoxyuridine ('control') or 5-(3-aminopropyl)-2'-deoxyuridine ('modified'). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the 'modified' duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15 degrees C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the 'control' duplex releases water while the 'modified' duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15 degrees C yielded a marginal differential DeltaG degrees term, positive DeltaDeltaH(ITC)-Delta(TDeltaS) compensation, negative DeltaDeltaV and a net release of counterions. The opposite signs of the differential enthalpy-entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the 'modified' duplex.

Energetic and conformational contributions to the stability of Okazaki fragments.

A combination of spectroscopic and calorimetric techniques was used to determine complete thermodynamic profiles accompanying the folding of a model Okazaki fragment with sequence 5'-r(gagga)d(ATCTTTG)-3'/5'-d(CAAAGATTCCTC)-3' and control DNA (with and without thymidine substitutions for uridine), RNA, and hybrid duplexes. Circular dichroism spectroscopy indicated that all DNA duplexes are in the B conformation, the RNA and hybrid duplexes are in the A conformation, and the Okazaki fragment exhibits a spectrum between the A and B conformations. Ultraviolet and differential scanning calorimetry melting experiments reveal that all duplexes unfold in two-state transitions with thermal stabilities that follow the order RNA > OKA > DNA (with thymidines) > hybrids > DNA (with uridines). The dependence of the transition temperature on salt concentration yielded counterion releases in the following order: DNA (with thymidines) > RNA > DNA (with uridines) > OKA > hybrids. Thus, Okazaki fragments have a conformation and charge density between those of its components DNA and hybrid segments. However, the presence of the RNA-DNA/DNA junction confers on them higher stabilities than their component hybrid and DNA segments. The binding of intercalators to an Okazaki hairpin of sequence 5'-r(gc)d(GCU5GCGC)-3' and to its control DNA hairpin has also been studied. The results show that the binding of intercalators to Okazaki fragments is accompanied with higher heats and lower binding affinities, compared with DNA duplexes. This suggests that the presence of an RNA/DNA junction yields a larger surface contact to interact with the phenanthroline ring of the intercalators, which may lead to a larger disruption of the flexible flanking bases of the junction. The overall results suggest that the presence of this junction stabilizes Okazaki fragments and provides a structural feature that can be exploited in the design of drugs to specifically target these molecules.