Isothermal titration calorimetry (ITC) and circular dichroism (CD) were used to study the thermodynamics of RPC·G-quadruplex DNA (G4) complex formation. The ruthenium polypyridyl complexes (RPCs) were [Ru(phen)3]2+ (12+), [Ru(phen)2(dppz)]2+ (22+), [Ru(phen)2(tatpp)]2+ (32+), and [Ru(phen)2(tatpp)(phen)2Ru]4+ (44+), and target DNAs were c-MYC NHE-III1 promoter sequence mutants forming 1-2-1 and 1-6-1 G-quadruplexes. Formation of the 2:1 RPC·G4 complexes is characterized by entropy driven RPC binding to the top and bottom of G-tetrad faces. 12+ appears to bind very weakly or not at all to G4 DNA. 22+ having a dipyridophenazine group to stack on the top and bottom of the G4 core, exhibits an average Ka = 6.7 × 104 m–1. 32+, with a larger G4 interactive tetraazatetrapyridopentacene group, binds with significantly higher affinity, Ka = 1.1 × 106 m–1. 22+ and 32+ appear to bind independently of G4 folding topology and RPC conformation. The thermograms for the titration of G4 DNA with rac-44+ are characterized by two binding modes exhibiting higher and lower affinity (Ka,1 = 3.6 × 107 m–1 and Ka,2 = 3.2 × 105 m–1). The two binding modes are attributed to preferential binding of one of the 44+ enantiomers (e.g. ΛΛ) over the other isomers (e.g. ΔΔ or ΔΛ). Tighter binding of the preferred 44+ enantiomer, in comparison to 32+, is due to additional favorable entropy for locating a second [(phen)2Ru–]2+ moiety in a G4 groove. Weaker binding of the disfavored 44+ isomers must be due to a poorer fit of these isomers with the G4 faces.

•H1.1 and H1.4 bind to DNA oligomers and to CT-DNA with favorable entropy changes.•The 9 °C increase in Tm for DNA in the H1.4/DNA complex indicates tight binding.•The H1.4 binding site size was found to be 36.9 bp in titrations of DNA oligomers.•The H1.1 binding site size was found to be 32 bp in titrations of CT-DNA.•Loss of water upon H1/DNA complex formation results in large negative ΔCp values.H1.1 and H1.4 bind tightly to both short DNA oligomers and to CT-DNA (Ka ≈ 1 × 107). Binding is accompanied by an unfavorable enthalpy change (∆H ≈ + 22 kcal/mol) and a favorable entropy change (− T∆S ≈ − 30 kcal/mol). The Tm for the H1.4/CT-DNA complex is increased by 9 °C over the Tm for the free DNA. H1.4 titrations of the DNA oligomers yield stoichiometries (H1/DNA) of 0.64, 0.96, 1.29, and 2.04 for 24, 36, 48, and 72-bp DNA oligomers. The stoichiometries are consistent with a binding site size of 37 ± 1 bp. CT-DNA titration data are consistent with binding site sizes of 32 bp for H1.1 and 36 bp for H1.4. The heat capacity changes, ΔCp, for formation of the H1.1 and H1.4/CT-DNA complexes are − 160 cal mol− 1 K− 1 and − 192 cal mol− 1 K− 1 respectively. The large negative ΔCp values indicate the loss of water from the protein DNA interface in the complex.

1H NMR and isothermal titration calorimetry (ITC) experiments were employed to obtain reliable thermodynamic data for the formation of the 1:1 inclusion complexes of fullerenes C60 and C70 with the buckycatcher (C60H28). NMR measurements were done in toluene-d8 and chlorobenzene-d5 at 288, 298, and 308 K, while the ITC titrations were performed in toluene, chlorobenzene, o-dichlorobenzene, anisole, and 1,1,2,2-tetrachloroethane at temperatures from 278 to 323 K. The association constants, Ka, obtained with both techniques are in very good agreement. The thermodynamic data obtained by ITC indicate that generally the host–guest association is enthalpy-driven. Interestingly, the entropy contributions are, with rare exceptions, slightly stabilizing or close to zero. Neither ΔH nor ΔS is constant over the temperature range studied, and these thermodynamic functions exhibit classical enthalpy/entropy compensation. The ΔCp values calculated from the temperature dependence of the calorimetric ΔH values are negative for the association of both fullerenes with the buckycatcher in toluene. The negative ΔCp values are consistent with some desolvation of the host-cavity and the guest in the inclusion complexes, C60@C60H28 and C70@C60H28.

Isothermal titration calorimetry (ITC) is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g., Keq (or ΔG), ΔH, ΔS, and n) for a ligand-binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combining equilibrium constant, mass balance, and charge balance equations for the system under study. Commercial ITC instruments are supplied with software that includes a number of simple interaction models, for example, one binding site, two binding sites, sequential sites, and n-independent binding sites. More complex models, for example, three or more binding sites, one site with multiple binding mechanisms, linked equilibria, or equilibria involving macromolecular conformational selection through ligand binding, need to be developed on a case-by-case basis by the ITC user. In this paper we provide an algorithm (and a link to our MATLAB program) for the nonlinear regression analysis of a multiple-binding-site model with up to four overlapping binding equilibria. Error analysis demonstrates that fitting ITC data for multiple parameters (e.g., up to nine parameters in the three-binding-site model) yields thermodynamic parameters with acceptable accuracy.

Histone H1 is a chromatin protein found in most eukaryotes. ITC and CD have been used to study the binding of H10 and its C-terminal, H10–C, and globular, H10–G, domains to a highly polymerized DNA. ITC results indicate that H10 and H10–C bind tightly to DNA (Ka ≈ 1 × 107), with an unfavorable ΔH (ΔH ≈ + 22 kcal/mol) and a favorable ΔS (− TΔS ≈ − 30 kcal/mol). Binding H10–G to DNA at 25 °C is calorimetrically silent. A multiple independent site model fits the ITC data, with the anomaly in the data near saturation attributed to rearrangement of bound H1, maximizing the number of binding sites. CD experiments indicate that H10/DNA and H10–C/DNA complexes form with little change in protein structure but with some DNA restructuring. Salt dependent ITC experiments indicate that the electrostatic contribution to binding H10 or H10–C is small ranging from 6% to 17% of the total ΔG.

The formation of two different minor groove complexes between netropsin and A2T2 DNA has been attributed to specific binding and hydration effects. In this study, we have examined the effect of added osmolyte (e.g., TEG or betaine) on the binding of netropsin to a hairpin DNA, d(CGCGAATTCGCGTC-TCCGCGAATTCGCG)-3, having a single A2T2 binding site. Netropsin binding to this DNA construct is described by a two fractional site model with a saturation stoichiometry of 1:1. Free energy changes, ΔGi, for formation of both complex I and complex II decrease continuously as osmolyte is added (e.g., ΔG1 decreases by 1.3 kcal/mol and ΔG2 decreases by 0.8 kcal/mol in 4 m osmolyte vs buffer). The negative ΔCp values for formation of both complexes, I and II, are largely unaffected by the addition of osmolyte. Formation of complex I is accompanied by the acquisition of 31 water molecules vs 19 waters for complex II. The most significant difference between the two osmolytes is that betaine diminishes the fractional formation of the complex II species, virtually eliminating complex II at 2 m. Addition of osmolyte or a decrease in the temperature have approximately the same effect on DNA hydration and on the thermodynamics of netropsin binding.

i-Motif-forming sequences are present in or near the regulatory regions of >40% of all genes, including known oncogenes. We report here the results of a biophysical characterization and computational study of an ensemble of intramolecular i-motifs that model the polypyrimidine sequence in the human c-MYC P1 promoter. Circular dichroism results demonstrate that the mutant sequence (5′-CTT TCC TAC CCTCCC TAC CCT AA-3′) can adopt multiple “i-motif-like,” classical i-motif, and single-stranded structures as a function of pH. The classical i-motif structures are predominant in the pH range 4.2–5.2. The “i-motif-like” and single-stranded structures are the most significant species in solution at pH higher and lower, respectively, than that range. Differential scanning calorimetry results demonstrate an equilibrium mixture of at least three i-motif folded conformations with Tm values of 38.1, 46.6, and 49.5°C at pH 5.0. The proposed ensemble of three folded conformations includes the three lowest-energy conformations obtained by computational modeling and two folded conformers that were proposed in a previous NMR study. The NMR study did not report the most stable conformer found in this study.

TMPyP4 (Mesotetra(N-methyl-4-pyridyl)porphine) is known to have a high affinity for G-quadruplex DNA. However, there is still some controversy over the exact site(s) and mode(s) of TMPyP4 binding to G-quadruplex DNA. We examined TMPyP4 interactions with seven G-quadruplex forming oligonucleotides. The parent oligonucleotide is a 27-mer with a wild-type (WT) G-rich sequence of the Bcl-2 P1 promoter mid-region (5′-d(CGG GCG CGG GAG GAA GGG GGC GGG AGC-3′)). This sequence folds into at least three unique loop isomer quadruplexes. The two mutant oligonucleotides used in this study are shorter (23-mer) sequences in which nonquadruplex core bases were eliminated and two different (-G-G-) → (-T-T-) substitutions were made to restrict the folding complexity. The four additional mutant oligonucleotides were labeled by substituting a 2-aminopurine (2-AP) base for an A or G in either the first three-base lateral loop or the second five- or seven-base lateral loop (depending on the G→T mutation positions). Spectroscopic and microcalorimetric studies indicate that four molecules of TMPyP4 can be bound to a single G-quadruplex. Binding of the first two moles of TMPyP4 appears to occur by an end or exterior mode (K ≈ 1 × 107 M−1), whereas binding of the third and fourth moles of TMPyP4 appears to occur by a weaker, intercalative binding mode (K ≈ 1 × 105 M−1). As the mid-loop size decreases from seven to five bases, end binding occurs with significantly increased affinity. 2-AP-labeled Bcl-2 promoter region quadruplexes show increased fluorescence of the 2-AP base on addition of TMPyP4. The change in fluorescence for 2-AP bases in the second half of the TMPyP4 titration lends support to our previous speculation regarding the intercalative nature of the weaker binding mode.

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P1 promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K+] and 130 mM [K+]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).