Break in the Heat Capacity Change at 303 K for Complex Binding of Netropsin to AATT Containing Hairpin DNA Constructs

Freyer, Matthew W.; Buscaglia, Robert; Hollingsworth, Amy; Ramos, Joseph; Blynn, Meredith; Pratt, Rachael; Wilson, W. David; Lewis, Edwin A.

doi:10.1529/biophysj.106.098723

Cited by 24 publications

(45 citation statements)

References 41 publications

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“…The first entropically driven binding event results in a binding stoichiometry ratio of 1:1 between 1 and DNA duplex while the second binding event is enthalpy driven and involves 2–4 molecules of ligand binding. Previous reports have characterized similar ITC profiles in ligand–DNA interaction …”

Section: Resultssupporting

confidence: 53%

Shape readout of AT‐rich DNA by carbohydrates

2014

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Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove-binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA “shape readout” properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT-rich) DNA. Isothermal Titration Calorimetry (ITC), Circular Dichroism (CD) titration, Ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT-rich DNA duplex d[5′-G2A6T6C2-3′]. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two-binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy driven with a binding constant of approximately 108 M−1. ITC-derived binding enthalpies were used to obtain the binding-induced change in heat capacity (ΔCp) of -225±19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT-tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT-rich DNA (d[5′-G2A6T6C2-3′]) > B form alternate AT-rich DNA (d[5′-G2(AT)6C2-3′]) > A form GC-rich DNA (d[5′-A2G6C6T2-3′]), demonstrating the preference of ligand 1 for B* form DNA.

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Section: Resultssupporting

confidence: 53%

Shape readout of AT‐rich DNA by carbohydrates

2014

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“…In the course of our experimental work, Freyer et al . ( 25–27 ) reported that the binding of some minor groove agents to several hairpin-forming DNA sequences is accompanied by ‘anomalous’ ITC curves that can be described in terms of a ‘two-fractional-sites’ model. The model assumes the existence of pairs of DNA structures, ligand structures or ligand–DNA structures that are separated by a large energy barrier so that paired structures are not in equilibrium, at least at temperatures and on the timescale of the ITC experiments.…”

Section: Resultsmentioning

confidence: 99%

What drives the binding of minor groove-directed ligands to DNA hairpins?

Lah

Drobnak

Dolinar

et al. 2007

Nucleic Acids Research

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Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.

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“…In our case, the decrease of the binding enthalpy should come exclusively from the annealing of the unannealed DNA basepairs, and protein folding should not be a factor. There are a few cases of unusual ITC curves for the binding of small ligands to DNA (40)(41)(42). For example, the titration of the minor-groove binder, netropsin, into a few DNA hairpins yielded rather complex ITC curves (40) that were fit and explained by a ''two-fractional-sites'' model.…”

Section: Discussionmentioning

confidence: 99%

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

Joynt

Morillo

Leng

2009

Biophysical Journal

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HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)(2) is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5'-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3' (FL-AT-1) and 5'-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3' (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to approximately 200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.

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Break in the Heat Capacity Change at 303 K for Complex Binding of Netropsin to AATT Containing Hairpin DNA Constructs

Cited by 24 publications

References 41 publications

Shape readout of AT‐rich DNA by carbohydrates

Shape readout of AT‐rich DNA by carbohydrates

What drives the binding of minor groove-directed ligands to DNA hairpins?

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

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