Contribution of Loops and Nicks to the Formation of DNA Dumbbells

Melting Behavior and Ligand Binding

Luis A Marky, James Ho, Luis A. Marky

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

We have evaluated the thermodynamic contribution of thymine loops and nicks to the overall stability of double-helical DNA by investigating (1) the melting behavior of two unligated DNA dumbbells and their corresponding core duplexes and (2) the association of netropsin to the central core of four A-T base pairs of these molecules. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of all four deoxyoligonucleotide duplexes. In 10 mM NaPi buffer at pH 7.0, all transitions were monophasic. The dumbbells melt with transition temperatures, Tm, independent of strand concentration, while each duplex melts with transition temperature dependence on strand concentration, characteristic of mono-and bimolecular processes, respectively. The Tm's for the dumbbells correspond to those of single hairpins containing only four base pairs in the stem. We obtain dTm/d log [Na+] values of 10.9-12.5 °C for these molecules, which correspond to similar counterion releases and suggest helical structures with similar charge densities and helical strandedness. Standard thermodynamics profiles at 5 °C reveal that the favorable free energy of forming these ordered structures results from the partial compensation of favorable enthalpies with unfavorable entropies. The stabilization of the dumbbells relative to the core duplexes is enthalpic, due to extra stacking of the nearest loop thymines on the G-C base pairs at both ends of the stem. The association of netropsin was used to thermodynamically probe the integrity of the base-pair stacking at the nick point in the center of the dumbbell molecules, GAT^TAC/GTAATC and CAT^TAG/CTAATG, by direct comparison with the similar sequences of the core duplexes without the nicks, GATTAC/GTAATC and CATTAG/CTAATG. In 10 mM NaPi buffer at pH 7.0, netropsin binds to the central core of A·T base pairs of these four sequences with similar binding affinities of ~108 and similar thermodynamic profiles: ΔG°b = −10.8 kcal·mol−1, ΔH°b = −10.6 kcal·mol−1, and TΔS°b = +0.2 kcal·mol−1, with binding enthalpies independent of salt concentration. Therefore, all four sequences constitute similar binding sites for netropsin. However, we obtained d ln Kb/d ln [Na+] values of −1.0 for the dumbbells and −1.1 for the duplexes, consistent with the lower charge density of these helical structures. The difference of 0.1 is attributed to the absence of a phosphate group at the nick point. In the dumbbells, the presence of the loops creates additional sites for netropsin, formed by two G-C pairs at each end of duplex and the constrained loop groups (forming additional stacked base pairs in a double helical geometry), with binding affinities of ~105 and ΔH°b of −5.1 kcal·mol−1. Both parameters are dependent on salt concentration. Our combined results show that the presence of a nick in the center of the dumbbells is not affecting the overall stacking of the T^T/AA base pair at the center of the dumbbells but interrupts the cooperative melting of the whole molecule.

Original languageEnglish (US)
Pages (from-to)2564-2572
Number of pages9
JournalBiochemistry
Volume32
Issue number10
DOIs
StatePublished - Mar 1 1993

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Netropsin
Nucleic Acid Denaturation
Base Pairing
Melting
Ligands
DNA
Molecules
Thymine
Thermodynamics
Charge density
Enthalpy
Transition Temperature
Buffers
Salts
Association reactions
Freezing
Free energy
Differential scanning calorimetry
Entropy
Differential Scanning Calorimetry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Contribution of Loops and Nicks to the Formation of DNA Dumbbells : Melting Behavior and Ligand Binding. / Marky, Luis A; Ho, James; Marky, Luis A.

In: Biochemistry, Vol. 32, No. 10, 01.03.1993, p. 2564-2572.

Research output: Contribution to journalArticle

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abstract = "We have evaluated the thermodynamic contribution of thymine loops and nicks to the overall stability of double-helical DNA by investigating (1) the melting behavior of two unligated DNA dumbbells and their corresponding core duplexes and (2) the association of netropsin to the central core of four A-T base pairs of these molecules. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of all four deoxyoligonucleotide duplexes. In 10 mM NaPi buffer at pH 7.0, all transitions were monophasic. The dumbbells melt with transition temperatures, Tm, independent of strand concentration, while each duplex melts with transition temperature dependence on strand concentration, characteristic of mono-and bimolecular processes, respectively. The Tm's for the dumbbells correspond to those of single hairpins containing only four base pairs in the stem. We obtain dTm/d log [Na+] values of 10.9-12.5 °C for these molecules, which correspond to similar counterion releases and suggest helical structures with similar charge densities and helical strandedness. Standard thermodynamics profiles at 5 °C reveal that the favorable free energy of forming these ordered structures results from the partial compensation of favorable enthalpies with unfavorable entropies. The stabilization of the dumbbells relative to the core duplexes is enthalpic, due to extra stacking of the nearest loop thymines on the G-C base pairs at both ends of the stem. The association of netropsin was used to thermodynamically probe the integrity of the base-pair stacking at the nick point in the center of the dumbbell molecules, GAT^TAC/GTAATC and CAT^TAG/CTAATG, by direct comparison with the similar sequences of the core duplexes without the nicks, GATTAC/GTAATC and CATTAG/CTAATG. In 10 mM NaPi buffer at pH 7.0, netropsin binds to the central core of A·T base pairs of these four sequences with similar binding affinities of ~108 and similar thermodynamic profiles: ΔG°b = −10.8 kcal·mol−1, ΔH°b = −10.6 kcal·mol−1, and TΔS°b = +0.2 kcal·mol−1, with binding enthalpies independent of salt concentration. Therefore, all four sequences constitute similar binding sites for netropsin. However, we obtained d ln Kb/d ln [Na+] values of −1.0 for the dumbbells and −1.1 for the duplexes, consistent with the lower charge density of these helical structures. The difference of 0.1 is attributed to the absence of a phosphate group at the nick point. In the dumbbells, the presence of the loops creates additional sites for netropsin, formed by two G-C pairs at each end of duplex and the constrained loop groups (forming additional stacked base pairs in a double helical geometry), with binding affinities of ~105 and ΔH°b of −5.1 kcal·mol−1. Both parameters are dependent on salt concentration. Our combined results show that the presence of a nick in the center of the dumbbells is not affecting the overall stacking of the T^T/AA base pair at the center of the dumbbells but interrupts the cooperative melting of the whole molecule.",
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N2 - We have evaluated the thermodynamic contribution of thymine loops and nicks to the overall stability of double-helical DNA by investigating (1) the melting behavior of two unligated DNA dumbbells and their corresponding core duplexes and (2) the association of netropsin to the central core of four A-T base pairs of these molecules. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of all four deoxyoligonucleotide duplexes. In 10 mM NaPi buffer at pH 7.0, all transitions were monophasic. The dumbbells melt with transition temperatures, Tm, independent of strand concentration, while each duplex melts with transition temperature dependence on strand concentration, characteristic of mono-and bimolecular processes, respectively. The Tm's for the dumbbells correspond to those of single hairpins containing only four base pairs in the stem. We obtain dTm/d log [Na+] values of 10.9-12.5 °C for these molecules, which correspond to similar counterion releases and suggest helical structures with similar charge densities and helical strandedness. Standard thermodynamics profiles at 5 °C reveal that the favorable free energy of forming these ordered structures results from the partial compensation of favorable enthalpies with unfavorable entropies. The stabilization of the dumbbells relative to the core duplexes is enthalpic, due to extra stacking of the nearest loop thymines on the G-C base pairs at both ends of the stem. The association of netropsin was used to thermodynamically probe the integrity of the base-pair stacking at the nick point in the center of the dumbbell molecules, GAT^TAC/GTAATC and CAT^TAG/CTAATG, by direct comparison with the similar sequences of the core duplexes without the nicks, GATTAC/GTAATC and CATTAG/CTAATG. In 10 mM NaPi buffer at pH 7.0, netropsin binds to the central core of A·T base pairs of these four sequences with similar binding affinities of ~108 and similar thermodynamic profiles: ΔG°b = −10.8 kcal·mol−1, ΔH°b = −10.6 kcal·mol−1, and TΔS°b = +0.2 kcal·mol−1, with binding enthalpies independent of salt concentration. Therefore, all four sequences constitute similar binding sites for netropsin. However, we obtained d ln Kb/d ln [Na+] values of −1.0 for the dumbbells and −1.1 for the duplexes, consistent with the lower charge density of these helical structures. The difference of 0.1 is attributed to the absence of a phosphate group at the nick point. In the dumbbells, the presence of the loops creates additional sites for netropsin, formed by two G-C pairs at each end of duplex and the constrained loop groups (forming additional stacked base pairs in a double helical geometry), with binding affinities of ~105 and ΔH°b of −5.1 kcal·mol−1. Both parameters are dependent on salt concentration. Our combined results show that the presence of a nick in the center of the dumbbells is not affecting the overall stacking of the T^T/AA base pair at the center of the dumbbells but interrupts the cooperative melting of the whole molecule.

AB - We have evaluated the thermodynamic contribution of thymine loops and nicks to the overall stability of double-helical DNA by investigating (1) the melting behavior of two unligated DNA dumbbells and their corresponding core duplexes and (2) the association of netropsin to the central core of four A-T base pairs of these molecules. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of all four deoxyoligonucleotide duplexes. In 10 mM NaPi buffer at pH 7.0, all transitions were monophasic. The dumbbells melt with transition temperatures, Tm, independent of strand concentration, while each duplex melts with transition temperature dependence on strand concentration, characteristic of mono-and bimolecular processes, respectively. The Tm's for the dumbbells correspond to those of single hairpins containing only four base pairs in the stem. We obtain dTm/d log [Na+] values of 10.9-12.5 °C for these molecules, which correspond to similar counterion releases and suggest helical structures with similar charge densities and helical strandedness. Standard thermodynamics profiles at 5 °C reveal that the favorable free energy of forming these ordered structures results from the partial compensation of favorable enthalpies with unfavorable entropies. The stabilization of the dumbbells relative to the core duplexes is enthalpic, due to extra stacking of the nearest loop thymines on the G-C base pairs at both ends of the stem. The association of netropsin was used to thermodynamically probe the integrity of the base-pair stacking at the nick point in the center of the dumbbell molecules, GAT^TAC/GTAATC and CAT^TAG/CTAATG, by direct comparison with the similar sequences of the core duplexes without the nicks, GATTAC/GTAATC and CATTAG/CTAATG. In 10 mM NaPi buffer at pH 7.0, netropsin binds to the central core of A·T base pairs of these four sequences with similar binding affinities of ~108 and similar thermodynamic profiles: ΔG°b = −10.8 kcal·mol−1, ΔH°b = −10.6 kcal·mol−1, and TΔS°b = +0.2 kcal·mol−1, with binding enthalpies independent of salt concentration. Therefore, all four sequences constitute similar binding sites for netropsin. However, we obtained d ln Kb/d ln [Na+] values of −1.0 for the dumbbells and −1.1 for the duplexes, consistent with the lower charge density of these helical structures. The difference of 0.1 is attributed to the absence of a phosphate group at the nick point. In the dumbbells, the presence of the loops creates additional sites for netropsin, formed by two G-C pairs at each end of duplex and the constrained loop groups (forming additional stacked base pairs in a double helical geometry), with binding affinities of ~105 and ΔH°b of −5.1 kcal·mol−1. Both parameters are dependent on salt concentration. Our combined results show that the presence of a nick in the center of the dumbbells is not affecting the overall stacking of the T^T/AA base pair at the center of the dumbbells but interrupts the cooperative melting of the whole molecule.

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