Unfolding thermodynamics of intramolecular G-quadruplexes: Base sequence contributions of the loops

Chris M. Olsen, Hui Ting Lee, Luis A Marky

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the "chair" conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (TMs) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with TMs in the range from 43.2 to 56.5 °C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.

Original languageEnglish (US)
Pages (from-to)2587-2595
Number of pages9
JournalJournal of Physical Chemistry B
Volume113
Issue number9
DOIs
StatePublished - Mar 5 2009

Fingerprint

Thermodynamics
thermodynamics
Differential scanning calorimetry
Enthalpy
Substitution reactions
Ions
Circular dichroism spectroscopy
Drug therapy
Molecules
Water
Fluorescence spectroscopy
Dichroism
Hydration
Superconducting transition temperature
Conformations
Melting
DNA
Aging of materials
dichroism
heat measurement

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

Unfolding thermodynamics of intramolecular G-quadruplexes : Base sequence contributions of the loops. / Olsen, Chris M.; Lee, Hui Ting; Marky, Luis A.

In: Journal of Physical Chemistry B, Vol. 113, No. 9, 05.03.2009, p. 2587-2595.

Research output: Contribution to journalArticle

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abstract = "G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the {"}chair{"} conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (TMs) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with TMs in the range from 43.2 to 56.5 °C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.",
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