Probing the temperature unfolding of a variety of DNA secondary structures using the fluorescence properties of 2-aminopurine

Hui Ting Lee, Lela Waters, Chris M. Olsen, Irine Khutsishvili, Luis A. Marky

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


The fluorescence probe 2-aminopurine (2AP) is widely used to monitor the molecular environment, including the local solvent environment, and overall dynamics of nucleic acids and nucleic acid-ligand complexes. This work reports on the temperature-induced conformational flexibility of a variety of secondary structures of nucleic acids using optical and calorimetric melting techniques, and evaluates the usefulness of fluorescence melting curves obtained from monitoring the fluorescence changes of 2AP as a function of temperature. Furthermore, the base stacking properties of 2AP are examined in these structures for a first time. Specifically, we incorporated single A → 2AP substitutions into a variety of DNA structures, such as a single strand (SS), a dodecamer duplex (Duplex), a hairpin loop (Hairpin), a G-quadruplex (G2), and an intramolecular triplex (Triplex). A combination of fluorescence, U V, and circular dichroism spectroscopies, and differential scanning calorimetric (DSC) techniques is used to investigate their temperature-induced unfolding. The melting curves of each molecule show monophasic transitions with similar T Ms and van't Hoff enthalpies indicating that all transitions are two-state and that the fluorescence changes for the unstacking of 2AP follow the unfolding of the whole molecule. The DSC thermodynamic profiles of each 2AP modified molecule, relative to their unmodified control molecules, yielded folding ΔΔG°s of 1.6 kcal/mol (Duplex), 3.1 kcal/mol (Hairpin), 1.6 kcal/mol (Triplex), and -1.7 kcal/mol (G2). These ΔΔG°s are driven by unfavorable differential enthalpies (Duplex and Hairpin), favorable differential enthalpy (G2), and by a favorable differential entropy term for Triplex. These enthalpy effects are explained in terms of stacking and hydration contributions, that are associated with the local environment that 2AP is experiencing. For example, the lower ΔΔH cal value of 8.7 kcal/mol (Hairpin), relative to Duplex, is due to weaker basepair stacks and higher hydration state of the stem of Hairpin. We conclude that the incorporation of 2AP in nucleic acids is a useful tool to monitor their temperature-induced unfolding; especially, when these sensitive fluorescent moieties are placed in the proper molecular environment of the nucleic acid.

Original languageEnglish (US)
Pages (from-to)443-453
Number of pages11
JournalActa Chimica Slovenica
Issue number3
StatePublished - 2012


  • 2 amino purine • T base-pair stacking
  • 2-Aminopurine
  • Differential scanning calorimetry
  • Fluorescence
  • Unfolding thermodynamics

ASJC Scopus subject areas

  • Chemistry(all)


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