Quadruplex to Watson–Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Abstract

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K⁺ and Na⁺ ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15 mer deoxynucleotide, d(G₂T₂G₂TGTG₂T₂G₂), labeled with 5′-fluorescein and 3′-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K⁺ ions bind with higher affinity than the Na⁺ ions. The enthalpy changes, ΔH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.