Investigation of the Melting Behavior of DNA Three-Way Junctions in the Closed and Open States

Abstract

Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell). The overall results show that both junctions are stable over a wide range of salt concentrations. However, Open-J is more stable due to a higher enthalpy contribution from the formation of a higher number of basepair stacks whereas Closed-J has a defined structure and retains the basepair stacking of all three stems. The comparison of the experimental results of Closed-J and Open-J with those of their component stem-loop motifs allowed us to be more specific about their cooperative unfolding. For instance, Closed-J sacrifices thermal stability of the Dumbbell structure to maintain an overall folded state. At higher salt concentration, the simultaneous unfolding of the above domains is lost, resulting in the unfolding of the three separate stems. In contrast, the junction of Open-J in low salt retains the thermal and enthalpic stability of the Dumbbell structure although sacrificing stability of the CTATC stem. The relative stability of Dumbbell is the primary reason for the higher ΔG°₍₅₎, or free energy, value seen for Open-J at low salt. Higher salt not only maintains thermal stability of the Dumbbell structure in Open-J but causes the CTATC stem to fully fold.

Figure 1

Cartoon of the sequences of DNA three-way junctions, drawn according to their hypothesized structure, with the Dumbbell forming the base of both three-way junctions upon which the different hairpins are stacked on top.

Figure 2

UV melting curves at 268 nm in 10 mM NaPi at pH 7.0 (left panels) and in 10 mM NaPi, 0.2 M NaCl at pH 7.0 (right panels); T_M (±0.5°C) using oligonucleotide concentrations of 2 μM. (Top panels) Shown here are melting curves of Closed-J (solid line) and Open-J (dashed line). (Bottom panels) Shown here are melting curves of Dumbbell (solid line), CTATC-Hp (dashed line), and GAAATT-Hp (dotted line).

Figure 3

Combined UV melting curves at 268 nm in 10 mM NaPi at pH 7.0 (left panels) and in 10 mM NaPi, 0.2 M NaCl at pH 7.0 (right panels) are shown. (Top panels) Given here are melting curves of Closed-J (solid line) and Dumbbell + GAAATT-Hp (dashed line). (Bottom panels) Given here are melting curves of Open-J (solid line) and Dumbbell + CTATC-Hp (dashed line).

Figure 4

(Left) CD spectra at 5°C in 10 mM NaPi at pH 7.0. (Top left) CD spectra of Closed-J (solid line) and Open-J (dashed line) are given. (Bottom left) CD spectra of Dumbbell (dotted line), CTATC-Hp (solid line), and GAAATT-Hp (dashed line) are given. (Top right) CD spectra of Closed-J (solid line) and Dumbbell + GAAATT-Hp (dashed line) are given. (Bottom right) Shown here are CD spectra of Open-J (solid line) and Dumbbell + CTATC-Hp (dashed line).

Figure 5

DSC curves of Closed-J (top) and Open-J (bottom) in 10 mM NaPi buffer at pH 7.0 (solid lines); with the addition of 0.1 M NaCl (squares); or with the addition of 0.2 M NaCl (triangles).

Figure 6

DSC curves are shown of Dumbbell (top), CTATC-Hp (middle), and GAAATT-Hp (bottom) in 10 mM NaPi buffer at pH 7.0 (solid lines); with the addition of 0.1 M NaCl (squares); or with the addition of 0.2 M NaCl (triangles).

Figure 7

Combined DSC thermograms in (left) 10 mM NaPi at pH 7.0 and (right) 10 mM NaPi, 0.2 M NaCl, pH 7.0; T_M (±0.5°C). (Top) Given here are combined DSC thermogram of Closed-J (solid line); Dumbbell + GAAATT-Hp (dotted line). (Bottom) Shown here is a DSC thermogram of Open-J (solid line), Dumbbell + CTATC-Hp (dotted line).

Figure 8

Cartoon of potential alternate structures of Closed-J and Open-J.