Detection of Mg2+-dependent, coaxial stacking rearrangements in a bulged three-way DNA junction by single-molecule FRET

Abstract

Three-way helical junctions (3WJs) arise in genetic processing, and they have architectural and functional roles in structured nucleic acids. An internal bulge at the junction core allows the helical domains to become oriented into two possible, coaxially stacked conformers. Here, the helical stacking arrangements for a series of bulged, DNA 3WJs were examined using ensemble fluorescence resonance energy transfer (FRET) and single-molecule FRET (smFRET) approaches. The 3WJs varied according to the GC content and sequence of the junction core as well as the pyrimidine content of the internal bulge. Mg 2+ titration experiments by ensemble FRET show that both stacking conformations have similar Mg 2+ requirements for folding. Strikingly, smFRET experiments reveal that a specific junction sequence can populate both conformers and that this junction undergoes continual interconversion between the two stacked conformers. These findings will support the development of folding principles for the rational design of functional DNA nanostructures.

Single–Molecule Study of DNAzyme Reveals Its Intrinsic Conformational Dynamics

“…Metal ions are involved in many critical biological processes, such as signal transduction and enzymatic activity. It is been found that metal–ions–dependent conformational changes play a critical role in the activities of proteins, DNA (DNAzymes [ 26 , 27 ] and Holliday junctions [ 39 , 40 ]), and RNA (ribozymes [ 41 ] and riboswitches [ 42 ]). The in vitro selected DNAzyme 39E has been isolated to have specificity to UO 2 2+ .…”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…In addition to the four-way HJ (4WHJ), smFRET has also been applied in determining the conformational dynamics of three-way junction (3WJ, Figure 6 B). Leveille et al [ 92 ] first reported the application of smFRET in the helical stacking arrangement for a series of bulged-in DNA 3WJs. Moreover, replication slippage is a frequent reason for the emergence of 3WJs.…”