Single-molecule
Förster resonance energy transfer microspectroscopic
methods are employed for real-time monitoring and to gain deeper insights
into the formation of the polypurine reverse Hoogsteen hairpin (PPRH)
and its triplex-forming activity. The heterogeneity in the behavior
of individual PPRHs has been documented, and it is seen that the degree
of anharmonic plasticity of the antiparallel hairpin is stabilized
by the formation of reverse Hoogsteen (RH) bonds. While being involved
in the hairpin formation, they flip reversibly between the open and
closed conformations, irrespective of the concentration of ions present
in their microenvironment. However, the nature of the cation present
in the buffer plays a crucial role in determining the structural stability.
The Watson Crick (WC) bonds are found to be more dynamic in the triplex
compared to that of the RH base pairs, indicating the involvement
of progressive WC bonds during the triplex motif formation by the
PPRH. The majority of the intact triplex DNA attained a semifolded
relaxed state before progressing toward a tightly folded state, emphasizing
the fact that the folding mechanism pursues an ambiguous path in the
mode of acquiring the final step of the triple helix motif. Moreover,
the presence of triplex-forming sequences in the regulatory regions
of the genome further provides an intricate link between the experimental
results and sequence occurrence.