jp5101413_si_001.pdf (3.15 MB)
Internal vs Fishhook Hairpin DNA: Unzipping Locations and Mechanisms in the α‑Hemolysin Nanopore
journal contribution
posted on 2015-12-17, 05:48 authored by Yun Ding, Aaron M. Fleming, Henry S. White, Cynthia J. BurrowsStudies
on the interaction of hairpin DNA with the α-hemolysin
(α-HL) nanopore have determined hairpin unzipping kinetics,
thermodynamics, and sequence-dependent DNA/protein interactions. Missing
from these results is a systematic study comparing the unzipping process
for fishhook (one-tail) vs internal (two-tail) hairpins when they
are electrophoretically driven from the cis to the trans side of α-HL via a 30-mer single-stranded tail.
In the current studies, fishhook hairpins showed long unzipping times
with one deep blockage current level. In contrast, the internal hairpins
demonstrated relatively fast unzipping and a characteristic pulse-like
current pattern. These differences were further explored with respect
to stem length and sequence context. Further, a series of internal
hairpins with asymmetric tails were studied, for which it was determined
that a second tail longer than 12 nucleotides results in internal
hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved
like fishhook hairpins. Interestingly, these studies were able to
resolve a current difference of ∼6% between hairpin DNA immobilized
in the nanopore waiting to unzip vs the translocating unzipped DNA,
with the latter showing a deeper current blockage level. This demonstration
of different currents for immobilized and translocating DNA has not
been described previously. These results were interpreted as fishhook
hairpins unzipping inside the vestibule, while the internal hairpins
unzip outside the vestibule of α-HL. Lastly, we used this knowledge
to study the unzipping of a long double-stranded DNA (>50 base
pairs)
outside the vestibule of α-HL. The conclusions drawn from these
studies are anticipated to be beneficial in future application of
nanopore analysis of nucleic acids.