Biomedical Engineering Reference
In-Depth Information
An approximately logarithmic dependence of the rupture force on the loading rate
was found, consistent with theoretical expectations [
14
,
15
].
Eco
RI binds most strongly to the sequence GAATTC; however, some variations
of this sequence still lead to different degrees of binding. Can MD predict the
strength of binding for sequences differing by one or two basepairs from the
cognate sequence?
The atomic-scale detail of the SMD simulations permitted us to identify the
order in which each nucleotide of the DNA within the enzyme dissociated during
rupture of the enzyme, thereby obtaining a qualitative estimate of the contribution
of each basepair to the strength of the
Eco
RI-DNA bond [
23
]. Although there was a
tendency for nucleotides nearest to the portion of the DNA to which force was
applied to dissociate first, the first and last basepairs of the cognate sequence
(
G
AATT
C
) were shown to be especially quick to dissociate. In accord with this,
mutation of one of these two basepairs was shown to have only a small effect on the
bulk free energy of dissociation (
DG
) - weakening it from the cognate value of
13.2 kcal/mol. The middle basepairs were slightly slower to dissociate
(GA
AT
TC), in agreement with the bulk
15.2 to
8.6 kcal/mol associated with the
mutation of one of them. The underlined basepairs of G
A
AT
T
C had a significant
tendency to dissociate later than the others and were therefore predicted to have the
greatest effect on
Eco
RI-DNA binding. Indeed, mutating just one of these two
basepairs weakens the binding dramatically:
DG
of
4.9 kcal/mol. Furthermore, no
specific binding at all was observed in experiments in which both were mutated.
Thus, the relative importance of each basepair to the binding predicted by simulation
was in agreement with experimental measurements of the binding free energy.
The results presented here emphasize the sharpness of the threshold voltage.
In fact, experiments seem to show that the dependence of the rupture rate on the
transmembrane voltage is much stronger than exponential and apparently stronger
than what is expected from theories of escape from a single well in the high-barrier
limit [
11
,
12
,
46
]. Interaction of DNA with surfaces in and around pores have been
implicated in modifying rupture kinetics from expected forms [
47
]. Furthermore,
simulations have shown that, in the presence of strong attraction between the DNA
and the pore walls, a threshold voltage for DNA translocation can exist even
without an enzyme. Much work remains to be done to understand the origin of
the sharpness of the threshold voltage and the small variance of the observed
rupture times.
DG¼
14.3 Unfolding of Hairpin DNA
Just as a bound enzyme can block passage of dsDNA through a nanopore unless a
sufficiently large force is applied to dissociate the enzyme, regions having helical
secondary structure on a DNA molecule can halt its translocation unless a suffi-
ciently large force is applied to unfold these regions. Hairpin DNA, having both a
double-helical portion and an overhanging coil portion, has been extensively
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