engineered, they can be obtained with high efficiency by applying a combinatorial
technique termed SELEX. The products of SELEX are so-called aptamers, i.e.,
nucleic acids that bind to a given target with high affinity. Thus, identified aptamers
hold great interest, e.g., in analytics, diagnostics, and potentially in material science.
However, as opposed to canonical Watson–Crick-based DNA structures [152], their
mechanical properties have hardly been investigated at all [153].
One particularly interesting problem in the context of aptamer science is the
detection of the binding of small molecules to macromolecules, and the impact of
binding on the mechanical properties of nucleic acid structures. Previous approaches
to this problem suffer from the need to chemically alter either the macromolecule or
the small molecule (hereafter called the “analyte”) in order to allow proper detection.
Because even the smallest alterations to the chemical structure of an analyte induce
significant changes to its physicochemical properties, we have devised a new
approach to measure the interaction of nucleic acid aptamers and small molecules
by atomic force spectroscopy (AFS). The approach combines both of the abovementioned structural features of DNA, fusing the structural domain of an aptamer
to the faithfully hybridizing stretches of Watson–Crick helices. Rather than
immobilizing the interaction partners in a traditional AFS experiment on opposing
surfaces (i.e., an aptamer on the AFM tip and the small molecule on a substrate
surface), we split an aptamer structure into two parts and equipped both halves with
flanking regions that would recognize the respective other half by Watson–Crick base
pairing. Neither half of the aptamer alone retains sufficient structure for binding the
analyte; instead, the fully competent binding pocket is transiently generated during
the short period of an AFS measurement cycle, in which both components are in
spatial proximity. Measurements in the absence and in the presence of an analyte
should then reveal whether this period is sufficiently long to allow detectable binding
events. Previous experiments had shown that transient binding motifs could in
principle be assembled from DNA strands in situ. Thus, binding of a third DNA
strand to a transient double-stranded DNA, as well as binding of intercalating small
molecules to such a triple helix, could indeed be detected by force spectroscopy
[154, 155]. As binding resulted in an increase of the most probable rupture force in
each case, this was also anticipated for the new aptamer approach. However, it was
entirely unclear whether the rather complicated binding pockets would form in a fast
and reproducible manner, and whether the binding of a single analyte molecule would
be strong enough to be detectable.
For a proof of concept, we used a DNA aptamer that binds adenosine
monophosphate (AMP) [156]. The sequence of the DNA aptamer was 3-ACT
GGAAGGAGGAGATGC-GCATCTAGGAGGTCCAGT-5 and provided two
binding pockets (underlined bases) for AMP. The structure suggests a contribution
of base stacking as well as of a total of five H-bonds to the binding derived
from NMR analysis [157]. Furthermore, the aptamer structure is symmetric and
thus provides binding pockets for two molecules of AMP, whose binding is
highly cooperative [157, 158]. For the force spectroscopy measurements, the
DNA sequence was split symmetrically in length between C–G. Then, the split
sequences were equipped with a poly-A tail at the 5-ends, a six-carbon spacer to
Mechanical Properties of Single Molecules and Polymer Aggregates
53
technique termed SELEX. The products of SELEX are so-called aptamers, i.e.,
nucleic acids that bind to a given target with high affinity. Thus, identified aptamers
hold great interest, e.g., in analytics, diagnostics, and potentially in material science.
However, as opposed to canonical Watson–Crick-based DNA structures [152], their
mechanical properties have hardly been investigated at all [153].
One particularly interesting problem in the context of aptamer science is the
detection of the binding of small molecules to macromolecules, and the impact of
binding on the mechanical properties of nucleic acid structures. Previous approaches
to this problem suffer from the need to chemically alter either the macromolecule or
the small molecule (hereafter called the “analyte”) in order to allow proper detection.
Because even the smallest alterations to the chemical structure of an analyte induce
significant changes to its physicochemical properties, we have devised a new
approach to measure the interaction of nucleic acid aptamers and small molecules
by atomic force spectroscopy (AFS). The approach combines both of the abovementioned structural features of DNA, fusing the structural domain of an aptamer
to the faithfully hybridizing stretches of Watson–Crick helices. Rather than
immobilizing the interaction partners in a traditional AFS experiment on opposing
surfaces (i.e., an aptamer on the AFM tip and the small molecule on a substrate
surface), we split an aptamer structure into two parts and equipped both halves with
flanking regions that would recognize the respective other half by Watson–Crick base
pairing. Neither half of the aptamer alone retains sufficient structure for binding the
analyte; instead, the fully competent binding pocket is transiently generated during
the short period of an AFS measurement cycle, in which both components are in
spatial proximity. Measurements in the absence and in the presence of an analyte
should then reveal whether this period is sufficiently long to allow detectable binding
events. Previous experiments had shown that transient binding motifs could in
principle be assembled from DNA strands in situ. Thus, binding of a third DNA
strand to a transient double-stranded DNA, as well as binding of intercalating small
molecules to such a triple helix, could indeed be detected by force spectroscopy
[154, 155]. As binding resulted in an increase of the most probable rupture force in
each case, this was also anticipated for the new aptamer approach. However, it was
entirely unclear whether the rather complicated binding pockets would form in a fast
and reproducible manner, and whether the binding of a single analyte molecule would
be strong enough to be detectable.
For a proof of concept, we used a DNA aptamer that binds adenosine
monophosphate (AMP) [156]. The sequence of the DNA aptamer was 3-ACT
GGAAGGAGGAGATGC-GCATCTAGGAGGTCCAGT-5 and provided two
binding pockets (underlined bases) for AMP. The structure suggests a contribution
of base stacking as well as of a total of five H-bonds to the binding derived
from NMR analysis [157]. Furthermore, the aptamer structure is symmetric and
thus provides binding pockets for two molecules of AMP, whose binding is
highly cooperative [157, 158]. For the force spectroscopy measurements, the
DNA sequence was split symmetrically in length between C–G. Then, the split
sequences were equipped with a poly-A tail at the 5-ends, a six-carbon spacer to
Mechanical Properties of Single Molecules and Polymer Aggregates
53