complexity. Worth mentioning is that flow cytometry is currently the only technique
enabling routing, standardized analyses of aptamer – cell interactions.
3 The Characterization Strategy: Aspects to Consider
The decision process for choosing the optimal aptamer from a pool of possible
molecules requires a suitable characterization strategy. Initially, the pool of aptamers
will be narrowed down to a few candidate aptamers which will be subsequently
characterized in more detail. This is often done by biophysical methods with a
suitable throughput, either ranking the candidates by affinity or off-rate (here MST
and SPR are the most suitable techniques). Alternatively, filter-binding assays can
rapidly give the required information on which aptamers bind to the target. It should
be noted that in an optimal case, already at this step, the downstream application of
the aptamer is considered. Choosing a therapeutic candidate aptamer, which shall
later be applied systemically, can have completely different requirements at the
initial selection step, compared to choosing the right candidate for a diagnostic
aptamer, immobilized on a point-of-care device.
After having chosen a few initial candidates, the subsequent detailed characterization must reflect which of the essential binding parameters – kinetics, affinity,
and/or thermodynamics – are key for the later application. A fast on-rate and a slow
off-rate may be most important if the aptamer needs to be tightly bound to a
therapeutic target. The characterization strategy should also keep the conditions in
mind at which the aptamer-target interaction will take place. Biophysical methods
allowing to study aptamer-target interactions in bioliquids (e.g., human serum or
plasma, bacterial cell lysate) may give important information on the behavior of a
therapeutic aptamer in such complex matrices or of a diagnostic aptamer in a
comparable sample. Suitable techniques for aptamer analysis in bioliquids are
MST, BLI, or SwitchSENSE. In general, it is recommended to apply a minimum
of two or three orthogonal methods describing the binding of an aptamer and its
target, to obtain a reliable and robust view on the binding event.
Having chosen one or two suitable aptamer candidates possessing the desired
binding parameters and characteristics, the next step in development is usually the
elucidation of structural binding information by methods such as X-ray crystallography or NMR (not described in this chapter).
4 Affinity Constant: Estimated by Biophysical Methods
As indicated by its name, “equilibrium constant,” the K D is a natural constant
describing the binding strength of two interaction partners in any possible system.
Therefore, the calculated affinity values of any biophysical analytical method should
theoretically be the same. However, biophysical methods can only help to obtain
Biophysical Characterization of Aptamer-Target Interactions
11
enabling routing, standardized analyses of aptamer – cell interactions.
3 The Characterization Strategy: Aspects to Consider
The decision process for choosing the optimal aptamer from a pool of possible
molecules requires a suitable characterization strategy. Initially, the pool of aptamers
will be narrowed down to a few candidate aptamers which will be subsequently
characterized in more detail. This is often done by biophysical methods with a
suitable throughput, either ranking the candidates by affinity or off-rate (here MST
and SPR are the most suitable techniques). Alternatively, filter-binding assays can
rapidly give the required information on which aptamers bind to the target. It should
be noted that in an optimal case, already at this step, the downstream application of
the aptamer is considered. Choosing a therapeutic candidate aptamer, which shall
later be applied systemically, can have completely different requirements at the
initial selection step, compared to choosing the right candidate for a diagnostic
aptamer, immobilized on a point-of-care device.
After having chosen a few initial candidates, the subsequent detailed characterization must reflect which of the essential binding parameters – kinetics, affinity,
and/or thermodynamics – are key for the later application. A fast on-rate and a slow
off-rate may be most important if the aptamer needs to be tightly bound to a
therapeutic target. The characterization strategy should also keep the conditions in
mind at which the aptamer-target interaction will take place. Biophysical methods
allowing to study aptamer-target interactions in bioliquids (e.g., human serum or
plasma, bacterial cell lysate) may give important information on the behavior of a
therapeutic aptamer in such complex matrices or of a diagnostic aptamer in a
comparable sample. Suitable techniques for aptamer analysis in bioliquids are
MST, BLI, or SwitchSENSE. In general, it is recommended to apply a minimum
of two or three orthogonal methods describing the binding of an aptamer and its
target, to obtain a reliable and robust view on the binding event.
Having chosen one or two suitable aptamer candidates possessing the desired
binding parameters and characteristics, the next step in development is usually the
elucidation of structural binding information by methods such as X-ray crystallography or NMR (not described in this chapter).
4 Affinity Constant: Estimated by Biophysical Methods
As indicated by its name, “equilibrium constant,” the K D is a natural constant
describing the binding strength of two interaction partners in any possible system.
Therefore, the calculated affinity values of any biophysical analytical method should
theoretically be the same. However, biophysical methods can only help to obtain
Biophysical Characterization of Aptamer-Target Interactions
11